DNA recombination induced by aflatoxin B1 activated by cytochrome P450 1A enzymes

Mutations in tumor suppressor genes are intricately associated with the etiology of neoplasia. Often, such mutations are followed by the loss of the second, functional alleles of tumor suppressor genes, a phenomenon known as loss of heterozygosity. Loss of heterozygosity may occur by different molecular mechanisms, including mitotic recombination, and it is conceivable that these molecular events are influenced by endogenous as well as exogenous factors. To test whether mitotic recombination is induced by certain carcinogens, we genetically engineered a Saccharomyces cerevisiae tester strain so that it metabolizes two important classes of carcinogens, polycyclic aromatic hydrocarbons and heterocyclic arylamines. This was accomplished by expressing human cDNA's coding for the cytochrome P450 (CYP) enzymes CYP1A1 or CYP1A2 in combination with NADPH-CYP oxidoreductase in a strain heterozygous for two mutations in the trp5 gene. Microsomes isolated from the transformed yeast strains activated various xenobiotics to powerful mutagens that were detected in the Ames test. Of these, the mycotoxin aflatoxin B1, when activated intracellularly in the strains containing either human CYP enzyme, significantly induced mitotic recombination. These results are discussed in light of possible mechanisms that are involved in aflatoxin B1-mediated hepatocarcinogenesis. Similarly, benzo[a]pyrene-trans-7,8-dihydrodiol and 3-amino-1-methyl-5H-pyrido[4,3-b]indole were activated to recombinagenic products, whereas benzo[a]pyrene and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline were negative in this assay. Our results argue that the constructed yeast strains may be a valuable tool for the investigation of drug-induced mitotic recombination

Reversible inhibition of mammalian tubulin assembly in vitro and effects in Saccharomyces cerevisiae D61.M by mitomycin C

Gaulden reported a novel and unexpected mitomycin C (MMC) effect, namely a pronounced retardation of very late prophase and loss of chromosome orientation in neuroblasts of the grasshopper Chortophaga viridifasciate. Because this effect may be due to interactions of MMC with non-DNA targets, MMC was tested for its interaction with porcine brain tubulin assembly in vitro and for the induction of chromosomal malsegregation in the diploid yeast Saccharomyces cerevisiae strain D61.M. A reversible dose-dependent inhibition of tubulin assembly was observed. Since no biological activation system was present in the incubation mixture this inhibition seems to result from an interaction of unactivated MMC with the assembly process. The possible chemical activation of MMC by reduction with 1, 4-dithioerythritol (DTE) was investigated by omission of this compound during isolation and polymerization of tubulin. The absence of DTE resulted in a strong reduction of the net tubulin assembly. Also under these conditions MMC led to a dose-dependent inhibition of the assembly, indicating that the effect of MMC on tubulin assembly is independent of a reductive chemical modification. In S.cerevisiae D61.M, MMC did not induce chromosome loss, but induced other genetic events (possibly mutations, deletions or mitotic recombination) as was detected by an increase of the total number and of the frequency of cycloheximide-resistant colonies. This effect could be observed with and without the addition of rat liver S9 as an exogenous activation syste

Environmental effects of genotoxins (eco-genotoxicology)

Genotoxic chemicals can damage the genetic material of humans as well as that of organisms living in the environment. With respect to adverse effects, alterations induced in the germ line, leading to alterations in the genetic make-up of populations, are of primary concern in ecosystems, because somatic changes, even if they lead to a loss of individuals, will not be critical in populations with a large reproductive surplus. This is different in human toxicology where genetic alterations in germ cells as well as in somatic cells of any individual are of concern. Increased frequencies of mutations and related genetic alterations in the gene pools of individual species or populations in ecosystems have to be judged against the background of spontaneous mutations that have enabled species to survive and adapt in changing environments since the beginning of life on our planet, and which have played an important role as the substrate for evolutionary developments. Examples of the selection of altered phenotypes (and genotypes) in response to environmental pollution and environmental stress are melanism in moth populations, metal resistance in plants, insecticide resistance in insects and malaria resistance in humans. Pollution, in general, can represent a stress factor selectively leading to a change in genetic make-up. In addition, environmental genotoxins can directly alter gene pools. A change in the genetic constitution may be advantageous for certain populations living in stressful conditions, but may present a disadvantage for others, including man. Examples are (i) the induction of (pesticide) resistance, (ii) the increased virulence of pathogens, (iii) alterations of host ranges of pathogenic forms or the appearance of new virus types and (iv) subtle changes in parasite—host or predator—prey relationships. Basically the release of genotoxins into the environment should be avoided because massive exposures may affect the reproductive capacity of many species, and modest exposures may lead to an enhanced instability of ecosystems and may provoke specific adaptations to stressful situations. Furthermore, the uncontrolled presence of genotoxins in any compartment of the natural environment is an unwanted situation, in particular also from a human point of view. In addition we need novel quantitative approaches in order to make quantitative risk estimates possibl

Characterization of the trp5-27 allele used to monitor drug-induced mitotic gene conversion in the Saccharomyces cerevisiae tester strain D7

Mitotic gene conversions, among other recombinagenic events, can play an important role in the multistep process of carcinogenesis. The ability of chemicals to induce such gene conversions can easily be monitored in the Saccharomyces cerevisiae tester strain YHE2, a derivative of strain D7. For the detection of drug-induced gene conversions, two mutations in the TRP5 locus are used, trp5-12 and trp5-27. Here we report on the characterization of the stable allele trp5-27. Our analysis revealed two relevant mutations in trp5-27: (a) a transition C to T at position 121 after ATG that results in an amber stop codon and abolishes gene expression and (b) a transversion A to T at position 1555 that creates an ochre stop codon. Simultaneous amber and ochre suppression with the suppressors SUP3 and SUP11, respectively, was capable of relieving the tryptophan-requiring phenotype of strains carrying the trp5-27 allele. These findings have implications on the length of gene conversion tracts in conversion events between trp5-12 and trp5-27: conversion tracts can cover several kilobases, if the site of the mutation in trp5-12 lies outside of the positions mutated in trp5-27. Conversely, the maximal length is limited to 1435 bp, if the mutation in trp5-12 is located between the positions mutated in trp5-27

Intergenerational transmission of nicotine within families: have e-cigarettes influenced passive smoking?

Plasmids were constructed to investigate homologous mitotic recombination in Drosophila cells. Heteroalleles containing truncated but overlapping segments of the bacterial β-galactosidase gene (lacZ) were positioned either on separate plasmids or as direct repeats on the same chromosome. Recombination reconstituted a functional lacZ gene leading to expression of LacZ+ activity detectable by histochemical staining. High extrachromosomal recombination (ECR) frequencies between unlinked heteroalleles were observed upon transient co-transfection into Drosophila melanogaster Schneider line 2 (S2) cells. Stably transfected cells containing the lacZ heteroalleles linked on a chromosome exhibited intra-chromosomal recombination (ICR) frequencies two orders of magnitude lower than ECR frequencies. Recombination was inducible by exposing the cells to ethyl methanesulphonate or mitomycin C. Recombination products were characterized by multiplex PCR analysis and unequal sister chromatid recombination was found as the predominant mechanism reconstituting the lacZ gene. To investigate recombination in vivo imaginal disc cells from transgenic larvae carrying the reporter gene on the X chromosome were isolated and stained for LacZ+ activity. The presence of a few LacZ+ clones indicated that mitotic recombination events occurred at frequencies two orders of magnitude lower than the corresponding event in cultured cells and late during larval developmen

Caffeine, estradiol, and progesterone interact with human CYP1A1 and CYP1A2 : evidence from cDNA-directed expression in Saccharomyces cerevisiae

Heterologous expression of cytochrome P-450 cDNAs in yeast is a potent instrument for the study of enzyme-specific parameters and can be used to answer questions with regard to substrate specificity as well as drug interaction in a background with no interfering activities. Two cDNAs of human CYP1A1 and CYP1A2 were expressed in yeast Saccharomyces cerevisiae, and microsomes of transformed strains contained substantial amounts of functional heterologous enzymes. Enzyme kinetics with 7-ethoxyresorufin as substrate resulted in KM values of 0.017 and 1.67 microM and Vmax values of 840 and 387 pmol/mg/min for CYP1A1 and CYP1A2, respectively. Both heterologous enzymes showed an overlapping substrate specificity pattern assayed with different phenoxazone ethers and caffeine. Caffeine was shown to be metabolized by CYP1A2 and CYP1A1. Both enzymes formed paraxanthine and minor amounts of theobromine; however, trimethyluric acid was exclusively formed by CYP1A1. The fact that theophylline was not formed by either enzyme anticipates the involvement of additional enzyme(s) in the primary metabolism of caffeine. Inhibition studies with caffeine, phenacetin, 17 beta-estradiol, and progesterone as inhibitors of the CYP1A1 and CYP1A2 catalyzed O-deethylation of 7-ethoxyresorufin suggest all compounds as possible substrates of CYP1A enzymes. 17 beta-estradiol inhibited CYP1A1-catalyzed paraxanthine and trimethyluric acid formation. In contrast 17 beta-estradiol did not inhibit CYP1A2-catalyzed formation of primary caffeine metabolites. These data clearly demonstrate the capacity of human CYP1A1 and CYP1A2 to metabolize caffeine. Furthermore, possible consequences of CYP1A enzyme inhibition by caffeine, phenacetin, 17 beta-estradiol, and progesterone will be discussed