The Ty1 transposition assay: a new short-term test for detection of carcinogens

An assay based on induction by carcinogens of Ty1 transposition in Saccharomyces cerevisiae is proposed. A tester strain was developed that contains a marked Ty1 element, which allows following the transposition in the genome as a whole and a mutation, which increases cellular permeability. Hypersensitivity to chemical agents, higher cell wall porosity and transformability with plasmid DNA evidenced an enhanced cellular permeability of the tester cells. The increased permeability resulted in higher sensitivity to carcinogens. The treatment with different laboratory carcinogens induced Ty1 transposition rates in the tester strain by a factor of 10 to 20, compared to the controls. The induction is not stress-generated by the cytotoxicity of carcinogens, since treatment with NaN3 at concentrations killing 50% of the cells did not increase the transposition rate. The increase of Ty1 transposition in tester cells is specific for active carcinogens and a positive response with procarcinogens was obtained only in presence of S9 mix. The Ty1 transposition test responded positively to a number of Ames-test or DEL-test negative carcinogens. The positive response of Ty1 test was statistically significant and verified in kinetics and concentration-dependent experiments. It is concluded that the Ty1 transposition test can be used, in addition to the Ames assay, as a short-term test for detection of carcinogens.     

Bacterial systems for carcinogenicity testing

During the past 30 years, bacterial test systems have been extensively refined in their ability to detect not only mutagenic agents but, in many cases, carcinogenic ones as well. Since many carcinogens are known to be activated within the mammalian body, major improvements in bacterial test systems were made when representative parts of mammalian metabolism were included as part of the test protocol. Presently, systems of great simplicity and convenience are available for the efficient detection of gene mutations, lysogenic induction of prophages, and differential DNA repair. These qualities render bacterial systems potentially useful in distinguishing between carcinogens and non-carcinogens, in characterizing induced mutation spectra, and possibly in quantifying mutagenic potency that may be used to predict tumor-initiating potency. Sensitive strains of Salmonella typhimurium. Escherichia coli and Bacillus subtilis with altered DNA-repair capacities have been constructed which accurately identify many carcinogens. Comparative studies have shown that techniques using these strains can be standardized to some extent and that the majority of carcinogens are active in all adequately sensitive genetic systems. Because of this redundancy, it may be sufficient to employ only one standardized set of tester strains and methodology. However, serveral classes of known carcinogens are undetected or underestimated when assayed in standard testing procedures. Some of these chemicals can be efficiently recognized as mutagens upon varying the methodology, the genetic endpoint, or the mammalian activation system. Thus, to modify and adjust the experimental protocol to the particular type of chemical under study and to calibrate the system with appropriate carcinogenic and non-carcinogenic reference compounds is advisable. It is noteworthy that chemical carcinogens which probably act by non-genotoxic mechanisms thus far remain undetected in bacterial tests. Newly developed systems which measure specific types of genetic events, such as transpositions of DNA segments and derepression of genes, presently are being tested for their ability to detect such carcinogens. A final matter of growing concern is the increasing number of environmental chemicals that are found to be mutagenic in bacteria but for which information about carcinogenic activity in vivo is insufficient. The possible use of bacteria for quantifying mutagenic potency and extrapolating this information to tumor-initiating potency can be envisaged in three ways: (i) direct extrapolation from standard in vitro tests, (ii) indirect extrapolation making use of an in vitro/in vivo comparison of induced effects (the parallelogram method) as devised by Sobels [138] on the basis of identical dose (to DNA), and (iii) host-mediated assays to assess mutagenic potency of carcinogens in selected organs of mammals...     

Induction of genetic tandem duplications in Salmonella by polycyclic aromatic hydrocarbon and amine carcinogens

6 polycyclic aromatic hydrocarbon or similar amine carcinogens were tested as inducers of genetic tandem duplications in a rough strain of Salmonella typhimurium. When metabolically activated by rat-liver microsomes, all 6 were active in inducing genetic tandem duplications, yielding from over 3 times to almost 14 times as many tandem duplicants per viable bacterium as did concurrent uninduced control cultures. These results extend the number and chemical diversity of carcinogens shown to induce genetic duplications in bacterial tester systems. We suggest that polycyclic hydrocarbon carcinogens may act in carcinogenesis by inducing genetic duplications or other genetic rearrangements. Duplication induction may be a useful genetic endpoint for screening potential carcinogens.     

Evaluation of the Escherichia coli K12 inductest for detection of potential chemical carcinogens

46 chemicals of diverse classes and structures, including 30 known animal carcinogens, were evaluated for prophage-inducing ability using the Escherichia coli inductest with lysogenic strain GY5027 envA - uvrB- and indicator strain GY4015 ampR . The inductest detected 9 of 30 known carcinogens as genotoxic agents, including 3 polycyclic hydrocarbons, 2 aflatoxins, and 2 antitumor antimicrobials. Among the 21 carcinogens ineffective as prophage inducers were 3 aromatic amines (other than 2-aminoanthracene), 3 azo-aminoazo compounds, 2 methanesulfonates, and 2 nitro aromatics. In contrast, 18 and 17 of the 30 animal carcinogens were detected as genotoxic agents in the Salmonella/Ames test and E. coli WP2/ WP100 rec assay, respectively. The threshold sensitivity of the inductest was less than that of the Salmonella/Ames test for chemicals genotoxic in both tests. The ineffectiveness of the inductest as a routine test for detecting potential chemical carcinogens may be related to the nature of the DNA damage lesions formed by various genotoxic agents.     

Deoxyribonucleic Acid Repair in Bacillus subtilis: Development of Competent Cells into a Tester for Carcinogens

The development of competent transformed Bacillus subtilis into a tester system for carcinogens is described. Precocious or noninduced activation of SOS functions occur in competent cells. Thus, lower doses or concentrations of SOS inducing agents are needed to cause cell death due to indigenous prophage activation and lysis of bacteria. The two known defective prophages in B. subtilis enhance the sensitivity of competent cells to the carcinogens ultraviolet light, mitomycin C, and methyl methanesulfonate. However, these same cells have no enhanced sensitivity for the non-carcinogenic ethyl methanesulfonate or for nalidixic acid. Therefore, competent B. subtilis appear to be a sensitive tester for carcinogens.     

Evaluation of the new system (umu-test) for the detection of environmental mutagens and carcinogens

The umu operon in Escherichia coli is responsible for chemical and radiation mutagenesis, and the expression of the operon itself is inducible by these DNA-damaging agents. The principle of the umu-test is based on the ability of the DNA-damaging agents, most of which are potential carcinogens, to induce the umu operon. A plasmid (pSK1002) carrying a fused gene umuC'-'lacZ was introduced into Salmonella typhimurium TA1535. The strain TA1535/pSK1002 enabled us to monitor the levels of umu operon expression by measuring the beta-galactosidase activity in the cells produced by the fusion gene. Using this strain, a simple, inexpensive, and sensitive system, the umu-test, for the screening of environmental mutagens and carcinogens was developed. 38 chemicals with different structures and modes of action, including 31 known animal carcinogens, were examined by the test to evaluate the system. The threshold sensitivity of the umu-test was approximately equal to that of the Ames test for chemicals genotoxic in both tests. By the umu-test, using the single tester strain, we detect many types of DNA-damaging agents for which the Ames test requires several tester strains. Furthermore, the umu-test provides a potential practical advantage for the screening of various environmental samples containing amino acids and nutrients such as urine, serum and foods.     

The 1996 Veylien Henderson Award of the Society of Toxicology of Canada. Current concepts: neutrophils and the activation of carcinogens in the breast and other organs

Many chemical carcinogens target epithelial tissues, but the biological and biochemical bases of carcinogen specificity remain largely unknown. Focusing on the mammary gland, we discuss the concept that neutrophils metabolize carcinogens to reactive species that damage adjacent epithelial cells. This mechanism may help to explain why epithelial cells are sensitive targets for chemical carcinogenesis, despite their limited bioactivation capacity.     

Mismatch repair processing of carcinogen-DNA adducts triggers apoptosis

The DNA mismatch repair pathway is well known for its role in correcting biosynthetic errors of DNA replication. We report here a novel role for mismatch repair in signaling programmed cell death in response to DNA damage induced by chemical carcinogens. Cells proficient in mismatch repair were highly sensitive to the cytotoxic effects of chemical carcinogens, while cells defective in either human MutS or MutL homologs were relatively insensitive. Since wild-type cells but not mutant cells underwent apoptosis upon treatment with chemical carcinogens, the apoptotic response is dependent on a functional mismatch repair system. By analyzing p53 expression in several pairs of cell lines, we found that the mismatch repair-dependent apoptotic response was mediated through both p53-dependent and p53-independent pathways. In vitro biochemical studies demonstrated that the human mismatch recognition proteins hMutSalpha and hMutSbeta efficiently recognized DNA damage induced by chemical carcinogens, suggesting a direct participation of mismatch repair proteins in mediating the apoptotic response. Taken together, these studies further elucidate the mechanism by which mismatch repair deficiency predisposes to cancer, i.e., the deficiency not only causes a failure to repair mismatches generated during DNA metabolism but also fails to direct damaged and mutation-prone cells to commit suicide.     

Activation of mammalian carcinogens to bacterial mutagens by microsomal enzymes from a pelecypod mollusk,Mercenaria mercenaria

Several kinds of neoplastic diseases have been described in mollusks collected from the field. The etiology of these lesions is unknown; however, the involvement of chemical carcinogens has been suggested. Experimental models for chemically-induced neoplasia in bivalves have yet to be developed. We have obtained data which suggest that aromatic amines may be more appropriate candidates than polycyclic aromatic hydrocarbons for putative molluscan carcinogens.Digestive gland enzymes from a bivalve mollusk, Mercenaria mercenaria, were found to be able to convert aromatic amines to frameshift mutagens as detected by the Ames Salmonella tester strains. Extensive mutagenesis was obtained with 2-aminoanthracene, 2-aminofluorene, 2-acetylaminofluorene and 4-amino-trans-stilbene which are all mammalian procarcinogens. Mutagenic activation of benzo[a]pyrene and 3-methylcholanthrene was minimal. Low levels of aromatic amine-activating enzyme(s) were found in all tissues examined, but activity was greatest in the digestive gland. Enzymatic activity was heat-labile, NADPH-dependent, and apparently not inducible by polychlorinated biphenyls (Aroclor 1254).     

Unmasking a killer: DNA O(6)-methylguanine and the cytotoxicity of methylating agents

Methylating agents are potent carcinogens that are mutagenic and cytotoxic towards bacteria and mammalian cells. Their effects can be ascribed to an ability to modify DNA covalently. Pioneering studies of the chemical reactivity of methylating agents towards DNA components and their effectiveness as animal carcinogens identified O(6)-methylguanine (O(6)meG) as a potentially important DNA lesion. Subsequent analysis of the effects of methylating carcinogens in bacteria and cultured mammalian cells - including the discovery of the inducible adaptive response to alkylating agents in Escherichia coli - have defined the contributions of O(6)meG and other methylated DNA bases to the biological effects of these chemicals. More recently, the role of O(6)meG in killing mammalian cells has been revealed by the lethal interaction between persistent DNA O(6)meG and the mismatch repair pathway. Here, we briefly review the results which led to the identification of the biological consequences of persistent DNA O(6)meG. We consider the possible consequences for a human cell of chronic exposure to low levels of a methylating agent. Such exposure may increase the probability that the cell's mismatch repair pathway becomes inactive. Loss of mismatch repair predisposes the cell to mutation induction, not only through uncorrected replication errors but also by methylating agents and other mutagens.     

A comparison of aflatoxin B1-induced cytotoxicity,mutagenicity and prophage induction in Salmonella typhimurium mutagen tester strains TA1535, TA1538, TA98 and TA100

Treatment of Ames mutagen tester strains with aflatoxin B1 (AFB1) and S9 mix results not only in the production of a poten mutagen, but induces a pathway that leads to the induction of prophages present in all Ames tester strains.Characterization of the prophage induction and mutagenic response following AFB1 treatment showed that plasmid pKM101 dramatically enhances mutagenesis, but suppressed prophage induction. Spontaneous release of phage by TA98 and TA100 was also lower than in TA1535 and TA1538.In addition to mutagenesis and prophage induction, survival of all 4 tester strains was quantitated after AFB1 treatment. The data show that the frameshift tester strains (TA1538 and TA98) are more sensitive to the bactericidal action of AFB1 than the base-pair tester strains (TA1535 and TA100), survival being significantly affected above 100 ng. One of several hypotheses examined was the difference in the number and types of prophages present in base-pair tester strains that are not detectable in the frame-shift tester strains.These data suggest that prophage induction can detect DNA damage that is non-mutagenic; and that it is important to characterize the lysogenic nature of the Ames strains since it may influence the observed histidine revertant rate and the survival of the tester strain.     

Hyperplastic foci in chemical carcinogenesis]

Morphological, histochemical and biochemical perculiarities of liver hyperplastic nodules induced by different chemical carcinogens are regarded. The presence of basophylic atypical cells in the hyperplastic nodules, the possibility of transplantation of some nodules, the irreversibility of some morphological and biochemical lesions in these allows to designate hyperplastic nodules as a neoplasm. Enzyme and isozyme patterns in hyperplastic vesicles, the appearance of alpha-fetoprotein in these, and some other metabolic findings suggest biochemical disdifferentiation occurring in the vesicles. These findings suggest that cells of hyperplastic nodules are intermediate between normal and malignant cells in the course of neoplastic transformation.     

Induction of mating type interconversion in a heterothallic strain of Saccharomyces cerevisiae by DNA damaging agents

Mating type interconversion of the yeast, Saccharomyces cerevisiae, is an example of a directed genome rearrangement leading to a change in gene expression and in the differentiation state of a cell. In heterothallic haploid cells this switching of the mating type from a to alpha or vice versa, which is accomplished by an intrachromosomal gene conversion mechanism, is a rare event, happening about once per 10(6) cells per generation. Those cells that have changed their mating type can be trapped as diploid colonies by making them mate with tester cells possessing complementary markers. We found that treating haploids with UV light or with chemical carcinogens before they could mate resulted in a significant and dose-dependent enhancement of the numbers of diploid colonies. By genetic as well as by DNA hybridization analyses, these diploid clones were proved to be descendants of haploids which had changed their mating type by the bona fide gene conversion process. Thus, the DNA damaging agents had caused the induction of a directed gene rearrangement. It is suggested that induction of genome rearrangements might be part of a general response to DNA damage, at least in yeast cells. If similar responses also took place in cell populations constituting multicellular organisms, induced gene rearrangements and a generally enhanced mobility of the genome as a consequence of DNA damage might play a determining role in chemical and radiation-induced carcinogenesis.     

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.     

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.     

Potential identification of chemical carcinogens in a viral transformation system

Chemical carcinogens from several diverse chemical classes i.e.; aromatic amines, polycyclic hydrocarbons, nitrosamines, hormonal derivatives, metals and direct alkylating agents cause a 6.2–60.5-fold increase in the frequency of murine sarcoma virus (MSV)-induced transformation in a normal rat kidney (NRK) cell system. Exogenous metabolic activation with a rat liver S-9 homogenate is required for expression of this activity by procarcinogens. Non-carcinogenic analogs of these compounds fail to cause significant increases in the transformation frequency either with or without prior metabolic activation. Iododeoxyuridine, a mutagen also does not cause enhancement of transformation. This system may serve as the basis for a rapid and quantifiable means of identifying chemical carcinogens while introducing a new model for the understanding of the interactions between oncorna-viruses and chemical carcinogens.     

Induction of prophage and mutagenic effects by alkyl alkylaminosulfonates, ethyl aminosulfonate and alkyl methanesulfonates

Prophage induction and mutation by alkylaminosulfonates, ethyl aminosulfonate and alkyl methanesulfonates were examined comparatively. Prophage induction was carried out with a lysozyme lysis technique on the lysogenic strain Micrococcus lysodeikticus 53-40 (N5). The sulfonic ester derivatives show a slight lysogenic induction. At higher concentrations their toxicity seems to mask phage detection. Only methyl isopropylaminosulfonate and ethyl aminosulfonate exhibit no or negligible toxic effects, and with these compounds at higher concentrations a strong prophage induction is found. Alkyl sulfonate derivatives induce mutations in the tester strain of Salmonella typhimurium TA1535. Methyl methylaminosulfonate and ethyl N-methyl-N-2-chloroethyl aminosulfonate show a mutagenicity comparable to that of the well-known methyl methanesulfonate or ethyl methanesulfonate. With ethyl aminosulfonate, however, which does not show inactivation, no significant mutagenic effect was observed. DNA alterations were found in the polymerase-deficient strain E. coli P3478. The results of prophage induction and mutagenicity are compared and discussed.     

A simple plate test for screening colicine-inducing substances as a tool for the detection of potential carcinogens.

A simple and rapid plate test is described for screening substances that induce colicine E2. By using chemicals activated with microsomal enzymes and a permeable (rfa) tester bacterium that is also deficient in DNA repair (uvrB), the range of inducing substances that can be detected has been extended. The possible correlation between colicine-inducing substances and carcinogens is discussed.     

Unmasking a killer: DNA O6-methylguanine and the cytotoxicity of methylating agents

Methylating agents are potent carcinogens that are mutagenic and cytotoxic towards bacteria and mammalian cells. Their effects can be ascribed to an ability to modify DNA covalently. Pioneering studies of the chemical reactivity of methylating agents towards DNA components and their effectiveness as animal carcinogens identified O⁶-methylguanine (O⁶meG) as a potentially important DNA lesion. Subsequent analysis of the effects of methylating carcinogens in bacteria and cultured mammalian cells — including the discovery of the inducible adaptive response to alkylating agents in Escherichia coli — have defined the contributions of O⁶meG and other methylated DNA bases to the biological effects of these chemicals. More recently, the role of O⁶meG in killing mammalian cells has been revealed by the lethal interaction between persistent DNA O⁶meG and the mismatch repair pathway. Here, we briefly review the results which led to the identification of the biological consequences of persistent DNA O⁶meG. We consider the possible consequences for a human cell of chronic exposure to low levels of a methylating agent. Such exposure may increase the probability that the cell's mismatch repair pathway becomes inactive. Loss of mismatch repair predisposes the cell to mutation induction, not only through uncorrected replication errors but also by methylating agents and other mutagens.     

Yeast DEL assay detects protection against radiation-induced cytotoxicity and genotoxicity: adaptation of a microtiter plate version

The DEL assay in yeast detects DNA deletions that are inducible by many carcinogens. Here we use the colorimetric agent MTS to adapt the yeast DEL assay for microwell plate measurement of ionizing radiation-induced cell killing and DNA deletions. Using the microwell-based DEL assay, cell killing and genotoxic DNA deletions both increased with radiation dose between 0 and 2000 Gy. We used the microwell-based DEL assay to assess the effectiveness of varying concentrations of five different radioprotectors, N-acetyl-l-cysteine, l-ascorbic acid, DMSO, Tempol and Amifostine, and one radiosensitizer, 5-bromo-2-deoxyuridine. The microwell format of the DEL assay was able to successfully detect protection against and sensitization to both radiation-induced cytotoxicity and genotoxicity. Such radioprotection and sensitization detected by the microwell-based DEL assay was validated and compared with similar measurements made using the traditional agar-based assay format. The yeast DEL assay in microwell format is an effective tool for rapidly detecting chemical protectors and sensitizers to ionizing radiation and is automatable for chemical high-throughput screening purposes.