The mouse bone marrow micronucleus test: evaluation of 21 drug candidates

The mouse bone-marrow micronucleus test is one of the most widely used genetic toxicology assays. In this report the results of testing 21 compounds in the micronucleus test are presented. Of the 21 compounds tested, 3 potential chemotherapeutic agents were identified as strongly clastogenic. In addition, one compound was identified as a weak inducer of micronuclei in the assay. Further testing of this compound in an in vivo bone marrow metaphase analysis failed to confirm this material as clastogenic. The remaining 17 compounds were classified as negative in the assay. In general the results of the micronucleus test agreed with the results of other genetic toxicology assays on this group of compounds.     

The in vitro unscheduled DNA synthesis (UDS) assay in rat primary hepatocytes: evaluation of 2-furoic acid and 7 drug candidates

The in vitro unscheduled DNA synthesis assay (UDS) is part of the routine genetic toxicology screening at The Upjohn Company. The purpose of this paper is to report results for 8 compounds which were tested in the in-house genetic toxicology program. These compounds represent diverse chemical structure and most of them entered the screening program because they are biologically active in efficacy screens. All tests were carried out under Good Laboratory Practices Regulations of the U.S. Food and Drug Administration. None of the materials reported here produced an increase in UDS and therefore the UDS results with these compounds do not suggest potential for genotoxicity.     

An evaluation of the L5178Y TK+/− mouse lymophoma forward mutation assay using 42 chemicals

The L5178YTK^+/? mouse lymphoma assay (MLA) has been utilized in several laboratories as a short-term test for chemical-induced forward mutation in cultured mammalian cells. In order to evaluate several technical modifications to the MLA, 42 chemicals representing 9 chemical classes were tested and the results were compared with those published elsewhere as well as with findings in a genetic toxicology test battery currently used in this laboratory. A positive response for the induction of TK^+/? mutants was obtained for 26 chemicals. With the exception of p-aminophenol, all of these compounds were recognized mutagens or carcinogens and were represented of direct-acting and activation-dependent genotoxins. 16 compounds did not induce IK^?/? mutanants and among these were 5 compounds that were considered to be mutagens or carcinogens. A comparison of the results of this study with those published elsewhere revealed a strong agreement among findings for this test irrespective of minor technical variations. It was concluded that th MLA is a useful system for identifying chemical mutagens in mammalian cells and can serve as a valuabel component in a genetic toxicology test battery.     

Mercapturic acids as biomarkers of exposure to electrophilic chemicals:applications to environmental and industrial chemicals

The use of mercapturic acids (N-acetyl-L-cysteine S-conjugates, MAs) in the biological monitoring of human exposure to environmental and industrial chemicals is receiving more and more attention. Mercapturic acids (MAs) are formed from glutathione (GSH) S-conjugates via the MA-pathway. Although this pathway can lead to different end-products, the formation of MAs is the predominant route in most species, including man. Two GSH S-transferases (GSTs) show genetic polymorphisms in humans and this can have major consequences for individual susceptibility to toxic effects and for MA formation. In occupational toxicology, adducts to biomacromolecules are also used as biomarkers. DNA adducts are a measure for the effective dose, while protein adducts are related to the dose at critical site. Both type of adducts are normally determined in blood, while MAs are determined in urine. Most MAs are excreted with relatively short half-lifes, allowing a direct evaluation of the occupational circumstances. For many compounds similar (linear) dose-dependency was found for MA excretion, formation of macromolecular adducts, and for various biomarkers of toxic effects. These relations together with fact that MAs relate to the electrophilic character of compounds, allows for the conclusion that MAs are biomarkers of toxicologically relevant internal doses of chemicals or their metabolites. An overview will be given here of the use of MAs in the assessment of internal human exposure to electrophilic environmental and industrial chemicals. Additionally, the formation of GSH S-conjugates, their catabolism to MAs and several of the frequently used analytical approaches are discussed. When appropriate, the influence of genetic polymorphisms on formation of MAs and on susceptibility to toxicity will be discussed for different chemicals as well.     

Mercapturic acids as biomarkers of exposure to electrophilic chemicals:applications to environmental and industrial chemicals

The use of mercapturic acids (N-acetyl-L-cysteine S-conjugates, MAs) in the biological monitoring of human exposure to environmental and industrial chemicals is receiving more and more attention. Mercapturic acids (MAs) are formed from glutathione (GSH) S-conjugates via the MA-pathway. Although this pathway can lead to different end-products, the formation of MAs is the predominant route in most species, including man. Two GSH S-transferases (GSTs) show genetic polymorphisms in humans and this can have major consequences for individual susceptibility to toxic effects and for MA formation. In occupational toxicology, adducts to biomacromolecules are also used as biomarkers. DNA adducts are a measure for the effective dose, while protein adducts are related to the dose at critical site. Both type of adducts are normally determined in blood, while MAs are determined in urine. Most MAs are excreted with relatively short half-lifes, allowing a direct evaluation of the occupational circumstances. For many compounds similar (linear) dose-dependency was found for MA excretion, formation of macromolecular adducts, and for various biomarkers of toxic effects. These relations together with fact that MAs relate to the electrophilic character of compounds, allows for the conclusion that MAs are biomarkers of toxicologically relevant internal doses of chemicals or their metabolites. An overview will be given here of the use of MAs in the assessment of internal human exposure to electrophilic environmental and industrial chemicals. Additionally, the formation of GSH S-conjugates, their catabolism to MAs and several of the frequently used analytical approaches are discussed. When appropriate, the influence of genetic polymorphisms on formation of MAs and on susceptibility to toxicity will be discussed for different chemicals as well.     

Enhancement of bleomycin-induced micronucleus formation in V79 cells as a rapid and sensitive screen for non-covalent DNA-binding compounds.

Non-covalent drug/DNA interactions are difficult to study and because of this, the significance of such interactions from a safety standpoint and their contribution to positive genetic toxicology test findings is poorly understood. It is shown in the present study that such interactions may be detected and quantified in Chinese hamster V79 cells by an adaptation of the bleomycin amplification assay. This assay measures the ability of a test compound to enhance the DNA damaging activity of the antibiotic bleomycin using micronucleus formation as an endpoint. Results are presented examining the bleomycin amplification activity of known intercalating agents, groove-binding agents and other structurally diverse classes of compounds for which intercalative status has not been reported. The assay reveals a strong and predictable SAR for amplification activity based on number and orientation of aromatic rings. Moreover, excellent correlations are observed between DNA binding (viscometric analyses) and DNA amplification in V79 cells for a series of seven experimental compounds. The assay is shown to be useful in understanding the genotoxicity of marketed antihistamines and to help explain genetic toxicology findings observed in a series of novel pharmaceutical entities. It is proposed that assessment of bleomycin amplification activity of novel compounds in early genotoxicity prescreening may provide important information upon which to base synthesis of compounds with minimal or no genotoxic liability.     

Actual problems of genetic toxicology

The review deals with current issues of genetic toxicology and aims to develop this science at the contemporary stage. We study general approaches to assessing the genotoxic and mutagenic activity of environmental factors; to constructing a regulatory system of chemical compounds that considers the mutagenic effect in Russia and abroad; and to determining modern methods for assessing the organ specificity of mutagens, alternative methods of genetic toxicology, the mutagenic action of various factors in the survey of population, and the abilities of toxicogenomics to identify the mutagenic properties of the environment.     

Practical aspects of mutagenicity testing strategy: an industrial perspective

Genetic toxicology studies play a central role in the development and marketing of new chemicals for pharmaceutical, agricultural, industrial, and consumer use. During the discovery phase of product development, rapid screening tests that require minimal amounts of test materials are used to assist in the design and prioritization of new molecules. At this stage, a modified Salmonella reverse mutation assay and an in vitro micronucleus test with mammalian cell culture are frequently used for screening. Regulatory genetic toxicology studies are conducted with a short list of compounds using protocols that conform to various international guidelines. A set of four assays usually constitutes the minimum test battery that satisfies global requirements. This set includes a bacterial reverse mutation assay, an in vitro cytogenetic test with mammalian cell culture, an in vitro gene mutation assay in mammalian cell cultures, and an in vivo rodent bone marrow micronucleus test. Supplementary studies are conducted in certain instances either as a follow-up to the findings from this initial testing battery and/or to satisfy a regulatory requirement. Currently available genetic toxicology assays have helped the scientific and industrial community over the past several decades in evaluating the mutagenic potential of chemical agents. The emerging field of toxicogenomics has the potential to redefine our ability to study the response of cells to genetic damage and hence our ability to study threshold phenomenon.     

Interphase cytogenetics in estimation of genomic mutations in somatic cells

The review considers the current state, possibilities, and perspectives of using interphase cytogenetics in the estimation of genomic mutations in human and animal somatic cells for aims of genetic toxicology and genetic instability analysis. Possible mechanisms underlying action of mutagens causing numeric chromosome aberrations are discussed.     

The automated bone marrow micronucleus test

A new technology is presented which offers high-quality slides enabling the fully automated scoring of large quantities of erythrocytic cells for micronuclei by computerized image analysis. The techniques are applicable to bone marrow specimens as well as to peripheral blood obtained from various species of laboratory animals as well as from man. The key steps leading to this improved slide quality are the total removal of nucleated hematopoietic cells and the production of 'flat' cells by cytocentrifugation on polylysine-coated slides. The new procedures also allow the quantitative elimination of artifact-producing leukocytic granules from the rat bone marrow, even for the Fischer-344 strain, thus making the rat micronucleus test an attractive system for routine purposes in genetic toxicology. In addition, the proportion of immature erythrocytes can, if desired, be increased to more than 90% by using a Percoll step-gradient. This greatly facilitates the peripheral blood micronucleus test in laboratory animals as well as in (splenectomized) humans. First results, using peripheral blood from 2 rat strains, indicate that the immature erythrocyte population is very useful for micronucleus analysis, which encourages the development of a rat peripheral blood micronucleus test. This is an interesting application because it allows repeated testing in the same animals, resulting in fewer rats being needed, as no separate control groups are necessary. A further advantage is the possibility of concomitantly using rats from an ongoing toxicological study for micronucleus testing. The present results demonstrate that the new methodology is a valuable tool for improved micronucleus testing. Possible consequences in the field of genetic toxicology are discussed.     

Follow-up testing of rodent carcinogens not positive in the standard genotoxicity testing battery: IWGT workgroup report

At the Plymouth Third International Workshop on Genotoxicity Testing in June 2002, a new expert group started a working process to provide guidance on a common strategy for genotoxicity testing beyond the current standard battery. The group identified amongst others "Follow-up testing of tumorigenic agents not positive in the standard genotoxicity test battery" as one subject for further consideration [L. Müller, D. Blakey, K.L. Dearfield, S. Galloway, P. Guzzie, M. Hayashi, P. Kasper, D. Kirkland, J.T. MacGregor, J.M. Parry, L. Schechtman, A. Smith, N. Tanaka, D. Tweats, H. Yamasaki, Strategy for genotoxicity testing and stratification of genotoxicity test results-report on initial activities of the IWGT Expert Group, Mutat. Res. 540 (2003) 177-181]. A workgroup devoted to this topic was formed and met on September 9-10, 2005, in San Francisco. This workgroup was devoted to the discussion of when it would be appropriate to conduct additional genetic toxicology studies, as well as what type of studies, if the initial standard battery of tests was negative, but tumor formation was observed in the rodent carcinogenicity assessment. The important role of the standard genetic toxicology testing to determine the mode of action (MOA) for carcinogenesis (genotoxic versus non-genotoxic) was discussed, but the limitations of the standard testing were also reviewed. The workgroup also acknowledged that the entire toxicological profile (e.g. structure-activity relationships, the nature of the tumor finding and metabolic profiles) of a compound needed to be taken into consideration before the conduct of any additional testing. As part of the meeting, case studies were discussed to understand the practical application of additional testing as well as to form a decision tree. Finally, suitable additional genetic toxicology assays to help determine the carcinogenic MOA or establish a weight of evidence (WOE) argument were discussed and formulated into a decision tree.     

Recombinant DNA approaches for the development of metabolic systems used in in vitro toxicology.

In the past few years there has been considerable progress in the development of mammalian cell systems for use in genetic toxicology by the stable transfer of genes/cDNAs coding for drug metabolizing enzymes directly into the target cell. Alternative approaches have also been developed in which mammalian cells are transiently transfected with cDNAs coding for drug-metabolizing enzymes and S9 preparations expressing a single metabolizing enzyme isolated and used for metabolic activation. Progress in these areas is reviewed here and the relative merits of the different approaches are discussed. Work to date has focused primarily on the cytochrome P450 family of enzymes, although other enzyme systems involved in xenobiotic metabolism have been used. The central theme of this review is the transfer of genetic information to improve the metabolic capability of cell systems used in genetic toxicology. However, a basic philosophy of the review is that genetic manipulation of cultured mammalian cells has the potential for developing systems to be used to better understand chemically induced toxicological effects.     

Genetic toxicology: can we design predictive in vivo assays?

The present analysis examines the assumptions in, the perceptions and predictivity of and the need for short-term tests (STTs) for genotoxicity in light of recent findings that most noncarcinogens from the National Toxicology Program are genotoxic (i.e., positive in one or more in vitro STTs). Reasonable assumptions about the prevalence for carcinogens (1-10% of all chemicals), the sensitivity of these STTs (ca. 90% of all carcinogens are genotoxic) and their estimated "false positive" incidence (60-75%) imply that the majority of chemicals elicit genotoxic responses and, consequently, that most in vitro genotoxins are likely to be noncarcinogenic. Thus, either the usual treatment conditions used in these in vitro STTS are producing a large proportion of artifactual and meaningless positive results or else in vitro mutagenicity is too common a property of chemicals to serve as a useful predictor of carcinogenicity or other human risk. In contrast, the limited data base on in vivo STTs suggests that the current versions of these assays may have low sensitivity which appears unlikely to improve without dropping either their 'short-term' aspect or the rodent carcinogenicity benchmark. It is suggested that in vivo genotoxicity protocols be modified to take into consideration both the fundamentals of toxicology as well as the lessons learned from in vitro genetic toxicology. In the meantime, while in vivo assays are undergoing rigorous validation, genetic toxicology, as currently practiced, should not be a formal aspect of chemical or drug development on the grounds that it is incapable of providing realistic and reliable information on human risk. It is urged that data generated in new, unvalidated in vivo genotoxicity assays be exempted from the normal regulatory reporting requirements in order to encourage industry to participate in the laborious and expensive development of this next phase of genetic toxicology.     

Vanadium in Biological Action: Chemical,Pharmacological Aspects,and Metabolic Implications in Diabetes Mellitus

Vanadium compounds have been primarily investigated as potential therapeutic agents for the treatment of various major health issues, including cancer, atherosclerosis, and diabetes. The translation of vanadium-based compounds into clinical trials and ultimately into disease treatments remains hampered by the absence of a basic pharmacological and metabolic comprehension of such compounds. In this review, we examine the development of vanadium-containing compounds in biological systems regarding the role of the physiological environment, dosage, intracellular interactions, metabolic transformations, modulation of signaling pathways, toxicology, and transport and tissue distribution as well as therapeutic implications. From our point of view, the toxicological and pharmacological aspects in animal models and humans are not understood completely, and thus, we introduced them in a physiological environment and dosage context. Different transport proteins in blood plasma and mechanistic transport determinants are discussed. Furthermore, an overview of different vanadium species and the role of physiological factors (i.e., pH, redox conditions, concentration, and so on) are considered. Mechanistic specifications about different signaling pathways are discussed, particularly the phosphatases and kinases that are modulated dynamically by vanadium compounds because until now, the focus only has been on protein tyrosine phosphatase 1B as a vanadium target. Particular emphasis is laid on the therapeutic ability of vanadium-based compounds and their role for the treatment of diabetes mellitus, specifically on that of vanadate- and polioxovanadate-containing compounds. We aim at shedding light on the prevailing gaps between primary scientific data and information from animal models and human studies.

     

Interphase cytogenetics in estimation of genomic mutations in somatic cells

The review considers the current state, possibilities, and perspectives of using interphase cytogenetics in the estimation of genomic mutations in human and animal somatic cells for aims of genetic toxicology and genetic instability analysis. Possible mechanisms underlying action of mutagens causing numeric chromosome aberrations are discussed.Translated from Genetika, Vol. 41, No. 1, 2005, pp. 5–16.Original Russian Text Copyright © 2005 by Timoshevsky, Nazarenko.     

Hormesis: changing view of the dose-response,a personal account of the history and current status

This paper provides a personal account of the history of the hormesis concept, and of the role of the dose response in toxicology and pharmacology. A careful evaluation of the toxicology and pharmacology literatures suggests that the biphasic dose response that characterizes hormesis may be much more widespread than is commonly recognized, and may come to rival our currently favored ideas about toxicological dose responses confined to the linear and threshold representations used in risk assessment. Although hormesis-like biphasic dose responses were already well-established in chemical and radiation toxicology by the early decades of the 20th century, they were all but expunged from mainstream toxicology in the 1930s. The reasons may be found in a complex set of unrelated problems of which difficulties in replication of low-dose stimulatory responses resulting from poor study designs, greater societal interest in high-dose effects, linking of the concept of hormesis to the practice of homeopathy, and perhaps most crucially a complete lack of strong leadership to advocate its acceptance in the right circles. I believe that if hormesis achieves widespread recognition as a valid and valuable interpretation of dose-response results, we would expect an increase in the breadth of evaluations of the dose-response relationship which could be of great value in hazard and risk assessment as well as in future approaches to drug development and/or chemotherapeutics.     

A review of the genotoxicity of marketed pharmaceuticals

Information in the 1999 Physician's Desk Reference as well as from the peer-reviewed published literature was used to evaluate the genotoxicity of marketed pharmaceuticals. This survey is a compendium of genotoxicity information and a means to gain perspective on the inherent genotoxicity of structurally diverse pharmaceuticals. Data from 467 marketed drugs were collected. Excluded from analysis were anti-cancer drugs and nucleosides, which are expected to be genotoxic, steroids, biologicals and peptide-based drugs. Of the 467 drugs, 115 had no published gene-tox data. This group was comprised largely of acutely administered drugs such as antibiotics, antifungals, antihistamines decongestants and anesthetics. The remaining 352 had at least one standard gene-tox assay result. Of these, 101 compounds (28.7%) had at least one positive assay result in the pre-ICH/OECD standard four-test battery (bacterial mutagenesis, in vitro cytogenetics, mouse lymphoma assay (MLA), in vivo cytogenetics). Per assay type, the percentage of positive compounds was: bacterial mutagenesis test, 27/323 (8.3%); in vitro cytogenetics 55/222 (24.8%); MLA 24/96 (25%); in vivo cytogenetics 29/252 (11.5%). Of the supplemental genetic toxicology test findings reported, the sister chromatid exchange (SCE) assay had the largest percentage of positives 17/39 (43.5%) and mammalian mutagenesis assays (excluding MLA) had the lowest percentage of positives 2/91 (2.2%). The predictive value of genetic toxicology findings for 2-year bioassay outcomes is difficult to assess since carcinogenicity can occur via non-genotoxic mechanisms. Nevertheless, the following survey findings were made: 201 drugs had both gene-tox data and rodent carcinogenicity data. Of these, 124 were negative and 77 were equivocal or positive for carcinogenicity in at least 1 gender/1 species. Of the 124 non-carcinogens, 100 had no positive gene-tox findings. Of the remaining 24, 19 were positive in in vitro cytogenetics assays. Among the 77 compounds that exhibited equivocal or positive effects in carcinogenesis studies, 26 were positive in gene-tox assays and 51 were negative. Of the 51 negatives, 47 had multiple negative gene-tox assay results suggesting that these are probably non-genotoxic carcinogens. Statistical analyses suggested that no combination of gene-tox assays provided a higher predictivity of rodent carcinogenesis than the bacterial mutagenicity test itself.     

浅论城市害蜂的危害与治理

蜂害是我国城市化发展过程中出现的新问题。该文综述了国内外螫人害蜂的种类、习性与危害 ,蜂毒的成分与毒理 ,被蜂螫伤者的救治及蜂害的治理方法 ,分析了目前我国所面临的城市蜂害问题 ,并提出了应采取的相应对策     

Genetic duplications in bacteria and their relevance for genetic toxicology

Tandem genetic duplications of various lengths occur at high frequency and at many chromosomal locations in bacteria. Most duplications are formed and lost by recombinational mechanisms. Since they readily give rise to haploid segregants, duplications are characteristically unstable. Various selection procedures permit measurements of duplication frequencies, and several mutagens have been shown to induce the formation of duplications in haploid and the loss of duplications from merodiploid bacteria. Although the data base is not extensive, it includes agents that interact with DNA by a variety of molecular mechanisms. Grounds on which the induction of genetic duplications in bacteria can be relevant for genetic toxicology are discussed.     

On key lesions and all that: a tribute to Paul Lohman

This paper is a tribute to Paul Lohman at the occasion of his retirement from the position of Professor in the Medical Faculty at the Leiden University in The Netherlands and as Director of its Department of Radiation Genetics and Chemical Mutagenesis. Paul's contributions to the science of genetic toxicology are discussed in the context of more recent insights as to how mammalian cells process DNA damage, and how this may lead to cancer and, possibly, aging. Starting with his work on the characterization of UV-induced DNA repair in cultured cells from xeroderma pigmentosum patients and the development of methodology for monitoring the removal of UV-induced lesions in human cells, the concept of the key lesion is introduced. Among the myriad of DNA lesions that can be induced in DNA as a consequence of exposure to a range of natural or synthetic mutagens, key lesions are the ones responsible for subsequent adverse effects, for example, because they give rise to mutation. The development of methods using immunofluorescence microscopy to detect and identify such key lesions and quantitate them at the single cell level, is one of the highlights of Paul's career. Based on the perceived need to evaluate mutational end points in vivo in relation to specific lesions identified by his immunofluorescence methods, Paul subsequently made crucial contributions to the development of the first transgenic mouse model to measure mutations in chromosomally integrated reporter genes. In parallel to his experimental work, Paul greatly contributed to genetic toxicology at the theoretical level by his work on the development and evaluation of methods for assessment or prediction of risks of exposure to environmental mutagens. Finally, Paul has served the discipline of genetic toxicology in a more administrative role in various ways, both locally as one of the founders of the Medical Genetics Center South-West Netherlands and internationally by playing a prominent role in organizations such as ICPEMC. Here, his numerous contributions to the journal Mutation Research, both as author on many papers and as Executive Managing Editor should not go unmentioned.