VOLUME 2 • NO.

1 • 2003 • ISSN 1303-3646

HAL‹Ç UNIVERSITY
‹STANBUL
FACULTY OF ARTS AND SCIENCES
1998

Journal of Cell and

Molecular Biology
 Journal of Cell and
M o l e c u l a r Biology
Haliç University
Published by Haliç University
Faculty of Arts and Sciences Faculty of Arts and Sciences
J o u r n a l o f C e l l a n d M o l e c u l a r Biology
Founder Editor
Prof. Dr. Gündüz GED‹KO⁄LU
Atilla ÖZALPAN
President of Board of Trustee
Rights held by
Prof. Dr. Ahmet YÜKSEL Associate Editor
Rector Narç›n PALAVAN ÜNSAL

Correspondence Address:
The Editorial Office Editorial Board
J o u r n a l o f C e l l a n d M o l e c u l a r Biology
Çimen ATAK
Haliç Üniversitesi, Fen-Edebiyat Fakültesi,
Ahmet Vefik Pafla Cad., No: 1, 34280, Atok OLGUN
F›nd›kzade, ‹stanbul-Turkey
P›nar ÖZKAN
Phone: 90 212 530 50 24
Fax: 90 212 530 35 35 Nihal BÜYÜKUSLU
E-mail: jcmb@halic.edu.tr Kürflat ÖZD‹LL‹
J o u r n a l o f C e l l a n d M o l e c u l a r Biology is Damla BÜYÜKTUNÇER
indexed in EBSCO database.
Özge EM‹RO⁄LU
Summaries of all articles in this journal are
available free of charge from www.halic.edu.tr Mehmet Ali TÜFEKÇ‹
ISSN 1303-3646 Merve ALO⁄LU
printed at yaflar printing house Asl› BAfiAR

Advisory Board
Igor ALEXANDROV, Dubna, Russia Ünal EGEL‹, Bursa, Turkey
Çetin ALGÜNEfi, Edirne, Turkey Candan JOHANSEN, ‹stanbul, Turkey
Aglaia ATHANASSIADOU, Patros, Greece As›m KADIO⁄LU, Trabzon, Turkey
fiehnaz BOLKENT, ‹stanbul, Turkey Valentine KEFEL‹, Pennsylvania, USA
Nihat BOZCUK, Ankara, Turkey Göksel OLGUN, Edirne, Turkey
‹smail ÇAKMAK, ‹stanbul, Turkey U¤ur ÖZBEK, ‹stanbul, Turkey
Adile ÇEV‹KBAfi, ‹stanbul, Turkey Zekiye SULUDERE, Ankara, Turkey
Beyaz›t ÇIRAKO⁄LU, ‹stanbul, Turkey ‹smail TÜRKAN, ‹zmir, Turkey
Ayfl›n ÇOTUK, ‹stanbul, Turkey Mehmet TOPAKTAfi, Adana, Turkey
Zihni DEM‹RBA⁄, Trabzon, Turkey Meral ÜNAL, ‹stanbul, Turkey
Mustafa DJAMGOZ, London, UK Mustafa YAT‹N, Boston, USA
Aglika EDREVA, Sofia, Bulgaria Ziya Z‹YLAN, ‹stanbul, Turkey
Journal of Cell and
M o l e c u l a r Biology

Volume 2/2003

Haliç University
Faculty of Arts and Sciences
‹stanbul-TURKEY
 Journal of Cell and
M o l e c u l a r Biology
Haliç University
Published by Haliç University
Faculty of Arts and Sciences Faculty of Arts and Sciences
J o u r n a l o f C e l l a n d M o l e c u l a r Biology
Founder Editor
Prof. Dr. Gündüz GED‹KO⁄LU
Atilla ÖZALPAN
President of Board of Trustee
Rights held by
Prof. Dr. Ahmet YÜKSEL Associate Editor
Rector Narç›n PALAVAN ÜNSAL

Correspondence Address:
The Editorial Office Editorial Board
J o u r n a l o f C e l l a n d M o l e c u l a r Biology
Çimen ATAK
Haliç Üniversitesi, Fen-Edebiyat Fakültesi,
Ahmet Vefik Pafla Cad., No: 1, 34280, Atok OLGUN
F›nd›kzade, ‹stanbul-Turkey
P›nar ÖZKAN
Phone: 90 212 530 50 24
Fax: 90 212 530 35 35 Nihal BÜYÜKUSLU
E-mail: jcmb@halic.edu.tr Kürflat ÖZD‹LL‹
J o u r n a l o f C e l l a n d M o l e c u l a r Biology is Damla BÜYÜKTUNÇER
indexed in EBSCO database.
Özge EM‹RO⁄LU
Summaries of all articles in this journal are
available free of charge from www.halic.edu.tr Mehmet Ali TÜFEKÇ‹
ISSN 1303-3646 Merve ALO⁄LU
printed at yaflar printing house Asl› BAfiAR

Advisory Board
Igor ALEXANDROV, Dubna, Russia Ünal EGEL‹, Bursa, Turkey
Çetin ALGÜNEfi, Edirne, Turkey Candan JOHANSEN, ‹stanbul, Turkey
Aglaia ATHANASSIADOU, Patros, Greece As›m KADIO⁄LU, Trabzon, Turkey
fiehnaz BOLKENT, ‹stanbul, Turkey Valentine KEFEL‹, Pennsylvania, USA
Nihat BOZCUK, Ankara, Turkey Göksel OLGUN, Edirne, Turkey
‹smail ÇAKMAK, ‹stanbul, Turkey U¤ur ÖZBEK, ‹stanbul, Turkey
Adile ÇEV‹KBAfi, ‹stanbul, Turkey Zekiye SULUDERE, Ankara, Turkey
Beyaz›t ÇIRAKO⁄LU, ‹stanbul, Turkey ‹smail TÜRKAN, ‹zmir, Turkey
Ayfl›n ÇOTUK, ‹stanbul, Turkey Mehmet TOPAKTAfi, Adana, Turkey
Zihni DEM‹RBA⁄, Trabzon, Turkey Meral ÜNAL, ‹stanbul, Turkey
Mustafa DJAMGOZ, London, UK Mustafa YAT‹N, Boston, USA
Aglika EDREVA, Sofia, Bulgaria Ziya Z‹YLAN, ‹stanbul, Turkey
J o u r n a l o f C e l l a n d M o l e c u l a r Biology

C O N T E N T S Volume 2, No.1, 2003

Dedication
Review articles
P o l y a m i n e s i n p l a n t s : An overview
Bitkilerde poliaminler: Genel bir bak›fl
R. Kaur-Sawhney, A.F. Tiburcio, T. Altabella, A.W. Galston 1-12
P h e n o l i c c y c l e i n p l a n t s a n d e n v i ro n m e n t
Bitkilerde fenolik döngü ve çevre
V. I. Kefeli, M. V. Kalevitch, B. Borsari 13-18
R e s e a rc h p a p e r s
The short-term effects of single toxic dose of citric acid in mice
Farelerde sitrik asidin tek toksik dozunun k›sa süreli etkileri
T. Aktaç, A. Kabo¤lu, E. Bakar, H. Karakafl 19-23
C h a r a c t e r i s a t i o n o f R P P 7 mutant lines of the col-5 ecotype of Arabidopsis thaliana
Arabidopsis thaliana’n›n col-5 ekotipinden elde edilen mutant hatlardan RPP7
geninin karakterizasyonu
C. Can, M. Özaslan, E. B. Holub 25-30
The effect of meta - t o p o l i n o n p r o t e i n p r o f i l e i n r a d i s h c o t y l e d o n s
Meta-topolinin turp kotiledonlar›nda protein profiline etkisi
S. Ça¤, N. Palavan-Ünsal 31-34

T h e e f f e c t o f e l e c t romagnetic fields on oxidative DNA d a m a g e

Elektromanyetik alan›n oksidatif DNA hasar› üzerindeki etkisi
S. ‹fller, G. Erdem 35-38
C h romosomes of a balanced translocation case evaluated with atomic force
m i c r oscopy
Dengeli translokasyon vakas›nda kromozomlar›n atomik güç mikroskobu ile
de¤erlendirilmesi
Z. Y›lmaz, M. A. Ergun, E. Tan 39-42
E f f e c t o f e p i r u b i c i n o n m i t o t i c i n d e x i n c u l t u r ed L-cells
Epirubisinin kültürdeki L-hücrelerinde mitotik indekse etkisi
G. Özcan Ar›can, M. Topçul 43-48
L e t t e r to editor 49-51
Book reviews 53
Instructions to authors 55-56
 This issue is dedicated to

P rof. Dr. A r t h u r W. Galston

for his invaluable contribution to plant biology
 A r t h u r W. G a l s t o n , C u r r i c u l u m Vitae

B o r n : April 21, 1920 Eaton Professor of Botany, Emeritus, Department of

Molecular, Cellular and Developmental Biology,
E d u c a t i o n : B.S. Cornell University, 1940; Yale University, New Haven, CT 06520-8103,
M.S. University of Illinois, 1942; Ph. D. 1943 Tel. (203) 432-6161; e-mail arthur.galston@yale.edu

H o n o r s : Elected to Phi Beta Kappa; Phi Kappa Phi; Sigma Xi; American Academy of Arts and Sciences, National
Sigma Xi Lecturer, 1966; National Phi Beta Kappa Visiting Scholar, 1972-1973; Award of the New York Academy
of Sciences, 1979; William Clyde De Vane Medal for lifelong teaching and scholarship, Yale University, 1994;
Honorary LL.D, 1980 Iona; Honorary Ph. D., Hebrew University of Jerusalem, 1992.

E x p e r i e n c e : Plant Physiologist, Emergency Rubber Project, California Institute of Technology 1943-1944; Instuctor
in Botany, Yale University, 1946-1947; Senior Research California Institute of Technology, 1947-1950; Associate
Professor of Biology, California Institute of Technology, 1951-1955. Professor of Plant Physiology, Department of
Botany, Yale University 1955-1961; Chairman, Department of Botany, 1961-1962; Director, Division of Biological
Sciences, Yale University, 1965-1966; Professor of Biology, 1962-1973; Eaton Professor of Botany, 1973-;
Chairman, Department of Biology 1985-1988; Eaton Professor Emeritus, 1990.

Fellow of the John Simon Guggenheim Memorial Foundation, Stockholm and Sheffield, 1950-1951; Fulbright
Fellow, Canberra, Australia, 1960-1961; National Science Foundation Faculty Fellow, London 1967-1968; Albert
Einstein Fellow and Visiting Professor, Hebrew University of Jerusalem, 1980; Visiting Fellow Wolfson College,
Cambridge, England, 1983; Visiting Scientist, RIKEN Institute, Wako, Saitama, Japan, 1988-1989.

Secretary, American Society of Plant Physiologists, 1955-1957; Vice President, 1957-1958; President, 1962-1963.
Secretary-Treasurer, International Association for Plant Physiology, 1962-1967. Vice-President
Botanical Society, 1967-1968; President 1968-1969; Award, 1970. Member, Commitee on Space Biology and
Medicine, National Research Council; Member Life Sciences Advisory Committee, NASA; also Long Range
Strategic Planning Committee in Life Sciences Advisory Committee, NASA; Member, NASA Disciplinary Working
Group for CELLS (Controlled Ecological Life Support Sytem).

P re s e n t o f p a s t E d i t o r i a l B o a r d M e m b e r : Plant Growth Regulation, Pesticide Physiology and Biochemistry,

Environment, Chemical and Engineering News, Science Year, Plant Physiology, Phsiology, Phytochemistry,
American Journal of Botany, Lloydia. Formerly regular columnist, Natural History Magazine.

F o r m e r M e m b e r : Metabolic and Regulatory Biology Panels, National Science Foundation; Executive Committee,
Growth Society; Life Science Advisory Committee, NASA; and Governing Boards, Biological Sciences Curriculum
Study, Commission on Undergraduate Education in the Biological Sciences and AIBS.
First American scientist to visit the People’s Rebuplic of China, 1971.

Books: ‘Principles of Plant Physiology’ (with J. Bonner), Freeman, 1952. ‘Life of the Green Plant’, Prentice Hall,
1961, 2nd Ed., 1964, 3rd Ed., 1980 (with P. J. Davies and R. L. Satter). ‘Control Mechanisms in Plant Development’, (with
P. J. Davies), Prentice Hall, 1970. ‘Daily Life in People’s China’, Crowell, 1973; Simon and Schuster, 1975. ‘Green
Wisdom’ Basic Books, Inc. NY, 1981; Putnam, 1983. ‘Life Processes in Plants’, Freeman (Scientific American
Library), 1994. ‘New Dimensions in Bioethics’, Arthur W. Galston and Emily G. Shurr, eds. Kluwer Academic
Publishers, Boston/Dordrecht/London, 2001.

More than 320 articles in referred scientific journal; approximately 60 general articles on problems of science and
society.
 Journal of Cell and Molecular Biology 2: 1-12, 2003. 1
Haliç University, Printed in Turkey.

P o l y a m i n e s i n p l a n t s : An overview
Ravindar Kaur-Sawhney1*, Antonio F. Tiburcio2, Teresa Altabella2, and Arthur W. Galston1
1
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT,
06520-8103, USA; 2Laboratori de Fisiologia Vegetal, Facultad de Farmacia, Universitat de Barcelona,
Spain (* author for correspondence)

Received 21 October 2002; Accepted 10 November 2002

Abstract

This article presents an overview of the role of polyamines (PAs) in plant growth and developmental processes. The
PAs, putrescine, spermidine and spermine are low molecular weight cations present in all living organisms. PAs and
their biosynthetic enzymes have been implicated in a wide range of metabolic processes in plants, ranging from cell
division and organogenesis to protection against stress. Because the PA pathway has now been molecularly and
biochemically elucidated, it is amenable to modulation by genetic approaches. Genes for several key biosynthetic
enzymes namely, arginine decarboxylase, ornithine decarboxylase and S-adenosyl methionine decarboxylase have
been cloned from different plant species, and antibodies to some genes are now available. Both over-expressed and
antisense transgenic approaches to PA biosynthetic genes have provided further evidence that PAs are required for
plant growth and development. However, molecular mechanisms underlying PA effects on these processes remain
unclear. Analysis of gene expression by using DNA microarray genomic techniques should help determine the precise
role of these compounds. The potential of proteomics to unravel the role of PAs in particular cellular processes has
also been examined. The extensive use of the two-hybrid system and other proteomic approaches will provide new
insights into the role of PAs in signal transduction. Furthermore, there is evidence that proteomics provides an
excellent tool for determining supramolecular organizations of PA metabolic enzymes which may help in
understanding homeostatic control of this metabolic pathway.

Key words: Polyamines, mutants, transgenic plants, genomics, proteomics

Bitkilerde poliaminler: Genel bir bak›fl

Özet

Bu makalede poliaminlerin (PA) bitki büyüme ve geliflme olaylar›ndaki rolüne genel bir bak›fl yap›lmaktad›r. PA ler
putresin, spermidin ve spermin, düflük molekül a¤›rl›kl› ve tüm canl› organizmalarda mevcut olan maddelerdir. PA
lerin ve bunlar›n biyosentetik enzimlerinin bitkileri strese karfl› korumaya yönelik olarak hücre bölünmesinden
organogeneze kadar de¤iflen genifl bir metabolik olaylar zincirinde yer ald›¤› ortaya konmufltur. Günümüzde PA yolu
moleküler ve biyokimyasal yönden aç›kl›¤a kavufltu¤u için genetik yaklafl›mlarla düzenlenmeye uygundur. Çeflitli
anahtar biyosentez enzimleri, arginin dekarboksilaz, ornitin dekarboksilaz ve S-adenozil metiyonin dekarboksilaz›n
genleri farkl› bitki türlerinde klonlanm›flt›r ve günümüzde baz› genlerin antikorlar›n› elde etmek mümkündür. PA
biyosentezi genlerine hem over-ekspres ve hem de antisens transgenik yaklafl›mlar PA lerin bitki büyüme geliflmesi
için gereklili¤ini daha da ortaya koymufltur. Bununla birlikte bu olaylardaki PA etkilerinin moleküler mekanizmas›
hala aç›kl›¤a kavuflmam›flt›r. DNA mikroarray genom teknikleri kullan›larak yap›lan gen ekspresyon analizleri bu
bilefliklerin rollerini kesin olarak belirlemeye yard›mc› olacakt›r. PA lerin özellikle hücresel olaylardaki rolünü ortaya
koymaya yönelik olarak proteomi¤in potansiyeli de araflt›r›lm›flt›r. ‹ki-hibrit sistemi ve di¤er proteomik yaklafl›mlar›n
yo¤un kullan›m›, PA lerin sinyal iletimindeki rolüne yeni bir bak›fl aç›s› getirecektir. Bundan baflka proteomi¤in, PA
metabolik yolunun homeostatik kontrolünü anlamaya yard›mc› olabilecek, PA metabolizma enzimlerinin
supramoleküler organizasyonunun belirlenmesinde çok önemli bir araç oldu¤u konusunda veriler mevcuttur.

Anahtar sözcükler: Poliaminler, mutantlar, transgenik bitkiler, genomik, proteomik

2 Ravindar Kaur-Sawhney et al.
1. I n t roduction
Polyamines (PAs) are low molecular weight
polycations found in all living organisms (Cohen,
1998). They are known to be essential for growth and
development in prokaryotes and eukaryotes (Tabor and
Tabor, 1984; Tiburcio et al., 1990). In plant cells, the
diamine putrescine (Put), triamine spermidine (Spd)
and tetramine spermine (Spm) constitute the major
PAs. They occur in the free form or as conjugates
bound to phenolic acids and other low molecular
weight compounds or to macromolecules such as
proteins and nucleic acids. As such, they stimulate
DNA replication, transcription and translation. They
have been implicated in a wide range of biological
processes in plant growth and development, including
senescence, environmental stress and infection by
fungi and viruses. Their biological activity is attributed
to their cationic nature. These findings have been
discussed in several recent review articles (Tiburcio et
al., 1993; Galston et al.,1997; Bais and Ravishankar,
2002).
The use of PA biosynthesis inhibitors has shown a
causal relationship between changes in endogenous PA
levels and growth responses in plants. These
observations led to further studies into undestanding
the mode of PA action. Some of the important
observations suggest that PAs can act by stabilizing
membranes, scavenging free radicals, affecting nucleic
acids and protein synthesis, RNAse, protease and other
enzyme activities, and interacting with hormones,
phytochrome, and ethylene biosynthesis (reviewed in
Slocum et al., 1984; Galston and Tiburcio, 1991).
Because of these numerous biological interactions of
PAs in plant systems, it has been difficult to determine
their precise role in plant growth and development.
In recent years, however, investigations into
molecular genetics of plant PAs have led to isolation of
a number of genes encoding PA biosynthetic enzymes
and development of antibodies to some of the genes.
Furthermore mutants and transgenic plants with altered
PA metabolism have also been developed. Genomic
and proteomic approaches are being used to further
gain an understanding into the role of PAs in plant
developmental processes. These findings will hopefully
lead to a better understanding of their specific functions
in plants. Several useful reviews on these aspects have
been published (Galston et al., 1997; Walden et al.,
1997; Malmberg et al., 1998; Martin-Tanguy, 2001;
Bais and Ravishankar, 2002).
This article presents an overview of the role of PAs
in plants with particular emphasis on recent
investigations using molecular and genetic
approaches.
2. Polyamine biosynthesis
The PA biosynthetic pathway in plants has been
thoroughly investigated and reviewed in detail (Evans
and Malmberg, 1989; Tiburcio et al., 1990; Slocum,
1991a; Martin-Tanguy, 2001). Briefly, PAs are
synthesized from arginine and ornithine by arginine
decarboxylase (ADC) and ornithine decarboxylase
(ODC) as illustrated in Figure 1. The intermediate
agmatine, synthesized from arginine, is converted to
Put, which is further transformed to Spd and Spm by
successive transfers of aminopropyl groups from
decarboxylated S-adenosylmethionine (dSAM)
catalysed by specific Spd and Spm synthases. The
aminopropyl groups are derived from methionine,
which is first converted to S-adenosylmethionine
(SAM), and then decarboxylated in a reaction
catalyzed by SAM decarboxylase (SAMDC). The
resulting decarboxylated SAM is utilized as an
aminopropyl donor. SAM is a common precursor for
both PAs and ethylene, and SAMDC regulates both
biosynthetic pathways as illustrated in Figure 1.
A number of investigators have used PA inhibitors
to modulate the cellular PA titer in order to determine
their role in various plant processes. Four commonly
used inhibitors of PA synthesis are: 1.
Difluoromethylornithine (DFMO), an irreversible
inhibitor of ODC (reviewed in Bey et al., 1987); 2.
Difluoromethylarginine (DFMA), an irreversible
inhibitor of ADC (Bitonti et al., 1987); 3. Methyl-
glyoxyl-bis guanylhydrazone (MGBG), a competitive
inhibitor of S-adenosyl-methionine decarboxylase
(SAMDC) (Williams-Ashman and Schenone,1972);
and 4. Cyclohexylamine (CHA), a competitive
inhibitor of spermidine synthase (Hibasami et al.,
1980). Common oxidases are diamine oxidase and
polyamine oxidase (PAO), as reviewed by Smith and
Marshall (1988). Each PA has been found to be
catabolized by a specific oxidase.
Several investigations have dealt with localization
of PAs and their biosynthetic enzymes in plants
(reviewed by Slocum, 1991b). However, paucity of
information regarding the exact cellular and
subcellular localization of these entities remains one of

Methionine
DFMO
MGBG
S - adenosylmethionine
SAMDC
ACC synthase
AVG
ACC
ACC oxidase
Ethylene
F i g u re 1: Polyamine biosynthetic pathway and its linkage to ethylene biosynthetis. Biosynthetic enzymes are ADC, ODC and
SAMDC and the inhibitor DFMA, DFMO and MGBG.
the obstacles in understanding their biological role.
Recent studies have shown that PAs are present in the
cell wall fractions, vacuole, mitochondria and
chloroplasts (Torrigiani et al., 1986; Slocum, 1991b;
Tiburcio et al., 1997). The biosynthetic enzymes,
ODC, SAMDC, and Spd synthase have been reported
to be localized in the cytoplasm, whereas ADC is
localized in the thylakoid membrane of chloroplast
(Borrell et al., 1996; Tiburcio et al., 1997) and PAO in
the cell wall (Kaur-Sawhney et al., 1981). ODC
activity has also been observed in the nucleus
(Slocum, 1991b). However, these findings have to be
interpreted with caution because various procedural
problems can mask the results. Despite these advances
in understanding the metabolic processes involving
PAs and their localization in plant cells, the precise
role of PAs in plant morphogenesis remains elusive.
Polyamines in plants 3
Ornithine Arginine
ODC ADC
DFMA
Agmatine
Putrescine
dSAM Spdsynthase
Spermidine
Spmsynthase
Spermine
3. Polyamines in plant growth and development
The availability of specific inhibitors of PA
biosynthesis has helped in investigating the
mechanisms involved in PA interactions to some extent,
providing a partial understanding of their physiological
role in plant growth and development. Clearly, PAs are
involved in many plant developmental processes,
including cell division, embryogenesis, reproductive
organ development, root growth, tuberization, floral
initiation and development, fruit development and
ripening as well as leaf senescence and abiotic stresses
(reviewed by Evans and Malmberg, 1989; Galston et
al., 1997; Bais and Ravishankar, 2002; Tiburcio et al.,
2002). Changes in free and conjugated PAs and their
biosynthetic enzymes, namely ADC, ODC, and
SAMDC have been found to occur during these
developmental processes. Earlier experiments had
shown that increases in PAs and their biosynthetic
enzymes are associated with rapid cell division in many
plant systems e.g., carrot embryogenesis (Montague
4 Ravindar Kaur-Sawhney et al.
and Koppenbrink, 1978; Feirer et al., 1984), tomato
ovaries (Heimer and Mizrahi, 1982), tobacco ovaries
(Slocum and Galston, 1985), and fruit development
(reviewed in Kakkar and Rai, 1993). Similar results
have been reported for many other plant species
(reviewed in Bais and Ravishankar, 2002). In contrast,
several other studies have suggested that correlations
between PAs and their biosynthetic enzymes and plant
growth processes, especially somatic embryogenesis,
are not universal and may be species specific (reviewed
in Evans and Malmberg, 1989; Galston et al., 1997;
Bais and Ravishankar, 2002).
In general, cells undergoing division contain high
levels of free PAs synthesized via ODC, and cells
undergoing expansion and elongation contain low
levels of free PAs synthesized via ADC (see review by
Galston and Kaur-Sawhney, 1995). High levels of
endogenous PAs and their conjugates have also been
found in apical shoots and meristems prior to
flowering (Cabbane et al., 1981) and flower parts of
many plants (Martin-Tanguy, 1985). Our experiments
using callus cultures derived from thin layer explants
of pedicels from tobacco inflorescence show that
endogenous Spd increased more rapidly than other
PAs in floral buds than in vegetative buds. Addition of
CHA, an inhibitor of Spd synthesis, to the culture
medium reduced flower formation in a dose dependent
manner and such inhibition was correlated with a
switch to initiation of vegetative instead of flower
buds. This inhibition was reversed by the addition of
exogenous Spd (Kaur-Sawhney et al., 1988). More
recently, we have found that higher levels of
endogenous PAs occur in flowers and siliques when
compared with their levels in leaves and bolts of
certain strains of Arabidopsis. Addition of the PA
biosynthetic inhibitors, DFMA and CHA to the culture
medium, at time of seed germination, inhibited bolting
and flower formation and this was partially reversed
by addition of exogenous Spd (Applewhite et al.,
2000). These results clearly show that Spd is involved
in flower initiation and development. Similar results
have been reported in other plants also (reviewed by
Galston et al.,1997; Bais and Ravishankar, 2002).
Many plant growth and development processes
known to be regulated by plant hormones, such as
auxins, 2,4-D, GA and ethylene, have also been
correlated with changes in PA metabolism. These
changes occur in both endogenous levels of PAs and
their biosynthetic enzymes and appear to be tissue
specific (reviewed by Galston and Kaur-
Sawhney,1995). Thus, PAs which may or may not be
mobile in plants (Young and Galston, 1983; Bagni and
Pistocchi, 1991) can serve as intracellular mediators of
hormone actions (Galston and Kaur-Sawhney, 1995).
Supporting evidence for this hypothesis has been
obtained in experiments using specific inhibitors of PA
biosynthesis (Bagni et al., 1981; Egea-Cortines and
Mizrahi, 1991; reviewed in Galston et al., 1997; Bais
and Ravishankar, 2002).
Of the major plant hormones, ethylene has been
most intensively investigated with respect to PA
metabolism. The two metabolites, PAs and ethylene,
play antagonistic roles in plant processes. While PAs
inhibit senescence of leaves (Kaur-Sawhney et al.,
1982), cell cultures of many monocot and dicot species
(Muhitch et al., 1983) and fruit ripening (Kakkar and
Rai, 1993), ethylene promotes these processes. The
most commonly held view is that PAs and ethylene
regulate each other’s synthesis, either directly or
through metabolic competition for SAM, a common
precursor for their biosynthesis (Figure 1). PAs inhibit
ethylene biosynthesis, perhaps by blocking the
conversion of SAM to ACC and of ACC to ethylene
(Apelbaum et al., 1981; Suttle, 1981; Even-Chen et al.,
1982; Furer et al., 1982). Ethylene, on the other hand,
is an effective inhibitor of ADC and SAMDC, key
enzymes in PA biosynthetic pathway (Apelbaum et al.,
1985; Icekson et al., 1985). Thus, PAs may affect
senescence and fruit ripening by modulating PA and
ethylene biosynthesis.
Apparently, PAs are essential members of an array
of internal metabolites required in many plant
developmental processes, but their precise role in these
processes has yet to be established. Whereas, specific
PAs at specific concentrations may be required at
critical stages of growth and morphogenetic events, no
definitive data are available to establish their role as
plant hormones.
4. Manipulation of the polyamine pathway
The PA pathway is ubiquitous in living organisms and
is relatively short (see Section 2) in terms of the
number of enzymes involved. Most of the genes
coding for enzymes involved in the pathway have been
cloned from different sources (Kumar et al., 1997;
Walden et al., 1997; Galston et al., 1997; Tiburcio et
al., 1997; Malmberg et al., 1998; Kumar and Minocha,
1998; Panicot et al., 2002b). Thus, the PA pathway

represents an excellent model to test various
hypotheses and to answer fundamental biological
questions derived from pathway manipulation (Thu-
Hang et al., 2002; Bhatnagar et al., 2002).
Initially, approaches to manipulate the PA pathway
made use of suicide inhibitors, but the effects of
DFMO and DFMA on ODC and ADC respectively, are
variable in different plant systems, ranging from
inhibition to stimulation or no effect and depending on
the concentration, plant system tested and the
existence of compensatory mechanisms (Slocum and
Galston, 1987). Therefore, alternative approaches to
manipulate polyamine metabolism have been
developed during the recent years.
4.1. Mutants
Mutants deficient in PA biosynthesis have been
isolated from several biological systems. Hafner et al.
(1979) isolated PA mutants in Escherichia coli
showing decreased growth and increased sensitivity to
paraquat (Milton et al., 1990). Yeast mutants
presenting ODC as the sole pathway, show reduced
growth and altered sporulation on PA deficient
medium (Cohn et al., 1980; Whitney and Morris,
1978). Chinese hamster ovary cells lacking ODC
activity do not grow in medium lacking PA (Steglich
and Scheffler, 1983) and a moderately reduced brood
size was observed in a Caenorhabditis elegans ODC
deletion mutant (Macrae et al., 1995). Mutations in
genes affecting Spd and Spm biosynthesis have also
been isolated in yeast. The spe3 Spd synthase mutation
causes a growth arrest, which can be complemented
with externally added Spd (Hamasaki-Katagiri et al.,
1997), while the yeast spe4 mutant is defective in Spm
biosynthesis (Hamasaki-Katagiri et al., 1998).
Less is known about mutants affecting PA
metabolism in plants. Mutants with high levels of
ADC activity have been identified in petunia because
of their abnormal morphology (Geerats et al., 1988),
but the basis of the mutation is still not known.
Screening for resistance to the SAMDC inhibitor
MGBG (Malmberg and Rose, 1987) or to inhibitory
concentrations of Spm (Mirza et al., 1997), yielded
mutants that showed reduced sensitivity to the
respective agent, but these mutants have not been
further exploited for the analysis of PA function.
Watson et al. (1998) isolated EMS mutants of A.
thaliana that are reduced in ADC activity. The mutants
fall into two complementation groups, spe1 and spe2,
Polyamines in plants 5
which may correspond to the two gene copies
encoding ADC, ADC1 and ADC2 (Watson et al.,
1998). The mutations have not been mapped and
therefore it cannot be excluded that other functions,
i.e. regulatory elements, are affected (Soyka and
Heyer, 1999). More recently, Hanzawa et al. (2000)
reported that the inactivation of the Arabidopsis
ACAULIS5 (ACL5) gene causes a defect in the
elongation of stem internodes by reducing cell
expansion. It was suggested that ACL5 encodes a Spm
synthase, but the possibility that ACL5 may exhibit
broad amine substrate specificities and be involved in
the synthesis of other polyamines could not be
excluded (Hanzawa et al., 2000).
Thus far the only well characterized plant
polyamine biosynthetic mutant has been generated by
using reverse genetics. The availability of mutant
collections generated either by transposon or T-DNA
tagging now facilitates the identification of knockouts
in any gene of interest using PCR-based mutant
screening techniques (Ferrando et al., 2002). By using
these techniques, Soyka and Heyer (2000) isolated an
Arabidopsis thaliana mutant line carrying an insertion
of the En-1 transposable element at the ADC2 locus
which should be regarded as a complete loss-of-
function or knockout mutation. The ADC2 knockout
mutant shows no obvious phenotype change under
normal growth conditions, but is completely devoid of
ADC induction by osmotic stress. As ADC1 gene
expression was not affected in the mutant, it was
concluded that ADC2 is the gene responsible for
induction of ADC and PA biosynthesis under osmotic
stress (Soyka and Heyer, 2000). More recently, Pérez-
Amador et al. (2002) have shown that ADC2 gene
expression is induced in response to mechanical
wounding and methyl jasmonate treatment in
Arabidopsis thaliana. All these observations appear to
indicate that ADC2 is a key gene involved in the PA
response to abiotic stress in Arabidopsis. We envisage
that the extensive use of functional genomics and
reverse genetic studies will facilitate the isolation of
novel knock-out mutants affected in other PA
biosynthetic genes.
4.2. Transgenic plants
With the availability of most of the genes involved in
PA metabolism, it has become possible to manipulate
this metabolic pathway using sense and antisense
transgenic approaches. Thus, cellular PA content has
6 Ravindar Kaur-Sawhney et al.
been modulated by overexpression or down regulation
of the key genes ODC, ADC or SAMDC (Kumar et al.,
1997; Walden et al., 1997; Malmberg et al., 1998;
Kumar and Minocha, 1998; Capell et al., 1998; Rajam
et al.,1998; Roy and Wu, 2001; Bhatnagar et al., 2002).
Most of the studies have used the constitutive 35S
promoter, but only few of them were successful in
using either inducible (Masgrau et al., 1997; Panicot et
al., 2002a; Mehta et al., 2002) or tissue-specific
promoters (Rafart-Pedros et al., 1999). Overexpression
of heterologous ODC or ADC cDNAs generally causes
the production of high levels of Put (DeScenzo and
Minocha, 1993; Bastola and Minocha, 1995; Masgrau
et al., 1997; Capell et al., 1998; Bhatnagar et al., 2002;
Panicot et al., 2002a), but in most cases only a small
increase or even no change in Spd and Spm has been
observed. This indicates that elevated levels of Put
resulting from genetic manipulation of a single step
located upstream of the PA biosynthetic pathway (i.e.
ODC or ADC) are not accompanied by an increase in
subsequent biosynthetic reactions (i.e. Spd and Spm
biosynthesis) (Bhatnagar et al., 2002). In contrast,
overexpression of genes located downstream of the
pathway (i.e. SAMDC or SPDS) generally lead to
increased levels of Spd or Spm or both (Thu-Hang et
al., 2002; Mehta et al., 2002). Taken together these
results suggest that the levels of Spd and Spm in the
cells are under a tight homeostatic regulation
(Bhatnagar et al., 2002), which possibly could be
related to a supramolecular organization of some of
these enzymes (see Section 5).
Discrepancies observed among different studies
may have several causes. These include: transgene
source, positional effects, recipient plant system, plant
material analyzed and type of promoter used. A
hierarchical accumulation of polyamines in different
transgenic tissues/organs has been observed (Lepri et
al., 2001). In general, less metabolically active tissues
accumulate higher levels of polyamines (Lepri et al.,
2001). These results are in line with experiments in
which metabolites such as vitamin A and
pharmaceutical antibodies accumulate at high levels in
seeds of different species. It is reasonable to assume
that dormant or less metabolically active tissues
provide a conducive environment for the accumulation
of transgenic products (Thu-Hang et al., 2002). In this
regard, it should be stressed that the most remarkable
results have been obtained by controlled expression of
transgenes using inducible or tissue-specific
promoters. For example, tissue-specific expression of
SAMDC gives rise to smaller potato tubers without
affecting tuber yield (Rafart-Pedros et al., 1999). The
distribution of tuber weights is of agronomic
importance, and generally a reduction of tuber-size
variation is economically advantageous, so that more
tubers fall into a given size grade either for seed or
ware (Rafart-Pedros et al., 1999). Similarly, fruit-
specific expression of heterologous SAMDC in tomato
resulted in ripening-specific accumulation of Spd and
Spm which led to an increase in lycopene, prolonged
vine life, and enhanced fruit juice quality (Mehta et al.,
2002). Besides the agronomic interest of this finding,
this latter study constitutes one of the most striking
evidence regarding the in vivo involvement of
polyamines in a particular developmental process, i.e.
fruit ripening (Mehta et al., 2002).
5. U n d e r s t a n d i n g t h e role of polyamines
Phenotypic analyses of mutants and transgenic plants
with altered PA levels gives further support to the
previous physiological studies (see Section 3) with
regard to the involvement of these compounds in
several plant processes (reviewed by Tiburcio et al.,
2002). These include somatic embryogenesis (Bastola
and Minocha, 1995), stem elongation and flowering
(Gerats et al., 1988; Masgrau et al., 1997; Hanzawa et
al., 2000; Panicot et al., 2002a), root growth (Watson
et al., 1998; Cordeiro et al., unpublished), tuber
development (Kumar et al., 1996; Rafart-Pedrós et al.,
1999), fruit ripening (Mehta et al., 1997; 2002), abiotic
stresses (Minocha and Sun, 1997; Soyka and Heyer,
1999; Roy and Nu, 2001). However, most of these
mutants and transgenic plants have not been further
exploited for the analysis of PA function. Application
of advanced genomic and proteomic approaches will
help to elucidate the role of PA in particular plant
processes.
5.1. Genomic approaches
The availability of complete genome sequences
permits the use of approaches to explore gene
expression variations on a large genome scale. Either
cDNAs or large oligonucleotide collections are
attached on surfaces to create a microarray. The
hybridisation of the microarray with fluorescent
labelled RNA or cDNA yields an overall image of gene
expression or ‘transcriptome’ (Lockhart and Winzeler,

2000). The global examination of gene expression
should reveal the coincidence of spatial and temporal
transcript expression profiles that may reflect a
requirement of co-ordinated gene product expression
in response to different type of signals. The technology
developed for the Arabidopsis genome has been
accelerated in the recent years both by public funding
through the Arabidopsis Functional Genomics
Consortium in the USA and the GARNet in the UK,
and also by private initiatives like Monsanto,
Affymetrix or Synteny/InCyte (Wisman and Ohlrogge,
2000).
Although there are already many examples in the
literature showing the utility of this approach for
unraveling complex plant responses and signal
transduction processes (Schena et al., 1995; Schaffer et
al., 2000), the use of this technology in our field is
unfortunately in its infancy. So far, DNA microarray
analysis has been used to reveal the induction of ADC
genes during drought stress (Ozturk et al., 2002) or in
response to wounding and methyl jasmonate treatment
(Sasaki et al., 2001; Pérez-Amador et al., 2002).
We envisage that global analysis of gene
expression in well characterized mutant and transgenic
plants with altered polyamine metabolism will provide
novel clues in the near future for understanding the
molecular mechanisms underlying polyamine effects
on plant growth and development.
5.2. Proteomic approaches
Proteomics’ uses biochemical approaches aimed at
systematically characterizing the ‘proteome’ or the
‘protein complement of the genome’ (Wasinger et al.,
1995) in a given organism, tissue, cell or subcellular
compartment. The means of proteome characterization
include protein localization, expression and most
importantly protein interaction maps. A plethora of
innovative procedures has been employed in recent
years for the large-scale analysis of protein signalling
pathways, including the yeast two-hybrid system
(Fields and Song, 1989), protein purification methods
linked to detection by mass spectrometry (Neubauer et
al., 1997; Verma et al., 2000); protein localization
(Ferrando et al., 2000; 2001; Farràs et al., 2001), and
protein microarray techniques (Zhu et al., 2001).
The yeast two-hybrid system is a genetic tool to
describe in vivo protein interactions using the yeast
cell as a test tube. Each separated module of the GAL4
transcription factor, either the DNA binding domain
Polyamines in plants 7
(DBD) or the transcriptional activation domain (AD),
is translationally fused to proteins of interest X or Y,
generating respectively the hybrid proteins X-DBD
(bait) and Y-AD (prey). A powerful aspect of the yeast
molecular genetics involves the facility to isolate the
corresponding cDNAs coding for proteins X or Y,
introduced in the form of plasmid DNA. This latter
feature immediately favored the use of this system to
identify interacting partners for a given bait protein X
using cDNA libraries as a prey (reviewed by Walhout
et al., 2000). The number of studies that have used
proteomics in our field is still scanty. Here we will
provide two examples that demonstrate the potential of
these techniques to (i) unravel the role of PA in
transcription; and (ii) to identify PA metabolons (see
below).
Although the potential role of PAs in affecting gene
expression had already been reported, the molecular
mechanisms underlying their effects were unknown
(Wang et al., 2002). The identification of a polyamine
responsive element and corresponding transacting
protein factors that respond to polyamines has opened
up an exciting new area to study the function of these
compounds in transcription (Wang et al., 1999). By
using the two-hybrid system, it was recently found that
the human homologue of the Arabidopsis subunit
COP9 signalosome complex binds to such transacting
protein factors with the potential to directly affect gene
expression (Wang et al., 2002). Remarkably, the COP9
signalosome proteins were first identified in
Arabidopsis and have been demonstrated to form a
regulatory complex involved in light-activated
development and playing a role in intracellular
signalling (Deng et al., 2000). We envisage that similar
type of experiments will be performed in the plant PA
field that hopefully will provide new insights into the
role of PAs in plant signal transduction.
Increasing number of reports document that many
metabolic reactions are catalysed by complexes of
sequentially acting enzymes that show highly ordered
structural organization (reviewed in Srere, 1987). In
such multienzyme complexes the metabolites pass
from one active enzyme site to the next through a
process termed ‘substrate channeling’. The
supramolecular arrangement of enzymes involved in
such metabolic reactions is referred to as ‘metabolon’.
Metabolons are multienzyme complexes in both
prokaryotes and eukaryotes that represent highly
organized assemblies of sequential enzymes in a
metabolic pathway and are thought to provide
8 Ravindar Kaur-Sawhney et al.
increased metabolic efficiency and higher substrate
selectivity. Metabolons may also help to coordinate the
activities of enzymes by sharing intermediates in a
given pathway, as well as to ensure protection of labile
substrates and sequestration of toxic intermediates
(Sugumaran et al., 2000). In addition, the formation of
multienzyme metabolon complexes may enhance
enzyme stability, improve enzymatic performance and
provide a means for adaptation to alterations of input
of metabolic reactions, especially during demanding
physiological conditions (Abadjieva et al., 2001).
The relevant information about intrinsic properties
of ‘metabolon’ formation can be acquired by studies of
protein-protein interactions using modern proteomic
approaches (Ferrando et al., 2002). In this regard, our
laboratory has recently analyzed possible interactions
between the SPDS and SPMS enzymes of polyamine
biosynthetic pathway in the yeast two-hybrid system
(Panicot et al., 2002b). Using the Arabidopsis
spermidine synthase as bait, two similar proteins were
identified to interact with SPDS2 that were named
SPDS1 and SPMS. Yeast and bacterial mutant
complementation tests revealed that SPDS1 encodes a
novel spermidine synthase, whereas SPMS displays
spermine synthase activity. The heterodimerization
capabilities of enzymes catalyzing the two last steps of
polyamine biosynthesis were also demonstrated in vivo
by co-immunoprecipitation using epitope tagged
SPDS1, SPDS2 and SPMS proteins (Ferrando et al.,
2000; Ferrando et al., 2001). Immunoaffinity
purification and size fractionation of SPDS and SPMS
enzymes labeled with different HA and c-Myc
epitopes revealed that the SPDS and SPMS proteins
co-purify with large multiprotein complexes of 650 to
750 kDa. Further analysis of subunits of isolated
SPDS-SPMS metabolon(s) by mass spectrometry is
expected to yield important information about yet
unknown regulatory subunits of SPDS-SPMS
metabolon in the PA biosynthesis pathway. The
available data support the conclusion that Spd
synthesized by SPDS is effectively channeled to
SPMS to control the formation of the end-product Spm
thereby regulating the synthesis of high molecular
weight polyamines (Panicot et al., 2002b).
6. Conclusions
Considerable evidence indicates that polyamines are
involved in a wide array of plant processes, including
DNA replication, transcription of genes, cell division,
organ development, fruit development and ripening,
leaf senescence and abiotic stresses. Despite ample
evidence of their involvement in these processes, their
precise role in these specific processes remains to be
established. Recent developments of PA-deficient
mutants and transgenic plants as well as of
molecular genetic investigations should further our
understanding of their role in plant growth and
development.
The polyamine pathway is now amenable to
modulation by genetic approaches because it has been
elucidated molecularly and biochemically in plants.
Reverse genetics has identified an Arabidopsis
knockout mutation of ADC2 gene which reveals
inducibility by osmotic stress. Extensive use of
functional genomics and reverse genetics studies will
facilitate the isolation of novel knockout mutants
affected in other polyamine metabolic genes. Sense
and antisense transgenic approaches have revealed the
feasibility of modulating cellular PA contents.
Generally, genetic manipulation of single steps located
upstream of the PA pathway (i.e. ODC or ADC) lead to
elevated levels of Put, but no changes occur in the
higher PAs, Spd and Spm. By contrast, overexpression
of genes located downstream of the pathway (i.e.
SAMDC or Spd synthase) generally leads to increased
levels of Spd and Spm, indicating that the levels of Spd
and Spm are under a tight homeostic cellular control.
Phenotypic analyses of mutants and transgenic plants
affected in polyamine metabolism further support
previous physiological evidence, but the molecular
mechanisms underlying PA effects on plant growth and
development remain to be elucidated. Global analysis
of gene expression by using the available DNA
microarray genomic techniques will help to understand
the role of these compounds. The potential of
proteomics to unravel the role of polyamines in
particular cellular processes is also examined. We
envisage that the extensive use of the two-hybrid
system and other proteomic approaches will provide
new insights into the role of PAs on plant signal
transduction. Furthermore, we provide evidence that
proteomics is an excellent tool to unravel
supramolecular organizations of PA metabolic
enzymes which may help to understand homeostatic
control of this metabolic pathway.

Acknowledgements
AFT acknowledges the grants from Ministerio de
Ciencia y Tecnología BIO-99-453 and BIO-2002-
04459-C02-02.
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Haliç University, Printed in Turkey.

P h e n o l i c c y c l e i n p l a n t s a n d e n v i ro n m e n t
Valentine I. Kefeli1, Maria V. Kalevitch2* and Bruno Borsari3
1
Slippery Rock Watershed Coalition, 3016 Unionville Rd., Cranberry Twp., PA 16066, USA; 2Robert
Morris University, 881 Narrows Run Rd., Moon Township PA 15108, USA; 3Slippery Rock University,
101 Eisenberg Bldg., Slippery Rock PA 16057, USA (* author for correspondence)

Received 30 October 2002; Accepted 15 November 2002

Abstract

Phenolic substances are synthesized in plants and in the soil. They exist in the form of polymers and monomers. The
latter group of phenolics is assembled within the chloroplasts of plant cells, whereas soil phenolics are associated
with the process of humus formation on the alumino-silicate matrix of the soil micelle. As plants grow, phenolics
accumulate in cell vacuoles, or polymerize into lignin, which strengthens the secondary cell walls. In addition to this,
phenolics possess also some physiological functions as they regulate cell elongation. When they are excreted from
plant root systems they exert inhibitory growth function within adjacent rhizospheres. This work presents the latest
experimental evidence of phenolic synthesis and transformation in the environment, while providing an
understanding of their effect in plant-soil relations.

Key words: Allelopathy, chloroplasts, humus, phenolics, soil micelle

B i t k i l e r d e f e n o l i k d ö n g ü v e ç e v re

Özet

Fenolik maddeler bitkilerde ve toprakta sentezlenir. Bunlar polimerler ve monomerler fleklinde bulunurlar.
Fenoliklerin monomer grubu bitki hücresinin kloroplastlar›nda biraraya gelirken, toprak fenolikleri toprak
misellerinin alumino-silikat matriksi üzerinde humus oluflum olay› ile uyumluluk gösterir. Bitki büyürken hücre
vakuollerinde fenolikler birikir veya sekonder hücre çeperlerine sa¤laml›k kazand›ran ligninlere polimerize olurlar.
Bunlara ilave olarak fenolikler hücre uzamas›n› düzenleyerek baz› fizyolojik ifllevlere de sahiptirler. Bitki kök
sistemlerinden sal›nd›klar› zaman hemen yak›n›ndaki rizosferlerde büyümeyi inhibe edici etki meydana getirirler. Bu
çal›flma fenolik sentezlerinin en son deneysel verilerini ve çevredeki dönüflümlerini sunarken, bitki-toprak
iliflkilerindeki etkilerini anlamam›za yard›m etmektedir.

Anahtar sözcükler: Allelopati, kloroplastlar, humus, fenolikler, toprak miseli

I n t ro d u c t i o n and their fragments contribute to the mineralization of

Phenolics are very stable products in plant organisms.
Generally, they are characterized by a benzene ring
and one hydroxyl group (-OH). They can be converted
into lignin which is the main phenolic polymer in
plants. Microorganisms break down these molecules
soil nitrogen and humus formation. Thus, humus
participates actively in fulfilling plants nutritional
needs and growth. Light enhances the biosynthesis of
phenolic substances in plant chloroplasts and these
constitute in addition to soil micelles (humus) a second
formation site for this diverse group of organic
14 Valentine I. Kefeli et al.

molecules. It should be mentioned however, that

COOH CHO
phenolics tend to accumulate in plant vacuoles in I
HCOH
C— O
relatively high amounts, or they deposit in the I HCOH
CH2
secondary cell wall as lignin. CH2O
Phosphoenol- Dehydroquinic acid
pyruvic acid 5-Dehydroshikimic acid Erythrose-4-phosphate
2-keto- COOH
3-desoxy-
C h l o ro p l a s t s a s c e n t e r s o f p h e n o l i c s b i o s y n t h e s i s 7-phospho- Shikimic acid
D-Araboheptonic acid
OH OH
Experiments with chloroplasts of willow (Salix spp.) OH
HOOC CH2-CO-COOH
leaves showed that the synthesis of phenol-carboxylic COOH
Chorismic acid
acids and flavonoids is strongly stimulated by light
exposure. Metabolic inhibitors that depress
OH NH2
photosynthetic activity (simazine, diurone, Prephenic acid Anthranilic acid
chloramphenicol), affect negatively the biosynthesis of
CH2-CH-COOH CHOH-CHOH-CH2O
flavonoids. Leaves chloroplasts have the capability to
localize phenol compounds, some of which are N
specific to these organelles only. The chloroplasts of
Phenylalanine Indolylglycerophosphate
spring willow leaves contain more phenols than the
chloroplasts of the same leaves in the autumn. Light is CH-CH-COOH
CH2-CO-COOH
a mandatory condition to initiate phenolics synthesis
and this is indicated also by the lack of such molecules N
I
in the protoplastids of etiolated willow shoots (Kefeli OH
H Indolyl-acetic acid
p-Coumaric acid IAA
and Kalevitch, 2002). Light appears also to induce
flavonols synthesis in the chloroplasts and cytoplasm. F i g u re 1: Phenol-propanoids in metabolic bifurcation.
Chalcone and phenolcarbonic acid present in etiolated
willow shoots can be considered metabolic precursors
of light-synthesized flavonols. In certain cell Muzafarov and collaborators (1992) investigated
compartments (vacuoles and cell wall) phenols are the functions of some phenolics in chloroplasts. They
contained in significant amounts (Lewis and assumed that the essence of the relationship between
Yamamoto, 1990). However, it is not clear yet how photosynthesis and phenolics biosynthesis is that
phenols are translocated within plant cells and how phenolics exert a direct and an indirect effect on the
they affect the function of cell organelles such as process of solar accumulation itself. From our point of
ribosomes and mitochondria. Phenolic substances that view, flavonoids as polyfunctional compounds in
inhibit plant growth (hydroxy derivatives of cinnamic green plastids fulfill three major functions as:
acid, coumarin and naringenin) are synthesized • substrates (use polyphenols and their catabolic
similarly to other phenolics. The synthesis of growth products for other kinds of biosynthesis);
inhibitor derivatives of hydroxycinnamic acids follows • energy sources (electron and proton transport, ion
the pathway: shikimic acid-chorismic acid-prephenic exchange and membrane potential, radicals
acid-cinnamic acid and p-coumaric acid. A theory of formation);
metabolism bifurcation among phenolic substances, • regulators (involvement in enzyme reactions as
some of which can inhibit growth and synthesis of inhibitors or activators).
indolic compounds has been proposed. According to During photosynthesis under light, flavonoids
this new approach, indolil-3-acetic acid (IAA) change the rate of electron transport and
becomes the main natural auxin (Kefeli, 1978; Kefeli photophosphorylation, bringing about the change of
and Dashek, 1994; Kefeli and Kalevitch, 2002). ATP/NADPH ratio. In the reactions of carbon
Therefore, indole auxins (IAA, indoleacetonitrile) as metabolism they can shift the dynamic equilibrium of
well as phenolic inhibitors (p-coumaric acid, pentosephosphate reduction cycle to enhance the
coumarin, naringenin and others) are derived from the synthesis of certain metabolites both due to the change
common precursors, shikimic and chorismic acids in energy substrate intake and to the interaction with
(Figure 1). enzymes of the cycle. Additionally, flavonoids
 Phenolic cycle 15

exercise a feedback control over their own

OH
biosynthesis, although this phenomenon is not clearly 2 3
6´ C CH = CH ´OH
understood. This questionable situation remains as the 5´
4´
1´
2´ O
1
6 5
4

HO 3´
biosynthesis of the entire flavonoid structure within O O6H10O5
Isosalipurposide
plastids has not been explained, nor the complete 1 2´, 4´, 6´, 4-tetroxychalcone-
enzymatic package of their biosynthesis has been -2´-glucoside of chalconarin-
O H genin (chalcone)
discovered yet. Lack of direct eveidence of flavonoids HO 8 1 C
2´ 3´
1´ 4´ OH
7 2
transport within the cell and through the whole plant 6
5 4
3
CH2
6´ 5´

constitues another challenge to a more accurate O

C
O
description of their functions. Noneless, a variety of C 6H 10O 3
Salipurposide
phenolic compounds, present simultaneously within naringenin-5-glucoside
cells appear to be capable of influencing the rate and 2 (Flavanone-glycoside)
direction of plants metabolic activities. Thus, any
change in the flavonoid structure, or qualitative O H
2´ 3´
HO 8 1 C OH
composition of the phenol complex result in a change 7
6
2
3
1´ 4´
6´ 5´
5 4 CH2
of the mechanism of its effect upon the processes of OH
C
cell energy exchange. O
3 Naringenin
Chalcone and phenolcarbonic acids present in (Flavanone)
etiolated willow shoots can be viewed as the potential Eriodictyol
precursors of light synthesized flavonoids. However, (Flavanone) 4
the use of paper chromatography to investigate Luteolin
isosalidpurposide transformation products did not (Flavone)

reveal the presence of any flavonols sensitive to

conventional reagents. Therefore, the transformation
of chalcone (isosalpurposide) in lightless vitro appears
to terminate at a second stage. The synthesis of
eriodyctiol and luteolin that occurred in willow leaves
evidently took place in vivo and under light exposure.
It should be pointed out however, that phloridzin and
isosalipurposide were decomposed from aglycone and
that phloridzin and phloretin produced yellow stains
on the chromatogram as well as flavonoids. It is known
that flavonoid glycosides are revealed as dark spots on
chromatograms exposed to UV light. Therefore, our
yellow stains were classified as flavanones, since they
did not react with AlCl3, nor Na2CO3 like flavonols,
that also form yellow spots. At the same time, similar
to chalcones and aurones, these floridzin
transformation products are yellow colored and they
turn into orange-pink when exposed to Na2CO3 or
NH4OH. Relatively easy transformations of
isosalipurposide and phloridzin into compounds of
other classes (flavanones, chalcones, or aurones)
evidenced the role of these products in the general
metabolism of flavonoids (Figure 2).
F i g u re 2: Flavonoid biosynthesis.
P h e n o l i c s u b s t a n c e s s e c re t e d b y ro o t s a n d l e a c h e d
f rom leaves
Plants contain and secrete a diverse group of growth
inhibiting substances that may affect other plants
development, if grown in their vicinity (allelopathy).
Leaf exudates of willow species such as Salix rubra or
Salix viminalis, contain phenolic inhibitors like
naringenin derivative isosalipurposide. Other species
instead like apple trees (Malus spp.) contain
phloridzin, which is a strong respiratory inhibitor.
Roots and leaves of the wild plant Nanaphyton native
to semi-desert regions of Mongolia contain also strong
phenolic inhibitors. Seed as well may secrete
allelochemicals. Tobacco seed (Nicotiana tabacum)
for example suppress germination of its own seed
when leachates come in contact with the seeds (Kefeli
and Kalevitch, 2002). Although the inhibition of
germination was observed at various levels of
intensity, this phenomenon demonstrates the
selectivity of these natural excreta, similar to the effect
of synthetic herbicides. Therefore, increasing evidence
indicates that phenolics and alkaloids play the role of
selective agents. Secondary compounds can be
modified in transgenic plants and genetic mutants.
16 Valentine I. Kefeli et al.

Hence, molecular genetics becomes a tool, which may
help to regulate the level of secondary metabolites in
plants. Therefore, there is a need continue the search
for botanical herbicides as a rise of ecological
concerns has clearly identified the environmental
impact of herbicides of synthesis.
Root exudates affect the germination of seeds of
different crops: monocots and dicots (Table 1 and
Table 2). However, it must be pointed out that only
some phenolics were studied in the exudates of willow
roots (1) which have no analogues in the roots (2) and
leaves found among the common allelopathogens.
Although some of these substances could be retained
by willow roots, others where excreted into an external
medium. Chromatography of these water exudates and
a subsequent investigation of their chromatograms
with UV-B light showed that most of these substances
are polyphenols such as coumarin, or phenolic acids.
The phenolic substances retained by cells had different
chemical properties than those located in the root
exudates. Thus, the data confirm the hypothesis that
excreted substances had an allelopathic nature and
were involved in developing ecological relationships
with adjacent plants of different species.
During the composting process water extracts
contain many inhibiting substances that might form
toxic exudates (Kefeli et al., 2001). Paper
chromatography reveals the presence of phenolic acids
and coumarins in water extracts. The highest
concentrations of these inhibitors was measured in
abscised leaves of red maple (Acer rubrum L.). One
gram of dry leaves was mixed in 29 ml of water to
prepare the extracts. The pH of the solution was
between 5.4 and 5.6 and the extracts were incubated
for a week at room temperature while the pH raised to
7.2. Further observations revealed that during
composting the amount of phenolics was drastically
reduced. Seed germination tests were performed with
these water extracts and pure water (control) on lettuce
and wheat seeds. Germination rate and seedling
lengths were measured to demonstrate that phenolics
decreased inhibiting properties after dilution, or after

Table 1: Effect of root exudation on germination of crop seeds (Non-concentrated exudates).

Variant % to tap water (control)

wheat clover lettuce mustard

Tap water 100 100 100 100

Spider plants (Chlorophytum) exudates 54 93 75 100
Willow (Salix vitaminalis) exudates 58 79 74 138

Stem length (5 tallest plants, mm)

Tap water 29 23 18 25
Spider plants (Chlorophytum) exudates 15 21 14 2
Willow (Salix vitaminalis) exudates 7 18 13.5 3.5

Table 2: Biological activity of willow root exudates after paper chromatography (Biological activity in % to control (water)).

Clover Lettuce

Rf Colour in Germination Stem length Germination Stem length

UV-B light

0 Blue 91 76 90 64
0.14 Blue 94 68 98 58
0.3 Violet 86 80 93 76
0.5 Blue 56 52 71 76
0.67 Yellow 87 68 89 88
0.88 Yellow 52 56 63 64
 Phenolic cycle 17

horizon (TSA (triple-soya-agar, 48 hours, room

Photosynthesis temperature). The topsoil (horizon A, 0-28 cm) was
dark gray in color, sandy, high organic matter content
(5.6%), with slightly alkaline pH=7.5. This horizon
Secondary Plant biomass
substances was also high in potassium, low in available nitrogen,
in living plants
and medium in phosphates content, while very high
was the microbial activity. Horizon E (28-52 cm) was
Active secretion
from roots ochric in color, it contained more loam, less organic
into the Biomass of matter, lower microbial activity and pH=7.7. Horizon
water and soil dead plants
B (52-62 cm) had no organic matter, microbial activity
was the lowest and pH=7.8. Water permeability was
Microorganisms Composting process also measured for each horizon to evaluate penetration
Composting process (phenolics and N-sources) times. The fastest penetration rate was measured in
horizon A (11 minutes), whereas it took 47 minutes for
horizon B and longer (more than 6 hours) below
Transformed phenolics and To alumo-silicate horizon B. Soil fertility conditions were also assessed
other secondary substances matrix with a wheat/clover germination test. A sand substrate
was used as control, which yielded 30-50%
Humus formation
germination. Horizon A had a germination of 80-82%,
horizon B 40-60%, horizon E/B (with lowest microbial
F i g u re 3: Secondary substances, plant biomass accumula- activity) yielded 30-70% germination rate (Kalevitch
tion and humus formation during allelopathic effects. et al., 2002). The results of these experiments appear to
indicate that topsoil (for its highest microbial activity)
contact with fungi. Therefore, the whole process of
allelopathogens formation in the environment could be
tightly connected with the formation of secondary
substances and plant biomass accumulation (Figure 3).

S o i l - m i c ro b i a l c o m p l e x f o r p h e n o l i c d e c o m p o s i t i o n

Phenolic substances are the most resistant metabolites

produced by plants. They undergo further
transformation in the soil, forming humus molecules,
strongly linked to the alumino-silicate matrix. Humus
is more or less a stable fraction of soil organic matter;
it adsorbs mineral elements that serve as important
nutrients for plant growth and development (Kefeli,
2002). The alumino-silicate matrix and humus form
primary soil units. Humus is formed by carbon-
nitrogen interaction. Potential sources of carbon
include cellulose and polyphenols from plant leaves,
or transformed lignin polymers.
In order to verify the efficacy of microbial activity
during the humification process, four different soil
horizons in a Grashem soil at the Macoskey Center of
Slippery Rock University of Pennsylvania, USA were
investigated. The presence and number of colonies of
heterotrophic soil microflora were determined in each F i g u re 4: Phenolic cycle.
18 Valentine I. Kefeli et al.

is an effective medium usable to facilitate composting Biochem Physiol Pflanzen. 184: 363-369, 1989.
of maple and sumac leaves, contaning nature phenolic Yamamoto T, Yokotani-Tomita K, Kosemura S, Yamada K
compounds. and Hasegava K. Allelopathic substances exuded from a
serious weed. J Plant Growth Reg. 18: 65-67, 1999.

Conclusion

Microorganisms have the capability to decompose

phenolic compounds to their monomers, being
deglicosidation of phenolic molecules, followed by
lignin decomposition the biochemical pathways of the
process. Leaves become a primary substrate for soil
microorganisms, while woody materials and sawdust
serve as secondary type of biomass and these
substrates play a major role in humus formation
(Figure 4).
The biosynthesis of phenolic substances within
chloroplasts and its further transformation on the
alumino-silicate matrix of soil micelles led us to
conclude about the existence of phenolics cycle in the
plant-soil system. Although many aspects remain
unknown, the ecological relevance of phenolic
substances in the environment has been amply
demonstrated as this cycle embrace lithosphere,
microsphere and biosphere.
These emerging concepts facilitate the
understanding of complexity within our living systems
and their physical habitat while reinforcing the idea of
interconnectedness among living species and
ecosystems.

R e f e r ences

Kalevitch MV, Kefeli VI, Borsari B and Liguory A. Soil

microflora and fabricated soils. American Society for
Microbiology. 103rd General Meeting. Washington DC.
2003 (In press).
Kefeli VI. Fabricated soil for landscape restoration. SME
Ann Meet. 02-142, 2002.
Kefeli VI and Dashek WV. Non hormonal stimulators and
inhibitors. Biol Rev Cambrige. 59: 273-288, 1984.
Kefeli VI, Borsari B and Welton S. The isolation of
inhibiting compounds from the leaves of the red maple
(Acer rubrum L.) for the germination and growth of
lettuce seeds (Lactuca sative L.) NE-Annual Meet of
ASPP J Plant Phys Abstr. 433.
Kefeli VI and Kalevitch MV. Natural Growth Inhibitors and
Phytohormones in Plant and Environment. Kluwer Acad
Publ. 1-310, 2002. In press.
Muzafarov EN and Zolotareva EV. Uncoupling effect of
hydrocinnamic acid derivatives in pea chloroplasts.
 Journal of Cell and Molecular Biology 2: 19-23, 2003. 19
Haliç University, Printed in Turkey.

The short-term effects of single toxic dose of citric acid in mice

Tülin Aktaç1*, Ayflegül Kabo¤lu1, Elvan Bakar1 and Hamiyet Karakafl2
1
University of Trakya, Faculty of Arts and Sciences, Department of Biology, 22080, Edirne-Turkey;
2
University of Trakya, Faculty of Medicine, Department of Biochemistry, 22080, Edirne-Turkey
(* author for correspondence)

Received 12 April 2002; Accepted 03 July 2002

Abstract

The effects of LD25 (480 mg/kg.bw.) dose of citric acid, a food preservative, were investigated on body weight, organ
weights (liver, kidney, spleen), creatin kinase (CK), lactate dehydrogenase (LDH), alanine aminotransferase (ALT)
and aspartate aminotransferase (AST) enzymes in the blood serum, and the liver tissue of mice after 10 days. Citric
acid (to experimental groups) and physiological saline (to control groups) were given intraperitoneally. The results
of enzyme activities were evaluated using autoanalyzer as IU/L. Even though significant decreases in the body
weights were noted when compared to those of the control group (p<0.001), generally an insignificant increase were
observed in the organ weights (liver: p>0.05, kidney: p>0.05, spleen: p>0.05) and serum enzyme levels
(CK: p>0.05, LDH,: p>0.05, ALT: p>0.05, AST: p>0.05). Microscopical examination of the liver showed
histopathological changes depending on the citric acid. These changes were tissue degeneration, cytoplasmic
vacuolisations, nuclear membrane invaginations, picnotic nucleus and necrosis of the hepatocytes.

Key words: Citric acid, food preservative, enzymes, mouse, liver

F a re l e r d e s i t r i k a s i d i n t e k t o k s i k d o z u n u n k › s a s ü re l i e t k i l e r i

Özet

Bir besin koruyucu olan sitrik asidin LD25 (480 mg/kg.va.) dozu farelere intraperitoneal yolla uyguland›. 10 gün
sonra hayvanlar›n vücut a¤›rl›klar›, organ a¤›rl›klar› (karaci¤er, böbrek, dalak), kreatin kinaz (CK), laktat
dehidrogenaz (LDH), alanin aminotransferaz (ALT) ve aspartat aminotransferaz (AST) enzimlerinin serum düzeyleri
ile, karaci¤er dokusu üzerinde sitrik asidin etkileri araflt›r›ld›. Otoanalizörde tayin edilen enzim aktiviteleri U/L
olarak de¤erlendirildi. Çal›flmada vücut a¤›rl›klar›nda kontrol grubuna k›yasla anlaml› bir azalma gözlenmesine
ra¤men (p<0.001), organ a¤›rl›klar›nda (karaci¤er: p>0.05 , böbrek: p>0.05, dalak: p>0.05) ve enzim aktivitelerinde
(CK: p>0.05, LDH: p>0.05, ALT: p>0.05, AST: p>0.05) anlaml› olmayan bir art›fl gözlendi. Karaci¤erin mikroskopik
incelenmesinde doku dejenerasyonu, sitoplazmik vakuolizasyon, nükleer zar çöküntüleri, piknotik nukleuslar ve
hepatositlerde nekroz gibi histopatolojik de¤ifliklikler gözlendi.

Anahtar sözcükler: Sitrik asit, besin koruyucu, enzimler, fare, karaci¤er

I n t ro d u c t i o n substances are antioxidants which are used as food

preservatives. However, peroxides of saturated fats
Humans are exposed daily to complex mixtures of and their secondary oxidation products, can be toxic
chemical compounds in their food. One of these and impair food quality (Würtzen, 1990). Thus despite
20 Tülin Aktaç et al.
their economic importance, they can have negative
effects on living organisms. Xenobiotics entering the
organism are held by intestine, kidney and liver cells
for detoxification. These cells contain important
detoxification enzymes. During the detoxification of
xenobiotics, free radicals are produced in
oxidation/reduction reactions, and these radicals can
have destructive effects on tissues.
The toxic effects of many food preservatives on
living organisms have been studied by many
researchers (Makoveç and Sindelar, 1984; Daniel,
1986; Cabel et al., 1988; Kagan et al., 1990; Jung et
al.,1992; Nijhoff and Peters, 1992; Fujitani, 1993;
Weemaes et al., 1997; Mc Farlene et al., 1997; Safer
and Nughamish, 1999; Kabo¤lu and Aktaç, 2002;
Aktaç et al., 2002). Although the citric acid and metal
salts (sodium or potassium citrat) are widely used in
food industry, there is no report on more detailed
effects of citric acid (or its salts) in liver. In addition,
soft drinks, cosmetics and drugs, in which citric acid is
approved for use, are consumed by most of humans
every day.
A way of analysing harmfull effects of foreign
materials entered to organism is to determine the
effects of the chemicals on the enzymes. Enzymes
have a very important role in the metabolical process
since they are biological catalysts. Thus, their abnormal
serum levels indicates various diseases. Among these
enzymes are, creatine kinase (CK), lactate
dehydrogenase (LDH), alanine aminotransferase (ALT)
and aspartate aminotransferase (AST) which are the
most important. Therefore, we studied short term
treatment of citric acid (10 days) in mice. In these
experiments, firstly we tested total body weigths,
organ weights (liver, kidney, spleen), and determined
the serum levels of creatin kinase (CK), lactate
dehydrogenase (LDH), alanin aminotransferase (ALT)
and aspartat aminotransferase (AST), and secondly the
liver tissue was investigated histopathologically.
Material and Methods
Male mice (Balb/C albino) weighing 25-30 g were
used in our experiments. Five mice were used control
group and ten mice were used the citric acid-treated
group. Animals were fed by pellet baits and water.
LD25 dose (480 mg/kg.bw.) of citric acid (Merck; in
physiological saline) were injected intraperitoneally to
experiment group mice, and the same amount of
physiological saline to control group mice. 10 days
after the injection, the mice were killed by cervical
dislocation and then the necessary studies were
commenced. The livers, kidneys and spleens dissected
out, weighed, liver samples were seperated for
microscopical examination. Blood samples were also
taken for enzyme assays. The serum levels of enzymes
were determined using a Merck Mega 600
autoanalyser with the aid of Diasis Kits. Data were
analyzed by M.Whitney U test for multiple
comparisons for the differences between the control
and treated groups. For histological examination, liver
samples were fixed with 10% buffered formalin,
processed and stained hematoxylin-eosin.
Results
The effects of citric acid injection on the body weight
and liver, kidney, spleen weights was shown Table 1
and 2. Although the liver, spleen and kidney weights
were not changed significantly (p>0.05), the body
weights were decreased significantly (p<0.001). CK,
LDH, ALT and AST activities were not changed
statistically (p >0.05) as shown in Table 2. The results
of the microscopic investigation showed that liver
of mice treated with citric acid has necrotic
changes, compare to the control group (Figure 1-6).
These changes were slightly degeneration of tissue
(Figure 2), cytoplasmic vacuolisation, nuclear
membran invaginations (Figure 3, 4) and picnotic nuclei
(Figure 5). In addition, we observed degeneration of the
blood vessel endothelium (Figure 6).
Discussion
The effects of xenobiotics in living organisms can
investigate in various ways. Among these are, short-
term toxicity tests which are used very commonly. In
these methods, many parameter are used to test the
effects of xenobiotics. Some of these parameters are
body weight, organ weights, blood profile, and
histopathological examination. In this study, the short-
term effects of citric acid applied intraperitoneally
were investigated. It was reported that the body weight
decreases in mouse (Würtzen, 1990), and in rats
(Nijhoff and Peters, 1992) by the effects of phenolic
antioxidant butylated hydroxytoluene (BHT) and
butylated hydroxyanisole (BHA) in chronic studies. In
 Short-term effects of citric acid 21

F i g u re 1: The control group of the liver tissue, bar F i g u r e 2: Citric acid group. Distortion of general
representes 20 µm. histological structure of the liver, v: blood vessel, bar
representes 10 µm.

F i g u re 3: Citric acid group. Nuclear invaginations (arrows),

vacuolisation (v), and damaged nucleus (n) in necrotic cells, F i g u re 4: Citric acid group. Invaginations of hypertrophic
bar representes 4 µm. cell nucleus (arrow), bar representes 4 µm.

F i g u re 5: Citric acid group. Picnotic nuclei (arrows) in F i g u re 6: Citric acid group. Degenerated endothelium
hepatocytes, bar representes 10 µm. (arrows) of blood vessel (v), bar representes 10 µm.

contrast, any significant change was seen in body of sodium benzoate) for ten days by Fujitani (1993).
weight in F344 rats (0.2, 2.5 and 3.0 % of sodium Similarly, Kabo¤lu and Aktaç (2002) were determined
benzoate) and in B6C3F1 mice (1.81, 2.09 and 2.4 % that a significant decrease obtained at 3.0 and 4.0 % of
22 Tülin Aktaç et al.

Table 1: Effect of citric acid on body weight in mice.

Body weight (g)

Before experiment Post experiment

(1. day) (10. days)

Citric acid (LD25 dose) 27.54 ± 0.813 24.76 ± 1.05 *

Control 26.04 ± 1.18 26.58 ± 2.65 **

Values are mean ± SD for ten mice of experiment group and five mice of control group.
(*) significant (p<0.001).
(**) not significant (p>0.05).

Table 2: Effects of citric acid on the organ weights and serum enzyme levels in mice.

Control Citric acid treated

Organ weight
Liver (g) 1.278 ± 0.085 1.257 ± 0.043 *
Kidney (g) 0.2260 ± 0.033 0.1910 ± 0.089 *
Spleen (g) 0.1620 ± 0.036 0.1250 ± 0.014 *
Serum enzyme levels
CK (IU/L) 572 ± 122 1050 ± 255 *
LDH (IU/L) 1296 ± 100 2245 ± 321 *
ALT (IU/L) 695 ± 6.84 101.0 ± 19 *
AST (IU/L) 177.8 ± 3.2 307.2 ± 46.6*

Abbrevations : CK = creatin kinase; LDH = lactate dehydrogenase; ALT = alanine aminotransferase; AST = aspartate aminotransferase.
Values are mean ± SD for ten mice of experiment group and five mice of control group.
(*) not significant (p>0.05).

sodium benzoate. Also, at the present study we
determined a significant decrease of body weight in
mice by the effect of citric acid (Table 1).
Some autors have shown that food preservatives
had increasing effects to organ weight. The effects of
BHT and BHA on the increasing of the liver and
thyroid weights were demonstrated in mice by
Würtzen (1990). Similarly, the effects of BHT on
increasing of the liver weight in rats was also shown
by Mc Farlene et al. (1997) and Safer and Nughamish
(1999). Fujitani (1993) was also obtained significant
increasing of the liver and kidney by the effects of
sodium benzoate in male rats. In our previous studies,
increasing of the total liver weight were seen oral
treatment of sodium benzoate (Kabo¤lu and Aktaç,
2002) and citric acid (Aktaç et al., 2002) but it was not
significant. Additionally, in the present study, we could
not find any significant change the liver, kidney and
spleen weights by the intraperitoneal injection of citric
acid (Table 2). According to our results, serum CK,
LDH, AST and ALT levels in the treated animals were
not significant to compare with the control. These
results were similar with findings obtained in F344 rats
and B6C3F1 mice by Fujitani (1993).
Although the organ weights and serum levels of
enzymes were not changed significantly, the
examination by light microscopy revealed
pathological changes in liver of mice, such as
vacuolisation and glassy cytoplasm in the hepatocyte,
nuclear membrane invaginations, picnotic nuclei.
Similarly, with the effect of sodium benzoate in the
rats and mice, high vacuolisation and glassy
appearance in hepatocyte cytoplasm was explained
(Fujitani, 1993). Again, similar findings were obtained
in the rats with oral treatment of BHT (Mc Farlene et
al., 1997; Safer and Nughamish, 1999), and with
sodium benzoate, benzoic acid and citric acid in mice
(Kabo¤lu and Aktaç, 2002; Aktaç et al., 2002). The
results of present study suggested that citric acid has
hepatotoxic effects and long term exposure may
induce severe damage in liver of mice. However, the
mechanism of damaging effects of citric acid need to
 Short-term effects of citric acid 23

be clarified by more detailed studies.

Finally, we can conclude that consumption of the
foodstuffs containing preservatives is important for the
human health.

R e f e r ences

Aktaç T, Kabo¤lu A, Ertan F, Ekinci F, Hüseyinova G. The

effects of citric acid (antioxidant) and benzoic acid
(antimicrobial agent) on the mouse liver: Biochemical
and histopathological study. Biologia Bratislava. 57(6):
2002. In press.
Cabel MC, Waldroup PW, Shermer WD, Calabotta DF
Effects of ethoxyquin feed preservative and peroxide
level on broiler performance. Poultry Science. 67: 1725-
1730, 1988.
Daniel JW. Metabolic aspects of antioxidants and food
preservatives. Xenobiotica. 16: 10-11, 1986.
Fujitani T. Short-term effect of sodium benzoate in F344 rats
and B6C3F1 mice. Toxicol Lett. 69: 171-179, 1993.
Jung R, Cojocel C, Müller W, Böttger D, Lück E. Evaluation
of the genotoxic potential of sorbic acid and potassium
sorbate. Food Chem Toxicol. 30: 1-7, 1992.
Kabo¤lu A. and Aktaç T.A study of the effects of the sodium
benzoate on the mouse liver. Biologia Bratislava. 57(3):
373-380, 2002.
Kagan VE, Serbinova EA, Packer L. Generation and
recycling of radicals from phenolic antioxidants. Arc
Biochem Biophysiol. 280: 33-39, 1990.
Makoveç P. and Sindelar L. The effect of phenolic
compounds on the activity of respiratory chain enzymes
and on respiration and phosphorylation activities of
potato tuber mitochondria. Biol Plant. 26: 415-422, 1984.
McFarlane M, Price SC, Cottrel S, Grasso P, Bremme JN,
Bomhard ME, Hinton HR. Hepatic and associated
response of rats to pregnancy, lactation and simultaneous
treatment with butylated hydroxytoluene. Food Chem
Toxicol. 35: 753-767, 1997.
Nijhoff WA and Peters WHM. Induction of rat hepatic and
intestinal glutathion S-transferases by butylated
hydroxyanisole. Biochem Pharmacol. 44: 596-600, 1992.
Safer AM and Nughamish AJ. Hepatotoxicity induced by the
antioxidant food additive butylated hydroxytoluene
(BHT) in rats: An electron microscopical study. Histol
Histopathol. 14: 391-406, 1999.
Weemaes CA, De-Cordt SV, Ludikhuyze LR, Van Den
Broeck I, Hendrickx ME, Tobback PP. Influenze of pH,
benzoic acid, EDTA, and glutathione on the pressure
and/or temperature inactivation kinetics of mashroom
polyphenoloxidase. Biotechnol Prog. 13: 25-32, 1997.
Würtzen G. Short comings of current strategy for toxicity
testing of food chemicals: Antioxidants. Food Chem
Toxicol. 28: 743-745, 1990.
 Journal of Cell and Molecular Biology 2: 25-30, 2003. 25
Haliç University, Printed in Turkey.

C h a r a c t e r i s a t i o n o f R P P 7 mutant lines of the col-5 ecotype of

Arabidopsis thaliana
Canan Can1, Mehmet Özaslan1*, Eric B. Holub2
1
University of Gaziantep, Faculty of Science & Arts, Department of Biology, 27310 Gaziantep; 2Plant
Genetics and Biotechnology Department, Horticulture Research International, Wellesbourne, Warwick,
CV35 9EF, England (* author for correspondance)

Received 21 May 2002; Accepted 20 November 2002

Abstract

In this study, phenotypic characterization of RPP7 that confers resistance to Hiks1 isolate of Peronospora parasitica,
deficient mutant lines of Col-5 ecotype of Arabidopsis thaliana was investigated. The Col-5 plants that exposed to
Fast Neutron (FN) were inoculated with 8 different P. Parasitica isolates and symptom development was
investigated. A total of 4 mutant lines were analyzed. It was found that the RPP7 gene present in the Col-5 ecotype
is a unique gene different from the other RPP genes present in Col-5.

Key words: Arabidopsis thaliana, Col-5, Hiks-1, Peronospora parasitica

Arabidopsis thaliana ’ n › n C o l - 5 e k o t i p i n d e n e l d e e d i l e n m u t a n t h a t l a r d a n R P P 7 g e n i n i n
karakterizasyonu

Özet

Bu çal›flmada, Arabidopsis thaliana’n›n Col-5 ekotipinde bulunan ve Peronospora parasitica’n›n Hiks-1 izolat›na
karfl› dayan›kl›l›¤› sa¤layan RPP7 geninde mutasyon içeren hatlar›n fenotipik olarak belirlenmesi üzerinde
araflt›rmalar gerçeklefltirilmifltir. Fast Nötron (FN) uygulamalar› ile mutasyon meydana getirilmifl Col-5
tohumlar›ndan geliflen bitkiler 8 farkl› P. parasitica izolat› ile inokule edilerek semptom geliflimleri incelenmifltir.
Toplam olarak 4 mutant hatta gerçeklefltirilen analizlerde, RPP7 geninin Col-5 ekotipinde bulunan ve farkl›
P. parasitica izolatlar›na karfl› dayan›kl›l›¤› sa¤layan genlerden ba¤›ms›z olarak fonksiyon gösteren bir gen oldu¤u
belirlenmifltir.

Anahtar sözcükler: Arabidopsis thaliana, Col-5, Hiks-1, Peronospora parasitica

I n t ro d u c t i o n rice wheat, tomato, pepper and some other important

Following the isolation of the Pseudomonas syringae
resistance genes (R-gene) from tomato, the research on
isolation and characterization of R-genes against plant
pathogens has been improved (Hammond-Kosack and
Jones, 1997). Recently many R-genes conferring
resistance to fungi, bacteria, nematode and viruses in
crops are isolated and the mechanism of resistance is
determined (Richter and Ronald, 2000). The mutant
lines with lack of R-genes have a potential importance
in this type of work (Mc-Dowel et al., 1998).
Arabidopsis thaliana is a member of cruciferae
family and it is known to have a small genome size of
120 Mb. It is a flowering plant and is a best model for
26 Canan Can et al.
the working on genome analyses, growth regulation,
hormons, flowering, disease resistance and
embryogenesis. Arabidopsis and tomato were used to
determine the mechanisms of disease resistance
(Thomas et al., 1997; Botella et al., 1998). It is also the
host of many pests which attacks to crop plants. Many
genes that provides resistance to bacteria and fungi
disease have been isolated and characterized from A.
thaliana (Dangl and Jones, 2001; Feys and Parker,
2000).
Peronospora parasitica is a causal agent of mildew
disease in the genus cabbage, turnip etc. of cruciferae
family. R-genes that determines resistance to P.
parasitica (RPP) were isolated and characterized
(Holub and Beynon, 1997; Parker et al., 1997; Botelli
et al., 1998; McDowell et al., 1998; Bittner-Eddy et al.,
2000). The researches on RPP genes have shown that
these genes are at the specific regions at certain places
of each chromosome called as “Major recognition
complexes-MRC” (Can, 1997; Holub and Beynon,
1997).
RPP7 gene is present in Col-5 ecotype and
recognized by the Hiks-1 isolate of P. parasitica. This
gene was placed onto the first chromosome between
the markers M421 and M213 by using the hybrid lines
of Col-5 and Nd1 ecotypes (Tor et al., 1994; Can et al.,
1995; Can, 1997). The Hiks1 isolate also recognizes
the RPP1 gene which is present in Nd-1 ecotype, and
has an epistatic effect on the RPP7 gene (Tor et al.,
1994).
The mutant lines that lack the R-genes were
studied in detail and has a wide area of interest such as
molecular and classical genetics. However, in order to
study the relationships between A. thaliana and the
RPP genes and to investigate the genome
organizations, some mutant lines were used (Parker et
al., 1996). The mutant lines lacking the RPP genes
were obtained from Ws-O that contain RPP14 gene
exhibiting resistance to No-Co2 isolate by using Ethyl
Methane Sulfate (EMS). The lines were then used to
separate the RPP10 and RPP1 genes, which were
allelic to RPP14 that is on the third chromosome. It
was found that the WsEDS line was susceptible to all
P. parasitica isolates tested and that the WsEDS locus
was necessary for the function of the RPP genes
(Parker et al., 1996; Bittner-Eddy and Beynon, 2001;
Falk et al., 1999). The npr (Non expressor of PR
protein) mutant lines of A. thaliana synthesize the
proteins which are related with pathogenesis. So,
systemic resistance is not seen following inoculation
with many isolates (Century et al., 1995; Aarts et al.,
1998). Similarly, in (Ethylene Intensitive) mutant lines
do have the ethylene synthesis. But it was found that
the P. syringae f.s.p. tomato resistance continued in
this mutant lines. This study showed that ethylene was
not important for A. thaliana and bacteria relationships
(Bent et al., 1994; Dong, 1998). Lsd (Lesions
Simulating Disease resistance response) and acd
(Accelerated Cell Death) mutant lines produce
Hypersensitive Resistance (HR) like symptoms
without a pathogen infection. These symptoms are
formed by the influence of external factors like heat
and light (Lam et al., 1999). So, it is accepted that lsd
and acd loci are negative regulators for HR formation
(Dietrich et al., 1994). In general, the presence of
different resistance mechanisms in A. thaliana which
are directed by RPP genes was found by the
characterization of mutant lines (Glazebrook et al.,
1997; McDowell et al., 2000).
In this study, the mutant lines of the Col-5 ecotype
of A. thaliana were characterized, to understand the
mechanisms of RPP7 gene that confers resistance to
Hiks-1 isolate of P. parasitica.
Material and methods
Plant and fungus material
In this study, Col-5 ecotype of A. thaliana lines having
MRC-B, MRC-C and MRC-H regions (Can, 1997;
Holub and Beynon, 1997) were used as wild type
ecotype. The Fast Neutron (FN) applied mutant lines
were obtained commercially and the selections of
mutant lines were performed by using the Hiks-1
isolate of P. parasitica. The Hiks-1 isolate recognizes
the RPP7 gene which is in the MRC-B region of wild
Col-5 ecotype, and 7 days after inoculation it induces
a resistance which is defined with HR. Four mutant
lines were used in this study denoted as FN3922,
FN3928, FN3929 and FN3930. The HR does not occur
in mutant lines, and the pathogen completes its life
cycle by sexual and asexual sporulation.
Regeneration of Hiks-1 isolate from oospore
population
The Hiks-1 isolate was regenerated by using oospore
population. To do this, the seeds of A. thaliana that
were susceptible to the Hiks-1 isolate were sown into

little plastic pots containing 4:1:1 (torf: perlit: sand) of
mixture for 40-50 seeds each. The pots were irrigated
to wet the seeds and 1-2 x 105 oospore/ ml were added
to the pots. The containers were held at 4 °C for 1-2
weeks to break the dormancy. Following this, the
containers were placed into the climated room at 18-20
°C, 10 h light and 14 hour dark period. Within 10-15
days following seed germination, some seedlings
having sporulation was collected and placed into
eppendorf tubes containing 200 µl dH2O. The
eppendorf tubes were shaked gently to allow the
conidia to pass to water. The conidia suspension was
used to inoculate 7 days old seedlings of EBH3529 and
Ksk-1 ecotypes, and the plants were placed into the
climate room. By this way, the regeneration of the
Hiks-1 isolate was done by subculturing 3-4 times. The
conidia were stored at –20 °C and were used when
needed.
The same procedure were applied for, Ahco-1,
Ahco-2, Ahco-7, Wand-1, Cand-5, Hind-2 and Hind-4
isolates using Nd-1 and Col-5 ecotypes (Can, 1997).
Characterization of P. parasitica isolates by using
different A. thaliana ecotypes
Regenerated P. parasitica isolates were inoculated into
Col-5, Ksk-1, Nd-1, Ws-3, Tsu-1, Ler-1, Oy-1 and
Wei-1 ecotypes in order to do phenotypic
characterization. The A. thaliana ecotypes were
obtained from Dr. Eric Holub (HRI- UK)
The conidia suspension was adjusted to 4-5 x 104
conidia/ml concentration for plant inoculations. The
cotyledons of 7-8 days of the A. thaliana ecotypes
were inoculated in such a way that it would be one
drop to each cotyledon. The plants were placed into
climated room with 18-20 °C, 10 h light, 14 h darkness
conditions after the inoculation and the plants were
checked at the end of 3. and 7. days. The evaluation
was done regarding the pathogen sporulation and
hypersensitive reaction types (the interaction
phenotypes). Phenotypic reactions were examined
under the fine group as; pitting with no pathogen
sporulation (PN), flecking with no pathogen
sporulation (FN), flecks with delate and moderate
pathogen sporulation, 1-20 sporangiophorus per each
cotyledon (DM), flecking with delate pathogen
sporulation, 5-10 sporangiophorus per each cotyledon
(FDL), early and heavy pathogen sporulation, 20>
sporangiophores per cotyledon (EH), (Holub et al.,
1994).
Characterisation of RPP7 gene 27
Microscopic analysis
Fungal development in plant tissue was examined
under light and fluorescence microscope. The infected
leaves were taken and put absolute methanol for 5-6
hours followed by saturated chloral hydrate solution
for 4-5 hours. Then, tissues were placed in 50 %
glycerol solution for microscopic analyses.
DNA analysis
Total plant genomic DNA was isolated with some
modifications by using the methods of Ausubel et al.,
(1994). Five to eight grams of plant material was
grounded in N2 and transferred to the tubes containing
15 ml buffers (100 mM Tris-HCl, 50 mM EDTA, 500
mM NaCl, 10 mM Mercaptoethanol, %25 SDS) with
100 mg/lt proteinase K. The solution was kept at 55 °C
for 1 hour. At the end of this time period, 5 ml of 5 M
potassium acetate was added and held in ice for 20
minutes, and the solution was centrifuged at 17000
rpm for 25 minutes. The supernatant was mixed with
0,6 volume of isopropanol and held at -20 °C for
minutes and the DNA was precipitated. Phenol-
chloroform was used to wash the DNA and a second
precipitation was done. The DNA was dissolved in
dH2O and stored at -20 °C. The isolated DNA was
diluted in such to 50-100 ng/µl to use in polimerase
chain reactions (PCR). For PCR reactions, the closest
marker to the RPP7 gene was used (Can, 1997). To do
this, the solution which contains 0.05 mm primer, 2
mm dNTPs, 25 mm MgCl2, 1 x Taq buffer and IU Taq
DNA polymerase was completed to 25 ml volume. The
PCR reactions were performed at 94 °C for 5 min
followed by 94 °C for 1 minute, 56 °C for 1 min, 72 °C
for 13 minute (35 cycles) and 72 °C for 10 min. The
samples were electrophoresed at 80 W for 4 hours.
Results and discussions
Characterization of P. parasitica isolates
In order to determine the changes at the RPP7 locus in
the mutant lines, Ahco-1, Ahco-2, Ahco-7, Wand-1,
Cand-5, Hind-2 and Hind-4 isolates were used. Ahco-
1, Ahco-2, Ahco-7 recognize MRC-B region which is
located at the first chromosome in the Nd-1 ecotype
(Can, 1997), and these isolates were presumed to
recognize RPP7 allele of Nd-1 ecotype. Wand-1,
28 Canan Can et al.

Table 1: Interaction phenotypes of different P. parasitica isolates on the A. thaliana ecotypes.

Interaction phenotypes on different A. thaliana ecotypes*

P. parasitica isolates Col-5 Ksk-1 Nd-1 Ws-3 Ler-1 Oy-1 Wei-1

Hiks-1 FN EH PN PN FN DM FDL
Ahco-1 DM FN FDL FN FDL FN FDL
Ahco-2 DM FN FDL FN FN FN DM
Ahco-7 DM FN FDL FN FDL FN EH
Wand-1 FN FN EH FN FN FN DM
Cand-5 FN EH EH FN CN FDL DL
Hind-2 FN FN EH PN FN FN EH
Hind-4 FR FN EH PN EH FN EH

*Necrotic pits (PN), necrotic flecks (FN), cavities (CN), flecks with delate and moderate pathogen sporulation, 1-20
sporangiophorus per each cotyledon (DM), flecking with delate pathogen sporulation, 5-10 sporangiophorus per each cotyledon
(FDL), early and heavy pathogen sporulation, 20> sporangiophores per cotyledon (EH).

Cand-5, Hind-2 and Hind-4 isolates recognize MRC-B
and MRC-C region which present at the second
chromosome in the Col-5 ecotype. The isolates
regenerated from the oospore populations were
inoculated on different A. thaliana ecotypes (Col-5,
Ksk-1, Nd-1, Ws-3, Ler-1, Oy-1 and Wei-1) to
determine if they were original. The results are shown
in Table1.
As it could be seen in Table 1, the isolates
generated from the oospore populations were found to
be as original, and there was no variation (Can, 1997).
Therefore these isolates were used to inoculate the
mutant lines recovered through inoculation with the
Hiks-1 isolate.
Phenotypic characterization of mutant lines
P. parasitica isolates were used to inoculate the Col-5
lines. The results were shown in Table 2.
As indicated in Table 1, DM and EH phenotypes
developed, following inoculation of FN3922, FN3928,
FN3929 and FN3930 mutant lines with the Hiks-1
isolate. These results revealed that the RPP7 gene is
not present in the mutant lines. However, Ahco-1,
Ahco-2 and Ahco-7 isolates exhibited the EH
phenotype compared to DM in the wild Col-5 ecotype.
This result showed that absence of the RPP7 gene
increased susceptibility. The important point in here
was that mutation of one R-gene could effect the
resistance in same plant to other isolate. The result

Table 2: Interaction phenotypes exhibited by Col-5 mutant lines following inoculation with different P. parasitica isolates.

The interaction phenotypes on wild type and mutant lines*

P. parasitica isolates Col-5 Nd-1 Ksk-1 FN3922 FN3928 FN3929 FN3930

Hiks-1 FN PN EH EH DM EH EH
Ahco-1 DM FDL FN EH EH EH EH
Ahco-2 DM FDL FN EH EH EH EH
Ahco-7 DM FDL FN EH EH EH EH
Wand-1 FN EH FN EH FN FN FDL
Cand-5 FN EH EH EH FN FN FN
Hind-2 FN EH FN FN FN FN FN
Hind-4 FR EH FN EH FN FN FN

*Necrotic pits (PN), necrotic flecks (FN), flecks with moderate and late pathogen sporulation, 1-20 sporangiophorus per each
cotyledon (DM), flecking with delate pathogen sporulation, 5-10 sporangiophorus per each cotyledon (FDL), early and heavy
pathogen sporulation, 20> sporangiophores per cotyledon (EH).

F i g u re 1: Microscopic reactions occurred in Col-5 ecotype
following inoculation with the Hiks-1 isolate. (A) Six hours
after inoculation. a. Haustorium formed in the mesofil cells.
b. Cell death and (B) Twelve hours after inoculation.
Hypersensitive reaction and cell death.
750 bp →
500 bp →
F i g u re 3: Distribution of g2a markers in mutant lines.
M indicates the 1 kb DNA marker.
may also show that when the RPP genes are allelic
they do function in coordination.
Cand-5 isolate recognizes MRC-B region which is
present in Col-5 and determined with FN interaction
phenotype. FN3922 mutant line exhibited the EH
phenotype with Cand-5 and FN with other isolates
tested (Table 2). This result may show that FN3922
may be a Col-5 contamination. On the other word it is
possible that in this line, many RPP genes present in
the MRC-B loci (Can, 1997) could be mutated.
Therefore, FN3922 may not be a specific mutant of the
RPP7 gene. As it is known the Fast Neutron
Characterisation of RPP7 gene 29
F i g u re 2: Microscopic reactions occured the FN3929
mutant line following inoculation with the Hiks-1 isolate.
Formation of fungal haustorium in the mesophyl cell (a) and
hif development (b), twelve hours after inoculation.
application results point mutations, so that at MRC-B
region in FN3922, many point mutations or deletions
may have been occurred.
Inoculations of the FN3928, FN3929 and FN3930
mutant lines with Wand-1, Hind-2, Hind-4 resulted FN
phenotype. This results shows that the RPP7 gene has
no correlation with the RPP genes present in seconder
chromosome at the MRC-C region (Table 2).
Microscopic characterization of mutant lines
The fungal development in plant tissue was examined.
Following inoculations at the first, third and seven
days the cotyledons from the mutant lines and the wild
type Col-5 were taken and prepared as described
before. At the first six hour of inoculation of Col-5
with the Hiks-1 isolate cell death was observed. The
susceptible genotypes and the mutant lines allowed the
penetration and mycelial development without any cell
(Figure 1, 2). Fungal development in the mutant lines
supported the macroscopic results (Mc Dowell et al.,
2000).
DNA analyses of mutant lines
nga280 and g2a markers present at the lower arm of
the first chromosome of A. thaliana were detected to
give less recombination with the RPP7 loci (Can,
1997). Therefore the mutant lines were subjected to
PCR analyses with these markers. Results are given in
Figure 3.
The distributions of the bands in mutant lines were
similar to those in the wild type Col-5. This result may
suggest that no deletions may occurred in the mutant
lines and that the loss of function could be due to a
point mutation.
30 Canan Can et al.
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Parker JE. Differential requirements for EDS1 and
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Smith JA and Struhl K. Current Protocols in Molecular
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Bent AF, Kunkel BN, Dahlbeck D, Brown KL, Schmidt R,
Giraudat J, Leung J and Staskawicz BJ. RPS2 of
Arabidopsis thaliana, A leucine-rich repeat class of plant
disease resistance genes. Science. 265 (5180): 1856-
1860, 1994.
Bittner-Eddy PD, Crute IR, Holub EB and Beynon JL.
RPP13 is a simple locus in Arabidopsis thaliana for
alleles that specify downy mildew resistance to different
avirulence determinants in Peronospora parasitica. The
Plant Journal. 21(2): 177-188, 2000.
Bittner-Eddy PD and Beynon JL. The Arabidopsis downy
mildew resistance gene, RPP13-Nd, functions
independently of NDR1 and EDS1 and does not require
the accumulation of salicylic acid. Mol Plant-Microbe
Interactions. 14: 416-421, 2001.
Botella MA, Parker JE, Frost LN, Bittner-Eddy PD,
Beynon JL, Daniels MJ, Holub EB and Jones JDG.
Three genes of the Arabidopsis RPP1 complex
resistance locus recognize distinct Peronospora
parasitica avirulence determinants. Plant Cell. 10: 1847-
1860, 1998.
Can C. Bittner-Eddy P, Tör M, Williams K, Gunn N, Bakht S,
Atkinson L, Debener T, Chimot P, Crute I, Beynon J and
Holub EB. Revealing the organization of RPP loci in the
Arabidopsis thaliana genome, Poster abstract in 6th
International Conference on Arabidopsis Research,
Medison, Winconsin. 7-11 June, 1995.
Can C. Genomic organisation of pathogen recognition genes
in Arabidopsis thaliana to Peronospora parasitica,
Ph. D. Thesis, University of London, Wye Collage, UK.
1997.
Century KS, Holub EB and Staskawicz BJ. NDR1, a locus of
Arabidopsis thaliana that is required for disease
resistance both a bacterial and a fungal pathogen. P roc
Natl Acad Sci USA. 92 (14): 6597-6601, 1995.
Dangl JL and Jones JDG. Plant pathogens and integrated
defense responses to infection. Nature. 411: 826-833,
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Dangl JL. A novel zinc finger protein is encoded by the
Arabidopsis LSD1 gene and functions as a negative
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Dong X. SA, JA, ethylene, and disease resistance in
plants. Curr Opin Plant Biol. 1: 316-323, 1998.
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 Journal of Cell and Molecular Biology 2: 31-34, 2003. 31
Haliç University, Printed in Turkey.

The effect of meta - t o p o l i n o n p r o t e i n p r o f i l e i n r a d i s h c o t y l e d o n s

Serap Ça¤1 and Narçin Palavan-Ünsal2*
1
Istanbul University, Department of Biology, Botany Section, Süleymaniye 34460, Istanbul-Turkey;
2
Haliç University; Department of Molecular Biology and Genetics, F›nd›kzade 34280, Istanbul-Turkey
(* author for correspondance)

Received 27 September 2002; Accepted 30 November 2002

Abstract

Meta-topolin (mT) has been established as an active aromatic cytokinin recently. The present investigation assessed
the effects of mT on radish cotyledon growth and protein content. 0.05 to 1 mM mT increased the cotyledon growth
about 2 fold in fresh weight basis. mT at 0.1, 0.25 and 0.5 mM concentrations caused an increase in soluble protein
levels compared to the control cotyledons almost in the same ratio by 3 %. Compared to control cotyledons analysis
of the soluble proteins displayed different electrophoretic pattern in mT treated cotyledons.

Key words: Cotyledon growth, meta-topolin, protein

Meta - t o p o l i n i n t u r p k o t i l e d o n l a r › n d a p r o t e i n p r ofiline etkisi

Özet

Son y›llarda meta-topolin (mT) aktif aromatik sitokinin olarak saptand›. Bu araflt›rma da mT’in turp kotiledonlar›n›n
büyüme ve protein içeri¤ine etkisi araflt›r›ld›. 0.05-1 mM mT kotiledon büyümesini taze a¤›rl›k baz›nda yaklafl›k 2
kat kadar teflvik etti. 0.05, 0.1 ve 0.25 mM mT çözünür protein düzeylerini kontrole oranla yaklafl›k % 3 oran›nda
artt›rd›. Çözünür proteinlerin analizleri, mT uygulanan kotiledonlarda kontrole oranla farkl› bir elektroforetik dizilim
gösterdi.

Anahtar sözcükler: Kotiledon büyümesi, meta-topolin, protein

I n t ro d u c t i o n Mok, 1994; Binns, 1994).

Cytokinins, N6-substituted adenine derivatives are a
class of plant hormones that were first identified as
factors that promoted cell division (Miller et al., 1955;
1956) and have been implicated in many other aspects
of plant growth and development including shoot
initiation and growth, apical dominance, senescence
and photomorphogenetic development (Letham,
1971; Thimann, 1980; Mok and Mok, 1994).
Although the physiological effects of cytokinins have
been well documented, the molecular mechanisms
underlying cytokinin action remain obscure (Mok and
Bioassays are used to establish the relative
biological activity of plant hormones compared with
others. The cytokinin bioassays used most frequently
depend on growth of tissues in sterile culture (Letham
1967). Such methods are extremely sensitive but it
needs at least 3 weeks to get final results. Letham
(1971) described a rapid bioassay for cytokinins based
on the ability of these compounds to promote
markedly the expansion of radish cotyledons excised
soon after seed germination.
To date the effects of common cytokinins i.e.
kinetin, benzyladenine (BA) and its riboside have been
32 Serap Ça¤ and Narçin Palavan-Ünsal
documented in radish cotyledons. A new active
aromatic cytokinin meta-topolin (mT) have been
determined by Strnad et al. (1997) in poplar. The
sensitivity of the radish cotyledon bioassay to mT has
been established by us before (Palavan-Ünsal et al.,
2002). This study will focus on the effect of mT on
soluble protein contents in radish cotyledons that has
not been studied before.
Material and methods
Plant material and bioassay
Radish (Raphanus sativus L.) seeds were germinated in
darkness for 4 days at 25 °C on moist filter paper in 5
cm petri dishes. Cotyledons were excised excluding
petiole tissues and four cotyledons were placed in each
petri dish after measuring the fresh weight. The
cotyledons were placed with their adaxial sides down
on the paper. They were incubated in a growth chamber
at 25°C ± 2°C and 12 h light-dark photoperiods. Three
ml mT was applied per petri dish at 0.05, 0.1, 0.25, 0.5
and 1.0 mM concentrations. Cotyledon growth was
measured by determining fresh and dry weights 3 days
after the application (Letham, 1971) and the data
presented here representative of 15 experiments.
Measurement of soluble protein content
Soluble protein content was determined as in Bradford
(1976) using bovine serum albumin as standard. Each
experiment was repeated four times and each treatment
included three replicates.
Electrophoresis for proteins
Sodium dodecylsulphate (SDS)-polyacrylamide slab
gel electrophoresis was performed according to
Laemmli (1970). Gel containing 3.0 % (stacking gel)
and 10.0 % (separation gel) acrylamide were prepared
from a stock solution of 30.0 % of acrylamide and 0.8
% N, N’-bis methylene acrylamide. The gels were
polymerized chemically by the addition of ammonium
persulphate. The mixture was completely dissociated
by immersing the samples for 3 min in boiling water.
Electrophoresis was carried out with a current of 150 V
per gel until the bromophenol blue marker reached the
bottom of the gel. The proteins were stained in the gel
with Coomassie brilliant blue solution for overnight at
room temperature. The gels were diffusion-destained
by repeated washing in the solution containing 7.5 %
acetic acide, 5 % methanole and 87.5 % distilled water.
Results and discussion
The early observations revealed that cytokinins exert
parallel effects in maintain protein or nucleic acid
levels while inhibiting senescence. Cytokinins
stimulate both structural and enzymatic protein
synthesis. They are selectively increasing the levels
of certain enzymes associated generally with
photosynthetic process (Feierabend, 1969). It is not
clear whether the enhanced activity is due to greater
synthesis, inhibition of degradation or activation of the
enzymes.
We already observed that new aromatic cytokinin
mT at 0.25 to 1 mM concentration range delayed
the senescence in excised wheat leaf segments
(Palavan et al., 2002). This concentration range was
high for radish cotyledon growth therefore lower
concentrations were examined (0.05 to 1 mM) in
addition.
Cotyledon growth increased with the treatments of
mT significantly (Figure 1). Stimulation of cotyledon
growth was closely related with increasing
concentrations of mT; 0.05 to 1 mM mT increased the
cotyledon growth about two fold in fresh weight basis
(p<0.05), while dry weights of cotyledons during the
growth were not effected by mT application (Palavan-
Ünsal et al., 2002).
Cytokinins promote cell enlargement in certain
tissues and organs. This effect is most clearly seen in
cotyledons. The expansion of cotyledons is resulted
F i g u re 1: The effect of meta-topolin on cotyledon growth in
radish (Values are average of 30 cotyledons).

F i g u re 2: The effect of meta-topolin on soluble protein con-
tent during the growth of radish cotyledons. Values are aver-
age of 4 experiments.
F i g u re 3: SDS-PAGE analysis of soluble proteins from
meta-topolin treated radish cotyledons. Gel was stained with
Coomassie blue. Lane 1: Control, Lane 2: 0.05 mM mT,
Lane 3: 0.1 mM mT, Lane 4: 0.25 mM mT, Lane 5: 0.5 mM
mT, Lane 6: 1 mM mT treatments. Molecular mass (kDa) of
markers are indicated on left hand margin.
from cell enlargement during cotyledon growth.
Cytokinin treatment promotes additional cell
expansion with no increase in the dry weight of the
treated cotyledons (Huff and Ross, 1975).
Letham (1971) reported the ability of cytokinins to
promote markedly the expansion of radish cotyledons
and explained this response by the promotion of cell
enlargement. mT also as a most active aromatic
meta-topolin effect on protein 33
cytokinin as reported by Strnad et al. (1997) caused to
cotyledon growth markedly as shown in Figure 1.
mT was found to increase the soluble protein
contents of radish cotyledons. Treatments with 0.1, 0.25
and 0.5 mM mT resulted an increase in soluble protein
content in the same ratio (by 3,4 and 3 % respectively)
compared to the control cotyledons (Figure 2).
These findings correlated with electrophoretic
determinations (Figure 3). Soluble proteins of mT
treated radish cotyledons were analyzed using SDS-
PAGE technique in order to test whether and
significant amount of difference in protein profile
occurred with mT treatments. Analysis of the soluble
proteins displayed different electrophoretic pattern in
mT treated cotyledons compared to control. Protein
bands were very sharp and dark in 0.05, 0.1 and 0.25
mM mT treated samples and their molecular masses
ranges between 66 to 45 kDa’s. Molecular mass of 45
to 29 kDa’s were weak in 0.5 and 1 mM mT treated
and in control cotyledons also. On the other hand
protein bands were very sharp and dark in 0.05, 0.1
and 0.25 mM mT treated cotyledons comparing with
0.5 and 1 mM mT treated and control cotyledons.
Obvious bands were also observed around 30 kDa in
cotyledons treated with 0.05, 0.1 and 0.25 mM mT.
Besides these there were additional bands in mT
treated samples different from controls and these
bands were weak in 0.5 and 1.0 mM mT treated
samples compared to the other applications around 24
kDa.
There is good evidence that cytokinins play a role
in regulating protein synthesis (Tepfer and Fosket,
1978). Cytokinins can not only increase the rate of
protein synthesis, but also change the spectrum of
proteins produced by plant tissues.
Results obtained in this study showed that, total
soluble protein content in radish cotyledons not
effected from exogenously applied mT. On the other
hand, when protein profile was examined
electrophoretically additional bands were observed in
mT treated samples. These can be explained by the fact
that mT stimulate new protein synthesis without
effecting total protein content.
In conclusion, natural aromatic cytokinin mT has
an important role in the control of cotyledon growth
and this response closely associated with protein
profile. The results of this research are exhibited mT as
a promising plant growth regulators in physiological
studies.
34 Serap Ça¤ and Narçin Palavan-Ünsal

Acknowledgement

We thank to Dr. M. Strnad and his colleagues for the

generous gift of aromatic cytokinins and to Damla
Büyüktunçer for technical assistance. This study was
supported by Istanbul University Research Fund
(Project number: B-430/13042000).

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 Journal of Cell and Molecular Biology 2: 35-38, 2003. 35
Haliç University, Printed in Turkey.

T h e e f f e c t o f e l e c t romagnetic fields on oxidative DNA d a m a g e

Serkan ‹fller1 and Günhan Erdem2*
1
Department of Biology, Institute of Applied Sciences, Çanakkale Onsekiz Mart University, Çanakkale,
Turkey; 2College of Health, Çanakkale Onsekiz Mart University, Çanakkale, Turkey (*author for
correspondence)

Received 30 September 2002; Accepted 26 December 2002

Abstract

Many recent studies have focused on the investigation of the biological effects of electromagnetic field. Although
the several types of biological effects of electromagnetic fields have been shown, the molecular mechanisms of these
effects have not been explained yet. Some epidemiological studies have suggested that exposure to ambient, low-
level 50-60 Hz electromagnetic fields increase risk of disease including cancer such as leukemia among children who
live close to power lines or among men whose jobs expose them to electromagnetic field, while others have
suggested that electromagnetic fields exposure could increase both the concentration of free radicals and oscillating
free radicals. Electromagnetic fields are known to affect radical pair recombination and they may increase the
concentration of oxygen free radicals in living cells. In this study, oxidative stress was formed by the oxidation of
ascorbic acid and the effect of 50 Hz, 0.3 mT electromagnetic fields on the oxidative DNA damage has been
investigated. The results of the study showed that extremely low-frequency electromagnetic fields enhanced the
effect of oxidative stress on DNA damage and supported the idea obtained from the previous studies on an increasing
effect of electromagnetic fields on the concentration and the life-time of free radicals.

Key words: Electromagnetic fields, DNA damage, ascorbic acid, vitamin C, oxidative stress

E l e k t ro m a n y e t i k a l a n › n o k s i d a t i f D N A h a s a r › ü z e r i n d e k i e t k i s i

Özet

Günümüzdeki birçok çal›flma, elektromanyetik alan›n biyolojik etkilerinin araflt›r›lmas› üzerinde odaklanm›flt›r.
Elektromanyetik alan›n biyolojik etkilerinin baz› türlerinin gösterilmifl olmas›na ra¤men, bu etkilerin moleküler
mekanizmalar› henüz aç›klanamam›flt›r. Baz› epidemiyolojik çal›flmalar, 50-60 Hz dolay›ndaki düflük düzeyli
elektromanyetik alana maruz kalman›n yüksek gerilim hatlar›na yak›n yaflamakta olan çocuklarda veya
elektromanyetik alana maruz kalarak çal›flanlarda görülen lösemi gibi kanser vakalar›n› kapsayan hastal›klara iliflkin
riski art›rd›¤›n› öne sürerken, baz› çal›flmalar ise elektromanyetik alan maruziyetinin serbest radikal
konsantrasyonunu ve serbest radikallerin izlenebilirli¤ini art›rabilece¤ini ileri sürmüfltür. Elektromanyetik alan›n
radikal çifti rekombinasyonunu etkiledi¤i bilinmektedir ve bu da, hücrelerdeki oksijene dayal› serbest radikal
konsantrasyonunu art›rabilir. Bu çal›flmada, askorbik asit oksidasyonu ile oksidatif stres oluflturulmufl ve 50 Hz, 0.3
mT düzeyindeki elektromanyetik alan›n, oksidatif DNA hasar› üzerindeki etkisi araflt›r›lm›flt›r. Bu çal›flman›n
sonuçlar›, oldukça düflük frekansl› elektromanyetik alan›n, oksidatif stresin DNA hasar› üzerindeki etkisini art›rd›¤›n›
göstermifl ve önceki araflt›rmalardan elde edilen, elektromanyetik alan›n serbest radikal konsantrasyonu ve yar› ömrü
üzerindeki art›r›c› etkisine dair düflünceleri desteklemifltir.

Anahtar sözcükler: Elektromanyetik alan, DNA hasar›, askorbik asit, C vitamini, oksidatif stres
36 Serkan ‹fller and Günhan Erdem
I n t ro d u c t i o n
There are many reports on the biological effects of
electromagnetic fields (EMF) and there have been
many attempts to develop a theoretical explanation of
this phenomenon. Some epidemiological studies have
suggested that exposure to ambient, low-level 50/60
Hz EMF increases risk of disease including cancer
such as leukemia among children who live close to
power lines or among men whose jobs expose them to
EMF (Wertheimer and Leeper, 1979; Tomenius, 1986;
Savitz et al., 1988; London et al., 1991). EMF firstly
affects the cell membrane. Some ion channels such as
Na-K ATPase have been affected according the level
of EMF. The alteration in the activity of these proteins
causes an increasing or decreasing intracellular
concentration of many ions such as Na+, K+, Mg2+ and
Ca2+ which plays very important roles in cell signaling.
Therefore, the biological effects of EMF expand
among the cellular systems (Goodman et al., 1995).
Although the several types of biological effects of
EMF have been shown, the molecular mechanisms of
these effects have not been explained yet. Some
studies have suggested that EMF exposure could be
due to both the increase in the concentration (Jajte,
2000) and oscillating of free radicals (Scaiano et al.,
1995). EMF is known to affect radical pair
recombination and they may increase the
concentration of oxygen free radicals in living cells
(Jajte, 2000). Increasing the concentration of free
radicals creates oxidative stress and some biological
reactions such as DNA damage occur under this
condition. Metabolic energy production or effects of
chemicals and radiation can form oxidative stress.
In this study, oxidative stress was formed by the
oxidation of ascorbic acid with Cu2+ ions and the effect
of 50 Hz, 0.3 mT EMF on the oxidative DNA damage
was investigated.
Materials and methods
DNA isolation
High molecular weight (app. 10 kb) human genomic
DNA was isolated from the white blood cells with the
modified method of Poncz et al. (1982) by using MBI
Fermentas genomic DNA isolation system. Molecular
weight and purity of DNA samples were controlled by
agarose gel electrophoresis. The concentration of
DNA samples was spectrophotometrically determined.
All DNA samples were free from proteins, RNAs and
solvents used for extraction.
Oxidative DNA cleavage reactions
Cleavage reactions were carried out in a medium
containing 0.5 µg DNA, 20 mM Tris-HCl (Sigma) pH
7.8, 0.25 mM ultra-pure ascorbic acid (Merck) and
CuCl2 (Sigma) in the final concentrations of 2.5, 5, 7.5
and 10 µM, in a final volume 10 µl. Other antioxidants
(glutathion, cystein and dithiothreitol, Sigma) and
metal chelator (EDTA, Merck) were added to the
reaction mixtures at a final concentration of 0.5 mM.
The mixtures were incubated at room temperature for
10, 20 and 30 minutes. Adding EDTA at a final
concentration of 25 mM stopped reactions. DNA
cleavages in the reaction mixtures were analyzed on
the 1% agarose gel (Promega) electrophoresis.
EMF exposure system
Electromagnetic fields were applied by using the
Helmholtz coil. The coil system was constructed by
using the polyester sphere that was surrounded by
copper wire with 0.75 cm diameter (Galt et al., 1995).
The diameter and height of the sphere were 16 and 26
cm, respectively. 50 Hz, 4.5 V electricity was applied
to coil system. As a result, 0.3 mT EMF was generated
at the center of the coil system which includes handles
for the sample tubes.
Results and discussion
The oxidative DNA damage was induced by the
concentration of cupric ions (Figure 1). In the constant
ascorbate concentration (0.25 mM), oxidative DNA
breakage was started in the presence of 2.5 µM
copper(II) ions and EMF also induced the DNA
breakage at this condition (Figure 1, lanes 4 and 10).
Therefore, main DNA band in the lane 10 of Figure 1
is thinner than the lane 4. In the presence of high
cupric ions concentration, excess scission of DNA
molecules occurred at the EMF when compared to
normal conditions (Figure 1, lanes 6 and 12).
Electromagnetic fields did not have an effect on the
oxidation of ascorbic acid in the absence of cupric ions
(specific data was not shown, but the sample in Figure
3, lane 12 had reflected this result, because EDTA was

1 2 3 4 5 6 7 8 9 10 11 12
F i g u re 1: The effect of EMF and Cu(II) concentrations on
oxidative DNA damage. All lanes include 0.5 µg DNA. The
DNA samples in the lanes from 1 to 6 were incubated under
normal condition, 7 to 12 were incubated in EMF at room
temperature in the presence of 0.25 mM ascorbic acid
except the 1 and 7 which were control lanes. Cu(II)
concentrations were 1.25 µM in 2 and 8, 2.5 µM in 3 and 9,
5 µM in 4 and 10, 7.5 µM in 5 and 11, 10 µM in 6 and 12
lanes. Incubation time was 30 min for all samples.
chelating cupric ions and eliminated their oxidative
effects).
The results of this study showed that the oxidative
DNA damage depends on the incubation time (Figure 2).
DNA breakages could be observed at the 20th minute of
incubation time (Figure 2, lanes 6 and 8). EMF
exposure enhances the oxidative DNA damage after
the 20th minute (Figure 2, lanes 2 and 4).
1 2 3 4 5 6 7 8 9 10 11 12
F i g u re 3: The effect of some antioxidants (glutathione,
cystein, dithiothreitol) and metal chelator (EDTA) on
excessive oxidative DNA damage in EMF. All lanes include
0.5 µg DNA. Ascorbic acid and Cu(II) concentrations were
0.25 mM and 7.5 µM in all lanes except the control DNA
lanes 1 and 7, respectively. Lanes 2 and 8 were scission
controls. Glutathione (3 and 9), cystein (4 and 10),
dithiothreitol (5 and 11) and EDTA (6 and 12)
concentrations were 0.5 mM. The samples in 1 to 6 were
incubated at normal condition. The others were incubated in
EMF. Incubation time was 30 min for all samples.
EMF and oxidative DNA damage 37
1 2 3 4 5 6 7 8 9 10 11 12
F i g u re 2: The effect of incubation time on oxidative DNA
damage depends in electromagnetic fields. All lanes include
0.5 µg DNA. Incubation times were 30 min from 1 to 4, 20
min from 5 to 8 and 10 min from 9 to 12. Ascorbic acid
concentration were 0.25 mM in all lanes. Cu(II)
concentrations were 2.5 µM in 1, 3, 5, 7, 9 and 11 while
5 µM in 2, 4, 6, 8, 10 and 12. The samples in 1, 2, 5, 6, 9 and
10 were incubated in EMF. Other samples were incubated at
normal conditions.
In the presence of EDTA as a cationic metal
chelator, oxidative DNA damage was not observed.
This result showed that ascorbate oxidation and
oxidative DNA damage depend on cupric ions as an
oxidizing agent (Figure 3, lanes 6 and 12). As an
antioxidant, cystein did not block the oxidative DNA
damage (Figure 3, lanes 4 and 10). Glutathione
reduced the oxidative stress. Therefore, the DNA
damage was formed as aggregation rather than
fragmentation in the presence of glutathione (Figure 3,
lanes 3 and 9). Dithiotreitol (DTT) was the most
effective antioxidant of all investigated but EMF
exposure inhibited the effectiveness of DTT (Figure 3,
lanes 5 and 11).
The oxidative species produced by ascorbate
oxidation in the presence of copper(II) ions damage
the DNA molecules (Figure 1). Previously DNA
damage depending on ascorbate oxidation had been
studied (Erdem et al., 1994; Zareie et al., 1996).
Oxidative DNA damage was observed as
fragmentation or aggregation. The degree of oxidative
DNA damage varies in the levels and reactivity of free
radicals produced in the reaction medium. In the
presence of oxygen, the hydroxyl and peroxyl radicals
such as superoxide anion and hydroperoxyl radical are
produced by the reaction between radical form of
ascorbic acid (ascorbyl radical) and molecular oxygen
(Fuchs et al., 1990).
These radicals attack to electrophilic nuclei on the
targets and create secondary carbon radicals. At the
38 Serkan ‹fller and Günhan Erdem
high level or high reactivity of these radicals, excess
formation of secondary carbon radicals on the same
DNA molecule causes a reaction between each other
and then the DNA damage occurs as fragmentation.
Therefore, DNA size became smaller and gave the
smeared patterns on gel electrophoresis (lanes 5 and 6
in Figure 1) However, at the low level or low reactivity
of oxygen species, the oxidative DNA damage results
in aggregation of the DNA molecules with the
intermolecular reaction of the secondary carbon
radicals. Thus, the DNA samples became heavier and
were retarded on the gel electrophoresis (lanes 3 and 9
in Figure 3).
Our results showed that extremely low-frequency
EMF enhanced the effect of oxidative stress on DNA
damage and supported the idea obtained from previous
studies on an increasing effect of EMF on the
concentration and the life-time of free radicals (Jajte,
2000; Scaiano et al., 1995; Jajte and ZmySlony, 2000).
Especially the comparisons of lane 2 to lane 4 in
Figure 2 and lane 5 to lane 11 in Figure 3, indicate that
the degree of the oxidative stress under the EMF is
greater than the normal condition.
In the brain cells of rats, an increase in DNA
single- and double-strand breaks had been found after
acute exposure to a sinusoidal 60 Hz magnetic field.
When the experiment was carried out in the presence
of melatonin or a radical scavenger compound N-tert-
butyl-alpha-phenylnitrone (PBN), the effect of
magnetic fields on brain cell DNA was not observed
(Lai and Singh, 1997). Melatonin is a neurohormone
and it is also an antioxidant and a free radical
scavenger. Therefore, this hormone could protect
biological systems against oxidative damage. The
increasing effect of EMF on the concentration of free
radicals has been suggested that melatonin suppression
in humans may increase the probability of mutagenic
and carcinogenic risk (Jajte and ZmySlony, 2000).
EMF (≥1 mT) increases the concentration of free
radicals that escape from the alkyl sulphate and
sulphonate micelles. The effect of extremely low-
frequency EMF on the radicals formed from singlet
precursors is larger than triplet precursors. Some
radicals such as hydroxyl and peroxyl radicals
generated in the biological reactions are formed from
singlet precursors (Eveson et al., 2000).
In conclusion, the results obtained from our study
suggest that the effects of extra low frequency EMF on
the concentration of free radicals and the
recombination of radical pairs might trigger the
carcinogenesis in the populations living close to the
overhead electric power distribution lines.
R e f e r ences
Erdem G, Öner C, Önal AM, K›sakürek D and Ö¤üfl A. Free
radical mediated interaction of ascorbic acid and
ascorbate/Cu(II) with viral and plasmid DNAs. J Biosci.
19: 9-17, 1994.
Eveson RW, Timmel CR, Brocklehurst B, Hore PJ and
McLauchland KA. The effects of weak magnetic fields
on radical recombination reactions in micelles. Int J
Radiation Biol. 76: 1509-1522, 2000.
Fuchs J, Mehlhron RJ and Packer L. Assay for free radical
reductase activity in biological tissue by electron spin
resonance spectroscopy. Methods in Enzymology.
186: 670-674, 1990.
Galt S, Whalstrom J, Hamnerius Y, Holmqvist D and
Johannesson T. Study of effects of 50 Hz magnetic fields
on chromosome aberration and growth-related enzyme
ODC in human amniotic cells. Bioelectrochemistry and
Bioenergetics. 36: 1-8, 1995.
Goodman EM, Greenebaum B and Marron MT. Effects of
electromagnetic fields on molecules and cells. In: Int
Rew Cytology, A Survey of Cell Biology. Jean KW and
Jarvik J (Ed). Academic Press. 158: 279-338, 1995.
Jajte J and ZmySlony M. The role of melatonin in the
molecular mechanism of weak, static and extremely low
frequency (50 Hz) magnetic fields (ELF). Medycyna
Pracy. 51: 51-57, 2000.
Jajte JM. Programmed cell death as a biological function of
electromagnetic fields at a frequency of (50/60 Hz).
Medycyna Pracy. 51: 383-389, 2000.
Lai H and Singh NP. Melatonin and N-tert-butyl-alpha-
phenylnitrone block 60-Hz magnetic field-induced DNA
single and double strand breaks in rat brain cells.
J Pineal Res. 22: 152-62, 1997.
London SJ, Thomas DC, Bowman JD, Sobel E, Cheng TC
and Peters JM. Exposure to residential electric and
magnetic fields and risk of childhood leukemia. Am J
Epidemiol. 134: 923-37, 1991.
Poncz M, Solowiejczyk D, Harpel B, Mory Y, Schwartz E
and Surrey S. Construction of human gene libraries from
small amounts of peripheral blood: Analysis of ß-like
globin genes. Hemoglobin. 6: 27-36, 1982.
Savitz DA, Wachtel H, Barnes FA, John EM and Tvrdik JG.
Case-control study of childhood cancer and exposure to
60-Hz magnetic fields. Am J Epidemiol. 128: 21-38, 1988.
Scaiano JC, Cozens FL and Mohtat N. Development of a
model and application of the radical pair mechanism to
radicals in micelles. Photochemistry and Photobiology.
62: 818-829, 1995.
Tomenius L. 50-Hz electromagnetic environment and the
incidence of childhood tumors in Stockholm County.
Bioelectromagnetics. 7: 191-207, 1986.
Wertheimer N and Leeper E. Electrical wiring configurations
and childhood cancer. Am J Epidemiol. 109: 273-284,
1979.
Zareie MH, Erdem G, Öner C, Öner R, Ö¤üfl A and Piflkin E.
Investigation of ascorbate-Cu (II) induced cleavage of
DNA by scanning tunneling microscopy. Int J Biol
Macromol. 19: 69-73, 1996.
 Journal of Cell and Molecular Biology 2: 39-42, 2003. 39
Haliç University, Printed in Turkey.

C h ro m o s o m e s o f a b a l a n c e d t r a n s l o c a t i o n c a s e e v a l u a t e d w i t h
a t o m i c f o r c e m i c r oscopy
Zerrin Y›lmaz1*, Mehmet Ali Ergun2, Erdal Tan3
1
Department of Medical Biology and Genetics, Baskent University, Faculty of Medicine, 06570,
Maltepe, Ankara, Turkey; 2Department of Medical Biology and Genetics, Gazi University, Faculty of
Medicine, 06510, Besevler, Ankara, Turkey; 3Materials Research Department, Ankara Nuclear Research
and Training Center, 06100, Besevler, Ankara, Turkey (*author for correspondence)

Received 2 December 2002; Accepted 30 December 2002

Abstract

A couple was referred to our genetics department for cytogenetic analysis because of two previous abortions. The
cytogenetic analysis of the male was found as 46, XY and the female revealed a balanced translocation; 46, XX,t
(7;12) (p21;q14) and also she had 14 cenh+ as her mother. Atomic force microscopy (AFM) is a useful method for
detecting detailed structures of chromosomes. With the help of this new technique the surface topography of human
chromosomes can be examined. We used AFM in order to analyse the surface topography of derivative chromosomes
of the patients, and found a 0.6 µm gap region. In this study, we aimed to examine the differences between the images
of the derivative chromosomes detected by light and atomic force microscopy analyses.

Key words: Balanced translocation, chromosome polymorphism, atomic force microscopy

D e n g e l i t r a n s l o k a s y o n v a k a s › n d a k ro m o z o m l a r › n a t o m i k g ü ç m i k roskobu ile
de¤erlendirilmesi

Özet

Ardarda iki gebelik kayb› nedeniyle departman›m›za yönlendirilen çiftin sitogenetik analizleri yap›lm›flt›r. Erkekte
normal kromozom kuruluflu 46, XY saptanm›fl ancak kad›nda dengeli translokasyonla birlikte
14. kromozoma ait sentromer art›fl› saptanm›flt›r; 46, XX, t (7;12) (p21;q14), 14 cenh+. Proband›n ailesinde yap›lan
sitogenetik çal›flma ile ayn› kromozom kuruluflunun proband›n annesinden kal›t›ld›¤› saptanm›flt›r. Atomik güç
mikroskobu kromozomlar›n yap›sal olarak detayl› incelenmesinde kullan›lmaktad›r. Bu yeni tekni¤in yard›m›yla
insan kromozomlar›n›n yüzey topografisi incelenebilmektedir. Biz de atomik güç mikroskobunu kullanarak derivatif
kromozomun yüzey topografisini araflt›rd›k ve 0.6 µm’lik bir gap bölgesi saptad›k. Bu çal›flmada probanda ait
derivatif kromozom yap›s›n› hem ›fl›k mikroskobu hem de atomik güç mikroskobu ile ayr› ayr› de¤erlendirerek
sonuçlar›m›z› karfl›laflt›rd›k.

Anahtar sözcükler: Dengeli translokasyon, kromozom polimorfizmi, atomik güç mikroskobu

I n t ro d u c t i o n cellular level. Human cytogenetics is almost always

concerned with light microscope studies of
Cytogenetics is the study of genetic material at the chromosomes. Staining procedures which provide a
40 Zerrin Y›lmaz et al.
uniform unbanded appearance to chromosomes are
referred to solid or covential staining. They can,
however, be useful for studies on chromosome
breakage as scoring gaps and breaks can be difficult in
lightly stained chromosome bands. Giemsa banding
(G-banding) has become the most widely used
technique for the routine staining of human
chromosomes. The chromosome banding patterns
obtained reflect both the structural and functional
composition of chromosomes. Consititutive
heterochromatin is the structural chromosomal
material seen as dark staining material in interphase as
well as during mitosis. It includes both repetetive
DNA, satellit DNA and some non-repetetive DNA.
C-banding can be used to demonstrate the repetetive
DNA (Benn and Perle, 1992).
Atomic force microscopy (AFM) is a diagnostic
tool for detecting detailed structures of the
chromosomes and the surface topography of human
chromosomes can be examined using this new
technique (Binning et al., 1986; Musio et al., 1997).
AFM could be considered as a tool for further
chromosomal studies.
In our previous studies using AFM, we showed
that, unbanded human metaphase chromosomes
displayed a banding pattern similar to G-bands, and for
the first time we have provided an AFM imaging of
chromosomes in trisomy 13, 21 and Klinefelter
Syndrome patients (Ergun et al., 1999). Besides, G and
C-banding patterns of chromosomes were also
investigated (Sahin et al., 2000; Tan et al., 2001).
In this study, we used AFM in order to analyse the
surface topography of derivative chromosomes of a
female patient whose daughter was referred to our
Genetics department with the chief complaints of
abortions.
Materials and methods
Case presentation
In this study we evaluated the chromosomes of a
family. This family was referred to our genetics
department for cytogenetic analysis because of two
previous abortions during the first trimester. They had
no live-born children after a marriage of 5 years. The
male was 37 years old and healthy, and his non-
consanguineous wife was 36 years old. Her physical
examination revealed no abnormalities in
genitourinary, endocrinological and other organ
systems; also laboratory findings were normal.
Light microscopy analysis
Metaphase chromosome preparation was obtained
from peripheral blood lymphocytes using standard
techniques (Verma and Babu, 1995). Conventional
cytogenetic analysis was carried out using GTG-
banding and C-banding techniques (Benn and Perle,
1992). The chromosome images were captured by
computer imaging (Cytovision system, Image
analysis, Applied Imaging, Saunderland, UK).
For each patient we analysed 20 metaphases, and
C-banding procedure was performed while
investigating 14 cenh+.
Atomic force microscopy and analysis
The AFM used in this study was TopoMetrix
TMX2000 Explorer, operating in contact mode and air.
Throughout the surface analysis, we have used
standard pyramidal tip (1520-00) with the radius of
curvature of approximately 1000 A°. During the
surface analysis, the metaphase region was primarily
determined and addressed by light microscopy. Later,
the region under consideration was scanned via AFM
at various scan ranges changing from 150 µm down to
10 µm or less to image the chromosomes in a good
manner. The applied force and the image resolution
were between 1 and 3 nN and 400x400 pixels (or
higher) respectively for each image acquisition. The
raw data gathered were analysed by using the software
of the microscopy system in two or three-dimensional
patterns.
In our study, the chromosomes of the patient were
spread on glass surface. Then, the metaphase spreads
were analysed by AFM. Line measure analysis was
performed on derivative chromosomes.
Results
The karyotype of the male revealed 46, XY, while the
cytogenetic analysis of his wife was karyotyped as
46, XX, t (7;12) (p 21;q 14); a balanced translocation.
She also had 14cenh+. In order to understand the origin
of this translocation chromosome, her mother was
karyotyped and she was also found to be a translocation
carrier; 46, XX, t (7;12) (p 21; q14) and 14 cenh+.

F i g u re 1: The partial karyotype of the daughter (a) and the
mother (b); derivative chromosomes 7 and 12 and
chromosomes 14 with C-banding images are shown.
F i g u re 2: AFM image of derivative chromosome 7 and line
measure analysis of the gap region.
The karyotypes of the mother and the daughter
were similar; 46, XX, t (7;12) (p 21; q 14). The partial
karyotype of the daughter (Figure 1a) and the mother
(Figure 1b) with the derivative chromosomes 7, 12 and
chromosomes 14 with C-banding images are shown.
We performed detailed measurements on the
derivative chromosomes of the mother and detected
the attached chromosome fragment from derivative
chromosome 12 to the derivative chromosome 7. Our
measurements revealed a 0.6 µm gap region (Figure 2).
Besides we also analysed the satellite region of
chromosome 14 (Figure 3) of the mother.
AFM in chromosome evaluation 41
F i g u re 3: AFM image of chromosome 14 indicated by an
arrow.
Discussion
In this study we evaluated the detailed structures of
translocated chromosomes of the mother with AFM.
Line measure analysis revealed a gap region on the
derivative chromosome 7, which was measured as 0.6
µm. It was equivalent to a mid-sized G-band region
(Figure 2).
In the previous studies, it was reported that these
gap regions correspond to narrowing and grooving
regions on chromosomes, and are considered as
negative G- band regions (Uehara et al., 1996). The
gap regions were also observed in fragile X (Harrison
et al., 1983) and in radiation exposed chromosomes
(Mullinger and Johnson, 1987). It was thought that,
these regions correspond to high-order structural
aberrations resulting from an incomplete or irregular
composition of chromatid fibres induced by a
translocation of a chromosomal fragment. The gap
regions were the results of chromosomal
rearrangements (Uehara et al., 1996).
Our second analysis was on the satellite region of
the chromosome 14 of the mother. First of all, GTG-
banding revealed an increase in the heterochromatin
region of the short arm of chromosome 14. Then, we
performed C-banding procedure to understand if this
region was belonging to a constitutive heterochromatin
region, or to an extra banding region. The results of
C-banding confirmed that these regions were
belonging to heterochromatin region. Our 3-
dimensional AFM analysis for the satellite region
showed an augmentation on the short arm of this
chromosome (Figure 3). These heterochromatin
regions are polymorphic regions, and they are highly
42 Zerrin Y›lmaz et al.

repetitive regions that are located on the centromeres Uehara S, Sasaki H, Takabayashi T and Yajima A. Structural
of chromosomes 1, 9, and 16 and on the distal arm of aberrations of metaphase derivative chromosomes from
Y chromosome (Burkholder and Duczek, 1980; Cook, reciprocal translocations as revealed by scanning
1995). Our AFM image also helps us to understand electron microscopy. Cytogenet Cell Genet. 74: 76-79,
1996.
that these regions were not belonging to G- banding
Verma RS and Babu A Banding techniques. In: Human
regions, as there was not a banding pattern (Tan et al., Chromosomes Principles and Techniques. Verma RS and
2001). Babu A (Ed). McGraw-Hill Inc. New York. 72-133,1995.
AFM can be considered as a novel technique for
analysing detailed structures of chromosomes for its
line measure analysis and 3-D image capture
capabilities. Reflecting these capabilities, AFM helped
us to investigate the gap region on the derivative
chromosome and this study is also novel by making
new implementations on the mechanism of
translocation. As a conclusion, the capability of AFM
for detecting chromosomal abnormalities will reflect
light into further studies.

R e f e r ences

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Ergun MA, Tan E, Sahin FI and Menevse A. Numerical
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Mullinger AM and Johnson RT. Scanning electron
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Musio A, Mariani T, Frediani C, Ascoli C and Sbrana I.
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Sahin FI, Ergun MA, Tan E and Menevse A. The mechanism
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Scanning. 22: 24-27, 2000.
Tan E, Sahin FI, Ergun MA, Ercan I and Menevse A.
C-banding visualised by AFM. Scanning. 23: 32-35,
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 Journal of Cell and Molecular Biology 2: 43-48, 2003. 43
Haliç University, Printed in Turkey.

E f f e c t o f e p i r u b i c i n o n m i t o t i c i n d e x i n c u l t u r ed L-cells
Gül Özcan Ar›can* and Mehmet Topçul
‹stanbul University, Faculty of Science, Department of Biology, 34459 Vezneciler, ‹stanbul, Turkey
(*author for correspondence)

Received 9 December 2002; Accepted 30 December 2002

Abstract

Cancer chemotherapy is an additional application to surgical operations and radiotherapy in the treatment of
widespread tumors. An anthracycline-derived antibiotic, epirubicin (EPI) is one of the clinically used antineoplastic
drugs. In this study the cytotoxic effects of EPI in transformed mouse fibroblasts (L-cell) were examined. EPI
concentrations of 0.001 µg/ml, 0.01 µg/ml and 0.1 µg/ml were applied to the cells for 2, 4, 8, 16 and 32 hours. The
results showed that EPI diminished mitotic index of L-cells depends upon time and applied concentrations. This
decrease was found statistically significant in each treatment group when compared to control (p<0.05 - p<0.001).

Key words: Epirubicin, L-cell, transformed fibroblast, mitotic index, in vitro

E p i r u b i s i n i n k ü l t ü r d e k i L - h ü c re l e r i n d e m i t o t i k i n d e k s e e t k i s i

Özet

Kanser kemoterapisi, yayg›n tümörlerin tedavisinde cerrahi uygulama ve radyoterapinin yan›nda gerçeklefltirilen ek
bir uygulamad›r. Antrasiklin türevi bir antibiyotik olan epirubisin (EPI) klinik olarak kullan›lan antineoplastik
ilaçlardan birisidir. Bu çal›flmada, EPI in sitotoksik etkileri, transforme edilmifl fare fibroblastlar›nda (L-hücreleri)
araflt›r›ld›. EPI in 0.001 µg/ml, 0.01 µg/ml ve 0.1 µg/ml konsantrasyonlar› 2, 4, 8, 16 ve 32 saat süresince hücrelere
uyguland›. Sonuçlar uygulanan zaman ve konsantrasyona ba¤l› olarak EPI in L-hücrelerinin mitotik indeks
de¤erlerini düflürdü¤ünü gösterdi. Bu düflüfl kontrol grubu ile karfl›laflt›r›ld›¤›nda, her bir deney grubunda istatistik
olarak anlaml› bulundu (p<0.05 - p<0.001).

Anahtar sözcükler: Epirubisin, L-hücreleri, transforme edilmifl fibroblast, mitotik indeks, in vitro

I n t ro d u c t i o n 1989; Zuckerman et al., 1993). This drug is commonly

Of the cancer drugs in clinical use, the anthracyclines
have a spectrum of antitumor activity and are clearly
the most useful cancer drugs among the natural
product (Chabner and Mayers, 1993).
Epirubicin (EPI) is the epimer of the anthracycline
antibiotic doxorubicin, with inversion of the 4’-
hydroxyl group on the sugar moiety, and has been used
alone or in combination with other cytotoxic agents in
the treatment of a variety of malignancies (Young,
used since it has an equivalent spectrum of antitumor
action as doxorubicin but with less systemic and
cardiac toxicity (El-Mahdy Sayed Othman, 2000).
The mechanism of anti-tumour action for EPI has
not been completely elucidated. Various studies have
revealed that EPI enters the cells rapidly and is
localised in nuclei and forms a complex with DNA by
intercalation between DNA strands (Di marco, 1984).
DNA replication and transcription have been shown to
be inhibited by this intercalation (Lollini et al., 1989;
44 Gül Özcan Ar›can and Mehmet Topçul
Skladonowski and Konopa, 1994). In addition,
topoisomerase-II has also been shown to be inactivated
by EPI (Robert and Gianni, 1993; Haldane et al.,
1993).
In vitro studies showed that EPI possesses
cytotoxicity at least equivalent to that of doxorubicin
against a variety of animal and human tumor cell lines
including those derived from breast, liver, lung,
gastric, colorectal, squamous cell, cervical, bladder,
ovarian carcinomas, neuroblastoma and leukaemia
(Bagnara et al., 1987; Zhang et al., 1992; Bartkowiak
et al., 1992).
EPI is a cell cycle phase non-specific
anthracycline, with maximal cytotoxic effects in the S
and G2 phases. Preliminary in vitro studies were
carried out on HeLa cells. The first tests demonstrated
that EPI and doxorubicin gave essentially the same
inhibition of HeLa cell colony formation (Di marco et
al., 1976). Similarly, EPI was as active as doxorubicin
on mouse embryo fibroblast proliferation (Di marco et
al., 1977), but was taken up in greater amount than
doxorubicin by L1210 leukemia cells in vitro (Wilson
et al., 1981).
There have been few studies about the effect of EPI
on mitotic index of rapidly proliferating cells. In this
study, we have therefore studied the effect at EPI,
employed in the concentrations of 0.001 µg/ml,
0.01 µg/ml and 0.1 µg/ml for a period of 2 to 32 hours,
on proliferation of transformed L-cells in culture
which was investigated by measuring mitotic index in
order to investigate the effectiveness of this drug in
chemotherapy.
Material and methods
Chemical
EPI (4’-epidoxorubicin), an anthracycline antibiotic, is
a doxorubicin stereoisomer, possessing the L-arabino
instead of the L-lyxo configuration of the sugar moiety
(Figure 1). In EPI therefore the hydroxyl group on the
sugar moiety, possessing the stable 1C4 conformation,
has an equatorial orientation (Plosker and Faulds,
1993).
Cell line
The cells used in this study were derived from mouse
fibroblast by in vitro malign transformation (Earle,
F i g u re 1: Structural formulae of EPI.
1943). Transformed L-cells obtained from mouse
subcutaneous connective tissue in 1943. They were
supplied by Dr. P.P. Dendy of Department of
Radiotherapeutics, Cambridge University, in 1975.
The cells were grown in Medium-199 (M-199, Gibco
lab.) containing 10% foetal bovine serum (FBS, Gibco
lab.), 100 µg/ml streptomycin and 100 IU/ml
penicillin, and were passaged twice a week in
appropriate number of 25 cm2 flasks and the volume of
the complete medium in each flask was completely to
12 ml. Cells were removed from the surface of culture
flasks by addition of 0.25% trypsine (Gibco lab.) and
centrifuged for 3 minutes at 1500 cycle/min.
Following the addition of M-199 on the cell
precipitate, the cells became ready for the experiment.
Cell doubling time (Tc) of L-cells was 22.8 hours
(Özcan and R›dvano¤ullar›, 1996). L-cells were
cultured on the cover-slips as 3.104 cells/ml in petri
dishes and incubated for 24 hours with 95% air and 5%
CO2 containing medium at 37°C with pH 7.2 in a
dessicator. At the end of this incubation medium was
removed, replaced with medium containing EPI
concentrations.
Drug application
Epirubicin (Farmorubicin, Carlo Erba) was dissolved
immediatedly before use in sterile medium (M-199) to
give the required concentration. We used 0.001 µg/ml,
0.01 µg/ml and 0.1 µg/ml concentrations of EPI. Cells
were treated with these doses for 2, 4, 8, 16 and 32
hours.
 Epirubicin effect on mitotic index 45

Mitotic index analysis Statistical analysis

Mitotic index were studied by the methods of Feulgen.
Before the cells were treated with Feulgen, they were
prepared with 1 N HCl at room temperature for 1
minute and then hydrolized with 1 N HCl for 10.5
minutes at 60°C. After slides were treated with
Feulgen, they were rinsed for few minutes in distilled
water and stained with 10% Giemsa stain solution pH
6.8, for 3 minutes and washed twice in phosphate
Mitotic index values which obtained from experiments
were calculated to evaluate the statistical analysis. The
differences between the percentage distrubition of M
phase of the various treatment groups and control were
compared by the Student-t test (n=25).
Results

The effect of EPI on mitotic index of L-cells

F i g u re 2: Mitosis in L-cells under the light microscope
(3.3x100).
buffer. After staining, the slides were rinsed in distilled
water. And then the slides were air dried. At last
mitotic index were calculated by counting metaphases,
anaphases and telophases for each tested drug
concentration and control (Figure 2). At least three
thousands cells were examined from each slide for
mitotic index.
in culture was investigated. EPI concentrations of
0.001 µg/ml, 0.01 µg/ml and 0.1 µg/ml were applied to
the cells for time periods of 2, 4, 8, 16 and 32 hours.
In this study, EPI diminished the mitotic index of L-
cells with increasing both treatment time and drug
concentration compared to controls (untreated group).
From the value of 2 hours treatment, we saw that all
EPI concentrations had a rapid effect. In subsequent
hours, this effect seemed to continue. The values of
mitotic index reached a minimum at EPI concentration
of 0.1 µg/ml with increasing drug concentration. Table
1 reveals that treatments of EPI decreased the
percentage of the cells at M phase. With increasing
time the differences among the effects of various drug
concentrations tended to be lower being very small at
2 to 8 hours applications. The inhibition of mitosis was
higher in 16 and 32 hours applications than those in 2,
4 and 8 hours EPI applications in Table 1 especially
with EPI concentration of 0.1 µg/ml. However, in the
treatment of 0.1 µg/ml concentration, mitotic
inhibition reached a maximum at 32 hours application.
The values of mitotic index of the cells treated with
EPI for 32 hours showed that mitotic index decreased
as drug concentrations were increased.

Table 1: Mitotic index values in cultures of L-cells treated with various concentrations of EPI, given in mean ± Standard devia-
tion (SD).

Mitotic index (%)

EPI 2 hours 4 hours 8 hours 16 hours 32 hours

concentrations

Control 1.44 ± 0.12 SD 1.93 ± 0.14 3.04 ± 0.08 3.39 ± 0.15 3.84 ± 0.21
0.001 µg/ml 1.35 ± 0.09 a 1.80 ± 0.10 a 2.72 ± 0.07 a 2.96 ± 0.04 b 3.10 ± 0.30 b
0.01 µg/ml 1.29 ± 0.11 a 1.79 ± 0.05 b 2.61 ± 0.06 b 2.70 ± 0.13 b 2.99 ± 0.16 b
0.1 µg/ml 0.94 ± 0.02 c 1.04 ± 0.01 c 1.77 ± 0.09 c 1.85 ± 0.08 c 1.02 ± 0.22 c
a
: p < 0.05, b: p < 0.01, c: p < 0.001
46 Gül Özcan Ar›can and Mehmet Topçul
EPI significantly decreased the mitotic index in
cultures of L-cells. The results show that EPI
decreased the mitotic index at significant level p<0.05
- p<0.01 for lower drug concentrations 0.01 µg/ml and
0.001 µg/ml, at highly significant level p< 0.001 for
0.1 µg/ml drug concentration when compared with the
control.
In addition, the reductions in mitosis of the cells
following different treatment times (2, 4, 8, 16 and 32
hours) with 0.1 µg/ml EPI concentration were
statistically significant (p<0.001) from each other.
However, this level of significance for the different
treatment times was not observed with 0.01 µg/ml and
0.001 µg/ml concentrations of EPI, respectively.
Discussion
Anthracycline antibiotics have been used extensively
in the treatment of wide variety of malignancies, and
are a standard component of many combination
chemotherapy regimens. EPI has been used alone or in
combination with other antineoplastic agents in the
treatment of a broad range of neoplasms. Studies in
understanding the mechanism of EPI have suggested
that EPI forms a complex with DNA by intercalation
between DNA strands, thus inhibiting DNA replication
and transcription (Özcan et al., 1997; Stewart et al.,
1997), and it increases DNA strand brekage (Cantoni et
al., 1990).
EPI induced differentiation of human
neuroblastoma cells in vitro, possibly related to a
reduction in the growth of surviving cells, thus
allowing activation of intrinsic differentiation
mechanisms. Following culture of human
neuroblastoma cell lines with EPI 10 or 100 nmol/L for
24 hours, outgrowth of neurite processes was
detectable 3 or 4 days after exposure, and maximal
morphological differentiation was achieved after 5 or 6
days (Rocchi et al., 1987).
EPI has also been shown to be effective in
inhibiting basement membrane degradation, a property
deemed necessary to prevent development of
metastases (Plosker and Faulds, 1993). In addition, EPI
has been shown to inhibit proliferation of all
neuroblastoma cell lines by 69 to 78 % relative to
controls (Rocchi et al., 1987), of a human alveolar
rhabdomyosarcoma cell clone (Lollini et al., 1989),
and of haemopoietic progenitor cells from several
human leukaemic cell lines in liquid culture (Bagnara
et al., 1987).
Although, in vitro studies with antitumour agents,
and with anthracyclines in particular, have not shown
to predict the antitumour activity in vivo (Sinha and
Politi, 1990; Nistico et al., 1999), in some studies,
significant correlations have been detected between the
in vitro activity of EPI and other anthracyclines against
various tumour specimens, and therapeutic response
(Bartkowiak et al., 1992; Plosker and Faulds, 1993).
Anthracyclines, including EPI, appear to result in
maximal cell death in the S and G2 phases of the cell
cycle, but cytotoxic effects may occur in the G1 and M
phases at higher drug concentrations (Plosker and
Faulds, 1993; Topçul et al., 2002). Maximal lethal
effects of EPI were demonstrated in the S and G2
phases of the cell cycle in murine and human tumour
cell lines (Hill and Whelan, 1982).
An important comparison between EPI and
doxorubicin in vitro was carried out by Hill and
Whelan (1982). The studies were performed on a wide
range of murine and human cell lines: NIL8 (Syrian
golden hamster cells); four human tumor lines (COLO-
206 and LOVO, derived from colon carcinomas; SCC-
T/G, derived from a squamous cell carcinoma from the
tongue; CHP 100, derived from a neuroblastoma);
L5178Y lymphoblastoid cell sub-lines. In the all cell
lines tested, both drugs showed comparable
cytotoxicity, which increased exponentially with drug
concentration and with duration of exposure. Of high
interest was the study carried out on NIL8 cells
synchronized by mitotic selection and treated for 1
hour with the drug. Results showed that maximum cell
kill was observed with both drug in the S phase, some
kill during early G2, but no kill in G1 and M if low
concentrations were used. Data from flow
microfluorimetry analyses and monitoring of mitotic
indices suggested population arrest in G2 for
doxorubicin (Casazza and Giuliani, 1984).
In the present study, treating L-cells with various
concentrations of EPI for 2, 4, 8, 16 and 32 hours,
decreased mitosis. The results showed that EPI
diminished mitotic index of L-cells, depending upon
time and applied concentrations. When compared to
the control, this decrease was found statistically
significant in each group (p<0.05-p<0.001). The values
of mitotic index reached a minimum at EPI
concentration of 0.1 µg/ml with increasing treatment
time. Increased concentrations resulted in a decrease
on the values of mitotic index, being statistically
significant (p<0.001). Briefly, EPI concentration of 0.1
µg/ml showed to possess relatively more effect on
proliferation of L-cells. Thus, the results of our study

seem to be concordant with the above mentioned
studies suggesting that cytotoxic effects of EPI might
occur in the G1 and M phases at higher drug
concentration.
In our study, decreases in the mitotic index of cells
with increasing both treatment time and EPI
concentration have confirmed that EPI is an effective
inhibitor of mitosis.
In conclusion, the results of this study declared the
cell kinetics and cytotoxic effects of the anticancer
drug, EPI, in treated cultures of L-cell line. Although
EPI has less systemic and cardiac toxicity than
doxorubicin and other anthracyclines with an
equivalent spectrum of antitumor action, it still has
cytotoxic effects.
Acknowledgment
We would like to thank Prof. Dr. Atilla Özalpan for his
kind help and critics.
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L e t t e r to editor
P ro s t a t e c a n c e r a n d i m p o r t a n c e o f
t u m o r m a r k e r studies
P ro s t a t k a n s e r i v e t ü m o r m a r k › r l a r › i l e
ilgili çal›flmalar›n önemi
Prostate cancer is the most commonly new diagnosed
noncutaneous malignancy in men in USA. In the year
2002, according to the health statistics 189,000 men in
the United States are expected to be diagnosed with the
disease and 30,200 men are expected to die of it.
Incidence varies greatly, with African Americans
having the highest incidence in the world (224 cases
per 100,000 population). The incidence of prostate
cancer in African Americans stands in stark contrast to
the incidence in white Americans (150 per 100,000)
and that in men in Western Europe (39.6 per 100,000),
Japan (8.5 per 100,000), and China (1.1 per 100,000).
Tumor markers are biological molecules that
indicate the presence of malignancy. They are
potentially useful in cancer screening, aiding
diagnosis, assessing prognosis, predicting in advance a
likely response to therapy, and monitoring patients
before and after diagnosis. Because of low prevalance
of most cancers in the general population and the
limited sensivity and spesificity of avaible markers,
these tests alone are generally of little value in
screening for cancer in healthy subjects. Currently,
however, prostate spesific antigen (PSA) in
combination with digital rectal examination (DRE) are
undergoing evaluation as screening modalities for
prostate cancer. Because of a lack of sensitivity and
spesificity markers are rarely of use in early diagnosis
of cancer. Also they can be used as monitoring disease
evaluation with therapy. The goal of future research
should be that development of the most specific, cheap
and easy markers for common cancer types as prostate
cancer.
P rostate spesific antigen: Screening prostate cancer
provides a dilemma unique among cancer sites. The
best strategy is determination of the ratio of the
prostate serum antigen (PSA) to the volume of the
prostate gland in prostate cancer diagnosis.
49
Determination of the free PSA (i.e., the percentage of
PSA that is unbound to serum proteins) has also been
suggested as a means of distinguishing malignancy
from benign hyperplasia. PSA has revolutionized the
management of prostate cancer since its development
in the 1980s. For unclear reasons, PSA derived from
malignant epithelial cells tends to bind more avidly to
serum proteins. Thus, in men with an elevated serum
PSA level, cancer is more likely to be present when the
percentage of free PSA is low. Because the relative
sensitivity versus specificity varies, depending on the
free PSA cutoff, the optimal cutoff value for free PSA
is still under debate. Prostate specific antigen (PSA)
represents the best serum marker for prostatic
carcinoma and is considered as most perfect tumor
marker available today. Nevertheless, the use of PSA
to detect prostate cancer is clinically imprecise since
benign and malignant prostate disease can cause
elevations in PSA. It is sensitive but spesificity is not
good to show tumor agressiveness, and so does not
benign prostatic hypertropy from invasive cancer.
Age-spesific cut-offs have been suggested to improve
spesificity, but there is still substantial overlap
between normals and those with cancer. Further
markers of tumor agressiviness, either measured in
serum or needle biopsy specimens are needed to
determine which patients are in need of curative
treatment.
S e r u m a c i d p h o s p h a t a s e : Serum acid phosphatase
(ACP) served as the only serum tumor marker for
prostate cancer between the 1930s and 1980s. more
sensitive serum tumor marker in detection of localized
disease and in monitoring response to therapy. In the
past two decades, the use of ACP has diminished
because of problems with lack of sensitivity and
specificity and because of the discovery of prostate-
specific antigen (PSA), a is an independent predictor
of biochemical recurrence in men who undergo
surgery. ACP level is independently predictive of
biochemical recurrence following radical retropubic
prostatectomy (RRP), when adjusted for other
predictive variables.
G r a n i n s : The nomenclature for chromogranin-A
continues to evolve; for simplicity,it is referred as
50
granin-A (GRN-A). GRN-A is a 49-kilodalton protein
that is produced exclusively by endocrine and
neurondocrine (NE) cells. It is costored and cosecreted
with the resident hormones of these cells, such as
catecholamines and calcitonin (CT). Although the
function of GRN-A is not known, it can serve as a
tissue and serum marker for a variety of endocrine
cells and tumors. There are several major cancer types
are characterized by NE differentiation. Recently, the
importance of NE differentiation and the attendant
expression of chromogranin-A has become
appreciated for prostate cancer. Clinical and basic
roles of chromogranin-A in human prostate cancer are
still studied. Although the function of GRN-A is not
known, several theories have emerged about its role:
(1) that it participates and perhaps regulates the storage
and secretion of its coresident hormones in secretory
vesicles; (2) that it inhibits proteolytic cleavage
enzymes; (3) that it binds calcium and thus regulates
the biologic effects of this ion; and (4) that it is a
precursor for peptides that have unique biologic effects
on the function and growth of its resident cells.
Function notwithstanding, the production of GRN-A in
NE prostate cancers has resulted in the availability of
a new serum and tissue marker for the tumors. The
clinical potential of GRN-A as a serum and tumor
marker in prostate cancer. It is now wellestablished
that GRN-A can be a marker for advanced disease.
More importantly, GRN-A may be a marker for early
and recurrent disease, even in the absence of abnormal
PSA. GRN-A serum levels may also have prognostic
significance, especially for androgen-independent
prostate cancer.
E - c a d h e r i n : In attempts to determine which cancers
of patients with clinically localized disease who
undergo radical prostatectomy will recur, the most
well-characterized and accepted predictors are model
equations that take into account preoperative serum
prostate-specific antigen (PSA), final Gleason score,
and final pathologic stage. Prediction of regression for
the individual patient using these statistical models,
however, is still not precise, and these models could
still be improved on. Thus, additional markers are
needed to more accurately target high-risk patients for
inclusion in clinical trials involving investigational
therapies for locally advanced prostate carcinoma.
Several other approaches show promise in this regard,
including nuclear morphometry, where the results have
been quite consistent. Other more controversial
markers include DNA ploidy and other biomarkers,
such as the amount of tumor angiogenesis, and
immunohistochemical levels of various markers,
including Ki-67, Bcl-2, p53, and E-cadherin. E-
cadherin as a biomarker to predict prognosis in
patients at risk of disease recurrence after radical
prostatectomy is warranted.
Serum total homocystein: Homocystein (Hcy) as a
tumor marker targets to reveal chemotherapy effects
on patients. It is largely derived from cellular
methionine, an essential amino acid drawn from
dietary intake. Intracellular homocysteine is normally
secreted extracellularly, at rapid rates. In the
circulating blood, the majority of the homocysteine
binds to albumin, forming a disulfide linkage.
Approximately 10% to 20% of the Hcy also exists as a
mixed disulfide with cysteine or with homocysteine
itself . Very little Hcy is present in the circulating
blood in a free reduced form (approximately
1%).Elevated serum tHcy (total homocysteine, free
and protein-bound) are detectable in patients with
malignant diseases. Finding increased circulating tHcy
in tumor cells may also be related to the so-called
‘‘methionine dependency’’ of many, but not all, tumor
cells. Many tumor cells are methionine dependent
because of their inability to convert homocysteine
(Hcy) to methionine by way of the remethylation
reaction. On the other hand, normal cells have no
problem obtaining methionine from homocysteine.
Folate is critical to the remethylation reaction. Any
folate deficiency will result in the impairment of
function of the remethylation reaction, causing
accumulation of Hcy. Therefore, it was generally
believed that the rapid proliferation rate of tumor cells,
such as in prostate cancer and in the so-called
methionine dependency of tumor cells, was due to the
depletion of folate by the rapid growing tumor cells
and changing levels of fLV (a form of folate) in 24 h
after therapy. In other words, with a better
understanding of the effects of various drugs, the rise
and fall of circulating tHcy could be used as a new
tumor marker to monitor cancer patients during
therapy, complementing commonly used tumor
markers. The general impression that elevated tHcy is
detectable in cancer patients derives from the fact that
many cancer patients take anti-folate drugs such as
methotrexate. It is important to know that the level of
tHcy reflects the tumor cell proliferation rate.
Regardless of the folate status, it is very likely given
our results and others that the rapid proliferation of
tumor cells is one of the major reasons that elevated
 51

circulating tHcy can be detected in cancer patients.

Conceivably, circulating tHcy could very well be used
as a marker to monitor cancer patients during therapy,
complementing the currently used tumor markers.
Choline kinase: Choline kinase (ChoK) is the first
enzyme in the Kennedy pathway, responsible for the
de novo synthesis of phosphatidylcholine (PC), one of
the basic lipid components of membranes. ChoK is
responsible of the generation of phosphorylcholine
(PCho) from its precursor, choline. Both ChoK and its
product, PCho, have been recently reported as
essential molecules in cell proliferation and
transformation. Generation of Pcho from ChoK
activity has been described as an essential event in
growth factor-induced mitogenesis in fibroblasts and
has been found to cooperate with several mitogens.
Furthermore, overexpression of several oncogenes
induces increased levels of ChoK and the intracellular
levels of PCho. A strong correlation can be established
between ChoK activity and cancer onset at least in
some human tumors. Additional evidence gives
support for a role of ChoK in the generation of human
tumors, since studies using nuclear magnetic
resonance (NMR) techniques have demonstrated
elevated levels of PCho in human tumoral tissues with
respect to the normal ones, including breast, prostate
carcinomas. ChoK is overexpressed with high
incidence in both, tumor- derived cell lines and
tumoral tissues, these results indicate the putative use
of ChoK as a tumor marker, potentially useful in
diagnosis and screening of the progression of tumors.
The recent findings show that overexpression of
the polycomb group transcriptional represor enhancer
zeste Gene (EZH2) in prostate cancer raises the
possibility that transcriptional regulation at the
chromatin level play a role in the development of the
metabolic phenotype and suggest new exploration
prespective on patient stratification, therapeutics and a
tumor marker identity.
Also proliferation markers in biopsies such as K67,
expression levels of mRNA and/or proteins for bcl2,
p53, p27 etc. and molecular changes in tumor
supressor genes such as PTEN or mutations in genes or
mutations in genes can be candidate markers. This is
urgently needed since radical surgery carries a high
morbidity leading to impotence and/or incontinence.

Serdar Ar›san
fiiflli Etfal State Hospital
1. Urology Clinics, fiiflli, ‹stanbul
 53

Book reviews

Ç e t i n ALGÜNEfi, Radyasyon Biyofizi¤i, Trakya The book is a valuable guide of radiation

Üniversitesi Yay›nlar›, Edirne, 135 sayfa, ISBN: 975- biophysics. On the other hand, this book is a good
374-051-4, 2002. document because there are very limited Turkish
publication in this area. For this reason, I
Kitapta atom ve çekirde¤inin yap›s›, karars›z recommended this book for the biologists, physisists,
çekirdekler, iyonizan radyasyon tipleri ve özellikleri, agriculturists and veterinarians who apply radiation on
radyasyonun madde ile etkileflmesi ve radyasyon living organisms for several purposes.
birimleri, iyonizasyona sebep olmayan radyasyonlar,
iyonizan radyasyonlar›n biyolojik etkileri ve
radyasyondan korunma konular› tart›fl›lm›flt›r. Atilla ÖZALPAN
Bölümlerin ayr›nt›l› incelenmesinde, kitab›n Haliç University,
diziliminin geleneksel tarzda, flekil ve tablolar›n Department of Molecular Biology and Genetics
geçtikleri yerlerde metin aras›nda verildi¤i
görülmektedir. Bütün konular, aç›klay›c› flekil, tablo ve
örneklerle desteklenmifltir.
Bu kitab›n radyasyon biyofizi¤i konusunda önemli
bilgiler kazand›rmas› aç›s›ndan çok yararl› bir rehber
olaca¤› kan›s›nday›m. Ayr›ca Türkiye’de bu alanlarda
kaynak oluflturacak Türkçe eserlerin say›lar› da son
derece s›n›rl› oldu¤u için, bu kitab› biyologlara,
radyobiyologlara, fizikçilere, radyasyon etkileri ile
ilgilenen ziraatç› ve veterinerlere öneririm.

Atilla ÖZALPAN
Haliç Üniversitesi,
Moleküler Biyoloji ve Genetik Bölümü

Çetin ALGÜNEfi, Radiation Biophysics, Published

by Trakya University, Edirne, 135 pp, ISBN: 975-374-
051-4, 2002.

In the book, atomic and nuclear structure, unstable

nuclei, types and properties of ionizing radiations,
interaction of radiation with matter, radiation units,
non-ionizing radiations, biological effects of ionizing
radiations and radiation protection are discussed.
In detail, the layout of the book has a traditional
format in that figures and tables have been integrated
into the text at appropriate places. All statement are
supported with a plenty of explenatory figures, tables
and examples.

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of the XRCCS gene and its role in DNA repair and
V (D) J recombination. Science. 265: 1442-1445,
1994.
Ohlrogge JB. Biochemistry of plant acyl carrier
proteins. In: The Biochemistry of Plants: A
Comprehensive Treatise. Stumpf PK and Conn EE
(Ed). Academic Press, New York. 137-157, 1987.
Weaver RF. Molecular Biology. WCB/Mc
Graw-Hill.1999.
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Redford IR. Evidence for a general relationship
between the induced level of DNA double-strand
breakage and cell killing after X-irradiation of
mammalian cells. Int J Radiat Biol. 49: 611- 620,
1986.
Taccioli CE, Cottlieb TM and Blund T. Ku 80: Product
Journal of Cell and Molecular Biology
CONTENTS Volume 2, No. 1, 2003
Dedication

Review articles

Polyamines in plants: An overview

Bitkilerde poliaminler: Genel bir bak›fl
R. Kaur-Sawhney, A.F. Tiburcio, T. Altabella, A.W. Galston 1-12

Phenolic cycle in plants and environment

Bitkilerde fenolik döngü ve çevre
V. I. Kefeli, M. V. Kalevitch, B. Borsari 13-18

Research papers

The short-term effects of single toxic dose of citric acid in mice

Farelerde sitrik asidin tek toksik dozunun k›sa süreli etkileri
T. Aktaç, A. Kabo¤lu, E. Bakar, H. Karakafl 19-23

Characterisation of RPP7 mutant lines of the col-5 ecotype of Arabidopsis thaliana

Arabidopsis thaliana’n›n col-5 ekotipinden elde edilen mutant hatlardan RPP7
geninin karakterizasyonu
C. Can, M. Özaslan, E. B. Holub 25-30

The effect of meta-topolin on protein profile in radish cotyledons

Meta-topolinin turp kotiledonlar›nda protein profiline etkisi
S. Ça¤, N. Palavan-Ünsal 31-34

The effect of electromagnetic fields on oxidative DNA damage

Elektromanyetik alan›n oksidatif DNA hasar› üzerindeki etkisi
S. ‹fller, G. Erdem 35-38

Chromosomes of a balanced translocation case evaluated with atomic force microscopy

Dengeli translokasyon vakas›nda kromozomlar›n atomik güç mikroskobu ile
de¤erlendirilmesi
Z. Y›lmaz, M. A. Ergun, E. Tan 39-42

Effect of epirubicin on mitotic index in cultured L-cells

Epirubisinin kültürdeki L-hücrelerinde mitotik indekse etkisi
G. Özcan Ar›can, M. Topçul 43-48

Letter to editor 49-51

Book reviews 53

Instructions to authors 55-56