Nephrotoxicity of mercapturic acids of three structurally related 2,2-difluoroethylenes in the rat. Indications for different bioactivation mechanisms.

The biotransformation and the hepato- and nephrotoxicity of the mercapturic acids (N-acetyl-1-cysteine S-conjugates) of three structurally related 2,2-difluoroethylenes were investigated in vivo in the rat. All mercapturic acids appeared to cause nephrotoxicity, without any measureable effect on the liver. The mercapturic acid of tetrafluoroethylene (TFE-NAC) appeared to be the most potent nephrotoxin, causing toxicity upon an i.p. dose of 50 μmol/kg. The mercapturic acids of 1,1-dichloro-2, 2-difluoroethylene (DCDFE-NAC) and 1, 1-dibromo-2,2-difluoroethylene (DBDFE-NAC) were nephrotoxic at slightly higher doses, i.e. at 75 and 100 μmol/kg, respectively. In the urine of TFE-NAC-treated rats significant amounts of difluoroacetic acid (DFAA) could be detected. With increasing doses, the relative amount of DFAA in urine increased progressively (5-18% of dose). In urine of rats treated with DCDFE-NAC and DBDFE-NAC, however, the corresponding dihaloacetic acids, dichloroacetic acid and dibromoacetic acid, could not be detected. Formation of DFAA and pyruvate could also be observed during in vitro metabolism of the cysteine conjugate of tetrafluoroethylene (TFE-CYS) by rat renal cytosol. Inhibition by aminooxyacetic acid (AOA) pointed to a β-lyase dependency for the DFAA-formation. Next to DFAA and pyruvate, also formation of hydrogen sulfide and thiosulfate could be detected. These results suggest that TFE-CYS is bioactivated to a significant extent to difluorothionoacyl fluoride, which most likely is subsequently hydrolysed to difluorothio(no)acetic acid and difluoroacetic acid. According to formation of pyruvate, the cysteine conjugates derived from DCDFE-NAC and DBDFE-NAC also were efficiently metabolized by rat renal β-lyase. However, the formation of corresponding dihaloacetic acids, dichloroacetic acid and dibromoacetic acid, could not be detected in vitro at all. Only very small amounts of hydrogen sulfide and thiosulfate were detected. These results suggest that bioactivation of the latter two conjugates to a dichloro- or dibromothionoacyl fluoride represents only a minor route. Because of better leaving group abilities of chloride and bromide compared to fluoride, rearrangement of the initially formed ethanethiol to a thiirane might be favoured. Based on the present in vivo and in vitro data, it is concluded that the nephrotoxicity of the structurally related mercapturic acids of 2,2-difluoroethylenes is dependent on halogen substitution and presumably the result of at least two different mechanisms of bioactivation. © 1988

Molecular mechanisms of toxic effects of fotemustine in rat hepatocytes and subcellular rat liver fractions.

Fotemustine is a clinically used DNA-alkylating 2-chloroethyl-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

Identification and quantitative determination of glutathione-related urinary metabolites of fotemustine, a new anti-cancer agent.

1. Potential sulphur-containing metabolites of the anticancer agent, fotemustine, were synthesized, namely thiodiacetic acid (TDA), S-2-hydroxyethyl N-acetyl-L-cysteine (2-HE-NAC), N-acetyl-L-cysteine (NAC), S-methyl N-acetyl-L-cysteine (M-NAC), S-carboxymethyl-L-cysteine (CM-Cys), S-carboxymethyl N-acetyl-L-cysteine (CM-NAC), their corresponding sulphoxides and sulphones. Their chemical structures and stabilities were confirmed and derivatization methods were developed for their analysis by sulphur-selective g.l.c. (g.l.c.-FPD) and g.l.c.-mass spectrometry. 2. Four methods for isolation of potential metabolites of fotemustine were developed. Quantification of metabolites, derived in various ways was carried out by g.l.c.-atomic emission detection (AED) or g.l.c.-mass spectrometry. 3. Male Wistar rats (n=4) were given a single i.p. dose of 40mg/kg fotemustine. Urine excretion of TDA (18.4+1.9% in 24h) and TDA sulphoxide (12.0+1.6% in 24h) was significant; 32.7 + 4.6% of the fotemustine dose was excreted as TDA, and TDA sulphoxide in 48 h. NAC was excreted in rat urine at 1% of the dose. No other potential glutathione-derived metabolites of fotemustine were excreted. 4. Male Wistar rats (n = 4) were also treated i.p. with fotemustine at 5,20 and 40 mg/kg, to investigate dose dependency and the time course of excretion of TDA. Excretion of TDA in 48 h urine decreased from 32 + 2 to 17 π 2% dose (mean π SD) with increasing dose of fotemustine. © 1993 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted