Chromosome aberration frequency in rat peripheral lymphocytes increases with repeated dosing with hexamethylphosphoramide or cyclophosphamide

Ann T. Doherty Julie Hayes Paul Holme 0 Mike O'Donovan 0 DMPK Department, AstraZeneca R&D , Alderley Park, Macclesfield, Cheshire SK10 4TG , UK TThheeAAuutthhoorr22001122..PPuubblilsishheeddbbyyOOxxfofordrdUUninvievresristiytyPrPersesssononbebheahlafloffotfhethUeKUKEnEvnirvoinromnemnetanltaMl uMtaugtaegneSnoScioectiye.ty. AAllll rriigghhttss rreesseerrvveedd.. FFoorr ppeerrmmiissssiioonnss,,pplleeaasseeee--mmaaiill::jjoouurrnnaallss..ppeerrmmiissssiioonnss@@oouupp..ccoomm.. - *To whom correspondence should be addressed. Tel: 44 1625 31285; Fax: 44 1625 231281; Email: Received on December 19, 2011; revised on March 2, 2012; accepted on March 7, 2012 Although there are several in vivo tests for potential genotoxicity, with the possible exception of the transgenic rodent mutation models, none is specifically intended to assess increasing damage with chronic administration. In principle, peripheral blood lymphocytes would be expected to accumulate DNA damage with repeated dosing because the majority are not in active division and appear to have limited DNA repair capability, and they are exposed to plasma levels of test materials and metabolites. However, there appear to be no published reports confirming this principle. Therefore, in the current study, after optimising culture conditions for rat lymphocytes in this laboratory, rats were given oral doses of cyclophosphamide or hexamethylphosphoramide (HMPA) for up to 28 days and peripheral lymphocytes analysed for chromosome aberrations at various time points. The results clearly show that, for both compounds, doses that gave no significant increases in aberration frequency after 2 days induced clear increases after 15 days with further damage detectable after 28 doses. With HMPA, it was shown that DNA damage persisted for at least 10 days after cessation of treatment. These data show that repeat dose studies in the rat measuring chromosome aberration frequency in lymphocytes can give a genuine indication that genotoxicity may increase with chronic administration and, therefore, maybe useful in assessing the risk of potentially genotoxic substances. Introduction Although there are several well-established in vivo tests for genotoxicity using acute dosing, with the exception of transgenic rodent mutation models (1), there is no test that routinely requires repeat dosing or in which genotoxic damage appears to increase with multiple administrations. Although incorporating genotoxicity endpoints into pivotal 14- to 28-day general toxicity studies is increasing and is an option in the revised International Conference of Harmonisation guideline (2), this is essentially intended to reduce animal numbers rather than to gain additional information on the effects of longer term administration of the test agent. For example, the bone marrow micronucleus endpoint is considered to report genetic damage induced only in 2448 h before sampling, irrespective of the length of the period of dosing up to that point. The FDA Guidance on the Recommended Approaches to Integration of Genetic Toxicology Study Results (3) identifies metaphase analysis of peripheral blood lymphocytes from repeat dose studies in rats as an additional in vivo assay that can be useful in clarifying in vitro positive results, but again it is not clear whether this is considered to be a genuine chronic genotoxicity assay. However, AstraZeneca does have a case where negative results from a rat 28-day chromosome aberration test were accepted as part of a package of studies to support administration to man for up to 1 month of a compound that had a genotoxic metabolite. Using peripheral lymphocytes as a marker in a chronic genotoxicity test is theoretically attractive because they are exposed to blood levels of drug and metabolites and damage might be expected to accumulate with time since the cell population is not in division. The lifespan of peripheral lymphocytes varies between species but, in the rat, the median survival time is 1 month and .5% have a lifespan of .9 months (4). However, there appear to be very few reports in the literature to support the hypothesis that DNA damage in them does actually increase with duration of treatment. In terms of DNA repair capability, in vitro studies using freshly isolated human lymphocytes have shown that DNA strand breaks induced by c-irradiation are rapidly repaired (5,6) and gene expression analysis has shown that all genes required for repair of damage induced by c-irradiation are expressed in both phytohaemagglutinin (PHA)-stimulated and resting lymphocytes (7). However, it has also been shown that non-cycling lymphocytes are much more sensitive to UV-B irradiation than those stimulated with PHA and this appears to be because they are functionally excision repair deficient due to very low intracellular deoxyribonucleotide pools (8). In terms of persistence, maximum levels of DNA adducts were seen 4 days after a single intraperitoneal injection of benzo[a]pyrene (BP) to rats and the apparent half-life of adducts in peripheral blood lymphocytes was 17 days, similar to those in liver and lung, 15 and 22 days, respectively (9). Also, increased frequencies of sister chromatid exchanges were still detectable in the lymphocytes of rats 28 days after a single dose of ethyl methanesulphonate (EMS) (10). These data indicate that, at least for BP and EMS, DNA adducts and damage would be expected to accumulate in lymphocytes with repeated dosing. The objective of this study, therefore, was to optimise a protocol for detecting chromosome breakage in the peripheral lymphocytes of rats in order to examine whether DNA damage accumulated during 28 days dosing with two reference genotoxins, cyclophosphamide (CPA) and hexamethylphosphoramide (HMPA). CPA was chosen as it is a common positive control used within our laboratory, and we had some knowledge of doses that would be tolerated for up to 28 days. HMPA was chosen for a number of reasons: we were looking for a compound to give genotoxic damage without immuosuppression of the lymphocytes; in addition, we were interested in the broader genotoxicity of this compound and also because HMPA could be dosed orally. CPA has been in widespread clinical use for over 50 years as a chemotherapeutic agent and immunosuppressant. It is a known human carcinogen and an increased incidence of chromosome aberrations has been seen in lymphocytes from patients treated for both malignant and non-malignant diseases (11,12). The genotoxicity of CPA has been reviewed (13) and it is active in numerous in vivo and in vitro systems and is also teratogenic; consequently, it has been used routinely as a positive control in various genotoxicity tests. It requires metabolic activation via cytochrome P450 (CYP)-mediated hydroxylation to 4-hydroxycyclophosphamide and subsequent breakdown to form two cytotoxic metabolites, phosphoramide mustard and acrolein in both humans and rats (Figure 1) (13). The systemic c (...truncated)