Modified suspension Ames test for testing proteinaceous substances: an initial step.

The basic Salmonella/microsome assay (Ames test) is a valuable primary tool by which to discriminate mutagens from non-mutagens. For a variety of chemical test substances this test is easily conducted according to international guidelines for genotoxicity testing. However, the testing of proteinaceous substances in the basic Ames test may generate false positives owing to the presence of growth-promoting constituents in the test sample, such as histidine or its precursors. It was hypothesized that the growth-promoting capacities of biological test samples might be overcome by testing according to the 'suspension variant' of the Ames test, which uses very rich growth conditions thereby overwhelming any growth-enhancing constituents present in a biological test sample. This hypothesis appeared to be correct, although several important modifications had to be made to the suspension assay. The most important aspect of this 'new suspension Ames test' appeared to be the plating of overnight regrown bacteria in the poorest way possible (by omitting histidine and nutrient broth from the overlay agar). This study may comprise an initial step in the development of a modified suspension Ames test for testing proteinaceous substances.

Effect of eugenol on the genotoxicity of established mutagens in the liver.

The influence of in vivo treatment with eugenol on established mutagens was studied to determine whether eugenol has antigenotoxic potential. The effects of eugenol in rats was investigated in the unscheduled DNA synthesis (UDS) assay with established mutagens and the Salmonella typhimurium mutagenicity assay. In addition, the effect of in vivo treatment with eugenol on benzo[a]pyrene (B[a]P)-induced genotoxicity in human hepatoma cell line Hep G2 was investigated in the single-cell gel electrophoresis assay. The mutagenicity of B[a]P in the S. typhimurium mutagenicity assay was lower in liver S-9 fractions from control rats. Incubation of liver S-9 fractions from eugenol-treated rats with dimethylbenzanthracene (DMBA) had no antimutagenic effect. Eugenol did not modify UDS activity in hepatocytes isolated from rats pretreated with eugenol orally after exposure of these cells in vitro to DMBA and aflatoxin B1. Four different treatment schemes of combinations of B[a]P and eugenol were examined in Hep G2 cells: pre-treatment with eugenol; simultaneous treatment with eugenol and B[a]P; a combination of these (pretreatment/simultaneous treatment); and post-treatment with eugenol. An increase in the genotoxicity of B[a]P was found in Hep G2 cells. No effect of eugenol on the genotoxicity of B[a]P was found with the pre- and post-treatments. It is concluded that the effect of eugenol on genotoxicity induced by established mutagens is not univocal; in vivo treatment of rats with eugenol resulted in a reduction of the mutagenicity of B[a]P in the S. typhimurium mutagenicity assay, while in the UDS assay no effect of eugenol was found. In vitro treatment of cultured cells with eugenol resulted in an increase in genotoxicity of B[a]P. These findings indicate that there is only limited support for the antigenotoxic potential of eugenol in vivo.

Effect of a vegan diet on biomarkers of chemoprevention in females.

1. In order to study the potential beneficial effects of a vegan diet, a cross-sectional study was performed and several biomarkers of chemoprevention were measured in a population of female 'living food' eaters ('vegans'; n = 20) vs matched omnivorous controls (n = 20). 2. White blood cells obtained from fresh blood samples were subjected to the single-cell gel-electrophoresis assay. There was no statistically significant difference between the vegans and controls in the parameters 'tail length' and 'tail moment'. However, the 'tail moment' was significantly lower in a subset of the vegans (i.e.in those who did not use any vitamin and/or mineral supplements). 3. Fresh blood samples were exposed in vitro to the mutagen mitomycin C just prior to culturing. After culturing the number of binucleated lymphocytes with micronuclei was scored. There was no difference between the controls and vegans in the incidence of baseline micronuclei, nor in the number of mitomycin C-induced micronuclei. However, a significant correlation (r = -0.64, P < 0.01) between the number of mitomycin C-induced micronuclei and the activity of erythrocyte superoxide dismutase was found in the vegans. The number of baseline micronuclei increased with age in both groups. These findings may be of biological relevance. 4. The content of glutathione-S-transferase-alpha in plasma was not different between the vegans (n = 12) and controls (n = 12). 5. The present data indicate a few differences in biomarkers of chemopreventive potential in strict vegans vs matched omnivorous controls. The significance of these changes as biologically relevant indicators of beneficial effects of vegan diets in humans needs to be determined in studies with a larger number of subjects.

Effect of short-term dietary administration of eugenol in humans.

1. In order to study the antigenotoxic potential of eugenol in humans, ten healthy non-smoking males ingested a daily amount of 150 mg eugenol or the placebo for seven consecutive days. After a washout period of one week, groups ingesting eugenol or the placebo were crossed and received the other treatment for seven consecutive days. 2. On days 8 and 22 blood samples were taken for the assessment of standard clinical biochemical parameters. To study the possible antigenotoxic effect of eugenol, on day 8 and 22 blood samples were collected and exposed in vitro to the established genotoxic agents mitomycin C and vinblastine. After exposure the percentage of cells with chromosome aberrations and micronuclei was determined in cultured white blood cells. On days 8 and 22 paracetamol (500 mg p.o.) was administered as test substance to measure phase-II biotransformation capacity. Glutathione-S-transferase (GST) activities were determined in erythrocytes and blood plasma. 3. No significant differences in the clinical biochemical parameters were detected between the eugenol-period and the placebo-period, indicating that daily administration of 150 mg eugenol for 7 days has no toxic affects. 4. No significant differences on the cytogenetic parameters were found after ingestion of eugenol. Thus, there are no indications for an antigenotoxic potential of eugenol in humans, consuming daily 150 mg eugenol for 7 days. 5. A significant reduction in alpha-class GSTs in plasma (P < 0.05), but not in the other measured biotransformation parameters, was found in volunteers during the eugenol-periods as compared to the placebo-period. This may either reflect GST-inhibition by eugenol or protection against background damage of liver cells by eugenol.