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DNA damage in testicular cells in vitro: The response of rat and human cells to known testicular toxicants
Abstracts/Mutation Research 360 (1996) 201-300 Netherlands, b Health Council of The Netherlands, The Hague, The Netherlands Health-based recommended exposure limits, safe doses for lifelong human exposure to chemicals, are estimated from dose-response data obtained in chronic animal experiments or, occasionally, from epidemiological data. These exposure limits are estimated by applying a safety factor to no-observed-adverse-effect levels derived from the data available. This approach is considered inappropriate to estimate limits for human exposure to genotoxic carcinogens. In the Netherlands, for genotoxic carcinogens linear non-threshold extrapolation is applied to the data to estimate the dose corresponding to a certain level of risk for lifelong exposure deemed acceptable by the authorities. Thus, this risk assessment addresses life-time low-dose exposure. The data that are presented address the cancer risk associated with peak exposure which can occur following accidents or calamities, to a genotoxic carcinogen. For the purpose of this study 'eak exposure' was defined as a single instantaneous exposure (lasting less than 24 h) to a high dose of a substance. The key question is: "What is the estimated cancer risk of peak exposure to a genotoxic carcinogen relative to the cancer risk of the same total dose of this carcinogen distributed over an entire lifetime?" For the purpose of answering this question a DoseRate Correction Factor (DRCF) was defined as " a factor by which the tumour incidence caused by a specific dose of a chemical carcinogen at low dose rates is multiplied to derive the tumour incidence at high dose rates", and the value of this DRCF was estimated. The information on the basis of which the key question was answered includes: (i) data which are relevant to this question but which did not allow calculation of a DRCF, and statements rather than evaluations of scientific data; (ii) theoretical calculations of the relative risk of peak exposure; and (iii) data on the relative risk of peak exposure to genotoxic chemical carcinogens and ionising radiation. The second and third categories allowed calculation of DRCF values. Some data from the first category were used for the evaluation of these values. The data reviewed show that the DRCF values for genotoxic carcinogens calculated from experimental
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studies vary from zero to 8.3. Theoretical calculations result in DRCF values that range from zero to 7.1. Epidemiological and experimental data on the effects of ionising radiation indicate that the DoseRate Effectiveness Factor (the radiation equivalent of the DRCF) ranges from 1 to 10. An important reason for the variation in DRCF values for genotoxic carcinogens is that the experiments we have based our conclusion on, experiments with genotoxic carcinogens, are limited in number and do not comply exactly with comparison of peak exposure with lifelong exposure to low doses. Furthermore, the experiments differed as to several aspects. Even though variations in the design of the experiments with the chemicals reviewed may explain the range of DRCF values observed, the additional data on kinetics, metabolism and DNA repair suggest that there is no universal DRCF value, but that the DRCF value is compound-specific. In view of the limitations of the data we regard a DRCF of 10 appropriate as a 'default' value if no or insufficient data are available to estimate a compound-specific DRCF ('worst-case approach'). 8-1
DNA damage in testicular cells in vitro: The response of rat and human cells to known testicular toxicants C. Bj0rge, G. Brunborg, T. Scholz a, R. Wiger, J.A. Holme, E. Dybing, E.J. SOderlund; Department of Environmental Medicine, National Institute of Public Health, Oslo, Norway and a Institute of Surgical Research, The National Hospital, Oslo, Norway Much attention has been given to recent reports claiming that mean sperm counts in man have declined by 40-50% during the last 50 years. Sperm count and quality can be influenced by many factors. Analysis of human sperm after radiation and chemotherapy indicates that exposure to DNA damaging agents may reduce sperm counts and cause infertility. Furthermore, DNA damage may lead to the production of mutated spermatozoa, which in turn may result in pregnancy loss, congenital malformations, mental retardation, or other heritable deseases.
288
Abstracts/Mutation Research 360 (1996) 201-300
Most information concerning reproductive toxicants have been derived from animal models. Thus, evaluation of human reproductive risk for drugs and chemicals depends on the extrapolation of animal data. In the present study, 15 known reproductive toxicants were studied with respect to their ability to induce single-strand DNA breaks and alkali-labile sites (ssDNA breaks) in vitro, in spermatogenic cells from rats and humans, measured by the alkaline filter elution technique. The compounds investigated were ethylene dibromide (1), 1,3-dinitrobenzene (2), aflatoxin B I (3), cadmium chloride (4), chromium (VI) (5), acrylamide (6), arylonitrile (7), cisplatin (8), styrene oxide (9), methoxychlor (10), thiram (11), benomyl (12), thiotepa (13), chlordecone (14) and 1,2-dibromo-3-chloropropane (15). In rat testicular cells, chemical 1, 2, 3, 5, 9, 11, 14 and 15 induced DNA damage, whereas the others induced no significant level of DNA damage. In human testicular cells, chemical 1, 3, 4, 5, 6, 9, 11 and 14 induced DNA damage, whereas the remaining compounds induced no or very low levels of DNA damage. Thus, chemical 2, 4, 6 and 15 gave different results in the two species. These species differences, may be related to differences in the capability of testicular cells to metabolize the compound to DNA reactive intermediates, or to differences between human and rat testicular cells in their ability to carry out DNA repair. By comparing in vivo data from rat testis and in vitro data from isolated rat testicular cells, it seems possible to identify human testicular genotoxicants with greater reliability. 8-2
On cancer chemoprevention by inducers of conjugation enzymes M. Paolini a-c, L. Pozzetti a, R. Mesirca a, A. Sapone a, p. Silingardi b, S. Grilli b, C. Dalla Croce d, G. Bronzetti d, G. Cantelli-Forti a; a Department of Pharmacology, b Institut of Cancerology, University of Bologna, c Department of Pharmacology and Biology, University of Bari and a Institut of Mutagenesis and Differentiation (CNR), Pisa, Italy
One of the major mechanisms of chemical protection against mutagenesis, carcinogenesis and other forms of toxicity is the induction of phase-II metabolizing enzymes such as UDP-glucuronosyl transferases, glutathione S-transferases and NAD(P)H quinone reductase. Over the past few years the use of selective inducers to conjugating enzymes revealed the possibility of reducing the expression of certain forms of malignance. However, contrary to customary statements, it should be taken into account that postoxidative enzymes, with their 'institutional' detoxicating role, are also able to activate many precarcinogens. Here, $9 fractions from Swiss Albino CD1 mice fed 7.5 g / k g BHA (powdered laboratory chow) for 3 weeks, show a clear pattern of induction (monofunctional inducer) with 2-3-fold increases in phase-II enzyme activities. In vitro DNA binding of the premutagen agents [14]l,2-dichlorobenzene, [14C]l,4_dichlorobenzene and [~4]l,4_dibromoben_ zene, mediated by such metabolic preparations (using either cytosolic or microsomal fractions, or both) showed a significant increase in specific activity. In some instances, specific activity was even more elevated when compared to that obtained with traditional (phase-I induced) $9 fractions. Together with DNA binding, genetic response of these chemicals on the diploid D 7 strain of Saccharomyces cerevisiae (as biological test system), revealed the ability of phase-II induced preparations to activate premutagens, as exemplified by the significant enhancement of mitotic gene-conversion, mitotic crossing-over and reverse point mutation. We concluded that the use of phase-II inducers in cancer chemoprevention, for their ability to simultaneously reduce or increase precarcinogen bioactivation, should be carefully reconsidered. 8-3
Induction of HGPRT" mutants by 1,2-dimethylhydrazine, azoximethane and Nnitrosodimethylamine in V79 Chinese hamster cell line G. Bronzetti, M. Cini, R. Fiorio; Istituto di Mutagenesi e differenziamento C.N.R. Pisa, Italy
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studies vary from zero to 8.3. Theoretical calculations result in DRCF values that range from zero to 7.1. Epidemiological and experimental data on the effects of ionising radiation indicate that the DoseRate Effectiveness Factor (the radiation equivalent of the DRCF) ranges from 1 to 10. An important reason for the variation in DRCF values for genotoxic carcinogens is that the experiments we have based our conclusion on, experiments with genotoxic carcinogens, are limited in number and do not comply exactly with comparison of peak exposure with lifelong exposure to low doses. Furthermore, the experiments differed as to several aspects. Even though variations in the design of the experiments with the chemicals reviewed may explain the range of DRCF values observed, the additional data on kinetics, metabolism and DNA repair suggest that there is no universal DRCF value, but that the DRCF value is compound-specific. In view of the limitations of the data we regard a DRCF of 10 appropriate as a 'default' value if no or insufficient data are available to estimate a compound-specific DRCF ('worst-case approach'). 8-1
DNA damage in testicular cells in vitro: The response of rat and human cells to known testicular toxicants C. Bj0rge, G. Brunborg, T. Scholz a, R. Wiger, J.A. Holme, E. Dybing, E.J. SOderlund; Department of Environmental Medicine, National Institute of Public Health, Oslo, Norway and a Institute of Surgical Research, The National Hospital, Oslo, Norway Much attention has been given to recent reports claiming that mean sperm counts in man have declined by 40-50% during the last 50 years. Sperm count and quality can be influenced by many factors. Analysis of human sperm after radiation and chemotherapy indicates that exposure to DNA damaging agents may reduce sperm counts and cause infertility. Furthermore, DNA damage may lead to the production of mutated spermatozoa, which in turn may result in pregnancy loss, congenital malformations, mental retardation, or other heritable deseases.
288
Abstracts/Mutation Research 360 (1996) 201-300
Most information concerning reproductive toxicants have been derived from animal models. Thus, evaluation of human reproductive risk for drugs and chemicals depends on the extrapolation of animal data. In the present study, 15 known reproductive toxicants were studied with respect to their ability to induce single-strand DNA breaks and alkali-labile sites (ssDNA breaks) in vitro, in spermatogenic cells from rats and humans, measured by the alkaline filter elution technique. The compounds investigated were ethylene dibromide (1), 1,3-dinitrobenzene (2), aflatoxin B I (3), cadmium chloride (4), chromium (VI) (5), acrylamide (6), arylonitrile (7), cisplatin (8), styrene oxide (9), methoxychlor (10), thiram (11), benomyl (12), thiotepa (13), chlordecone (14) and 1,2-dibromo-3-chloropropane (15). In rat testicular cells, chemical 1, 2, 3, 5, 9, 11, 14 and 15 induced DNA damage, whereas the others induced no significant level of DNA damage. In human testicular cells, chemical 1, 3, 4, 5, 6, 9, 11 and 14 induced DNA damage, whereas the remaining compounds induced no or very low levels of DNA damage. Thus, chemical 2, 4, 6 and 15 gave different results in the two species. These species differences, may be related to differences in the capability of testicular cells to metabolize the compound to DNA reactive intermediates, or to differences between human and rat testicular cells in their ability to carry out DNA repair. By comparing in vivo data from rat testis and in vitro data from isolated rat testicular cells, it seems possible to identify human testicular genotoxicants with greater reliability. 8-2
On cancer chemoprevention by inducers of conjugation enzymes M. Paolini a-c, L. Pozzetti a, R. Mesirca a, A. Sapone a, p. Silingardi b, S. Grilli b, C. Dalla Croce d, G. Bronzetti d, G. Cantelli-Forti a; a Department of Pharmacology, b Institut of Cancerology, University of Bologna, c Department of Pharmacology and Biology, University of Bari and a Institut of Mutagenesis and Differentiation (CNR), Pisa, Italy
One of the major mechanisms of chemical protection against mutagenesis, carcinogenesis and other forms of toxicity is the induction of phase-II metabolizing enzymes such as UDP-glucuronosyl transferases, glutathione S-transferases and NAD(P)H quinone reductase. Over the past few years the use of selective inducers to conjugating enzymes revealed the possibility of reducing the expression of certain forms of malignance. However, contrary to customary statements, it should be taken into account that postoxidative enzymes, with their 'institutional' detoxicating role, are also able to activate many precarcinogens. Here, $9 fractions from Swiss Albino CD1 mice fed 7.5 g / k g BHA (powdered laboratory chow) for 3 weeks, show a clear pattern of induction (monofunctional inducer) with 2-3-fold increases in phase-II enzyme activities. In vitro DNA binding of the premutagen agents [14]l,2-dichlorobenzene, [14C]l,4_dichlorobenzene and [~4]l,4_dibromoben_ zene, mediated by such metabolic preparations (using either cytosolic or microsomal fractions, or both) showed a significant increase in specific activity. In some instances, specific activity was even more elevated when compared to that obtained with traditional (phase-I induced) $9 fractions. Together with DNA binding, genetic response of these chemicals on the diploid D 7 strain of Saccharomyces cerevisiae (as biological test system), revealed the ability of phase-II induced preparations to activate premutagens, as exemplified by the significant enhancement of mitotic gene-conversion, mitotic crossing-over and reverse point mutation. We concluded that the use of phase-II inducers in cancer chemoprevention, for their ability to simultaneously reduce or increase precarcinogen bioactivation, should be carefully reconsidered. 8-3
Induction of HGPRT" mutants by 1,2-dimethylhydrazine, azoximethane and Nnitrosodimethylamine in V79 Chinese hamster cell line G. Bronzetti, M. Cini, R. Fiorio; Istituto di Mutagenesi e differenziamento C.N.R. Pisa, Italy