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Mutation Research, 48 (1977) 327--336 © Elsevier/North-Holland Biomedical Press

VINYL CHLORIDE MUTAGENESIS IN DROSOPHILA M E L A N O G A S T E R

F.G. VERBURGT and E. VOGEL

Department of Radiation Genetics and Chemical Mutagenesis, State Unversity of Leiden, Sylvius Laboratories, Leiden (The Netherlands) (Received October 26th, 1976) (Revision received January 20th, 1977) (Accepted February 2nd, 1977)

Summary In inhalation experiments, Drosophila males were exposed to vinyl chloride at concentrations of 200, 850, 10,000, 30,000, or 50,000 ppm for 2 days, and to 30 or 850 ppm for 17 days. VCM was mutagenic in the recessive-lethal test both after short-term and long-term exposures. The lowest effective concentration (LEC) was 850 ppm after 2 day exposure, and this value could be lowered to 30 ppm by prolonging the exposure time to 17 days. With the concentration levels tested, the mutation frequency increased with concentrations and reached a plateau at 10,000 ppm. This indicates a substrate saturation effect. In contrast with the recessive lethal assay, negative results were obtained when tests on dominant lethals, translocations, entire and partial sex~hromosome loss were carried out with VCM at 30,000 ppm for 2 days. This finding of a false negative seems a logical consequence of the observed saturation effect, and strengthens the concept that there exist two effective concentrations for point mutations vs the induction of chromosome breakage events. Vinyl chloride monomer provides another example to support our view that chromosome breakage is not a reliable measure of mutagenic activity.

Introduction

A 1975 survey of the top fifty chemical products [1] lists vinyl chloride monomer at position 23, thereby expressing its great economic importance. Over the past two years it has become clear that VCM can cause late toxic effects in experimental animals and man. Several reports indicate that VCM is

Abbreviations: ER, endoplasmic reticulum; PyDMT, 3,3-dimethyl-l-(3-pyridyl) triazene; PyODMT, 3,3-dimethyl-l-(3-pyridyl-N-oxide) triazene; VCM, vinyl chloride monomer.

328 mutagenic in Salmonella typhimurium [2,3,13,16], Escherichia coli [7], and Schizosaccharomyces pombe [11]. Ducatman et al. [5], Funes-Cravioto et al. [6], and Purchase et al. [15] have found chromosomal aberrations in workers exposed to VCM. A study of Infante et al. [8] on pregnancy among wives of workers exposed to vinyl chloride has indicated a significantly raised fetal loss, in comparison with controls. Systemic carcinogenic activity of VCM was observed in rats, mice and hamsters [14,19]. All these findings have raised serious concern a b o u t the genetic and carcinogenic risks of VCM to man. Vinyl chloride as a chlorinated ethylene shows low reactivity, and its systemic action suggests that the biological effects of VCM are dependent on its biotransformation. This is in line with the failure of VCM to exert mutagenic activity in microorganisms in the absence of active microsomal enzymes [2,3,7,13,16]. Our interest in VCM stems from the observation that Drosophila can execute the essential activation steps needed to convert such pre-carcinogens into electrophilic species [24]. Its mode of action and its chemical properties made VCM a suitable material for further investigating the ability of the Drosophila system to detect different types of environmental mutagens. Preliminary data from our group [20] and the work b y Magnusson and Ramel [12] demonstrate that Drosophila is capable of activating VCM. In this study we report the mutagenicity of VCM in Drosophila at low and high concentrations, by comparing short-term vs long-term exposure, and by using different genetic end-points. It will be seen from the data that the detection of the mutagenic activity of VCM is dependent on the category of genetic damage used as a diagnostic criterion. Materials and methods

Trea tmen t procedure VCM, produced b y Matheson Gas Products (supplied by Hoek Loos, Amsterdam), was contaminated with acetylene (1--2 ppm), water (1 ppm) and phenol (82 ppm). For exposure, groups of 50 males (2 days old) of our tester strain Berlin K were continuously exposed to VCM in 1-1 bottles for a period of 2--17 days. Each bottle contained about 10 g of food (normal food with killed yeast) and was air-tight sealed with a rubber screw-cap. A known quantity of pure VCM gas was injected with a disposable syringe through this cap into the bottle. A Packard gas chromatograph, model 417, with a stainless steel column (4 m, 3 mm, filled with poropack Q, 200 mesh) was used to exactly determine the initial quantity of VCM injected into each bottle. The temperature of the injection port, the column and the flame ionization detecter was 200°C. N2 with a flow of 1.5 1/h was used as the carrier gas. When low VCM concentrations were used, a known quantity of pure VCM gas was injected into a 35-ml bottle. From the resulting air-VCM mixture a sample was transferred into a new 1-1 bottle, to obtain the desired concentration o f VCM. The final concentration of VCM in the bottles was measured by gas chromatography analysis. This analysis show that the deviation in VCM content obtained b y this simple device was less than 10%, except for the lowest concentration of 30 p p m for which the error was larger, 30 ppm + 10 ppm.

329 The survival rate was 95% for males exposed to 200--50,000 ppm for 2 or 4 days. At the highest concentration of 50,000 ppm, the mortality rate was not enhanced, though the files seemed slightly narcotized. No killing effect could be detected even in the long-term experiments of 17 days duration. The survical rate was equal (80--90%) for both the treated and the control groups. In these experiments the males were transferred to freshly prepared bottles every 3 or 4 days. M u tagenicity assays S e x - l i n k e d recessive lethals

Berlin K males, 2 days old, were exposed to increasing concentrations of VCM (30--50,000 ppm), for 2 or 4 days and then individually mated to 3-4 Basc (In (1) sc slL sc sR ÷ S, sc 1 sc s w a B ) virgins [10]. The mutagenic response to VCM of successive stages of spermatogenesis was tested by brood pattern analysis, with breeding intervals of 2 or 3 days each and total breeding period of 12 days. All suspected recessive lethals were verified by re-tests. D o m i n a n t lethals

The induction of dominant lethals byVCM at 30,000 ppm was tested for in mature sperm and spermatids by the technique developed by Sankaranarayanan [17]. To sample mature sperm, 2~lay~ld Berlin K males were exposed to VCM (30,000 ppm) for 2 days and then mated with one 4

Vinyl chloride mutagenesis in Drosophila melanogaster.

327 Mutation Research, 48 (1977) 327--336 © Elsevier/North-Holland Biomedical Press VINYL CHLORIDE MUTAGENESIS IN DROSOPHILA M E L A N O G A S T E R...
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