Assessment of genotoxic effects by lindane.

The well known and previously widespread insecticide lindane has been re-assessed for DNA-damaging activity. A first group of investigations using standard in vitro and in vivo mutagenicity assays did not indicate any genotoxic effects of lindane at all. The assay systems used were for the induction of HPRT mutations and sister chromatid exchanges in CHO cells cultured in vitro, and for micronuclei induction in vivo in bone marrow cells of rats, hamsters and mice. Also, lindane was assessed for its potential to induce sister chromatid exchanges in vivo in the bone marrow of Chinese hamsters. These specific assay systems had not been used previously for elucidating the genotoxic effects of this compound, but they are basically similar to other standard mutagenicity assays in which lindane has been shown to be devoid of genotoxic activity. The second part of the investigations was directed at re-evaluating a previously reported positive effect of the compound in primary rat hepatocytes in vitro. We performed in vitro and in vivo studies with hepatocytes from the rat liver and used alkaline elution to detect DNA damage. However, we could not demonstrate that lindane induced genotoxicity, unless considerable concomitant cytotoxicity was apparent as well. Finally, since lindane can be ingested and inhaled by humans, we also measured the induction of DNA damage in local target organs of absorption using single cell micro-gel-electrophoresis (the comet assay). In these cases lindane was genotoxic in cells of the gastric and nasal mucosa in vitro and also in vivo following appropriate routes of application (oral and inhalational exposure).

The possible role of probiotics as dietary antimutagen.

Possible antimutagenic actions of probiotics--mainly lactic acid bacteria--were examined using in vitro and in vivo test systems. In the Ames test with Salmonella typhimurium TA1538 beef extract and nitrosated beef extract were used as mutagens. L. casei showed high antimutagenic activity on mutagenicity induced by nitrosated beef extract only without S9 mix, whereas Omniflora (a lyophilized preparation of lactobacilli and E. coli) and its cell-free culture broth exhibited antimutagenic action only on beef extract. The actions of probiotics were more homogeneous when living animals were used in the tests. Using busulfan as a mutagen both the chromosome aberration test (with Chinese hamster bone marrow cells) and the micronucleus test (with bone marrow cells of Chinese hamsters and mice) showed strong anticlastogenic action when L. casei, Omniflora or yoghurt (with living bifiobacteria) were given orally at the same time as the mutagen. Lactobacilli were effective also after i.p. injection. Cell-free culture broths had no or only weak antimutagenic effects. Mutagen-induced chromosome aberrations and micronuclei were reduced by up to 80% by the lactobacilli.

Re-examination of potassium sorbate and sodium sorbate for possible genotoxic potential.

Potassium sorbate and sodium sorbate were investigated for possible genotoxic actions using the Salmonella/mammalian-microsome test, HGPRT and sister chromatid exchange (SCE) test with Chinese hamster ovary cells, the micronucleus test on bone marrow cells of mice and Chinese hamsters, and the chromosome aberration and SCE test on Chinese hamsters. In all the in vitro tests no signs of genotoxicity were detected. Whereas no in vivo mutagenicity of potassium sorbate and sodium sorbate with freshly prepared aqueous solutions and with stored potassium sorbate was found, investigations with stored sodium sorbate revealed weak clastogenic activity by increased chromosome aberrations and elevated numbers of micronuclei at doses of 200 mg/kg body weight, but no induction of SCEs.

In vivo effects of single or combined dietary antimutagens on mutagen-induced chromosomal aberrations.

The food components chlorophyllin, beta-carotene and alpha-linolenic acid (in its methyl ester form) were tested in Chinese hamsters for antimutagenic activity on the powerful mutagen thio-tepa. Each of these natural protective compounds inhibited by 70-85% the clastogenic effects induced by the mutagen. When 2 or 3 of these antimutagens were administered simultaneously no additive effects were observed. alpha-Linolenic acid methyl ester was the most effective antimutagen under the experimental conditions.

Genotoxic investigations of tobacco protein using microbial and mammalian test systems.

Tobacco protein was assayed for mutagenicity using the Ames test and three in vivo tests. In the Salmonella strains TA 98 and TA 100 methanolic extracts of the tobacco protein and urine of rats fed tobacco protein exhibited increased revertant numbers, but extracts of feces did not. Using the micronucleus test throughout, weak mutagenic effects after feeding the tobacco protein were detected in Chinese hamsters and two inbred strains of mice, and again in Chinese hamsters when the chromosome aberration test and the SCE test were applied. The analytical specifications of the tobacco protein listed nicotine, chlorogenic acid and rutin as components. These were examined separately in a chromosome aberration test, and nicotine was discovered to be the factor or a factor responsible for the weak positive test results.

Testing of sunset yellow and orange II for genotoxicity in different laboratory animal species.

The azo dyes Sunset Yellow and Orange II were gavaged to rodent species to check bile, urine, and fecal extracts for possible mutagenic activity in the Ames test or in bone marrow cells for clastogenicity using cytogenetic test systems. After oral application the dyes showed a negative response in bile, excrements, and bone marrow. When an exogenous metabolic activation was performed, increased revertant numbers using Salmonella strain TA100 were obtained only in fecal extracts of Sunset Yellow-treated animals. It is concluded that no genotoxic harm is to be expected from the ingestion of Sunset-Yellow or Orange II.

The anticlastogenic potential of fatty acid methyl esters.

To test for possible anticlastogenic effects of fatty acids, the methyl esters of fatty acids--short-chain to long-chain--were examined on busulfan in Chinese hamster bone-marrow cells using the chromosome aberration test. When the experimental animals were treated with fatty acid esters and the mutagen, the chromosome-breaking actions of busulfan were not modulated by the short-chain fatty acids, but the fatty acids from lauric acid (C12) up to nonadecanoic acid (C19) reduced the rate of aberrant metaphases from 9.4 to about 3% at doses of 100 mg/kg and less. Other chemical properties of the fatty acids (saturated or not, number of double bonds, even- or odd-numbered) had no influence on the anticlastogenic effects. The only exceptions to this rule were arachidonic acid, which had no effect, and gamma-linolenic acid, which had no consistent effect on the action of busulfan.

Anticlastogenic effect of beta-carotene in Chinese hamsters. Time and dose response studies with different mutagens.

beta-Carotene exhibited dose-dependent anticlastogenic effects on aberrations induced by the direct-acting mutagens thio-TEPA, methyl methanesulfonate and busulfan in the in vivo chromosome aberration test (bone marrow cells, Chinese hamsters). No effect was seen when retinol was used. Apparent differences in the action of beta-carotene on aberrations induced by the three applied mutagens may be due to differences in the solubility of the compounds and to the different routes of administration.

Antimutagenic effects on male germ cells of mice.

The alkylating agent cyclophosphamide (CPA) and the antioxidant ethoxyquin (EQ) were administered perorally to NMRI mice. The strong clastogenic action of CPA on spermatogonia was diminished by simultaneous doses of EQ. Higher doses of the antioxidant produced greater anticlastogenic action. Furthermore, the action of the mutagen and the antioxidant on the late spermatids and the spermatozoa was observed using the dominant lethal test. The antioxidant had only a weak influence on these postmeiotic stages.

Antimutagenic effect of an antioxidant in mammals.

To test a possible antimutagenic activity of ethoxyquin (EQ) in bone-marrow cells, 3 cytogenetic tests with distinct genetic end-points were applied. Cyclophosphamide (CPA), serving as test mutagen, and the antioxidant EQ were administered immediately following each other to Chinese hamsters by stomach tube. Whereas the CPA dose was the same in each test, the EQ doses were increased up to a ratio of CPA/EQ = 1:25, in some cases up to 1:50. The formation of SCEs induced by CPA was not influenced by EQ--even at the highest dose. In the micronucleus test, however, EQ drastically reduced the micronucleus rate even at the lowest dose applied (20 mg/kg) and abolished the CPA effects at a dose of 100 mg/kg. This action of EQ against CPA was also found in the rat and mouse in the micronucleus test system. Two inbred strains of mouse showed similar reactions, but the induced micronucleus rate was higher and its decrease in response to increasing doses of EQ was more delayed. In the chromosome aberration test, EQ also showed a distinct anticlastogenic response. At higher EQ doses, all CPA-induced chromosomal damage was reduced down to the level of spontaneous rates. The anticlastogenic effect of EQ was quantitatively similar in the micronucleus and chromosome aberration tests. Only minor qualitative differences were recognizable.

Attempts to induce cytogenetic effects with sulphite in sulphite oxidase-deficient Chinese hamsters and mice.

Chinese hamsters and mice were made sulphite-oxidase deficient by the feeding of a low-molybdenum diet with sodium tungstate as a drinking-water supplement. Hepatic sulphite-oxidase activity was checked spectrophotometrically. Under normal conditions, sulphite-oxidase activity is high in the mouse and low in the Chinese hamster. Sulphite (SO3--) was given in a single or double oral dose in aqueous solution or dissolved in fruit juice or by repeated subcutaneous injections up to the maximum tolerated doses. Possible cytogenetic effects were studied in bone-marrow cells using three test systems--the sister chromatid exchange, chromosome aberration and micronucleus tests. No induction of cytogenetic effects was observed with any of the three tests in either species, indicating that no damage at the chromosomal level was induced by sulphite in these animals, even when sulphite-oxidase activity was reduced to a very low level.

Mutagenicity testing of quinine with submammalian and mammalian systems.

Quinine hydrochloride was assayed for genotoxic activity by using 4 different test systems with distinct genetic endpoints. No indications for point mutations were observed in the Ames system. In 3 cytogenetic tests performed on small rodents, Chinese hamsters showed no genotoxic activity, while inbred strains of mice revealed a dose dependent increase of SCEs, enhanced incidence of micronuclei and elevated chromatid breaks.

Genotoxicity of brown-colored polymerization products formed in smoke flavors.

Smoke aroma essences, which are prepared from smokehouse smoke by condensation and purification, are used for flavoring raw food products. The essences spontaneous decompose and produce brown-colored polymerization products, which may react with protein and be liberated within the acidic environment of the human stomach. The potential of these products to cause DNA damage was studied in two microbial and two in vivo assay systems. The polymerization products induced his+ reversion in Salmonella typhimurium TA 100 after metabolic activation by liver enzymes. There was no significant activity in a differential killing assay with repair-deficient strains of Escherichia coli WP2. In vivo tests demonstrated significant increases in the rate of sister chromatid exchanges in bone marrow cells of Chinese hamsters, but no increase in micronuclei was detectable. Thus, genotoxic components may be present in the brown-colored fractions of smoke aroma essences, but further study is needed.

An investigation of the genetic toxicology of irradiated foodstuffs using short-term test systems. III--In vivo tests in small rodents and in Drosophila melanogaster.

Six in vivo genetic toxicity tests were carried out on irradiated or unirradiated cooked chicken, dried dates and cooked fish. The tests were as follows: sex-linked recessive lethal mutations in Drosophila melanogaster (dried dates only), chromosome aberrations in bone marrow of Chinese hamsters, micronucleus test in rats, mice and Chinese hamsters, sister-chromatid exchange in bone marrow of mice and Chinese hamsters and in spermatogonia of mice, and DNA metabolism in spleen cells of Chinese hamsters. None of the tests provided any evidence of genetic toxicity induced by irradiation. However, dried dates, whether irradiated or not, showed evidence of some genetic toxicity in their effect on DNA metabolism in spleen cells and SCE induction in bone marrow. Feeding irradiated fish affected DNA metabolism in the spleen cells of Chinese hamsters. This effect could be interpreted as an induction of an immunoactive compound, although it could also be explained by the persistence of an immunoactive compound due to the removal by irradiation of spoilage organisms that would normally degrade it.

Genotoxicity of cocoa examined by microbial and mammalian systems.

Unroasted or roasted cocoa powder dispersed in water and applied to Chinese hamsters by stomach tube caused elevated numbers of SCEs in the sister-chromatid exchange test (bone-marrow cells). Roasted cocoa freed from fat produced distinctly higher SCE values with a linear dose-response relationship, whereas cocoa butter had no influence on SCE levels. Positive results in the SCE test (1.5-fold values of the controls) were obtained after application of about 5 g cocoa/kg b.w. Presumably, because of the smaller quantities that could be administered in this way, positive test results were not found when cocoa was given in the diet instead of being administered by stomach tube. Cocoa from which theobromine was extracted by chloroform did not affect SCE levels. Pure theobromine increased SCE levels in a dose-dependent way. Theobromine was also positive in the micronucleus test at 2 X 40 mg/animal and negative in the chromosome aberration test at 1 X 40 mg/animal. Cocoa and the theobromine were negative in the Salmonella/mammalian microsome mutagenicity test both with and without metabolic activation.