Acid‐labile adducts to protein can be used as indicators of the cysteineS‐conjugate pathway of trichloroethene metabolism

Abstract

Covalent binding of radiolabel to tissue proteins following [14C]trichloroethene (TRI) exposure has been used as a measure of TRI activation. Gross binding of 14C label does not differentiate between alternate routes of metabolism and can be confounded when there is significant metabolic incorporation of radiolabel. We examined the covalent association of 14C label to hepatic and renal proteins in male F344 rats and B6C3F1 mice following oral treatment with [14C]TRI and three metabolites of TRI: [14C]trichloroacetate (TCA), [14C]dichloroacetate (DCA), and [14C]dichlorovinylcysteine (DCVC) in vivo. Association of radiolabel from [14C]TRI with hepatic proteins reached a maximum at 2 and 4 h in mouse and rat hepatic proteins, respectively. Association of radiolabel with renal proteins reached a maximum at 8 h in both species. An approach was developed based upon formation of protein adducts that release acetate and monochloroacetate (MCA) on acid hydrolysis. These adducts were found to be specifically associated with the activation of DCVC to reactive intermediates. Acetate and MCA were identified by using two different conditions of high-performance liquid chromatography (HPLC) separation with differing selectivity. Diethylmaleate and aminooxyacetic acid pretreatment inhibited the formation of these adducts from TRI, consistent with requirements for glutathione and beta-lyase. No evidence of these adducts was detected following [14C]TCA and [14C]DCA treatment. Renal acid-labile adduct formation from 25 mg/kg DCVC was approximately 12-fold greater in male B6C3F1 mice than in male F344 rats. They accounted for 7.8 and 4.6% of the total adducts to renal protein in rats and mice, respectively. Acid-labile adducts formed from 1000 mg/kg TRI were approximately two times greater in mice than rats. In this case, they accounted for 1.4 and 3.3% of the total adduct formed in renal proteins from TRI (corrected for metabolic incorporation), respectively. This greater dilution of adducts associated with DCVC in renal proteins of the rat suggests that covalent binding of TRI has less specificity for the DCVC pathway in rats than in mice.

Metabolism of trichloroethylene.

“…While this would seem to make sense, as it is the quantity of the reactive species that is formed that can produce cellular injury and not, as discussed below, the quantity of stable endproducts that are excreted into the urine (e.g., mercapturates), the methodology is potentially complicated by artifacts due to chemical instability of the molecules being quantified. Additionally, Eyre et al (122,123) found that apparent net activation of TCE by the 1-lyase pathway and apparent acid-labile adduct formation were greater in mice than in rats, which does not correspond to the higher susceptibility of rats to renal toxicity and carcinogenesis, although adduct formation did correspond to increased cell replication rates. Hence, the relationship between adduct formation and effects on cellular function and homeostasis are very complex and require further study.…”

“…The initial 3-lyase activities that were purified from rat (115) and human (113) (122,123) suggested that measurement of the acid-labile adducts could be used as an index of flux through the ,B-lyase pathway. While this would seem to make sense, as it is the quantity of the reactive species that is formed that can produce cellular injury and not, as discussed below, the quantity of stable endproducts that are excreted into the urine (e.g., mercapturates), the methodology is potentially complicated by artifacts due to chemical instability of the molecules being quantified.…”

Development of a Physiologically-Based Pharmacokinetic Model of Trichloroethylene and Its Metabolites for Use in Risk Assessment

“…The significance of this result is enhanced by the observation that activation of DCVC in the kidney appears to be much greater in the mouse than in the rat, and the mouse also appears to be more responsive to the induction of cell proliferation by DCVC than the rat (138). Moreover, measurements of acid-labile protein adducts in the kidney associated with DCVC suggest that the production of DCVC-derived reactive species in the kidney resulting from an oral dose of 1,000 mg/kg TCE may actually be greater in mice than in rats (137,138), and mice but not rats showed increased cell proliferation in the kidney in response to treatment with TCE at 1,000 mg/kg. Other studies in the rat also fail to support the suggestion that significant hyperplasia is produced in the kidney from exposure of rats to TCE (163).…”

“…In a study with PERC (136), it was determined that the excretion of the N-acetyl derivative was dose related (a higher fraction of N-acetyl derivative was excreted at doses where the oxidative pathway was saturated) and was significantly greater in the rat than in the mouse. However, measurements of acid-labile protein adducts associated with DCVC suggest that the activation of DCVC in the kidney may be as much as 12-fold greater in mice than in rats and that the kidney tissue exposure to DCVC-derived reactive species from oral dosing with TCE may be twice as great in the mouse as in the rat (137,138).…”

Effects of Genetic Polymorphisms of N-Acetyltransferase on Trichloroethylene-Induced Hypersensitivity Dermatitis among Exposed Workers

“…The effect of NAT2 genetic polymorphism on individual susceptibility to TCE-induced hypersensitivity dermatitis may be due to the lower NAT2 activity caused by gene mutation increase the metabolic flux through bioactivation catalyzed by β-lyase to produced large quantities of reactive intermediate. Eyre 36,37) . It is suggested that adducts could act as one of effective immune antigen which trigger abnormal immunological response and induced the hypersensitivity dermatitis.…”