Toxic and mutagenic effects of carcinogens on the mitochondria of Saccharomyces cerevisiae.

Summary Nineteen haploid yeast (Saccharomyces cerevisiae) strains were used to assess the relative growth inhibitory potencies on fermentable vs. non-fermentable media of a collection of carcinogenic and noncarcinogenic chemicals. The majority of carcinogens were distinctly more potent on the non-fermentable (glycerol) medium, where mitochondrial function is required for growth, than on the fermentable medium, where it is not. The anti-mitochondrial selectivity indicated by these growth tests was much slighter for the non-carcinogens. Similarly most carcinogens induced the cytoplasmic petite mutation whereas the non-carcinogens did not.Five carcinogens which were tested impaired the development of cytochromes aa ₃ and b in glucose cultures.Six carcinogens, when tested, inhibited growth on three fermentable sugars, the utilisation of which requires mitochondrial function.Out of five carcinogens which were examined, four suppressed the surface-dependent phenomenon of flocculence in a flocculating strain of yeast, at concentrations primarily affecting the mitochondrial system; the fifth had a similar but less pronounced effect.     

NMR relaxation study of water protons in Syrian hamster fetal cells at 300 MHz

TheT ₁ andT ₂ relaxation times of water protons in two cell types in culture derived from Syrian hamster fetuses (normal primary or secondary fetal cells vs BP6T tumor cells derived from the normal cells transformed by carcinogens) were measured at 7.05 Tesla magnetic field (proton frequency =300 MHz). TheT ₁/T ₂ ratios and the correlation time, τ _c , calculated from theT ₁/T ₂ ratio of cellular water protons, are significantly different in these two fibroblastic cell types of the same biological origin and with similar morphologies and growth rates in culture.     

Sister-chromatid exchange induction by carcinogens in HTC cells An in vitro system which does not require addition of activating factors

The hepatic tumor cell line (HTC) was tested for the ability to produce sister chromatid exchanges (SCEs) in response to chemical carcinogens which require activation. Without the addition of exogenous microsomal enzyme preparations, cyclophosphamide, N-nitrosodiethylamine (DEN) and aflatoxin B₁ (AFB₁) induced significant levels of SCEs in these cells. Mitomycin C (MMC) and ultraviolet light, which do not require activation, also produced significant levels of SCEs. The induction of SCEs in HTC cells by AFB₁ was shown to be inhibited by estradiol, a known inhibitor of microsomal activating enzymes. For the carcinogens tested, the HTC cell SCE assay was quite sensitive and comparable to other mammalian test systems. Exceptional sensitivity was found in the case of AFB₁. SCE analysis of HTC cells offers a simplified system of detecting carcinogens requiring activation. This system also has the potential of investigating interactions between agents such as steroid hormones and carcinogens.     

Effects of incubation in an atmosphere of 20% CO2 in air on the syrian hamster embryo clonal transformation assay

Summary An atmosphere containing 10% CO₂ has been generally accepted as optimal for the growth of Syrian hamster embryo cells in a clonal transformation assay. Data presented in this paper show that 10% CO₂ may not be the optimum environment for this assay. Using 10 or 20% (analytically measured) CO₂ in air (1 atm pressure), hamster embryo cell pools were examined for clonal growth characteristics and transformability using five known carcinogens and a single noncarcinogenic compound. At 10% CO₂, only 2 of 11 pools weee transformed by the five carcinogens but not by the noncarcinogen. At 20% CO₂, six of seven pools were transformed by the five carcinogens and not by the noncarcinogen. Further, the transformation frequencies were found to be greater in cultures incubated in an atmosphere consisting of 20% CO₂ in air. The data also show that 20% CO₂ increased the cloning efficiency of these cells. A comparison of the 10 and 20% CO₂ data to results reported from other conflicting interlaboratory results with this assay system may be due, in part, to variations of CO₂ concentrations. In some instances, the CO₂ levels indicated by incubator flow meters vary considerably from analytically determined CO₂ values. To prevent these CO₂ discrepancies and their resultant effects on transformation and cloning efficiency, methods for monitoring the CO₂ environment other than flow meters are recommended. The observation of increased cloning efficiencies and transformation rates strongly suggests that culture incubation at 20% CO₂ is a preferred environment for the conduct of this assay.     

Use of four new human-derived liver-cell lines for the detection of genotoxic compounds in the single-cell gel electrophoresis (SCGE) assay

One of the main problems of in vitro genotoxicity assays is that the lack of adequate representation of drug-metabolising enzymes in indicator cell lines that are currently used in routine testing may lead to false results. In the present study, we investigated the ability of four new human-derived livercell lines to detect the DNA-damaging effects of representatives of different classes of genotoxic carcinogens that require metabolic activation, namely the nitrosamine N-nitrosodimethylamine (NDMA), the heterocyclic aromatic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), the polycyclic aromatic hydrocarbon benzo(a)pyrene (B(a)P) and the mycotoxin aflatoxin B₁ (AFB₁). Hydrogen peroxide (H₂O₂) was used in all experimental series as a positive control and parallel experiments were carried out with human HepG2 cells, which have been used in earlier studies. DNA damage was monitored in single-cell gel electrophoresis (SCGE) assays. Furthermore, RT-PCR experiments were carried out to study the expression of genes encoding for a panel of different phase-I and phase-II enzymes, which are involved in the activation/detoxification of genotoxic carcinogens. With one of the newly isolated hepatocellular lines, HCC1.2, positive results were obtained with all model compounds, two other new lines (HCC2 and HCC3), HepG2 and the virally immortalized line NKNT-3 were less sensitive and/or failed to detect some of the genotoxins. PCR analyses showed that all cell lines express genes coding for a variety of xenobiotic drug-metabolising enzymes. The highest levels were found in general in HCC1.2, while in NKNT-3 cells some genes were not transcribed. Overall, our results indicate that the line HCC1.2 may be useful for the development of improved in vitro genotoxicity test systems.     

Effects of selenium on chemical carcinogenesis

Chemical carcinogenesis can be characterized by a sequence of events leading to the development of tumors. Selenium (Se) inhibition of colon, liver, and lung carcinogens is demonstrated. Using the male Sprague Dawley rat model Se inhibited the colon tumor incidence in 1,2-dimethylhydrazine (DMH) treated rats and reduced the total number of colon tumors in methylazoxymethanol (MAM) treated rats. Selenium inhibited 2-acetylaminofluorene (AAF) and 3′-methyl-4-dimethylaminoazobenzene (3′-MeDAB) hepatocarcinogenesis. The hepatic tumor incidence induced by 3′-MeDAB was reduced by both inorganic Se (Na₂SeO₃) and by organic Se (Se-yeast) supplements. In vitro systems have been studied in an effort to decipher the inhibitory properties of Se on the multistage origin of tumors induced by chemical carcinogens. Current studies suggest that the protective effect of Se against AAF hepatocarcinogenesis may be correlated with a change in AAF metabolism. The mutagenicity of AAF and AAF metabolites inSalmonella typhimurium TA1538 is decreased by Se. Additionally, Se reduced N-t-OH−AAF induction of sister chromatid exchange (SCE) frequencies in whole blood cultures, and also reduced aryl hydrocarbon hydroxylase activity using benzo(a) pyrene as substrate. The comparative effects of antioxidants on DMH induction of colon tumors are presented in detail. Supplements of 4 ppm Se to the drinking water, 1.2% ascorbic acid (V _c ) to the diet or 0.5% butylated hydroxytoluene (BHT) to the diet of DMH-treated rats reduced the colon tumor incidence of DMH controls from 64 to 31% (Se), 38% (V _c ), and 43% (BHT). The colon tumor incidence in DMH-treated rats receiving a combination of Se+V _c increased to 83%, while the combination of Se+BHT decreased the colon tumor incidence to 55%. The growth and survival of rats provided long-term supplements of 4 ppm Se in the drinking water are compared with untreated controls.     

Regional mapping of short tandem repeats on humanchromosome 10: Cytochrome P450 gene CYP2E, D10S196, D10S220, and D10S225

Human CYP2E encodes an ethanol-inducible cytochrome P450 monooxygenase that metabolizes various carcinogens and may therefore play a role in cancer susceptibility. An intronic (GGAT)_n · (CCTA)_n repeat element was found to display limited polymorphism in Caucasoids and was used as a sequence-tagged site for genomic amplification from somatic cell hybrids to localize CYP2E to 10q24.3-qter; using the same panel, three microsatellite markers, D10S196, D10S220, and D10S225, were mapped to 10q21. The close synteny of CYP2E, CYP2C, and CYP17 belonging to two different cytochrome P450 families suggests a central role for the long arm of chromosome 10 in the evolution of this large gene superfamily.     

Transgenic tumor models for carcinogen identification: the heterozygous Trp53-deficient and RasH2 mouse lines

Genetically altered mouse models (GAMM) for human cancers have been critical to the investigation and characterization of oncogene and tumor suppressor gene expression and function and the associated cancer phenotype. Similarly, several of the mouse models with defined genetic alterations have shown promise for identification of potential human carcinogens and investigation of mechanisms of carcinogen–gene interactions and tumorigenesis. In particular, both the B6.129N5-Trp53 mouse, heterozygous for a p53 null allele, and the CB6F₁-RasH2 mouse, hemizygous for the human H-ras transgene, have been extensively investigated. Using 26-week exposure protocols at or approaching the maximum tolerated dose, the summary results to date indicate the potential for GAMM to identify and, possibly, classify chemicals of potential risk to humans using short-term carcinogenicity experiments. This IWGT session focused on: (1) the development of recommendations for genetic/molecular characterization required in animals, tissues, and tumors before and after treatment for identification of presumptive human carcinogens based on the current state of knowledge, (2) identification of data gaps in our current state of knowledge, and (3) development of recommendations for research strategies for further development of our knowledge base of these particular models. By optimization of protocols and identification of significant outcomes and responses to chemical exposure in appropriate short-term mechanism-based genetically altered rodent models, strategies for prevention and intervention may be developed and employed to the benefit of public health.     

Helical polysomes induced by aflatoxin B1 in vivo : A new hypothesis for helix formation by chemicals and carcinogens

We have studied the induction of helical polysomes by aflatoxin B₁ in liver and kidney cells from rat and mouse. We succeeded in giving to reticulocyte polysomes a shape resembling helices after in vitro treatment with O-methylthreonine which is used as an inhibitor of polypeptide chain termination. From this and knowing the site of action of aflatoxin B₁ on rat liver polysomes, we hypothesize that the induction of helical polysomes in tissues from adult animals treated by chemicals or carcinogens is due to the inhibition of release of ribosomes from the messenger RNA (mRNA). Theoretical studies of protein synthesis inhibition are in agreement with this new hypothesis.     

Evaluation of different genotypes of nontoxigenic Aspergillus flavus for their ability to reduce aflatoxin contamination in peanuts

Aflatoxins produced by the fungus Aspergillus flavus are potent carcinogens and account for large monetary losses worldwide in peanuts, maize, and cottonseed. Biological control in which a nontoxigenic strain of A. flavus is applied to crops at high concentrations effectively reduces aflatoxins through competition with native aflatoxigenic populations. In this study, eight nontoxigenic strains of A. flavus belonging to different vegetative compatibility groups and differing in deletion patterns within the aflatoxin gene cluster were evaluated for their ability to reduce aflatoxin B₁ when paired with eight aflatoxigenic strains on individual peanut seeds. Inoculation of wounded viable peanut seeds with conidia demonstrated that nontoxigenic strains differed in their ability to reduce aflatoxin B₁. Reductions in aflatoxin B₁ often exceeded expected reductions based on a 50:50 mixture of the two A. flavus strains, although one nontoxigenic strain significantly increased aflatoxin B₁ when paired with an aflatoxigenic strain. Therefore, nontoxigenicity alone is insufficient for selecting a biocontrol agent and it is also necessary to test the effectiveness of a nontoxigenic strain against a variety of aflatoxigenic strains.     

Horseradish peroxidase for the removal of carcinogenic aromatic amines from water

A new method has been developed for the removal of carcinogenic aromatic amines from industrial aqueous effluents. It includes the treatment of aqueous solutions containing the carcinogens with horseradish peroxidase and hydrogen peroxide. Such treatment results in a nearly complete precipitation of carcinogenic aromatic amines from water due to enzymatic crosslinking. This method was used to remove ten recognized human carcinogens from water: benzidine and its derivatives, naphthylamines, 4-aminobiphenyl, and p-phenylazoaniline. The dependence of the removal efficiency of the peroxidase treatment on the concentrations of the enzyme, H₂O₂ and a carcinogen and also on pH and the duration of the treatment was studied. The enzymatic removal of carcinogens from water was confirmed by both chemical and toxicological assays.     

A comparison of the DNA binding,cytotoxicity and repair synthesis induced in human fibroblasts by reactive derivatives of aromatic amide carcinogens

The cytotoxicity of three structurally-related direct-acting carcinogens, N-acetoxy-2-acetylaminofluorene, N-acetoxy-2-acetylaminophenanthrene and N-acetoxy-4-acetylaminobiphenyl, was compared in normal cells and in excision repair deficient xeroderma pigmentosum cells (XP12BE). All three proved significantly more cytotoxic to the XP cells than to the normal cells. At equicytoxic levels, substantially more residues were initially bound to the DNA of the normal cells than to the XP cells, suggesting that the former are able to remove a large percentage of the DNA bound residues before these can result in cell death. The ability of these cell strains to remove bound residues from DNA, to incorporate thymidine into parental strands of DNA during repair replication, and to recover from potentially lethal damage if held in the non-replicating, density-inhibited G_o state was compared as a function of dose and time. The XP12BE cells proved virtually incapable of excision repair of DNA damage induced by these carcinogens and of recovery. In contrast, normal cells recovered from the potentially lethal effects of these three compounds and did so at a rate comparable to their rate of removal of bound residues and of repair synthesis. In the excision-deficient XP12BE cells, DNA adducts induced by N-acetoxy-2-acetylaminophenanthrene proved 3- to 6-fold more cytotoxic than adducts induced by the other two carcinogens.     

Neoplastic transformation of BALB/3T3 cells by metals and the quest for induction of a metastatic phenotype

In the mouse embryo cell line BALB/3T3 Clone A31-1-1, dose-dependent morphologic neoplastic transformation was obtained with NaAsO₂, Na₂HAsO₄, CdCl₂, and K₂CrO₄. Cellular uptake was four fold higher for As³⁺ than for As⁵⁺, and As⁵⁺ was metabolized to As³⁺ in cytosol. Cytotoxicity and transformation rates were four fold higher for As³⁺ than As⁵⁺, but when correlated to cellular As burden they were equivalent. As³⁺ appears responsible for the transforming activity. The foci transformed by metals (or by other carcinogens) gave rise to tumorigenic cell lines (sc sarcomas in nude mice), none of which, however, induced metastases when tested by sc or by iv injection in nude mice. Thus carcinogens change this aneuploid cell line from a preneoplastic stage to the expression of malignant growth but not of metastatic activity. Metastatic and type IV collagenolytic activities can be induced by transfection of the c-Ha-ras oncogene and inhibited by the Ad2-E1a gene (so far shown in other cell types). It remains to be seen whether metal or other carcinogens can induce the nonmetastatic phenotype to become metastatic. The molecular mechanisms of metal carcinogenesis, studied in cell culture systems, in combination with other factors or oncogenes, may reveal the effect of individual metal carcinogens on discrete steps of the complex process of carcinogenesis.     

Induction of tumor clones in D. melanogaster wts/+ heterozygotes with chemical carcinogens

Ten chemicals were assessed for blastomogenic activity in adult wts/+ heterozygotes of D. melanogaster. All of the strong mammalian carcinogens tested (benzo(a)pyrene (B(a)P), pyrene, aflatoxin B₁, 2-acetylaminofluorene (2-AAF) and cis-dichlorodihydroxydiamminoplatinum IV) were also shown to be strong Drosophila blastomogens. They induced several times more tumors than their counterparts that are less carcinogenic for mammals (4-acetylaminofluorene (4-AAF), aflatoxins B₂ and G₂) and 4-(methylnitrosamino)-1-(-3-pyridine)-1-butanone (NNK). Benzo(e)pyrene (B(e)P) and pyrene demonstrated minor effects. Most tumors were localized on the wing and notum, which are the derivatives of the wing disc. Humeri derived from dorsal prothoracic disc and the abdominal tergites and sternites had the lowest number of tumors. The tumor frequency in the cross of the wild type females with wts^P2/TM6B males was different from that in the reciprocal cross. The former type of cross exhibited consistently higher tumor frequency both in the experimental and control series.     

In vivo Comet assay on isolated kidney cells to distinguish genotoxic carcinogens from epigenetic carcinogens or cytotoxic compounds

The objective of this study was to determine the ability of the alkaline in vivo Comet assay (pH > 13) to distinguish genotoxic carcinogens from epigenetic carcinogens when performed on freshly isolated kidney cells and to determine the possible interference of cytotoxicity by assessing DNA damage induced by renal genotoxic, epigenetic or toxic compounds after enzymatic isolation of kidney cells from OFA Sprague–Dawley male rats. The ability of the Comet assay to distinguish (1) genotoxicity versus cytotoxicity and (2) genotoxic versus non-genotoxic (epigenetic) carcinogens, was thus investigated by studying five known genotoxic renal carcinogens acting through diverse mechanisms of action, i.e. streptozotocin, aristolochic acids, 2-nitroanisole, potassium bromate and cisplatin, two rodent renal epigenetic carcinogens: d-limonene and ciclosporine and two nephrotoxic compounds: streptomycin and indomethacin. Animals were treated once with the test compound by the appropriate route of administration and genotoxic effects were measured at the two sampling times of 3–6 and 22–26 h after treatment. Regarding the tissue processing, the limited background level of DNA migration observed in the negative control groups throughout all experiments demonstrated that the enzymatic isolation method implemented in the current study is appropriate. On the other hand, streptozotocin, 20 mg/kg, used as positive reference control concurrently to each assay, caused a clear increase in the mean Olive Tail Moment median value, which allows validating the current methodology.Under these experimental conditions, the in vivo rodent Comet assay demonstrated good sensitivity and good specificity: all the five renal genotoxic carcinogens were clearly detected in at least one expression period either directly or indirectly, as in the case of cisplatin: for this cross-linking agent, the significant decrease in DNA migration observed under standard electrophoresis conditions was clearly amplified when the duration of electrophoresis was increased up to 40 min. In contrast, epigenetic and nephrotoxic compounds failed to induce any signifcant increase in DNA migration. In conclusion, the in vivo rodent Comet assay performed on isolated kidney cells could be used as a tool to investigate the genotoxic potential of a test compound if neoplasic/preneoplasic changes occur after subchronic or chronic treatments, in order to determine the role of genotoxicity in tumor induction. Moreover, the epigenetic carcinogens and cytotoxic compounds displayed clearly negative responses in this study. These results allow excluding a DNA direct-acting mechanism of action and can thus suggest that a threshold exists. Therefore, the current in vivo rodent Comet assay could contribute to elucidate an epigenetic mechanism and thus, to undertake a risk assessment associated with human use, depending on the exposure level.     

Spin trapping and its application in the study of lipid peroxidation and free radical production with liver microsomes.

Lipid peroxidation in microsomes was studied using a spin-trapping technique. Free radical adducts of phenyltertiarybutylnitrone (PBN) were produced as detected by electron spin resonance during induced lipid peroxidation of microsomes with a system consisting of NADPH, Fe²⁺, and pyrophosphate. The adducts were identified as intermediates of the substrates added to the microsomal system and not OH · or HO₂ radicals. The production of the adduct parallels the NADPH-dependent formation of malondialdehyde (MDA). Analyses of the electron spin resonance hyperfine splitting constants allowed in some instances identification of the adducts. Purified preparations of cytochrome P-450 mimic the results of the microsomes. The carcinogens dimethyl and diethylnitrosoamine were metabolized in this system yelding reactive free radicals and free NO, suggesting an alternate mechanism for the activity of these compounds as ultimate carcinogens.     

Phytuberol,from Japanese Domestic Tobacco,Nicotiana tabacum cv. Suifu

Carcinogenesis is believed to be induced through the oxidative damage of DNA, and antioxidants are expected to suppress it. So, the polyphenolic antioxidants in daily foods were investigated to see whether they protect against genetic damage by active oxygen. In the evaluation, we used a bioassay and a chemical determination, a Salmonella mutagenicity test for mutation by a N-hydroxyl radical from one of the dietary carcinogens 3-amino-1-methyl-5H-pyrido[4,3-b]indole and the formation of 8-hydroxyl (8-OHdG) from 2′-deoxyguanosine (2′-dG) in a Fenton OH-radical generating system. Thirty-one antioxidants including flavonoids were compared in terms of radical-trapping activity with bacterial DNA and 2′-dG. Antioxidants inhibited the mutation but the IC₅₀ values were in the mM order. Against 8-OHdG formation, only α-tocopherol had a suppressive effect with an IC₅₀ of 1.5 μM. Thus, except α-tocopherol, the dietary antioxidants did not scavenge the biological radicals faster than bacterial DNA and intact 2′-dG, indicating that they failed to prevent oxidative gene damage and probably carcinogenesis.     

The detection of carcinogen-induced sperm head abnormalities in mice

The (CBA × BALB/c)F₁ male mouse is sensitive to the induction of sperm-head abnormalities after exposure to a range of chemical mutagens and carcinogens. 8 carcinogens including ethionine and diethyl stilboestrol were correctly identified. 23 non-carcinogens and compounds of unknown carcinogenicity including a range of 13 substituted anilines and methionine did not induce sperm-head abnormalities. 4-Aminophenol induced an increase in sperm-head abnormalities.The utility of the procedure for identifying genotoxic compounds is discussed.     

Evaluation of a DNA polymerase-deficient mutant of E. coli for the rapid detection of carcinogens

Differential growth inhibition of two E. coli cultures was evaluated as a rapid screening technique for chemical carcinogens. Of the carcinogens tested, only “direct acting” carcinogens produced positive results. Furthermore, this test is not a quantitative assay in that neither was a dose—response relationship seen nor did potent carcinogens necessarily show a greater response than weaker carcinogens.Most of the carcinogens tested are considered to require metabolic activation in order to exert their carcinogenic action. Despite many attempts, including several variations of reaction conditions, metabolic activation by rat liver fractions was not apparent. Many of these carcinogens are insoluble in water and may not diffuse through the agar and therefore not reach the indicator organism.A number of chemicals that are not carcinogenic produced positive results with this assay. Many of these substances are oxidants or oxidation products which are highly reactive with DNA as well as with other cellular constituents. Therefore, it is possible that the toxicity exhibited by these chemicals was caused by a reaction with some essential cellular constituent other than DNA and such damage would not be repairable by DNA polymerase. These observations limit the usefulness of the P3478 E. coli technique in its present form as a prescreen for chemical carcinogens.     

Genetic metabolic polymorphisms and the risk of cancer: a review of the literature

Abstract

The purpose of this paper is to systematically analyse the design and results of epidemiological studies on the association between various types of cancer (lung, bladder, breast, colon, stomach) and four genetically-based metabolic polymorphisms, involved in the metabolism of several carcinogens (glutathione-S-transferase M1, debrisoquine hydroxylase, N acetyltransferase, aryl hydrocarbon hydroxylase). These inherited polymorphisms usually cause modifications in the quality or quantity of the relevant enzymes. Such enzymes are involved in the activation/inactivation of known carcinogens and seem to modify the extent to which carcinogens interact with DNA in target tissues. Two enzymes, debrisoquine hydroxylase and aryl hydrocarbon hydroxylase, activate procarcinogens to carcinogens (phase I enzymes). The other two, glutathione-S-transferase M1 and N-acetyltransferase, mainly detoxity carcinogenic substances (phase II enzymes). Because of their role as host factors (modulating the action of carcinogens), it has been hypothesized that subjects presenting a specific phenotype for such polymorphisms could be at a greater risk of developing various types of cancer. A number of epidemiological studies have investigated such associations, often with discordant results. We examine and discuss the design of the studies, and present a meta-analysis of the available data.