Peroxidative activation of 3,3'-dichlorobenzidine to mutagenic products in the Salmonella typhimurium test

The direct and H2O2-dependent mutagenicity of 3,3'-dichlorobenzidine (DCB) were compared in Salmonella tester strains TA98, TA98/1,8-DNP6, TA100 and TA102 using the Ames test. DCB exhibited both direct and H2O2-dependent mutagenicity to both tester strains TA98 and TA98/1,8-DNP6. This H2O2-dependent mutagenicity of DCB was prevented by horseradish peroxidase. DCB, in contrast to its effects in tester strains TA98, was not mutagenic to TA100 and TA102 either directly or in the presence of H2O2. These results suggest that mechanisms, perhaps enzymes endogenous to tester strains TA98, may play a role in the activation of DCB.     

Comparative activation of 3,3'-dichlorobenzidine and related benzidines to mutagens in the Salmonella typhimurium assay by hepatic S9 and microsomes from rats pretreated with different inducers of cytochrome P-450

The metabolic basis of the differential activation of 4 benzidines--3,3'-dichlorobenzidine (DCB), benzidine (BZ), o-tolidine (TOL) and o-dianisidine (DIN)--to mutagens was examined in the Ames test, using Salmonella typhimurium TA98. For each benzidine congener, the comparative activation by 3 rat liver enzyme systems--(i) postmitochondrial supernatant (S9), (ii) S9 + acetylcoenzyme A (S9-Ac) and (iii) microsomes--and the effect thereon of animal pretreatment with 3 cytochrome P-450 inducers--DCB, 3-methylcholanthrene (MC) and phenobarbital (PB)--were examined. DCB was the most activated of the benzidines, with activation by the 3 systems being in the order: S9 = S9-Ac greater than microsomes, whereas dianisidine and tolidine were the least activated. Benzidine was activated only in the S9 systems but the S9-catalyzed activation of benzidine, unlike that of DCB, was enhanced by added acetylcoenzyme A. Pretreatment with either DCB, MC or PB enhanced the activation of DCB, decreased that of benzidine, and had no effect on that of tolidine or dianisidine. The enhanced DCB activation was most pronounced with DCB pretreatment and was 2.5-fold, 2-fold, and 3-fold, in S9-Ac, S9, and microsomes, respectively. The microsomal-catalyzed DCB activation was inhibited by the cytochrome P-450 inhibitors 2,4-dichloro-6-phenylphenoxyethylamine and alpha-naphthoflavone by 93% and 48%, respectively. DCB, but not its congeners, elicited NADPH-dependent metabolite complex formation with microsomal cytochrome P-450. The results show that DCB is the most mutagenic of the 4 benzidines under conditions of cytochrome-P-450-catalyzed activation and suggest that the DCB activation may be catalyzed most effectively by cytochrome P-450 species induced specifically by the compound itself.     

The metabolism of N4-hydroxycytidine-a mutagen for Salmonella typhimurium.

Salmonella typhimurium cells were grown in the presence of (14C)-N4-hydroxycytidine (N4OHcyd), a mutagenic nucleoside, and labelling in DNA and RNA digest was traced. The results show that this analogue is incorporated into RNA at a level of 40 mug/100 mg, and into DNA with a yield at least 100 times smaller. Some N-minus4OHcyd was rapidly metabolized and labelling was found in all ribo- and deoxyribo-nucleosides.     

A mutagen assay detecting forward mutations in an arabinose-sensitive strain of Salmonella typhimurium.

Strain SV3 of Salmonella typhimurium is sensitive to arabinose, that is, unable to grow in a medium containing arabinose plus glycerol as carbon source. Arabinose resistance is the consequence of the mutational inactivation of one of at least three different genes. The selection of arabinose-resistant mutants provides a simple and sensitive assay for the detection of weak mutagens and for refined quantitative studies of strong ones. The assay is not influenced by experimental artifacts derived from physiological or lethal effects or from differences in plating density. Such artifacts are common with other bacterial mutagen assays, including those using strains analogous to SV3. As practical examples, the assay was used with N-methyl-N'-nitro-N-nitrosoguanidine and the fungicide captafol.     

Assessing the use of known mutagens to calibrate the Salmonella typhimurium mutagenicity assay: I. Without exogenous activation

There has been an increasing need in genetic toxicology to progress from strictly qualitative tests to more quantitative tests. This, in turn, has increased the need to develop better quality assurance and comparative bioassay methods. In this paper, two laboratories tested 10 Salmonella mutagens in order to determine the usefulness of selected chemicals as potential reference materials to calibrate the Salmonella assay. If variance within a bioassay is sufficiently low and the rankings of the compounds are of acceptable consistency, the chemicals later could be evaluated for use as standard control compounds, as audit materials, and as standard reference materials for comparative bioassay efforts. The results demonstrated that the chosen chemicals (with the possible exception of dimethylcarbamylchloride) provide such consistent results in the Salmonella mutagenicity bioassay that they can be used for semi-quantitative calibration and as possible bioassay controls, special audit chemicals, and potentially as reference standards in comparative bioassay efforts. Reference standards, whether used as audit materials or in comparative bioassays, must be used concurrently with the test substances of interest; used without bias; used in a standardized, highly controlled bioassay; and be tested across an appropriate dose range. The study also shows that when these compounds are used as reference standards much care must be given to the number and spacing of doses if highly reproducible slope values are to be generated. We recommend use of a pilot test to establish a dose range for definitive tests and the placement of doses for the definitive tests within the first half of the linear dose-response curve. For appropriate comparisons, one should replicate the tests using the defined dose range and analyze the results in a non-biased statistical manner.     

Intracellular activation of albomycin in Escherichia coli and Salmonella typhimurium.

The antibiotic albomycin is actively taken up by Escherichia coli via the transport system for the structurally similar iron complex ferrichrome. Albomycin is cleaved, and the antibiotically active moiety is released into the cytoplasm, whereas the iron carrier moiety appears in the medium. Besides transport-negative mutants, additional albomycin-resistant mutants were isolated. The mutations were mapped outside the transport genes close to the pyrD gene at 21 min. The mutants were devoid of peptidase N activity. The molecular weight, sensitivity to inhibitors, and cytoplasmic location of the enzyme hydrolyzing albomycin in vitro corresponded to the known properties of peptidase N. The aminoacyl thioribosyl pyrimidine moiety of albomycin apparently has to be cleaved off the iron chelate transport vehicle to inhibit growth. Peptidase N is the major hydrolyzing enzyme. In Salmonella typhimurium peptidase N and peptidase A were equally active in hydrolyzing and activating albomycin.     

Purification of pseudouridylate synthetase I from Salmonella typhimurium.

Pseudouridylate synthetase from Salmonella typhimurium has been purified 1,000 fold and is about 90% pure. The enzyme has a molecular weight of 50,000 daltons. In the presence of tRNA there is a change in molecular weight from 50.000 to 100.000. This change does not seem to be due to the formation of a tRNA-enzyme complex but rather to a tRNA induced dimerization. Other properties of the enzyme are described.     

Differing activation pathways for 2-acetylaminofluorene to a mutagen in vitro

The mutagenicity of 2-acetylaminofluorene (AAF) in S. typhimurium TA 1538 was investigated using Ames' test and activation systems based upon rat- or cotton rat-liver post-mitochondrial supernatant (S9) fractions. Part of this study involved sub-fractionation of S9 into microsomes (M) and 100,000 X g supernatant (S100) fractions. With a rat liver-derived fractions, mos activity was associated with S100; M-activating potential was never greater than that achieved with S9. In cotton rats, most activating potential was associated with S9. This activity was greater than could be accounted for by the separated cotton-rat M and S100 components. Reconstituted, cross-species 'S9' fraction studies showed that the dominant determinant of S9 properties was the M fraction in both rats and cotton rats. The principal co-factor required in the activation reactions was NADPH, but it could be largely replaced by NADH. 7,8-Benzoflavone inhibited activation both in M and S100 whereas paraoxon had no effect upon rat S100 activation, but had a marked effect upon cotton-rat M activation.     

The metabolic activation of dichloromethane and chlorofluoromethane in a bacterial mutation assay using Salmonella typhimurium

The metabolic activation and mutagenicity of dichloromethane and chlorofluoromethane were investigated using rat liver fractions and Salmonella typhimurium strain TA100. Both dihalomethanes gave a mutagenic response without the addition of rat-liver fractions. This response has been shown to be due to bacterial metabolism of the test compounds by pathways believed to be similar to those known in the rat. When rat-liver post-mitochondrial supernatant was added to the mutagenicity assay, there was no significant increase in the mutagenicity of dichloromethane, whereas a 2-fold increase was observed for chlorofluoromethane under the same conditions. This increase was derived both from glutathione conjugation and cytochrome P450 oxidative dehydrochlorination. A significant increase in dichloromethane mutagenicity could only be achieved by increasing the concentration of post-mitochondrial supernatant. Under these conditions the increase in mutagenicity was derived solely from glutathione conjugation of dichloromethane. The difference in mutagenic response after the addition of rat-liver fractions can be explained by differences in the half lives of the reactive intermediates rather than a difference in overall metabolic rate between the two compounds.     

Susceptibility of Salmonella typhimurium and Salmonella typhi to oxygen metabolites

The susceptibility of Salmonella typhimurium LT2 and S. typhi 1079 to oxygen metabolites were compared. S. typhimurium LT2 and S. typhi 1079 were killed to an equal extent (about 40%) by the xanthine-xanthine oxidase (200 mU/ml) system. Among the various scavengers of oxygen metabolites, catalase alone inhibited the killing of S. typhimurium LT2 and S. typhi 1079 by the xanthine-xanthine oxidase system, indicating that hydrogen peroxide contributed to the killing of Salmonellae. The respiratory burst of murine macrophages was efficiently triggered by the ingestion of S. typhimurium LT2, S. typhimurium SL1102, and S. typhi 1079 and all to the same extent. However, in the range of the concentration of hydrogen peroxide produced by murine macrophages, neither S. typhimurium LT2 nor S. typhi 1079 were killed. Only S. typhimurium SL1102, a rough mutant of S. typhimurium LT2, was markedly susceptible under these conditions. The findings suggest that both S. typhimurium LT2 and S. typhi 1079 are resistant to oxygen-dependent killing mechanisms.     

Susceptibility of Salmonella typhimurium and Salmonella typhi to oxygen metabolites

Abstract The susceptibility of Salmonella typhimurium LT2 and of S. typhi 1079 to oxygen metabolites were compared. S. typhimurium LT2 and S. typhi 1079 were killed to an equal extent (about 40%) by the xanthine-xanthine oxidase (200 mU/ml) system. Among the various scavengers of oxygen metabolites, catalase alone inhibited the killing of S. typhimurium LT2 and S. typhi 1079 by the xanthine-xanthine oxidase system, indicating that hydrogen peroxide contributed to the killing of Salmonellae . The respiratory burst of murine macrophages was efficiently triggered by the ingestion of S. typhimurium LT2, S. typhimurium SL1102, and S. typhi 1079 and all to the same extent. However, in the range of the concentration of hydrogen peroxide produced by murine macrophages, neither S. typhimurium LT2 nor S. typhi 1079 were killed. Only S. typhimurium SL1102, a rough mutant of S. typhimurium LT2, was markedly susceptible under these conditions. The findings suggest that both S. typhimurium LT2 and S. typhi 1079 are resistant to oxygen-dependent killing mechanisms.     

Experimental adaptation of Salmonella typhimurium to mice

Experimental evolution is a powerful approach to study the dynamics and mechanisms of bacterial niche specialization. By serial passage in mice, we evolved 18 independent lineages of Salmonella typhimurium LT2 and examined the rate and extent of adaptation to a mainly reticuloendothelial host environment. Bacterial mutation rates and population sizes were varied by using wild-type and DNA repair-defective mutator (mutS) strains with normal and high mutation rates, respectively, and by varying the number of bacteria intraperitoneally injected into mice. After <200 generations of adaptation all lineages showed an increased fitness as measured by a faster growth rate in mice (selection coefficients 0.11-0.58). Using a generally applicable mathematical model we calculated the adaptive mutation rate for the wild-type bacterium to be >10(-6)/cell/generation, suggesting that the majority of adaptive mutations are not simple point mutations. For the mutator lineages, adaptation to mice was associated with a loss of fitness in secondary environments as seen by a reduced metabolic capability. During adaptation there was no indication that a high mutation rate was counterselected. These data show that S. typhimurium can rapidly and extensively increase its fitness in mice but this niche specialization is, at least in mutators, associated with a cost.