Mutation Research 402 1998.

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Antigenotoxicity studies in Drosophila melanogaster

Ulrich Graf
a

a,)

, Suresh K. Abraham b, Judith Guzman-Rincon c , Friedrich E. Wurgler a

Institute of Toxicology, Swiss Federal Institute of Technology (ETH) and Uniersity of Zurich, Zurich CH-8603, Schwerzenbach, Switzerland b School of Life Sciences, J. Nehru Uniersity, New Delhi, India Departamento de Genetica, Instituto Nacional de Inestigaciones Nucleares (ININ), Mexico D.F., Mexico Received 3 June 1997; revised 7 July 1997; accepted 7 July 1997

Abstract The fruit fly Drosophila melanogaster with its well developed array of genotoxicity test systems has been used in a number of studies on antigenotoxicity of various compounds and mixtures. In recent years, the newly developed Somatic Mutation and Recombination Tests SMART. have mainly been employed. These one-generation tests make use of the wing or eye imaginal disc cells in larvae and have proven to be very efficient and sensitive. They are based on the principle that the loss of heterozygosity of suitable recessive markers can lead to the formation of mutant clones of cells that are then expressed as spots on the wings or eyes of the adult flies. We have employed the wing spot test with the two markers multiple wing hairs mwh, 30.3. and flare flr, 338.8.. Three-day-old larvae, trans-heterozygous for these markers, are treated chronically or acutely by oral administration with the test compounds. or complex mixtures. For antigenotoxicity studies, chronic co-treatments can be used, as well as separate pre-treatments with an antigenotoxic agent followed by a chronic treatment with a genotoxin. After eclosion, the wings of the adult flies are scored for the presence of single and twin spots. These spots can be due to different genotoxic events: either mitotic recombination or mutation deletion, point mutation, specific types of translocation, etc... The analysis of two different genotypes one with structurally normal chromosomes, one with a multiply inverted balancer chromosome. allows for a quantitative determination of the recombinagenic activity of genotoxins. Results of two separate studies are presented: 1. instant coffee has antirecombinagenic but not antimutagenic activity in the wing spot test; and 2. ascorbic acid and catechin are able to protect against in vivo nitrosation products of methyl urea in combination with sodium nitrite. q 1998 Elsevier Science B.V. All rights reserved.
Keywords: Drosophila melanogaster; Somatic cell; Recombination; Antigenotoxicity; Nitrosation; Coffee; Catechin; Urethane

1. Introduction There are several advantages that make the fruit fly Drosophila melanogaster an ideal species for antigenotoxicity studies: the somatic genotoxicity assays which have been developed in Drosophila, i.e., the somatic mutation and recombination tests

) Corresponding author. Institute of Toxicology, ETH and University of Zurich, Schorenstrasse 16, CH-8603 Schwerzenbach, Switzerland. Tel.: q41-1-825-74-40; fax: q41-1-825-04-76; E-mail: graf@toxi.biol.ethz.ch

0027-5107r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 9 7 . 0 0 2 9 8 - 4

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SMART., are able to detect a wide spectrum of genetic end points such as point mutations, deletions, certain types of chromosome aberrations, as well as mitotic recombination and gene conversion w13x. Two different test systems have been amply explored, i.e., the wing spot test and the eye spot test. Both are based on the fact that during early embryonic development, groups of cells imaginal discs. are set apart. They proliferate mitotically during the larval development until they differentiate during metamorphosis into structures of the body of the adult fly eyes, wings, etc... The somatic assays take advantage of the possibility to expose such large populations of mitotically growing cells in the imaginal discs of larvae. If a genetic alteration occurs in one of these imaginal disc cells, this alteration will be present in all the descendant cells and will form a clone of mutant cells. If the alteration causes a visible change in the phenotype, the mutant cell clone can be detected as a spot of mutant cells on the body surface of the adult flies. The SMART assays were developed to detect the loss of heterozygosity of suitable gene markers that determine detectable phenotypes expressed on the eyes or the wings of the flies. The somatic assays can be performed in only one fly generation in contrast to the classical test for sex-linked recessive lethals in germ cells that needs at least one month for completion w4x. Furthermore, for antigenotoxicity studies, the SMART assays offer a great variety of and also flexibility in the protocols for the application of the test compounds. Both simultaneous co-treatments and pre- or post-treatments of various durations with two or more compounds are feasible see below.. Owing to these advantages, the SMART assays have become a very suitable approach for genotoxicity and antigenotoxicity testing of chemical and physical agents w3,5,6x. At present, over 400 compounds have been tested in the eye and the wing SMARTs of D. melanogaster Frei and Graf, unpublished.. A number of studies have been published, where these somatic test systems have been used for investigations on antigenotoxicity of single compounds or complex mixtures. The first paper, published in 1989 by Negishi et al. w7x, showed that chlorophyllin inhibits the genotoxicity of Trp-P-2 3-amino-1methyl-5H-pyridow4,3-b xindole. in the wing spot test. In the same year, Katz w8x demonstrated that sodium

thiosulfate is an efficient inhibitor of cisplatin-induced mutagenesis, also using the wing SMART. A series of papers were then published on the protective effects of chlorophyllin against gamma rays w9x, chromiumVI. oxide w10x, and a number of carcinogens w11x. Suppression of genotoxicity of carcinogens in the wing spot test by y.-epigallocatechin gallate, a flavonoid compound, was shown by Hayatsu et al. w12x. In addition, the protective effect of ascorbic acid vitamin C. against the genotoxicity of gamma rays and chromiumVI. oxide in wing somatic cells has also been demonstrated w13x. A further study gave evidence for bio-antimutagens against chlorambucil and methotrexate w14x. Finally, it was shown that several antipyretic analgesics have antimutagenic activity against mitomycin C in the wing spot test w15x. With respect to the antigenotoxic activity of complex mixtures, Abraham w16x and Abraham and Graf w17x have given evidence that instant coffee exhibits protective effects against a series of known mutagens and carcinogens. In addition to these antigenotoxicity studies, the group of Cederberg and Ramel w18x, Magnusson and Ramel w19x, Romert et al. w20x, Ramel and Magnusson w21x, and de la Rosa et al. w22x have published several papers on the modulation of genotoxicity in wing somatic cells by various modulating agents such as enzyme inhibitors or inducers. Another study has also shown that tannic acid has modulating effects on the genotoxicity of three mutagens using the eye wrwq system w23x. In the following, we will give a short overview on the test protocol of the wing SMART and its application for antigenotoxicity studies. This will then be illustrated with results recently obtained in two different investigations. 1. Instant coffee protects rather against the recombinagenic than the mutagenic activity of urethane ethyl carbamate.. 2. Ascorbic acid vitamin C. and catechin have an inhibitory effect on the in vivo nitrosation of methyl urea in combination with sodium nitrite. 2. Wing spot test 2.1. Markers and strains The marker multiple wing hairs mwh, 30.3. is a recessive, homozygously viable mutation and pro-

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duces multiple trichomes per cell instead of the normally unique trichome. The marker flare 3 flr 3 , 338.8. is a recessive mutation that produces malformed wing hairs that have the shape of a flare. All three mutant alleles of flr are recessive zygotic lethals. However, homozygous cells in the wing imaginal discs are viable and lead to mutant wing cells. The flr 3 allele is kept over a balancer chromosome carrying multiple inversions and a dominant marker that is a homozygous lethal flr 3rTM3, Bd S : Third Multiple 3, Beaded-Serrate.. More detailed descriptions of the genetic markers and the balancer chromosome can be found in Ref. w24x. For the standard ST. cross, virgin females are collected from the flr 3rTM3, Bd S strain and crossed with mwh males. To improve the performance of the wing spot test in the case of promutagens activated via cytochrome P-450-dependent metabolic pathways, Frolich and Wurgler w25x constructed new tester strains. These strains carry chromosomes 1 and 2 from a DDT-resistant Oregon RR. line which is characterised by a constitutively high level of cytochrome P-450 w26x. A number of promutagens show increased genotoxicity when the high bioactivation HB. cross ORRrORR; flr 3rTM3, Bd S females and mwh males. is employed w27,28x. 2.2. Treatment of larae The flies of the ST or the HB cross are used to collect eggs during 8 h in culture bottles containing a solid agar base 4% wrv. and a thick layer of live fermenting yeast supplemented with sucrose. At a pre-selected age of the cultures, the larvae are washed from the bottles with tap water and collected in a strainer. The larvae can be treated with the test compounds chronically 24 to 96 h. or acutely for periods of 1 to 6 h. by oral administration or inhalation see Fig. 1.. In special cases, injection of larvae with test solutions may also be used w1x. In the standard protocol for the wing spot test, three-day-old larvae are fed for the rest of the larval development approx. 48 h. w29x. For this chronic feeding, the larvae are put into vials with instant medium or any other type of medium ordinary cornmeal medium, mashed potatoes, agaryeast medium, etc.. containing the test compounds.. For antigenotoxicity studies, the simplest protocol is a chronic co-treatment

Fig. 1. Administration protocols for feeding larvae of the wing spot test of D. melanogaster. L1, L2, L3: Larval stages 1 to 3.

with the compounds under study w8,1619x. However, it is also feasible to apply two separate treatments. For example, two-day-old larvae can first be pre-treated with an antigenotoxic agent for 24 h. Then they are collected again and transferred to vials for a second treatment for 48 h with the genotoxin see Fig. 1. w17,18x. Similarly, it is also possible to apply post-treatments. Finally, an acute treatment can be combined with chronic feeding see Fig. 1.. All the experiments are carried out at 258C and 65% rel. humidity. Strict control of temperature is essential as it affects the frequencies of spots w30,31x. 2.3. Scoring of wings After eclosion, the adult flies are collected from the exposure vials and stored in 70% ethanol. Both the ST and the HB cross produce two types of progeny that can be distinguished phenotypically based on the Bd S marker: 1. marker-trans-heterozygous flies mwh flrqrmwhq flr 3, phenotypically wild type wings. and 2. balancer-heterozygous flies mwh flrqrTM3, Bd S, phenotypically serrate wings.. The wings are mounted in Faures solution gum arabic 30 g, glycerol 20 ml, chloral hydrate 50 g, water 50 ml.. Both the dorsal and the ventral surfaces of the wings are analysed under a compound microscope at 400 = magnification for the occurrence of single spots mwh or flr phenotype. or twin

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spots mwh clone adjacent to flr clone.. Because there is a correlation between the time of induction of a genetic change in the somatic cells and the size of the resulting spot w29x, spot sizes are also recorded in addition to spot frequencies. The different types of spots are due to different genotoxic mechanisms: mutational events point mutation, deletion, or specific types of translocation., as well as mitotic recombination, and perhaps also monosomy w1x. Moreover, the analysis of the two different genotypes mwhrflr and mwhrTM3. allows for a quantitative determination of the recombinagenic activity of genotoxins. In individuals with structurally normal chromosomes, both mutational and recombinational events lead to the formation of single andror twin spots. In contrast, in the balancer-heterozygous individuals, all recombinational events are eliminated due to the multiple inversions, and only mutational events can give rise to single mwh. spots w3234x. 2.4. Statistical analysis For the evaluation of the genotoxic effects recorded, the frequencies of spots per wing of a treated series are compared to its concurrent negative solvent. control series. A multiple-decision procedure is used to decide whether a result is positive, weakly positive, inconclusive, or negative w35,36x.

pre-treated for 24 h with coffee and then exposed to urethane for 48 h w17x. All these experiments were performed by using one fixed concentration of the genotoxic agent. In a new series of experiments, we have administered different concentrations of urethane without or with 5% coffee for 48 h. First results obtained for 10 mM and 20 mM urethane are shown in Fig. 2. In addition to scoring the wings from normal i.e., marker-trans-heterozygous. flies, we have also analysed those from balancer-heterozygous flies in which all recombinational events are eliminated. To allow a quantitative comparison, the frequencies of mwh clones i.e., mwh single spots and the mwh clone of twin spots. per wing are plotted in Fig. 2. Two points are evident from Fig. 2. 1. A comparison of the clone frequencies observed in the treatments without coffee in the two genotypes shows that approximately 50% of the genotoxic events induced by urethane are due to recombination and 50% to mutation. This pattern is similar to those determined for the two monofunctional alkylating agents N-dimethylnitrosamine and N-diethylnitrosamine w34x. 2. The protective effect of coffee co-administration is only observed in the normal individuals but not in the balancer-heterozygous in-

3. Antigenotoxicity of instant coffee Abraham w16x has demonstrated that the chronic co-administration of 2% instant coffee to larvae of the ST cross was effective in significantly reducing the frequencies of single and of twin spots induced by cyclophosphamide, diethylnitrosamine, mitomycin C and urethane by a 48 h feeding. We have continued this study by using larvae of both the ST and the HB cross and by assaying different concentrations of coffee for their protective effect. Co-administration of coffee with mitomycin C or urethane showed a dose-dependent inhibition of genotoxicity in larvae of both crosses. Therefore, the protective effect of coffee is independent of the metabolic capacity of the larvae used for treatment. Furthermore, we were able to demonstrate that the protective effect is also observed when the larvae are first

Fig. 2. Antirecombinagenic activity of coffee in the wing spot test of D. melanogaster. Feeding of three-day-old high bioactivation larvae with urethane on instant medium for 48 h. Hatched columns: without coffee; black columns: with 5% instant coffee.

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dividuals. Although single recombinational events do not lead to spot formation in the balancer-heterozygous individuals, it is possible that a small fraction of the spots may be due to multiple recombinational events. However, the majority of the observed spots originate from mutational events. Therefore, one possible interpretation for the absence of a protective effect in these individuals is that coffee may have primarily antirecombinagenic rather than antimutagenic activity. This hypothesis will be tested experimentally by evaluating the antigenotoxicity of coffee against a series of compounds with different proportions of recombinagenic activity w34x.

4. Inhibition of in vivo nitrosation We have also performed experiments for investigating the in vivo nitrosation capacity of third instar larvae of the wing spot assay. In this case, three-dayold larvae of the ST cross and the HB cross were used. The nitrosation reaction chosen is the combination of methyl urea and sodium nitrite, which produces methylnitrosourea at low pH. The latter compound is one of the most potent genotoxic and carcinogenic agents. Methyl urea alone was negative, whereas sodium nitrite was weakly genotoxic in both crosses. However, the combination of both compounds produced highly increased frequencies of spots particularly in the HB cross. With the combined treatment, the frequency of total spots per wing was more than twice the sum of the frequencies of the two single treatments w37x. The genotoxic effects produced by the combined treatments were considerably increased when mashed potatoes were used for treatment instead of the standard instant medium. Treatments of larvae with the products. obtained after in vitro nitrosation with the two compounds also resulted in high frequencies of induced spots comparable to those recorded with methylnitrosourea. Further experiments showed that co-treatment with ascorbic acid has an inhibitory effect on the in vivo nitrosation reaction w38x. These results are shown in Fig. 3. Feeding of 30 mM methyl urea plus 30 mM sodium nitrite in mashed potatoes for 48 h to HB larvae gave a frequency of total spots per wing of 18.30. The co-administration of 17 mM ascorbic acid reduced this frequency to 5.70. Similar experi-

Fig. 3. Protective effects of ascorbic acid and catechin against in vivo nitrosation in the wing spot test of D. melanogaster. Feeding of three-day-old high bioactivation larvae on mashed potatoes for 48 h. Concentrations used: 30 mM methyl urea; 30 mM sodium nitrite; 17 mM ascorbic acid; 20 mM catechin.

ments were performed with catechin, a bioflavonoid. The results are also given in Fig. 3. In these experiments, the frequency of spots induced by the same concentrations of methyl urea and sodium nitrite was 21.19. When 20 mM catechin were co-administered, this frequency dropped to only 4.38. The results presented here show that ascorbic acid and catechin have a protective effect against the products. of in vivo nitrosation of methyl urea with sodium nitrite. For this reason, the wing spot test is well suited to study genotoxicity resulting from in vivo nitrosation processes and their modulation by other compounds or complex mixtures. 5. Conclusions We have shown here that the wing spot test in D. melanogaster is a versatile and efficient eukaryotic

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U. Graf et al.r Mutation Research 402 (1998) 203209 w12x H. Hayatsu, N. Inade, T. Kakutani, S. Arimoto, T. Negishi, K. Mori, T. Okuda, I. Sakata, Suppression of genotoxicity of carcinogens by y.-epigallocatechin gallate, Prev. Med. 21 1992. 370376. w13x O. Olvera, S. Zimmering, C. Arceo, J. Guzman, M.E. de la Rosa, Evidence for the protective effect of ascorbic acid vitamin C. in treatment with gamma-rays and chromiumVI. oxide CrO 3 . in somatic cells of Drosophila, Mutat. Res. 346 1995. 1921. w14x T. Sato, H. Nishino, H. Nagase, M. Niikawa, H. Kito, The effect of bio-antimutagens on chlorambucil and methotrexate using the wing spot test in Drosophila melanogaster, Jpn. J. Toxicol. Environ. Health 41 1995. 452457. w15x T. Sato, K. Nagaoka, H. Nagase, M. Niikawa, H. Kito, The effect of several antipyretic analgesics on mitomycin C-induced mutagenesis using the wing spot test in Drosophila melanogaster, Jpn. J. Toxicol. Environ. Health 42 1996. 136141. w16x S.K. Abraham, Antigenotoxicity of coffee in the Drosophila assay for somatic mutation and recombination, Mutagenesis 9 1994. 383386. w17x S.K. Abraham, U. Graf, Protection by coffee against somatic genotoxicity in Drosophila: role of bioactivation capacity, Food Chem. Toxicol. 34 1996. 114. w18x H. Cederberg, C. Ramel, Modification of the effect of bleomycin in the somatic mutation and recombination test in Drosophila melanogaster, Mutat. Res. 214 1989. 6980. w19x J. Magnusson, C. Ramel, Inhibitor of polyADP-ribose.transferase potentiates the recombinogenic but not the mutagenic action of alkylating agents in somatic cells in vivo in Drosophila melanogaster, Mutagenesis 5 1990. 511514. w20x L. Romert, J. Magnusson, C. Ramel, The importance of glutathione and glutathione transferase for somatic mutations in Drosophila melanogaster induced in vivo by 1,2-dichloroethane, Carcinogenesis 11 1990. 13991402. w21x C. Ramel, J. Magnusson, Modulation of genotoxicity in Drosophila, Mutat. Res. 267 1992. 221227. w22x M.E. de la Rosa, J. Magnusson, C. Ramel, R. Nilsson, Modulating influence of inorganic arsenic on the recombinogenic and mutagenic action of ionizing radiation and alkylating agents in Drosophila melanogaster, Mutat. Res. 318 1994. 6571. w23x A. Szakmary, S. Knasmuller, Effects of tannic acid on spontaneous and induced somatic mutations in Drosophila melanogaster, Mutagenesis 6 1991. 225228. w24x D.L. Lindsley, G.G. Zimm Eds.., The Genome of Drosophila melanogaster, Academic Press, San Diego, CA, 1992, 1133 pp. w25x A. Frolich, F.E. Wurgler, New tester strains with improved high bioactivation capacity for the Drosophila wing spot test, Mutat. Res. 216 1989. 179187. w26x C. Saner, B. Weibel, F.E. Wurgler, C. Sengstag, Metabolism of promutagens catalyzed by Drosophila melanogaster CYP6A2 enzyme in Saccharomyces cereisiae, Environ. Mol. Mutagen. 27 1996. 4658. w27x U. Graf, D. Singer, Genotoxicity testing of promutagens in the wing somatic mutation and recombination test in

in vivo genotoxicity assay with a great flexibility in the administration protocols. It lends itself not only to the testing of single pure compounds, but also to the evaluation of complex mixtures. For this reason, it is ideally suited also for antigenotoxicity studies, as well as for investigations into modulation of genotoxicity. Our own results show that in the wing spot test, instant coffee has primarily antirecombinagenic rather than antimutagenic activity. Furthermore, ascorbic acid and catechin are able to protect against in vivo nitrosation.

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