title: Molecular mechanisms of toxic effects of fotemustine in rat hepatocytes and subcellular rat liver fractions.
abstract: Fotemustine is a clinically used DNA-alkylating 2-chloro-ethyl-substituted N-nitrosourea, which sometimes shows signs of haematotoxicity and reversible liver and renal toxicity as toxic side-effects. Mechanistic data on these side-effects are scarce and incomplete. In this study, firstly the cytotoxicity of fotemustine in freshly isolated rat hepatocytes was investigated and secondly the metabolism of fotemustine and possible mechanisms involved in the observed cytotoxicity. Fotemustine caused concentration- and time-dependent cytotoxic effects in rat hepatocytes. Extensive GSH-depletion and formation of GSSG were first observed, followed by lipid peroxidation and finally by cell death measured as LDH-leakage. 2-Chloroethyl analogues of fotemustine, which in contrast to fotemustine have no carbamoylating potency, were not toxic to rat hepatocytes. The data suggest that the cytotoxicity of fotemustine is resulting from its reactive decomposition product, DEP-isocyanate. GSH-conjugation of DEP-isocyanate was shown to protect against the cytotoxicity of fotemustine, however, only temporary and not completely. Synthetical DEP-SG, the GSH-conjugate of DEP-isocyanate, was also toxic to rat hepatocytes, albeit to a significantly lesser extent than fotemustine. In rat liver microsomes, no fotemustine-induced LPO was observed, suggesting that reactive decomposition products of fotemustine do not directly cause peroxidation of cellular membranes. Fotemustine did not affect the antioxidant enzymes superoxide dismutase, catalase, GSH-peroxidase, GSSG-reductase and GSH S-transferases. Thus, direct effects on these antioxidant enzymes are not likely to explain the cytotoxic effects of fotemustine in hepatocytes. In conclusion, it is proposed that the cytotoxicity of fotemustine in rat hepatocytes is caused by rapid and extensive depletion of GSH by DEP-isocyanate, a reactive decomposition product of fotemustine, consequently hampering the endogenous protection against its own toxicity. Knowledge of molecular mechanisms of the cytotoxicity of fotemustine may contribute to a more rational design of selective protection against toxic side-effects which occur upon therapy of patients with fotemustine.
authors.affiliation: Leiden/Amsterdam Center for Drug Research, Division of Molecular Toxicology, Free University, The Netherlands.
authors.forename: J P
authors.initials: JP
authors.lastname: Brakenhoff
authors.forename: J N
authors.initials: JN
authors.lastname: Commandeur
authors.forename: L W
authors.initials: LW
authors.lastname: Wormhoudt
authors.forename: E J
authors.initials: EJ
authors.lastname: Groot
authors.forename: N P
authors.initials: NP
authors.lastname: Vermeulen
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity*; D002374:Catalase / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000172:deficiency; D019803:Glutathione Disulfide; D005979:Glutathione Peroxidase / Q000378:metabolism; D005980:Glutathione Reductase / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D015227:Lipid Peroxidation; D008099:Liver / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D009607:Nitrosourea Compounds / Q000633:toxicity*; D009943:Organophosphorus Compounds / Q000633:toxicity*; D051381:Rats; D013347:Subcellular Fractions / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D009607:Nitrosourea Compounds; D009943:Organophosphorus Compounds; D007770:L-Lactate Dehydrogenase; D002374:Catalase; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; D005980:Glutathione Reductase; D005982:Glutathione Transferase; D005978:Glutathione; C054368:fotemustine; D019803:Glutathione Disulfide