In vivo detection and characterization of protein adducts resulting from bioactivation of haloethene cysteine S-conjugates by fluorine-19 NMR: chlorotrifluoroethene and tetrafluoroethene

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Abstract
Several haloalkenes are selective nephrotoxins. The bioactivation of nephrotoxic haloalkenes involves hepatic glutathione S-conjugate formation, peptidase-catalyzed metabolism of the glutathione S-conjugates to the corresponding cysteine S-conjugates, uptake of cysteine S-conjugates by the kidneys, and renal cysteine conjugate beta-lyase-catalyzed beta-elimination of a thiol. The haloalkyl and haloalkenyl thiols thus released are unstable and yield reactive intermediates whose interactions with cellular constituents are thought to contribute to the observed toxicity of S-conjugates. Tetrafluoroethene and chlorotrifluoroethene are metabolized to the cysteine S-conjugates S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC), respectively. Administration of TFEC (1.0 mmol/kg) or CTFC (1.0 mmol/kg) to rats resulted in acylation of renal proteins, as demonstrated with F-19 nuclear magnetic resonance spectroscopy. Single, broad resonances near 41 or 56 ppm were found in spectra of renal proteins from TFEC- or CTFC-treated rats, respectively, and these resonances were not lost on dialysis. Renal protein incubated with 2-chloro-1,1,2-trifluoroethyl 2-nitrophenyl disulfide, a proreactive intermediate that yields 2-chloro-1,1,2-trifluoroethanethiol, showed the same F-19 NMR spectrum as was found with CTFC-treated rats. In vitro incubation of various N(alpha)-blocked amino acids with this proreactive intermediate indicated that only lysine is stably adducted, whereas histidine is transiently acylated. In each case, proteolysis of modified protein converted a single broad NMR resonance to a doublet with little change in chemical shift and with clearly resolved, characteristic H-F couplings. The single, stable amino acid adduct formed with renal proteins of rats given CTFC or TFEC was N(epsilon)-(chlorofluorothioacetyl)lysine and N(epsilon)-(difluorothioacetyl)lysine, respectively.

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