Cytotoxic and mutagenic effects of specific carcinogen-DNA adducts in diploid human fibroblasts (original) (raw)
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The combined action of chemical carcinogens on DNA repair in human cells
Radiation and Environmental Biophysics, 1980
Excision repair was studied in normal human and ataxia telangiectasia (AT) cells proficient in repair of UV and its mimetic chemicals, and in xeroderma pigmentosum group C (XP C) cells (deficient in repair of UV and its mimetics), after treatment with several combinations of chemical carcinogens, by the photolysis of bromodeoxyuridine incorporated into parental DNA during repair. Results indicate that repair was additive in AT, and XP C cells treated with N-acetoxy-2acetylaminofluorene (AAAF) plus ethylmethanesulfonate (EMS) or methyl methanesulfonate (MMS) indicating that there are different rate limiting steps for removal of both types of damage. Data on the combinations of 4-nitroquinoline 1oxide (4NQO) plus MMS or EMS are difficult to interpret, but they do not indicate inhibition of DNA repair.
DNA Repair and Human Health, 2011
Introduction 1.1 Environmental endogenous DNA damages Cancer development is a long-term, multistep process with a complex interplay between genes and environment. The magnitude of environmental effects depends on the presence or absence of genetic susceptibility of the subjects to certain cancer types. Molecular epidemiological studies in cancer have proved that besides target cell genetic instability, the presence of triggering environmental exposure is critical in cancer development [Albertini & Hayes 1997, Newby & Howard 2005]. The biomarker responses, exposure character and the route of exposure of different environmental factors (chemicals, physical agents and biological agents) are also important in causing tumors especially in the cases of occupational cancer [Ward 1995]. The EPA Guidelines for carcinogen Risk Assessment [EPA 2005] is based on the mode of action of chemicals, such as interaction with DNA, cytotoxicity, or binding to the receptors modifying signal pathways. There are several natural compounds-so called chemopreventive agents-which are able to modify the genotoxic or mutagenic response (Ames 1983) in different organisms. These vitamins, antioxidants, phytochemicals, micro nutrients are available on the market without knowing their mode of action. Mutagenesis caused by environmental chemicals or physical agents can be prevented by protection of the cell's DNA replication, increasing the repair capacity or delaying cell replication to allow enough time to make a complete repair of damaged cells. Antioxidants are able to protect the cells from oxidative stress, and stimulate the phase I reactions including oxidation, reduction, and hydrolysis of xenobiotics by the monoxigenase detoxicating key enzymes, such as CYP450 [Xu et al. 1996, Poulsen & Loft 1998]. These changes increase the polarity of these molecules and help to conjugate them in phase II to glucuronic acid, acetic acid and sulfuric acid which are the physiological ways to eliminate active metabolites that are genotoxic to the target cells. The best studied crucial early event in carcinogenesis is chromosomal aberration, including microsatellite instability, abnormal number of chromosomes (aneuploidy), gene amplification or the loss of heterozygosity of tumor suppressor genes. By reducing chromosomal mutation via chemoprevention, the cell may be able to survive the genotoxic effects without any permanent damage, or it is able to go through the physiological pathway of apoptosis, without mutation occurring in the P53 gene [Lowe & Lin 2000]. www.intechopen.com DNA Repair and Human Health 308 1.2 The role of DNA repair in gene-environmental interactions The measurement of UV-induced DNA repair is recommended in the risk assessment of environmental exposure to harmful chemicals (Reg. 440/2008/EC). Data obtained on prokaryota organisms suggest that exposure to chemicals as e.g. free oxygen radicals can interact with UV-induced DNA repair mechanisms (Chandor-Proust et al, 2008). Among the repair mechanisms existing in higher eukaryota, base excision repair (BER) seems to be the main mechanism involved in the removal of lesions produced by alkylation, deamination or oxidation (Rastogi et al, 2010). Orelli et al. (2009) demonstrated recently that nucleotide excision repair (NER) also plays an important role in the development of cisplatin resistance. UV-induced DNA damages can induce the so called three prime exonuclease1 (trex1), as a response to genotoxic stress. Beside thymine dimer production, UV irradiation can also produce reactive oxygen species. Benzo(a)pyrene (BaP) and hydrogen peroxide may, similarly to UV, induce the so-called three prime exonuclease1 (trex1) involved in the repair pathways of UV-induced DNA lesions, and cells deficient in trex1 show reduced recovery from UV and BaP replication inhibition, and increased sensitivity to towards genotoxins compared to the isogenic control (Christmann et al, 2010). These data suggest that both main mechanisms can be involved in the total repair of environmental chemical-induced genotoxic stress. Such mechanisms can probably explain the observed UDS reduction in some of our groups exposed to various chemicals but not UV. A second question is whether decreased UDS can be related to an increase in apoptotic capacity? Cells deficient in the repair of UV-induced DNA damage can be more susceptible to a G1 arrest after UV treatment than cells with normal repair capacity or those cells which have completed their DNA repair prior to movement from G1 to S phase (Geyer et al, 2000). Zampetti-Bosseler and Scott (1981) demonstrated a prolonged mitotic delay in repair deficient ataxia teleangiectasia and retinoblastoma fibroblasts after X-ray irradiation compared to normal human fibroblasts, also suggesting a general key role of cell cycle check points beside DNA repair in preservation of genome stability (Kaufman, 1995). Skin fibroblasts from derived ataxia teleangiectasia patients are also more sensitive to UVinduced mutagenesis than those taken from healthy subjects (Hannan et al, 2002), and their results suggested a relationship between cell cycle control and DNA repair pathways in human cells. Genotoxic chemicals can also delay cellular proliferation in DNA repairdeficient cell clones more significantly than in wild type cells, by interfering with DNA replication, thereby inducing DNA damage (Kyunghee et al, 2009). The recently discovered cell cycle checkpoint activation mechanisms are discussed in detail by Rastogi et al (2010). In the present study the so-called premature centromere division (PCD) was used as a cytogenetic indicator of abnormalities in cell cycle regulation (Méhes 1978, Vig, 1981, Major et al, 1999). PCD yields were increased among cytostatic drug producers, anesthesiologists using halothane, and in exposures to formaldehyde, benzene and PAHs. PCD can be involved in the pathomechanism of aneuploidy, it seems to be a possible manifestation of chromosome instability also in human chromosome breakage syndromes and it can be connected with carcinogenesis (for review, c.f. Major et al, 1999). 2. Cancer development and DNA repair We don't know exactly what the cause of cancer is; therefore we have several mechanisms and theories to explain it. One of them is shown in Fig.1. www.intechopen.com Application of UV-Induced Unscheduled DNA-Synthesis Measurements in Human Genotoxicological Risk Assessment 315 6. Analysis of biomarkers in blood samples Biomarkers for DNA damages and risk assessment Considering the basic mechanism of cancer development, the most acceptable predictors of cancer risks are the DNA-damage biomarkers (see Table 3.). These damages can be provoked by exogenous or end o g e n o u s a g e n t s w h e n D N A r e p a i r o r m i s-r e p a i r i s i n dysfunction. The unrepaired DNA damage can reduce the basic cell functions eg. maintenance of genetic integrity, triggering of cell cycle arrest, apoptosis, uncontrolled growth and other functionalities. Ultimately, damaged repair capacity leads to an increase in somatic mutations and cancer. Any living cells Mitochondrial DNA mutation Any living cells Point mutation (HPRT) Any living cells Nuclear p53 Lymphocytes, germ cells DNA-adducts and oxidation, methylation Any living cells Telomere shortening Tumor cells, lymphocytes, germ cells Aneuploidy Lymphocytes, bone marrow cells Micronucleus assay Tumor cells, lymphocytes, germ cells Chromosomal aberrations Any living cells, germ cells DNA strand breaks (SGE or Comet assay) Lymphocytes, hepatocytes Unscheduled DNA synthesis (UDS) Target cells Methods Any living cells Mitochondrial DNA mutation Any living cells Point mutation (HPRT) Any living cells Nuclear p53 Lymphocytes, germ cells DNA-adducts and oxidation, methylation Any living cells Telomere shortening Tumor cells, lymphocytes, germ cells Aneuploidy Lymphocytes, bone marrow cells Micronucleus assay Tumor cells, lymphocytes, germ cells Chromosomal aberrations Any living cells, germ cells DNA strand breaks (SGE or Comet assay) Lymphocytes, hepatocytes Unscheduled DNA synthesis (UDS)
Somatic Cell and Molecular Genetics, 1984
CHO cells of normal or UV-sensitive phenotypes were analyzed for their ability to remove DNA adducts produced by the carcinogen 7-BrMeBA. At a dose of O.1 tzM, which reduced the survival of the normal AA8 cells to -90 % and the mutant UV5 cells to -20 %, the frequency of adducts was 5-6 per 106 nucleotides for both cell types, and AA8 cells removed -30% of the adducts in 8 h and -55% in 24 h. In contrast, UV5 and mutants from four other genetic complementation groups had no significant removal. Binding of 7-BrMeBA did not vary through the cell cycle in synchronous cultures. At a dose of mutagen (0.07 aM) resulting in -25% survival of asynchronous UV5, the survival of synchronous cultures rose about threefold from early GI to early S phase and then decreased somewhat in late S/G:. At a dose (0.28 #M) producing similar survival of asynchronous cultures, AA8 cells differed qualita.
Methods in Pharmacology and Toxicology, 2014
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.
Toxicologic Pathology, 1984
Mutagenesis and neoplastic transformation assays on mammalian cells in culture have been extensively used for quantitative estimates of the activity of carcinogens, in spite of the limitations that such in vitro systems have when compared with in vivo systems for tumor induction. In order to assess the validity of these correlations, a series of studies was undertaken in our laboratory with the BALB/3T3 CI A31-1-1 mouse embryo cell line. Different carcinogens were found to induce dose-dependent frequencies of transformation, including the direct-acting alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and carcinogens that were metabolically activated by these cells through different pathways (benzo[a]pyrene, 3-methylcholanthrene, aflatoxin B,, and benzidine). Their respective level of activity on a molar basis was different from that obtained in standard Salmonella + S9 mutagenesis tests. Studies currently underway indicate the possibility of lowering the serum content in the medium considerably, thereby reducing a major variable in the assay. Methods were established for the induction of ouabain-resistant (oua') mutants in these cells. Studies were conducted by applying 30-min MNNG exposures to cells that were synchronized by serum deprivation followed by serum-induced release from growth block. While maximal induction of mutants occurred in the S phase, the transformation frequency remained constant for treatments in GI and early or late S. In subsequent studies, cytotoxicity, alkali-labile DNA lesions, oua' mutations, and neoplastic transformation were analyzed concurrently in this cell line after cells were exposed to two concentrations of MNNC and the exposures were protracted for different time periods (30, 60, 90, 120, and 240 min; 24, 48, and 72 hr). A marked temporal dissociation was found in the exposure times required to induce maximal frequencies of mutations and of transformation. Cytotoxicity increased for periods up to 100-200 min; mutations reached a maximal induction level after a much shorter exposure time (30-60 min); DNA damage detected by alkaline elution was already maximal by 30 min. Transformation frequencies, however, reached maximal levels only after exposure periods 1-3 hr longer than those required for maximal mutation. The ratio of transformation to oua' mutation frequencies was 3.7 for short treatment times (30-60 min), but it increased to more than 20 for exposure times of 240 min or longer. These studies support the hypothesis that a single gene mutational event is not sufficient to account for the expression of neoplastic transformation. Presented at the Second International Symposium sponsored by the Universities of Sassari and Cagliari, Session V: "Risk Assessment." October 12-15. 1983. Alghero. Italy. This Symposium section completes the series of papers published in Volume 12.1984.
Mutations and Homologous Recombination Induced by N-Substituted Aryl Compounds in Mammalian Cells
Springer eBooks, 1990
Metabolites of two structurally related chemical carcinogens, benzo[a]pyrene and 1-nitropyrene, were compared for their ability to cause cytotoxicity and induce mutations in normally repairing or nucleotide excision repair-deficient diploid human fibroblasts; for their ability to induce mutations in a defined gene sequence, supF, when a plasmid containing adducts formed by these carcinogens replicates in human 293 cells; and for their ability to induce homologous recombination between duplicated genes in mouse L cells. Both of the metabolites tested, i.e., (±)-73,8a-dihydroxy-9a,1Oa, epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and 1-nitrosopyrene (1-NOP), form adducts on guanine. BPDE binds principally at the N2 position of guanine; 1-NOP binds to guanine at the C8 position. Results of the studies in diploid human cells indicated that when compared on the basis of equal numbers of DNA adducts, BPDE is more effective than 1-NOP in inducing mutations in DNA repair-proficient cells, but when compared in repair-deficient xeroderma pigmentosum human cells that do not remove such adducts from their DNA, the frequency of mutants induced per adduct is equal. These results suggest that during the time available for repair of potentially mutagenic lesions, repair-proficient human cells excise 1-NOP adducts more rapidly than they excise BPDE adducts. Molecular analysis. of the specific kinds of mutations induced when a plasmid containing BPDE residues was allowed to replicate in human cells showed that BPDE induces mainly base substitution mutations, predominantly G:C to T:A transversions. Preliminary results with 1-NOPtreated plasmids in that system indicate that 1-NOP is not as mutagenic as BPDE when the two carcinogens are compared on the basis of equal initial numbers of adducts per plasmid. Just as was the case for mutation induction in the genome of diploid human fibroblasts discussed above, the difference in mutagenicity per adduct may reflect a difference in excision repair rate by the human cell line 293. This question is under investigation. In addition, preliminary results of a study comparing 1-NOP with BPDE for ability to induce homologous recombination in mouse L cells indicate that BPDE is more effective than 1-NOP.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1996
Previous studies on structure-activity relationships (SARs) between types of DNA modifications and tumour incidence revealed linear positive relationships between the log TDso estimates and s-values for a series of mostly monofunctional alkylating agents. The overall objective of this STEP project was to further elucidate the mechanistic principles underlying these correlations, because detailed knowledge on mechanisms underlying the formation of genotoxic damage is an absolute necessity for establishing guidance values for exposures to genotoxic agents. The analysis included: (1) the re-calculation and further extension of TDso values in mmol/kg body weight for chemicals carcinogenic in rodents. This part further included the checking up data for Swain-Scott s-values and the use of the covalent binding index (CBI); (2) the elaboration of genetic toxicity including an analysis of induced mutation spectra in specific genes at the DNA level, i.e., the vermilion gene of Drosophila, a plasmid system (pX2 assay) and the HPRT gene in cultured mammalian cells (CHO-9); and (3) the measurement of specific DNA alkylation adducts in animal models (mouse, rat, hamster) and mammalian cells in culture. The analysis of mechanisms controlling the expression of mammalian DNA repair genes (alkyltransferases, glycosylases) as a function of the cell type, differentiation stage, and cellular microenvironment in mammalian cells. The 3 classes of genotoxic carcinogens selected for the project were: (1) chemicals forming monoalkyl adducts upon interaction with DNA;
The mutation studies of mutagen-sensitive and dna repair mutants of chinese hamster fibroblasts
Environmental Mutagenesis, 1981
We have previously reported the isolation and partial characterization of DNA repair and/or mutagen-sensitive mutant Chinese hamster cell strains. Here we present the results of a detailed study of the ultraviolet light (UV)-induced mutability of one of these strains, UVS-7, and provide preliminary mutability data on two additional lines, UVI-23 and UVS-40. UVS-7 is extremely deficient in unscheduled DNA synthesis (UDS) but only slightly more sensitive to UV than the parental line. When examined for the UV-inducibility of mutants resistant t o ouabain, 6-thioguanine, or diphtheria toxin, UVS-7 was found t o be hypermutable at all three loci as compared t o the parental line. The degree of hypermutability was not the same for any two loci. UVS-40, a highly UVsensitive strain, was also found t o be hypermutable at the ouabain-resistant (ouar) locus. UV*-23, which is UV-resistant and more proficient at UDS than the parental line, appeared to exhibit a tendency toward hypomutability at both the ouabain(ouar) and 6-thioguanine-resistant (6TG') loci. Further characterization of all these lines should aid in delineating mammalian mechanisms of DNA repair and mutagenesis.