Richard J. Brennan

Cadmium is an inducer of oxidative stress in yeast

The heavy metal cadmium is a carcinogen in long-term rodent studies and is a suspect human carcinogen. Cadmium scores negative in the Ames Salmonella mutagenicity assay and in most other short-term genotoxicity assays, but induces deletions in the yeast Saccharomyces cerevisiae. We have investigated whether cadmium induces an oxidative stress in S. cerevisiae which may be responsible for its recombinagenic activity. The free radical scavenger N-acetylcysteine blocked toxicity and recombination induced in S. cerevisiae by cadmium. Yeast strains deficient in the antioxidant defense enzymes superoxide dismutase or gamma-glutamylcysteine synthetase were hypersensitive to cadmium toxicity. Cells grown in the absence of oxygen were more resistant to cadmium. An intracellular free radical-sensitive reporter compound was activated in S. cerevisiae exposed to cadmium. Toxicity or recombination induced by the mutagenic carcinogen methyl methanesulfonate were unaffected in any of the above experiments. These results suggest that the toxicity and recombinagenic activity of cadmium in S. cerevisiae is caused by oxidative stress.

Oxidative mutagens induce intrachromosomal recombination in yeast

Active oxygen species are thought to be involved in the causation of a number of diseases including cancers. We have investigated the effect of 5 oxidative mutagens, methyl viologen (paraquat), mitomycin C, phenylhydrazine, cumene hydroperoxide and hydrogen peroxide, on the frequency of both intrachromosomal recombination and interchromosomal recombination in the yeast Saccharomyces cerevisiae. All of the chemicals significantly increased the frequency of intrachromosomal recombination in a dose-dependent manner. Only hydrogen peroxide increased the frequency of interchromosomal recombination at the doses tested in this study. A role for hydroxyl radical (.OH) in the effect of H2O2 on recombination is indicated by the ability of the radical scavenger dimethyl sulfoxide (DMSO) to significantly inhibit the induction of both intrachromosomal and interchromosomal recombination by H2O2. The results presented here give further support for the suitability of intrachromosomal recombination measurements as a short-term test for the detection of mutagens and carcinogens.

Free radicals generated in yeast by the Salmonella test-negative carcinogens benzene, urethane, thiourea and auramine O

A large fraction of carcinogens score negative in short-term genotoxicity assays such as the Salmonella reverse mutation (Ames) assay. More information is needed about the mechanism of action of such Salmonella-negative carcinogens. Many Salmonella-negative carcinogens induce deletions due to intrachromosomal recombination in Saccharomyces cerevisiae with an apparent threshold. We have previously shown that the Salmonella-negative carcinogens cadmium, aniline, chloroform and carbon tetrachloride generate free radical species in S. cerevisiae. We have further investigated the possible generation of intracellular free radical species by the diverse Salmonella-negative carcinogens benzene, urethane, thiourea and auramine O. The toxicity and recombinagenicity of thiourea and auramine O was reduced in the presence of the free radical scavenger N-acetyl cysteine. N-acetyl cysteine did not protect against toxicity or recombination induced by the Salmonella-positive carcinogens ethyl methane sulfonate, methyl methane sulfonate or nitroquinoline-N-oxide. A strain deficient in the enzyme superoxide dismutase, which catalyses the dismutation of superoxide anion radical, was hypersensitive to killing by benzene, urethane and thiourea. The sod- strain was only slightly more sensitive to the Salmonella-positive carcinogens. Intracellular oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate was increased in yeast cultures exposed to benzene, urethane and auramine O; again, the Salmonella mutagens had no effect on oxidation of the dye. These data show that free radical species are produced in Saccharomyces cerevisiae following exposure to benzene, urethane, thiourea and auramine O, and suggest a possible role for oxidative stress is recombination induced by these carcinogens.

Chloroform and carbon tetrachloride induce intrachromosomal recombination and oxidative free radicals in Saccharomyces cerevisiae.

Chlorination of drinking water results in the generation of low levels of numerous chlorinated hydrocarbons due to the reaction of chlorine with naturally occurring organic compounds in the water. Concern has been raised about the safety of these chlorinated contaminants as several of them, most notably chloroform (trichloromethane), have been shown to be carcinogenic in long-term rodent bioassays and weak correlations between trihalomethane levels in drinking water and an increased risk of bladder and colorectal cancer in humans have been found. Chloroform and carbon tetrachloride induce liver cancer in rats and mice only at doses where significant hepatotoxicity is observed and have been classed as non-genotoxic carcinogens. We have investigated the ability of chloroform, carbon tetrachloride and 1,1,1-trichloroethane to induce deletions via intrachromosomal recombination in the yeast Saccharomyces cerevisiae. Chloroform and carbon tetrachloride induced this genotoxic recombination event at similar doses, 1,1,1-Trichloroethane gave only a weak response in the DEL recombination assay and only at the highest dose. We further show that chloroform and carbon tetrachloride, but not trichloroethane, induced oxidative free radical species in our yeast strain. The free radical scavenger N-acetylcysteine reduced chloroform-induced toxicity and recombination, and both chloroform and carbon tetrachloride were able to oxidize the free radical-sensitive reporter compound dichlorofluorescein diacetate in vivo. The implications of these findings to the carcinogenic activities of the three compounds are discussed.

Detecting Carcinogens With the Yeast DEL Assay

The yeast DEL assay is a simple, rapid method for measuring the frequency of reversion of a disrupted his3 gene by homologous intrachromosomal recombination. Reversion to histidine prototrophy results in deletion (DEL) of the disrupting sequence. The DEL assay has been used to study the effects of various DNA-damaging treatments on the frequency of deletion-recombination and has been shown to have a high level of sensitivity and specificity toward carcinogens, many of which are poorly detected by bacterial mutagenicity and other short-term genotoxicity assays. The DEL assay therefore is a useful addition to the arsenal of predictive tests for genotoxicity and carcinogenicity. This chapter provides an in-depth description of materials and methods for the yeast DEL assay from a users prospective and should allow the assay to be successfully deployed in any laboratory with basic microbiological capability and minimal user training.

The aromatic amine carcinogens o-toluidine and o-anisidine induce free radicals and intrachromosomal recombination in Saccharomyces cerevisiae.

Aniline-based aromatic amine carcinogens are poorly detected in short-term mutagenicity assays such as the Salmonella reverse mutation (Ames) assay. More information on the mechanism of toxicity of such Salmonella-negative carcinogens is needed. Aniline and o-toluidine are negative in the Ames assay, but induce deletions (DEL) due to intrachromosomal recombination in Saccharomyces cerevisiae with an apparent threshold. We show here that the DEL assay also detects the genotoxic activity of another aromatic amine carcinogen, o-anisidine, which is also negative in the Salmonella assay. We also show that the DEL assay distinguishes between o-anisidine and its non-carcinogenic structural analog 2, 4-dimethoxyaniline. We have investigated whether the ability of the DEL assay to detect the carcinogens and to distinguish between the carcinogen/non-carcinogen pair is linked to rises in intracellular free radical species following exposure to the carcinogens. Toxicity induced by all three compounds was reduced in the presence of the free radical scavenger and antioxidant N-acetyl cysteine, recombination induced by o-anisidine and o-toluidine was also reduced by N-acetyl cysteine. All three compounds induced oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate. Superoxide dismutase-deficient strains, however, were hypersensitive to cytotoxicity induced by o-toluidine and o-anisidine but not by the non-carcinogen 2,4-dimethoxyaniline, indicating a different potential for generating superoxide radical between the carcinogens and the non-carcinogen analog. The results indicate that the yeast DEL assay is a useful tool for investigating the genotoxic activity of aromatic amine carcinogens.

Persistent Genomic Instability in the Yeast Saccharomyces cerevisiae Induced by Ionizing Radiation and DNA-Damaging Agents

Abstract Brennan, R. J. and Schiestl, R. H. Persistent Genomic Instability in the Yeast Saccharomyces cerevisiae Induced by Ionizing Radiation and DNA-Damaging Agents. Radiat. Res. 155, 766–775 (2001). A “hypermutable” genome is a common characteristic of cancer cells, and it may contribute to the progressive accumulation of mutations required for the development of cancer. It has been reported that mammalian cells surviving exposure to γ radiation display several highly persistent genomic instability phenotypes which may reflect a hypermutability similar to that seen in cancer. These phenotypes include an increased mutation frequency and a decreased plating efficiency, and they continue to be observed many generations after the radiation exposure. The underlying causes of this genomic instability have not been fully determined. We show here that exposure to γ radiation and other DNA-damaging treatments induces a similar genomic instability in the yeast Saccharomyces cerevisiae. A dose-dependent increase in intrachromosomal recombination was observed in cultures derived from cells surviving γ irradiation as many as 50 generations after the exposure. Increased forward mutation frequencies and low colony-forming efficiencies were also observed. Persistently elevated recombination frequencies in haploid cells were dominant after these cells were mated to nonirradiated partners, and the elevated recombination phenotype was also observed after treatment with the DNA-damaging agents ultraviolet light, hydrogen peroxide, and ethyl methanesulfonate. Radiation-induced genomic instability in yeast may represent a convenient model for the hypermutability observed in cancer cells.

Saturated and monofluoro analogs of the oriental fruit fly attractant methyl eugenol show reduced genotoxic activities in yeast

Methyl eugenol, is a commercially used fruit fly attractant and a suspected carcinogen. Several phenylpropenes, including methyl eugenol and the known carcinogen safrole, score negative in the Salmonella assay but score positive in the yeast DEL assay that selects for intrachromosomal recombination events in the yeast Saccharomyces cerevisiae. In an attempt to dissociate the beneficial properties of methyl eugenol from its genotoxic properties, saturated or fluorinated analogs were evaluated for their ability to induce intrachromosomal (DEL) recombination in yeast. Field tests have previously shown that all of the analogs used have appreciable properties as fruit fly attractants. The analogs 1,2-dimethoxy-4-ethylbenzene, 1,2-dimethoxy-4-(2-fluoro-2-propenyl)benzene, 1,2-dimethoxy-4-(2-fluoroethyl)benzene and 1,2-dimethoxy-4-(3-fluoro-2-propenyl)benzene all showed reduced toxicity and reduced recombinagenicity in yeast compared to methyl eugenol. These results confirm the validity of fluorination and/or removal of the 2-propenyl moiety in reducing the toxicity and recombinagenicity of methyl eugenol derivatives.

Network and pathway analysis of compound-protein interactions.

We describe an integrated system that brings together predictive chemical analyses based on compound structure, knowledge bases of chemogenomics data associating compounds to biological, pharmacological and toxicological properties, and a systems biology functional data analysis and network reconstruction approach, to provide an in silico evaluation of the possible effects of xenobiotics on biological systems. We demonstrate the combination of drug and xenobiotic metabolism prediction, quantitative structure-activity relationship (QSAR) models and structural similarity searching to generate a list of similar compounds to, and possible targets for novel compounds. These lists of compounds and proteins are mapped to functional ontologies such as gene-disease associations, biological processes, and mechanisms of toxicity, and can be used to reconstruct biological networks linking together the component nodes into biologically-meaningful clusters. Thus, an assessment of biological effects can be made early in the discovery and development process that can be used to prioritize the best compounds for additional testing or development, or to direct efforts in medicinal chemistry to improve compound activity profiles.

Diaminotoluenes induce intrachromosomal recombination and free radicals in Saccharomyces cerevisiae

The carcinogenicity of aniline-based aromatic amines is poorly reflected by their activity in short-term mutagenicity assays such as the Salmonella typhimurium reverse mutation (Ames) assay. More information about the mechanism of action of such carcinogens is needed. Here we report the effects on DEL recombination in Saccharomyces cerevisiae of the carcinogen 2,4-diaminotoluene and its structural isomer 2,6-diaminotoluene, which is reported to be non-carcinogenic. Both compounds are detected as equally mutagenic in the Salmonella assay. In the absence of any external metabolizing system both compounds were recombinagenic in the DEL assay with the carcinogen being a more potent inducer of deletions than the non-carcinogen. In the presence of Aroclor-induced rat liver S9, however, the carcinogen 2,4-diaminotoluene became a 2-fold more potent inducer of deletions, and the non-carcinogen 2,6-diaminotoluene was rendered less toxic and no induced recombination was observed. 2,4-Diaminotoluene is distinguished from its non-carcinogen analog in the DEL assay, therefore, on the basis of a preferential activation of the carcinogen in the presence of a rat liver microsomal metabolizing system. Free radical species are produced by several carcinogens and have been implicated in carcinogenesis. We further investigated whether exposure of yeast to either 2,4-diaminotoluene or 2,6-diaminotoluene resulted in a rise in intracellular free radical species. The effects of the free radical scavenger N-acetylcysteine on toxicity and recombination induced by the two compounds and intracellular oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate were studied. Both 2,4- and 2,6-diaminotoluene produced tree radical species in yeast, indicating that the reason for the differential activity of the compounds for induced deletions is not reflected in any difference in the production of free radical species.