Mutagenicity of lead chromate in Drosophila melanogaster in the presence of nitrilotriacetic acid (NTA)

Mutation Research, 204 (1988) 257-261 Elsevier

257

MTR 01265

Mutagenicity of lead chromate in Drosophilamelanogaster in the presence of nitrilotriacetic acid (NTA) Rodolfo Costa, Giuliana Strolego and Angelo Gino Levis Department of Biology, University of Padua, Via Loredan 10, 35131 Padova (Italy) (Received 7 April 1987) (Revision received 6 October 1987) (Accepted 9 October 1987)

Keywords: D. melanogaster; Sex-linked recessive lethals; Lead chromate; NTA.

Summa~ By using the sex-linked recessive lethal mutation test in Drosophila melanogaster (standard Basc scheme) we analysed the mutagenic effects of treatments by feeding with nitrilotriacetic acid (NTA: 5 x 10 -2 M), with the insoluble Cr(VI) compound lead chromate, PbCrO 4 (siapernatant of 4.6 x 10-4-M suspension in which the actual concentration was 0.06 T / m l as Cr(VI)) and with both compounds preincubated at 3 relative ratios (NTA: 5 x 10 -2 M; P b C r O 4 : 4 . 6 x 10 - 4 , 4.6 x 10 -3 and 9.2 x 10 - 6 M, respectively). The estimation of mutation frequencies was done at different developmental stages of the germ cells, namely spermatozoa, spermatids and spermatocytes. Ethyl methanesulphonate (EMS: 5 x 10-3 M) was used as the reference positive control, with clearly mutagenic results. Treatments with NTA or with PbCrO4 alone did not induce any significant increase of the mutation frequency. PbCrO 4 at the 3 concentrations tested was completely soluble in the 5 x 10-2-M N T A solution, and the mixture of NTA and PbCrO4 induced significant increases of the frequency of sex-linked lethal mutations, with a significant dose-effect relationship with respect to PbCrO 4, apparently as a result of the interaction of the compounds and subsequent release of the genotoxic heavy-metal Cr(VI) ions. This result indicates an important synergistic action of NTA with PbCrO 4 under the conditions described.

Nitrilotriacetic acid (NTA), a substitute for polyphosphates in household detergents which may have a great environmental impact (Perry et al., 1984), is an established animal carcinogen (Anderson et al., 1985) but its mechanism of action is still to be clarified (Environmental Protection Agency, 1980; New York State, 1984). NTA is inactive in the majority of the mutagenicity

Correspondence: Dr. R. Costa, Department of Biology, University of Padua, Via Loredan 10, 35131 Padova (Italy).

short-term tests, but some positive results have been obtained concerning the ability of N T A to induce different kinds of genotoxic effects, such as binding to DNA, D N A damage and repair, gene mutations, chromosomal effects and in vitro mammalian cell transformation (reviewed by Venier et al., 1987). In Drosophila melanogaster mutagenicity of NTA was studied so far by Kramers (1976) and Woodruff et al. (1985). When the route of administration was feeding, the first report suggested a possible increase in the mutation frequency in one

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258 experiment ( S L R L test), in broods 1 and 2, but this result was not reproducible in 2 others. In the second report questionable or equivocal results were also obtained. In both studies, when the route of administration was injection instead of feeding, negative results were obtained. Due to the uncertainty of the above results we planned new tests on the mutagenic effects of N T A administered to Drosophila by feeding. Moreover, as N T A is k n o w n to complex insoluble heavy metals by increasing or even eliciting their ability to induce gene mutations in bacteria (Loprieno et al., 1985; Venier et al., 1985) and in m a m m a l i a n cells (Celotti et al., 1987), and chromosomal effects (Montaldi et al., 1985) and cell transformation (Lanfranchi et al., 1988) in cultured m a m m a l i a n cells, we also determined the mutagenic activity in Drosophila of an insoluble Cr(VI) c o m p o u n d (lead chromate) alone or in association with N T A . Materials and methods Stocks

Males from a ' C a s t - P D ' stock maintained in our laboratory were used in sex-linked recessive lethal experiments. The ' C a s t - P D ' stock was

established in 1984 f r o m a sample collected in the wild in the n e i g h b o u r h o o d of Padua. The balancer X - c h r o m o s o m e stock used in the lethal tests was Basc ( I n ( l ) sc slL scS R+ S, sc sl scSw a B ) (see Lindsley and Grell, 1968). Stocks were cultured and tests performed on a sucrose-yeast m e d i u m (Mittler and Bennet, 1962). A standard temperature (23-25 ° C) was adopted throughout all the experiments.

Solvent and treatments

For the experiments performed, adult feeding was the route of administration and the solvent of choice was a sterile solution of 5% sucrose in distilled water. At the concentrations tested in the experiments, E M S (ethyl m e t h a n e s u l p h o n a t e ; E a s t m a n - K o d a k , U.S.A.), a direct m u t a g e n used as positive control, and N T A (trisodium salt of nitrilotriacetic acid; B.H. Shilling, F.R.G.) were completely soluble. Lead c h r o m a t e (PbCrO4; Merck, Darmstadt, F.R.G.) is a highly water-insoluble Cr(VI) c o m p o u n d but it was completely soluble in a 5 × 1 0 - 2 - M N T A solution at the 3 different concentrations tested. All the c o m p o u n d s studied were stirred for 1 h in order to obtain complete or partial solubilisation or complex formation and then centrifuged at

TABLE 1 INDUCTION OF SEX-LINKED RECESSIVE LETHAL MUTATIONS BY EMS, NTA, PbCrO4 AND PbCrO4 PLUS NTA Treatment

Dose (M)

Control a EMS NTA PbCrO4 NTAplus PbCrO4 NTAplus PbCrO4 NTAplus PbCrO4 c

0 5 X10 -3 5 Xl0 -2 _ d 5 xl0 -2 9.28 x 10 - 6 5 ×10 -2 4.64 × 1 0 - 5 5 ×10 -2 4.64x 10-4

Sterility b Lethals: brood (%) 1 2 3 0 0 0.71 2.71 0 13.71

Tests: brood

Total Total lethals t e s t s

Percent Induced mutation lethals frequency

1

2

3

7 352 5 5 5

6 316 4 4 9

7 309 4 5 8

5035 2192 2359 2053 2060

5168 2400 2347 2136 2124

4943 2159 2052 2082 2146

20 977 13 14 22

15146 6751 6758 6271 6330

0.13 14.47 * 0.19 0.22 0.35 *

13

15

13

2024

2110

2165

41

6299

0.65 *

0.52X10 -2

28

40

19

2112

1935

1731

87

5778

1.51"

1.37X10 -2

14.34X10 0.06×10 0.09X10 0.22X10

-2 -2 -2 -2

a Control treatment was a sterile solution of 5% sucrose. b Percent sterility: induced sterility in percent (calculated as the percentage of chemically treated males that were sterile minus the percent of control males that were sterile). c Two clusters of 5 lethals, 2 clusters of 6 lethals and 1 cluster of 10 lethals were excluded from the analysis. d The actual concentration of Cr(VI) in the supernatant was 0.06 3'/mi. * Significant at the 1% level.

259 2000 rpm before using the supernatants for adult feeding treatments. In Table 1 the measured concentrations are indicated which correspond to the originally used doses, except for PbCrO 4 alone, for which the actual concentration of Cr(VI) in the supernatant, used in feeding treatment, is reported. To measure the level of solubilisation of PbCrO 4 and to verify the complete solubilisation of PbCrO4 by the N T A concentration used for the treatments, Cr(VI) levels were determined in the supernatant of the treatment solutions both spectrophotometrically and by atomic absorption spectrometry, following the methods indicated by Loprieno et al. (1985). Feeding treatments ' C a s t - P D ' males, 24 h old, were treated in glass vials containing a porous membrane maintained constantly wet with the feeding solution. Mortality was recorded after treatment and the males were removed and mated. Sex-linked recessive lethal tests Treated and control males, 3 days old, were mated individually with 3 (3-5 days old) Base females to produce 3 broods: brood 1 (3 days), brood 2 (2 days) and brood 3 (2 days), so that mainly postmeiotic germ cells were sampled in this scheme (Chandley and Bateman, 1962). No more than 90 F 1 females were individually mated for each Pa male. The presence of a lethal in a culture was ascertained by the absence of wild-type males among 20 or more individuals in each F 2 progeny. Stat&tical analys& The data were analysed by the K a s t e n b a u m Bowman (1970) test, except for EMS treatment, where the comparison to controls was made using the chi-square test. To identify clusters when more than 2 lethals deriving from a single parental male were observed, a comparison was made with the expectation based on a Poisson distribution. When the expectation was less than 0.01, a cluster of sperm cells due to a premeiotic event was hypothesised and data from that male were excluded from the analysis. Due to the high mutagenic

activity of EMS and the consequently high incidence of more than 2 mutations per male, cluster analysis was not done in this case.

Results Whereas N T A and the supernatant containing negligible amounts of the insoluble PbCrO4 (Cr(VI) = 0.06 ~,/ml) were not toxic when separately tested, preliminary assays to determine toxicity of the PbCrO 4 plus N T A treatment showed that at concentrations of N T A 5 X 10 2 M plus PbCrO 4 1.16 x 10 -3 M, and N T A 5 x 10 -2 M plus PbCrO 4 2.32 x 10 -3 M, the percentage of mortality of treated males was 61.66 and 68.80, respectively. It was thus decided to test the mutagenicity of the highest non-toxic dose of PbCrO4 solubilised by NTA, corresponding to 4.64 x 10 - 4 M PbCrO4, as well as 2 lower concentrations (4.64 × 10 5 M and 9.28 X 10 - 6 M). The detailed results of the sex-linked recessive lethal mutation tests are given in Table 1. No mortality was observed. At the concentrations tested in the experiments the positive control mutagen EMS produced a very significant increase of mutation frequency, whereas neither N T A nor PbCrO 4 ( C r ( V I ) = 0.06 -f/ml in the supernatant used for treatment) was found to be mutagenic per se after feeding. In the case of PbCrO 4 the negative results were obviously due to the extremely low amount of Cr(VI) solubilised in the absence of NTA. PbCrO 4 at the 3 tested concentrations was on the contrary completely soluble in a 5 x 10-2-M N T A solution and treatments with PbCrO 4 solubilised by N T A induced highly significant increases in the mutation frequency. Moreover, significant PbCrO 4 dose-related effects were observed in the series treated with N T A + PbCrO 4 (t[2 j in the test of significance in regression= 6.621; P < 0.05). Two clusters of 5 lethals, 2 clusters of 6 lethals and 1 cluster of 10 lethals were observed in the N T A 5 x 10-2-M plus PbCrO 4 4.64 x 10-4-M experiments and data from the corresponding parental males were excluded from the analysis (see Methods).

260

Discussion Urinary tumours, which are produced by chronic high exposure of rodents to NTA, were referred to chronic toxicity that was caused by changes in Zn and Ca distribution between the urinary tract tissues and urine at high doses of NTA, and a threshold for the effects of N T A on Zn and Ca distribution was hypothesised (Anderson et al., 1985). Although the true mechanism of the carcinogenic action of N T A is still to be clarified (Environmental Protection Agency, 1980; New York State, 1984), the general lack of genotoxic effects of N T A in short-term tests (reviewed by Venier et al., 1987) led to consider N T A as an indirect, non-mutagenic carcinogen, such as a promoter (Hodges, 1982). As a matter of fact N T A was observed to inhibit metabolic cooperation between cultured mammalian cells, which may result in tumour promotion (Malcolm et al., 1983). Promoting action of N T A in tumour formation was indeed observed in animals treated with N T A and nitrosamine (Hiasa et al., 1985) and acetamide (Lipsky, 1984) derivatives, as well as with an N T A - F e complex (Okada and Midorikawa, 1982). The present results, which clearly show that recessive lethal mutations are not induced in Drosophila fed 5 × 10 2 M NTA, definitively rule out any doubt left by previous studies performed with the same test and corresponding N T A concentrations ( K r a m e r s , 1976: 5 M 10 - 2 M ; Woodruff et al., 1985: from 4.7 × 10 -3 M to 10 -2 M), and confirm the general lack of activity of this compound in the short-term mutagenicity assays. Our results also indicate that N T A elicits genetic activity from an otherwise inactive Cr(VI) salt (PbCrO4), thus in line with the ability of different Cr(VI) compounds, as well as of insoluble salts of other metals such as Cd(II), Hg(I), Ni(II) and Pb(II), to induce gene mutations (Loprieno et al., 1985; Venier et al., 1985; Celotti et al., 1987), chromosomal effects (Montaldi et al., 1985) and in vitro mammalian cell transformation (Lanfranchi et al., 1988) when tested in the presence of NTA. The activity of PbCrO4 in the presence of N T A is due to the formation of a P b - N T A complex and the release of soluble chromate anion (Loprieno et al., 1985; Venier et al., 1985). Chromate, i.e., the Cr(VI) anion, is certainly the most

studied among genotoxic metals and it was found to be mutagenic in an extremely wide range of test systems (reviewed by Bianchi and Levis, 1984), however, until now only 1 abstract (De La Rosa et al., 1985) reported a variety of germinal genetic effects induced in Drosophila by chromium (oxidation state and chemical composition of the tested compound were unspecified). Results regarding the genotoxic activity of Pb(II) compounds are much more contradictory (reviewed by Gerber et al., 1980; Sunderman, 1984; Montaldi et al., 1985), however, also some Pb(II) compounds are mutagenic in different test systems, in particular they were found able to induce germinal genetic effects (De La Rosa et al., 1985) but not somatic mutations (Rasmuson, 1985) in Drosophila me-

lanogaster. In our previous studies (Loprieno et al., 1985; Venier et al., 1985) we were able to demonstrate, by means of Cr(VI) determinations and mutagenicity titration curves with totally soluble Cr(VI) (K2Cr207), that the mutagenicity of PbCrO 4 elicited by N T A was totally due to the activity of the solubilised chromate anion. In the present experiments the assessment of the relative contribution of Cr(VI) and Pb(II) to the mutagenicity of PbCrO 4 observed in the presence of N T A requires a study of the mutagenicity in the same test system of soluble Cr(VI), alone or in association with NTA, which we are carrying on. It must be stressed that the solubilisation of genotoxic heavy metals by N T A seems to represent the major hazard deriving from a massive introduction of N T A in the environment.

Acknowledgements This work was supported by grants from the National Research Council of Italy (C.N.R., P.F. 'Oncologia'), the Venetia Region and the Italian Ministry of Health.

References Anderson, R.L., W.E. Bishop and R.L. Campbell (1985) A review of environmental and mammalian toxicology of nitrilotriacetic acid, CRC Crit. Rev. Toxicol., 15, 1-102. Bianchi, V., and A.G. Levis (1984) Mechanisms of chromium genotoxicity, Toxicol. Environ. Chem., 9, 1-25.

261 Celotti, L., D. Furlan, L. Seccati and A.G. Levis (1987) Interactions of nitrilotriacetic acid (NTA) with Cr(VI) compounds in the induction of gene mutations in cultured mammalian cells, Mutation Res., 190, 35-39. Chandley, A.C., and A.J.H. Bateman (1962) Timing of spermatogenesis in Drosophila melanogaster using tritiated thymidine, Nature (London), 193, 299-300. De La Rosa, D.M.E., and E.R. Felix (1985) Genetic effects induced by heavy metals in Drosophila melanogaster. 4th Intern. Conf. Environ. Mut., Stockholm, June 1985, p. 108 (abstract). Environmental Protection Agency (1980) Results of EPA's risk assessment of the use of nitrilotriacetic acid (NTA) in laundry detergents, United States Environmental Protection Agency, Washington, DC. Gerber, G.B., A. Leonard and P. Jacquet (1980) Toxicity, mutagenicity and teratogenicity of lead, Mutation Res., 76, 115-141. Hiasa, Y., Y. Kitahori, N. Konishi, T. Shimoyama and A. Miyashiro (1985) Trisodium nitriloacetate monohydrate: promoting effect in urinary bladder carcinogenesis in rats treated with N-butyl-N-(4-hydroxybutyl)hitrosamine, J. Natl. Cancer Inst., 74, 235-239. Hodges, M.E. (1982) NTA. Epigenetic tumorigenicity?, Fd. Chem. Toxic., 20, 480-483. Kastenbaum, M.A., and K.O. Bowman (1970) Tables for determining the statistical significance of mutation frequencies, Mutation Res., 9, 527-549. Kramers, P.G.N. (1976) Mutagenicity studies with hitrilotriacetic acid (NTA) and citrex S-5 in Drosophila, Mutation Res., 40, 277-280. Lanfranchi, S., S. Paglialunga and A.G. Levis (1988) Mammalian cell transformation induced by Cr(VI) compounds in the presence of hitrilotriacetic acid (NTA), J. Toxicol. Environ. Health, in press. Lindsley, D.L., and E.H. Grell (1968) Genetic variations of Drosophila melanogaster, Carnegie Institution of Washington, Publ. No. 627, 471 pp. Lipsky, M.M. (1984) Progression of 4'-(fluoro-4-biphenyl) acetamide (FBPA)-induced premalignant renal lesions after cessation of exposure and nitrilotriacetic acid (NTA) treatment, Proc. Am. Ass. Cancer Res., 25, 151 (abstract). Loprieno, N., G. Boncristiani, P. Venier, A. Montaldi, F. Majone, V. Bianchi, S. Paglialunga and A.G. Levis (1985)

Increased mutagenicity of chromium compounds by nitrilotriacetic acid, Environ. Mutagen., 7, 185-200. Malcolm, A.R., L.J. Mills and E.J. McKenna (1983) Inhibition of metabolic cooperation between Chinese hamster V79 cells by tumor promoters and other chemicals, Ann. N.Y. Acad. Sci., 407, 448-450. Mittler, S., and J. Bennet (1962) A single food medium that requires no hve yeast with the minimum of variables, Drosophila Inform. Serv., 36, 131-132. Montaldi, A., L. Zentilin, P. Venier, I. Gola, V. Bianchi, S. Paglialunga and A.G. Levis (1985) Interaction of nitrilotriacetic acid with heavy metals in the induction of sister chromatid exchanges in cultured mammalian cells, Environ. Mutagen., 7, 381-390. New York State (1984) Draft: Environmental impact statement and regulatory impact statement on the proposed regulation banning the use of nitrilotriacetic acid (NTA) in New York State, New York State Dept. of Environmental Conservation, 1.8.1984. Okada, S., and O. Midorikawa (1982) Induction of the rat renal adenocarcinoma by Fe-nitrilotriacetate (Fe-NTA), Nai Ka Hou Kan., 29, 485-491. In Japanese (English summary). Perry, R., P.W.W. Kirk, T. Stephenson and J.N. Lester (1984) Environmental aspects of the use of NTA as a detergent builder, Water Res., 18, 255-276. Rasmuson, A. (1985) Mutagenic effects of some water-soluble metal compounds in a somatic eye-color test system in Drosophila melanogaster, Mutation Res., 157, 157-162. Sunderman, Jr., F.W. (1984) Recent advances in metal carcinogenesis, Ann. Clin. Lab. Sci., 14, 93-122. Venier, P., A. Montaldi, C. Gava, L. Zentilin, G. Tecchio, V. Bianchi, S. Paglialunga and A.G. Levis (1985) Effects of nitrilotriacetic acid on the induction of gene mutations and sister-chromatid exchanges by insoluble chromium compounds, Mutation Res., 156, 219-228. Venier, P., S. De Flora, C. Gava, C. Bennicelli, A. Camoirano, M. Zordan, V. Bianchi and A.G. Levis (1987) Interactions of chromium with nitrilotriacetic acid (NTA) in the induction of genetic effects in bacteria, Toxicol. Environ. Chem., 14, 201-218. Woodruff, R.C., J.M. Mason, R. Valencia and S. Zimmering (1985) Chemical mutagenesis testing in Drosophila. V. Resuits of 53 coded compounds tested for the National Toxicology Program, Environ. Mutagen., 7, 677-702.