NEW IN VITRO APPROACHES TO EXPLORE CELLULAR AND MOLECULAR EVENTS RELATED TO CARCINOGENESIS (original) (raw)
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Regulatory Toxicology and Pharmacology, 1996
The phenomena associated with nongenotoxic carcisubsequent marketing. The responsibility for making nogenesis are multifaceted and complex. Nongenotoxic carcinogens stimulate cell replication in the this evaluation rests with the manufacturer and depresence or the absence of cytotoxicity. Cell prolifera-pends upon information derived from a diverse array tion is pivotal in the neoplastic process, but the extent of in vitro and in vivo experiments in bacterial and of its contribution to the development of xenobioticmammalian species that are demanded by regulatory induced cancer remains an open question. The search prescription. Identification of a xenobiotic as a carcinofor a better understanding of this process has genergen follows its chronic administration to rodents during ated considerable interest and effort, often with the their lifetime and is based on tumor incidence in target objective of obtaining useful predictors of the tumourorgans above that which would be expected in unigenic potential of xenobiotics. Alterations in the natutreated animals. Many xenobiotics may be predicted to ral balance of endogenous humoural agents that mainbe carcinogens based on their reactivity with genomic tain replicative homeostasis results in proliferative DNA in prokaryotic or eukaryotic cells in vitro. The stimulation (or inhibition) which may be transient or covalent binding of these genotoxic carcinogens or their sustained. The bases for the molecular interaction of metabolites to DNA in the absence of lesion repair rethese mediators with cellular receptors, trans-cytosults in mutation of the genome, designating these subplasmic message conveyance, and subsequent nuclear stances as mutagens (Ames and Gold, 1990a,b; Shaw responses leading to xenobiotic-induced mitosis are and Jones, 1994). Such in vitro tests are regarded as becoming better understood. Assessment of tissue reppredictive for genotoxic carcinogens which would be licative status has now become established and utilizes anticipated to be carcinogenic in mammalian species biochemical and histological methodology in a routine following chronic exposure. For a given DNA-reactive manner. The increasingly challenging international (genotoxic) carcinogen, all of the complex pathways of regulatory environment is demanding greater undermetabolic activation, specificity of DNA binding, DNA standing of the mechanisms that underlie fundamenrepair, conversion of adducts to mutations and the contal phenomena and the influences exerted by xenobiotics prior to their registration. While the precise mode sequences of altered oncogene and tumor suppressor of action of an individual xenobiotic may not be gene function may not be understood, but in general, known, sound interpretation of toxicological data, inthe belief that DNA reactivity resulting in mutations cluding the contribution made by cell replication, creis an important mode of action 1 is a useful concept in ates greater confidence of its safety in the scientific, classification of carcinogens and has gained widespread regulatory, and commercial communities. This article acceptance. Identification of genotoxic activity provides offers a view of cell proliferation from molecular interimportant information for determination of carcinoactions at the cellular level, through practical assessgenic potential and influences the construction of an ment of cell and tissue replicative status to its utility appropriate risk assessment model. in contributing to the registration of new drugs and However, many xenobiotics are carcinogenic in vivo chemicals.
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.
Comparison of target organs of carcinogenicity for mutagenic and non-mutagenic chemicals
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1993
A comparison of target organs for mutagens and non-mutagens is presented for 351 rodent carcinogens in the Carcinogenic Potency Database (CPDB) with mutagenicity evaluations in Salmonella. Results are consistent with the hypotheses that in high-dose rodent tests mitogenesis is important in the carcinogenic response for mutagens and non-mutagens alike, and that mutagens have a multiplicative interaction for carcinogenicity because they can both damage DNA directly and cause cell division at high doses. These hypotheses would lead one to expect several results that are found in the analysis:
Food and Chemical Toxicology, 1986
The interdisciplinary evaluation of risks from carcinogens utilizes, inter alia, data on the activities of the compounds in short-term assays. A systematic approach is being used to determine (a) mutagenesis in bacteria (the study of direct activities and specific modes of metabolic activation), (b) DNA damage within primary mammalian cells (DNA single-strand breaks and persistence of damage, by a method extendable to the in vivo situation) and (c) amplified DNA sequences in cultured cells (as an endpoint probably relevant to carcinogenesis). This test combination was expected to reduce some of the shortcomings of other batteries of tests, which suffer from a lack of appropriate metabolic conversion of compounds, irrelevancy of genetic endpoints and pharmacokinetic limitations. Furthermore, as each assay in the test strategy differs from the others only by one of the parameters described above, a reasonable understanding of divergent test results from assay to assay was anticipated. Several substances were investigated to elucidate why their activities in short-term assays and in carcinogenesis experiments do not correlate. The substances were N-nitrodimethylamine, for which formaldehyde is the reactive intermediate in bacterial mutagenesis but not in mammalian cells or in vivo, N-nitrosodiethanolamine, a carcinogen that must be activated by external alcohol dehydrogenase to be mutagenic in bacteria, N-nitrosodialkylamines, with unique organotropism in vivo for which organ-specific activation was studied in vitro, N-nitroso compounds that are inactivated in vivo but not in vitro, and components of the aristolocbic acid mixture which may be metabolized oxidatively or reductively, as well as numerous miscellaneous compounds that were expected to be genotoxins on account of their chemical structure. In addition to the assessment of genotoxicity, the results obtained in individual tests of this strategy yield important data on mechanisms of activity, such as organ-specific activation and deactivation, species variations, in vitro~in vivo correlation and persistence or repair of damage.
Quantitative Approaches to Assess Key Carcinogenic Events of Genotoxic Carcinogens
Toxicological research, 2018
Chemical carcinogenesis is a multistep process. Genotoxic carcinogens, which are DNA-reactive, induce DNA adduct formation and genetic alterations in target cells, thereby generating mutated cells (initiation). Subsequently, preneoplastic lesions appear through clonal proliferation of the mutated cells and transform into tumors (promotion and progression). Many factors may influence these processes in a dose-dependent manner. Therefore, quantitative analysis plays an important role in studies on the carcinogenic threshold of genotoxic carcinogens. Herein, we present data on the relationship between key carcinogenic events and their deriving point of departure (PoD). Their PoDs were also compared to those of the carcinogenesis pathway. In an experiment, the liver of rats exposed to 2-amino-3,8-dimethylimidazo-(4,5-)quinoxaline (MeIQx) was examined to determine the formation of MeIQx-DNA adducts, generation of mutations at transgene, and induction of preneoplastic glutathione -transfe...
Regulatory Toxicology and Pharmacology, 1985
Based on the published guidelines of regulatory agencies and the latest scientific findings, an experimental strategy for the routine testing of the mutagenic activity of industrial chemicals in mammalian cells in vitro is designed. The strategy includes three individual experiments: (1) a range-finding experiment for cytotoxicity to determine the appropriate doses to be used in subsequent mutagenicity testing; (2) mutagenicity testing at limited doses with different concentrations of Aroclor 1254-induced rat liver homogenate (S9); and (3) a more extensive dose-response experiment using an S9 concentration determined to be optimum in mutagenicity. Besides satisfying the guidelines of regulatory agencies, this strategy should yield data that allow sound scientific judgment on the mutagenicity of the chemicals tested. o 1985 Academic press, IX.
Mutagenesis, 2011
The need for tools able to predict chemical carcinogens in less time and at a lower cost in terms of animal lives and money is still a research priority, even after several decades of effort in that direction. Now, new regulatory requirements (e.g. the Registration, Evaluation, Authorisation and Restriction of Chemical substances recently implemented in Europe) have even increased the pressure to develop new tools in this field. Drawbacks of the present testing strategies have come to light again recently especially in view of new requirements in worldwide regulations. Among these are (i) the lack of assays able to identify non-genotoxic carcinogens, (ii) the exaggerated rate of misleading (false) positive results of the in vitro mammalian cell-based short-term mutagenicity tests and (iii) the extremely low sensitivity of in vivo short-term mutagenicity tests. Within this perspective, we analyse the contribution of cell transformation assays (CTAs), and we show that they are a valid complement to tools able to detect DNA-reactive carcinogens. We also show that a tiered strategy, with inexpensive and fast tests in Tier 1 (e.g. the Ames test or structural alerts) and the Syrian hamster embryo CTA in Tier 2, is able to identify up to 90% of carcinogens.