ToxSci Advance Access originally published online on December 28, 2005
Toxicological Sciences 2006 90(2):500-509; doi:10.1093/toxsci/kfj086
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Characterizing the Ovotoxicity of Cyclophosphamide Metabolites on Cultured Mouse Ovaries
Université du Québec, Institut national de la recherche scientifique, Institut Armand-Frappier, Pointe Claire, Québec, H9R 1G6 Canada
Received October 25, 2005; accepted December 20, 2005
Cyclophosphamide (CPA) is reported to target dormant primordial ovarian follicles in rodents and humans. However, mechanistic studies are complicated due to the complex ovarian structure. We present here the characterization of the sensitivity of ovaries to CPA metabolites and the timing of morphological alterations induced by phosphoramide mustard (PM) in an in vitro system. Intact mouse ovaries (postnatal-day-4) were cultured in vitro and exposed to multiple breakdown products of CPA on day 0 (d0). Tissues were cultured up to d8, and then follicle counts and immunohistochemistry were performed. 4-Hydroperoxy-CPA (4-HC), a precursor of an activated form of CPA, and PM depleted primordial and primary follicles (
1 µM and 3 µM, respectively, p < 0.05); acrolein had effects on follicle numbers only under continuous exposure (30 µM); carboxycyclophosphamide and 4-ketocyclophosphamide reduced primordial and small primary follicles only at high concentrations (100 µM). PM-induced follicle loss became significant (p < 0.05) by d1 or d2 following exposures to 10 µM or 3 µM PM, respectively, as determined by the numbers of pyknotic or TUNEL-positive follicles. Cellular targets were oocytes in the smallest follicles, but granulosa cells in large primary follicles. TUNEL staining was observed in both cell types, but caspase-3, a marker of apoptosis, was absent from primordial follicles. In addition, a pan-caspase inhibitor could not prevent follicle losses when administered prior to PM. Thus, brief exposures to 4-HC or PM are sufficient to induce permanent follicle loss in ovaries, and PM is likely the ultimate ovotoxicant. Furthermore, the cell death pathway is likely caspase-independent.
Key Words: cyclophosphamide; chemotherapy; phosphoramide mustard; ovarian toxicity; in vitro; mouse; follicle.
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