Mutation Research, 204 (1988) 257-261 257 Elsevier

MTR 01265

Mutagenicity of lead chromate in Drosophila melanogaster 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 /ml 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 NTA 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 ac- tion is still to be clarified (Environmental Protec- tion 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 NTA to induce different kinds of genotoxic effects, such as binding to DNA, DNA damage and repair, gene mutations, chromosomal effects and in vitro mam- malian 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 admin- istration was feeding, the first report suggested a possible increase in the mutation frequency in one

0165-1218/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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experiment (SLRL 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 administrat ion 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 Drosophi la by feeding. Moreover, as N T A is known to complex insoluble heavy metals by increasing or even eliciting their ability to induce gene mutat ions in bacteria (Loprieno et al., 1985; Venier et al., 1985) and in mammal ian cells (Celotti et al., 1987), and chro- mosomal effects (Montaldi et al., 1985) and cell t ransformation (Lanfranchi et al., 1988) in cul- tured mammal ian cells, we also determined the mutagenic activity in Drosophi la of an insoluble Cr(VI) compound (lead chromate) alone or in association with NTA.

Materials and methods

Stocks

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

established in 1984 f rom a sample collected in the wild in the ne ighbourhood of Padua. The balancer X-chromosome stock used in the lethal tests was Basc ( I n ( l ) sc slL scSR+ S, sc sl scSw a B ) (see Lindsley and Grell, 1968).

Stocks were cultured and tests performed on a sucrose-yeast medium (Mittler and Bennet, 1962). A s tandard temperature (23-25 ° C) was adopted throughout all the experiments.

Solvent and treatments

For the experiments performed, adult feeding was the route of adminis t rat ion and the solvent of choice was a sterile solution of 5% sucrose in distilled water. At the concentrat ions tested in the experiments, EMS (ethyl methanesulphonate ; Eas tman-Kodak , U.S.A.), a direct mutagen used as positive control, and N T A (trisodium salt of nitrilotriacetic acid; B.H. Shilling, F.R.G.) were completely soluble. Lead chromate (PbCrO4; Merck, Darmstadt , F .R.G.) is a highly water-in- soluble Cr(VI) c o m p o u n d but it was completely soluble in a 5 × 10 -2 -M N T A solution at the 3 different concentra t ions tested.

All the compounds studied were stirred for 1 h in order to obtain complete or partial solubilisa- t ion or complex format ion and then centrifuged at

TABLE 1

INDUCTION OF SEX-LINKED RECESSIVE LETHAL MUTATIONS BY EMS, NTA, PbCrO 4 AND PbCrO 4 PLUS NTA

Treatment Dose Sterility b Lethals: brood

(M) (%) 1 2 3

Tests: brood

1 2 3

Total Total Percent Induced mutation lethals t e s t s lethals frequency

C o n t r o l a 0 - 7 6 7 5035 5 1 6 8 4 9 4 3 20 1 5 1 4 6 0.13 - E M S 5 X 1 0 - 3 0 352 316 309 2 1 9 2 2 4 0 0 2159 977 6751 14.47 * 1 4 . 3 4 X 1 0 - 2

N T A 5 X l 0 - 2 0 5 4 4 2 3 5 9 2 3 4 7 2 0 5 2 13 6 7 5 8 0.19 0 . 0 6 × 1 0 - 2

P b C r O 4 _ d 0.71 5 4 5 2053 2 1 3 6 2 0 8 2 14 6 2 7 1 0.22 0 . 0 9 X 1 0 - 2

N T A p l u s 5 x l 0 - 2 2.71 5 9 8 2 0 6 0 2 1 2 4 2 1 4 6 22 6 3 3 0 0.35 * 0 . 2 2 X 1 0 - 2

P b C r O 4 9.28 x 10 - 6 N T A p l u s 5 × 1 0 - 2 0 13 15 13 2 0 2 4 2 1 1 0 2165 41 6 2 9 9 0.65 * 0 . 5 2 X 1 0 - 2

P b C r O 4 4.64 × 1 0 - 5

N T A p l u s 5 × 1 0 - 2 13.71 28 40 19 2 1 1 2 1935 1731 87 5 7 7 8 1 . 5 1 " 1 . 3 7 X 1 0 - 2

P b C r O 4 c 4 . 6 4 x 1 0 - 4

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.

2000 rpm before using the supernatants for adult feeding treatments. In Table 1 the measured con- centrations 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 re- ported.

To measure the level of solubilisation of PbCrO 4 and to verify the complete solubilisation of PbCrO4 by the NTA concentration used for the treat- ments, Cr(VI) levels were determined in the super- natant of the treatment solutions both spectropho- tometrically and by atomic absorption spectrome- try, following the methods indicated by Loprieno et al. (1985).

Feeding treatments 'Cas t -PD' males, 24 h old, were treated in glass

vials containing a porous membrane maintained constantly wet with the feeding solution. Mortal- ity 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 Kas t enbaum-

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 hy- pothesised and data from that male were excluded from the analysis. Due to the high mutagenic

259

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

Results

Whereas NTA and the supernatant containing negligible amounts of the insoluble PbCrO4 (Cr(VI) = 0.06 ~,/ml) were not toxic when sep- arately tested, preliminary assays to determine toxicity of the PbCrO 4 plus NTA treatment showed that at concentrations of NTA 5 X 10 2 M plus PbCrO 4 1.16 x 10 -3 M, and NTA 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 muta- genicity 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 in- crease of mutation frequency, whereas neither N T A nor PbCrO 4 (Cr (VI )= 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 in- creases in the mutat ion frequency. Moreover, sig- nificant PbCrO 4 dose-related effects were ob- served in the series treated with NTA + 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 ex- periments and data from the corresponding paren- tal 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 NTA on Zn and Ca distribution was hypothesised (Ander- son 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 geno- toxic effects of NTA in short-term tests (reviewed by Venier et al., 1987) led to consider NTA as an indirect, non-mutagenic carcinogen, such as a pro- moter (Hodges, 1982). As a matter of fact NTA was observed to inhibit metabolic cooperation be- tween cultured mammalian cells, which may result in tumour promotion (Malcolm et al., 1983). Pro- moting action of NTA in tumour formation was indeed observed in animals treated with NTA 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 NTA con- centrat ions (Kramers , 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 NTA 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 NTA 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). Chro- mate, 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 re- garding the genotoxic activity of Pb(II) com- pounds 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 particu- lar 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 mutagen- icity 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 contri- bution of Cr(VI) and Pb(II) to the mutagenicity of PbCrO 4 observed in the presence of NTA requires a study of the mutagenicity in the same test sys- tem 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 NTA seems to repre- sent the major hazard deriving from a massive introduction of NTA 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.

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