Inactivation of electrophilic metabolites by glutathione S-transferases and limitation of the system due to subcellular localization

Benzo(a)pyrene was activated to metabolites mutagenic for Salmonella typhimurium TA 98 by liver microsomes from control and phenobarbital treated mice. Under these conditions benzo(a)pyrene 4,5-oxide accounts for most of the mutagenicity. We have therefore investigated (1) the conjugation of benzo(a)pyrene 4,5-oxide with glutathione and (2) the effect of glutathione on the mutagenicity of benzo(a)pyrene.The spontaneous conjugation occurred only very slowly. The rate of this reaction was slightly augmented by microsomes and very greatly augmented by the cytosol fraction of liver homogenate. With respect to the mutagenicity of benzo(a)pyrene, glutathione had only a weak effect when benzo(a)pyrene was activated by microsomes in the absence of the cytosol fraction. In its presence, however, glutathione was able to strongly reduce the mutagenicity. But this reduction depended on the spatial relationship between microsomes and bacteria. The strongest inactivation was found when bacteria and microsomes were in separate agar layers. In contrast, no inactivation was observed when all the microsomes were in direct contact with the bacteria. When the test was performed according to the Ames procedure the topographical situation was intermediate: some microsomes were adsorbed onto the bacteria and some were free. Accordingly, the effect of glutathione was intermediate. When the premutagen trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene was activated in the presence of the cytosol fraction, glutathione again reduced the mutagenicity, when microsomes and bacteria were separated from each other, but did not reduce the mutagenicity, when all the microsomes were bound to the bacteria.Obviously in the situation where a direct diffusion within the lipophilic environment from the site of formation to the target bacteria was physically possible the mutagenic metabolites diffused preferentially directly to the bacteria and not through the hydrophilic environment of the medium. Therefore they could not be inactivated by components of the cytosol fraction. This could be of significance also for the situation in the eucaryotic cell, since the endoplasmic reticulum is in direct contact with other cell structures such as the nuclear envelope. Thus, hydrophobic metabolites generated in the endoplasmic reticulum could reach such sites by lateral diffusion within the membranes. The observation that benzo(a)pyrene 4,5-oxide was a very good substrate for the cytosol localized glutathione S-transferase, but that it was not inactivated by this system when bacteria and microsomes were in direct contact, indicates that a severe limitation for the inactivation of benzo(a)pyrene metabolites by this enzyme is imposed by its localization in the cytosol.Presented at the Symposium Influence of Metabolic Activations and Inactivations on Toxic Effects held at the 18th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Section Toxicology, D-6500 Mainz, March 15, 1977     

172G>T variant in the 5' untranslated region of DNA repair gene RAD51 reduces risk of squamous cell carcinoma of the head and neck and interacts with a P53 codon 72 variant

RAD51 participates in homologous recombination (HR) repair of double-stranded DNA breaks (DSBs) that may cause genomic instability and cancer. Two single-nucleotide polymorphisms (SNPs) and three P53 binding sites have been found in the RAD51 promoter and 5' untranslated region. We hypothesized that RAD51 and P53 SNPs may interact and alter risk of squamous cell carcinoma of the head and neck (SCCHN) and we genotyped for RAD51 135G>C and 172G>T and P53 Arg72Pro SNPs in 716 SCCHN patients and 719 matched controls (all non-Hispanic whites) and evaluated their effects on gamma radiation-induced mutagen sensitivity. We found that RAD51 172TT homozygotes had a significantly decreased risk [adjusted odds ratio (OR) = 0.66, 95% confidence interval (CI) = 0.50-0.87] of SCCHN, compared with carriers of other genotypes, particularly in P53 Arg72Arg homozygotes (adjusted OR = 0.60, 95% CI = 0.41-0.89) (homogeneity test P = 0.047), although no alterations in the risk were associated with the RAD51 135G>C and P53 Arg72Pro SNPs. Consistent with a protective effect of the 172TT genotype, significantly fewer gamma radiation-induced chromatid breaks per cell were present in 172TT homozygotes (mean +/- SD = 0.36 +/- 0.13) than in subjects with other genotypes (mean +/- SD = 0.46 +/- 0.13, P < 0.001) among 148 control subjects we tested. The finding that the functional RAD51 172G>T SNP, particularly in the presence of the P53 Arg72Arg genotype, may be a marker of susceptibility to SCCHN needs to be validated by larger studies of different ethnic populations.     

The Induction of Enhanced Antitumor Effect against a Nonimmunogenic Tumor by Highly Immunogenic Variants Obtained by Mutagen Treatment

Nonimmunogenic 1767-3 fibrosarcoma was treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine, and stable variant cell clones (M-clones) were obtained that were able to elicit an immunological rejection response in syngenic C3H mice. Mice immunized with some M-clones were protected against a challenge from the original nonimmunogenic fibrosarcoma. Furthermore, when spleen cells of immunized syngenic mice were restimulated in vitro with M-clones, cytotoxic T lymphocytes (CTL) were obtained that were able to lyse not only M-clones but also the original nonimmunogenic tumor. These in vivo and in vitro results demonstrate the immunogenicity of M-clones and the existence of a singular antigenic specificity between the original nonimmunogenic tumor and M-clones. For the purpose of application of this mutagen treatment to cancer therapy, we combined it with lymphokine-activated killer (LAK) adoptive immunotherapy (AIT). With interleukin 2 and in vitro stimulation with highly immunogenic variant clones, we tried to induce transfer cells that had not only nonspecific LAK cells but also CTL with specific immunity against the original nonimmunogenic tumor. Successful results were obtained in the LAK AIT models. These findings indicate that an immunotherapy of human cancers that are thought to be weakly or nonimmunogenic may be possible by the application of this approach to LAK AIT.     

Methods for measuring mutagenicity in urine of rats dosed with [14C]di(2-ethylhexyl)phthalate

Di(2-ethylhexyl)phthalate (DEHP) is extensively used as a plasticizer for vinyl plastic articles. It has been found to be positive in an NCI rodent bioassay but has generally given negative results in in vitro genotoxicity tests. We therefore decided to test the urine of rats fed [14C]DEHP for mutagenic activity in the Ames Salmonella test. The recovery of radioactivity from the urine of rats dosed with [14C]DEHP was examined by solvent extraction and XAD-2 resin absorption procedures. Both of these procedures were inadequate for quantitative recovery of urinary metabolites required for subsequent mutagenicity testing using the Ames Salmonella/microsome procedure. Recoveries of less than 5% were observed using standard solvent extraction techniques whereas the XAD-2 adsorption technique gave about 67% at high resin/urine ratios. Treatment of the urine with beta-glucuronidase/aryl sulfatase did not affect these recoveries. The direct urine plating procedure represents a viable alternative to the above concentration procedures for this phthalate ester. The effects of L-histidine and the beta-glucuronidase/aryl sulfatase preparation on the background reversion frequencies of the Ames tester strains is discussed.     

The road to linearity: why linearity at low doses became the basis for carcinogen risk assessment

This article assesses the historical foundations of how linearity at low dose became accepted by the scientific/regulatory communities. While the threshold model was used in the 1920s/1930s in establishing radiation health standards, its foundations were challenged by the genetics community who argued that radiation induced mutations in reproductive cells followed a linear response, were cumulative and deleterious. Scientific foundations of linearity for gonadal mutations were based on non-conclusive evidence as well as not being conducted at low doses. Following years of debate, leaders in the genetics community participated in the U.S. National Academy of Sciences (NAS) (1956) Biological Effects of Atomic Radiation (BEAR) BEAR I Committee, getting their perspectives accepted, incorporating linearity for radiation-induced mutational effects in risk assessment. Overtime the concept of linearity was generalized to include somatic effects induced by radiation based on a protectionist philosophy. This affected the course of radiation-induced and later chemically-induced carcinogen risk assessment. Acceptance of linearity at low dose from chemical carcinogens was strongly influenced by the NAS Safe Drinking Water Committee report of 1977 which provided the critical guidance to the U.S. EPA to adopt linear at low dose modeling for risk assessment for chemical carcinogens with little supportive data, much of which has been either discredited or seriously weakened over the past 3 decades. Nonetheless, there has been little practical change of regulatory policy concerning carcinogen risk assessment. These observations suggest that while scientific disciplines are self correcting, that regulatory ‘science’ fails to display the same self-correcting mechanism despite contradictory data.     

阻遏诱变剂镉在海洋生物中传递的物质及作用机理的研究（Ⅰ）褐藻酸钠

镉是海洋中重要的诱变剂，海洋生物体内的镉随食物链流动最后进入人体，本文首次报道了海藻多糖褐藻酸钠不仅能将进入中国对虾体内起致毒作用的镉从体内排至体外，从而降低了肠道对镉的吸收，而且还能在一定程度上降低镉在机体内的遗传毒性，遏制镉的遗传毒性作用。上述结果为化学和生物二方面平行实验所证实。     

History of the science of mutagenesis from a personal perspective

A career in the study of mutagenesis spanning 50 years is a gift few scientists have been bestowed. My tenure in the field started in 1953, the year the structure of DNA became known (Watson and Crick [1953]: Nature 171:737). Before that time, it was suspected that DNA was the genetic material based on the research of Oswald T. Avery (Avery et al. [1944]: J Exp Med 79:137), but many scientists still believed that proteins or polysaccharides could be the genetic material. The present article describes a lifetime of personal experience in the field of chemical mutagenesis. The methods used to treat viruses with chemical mutagens were well developed in the 1950s. Here I review the early use of nitrous acid and hydroxylamine as mutagens in eukaryotes, the development of methods for the metabolic activation of mutagens by microsomal preparations, and the selection of a mutant tester set for the qualitative characterization of the mutagenic activity of chemicals. These studies provided critical background information that was used by Bruce Ames in the development of his Salmonella/microsome assay, widely known as the Ames test (Ames et al. [1973]: Proc Nat Acad Sci USA 70:2281-2285). This article also describes how a set of diagnostic chemical mutagens was selected and used to identify the molecular nature of gene mutations. Today, DNA sequencing has replaced the use of diagnostic mutagens, but studies of this kind formed the foundation of modern mutation research. They also helped set the stage for the organization of the Environmental Mutagen Society and the Environmental Mutagen Information Center, which are described. The article ends with the development of mammalian single-cell mutation assays, the first system for studying in vivo mutagenesis using recoverable vectors in transgenic animals, other mutation assays in intact mammals, and my thoughts on the critically important area of germ cell mutagenesis. This narrative is not a complete autobiographical account, in that I have selected only those experiences that I feel are important for the history of the field and the edification of today's students. I hope I have shown that science not only is a valuable pursuit but can also be fun, stimulating, and satisfying. A good sense of humor and the knowledge that many discoveries come by serendipity are essential.     

Induction and isolation of mutants in fungi at low mutagen doses

Summary Since the yield of mutants per surviving cell increases in general with increasing dose of mutagen, it has often been concluded in the literature that it is the most efficient to apply high mutagen doses so that most spores are killed. As high doses of mutagen produce chromosome rearrangements and unnoticed mutations which disturb the genetic background, the relationship between mutant frequency and survival was analyzed with Aspergillus nidulans as a model. It is shown that for different types of mutants the highest mutant yield is obtained at low mutagen doses (20–50% survival). Mutant frequency increases with increasing dose of mutagen but levels off and even decreases at higher dosages. There is no simple linear relationship between mutant frequency and the logarithm of the mutagen dose or the logarithm of the surviving fraction. If appropriate enrichment procedures are also available auxotrophic mutants can best be isolated at low doses of mutagen. Taking into account the disturbance of the genetic background, mutation induction should be done preferentially at a survival level of at least 70%.     

吖啶诱变剂ICR-170致弱日本血吸虫尾蚴诱导的保护性免疫效应观察

为了观察吖啶诱变无产卵能力的血吸虫是否能诱生抗感染的保护性免疫力,用 １０ μｇ／ｍ ｌ吖啶诱变剂 Ｉ Ｃ Ｒ１７０ 致弱日本血吸虫尾蚴作免疫原,免疫 Ｃ５７ Ｂ Ｌ／６ Ｎ 小鼠两次,分别于初次免疫后６、８、１０ ｗ ｋ 用正常尾蚴作攻击感染。结果免疫鼠成虫减少率为６８．９％ ,肝组织虫卵减少率为７４．９％ 。动态观察显示,初次免疫后６ ｗ ｋ 攻击感染的减虫率最高,减卵率高峰在６～８ ｗ ｋ。表明诱变剂 Ｉ Ｃ Ｒ１７０ 致弱日本血吸虫尾蚴发育的变异成虫具有较高的免疫原性,可诱导较高的抗攻击感染的保护性免疫力。