Fluoride 2004;37(4):249–256 Research report 249

MITIGATION OF GENOTOXIC EFFECTS OF FLUORIDE AND ARSENIC

BY ASCORBIC ACID IN HUMAN LYMPHOCYTE CULTURES
SB Nair, DD Jhala, NJ Chinoya
Ahmedabad, India

SUMMARY: The in vitro effects of arsenic and fluoride added, individually and in
combination, to human lymphocytes on the cell cycle proliferative index (CCPI)/
replicative index (RI) and sister chromatid exchange (SCE) using fluorescence plus
Giemsa staining were examined. A significant increase occurred in SCE per
metaphase (SCE/chromosome and SCE/cell) and inhibition of proliferative kinetics,
which resulted in a decline of the replicative index in comparison to the controls.
The treated lymphocytes also exhibited a reduction in % M1 and % M2 plates but an
increase in % M3 plates. The beneficial effects of adding ascorbic acid were clearly
evident with recovery in the replicative index. Genotoxic effects induced by fluoride
and by arsenic were therefore reversed by ascorbic acid.
Key words:Arsenic and genotoxicity; Ascorbic acid; Cell cycle proliferation index (CCPI); Fluo-
ride and genotoxicity; Genotoxicity in vitro; Sister chromatid exchange (SCE).
INTRODUCTION
There are conflicting reports in the literature regarding genotoxic effects of
fluoride.1,2 Jachimczak and Skotarczak3 found that sodium fluoride induces
chromosome aberrations in cultured human leucocytes, whereas others4.5
observed no significant changes in SCE in bone marrow cells after ingestion
of fluoride by mice and rats. Thompson et al6 also found that fluoride did not
increase the frequencies of chromosomal aberrations or sister chromatid
exchanges (SCEs) in human lymphocyte cultures. However, Sheth et al7
observed an increase in the frequency of SCEs in endemic human population of
North Gujarat, India, as compared to control population. Joseph and Gadhia8 also
demonstrated that the rates of SCEs and chromosome aberrations in persons in an
endemic village with high fluoride water in South Gujarat, India, were signifi-
cantly higher than in another village with low fluoride.
Investigations of genotoxic effects of ingested arsenic have yielded mixed
results. In humans exposed to Fowler’s solution (1% KAsO2 in H2O), increased
SCEs but no increase in chromosomal aberrations was found,9 while another
study10 reported increased chromosomal aberrations. Clearly, the foregoing
information indicates there is controversy regarding the genotoxic effects of fluo-
ride and arsenic, and further investigations are needed to clarify this important
issue.
There are therapeutic agents capable of minimizing the genotoxicity of various
natural and man-made mutagens in our daily life. These agents, which have
gained increasing importance, include vitamins, sulfhydryl substances, and plant
products.11,12 Here the protective effects of ascorbic acid (vitamin C) on geno-
aForcorrespondence: Cell Biology and Human Cytogenetics Unit, Department of Zoology,
School of Sciences, Gujarat University, Ahmedabad 380 009, INDIA.
E-mail: zooldeptgu@satyam.net.in

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250 Nair, Jhala, Chinoy

toxicity of fluoride and arsenic treated cultures were investigated since there are
reports suggesting it has ameliorative effects on other sources of chemical toxic-
ity in lymphocyte cultures.13,14
MATERIALS AND METHODS
Subjects: Venous blood from ten normal, healthy, non-smoking, adult Indian
volunteers, 20–25 years old (5 males and 5 females), was collected after obtain-
ing their consent, in sterile heparinized syringes at our departmental clinical facil-
ity.
Peripheral blood lymphocyte culture (PBLC): After collection of peripheral
blood, microcultures were set up in duplicate for each individual according to the
standard protocol of Hungerford.15 Seven milliliters of RPMI 1640 culture
medium (Hi-media, Mumbai, India, pH 7.4) pre-supplemented with 10% fetal
calf serum (Centron Laboratories, Mumbai, India) and antibiotics (benzyl peni-
cillin and streptomycin) were dispensed into dark culture vials along with four
drops of heparin (Biological E. Ltd, Hyderabad, India) and 100 µL of phytohe-
maglutinin (5 mg/5 mL distilled water) (Sigma-Aldrich Chemicals, St Louis,
MO, USA). After gently shaking the blood samples, 0.5 mL of the whole blood
and the following test chemicals (µg/7 mL culture media as listed in Table 1)
were added during the time of culture setting: sodium fluoride (Loba Chemie,
Mumbai, 99% purity), arsenic trioxide (Maniyar Chemie, Mumbai, 99% purity),
and ascorbic acid (Loba Chemie, Mumbai, 99% purity).

Table 1. Experimental protocol

Group Treatment Dose

(µg/7 mL of culture media)

I Control, Untreated (Control + distilled water) (Con) -

II Positive control (Control + ascorbic acid) (AA) 12

III Sodium fluoride (NaF) 10

IV Arsenic trioxide (As2O3) 0.001

V NaF + As2O3 10 + 0.001

VI NaF + As2O3 + AA 10 + 0.001 + 12

All the test compounds were added at ‘0’ hour i.e. during the time of culture setting.
Number of volunteers = 10 (5 males; 5 females)
Groups I to VI – Duration of exposure in each group was 69 hr.

For differential staining of sister chromatids, 80 µL bromodeoxyuridine (1 mg/

mL distilled water) (Sigma-Aldrich Chemicals, St Louis, MO, USA) was added,
mixed gently, and cultures were incubated at 37 ºC for 72 hr in complete dark-
ness. At the 69th hour, 30 µL of colchicine (1 mg/5 mL distilled water) (Sigma-

Fluoride 2004;37(4)
 Mitigation of F and As genotoxicity by ascorbic acid 251

Aldrich Chemicals, St Louis, MO, USA) and 80 µL ethidium bromide (1 mg/mL

distilled water) (Himedia, Mumbai, India) were added to cultures to arrest the cell
division at metaphase stage. The cells were collected by centrifugation and then
exposed to hypotonic solution (0.075 M KCl; pH 7) for 45 min and fixed in a
mixture of acetic acid and methanol (1:3). Three changes of fixative were given
prior to the preparation of the final cell suspension. A few drops of this concen-
trated cell suspension were placed on clean and chilled microslides and allowed
to dry on a hotplate at 50 ºC.
The slides were stained according to the fluorescence-plus-Giemsa (FPG)
method of Perry and Wolff16 for scoring of metaphase plates to analyse the sister
chromatid exchange (SCE) and cell cycle proliferative index (CCPI).
Analysis of sister chromatid exchanges (SCEs): Metaphases in their second in
vitro division (M2) were selected for scoring on the basis of the spreading of
chromosomes and differentiation of chromatids. At least 30 metaphases from
each culture that were in their second in vitro division (M2) were analysed for
SCE. In these preparations cells dividing for the first (M1), second (M2), third or
successive (M3+) divisions in culture containing bromodeoxy uridine (BrdU)
were designated by the differential staining pattern of sister chromatids. First
division (M1) cells contained chromosomes with both sister chromatids stained
uniformly dark. Second division (M2) cells contained only differentially stained
chromatids with one chromatid darkly stained and its sister chromatid light
stained, whereas, third or subsequent division (M3+) cells had both sister chroma-
tids stained lightly.
Analysis of cell cycle kinetics (CCPI) or Replicative index (RI): Differentially
stained slides were scored for analysis of CCPI based on the staining pattern of
chromosomes. At least 100 consecutive metaphases were analysed for each indi-
vidual, classifying them as first (M1), second (M2), or third and subsequent
(M3+) generation cells. The CCPI for each individual was calculated according to
the formula:)
1 x (M1) + 2 x (M2) + 3 x (M3+)
CCPI =
100

Statistical analysis: Results are expressed as means ± SE. The treated groups
were compared with controls, and the antidote group was compared with com-
bined treatment group by Student’s t test. Probability values of 0.05 or less were
considered significant.
RESULTS
The SCE showed an increase in treated groups (Groups III–V) as compared to
controls (Groups I and II) (Table 2). Reduction in the frequency of SCE was
observed in Group VI in which ascorbic acid was added with fluoride and arsenic
at ‘0’ hour as compared to the treated Groups III-V (Table 2).

Fluoride 2004;37(4)
252 Nair, Jhala, Chinoy

Table 2. Frequency of sister chromatid exchange (SCEs) in

lymphocyte culturesa

Group Female Male

Total M2 Total % SCE Total M2 Total % SCE

SCEs SCEs
I 136 644 473.53 140 624 445.72
II 135 591 437.78 130 559 430.00
III 127 660 519.69 131 659 503.05
IV 134 907 676.87 130 899 691.54
V 117 1103 942.74 109 1072 983.49
VI 146 695 451.37 128 671 524.22
aData are expressed as mean ± S.E. P<0.05; * P<0.02; †
P<0.01; ‡
§P<0.001; no sign = Not significant.
Total metaphase counted in each individual of all groups = 100.
Comparisons between: Group I with Groups III or IV or V individually;
Group V with Group VI.

Table 3. Frequency of SCE/ cell, SCE/ chromosome, and cell cycle proliferative index
(CCPI) in lymphocyte culturesa

Group SCE/Cell SCE/Chromosome CCPI

Female Male Female Male Female Male

I 4.77±0.30 4.46±0.60 0.10±0.01 0.10± 0.004 1.48±0.02 1.46±0.02

II 4.37±0.08 4.30±0.11 0.10±0.04 0.09± 0.002 1.48±0.02 1.47±0.02
III 5.20±0.11 5.02±0.12† 0.11±0.02 0.11± 0.002† 1.40±0.02† 1.39±0.01‡
IV 6.80±0.22§ 6.86±0.21§ 0.15±0.01§ 0.15± 0.005§ 1.31±0.01§ 1.33±0.02§
V 9.48±0.29§ 9.85±0.18§ 0.21±0.01§ 0.22± 0.004§ 1.28±0.01§ 1.28±0.01§
VI 4.78±0.19§ 5.24±0.10§ 0.11±0.01§ 0.12± 0.003§ 1.46±0.01§ 1.42±0.01§
aData are expressed as mean ± S.E. * P<0.05; † P<0.02; ‡ P<0.01; § P<0.001; no sign = Not
significant.
Total metaphase counted in each individual of all groups = 100.
Comparisons between: Group I with Groups III or IV or V individually; Group V with Group VI.

The mean SCE/cell and SCE/chromosome ratios showed a nonsignificant

increase in females but a significant increase (p<0.02) in males in the NaF treated
Group III. A highly significant increase (p<0.001) occurred in both females and
males in the As2O3 and NaF + As2O3 treated Groups IV and V as compared to
the control Groups I and II. Group VI with ascorbic acid present showed a signif-

Fluoride 2004;37(4)
 Mitigation of F and As genotoxicity by ascorbic acid 253

icant (p<0.001) decline in the frequency of SCE/cell and SCE/chromosome ratios

as compared to Group V and were comparable to control values (Table 3).
The CCPI was significantly decreased in females (p<0.02) and in males
(p<0.01) in the NaF and As2O3 treated Groups III and IV, while combined treat-
ment with NaF and As2O3 (Group V) showed highly significant decline
(P<0.001) in CCPI as compared to control Groups I and II. The CCPI showed a
significant (P<0.001) increase in Group VI by addition of ascorbic acid, and the
values were comparable to control (Table 3).

DISCUSSION
The DNA molecule is a target site of most, if not all, carcinogenic and
mutagenic agents. Additionally, a number of agents like fluoride and arsenic are
cytotoxic to the DNA molecule.10,17,18 Tsutsui et al19 demonstrated that sodium
fluoride induced an increase in SCEs in Syrian hamster embryo cells. Likewise,
NaF + AlCl3 induced an increase in SCEs in human lymphocytes, respectively, in
culture.20 Some authors have reported an increase in the frequency of SCEs in
fluoride endemic human populations of North Gujarat7 and South Gujarat,8 India,
as compared to control populations and in peripheral blood lymphocyte cultures
of volunteers having a concentration of 4–15 mg/L of fluoride in drinking water
in China.21
Zanzoni and Jung22 reported that addition of inorganic trivalent arsenic elevates
the SCE rate in human lymphocyte cultures. Similarly, Burgdorf et al9 observed
an increase in the rate of SCEs in lymphocytes of patients treated with arsenic.
Others23,24 found similar results, presumably mainly from exposure to fluoride,
in lymphocytes of workers at a phosphate fertilizer factory in northern China and
in workers of an aluminium factory. The results of the present study are in agree-
ment with the above findings. However, Thompson et al6 and Gadhia and
Joseph25 however, observed no increase in SCE frequency or chromosomal aber-
rations in peripheral blood lymphocytes cultured with fluoride.
The cell cycle proliferative index declined when compared to the controls, in
agreement with data of Sivikova and Dianovsky.24 He et al26 reported that NaF
and fluoroacetamide influence the cell cycle kinetics, chromosomal aberrations,
and SCE frequencies in cultured red Muntjac cells. The percentage of M1 cells
increased, while that of M2 and M3 decreased significantly by NaF. In the present
study, a significant lag in the cell cycle was observed which was similar to the
foregoing report. Hayashi and Tsutsui27 also found that cytotoxicity and clastoge-
nicity of NaF to cultured human diploid fibroblast are cell-cycle dependent, and
cells in early and middle S phase are more sensitive to the effects of toxicant.
Other workers have also reported cell mitotic delays.23
Arsenic treatment likewise exhibits reduced mitotic activity and chromosomal
alterations.28-30 The increase in SCE and lag in cell cycle proliferation suggests
that fluoride and arsenic are genotoxic agents and may cause mutagenesis.
Fluoride may not cause direct damage to DNA. It could be indirectly involved
in disruption of normal DNA replication. The actual mechanism could be quite

Fluoride 2004;37(4)
254 Nair, Jhala, Chinoy

complex. The high electronegativity of the fluoride ion allows it to interact with
both organic and inorganic compounds of cells, so its physiological effects could
be expected to be more pronounced and varied than those caused by any other
elemental ion. Thus fluoride has a great affinity for Ca2+, Mg2+, and phosphate,
all of which are present in the culture medium and within the cells, so the active
agent could be a magnesium-fluorophosphate, which is known to interfere with
DNA polymerase and RNAase activities.31,32 Fluoride ion also forms a strong
hydrogen bonds (NH---F–-) with purine and pyrimidine bases,33 which could dis-
rupt the structures of both DNA and RNA and interfere with their synthesis or
enhance the frequency of base-pair error during DNA replication. Mutagenic
activity of NaF, KF, and NaHF2 has also been demonstrated by Caspary et al33
Similarly, chromosome aberrations were obtained in rat bone marrow cells
treated with inorganic fluoride.34
The biochemical mechanism by which arsenicals exert their chromosome dam-
aging properties is not yet clear, but arsenic is known to interfere with several
enzymic functions by blocking sulfhydryl groups,35 by replacing phosphorus in
the phosphate group of DNA, and by forming a weak bond in the DNA chain.
Ascorbic acid (AA, vitamin C) has a role in maintaining the growth and integ-
rity of leucocytes and has antioxidant and detoxification properties.36,37 In the
present study, when AA was added to the cultures along with NaF and As2O3 at
‘0’ hour, a significant protective effect by AA was manifested by decreasing the
genotoxicity. Fluoride and arsenic alone and in combination induce genotoxicity
in various cell lines, which was probably triggered by the generation of free radi-
cals and increased lipid peroxidation in vitro. Consequently, some chemothera-
peutic approaches have been proposed that include the use of radical scavenging
agents and antioxidant vitamins like vitamin C.38,39 Earlier studies from our lab-
oratory have shown a protective effect of vitamin C on chemical induced toxicity
in vitro similar to the present study.14,20,39
This work was presented at the XXth National Symposium on Reproductive
Biology and Comparative Endocrinology, Department of Animal Science, Bhar-
athidasan University, Tiruchirappalli, India, 7–9 January 2002. (Abstract No. RT-
P-9, p. 94).
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