The kidney as a novel target tissue for protein adduct formation associated with metabolism of halothane and the candidate chlorofluorocarbon replacement 2,2‐dichloro‐1,1,1‐trifluoroethane

Abstract

Hydrochlorofluorocarbons (HCFCs) have been identified as chemical replacements of the widely used chlorofluorocarbons (CFCs) that are implicated in stratospheric ozone depletion. Many HCFCs are structural analogues of the anesthetic agent halothane and may follow a common pathway of biotransformation and formation of adducts to protein-centered and other cellular nucleophiles. Exposure of rats to a single dose of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) or of the candidate CFC substitute HCFC 123 (2,2-dichloro-I ,1 ,1-trifluoroethane) led to the formation of trifluoroacetylated protein adducts (CF3CO-proteins) not only in the liver, but also in the kidney as a novel target tissue for protein trifluoroacetylation. CF,CO-proteins in the kidney amounted to about 5% of those formed in the liver of the same animal. The amount of CF,CO-proteins formed within the kidney was roughly reflected by the capacity of metabolism of halothane or HCFC 123 by rat kidney microsomes in vitro which amounted to about 10% of that observed with liver microsomes. By immunohistochemistry, CF3CO-proteins in the kidney were mainly localized in the tubular segments of the cortex. In the liver, the density of CF,CO-proteins decreased from the central vein towards the portal triad. In vitro incubation of rat liver microsomes with halothane or HCFC 123 resulted in extensive formation of CF3CO-proteins and reproduced faithfully the pattern of liver CF,CO-proteins obtained in vivo. CF,CO-proteins generated in vitro were immunochemically not discernible from those generated in vivo. Glutathione (5 mM) and cysteine ( 5 mM) virtually abolished CF,CO-protein formation; the release of Br-from halothane and C1-from HCFC 123 was reduced to much lesser a degree. S-Methyl-glutathione, N-acetyl-cysteine, methionine, and N-acetyl-methionine only slightly affected the formation of CF,CO-proteins or metabolism of either substrate. The data suggest that metabolism and concomitant CF,CO-protein formation of halothane or of candidate CFC replacements like HCFC 123 is not restricted to the liver but also takes place in the kidney. Furthermore, an in vitro system for CF,CO-protein formation has been developed and used to show that protein-centered and glutathione-centered nucleophilic sites compete for intermediates of metabolism of halothane or of HCFC 123.Chlorofluorocarbons (CFCs) have a high ozone-depleting potential [I] and are held responsible for seasonal depleting of stratospheric ozone layers over the Antarctic continent [2]. Ozone destruction leads to an increase of the amount of Correspondence to J. Gut,

Identification of the dihydrolipoamide acetyltransferase subunit of the human pyruvate dehydrogenase complex as an autoantigen in halothane hepatitis

“…Overall, Immunoblots of Llip (1 pg/cm slot width) were incubated in presence of CF,CO-Lys (100 mM), (6RS)-lipoic acid (100 mM), the peptide ETDK(,,,,,,,ATIG (1 mM), the peptide IETD-KATIGFE (1 mM), and L-lysine (100 mM). Immunoblots were developed with the ECL detection system (A, B) followed (B) by scanning densitometry of the obtained films as described [6] and relative signals (as a percentage of the control, set at 100%) were plotted. these data suggest that molecular shape recognition by anti-CF,CO antibody is one, but not the sole, determinant for molecular mimicry of CF,CO-Lys by (6RS)-lipoic acid and related structures.…”

“…In halothane-exposed rats, CF,CO-proteins are found in centrilobular liver sections [6], on the surface of hepatocytes [8], within Kupffer cells [14], in liver microsomal membranes [6, 1.51 and, at low levels, disseminated in the kidneys [6], the heart [16] and the testes [17]. Furthermore, exposure of rats to the newer volatile anesthetics enflurane and isoflurane [18, 191, or to pentahaloethane-based candidate replacements of chlorofluorocarbons such as HCFC 123 , which are close structural analogues of halothane, has been shown to result in the formation of metabolite-protein adducts not discernible immunochemically from CF,CO-proteins.…”

“…Trifluoroacetyl-protein adducts (CF,CO-proteins) arise through covalent modification of target proteins by the acyl halide intermediate CF,COCl that is elicited upon oxidative, cytochrome-P450-dependent metabolism of halothane and also other structurally closely related compounds [3 -71. In fact, with halothane and 2,2-dichloro-l,l,l-trifluoroethane (HCFC 123) as substrates, the predominant adduct formed in the process has been identified by 19F-NMR as W-trifluoroacetyl-L-lysine (CF,CO-Lys detected in the livers of halothane-exposed rats, rabbits, guinea pigs and mice [6,[8][9][10][11] and also in liver biopsies of halothane-exposed humans [12,13]. In halothane-exposed rats, CF,CO-proteins are found in centrilobular liver sections [6], on the surface of hepatocytes [8], within Kupffer cells [14], in liver microsomal membranes [6, 1.51 and, at low levels, disseminated in the kidneys [6], the heart [16] and the testes [17].…”

“…In fact, with halothane and 2,2-dichloro-l,l,l-trifluoroethane (HCFC 123) as substrates, the predominant adduct formed in the process has been identified by 19F-NMR as W-trifluoroacetyl-L-lysine (CF,CO-Lys detected in the livers of halothane-exposed rats, rabbits, guinea pigs and mice [6,[8][9][10][11] and also in liver biopsies of halothane-exposed humans [12,13]. In halothane-exposed rats, CF,CO-proteins are found in centrilobular liver sections [6], on the surface of hepatocytes [8], within Kupffer cells [14], in liver microsomal membranes [6, 1.51 and, at low levels, disseminated in the kidneys [6], the heart [16] and the testes [17]. Furthermore, exposure of rats to the newer volatile anesthetics enflurane and isoflurane [18, 191, or to pentahaloethane-based candidate replacements of chlorofluorocarbons such as HCFC 123 [5-71, which are close structural analogues of halothane, has been shown to result in the formation of metabolite-protein adducts not discernible immunochemically from CF,CO-proteins.…”

“…detected in the livers of halothane-exposed rats, rabbits, guinea pigs and mice [6,[8][9][10][11] and also in liver biopsies of halothane-exposed humans [12,13]. In halothane-exposed rats, CF,CO-proteins are found in centrilobular liver sections [6], on the surface of hepatocytes [8], within Kupffer cells [14], in liver microsomal membranes [6, 1.51 and, at low levels, disseminated in the kidneys [6], the heart [16] and the testes [17].…”