In vitro genotoxicity testing needs to include tests in both bacterial and mammalian cells, and be able to detect gene mutations, chromosomal damage and aneuploidy. This may be achieved by a combination of the Ames test (detects gene mutations) and the in vitro micronucleus test (MNvit), since the latter detects both chromosomal aberrations and aneuploidy. In this paper we therefore present an analysis of an existing database of rodent carcinogens and a new database of in vivo genotoxins in terms of the in vitro genotoxicity tests needed to detect their in vivo activity. Published in vitro data from at least one test system (most were from the Ames test) were available for 557 carcinogens and 405 in vivo genotoxins. Because there are fewer publications on the MNvit than for other mammalian cell tests, and because the concordance between the MNvit and the in vitro chromosomal aberration (CAvit) test is so high for clastogenic activity, positive results in the CAvit test were taken as indicative of a positive result in the MNvit where there were no, or only inadequate data for the latter. Also, because Hprt and Tk loci both detect gene-mutation activity, a positive Hprt test was taken as indicative of a mouse-lymphoma Tk assay (MLA)-positive, where there were no data for the latter. Almost all of the 962 rodent carcinogens and in vivo genotoxins were detected by an in vitro battery comprising Ames+MNvit. An additional 11 carcinogens and six in vivo genotoxins would apparently be detected by the MLA, but many of these had not been tested in the MNvit or CAvit tests. Only four chemicals emerge as potentially being more readily detected in MLA than in Ames+MNvit--benzyl acetate, toluene, morphine and thiabendazole--and none of these are convincing cases to argue for the inclusion of the MLA in addition to Ames+MNvit. Thus, there is no convincing evidence that any genotoxic rodent carcinogens or in vivo genotoxins would remain undetected in an in vitro test battery consisting of Ames+MNvit.

A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins.

Introduction: Uses of methotrexate (MTX) are well established for the treatment of various types of malignancy, psoriasis, rheumatological diseases and the medical termination of pregnancy. Formulation and targeting approaches for MTX with controlled release carriers, multiparticulate systems, prodrug and drug conjugates have been found to improve bioavailability, reduce adverse effects and maximize clinical efficacy, compared with conventional methods. Areas covered: This exhaustive literature survey on different electronic databases covers drug delivery and clinical trials on MTX. This review deals with the challenges and achievements of controlled release, multiparticulate, prodrug and drug conjugate systems of MTX. Expert opinion: Therapeutic drug monitoring of MTX is crucial to attain a good efficacy. In spite of the advantages of multiparticulate, prodrug and drug conjugates, clinical applications of such formulations of MTX are still under infancy. These drug delivery systems require the special at...

Methotrexate: a detailed review on drug delivery and clinical aspects.

At a recent ECVAM workshop considering ways to reduce the frequency of irrelevant positive results in mammalian cell genotoxicity tests [D. Kirkland, S. Pfuhler, D. Tweats, M. Aardema, R. Corvi, F. Darroudi, A. Elhajouji, H.-R. Glatt, P. Hastwell, M. Hayashi, P. Kasper, S. Kirchner, A. Lynch, D. Marzin, D. Maurici, J.-R. Meunier, L. Muller, G. Nohynek, J. Parry, E. Parry, V. Thybaud, R. Tice, J. van Benthem, P. Vanparys, P. White, How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary followup animal tests: Report of an ECVAM Workshop, Mutat. Res. 628 (2007) 31-55], recommendations for improvements/modifications to existing tests, and suggestions for new assays were made. Following on from this, it was important to identify chemicals that could be used in the evaluation of modified or new assays. An expert panel was therefore convened and recommendations made for chemicals to fit three different sets of characteristics, namely: This paper therefore contains these three recommended lists of chemicals and describes how these should be used for any test-evaluation programme.

Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop.

A number of drugs have been withdrawn from the market or severely restricted in their use because of unexpected toxicities that become apparent only after the launch of new drug entities. Circumstantial evidence suggests that, in most cases, reactive metabolites are responsible for these unexpected toxicities. In this review, a general overview of the types of reactive metabolites and the consequences of their formation are presented. The current approaches to evaluate bioactivation potential of new compounds with particular emphasis on the advantages and limitation of these procedures will be discussed. Reasonable reasons for the excellent safety record of certain drugs susceptible to bioactivation will also be explored and should provide valuable guidance in the use of reactive-metabolite assessments when nominating drug candidates for development. This will, in turn, help us to design and bring safer drugs to the market.

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Deleterious effects of reactive metabolites.

In 2008 we published recommendations on chemicals that would be appropriate to evaluate the sensitivity and specificity of new/modified mammalian cell genotoxicity tests, in particular to avoid misleading positive results. In light of new data it is appropriate to update these lists of chemicals. An expert panel was convened and has revised the recommended chemicals to fit the following different sets of characteristics: • Group 1: chemicals that should be detected as positive in in vitro mammalian cell genotoxicity tests. Chemicals in this group are all in vivo genotoxins at one or more endpoints, either due to DNA-reactive or non DNA-reactive mechanisms. Many are known carcinogens with a mutagenic mode of action, but a sub-class of probable aneugens has been introduced. • Group 2: chemicals that should give negative results in in vitro mammalian cell genotoxicity tests. Chemicals in this group are usually negative in vivo and non-DNA-reactive. They are either non-carcinogenic or rodent carcinogens with a non-mutagenic mode of action. • Group 3: chemicals that should give negative results in in vitro mammalian cell genotoxicity tests, but have been reported to induce gene mutations in mouse lymphoma cells, chromosomal aberrations or micronuclei, often at high concentrations or at high levels of cytotoxicity. Chemicals in this group are generally negative in vivo and negative in the Ames test. They are either non-carcinogenic or rodent carcinogens with an accepted non-mutagenic mode of action. This group contains comments as to any conditions that can be identified under which misleading positive results are likely to occur. This paper, therefore, updates these three recommended lists of chemicals and describes how these should be used for any test evaluation program.

Updated recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests.

Cytogenetic toxicity of methotrexate in mouse bone marrow.

Modulatory effects of caffeine on methotrexate-induced cytogenotoxicity in mouse bone marrow.

Increased clinical applications of the anticancer drug etoposide (a non-intercalative epipodophyllotoxin derivative) and the frequent induction of a second malignancy, particularly leukaemia, in post-etoposide-treated cancer survivors warrant detailed genotoxicity testing of etoposide. The genotoxicity test results available on etoposide are either primarily in in vitro test systems or in lower organisms after treatment with unusually high doses, or after chronic exposures, having little extrapolative value to humans. Therefore, a cytogenetic risk assessment study on etoposide in mouse in vivo was undertaken after a low dose (in accordance with the human therapeutic dose) single exposure. The cytogenetic toxicity of etoposide was assessed from bone marrow of mouse at three separate endpoints: chromosomal aberration and mitotic index studies at 24 h post-treatment and the micronucleus test (MNT) at 30 h post-treatment. The flame drying technique using colchicine, hypotonic sodium citrate, methanol-glacial acetic acid and Giemsa was followed for the preparation of slides for the metaphase chromosomal aberration and mitotic index studies and a simple technique was followed for the MNT. Although induction of chromosomal aberrations, excluding gaps, per 100 metaphases by 10 and 15 mg kg(-1) etoposide was not significant statistically, 20 mg kg(-1) of etoposide induced a significantly higher number of chromosomal aberrations in female (P < or = 0.01) and male (P < or = 0.05) mice. There was no significant change in the induced percentages of dividing cells by any of the doses of etoposide tested. The micronucleus induction also was not significant statistically with the lowest dose but it was significant in female (P </= 0.01) and male (P </= 0.05) mice that received 15 mg kg(-1) etoposide and was highly significant (P </= 0.01) in both female and male mice that received 20 mg kg(-1) etoposide. The results indicated the clastogenic action of etoposide in mouse bone marrow after a single treatment with such low doses. However, the drug did not interfere with cell cycle progression. Although it is a DNA-non-intercalating agent, etoposide is known for its interference in the activity of DNA topoisomerase IIalpha enzyme, particularly in the proliferative cells where the concentration and activity of the enzyme are greater. This might be the reason for the induction of leukaemia in post-etoposide-treated cancer survivors. Therefore, it has become absolutely necessary to make etoposide target-specific, i.e. specific to the topoisomerase II enzymes of cancerous cells.

Cytogenetic risk assessment of etoposide from mouse bone marrow.

Potential transmission of the cytogenetic toxic effects of methotrexate in the male germline cells of Swiss mice.

Cytosine arabinoside (Ara-C), a widely prescribed antineoplastic drug, especially for the treatment of acute myeloid leukaemia, is a pyrimidine analog, in which the ribose sugar of cytidine is replaced by arabinose moiety. Ara-C reportedly competes with dCTP for incorporation into DNA during synthesis and exhibits various cytogenotoxic effects. In the present study, single intraperitoneal treatment of three different doses of Ara-C, 100, 150 and 200 mg/kg b.w. of mice, selected in accordance with its human therapeutic dose, induced statistically significant (p < or = 0.01) and dose dependent increase in the percentages of aberrant metaphases and chromosomal aberrations (CAs) at 24h post-treatment, and micronuclei (MN) in polychromatic erythrocytes (PCEs) at 30 h post-treatment. However, there was no significant change in the mitotic index (MI) at 24h post-treatment, when compared to that of the control mice. In the male germline, all the three doses of Ara-C induced statistically significant (p < or = 0.05 or p < or = 0.01) and dose dependent increase in the percentages of aberrant spermatogonial metaphases and CAs at 24h post-treatment and statistically significant (p < or = 0.01) and dose dependent increase in the percentages of aberrant primary spermatocytes at week 4 post-treatment. However, the induction of abnormal sperm at week 8 post-treatment was decreased, although significantly. The results indicated that Ara-C was clastogenic to both bone marrow and spermatogonial cells of mice, and some of its cytogenotoxic effects were found transmitted through the male germline, at least up to the formation of primary spermatocytes. As the drug is not target specific, the induced cytogenotoxic effects of Ara-C on non-cancerous cells of cancer patients retain possible risk of recurrence of second malignancy among the post-chemotherapeutic cancer survivors. In addition, there is every risk of transmission of some induced genetic alterations to the next generation through the gametes of the cancer survivors pre-treated with this drug. Therefore, Ara-C essentially be made target specific.

Anil K. Palo

Papers

Cytogenetic toxicity of methotrexate in mouse bone marrow.

Modulatory effects of caffeine on methotrexate-induced cytogenotoxicity in mouse bone marrow.

Cytogenetic risk assessment of etoposide from mouse bone marrow.

Potential transmission of the cytogenetic toxic effects of methotrexate in the male germline cells of Swiss mice.

Cytosine arabinoside-induced cytogenotoxicity in bone marrow and spermatogonial cells of mice and its potential transmission through the male germline