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Publications and source records attributed to E Zeiger.
Three hundred chemicals were tested for mutagenicity, under code, in Salmonella typhimurium, using a preincubation protocol. All tests were performed in the absence of exogenous metabolic activation, and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters. The results and data from these tests are presented.
Four widely used in vitro assays for genetic toxicity were evaluated for their ability to predict the carcinogenicity of selected chemicals in rodents. These assays were mutagenesis in Salmonella and mouse lymphoma cells and chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells. Seventy-three chemicals recently tested in 2-year carcinogenicity studies conducted by the National Cancer Institute and the National Toxicology Program were used in this evaluation. Test results from the four in vitro assays did not show significant differences in individual concordance with the rodent carcinogenicity results; the concordance of each assay was approximately 60 percent. Within the limits of this study there was no evidence of complementarity among the four assays, and no battery of tests constructed from these assays improved substantially on the overall performance of the Salmonella assay. The in vitro assays which represented a range of three cell types and four end points did show substantial agreement among themselves, indicating that chemicals positive in one in vitro assay tended to be positive in the other in vitro assays.
A total of 224 chemicals that have been tested in long-term studies for carcinogenicity in rats and mice by the National Cancer Institute and the National Toxicology Program were tested for mutagenicity in Salmonella typhimurium. Correlations between mutagenicity and carcinogenicity were examined. The influences of chemical structure, rodent species and organ responses, and bacterial strain responses on the carcinogenesis/mutagenesis correlations were also examined. Not all carcinogens induced tumors in both rats and mice. A clear mutagenic or equivocal mutagenic response in Salmonella was predictive for 77% of the carcinogens or equivocal carcinogens, although only 54% of the 149 carcinogens or equivocal carcinogens were mutagens, and 58% of the nonmutagens were carcinogens or equivocal carcinogens. The proportion of mutagens and equivocal mutagens that were not carcinogenic or equivocal was 23%. There was no apparent way to distinguish the mutagenic carcinogens from the mutagenic noncarcinogens by the responses of the specific Salmonella strains. The proportions of different chemical classes in the data base strongly affected the correlations; 40% of the chlorinated carcinogens were mutagens, whereas 75% of the amines and 100% of the nitro-containing carcinogens were mutagens. Because 29% of the chemicals (30% of the carcinogens) were chlorinated, the poor correlation of this class was reflected in the overall correlation. It is concluded that the use of the Salmonella mutagenicity assay is warranted for the identification of carcinogens, but not for noncarcinogens. The proportion of carcinogens detected as mutagens is dependent on the specific classes of chemicals tested and on the rodent species used to define the carcinogens.
The genotoxic effects of methyl isocyanate (MIC) were investigated using four short-term tests: the Salmonella reversion assay (Ames test), the Drosophila sex-linked recessive lethal assay, and the sister chromatid exchange (SCE) and chromosomal aberration assays in cultured Chinese hamster ovary (CHO) cells. No evidence was found for the induction of mutations in either Salmonella or Drosophila. MIC did, however, induce SCEs and chromosomal aberrations in CHO cells both in the presence and absence of Aroclor-induced rat liver S-9.
5-(4-Nitrophenyl)-2,4-pentadien-1-al (NPPD; spy dust) was tested for mutagenicity in Salmonella and for its ability to induce chromosome aberrations and sister chromatid exchanges (SCE) in cultured Chinese hamster ovary (CHO) cells and mouse bone marrow. Two metabolites of NPPD produced by the rat, 4-nitrobenzoic acid (NBA) and 4-nitrohippuric acid (NHA), were also tested in Salmonella and CHO cells. NPPD was mutagenic in Salmonella, and induced low-level increases in chromosome aberrations and SCE in bone marrow. It did not induce aberrations in CHO cells. NBA was positive in Salmonella and CHO cells, while NHA was negative. The mutagenicity of NPPD in Salmonella was partially, but not completely, eliminated in a strain lacking one of the bacterial nitroreductases.
Glutathione was mutagenic in Salmonella typhimurium strain TA100 in the presence of purified mammalian gamma-glutamyltranspeptidase. Glutathione disulfide, gamma-glutamyl glutamic acid, and S-methyl-glutathione were not mutagenic under the same conditions. Glutathione-mediated, gamma-glutamyltranspeptidase-dependent mutagenesis of TA100 cells was inhibited by serine-borate complex, a known gamma-glutamyltranspeptidase inhibitor, and potentiated by glycylglycine, a known gamma-glutamyltranspeptidase enhancer. It is concluded that this enzyme is necessary and sufficient to activate glutathione to a mutagen.
Sodium bisulfite is a weak mutagen at pH 5 and 6 in S. typhimurium strains carrying the hisG46 and hisD6610 mutations, but is not mutagenic in strains with the hisC3076 or hisD3052 mutations. The bisulfite-induced base-pair substitution mutations were slightly enhanced by the presence of the plasmid, pKM101, but inhibited by the presence of the uvrB and rfa mutations. The hisO1242 mutation which causes constitutive expression of the histidine operon, produced a slight enhancement of frameshift (hisD6610), but not base-pair substitution (hisG46) mutations. Bisulfite-induced mutations appear to be the result of two different mechanisms which may be a function of the repair capacity of the strains. The data suggest that the deamination of cytosine may not be responsible for frameshift mutations, but may be responsible for base-pair substitution mutagenesis. Because the rate of bisulfite autooxidation appears to play a role in the mutagenic process, we are suggesting that the deamination of cytosine may be the result of oxidative damage rather than through the direct formation of a cytosine-bisulfite adduct. This is further supported by the much lower concentrations of bisulfite needed to cause mutagenicity than the 1 M concentrations cited to produce cytosine-bisulfite adducts.
Since its development by Dr. Bruce Ames and his coworkers, the Salmonella typhimurium/mammalian microsome mutagenicity assay has been used widely throughout the world. Many authors have suggested various modifications and made recommendations in regards to this assay. Although the recommendations of a panel of experts was published in 1979 by de Serres and Shelby, a committee of members of the Environmental Mutagen Society (EMS) initiated this effort in response to the encouragement by the American Society of Testing and Materials (Committee E47.09.01) and because of new developments within the field of microbial mutagenesis testing. Its purpose is to provide a guide for people who perform or evaluate microbial mutagenesis tests, but it is not intended for these recommendations to replace or diminish the usefulness of presently available protocols and procedures.
Suspensions of dark-adapted guard cell protoplasts of Vicia faba L. alkalinized their medium in response to irradiation with red light. The alkalinization peaked within about 50 minutes and reached steady state shortly thereafter. Simultaneous measurements of O(2) concentrations and medium pH showed that oxygen evolved in parallel with the red light-induced alkalinization. When the protoplasts were returned to darkness, they acidified their medium and consumed oxygen. Both oxygen evolution and medium alkalinization were inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). In photosynthetically competent preparations, light-dependent medium alkalinization is diagnostic for photosynthetic carbon fixation, indicating that guard cell chloroplasts have that capacity. The striking contrast between the responses of guard cell protoplasts to red light, which induces alkalinization, and that to blue light, which activates proton extrusion, suggests that proton pumping and photosynthesis in guard cells are regulated by light quality.
The National Cancer Institute (NCI) and the National Toxicology Program (NTP) have carried out a number of laboratory animal carcinogenicity studies and presented the results of these experiments in a series of Technical Reports. This paper tabulates the results of the 327 NCI/NTP studies carried out to date on 308 distinct chemicals, and discusses certain issues relevant to the evaluation of carcinogenicity in these experiments. This compilation of results from NCI/NTP carcinogenicity experiments provides a large database that can be used to study structure-activity correlations, interspecies concordance, and associations between laboratory animal carcinogenicity and other toxicological effects.
The results and data from the testing of 255 chemicals for mutagenicity in Salmonella are presented. All chemicals were tested under code using a preincubation modification of the Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters.
It is a widely held view that objective statistical criteria are needed for the evaluation of genetic toxicity assays. This paper presents statistical methods for the analysis of data from in vitro sister chromatid exchange (SCE) and chromosome aberration tests that use Chinese hamster ovary cells. For SCEs, an extensive study of solvent control results demonstrated that there is a substantial interday component of variability in the data, and that a Poisson sampling model is applicable to data generated via the protocol of Galloway et al [1985]. Consequently, a trend test for evidence of a dose response is proposed for such SCE data. As an illustration of this statistical method, analysis of data previously considered to be negative [Gulati et al, 1985] indicates that di(2-ethyl-hexyl) phthalate induces a weak, but reproducible, SCE dose response in CHO cells. Monte Carlo methods are used to show that the trend test is more sensitive than four other statistical procedures considered for the analysis of Poisson-distributed SCEs. A similar trend test for dose response in proportions is proposed for chromosome aberration data, where the percent of cells with chromosome aberrations is the response of interest. Sensitivity (or power) studies indicate that three doses and a control with 50 cells/dose point is a reasonable design for an in vitro SCE study that uses the Galloway et al protocol. For in vitro chromosome aberrations, however, three doses and a control with 100 cells/dose point appears to produce too insensitive an assay; an increase to 200 cells/dose point in the Galloway et al protocol seems worthy of serious consideration.
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37 aliphatic epoxides comprising 6 subclasses (unsubstituted aliphatic epoxides, halogenated aliphatic epoxides, glycidyl esters, glycidates, glycidyl ethers and diglycidyl ethers) were tested, under code, for mutagenicity in Salmonella strains TA98, TA100, TA1535 and TA1537 and/or TA97 with and without metabolic activation using a standardized protocol. The 4 halogenated aliphatic epoxides and the 4 diglycidyl ethers were all mutagenic. The 2 glycidates were negative in all strain/activation systems used while all 5 glycidyl esters were mutagenic. 3 of the 8 unsubstituted aliphatic epoxides and 11 of the 12 glycidyl ethers were mutagenic. Glycidol also was mutagenic whereas 9,10-epoxyoctadecanoic acid, 2-ethylhexyl ester was not mutagenic. Of the 28 mutagenic compounds, all but neodecanoic acid, 2,3-epoxypropyl ester and 2-ethylhexyl glycidyl ether were detected in TA100 without activation. The latter two were detected only with activation in TA100 and TA1535. The majority of the other 26 chemicals were also mutagenic in TA1535 without activation. Good intra- and interlaboratory reproducibility was seen in the results of each of the 4 chemicals tested in more than one set of experiments. The current results confirm and extend the observations of other investigators regarding structural effects on the mutagenicity of members of the aliphatic epoxide class of chemicals.
Stomatal responses to light and humidity (vapor pressure difference, VPD) are important determinants of stomatal conductance. Stomatal movements induced by light are the result of a transduction of the light stimulus into modulated ion fluxes in guard cells and concomitant osmotic adjustments and turgor changes. It is generally assumed that this transduction process is a general stomatal property, with different environmental stimuli integrated into guard cell metabolism through their modulation of ion fluxes. In contrast with this notion, the VPD response, which is unique because both its triggering signal and the turgor changes required for aperture modulations involve water molecules, has been considered to be hydropassive and thus independent of guard cell metabolism. We used a kinetic approach to compare the light and VPD responses in order to test the hypothesis that hydropassive changes in guard cell turgor could be faster than the metabolism-dependent light responses. Changes in stomatal conductance in intact leaves of sugarcane and soybean were measured after application of step changes in VPD and in light. In spite of a 5-fold difference in overall rates between the two species, the response rates following light or VPD steps were similar. Although a coincidental kinetic similarity between two mechanistically different responses cannot be ruled out, the data suggest a common mechanism controlling stomatal movements, with the VPD stimulus inducing metabolic modulations of ion fluxes analogous to other stomatal responses.
This article is an expanded summary of the workshop discussions. Its objective is to add perspective and future orientation to the scientific symposium presented in the previous articles of this volume.
A series of K-region, N-substituted phenanthrene imines were tested for mutagenicity in Salmonella typhimurium TA100. All chemicals were mutagenic in the absence of an exogenous metabolic activation system. The apparent decay times of the mutagenic species in diffusion plates and their alkylating activities were also measured. The unsubstituted phenanthrene-9,10-imine was approximately 70-fold more mutagenic than the corresponding phenanthrene-9,10-oxide. N-substitution with electron-releasing groups resulted in chemicals that were more mutagenic than those substituted with electron-withdrawing groups. The mutagenic activity of the latter group of chemicals was comparable with that of phenanthrene-9,10-oxide. Except for N-chlorophenanthrene imine, both alkylation of p-nitrothiophenol and apparent decay times in diffusion plates were inversely correlated with mutagenicity. It is hypothesized that reactivity towards p-nitrothiophenol (alkylating activity) and mutagenicity reflect different reactions, in contrast to other chemical mutagens. The results suggest that the high potency of phenanthrene imines as mutagens is possibly due to DNA binding via an aziridinium ion rather than a carbonium ion.
This publication includes data of Salmonella mutagenicity results on 270 coded chemicals, encompassing 329 tests performed by three laboratories under contract to the National Toxicology Program (NTP). The preincubation modification of the Salmonella/mammalian microsome assay was used to test chemicals in up to five Salmonella strains in the presence and absence of rat and hamster liver S-9. With a few exceptions, inter- and intralaboratory reproducibility was good.