Mutagenicity studies with x-ray-contrast media, analgesics, antipyretics, antirheumatics and some other pharmaceutical drugs in bacterial, Drosophila and mammalian test systems.

As part of our investigation into mutagenic effects of environmental compounds, we studied 21 pharmaceuticals most frequently sold in West Germany: 6 X-ray-contrast media, 13 analgesics, antipyretics and antirheumatics, 1 central stimulant, and 1 antidepressant. They were studied in different bacterial, Drosophila and mammalian test systems. 4 of these 21 compounds could be detected as mutagens in one of the test systems. namely: 1,2-dichloroethane induced an increase in the frequency of recessive sex-linked lethal mutations in Drosophila melanogaster, quinine dihydrochloride and dimethylaminophenazone were mutagenic in the Salmonella typhimurium tester strain TA98 in the presence of S-9 liver fraction derived from Aroclor-induced rats, and trilithium citrate caused a significant effect in the micronucleus test on bone marrow of NMRI mice.     

A negative test for mutagenic action of microwave radiation in Drosophila melanogaster.

Microwave radiation (2450 MHz CW) was tested for mutagenicity in Drosophila melanogaster. Embryos in water were exposed to the electromagnetic field with a mean specific absorption rate of 100 W/kg. A sensitive somatic test system was used, in which mutagenicity was measured as the frequency of somatic mutations for eye pigmentation. With the test system used, microwaves did not show any mutagenic activity.     

Differential mutagenic activity of IQ (2-amino-3-methylimidazo[4,5-f]quinoline) in Salmonella typhimurium strains in vitro and in vivo, in Drosophila, and in mice

IQ, a heterocyclic aromatic amine which is formed during the frying of meat, was prepared by chemical synthesis. Its genotoxic potential was studied in bacteria, Drosophila and in mice. A mutagenic effect of IQ (frameshift induction) was detected in Salmonella typhimurium in experiments without metabolic activation; this effect was several orders of magnitude lower than that observed in the presence of an activation system. Ames tests with liver-homogenate S9 fraction from PCB-induced mice and rats confirmed the high mutagenic potency of IQ metabolites (Kasai et al., 1980a). Comparative studies on diagnostic Salmonella strains revealed that the high frameshift-inducing activity is independent of the plasmid pkM101; it is, however, greatly reduced by an intact excision-repair system for DNA lesions. The mutagenic activity of the metabolite(s) formed in vitro by S9 mix has a half-life of ca. 14 min. In the fruit fly, Drosophila melanogaster, IQ induced when used at sublethal concentrations, X-chromosomal recessive lethal mutations in male germ cells in a dose-dependent manner. In mice, tests were performed to detect somatic mutations: chromosomal anomalies (micronuclei) in bone marrow, and gene mutations (affecting coat pigmentation) in mice exposed to IQ in utero. No genotoxic effects were observed in these assays. However, the formation of mutagenic metabolites in the liver of IQ-treated mice was unequivocally demonstrated in host-mediated assays using Salmonella as mutagen probes in mice. The data demonstrate genotoxic activity of IQ in prokaryotic and eukaryotic organisms. The possible reasons for the different response of mammalian systems in vivo and the Salmonella system are discussed.     

Activation or detoxification of mutagenic and carcinogenic compounds in transgenic Drosophila expressing human glutathione S-transferase.

Sensitivity of transgenic Drosophila melanogaster with expression of a human gene encoding the glutathione S-transferase alpha subunit (GSTA1-1) to 1,2:5,6-dibenzanthracene (DBA) and 1,2-dichloroethane (DCE) was investigated in the somatic mutation and recombination test (SMART). We performed the same assay in control transgenic flies expressing the bacterial lacZ gene. Three types of transgenic Drosophila strains carrying GSTA1-1 were used: two transgenic strains homozygous for the second chromosome with a single-copy transgene insertion and one strain with two transgene insertions. Larvae carrying the lacZ gene were significantly more sensitive to genotoxic effects of DBA than those carrying three copies of the GSTA1-1 gene. The larvae with lacZ expression showed significantly lower sensitivity to DCE compared with those expressing GSTA1-1. Finally, a pretreatment with buthionine-sulphoximine (BSO) in experiment with DCE significantly decreased the frequency of mutation events in larvae with three GSTA1-1 copies in comparison with others.     

Mutagenicity of organophosphorus compounds in bacteria and drosophila

140 Organophosphorus compounds (OP's) have been tested for mutagenic activity in bacteria, principally by using two specially constructed sets of tester strains of the bacteria Salmonella typhimurium and Escherichia coli. It was found that 20% gave positive mutagenic responses and that this group of chemicals produce base substitutions rather than frame-shift mutations. In most cases the DNA repair genes exrA⁺ and recA⁺ were for mutagenic activity.Seven compounds were further tested in Drosophila melanogaster for the ability to induce recessive lethal mutations. In some of these cases the doses administered to the flies had to be very low due to the highly toxic nature of the compounds. To overcome this problem, the accumulation of recessive lethal mutations was measured in populations which were continually exposed to the compounds over a period of some 18 months. During this time the populations developed increased resistance to the compound and so the dose administered could gradually be increased. Six of the compounds were mutagenic.Of the compounds tested in both systems, those showing mutagenic activity in bacteria were also mutaganic in Drosophila, those mutagenic in bacteria were not mutagenic in Drosophila.     

Some aspects of the detection of potential mutagenic agents in Drosophila.

The Drosophila system is a valuable test for detecting and characterizing mutagenic agents. Tester strains are available or can be synthesized for determining almost all types of genetical change ranging from gene mutations to chromosome rearrangements in a great variety of cell types of both sexes. Metabolic activation of all groups of indirect mutagens tested so far (aryldialkyltriazenes, cyclophosphamides, nitrosamines, azo-, hydrazo- and azoxyalkanes, aflatoxins, and polycyclic hydrocarbons; about 35 representatives in all), gives strong although indirect support for the considerable metabolizing ability of Drosophila. This capability would be expected from comprehensive biochemical data on bioactivation of foreign compounds in other insects. From a comparison of which types of genetical change are induced at high, low and threshold concentrations, it appears that lethal tests remain the most reliable method for any screening program. Mutagenic agents such as diethylnitrosamine, hycanthone and certain triazenes, which are highly efficient in the induction of recessive lethals (gene mutations and/or deficiencies), would not have been detected in Drosophila if chromosome breakage were the only indicator for mutagenic activity. Moreover, for several mono- and polyfunctional agents, the lowest dose which is still genetically active was definitely lowest for recessive lethals when compared with dominant lethals, chromosome rearrangements or loss. If a new mutagen is discovered by a screening procedure using Drosophila, an accurate picture of its ability to cause either or both gene mutations and chromosome aberrations can be drawn. Such work will be valuable in helping to clarify similar problems in mammalian systems. For instance, it was important to learn that mutagens of the nitrosamine type apparently fail to produce breakage events in Drosophila. Similarly, three cyclophosphamides appeared not to have chromosome breaking ability. However, from a more detailed study, in which a series of concentrations was used, it became obvious that a penetration effect or, more likely, a rate-limiting factor in bioactivation, was the cause of the negative results obtained with these agents.     

The mutagenic effect of 1,2-dichloroethane on Salmonella typhimurium. II. Activation by the isolated perfused rat liver.

In this investigation Salmonella typhimurium strain TA 1530 and TA 1535 were combined with isolated perfused rat liver. Samples of perfusate and bile produced were tested for mutagenicity after treatment with 1,2-dichloroethane (DCE), 1,2-dibromoethane (DBE) or 2-chloroethanol. The results are in good agreement with our previous experiments which indicate that both DEC and DBE are activated through conjugation with glutathione (GSH). Most GSH conjugates are normally excreted in bile. Following liver perfusion the bile was highly mutagenic after DCE and DBE treatments, while 2-chloroethanol did not have this effect. The highest mutagenic effect was seen 15--30 min after the addition of DCE or DBE. The production of mutagenic bile also occurred in mice treated in vivo with DCE. One possible metabolic endproduct of a GSH conjugate is the corresponding mercapturic acid. Thus synthetic N-acetyl-S-(2-chloroethyl)-L-cysteine was tested on TA 1535 and found to be as mutagenic as S-(2-chloroethyl)-L-cysteine in the concentration range 0.2--0.6 mumol/plate. Differences and similarities in the metabolism of DCE and vinyl chloride are discussed on the basis of these results.     

Involvement of a large plasmid in the degradation of 1,2-dichloroethane by Xanthobacter autotrophicus.

Xanthobacter autotrophicus GJ10 is a bacterium that can degrade short-chain halogenated aliphatic compounds such as 1,2-dichloroethane. A 200-kb plasmid, pXAU1, was isolated from this strain and shown to contain the dhlA gene, which codes for haloalkane dehalogenase, the first enzyme in the degradation pathway of 1,2-dichloroethane by GJ10. Loss of pXAU1 resulted in loss of haloalkane dehalogenase activity, significantly decreased chloroacetaldehyde dehydrogenase activity, and loss of resistance to mercuric chloride but did not affect the activity level of haloalkanoate dehalogenase, the second dehalogenase in the degradation of 1,2-dichloroethane.     

HpaII methyltransferase is mutagenic in Escherichia coli.

A genetic reversion assay to study C-to-T mutations within CG sites in DNA is described. It was used to demonstrate that the presence of HpaII methyltransferase (MTase) in Escherichia coli causes a substantial increase in C-to-T mutations at CG sites. This is similar to the known mutagenic effects of E. coli MTase Dcm within its own recognition sequence. With this genetic system, a homolog of an E. coli DNA repair gene in Haemophilus parainfluenzae was tested for antimutagenic activity. Unexpectedly, the homolog was found to have little effect on the reversion frequency. The system was also used to show that HpaII and SssI MTases can convert cytosine to uracil in vitro. These studies define 5-methylcytosine as an intrinsic mutagen and further elaborate the mutagenic potential of cytosine MTases.     

Testing the mutagenicity of malondialdehyde and formaldehyde by the Drosophila mosaic and the sex-linked recessive lethal tests

The mutagenicities of malondialdehyde and formaldehyde were tested by screening each for genetic mosaics of Drosophila melanogaster and by the Muller-5 test for sex-linked recessive lethal mutations. For comparison, the effects of X-rays were also assayed by the above technique. Malondialdehyde, a degradation product of polyunsaturated fatty acids, was found to be a weak mutagen by the above criteria; it induced point mutations and chromosome exchanges at low frequency, as proved by the mosaic test, but failed to induce detectable sex-linked lethality. Formaldehyde was more mutagenic than malondialdehyde; beside induction of mosaic spots it induced sex-linked recessive lethal mutations, but only in the larval testes of Drosophila. Formaldehyde also induced disintegration of the clones. Formaldehyde treatment (feeding larvae with formaldehyde-containing food for about 4 days) was 5 times more mutagenic than malondialdehyde treatment and 5 times less effective than irradiation by 1000 R of X-rays. Wing mosaicism offers a more sensitive way to detect mutagenesis as compared with eye mosaicism. It is suggested that aldehyde-induced mosaic spots derive from mitotic recombination and point mutations.     

Influence of metabolic factors on the mutagenic effectiveness of cyclophosphamide in Drosophila melanogaster

This paper describes the influence of changes in metabolic activity on the in-vivo mutagenic effectiveness of cyclophosphamide in Drosophila melanogaster. A dose-dependent increase in mutagenicity was observed until a plateau value is reached which was increased only slightly after enzyme induction with Aroclor 1254, whereas induction with phenobarbital resulted in a decrease, especially when cyclophosphamide was applied by injection. Treatment of the adult males with inhibitors of the monoamine oxidase (MAO, EC 1.4.3.4), such as iproniazid (Ipr), benzimidazole or tryptamine, led to a marked increase of the mutagenic effectiveness of cyclophosphamide especially in spermatocytes. This indicates the importance of metabolic de-activation processes for the limited mutagenicity of cyclophosphamide in Drosophila. The principal active metabolite of cyclophosphamide, phosphoramide mustard, is extensively de-activated by enzymes that can be inhibited by 1-phenylimidazole (PhI), presumably cytochrome P-450 (EC 1.14.14.1), but not by those blocked by MAO inhibitors. Inhibition of the FAD-containing dimethylaniline monooxygenase (FDMAM, EC 1.14.13.8) by N,N-dimethylbenzylamine (N,N-DMB) resulted in some increase in cyclophosphamide mutagenicity only in spermatids. The marginal mutagenicity of cyclophosphamide in Drosophila larvae could not be increased either by cytochrome P-450 induction with phenobarbital or by MAO inhibition with Ipr. In contrast to the failure of cyclophosphamide to induce rod-chromosome loss, a considerable activity was found when a ring-shaped chromosome was used. Similar to the sex-linked recessive lethal (SLRL) test, ring-X loss frequency could be enhanced by simultaneous treatment with MAO inhibitors. The observed ring-X loss frequency declined when males treated with cyclophosphamide were mated to DNA-repair deficient mei-9L1 females. Cyclophosphamide produces chromosome breaks, detected as 2-3 translocations, in Drosophila spermatocytes, the stage in spermatogenesis that is also the most sensitive to the induction of SLRL mutations.     

Genotoxic activity of 1,3-butadiene and nitrogen dioxide and their photochemical reaction products in Drosophila and in the mouse bone marrow micronucleus assay.

The genotoxic activity of a photochemical reaction mixture of 1,3-butadiene and nitrogen dioxide was investigated in vivo in the mouse bone marrow micronucleus assay and the somatic mutation and recombination test in Drosophila (the wing spot test). Butadiene alone was not mutagenic in Drosophila, but induced micronuclei in mice at 10 ppm after 23 h of exposure. Nitrogen dioxide was not genotoxic in either test system. The photochemical reaction products were toxic but probably not mutagenic in Drosophila and not genotoxic in mouse bone marrow. The in vivo results do not confirm earlier in vitro results that demonstrated a strong direct-acting mutagenic activity of the photochemical products in Salmonella.     

Comparison of the mutagenic potency of 2-chloroethanol, 2-bromoethanol, 1,2-epoxybutane, epichlorohydrin and glycidaldehyde in Klebsiella pneumoniae, Drosophila melanogaster and L5178Y mouse lymphoma cells

A series of 2 haloethanols and 3 epoxides was investigated in 3 mutagenicity test systems, namely (1) the fluctuation test in Klebsiella pneumoniae, (2) the sex-linked recessive lethal test in Drosophila melanogaster, and (3) the HGPRT test with L5178Y mouse lymphoma cells. The order of mutagenic potency was, in Klebsiella: glycidaldehyde greater than 2-bromoethanol = epichlorohydrin greater than 1,2-epoxybutane greater than 2-chloroethanol; in Drosophila: glycidaldehyde = epichlorohydrin greater than 1,2-epoxybutane; in mouse lymphoma cells: epichlorohydrin greater than 1,2-epoxybutane. The haloethanols were non-mutagenic in Drosophila. 2-Chloroethanol and glycidaldehyde were negative in mouse lymphoma cells. The high mutagenic potency of epichlorohydrin as compared with 1,2-epoxybutane was consistent in all systems, and with published data.     

Comparative induction of somatic eye-color mutations and sex-linked recessive lethals in Drosophila melanogaster by tryptophan pyrolysates

The mutagenicities of the products of pyrolysis of tryptophan, Trp-P-1 and Trp-P-2, on Drosophila melanogaster were examined by measuring the effects of these compounds in inducing recessive lethals and somatic eye-color mutations. Since negative results have already been obtained by the standard procedure in males, Trp-P-1 and Trp-P-2 (0.75 to 6 mg/ml) in sucrose solution were given to females for assay of recessive lethal mutations in X-chromosomes. These compounds caused a marginal increase above the control level in the mutation frequency. For the assay of effects on somatic eye-color mutations, Trp-P-1 (200 and 400 ppm) and Trp-P-2 (400 and 800 ppm) were fed to male larvae of a tester strain carrying a genetically unstable marker set of z and w+ on the X-chromosome. These compounds caused dose-dependent increases above the control level in somatic eye-color mutations in adults. It is concluded that, under the conditions used, the somatic eye-color mutation system was more sensitive than the recessive lethal system to the mutagenic effects of tryptophan pyrolysates.     

Mutagenic activity of dioxydine]

A previous evaluation of mutagenic activity of some drugs and perspective substances is carried out using indicator microorganisms. The mutagenicity of dioxydine, a drag with discovered antibacterial activity, is investigated. Dioxydine is shown to induce reversions in mutant of Salmonella typhimurium TA-1950, the indicator strain which demonstrates mutagenic activity of agents, producing mutations of base pair substitution type. Dioxydine proved to affect logariphmiically growing bacterial culture with great activity. Mutageni effect of dioxydine is not modified itself in microsomal oxidation system in vitro. Some data concerning participation of excision reparation enzyme (uvr-B+ gene product) in repair of lethal damages induced by dioxydine, have been obtained. The dioxydine ability to cause bacterial gene mutations in host mediated assay as well as dominant and recessive sex-linked lethal mutations in Drosophila is demonstrated. Dioxydine is capable of inducing chromosome aberrations in bone marrow cells and dominant lethal mutations in mouse germ cells.     

Vinyl chloride mutagenesis in Drosophila melanogaster.

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-chromosome 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.     

Purification and characterization of hydrolytic haloalkane dehalogenase from Xanthobacter autotrophicus GJ10.

A new enzyme, haloalkane dehalogenase, was isolated from the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ10. The purified enzyme catalyzed the hydrolytic dehalogenation of n-halogenated C1 to C4 alkanes, including chlorinated, brominated, and iodinated compounds. The highest activity was found with 1,2-dichloroethane, 1,3-dichloropropane, and 1,2-dibromoethane. The enzyme followed Michaelis-Menten kinetics, and the Km for 1,2-dichloroethane was 1.1 mM. Maximum activity was found at pH 8.2 and 37 degrees C. Thiol reagents such as p-chloromercuribenzoate and iodoacetamide rapidly inhibited the enzyme. The protein consists of a single polypeptide chain of a molecular weight of 36,000, and its amino acid composition and N-terminal sequence are given.     

Effects of N-nitrosopiperidine substitutions on mutagenicity in Drosophila melanogaster.

N-Nitrosopiperidine (NP) and various derivatives were fed to Drosophila melanogaster males over a wide concentration range in order to assess their mutagenic potency in the induction of X-linked recessive lethals and chromosome loss. NP was effective in inducing lethals, as were its halogen and methyl-substituted derivatives, with the exception of 2,6-dimethyl NP. (Methyl substitutions at the alpha carbon atoms reduce or eliminate mutagenic activity.) Substitution of halogen groups on the piperidine ring enhanced the mutagenic activity, with the 3-chloro compound being the most mutagenic. In contrast, substitutions with a hydroxyl, carboxyl, or keto group resulted in a loss of mutagenicity. None of the compounds tested increased the frequency of chromosome loss or breakage in mature sperm.     

Involvement of a large plasmid in the degradation of 1,2-dichloroethane by Xanthobacter autotrophicus

Xanthobacter autotrophicus GJ10 is a bacterium that can degrade short-chain halogenated aliphatic compounds such as 1,2-dichloroethane. A 200-kb plasmid, pXAU1, was isolated from this strain and shown to contain the dhlA gene, which codes for haloalkane dehalogenase, the first enzyme in the degradation pathway of 1,2-dichloroethane by GJ10. Loss of pXAU1 resulted in loss of haloalkane dehalogenase activity, significantly decreased chloroacetaldehyde dehydrogenase activity, and loss of resistance to mercuric chloride but did not affect the activity level of haloalkanoate dehalogenase, the second dehalogenase in the degradation of 1,2-dichloroethane.