Liver Protein Targets of Hepatotoxic 4‑Bromophenol Metabolites

The hepatotoxicity of bromobenzene (BB) is directly related to the covalent binding of both initially formed epoxide and secondary quinone metabolites to at least 45 different liver proteins. 4-Bromophenol (4BP) is a significant BB metabolite and a precursor to reactive quinone metabolites; yet, when administered exogenously, it has negligible hepatotoxicity as compared to BB. The protein adducts of 4BP were thus labeled as nontoxic [Monks, T. J., Hinson, J. A., and Gillette, J. R. (1982) Life Sci. 30, 841−848]. To help identify which BB-derived adducts might be related to its cytotoxicity, we sought to identify the supposedly nontoxic adducts of 4BP and eliminate them from the BB target protein list. Administration of [¹⁴C]-4BP to phenobarbital-induced rats resulted in covalent binding of 0.25, 0.33, and 0.42 nmol equiv 4BP/mg protein in the mitochondrial, microsomal, and cytosolic fractions, respectively. These values may be compared to published values of 3–6 nmol/mg protein from a comparable dose of [¹⁴C]-BB. After subcellular fractionation and 2D electrophoresis, 47 radioactive spots on 2D gels of the mitochondrial, microsomal, and cytosolic fractions were excised, digested, and analyzed by LC-MS/MS. Twenty-nine of these spots contained apparently single proteins, of which 14 were nonredundant. Nine of the 14 are known BB targets. Incubating freshly isolated rat hepatocytes with 4BP (0.1–0.5 mM) produced time- and concentration-dependent increases in lactate dehydrogenase release and changes in cellular morphology. LC-MS/MS analysis of the cell culture medium revealed rapid and extensive sulfation and glucuronidation of 4BP as well as formation of a quinone-derived glutathione conjugate. Studies with 7-hydroxycoumarin, (−)-borneol, or d-(+)-galactosamine showed that inhibiting the glucuronidation/sulfation of 4BP increased the formation of a GSH-bromoquinone adduct, increased covalent binding of 4BP to hepatocyte proteins, and potentiated its cytotoxicity. Taken together, our data demonstrate that protein adduction by 4BP metabolites can be toxicologically consequential and provide a mechanistic explanation for the failure of exogenously administered 4BP to cause hepatotoxicity. Thus, the probable reason for the low toxicity of 4BP in vivo is that rapid conjugation limits its oxidation and covalent binding and thus its toxicity

Metabolism and Toxicity of Thioacetamide and Thioacetamide <i>S</i>‑Oxide in Rat Hepatocytes

The hepatotoxicity of thioacetamide (TA) has been known since 1948. In rats, single doses cause centrolobular necrosis accompanied by increases in plasma transaminases and bilirubin. To elicit these effects, TA requires oxidative bioactivation, leading first to its <i>S</i>-oxide (TASO) and then to its chemically reactive <i>S</i>,<i>S</i>-dioxide (TASO₂), which ultimately modifies amine-lipids and proteins. To generate a suite of liver proteins adducted by TA metabolites for proteomic analysis and to reduce the need for both animals and labeled compounds, we treated isolated hepatocytes directly with TA. Surprisingly, TA was not toxic at concentrations up to 50 mM for 40 h. On the other hand, TASO was highly toxic to isolated hepatocytes as indicated by LDH release, cellular morphology, and vital staining with Hoechst 33342/propidium iodide. TASO toxicity was partially blocked by the CYP2E1 inhibitors diallyl sulfide and 4-methylpyrazole and was strongly inhibited by TA. Significantly, we found that hepatocytes produce TA from TASO relatively efficiently by back-reduction. The covalent binding of [¹⁴C]-TASO is inhibited by unlabeled TA, which acts as a “cold-trap” for [¹⁴C]-TA and prevents its reoxidation to [¹⁴C]-TASO. This in turn <i>increases</i> the net consumption of [¹⁴C]-TASO despite the fact that its oxidation to TASO₂ is inhibited. The potent inhibition of TASO oxidation by TA, coupled with the back-reduction of TASO and its futile redox cycling with TA, may help explain phenomena previously interpreted as “saturation toxicokinetics” in the in vivo metabolism and toxicity of TA and TASO. The improved understanding of the metabolism and covalent binding of TA and TASO facilitates the use of hepatocytes to prepare protein adducts for target protein identification

Metabolism and Toxicity of Thioacetamide and Thioacetamide <i>S</i>‑Oxide in Rat Hepatocytes

The hepatotoxicity of thioacetamide (TA) has been known since 1948. In rats, single doses cause centrolobular necrosis accompanied by increases in plasma transaminases and bilirubin. To elicit these effects, TA requires oxidative bioactivation, leading first to its <i>S</i>-oxide (TASO) and then to its chemically reactive <i>S</i>,<i>S</i>-dioxide (TASO₂), which ultimately modifies amine-lipids and proteins. To generate a suite of liver proteins adducted by TA metabolites for proteomic analysis and to reduce the need for both animals and labeled compounds, we treated isolated hepatocytes directly with TA. Surprisingly, TA was not toxic at concentrations up to 50 mM for 40 h. On the other hand, TASO was highly toxic to isolated hepatocytes as indicated by LDH release, cellular morphology, and vital staining with Hoechst 33342/propidium iodide. TASO toxicity was partially blocked by the CYP2E1 inhibitors diallyl sulfide and 4-methylpyrazole and was strongly inhibited by TA. Significantly, we found that hepatocytes produce TA from TASO relatively efficiently by back-reduction. The covalent binding of [¹⁴C]-TASO is inhibited by unlabeled TA, which acts as a “cold-trap” for [¹⁴C]-TA and prevents its reoxidation to [¹⁴C]-TASO. This in turn <i>increases</i> the net consumption of [¹⁴C]-TASO despite the fact that its oxidation to TASO₂ is inhibited. The potent inhibition of TASO oxidation by TA, coupled with the back-reduction of TASO and its futile redox cycling with TA, may help explain phenomena previously interpreted as “saturation toxicokinetics” in the in vivo metabolism and toxicity of TA and TASO. The improved understanding of the metabolism and covalent binding of TA and TASO facilitates the use of hepatocytes to prepare protein adducts for target protein identification

Metabolism and Toxicity of Thioacetamide and Thioacetamide <i>S</i>‑Oxide in Rat Hepatocytes

The hepatotoxicity of thioacetamide (TA) has been known since 1948. In rats, single doses cause centrolobular necrosis accompanied by increases in plasma transaminases and bilirubin. To elicit these effects, TA requires oxidative bioactivation, leading first to its <i>S</i>-oxide (TASO) and then to its chemically reactive <i>S</i>,<i>S</i>-dioxide (TASO₂), which ultimately modifies amine-lipids and proteins. To generate a suite of liver proteins adducted by TA metabolites for proteomic analysis and to reduce the need for both animals and labeled compounds, we treated isolated hepatocytes directly with TA. Surprisingly, TA was not toxic at concentrations up to 50 mM for 40 h. On the other hand, TASO was highly toxic to isolated hepatocytes as indicated by LDH release, cellular morphology, and vital staining with Hoechst 33342/propidium iodide. TASO toxicity was partially blocked by the CYP2E1 inhibitors diallyl sulfide and 4-methylpyrazole and was strongly inhibited by TA. Significantly, we found that hepatocytes produce TA from TASO relatively efficiently by back-reduction. The covalent binding of [¹⁴C]-TASO is inhibited by unlabeled TA, which acts as a “cold-trap” for [¹⁴C]-TA and prevents its reoxidation to [¹⁴C]-TASO. This in turn <i>increases</i> the net consumption of [¹⁴C]-TASO despite the fact that its oxidation to TASO₂ is inhibited. The potent inhibition of TASO oxidation by TA, coupled with the back-reduction of TASO and its futile redox cycling with TA, may help explain phenomena previously interpreted as “saturation toxicokinetics” in the in vivo metabolism and toxicity of TA and TASO. The improved understanding of the metabolism and covalent binding of TA and TASO facilitates the use of hepatocytes to prepare protein adducts for target protein identification

Metabolism and Toxicity of Thioacetamide and Thioacetamide <i>S</i>‑Oxide in Rat Hepatocytes

The hepatotoxicity of thioacetamide (TA) has been known since 1948. In rats, single doses cause centrolobular necrosis accompanied by increases in plasma transaminases and bilirubin. To elicit these effects, TA requires oxidative bioactivation, leading first to its <i>S</i>-oxide (TASO) and then to its chemically reactive <i>S</i>,<i>S</i>-dioxide (TASO₂), which ultimately modifies amine-lipids and proteins. To generate a suite of liver proteins adducted by TA metabolites for proteomic analysis and to reduce the need for both animals and labeled compounds, we treated isolated hepatocytes directly with TA. Surprisingly, TA was not toxic at concentrations up to 50 mM for 40 h. On the other hand, TASO was highly toxic to isolated hepatocytes as indicated by LDH release, cellular morphology, and vital staining with Hoechst 33342/propidium iodide. TASO toxicity was partially blocked by the CYP2E1 inhibitors diallyl sulfide and 4-methylpyrazole and was strongly inhibited by TA. Significantly, we found that hepatocytes produce TA from TASO relatively efficiently by back-reduction. The covalent binding of [¹⁴C]-TASO is inhibited by unlabeled TA, which acts as a “cold-trap” for [¹⁴C]-TA and prevents its reoxidation to [¹⁴C]-TASO. This in turn <i>increases</i> the net consumption of [¹⁴C]-TASO despite the fact that its oxidation to TASO₂ is inhibited. The potent inhibition of TASO oxidation by TA, coupled with the back-reduction of TASO and its futile redox cycling with TA, may help explain phenomena previously interpreted as “saturation toxicokinetics” in the in vivo metabolism and toxicity of TA and TASO. The improved understanding of the metabolism and covalent binding of TA and TASO facilitates the use of hepatocytes to prepare protein adducts for target protein identification

Metabolism and Toxicity of Thioacetamide and Thioacetamide <i>S</i>‑Oxide in Rat Hepatocytes

The hepatotoxicity of thioacetamide (TA) has been known since 1948. In rats, single doses cause centrolobular necrosis accompanied by increases in plasma transaminases and bilirubin. To elicit these effects, TA requires oxidative bioactivation, leading first to its <i>S</i>-oxide (TASO) and then to its chemically reactive <i>S</i>,<i>S</i>-dioxide (TASO₂), which ultimately modifies amine-lipids and proteins. To generate a suite of liver proteins adducted by TA metabolites for proteomic analysis and to reduce the need for both animals and labeled compounds, we treated isolated hepatocytes directly with TA. Surprisingly, TA was not toxic at concentrations up to 50 mM for 40 h. On the other hand, TASO was highly toxic to isolated hepatocytes as indicated by LDH release, cellular morphology, and vital staining with Hoechst 33342/propidium iodide. TASO toxicity was partially blocked by the CYP2E1 inhibitors diallyl sulfide and 4-methylpyrazole and was strongly inhibited by TA. Significantly, we found that hepatocytes produce TA from TASO relatively efficiently by back-reduction. The covalent binding of [¹⁴C]-TASO is inhibited by unlabeled TA, which acts as a “cold-trap” for [¹⁴C]-TA and prevents its reoxidation to [¹⁴C]-TASO. This in turn <i>increases</i> the net consumption of [¹⁴C]-TASO despite the fact that its oxidation to TASO₂ is inhibited. The potent inhibition of TASO oxidation by TA, coupled with the back-reduction of TASO and its futile redox cycling with TA, may help explain phenomena previously interpreted as “saturation toxicokinetics” in the in vivo metabolism and toxicity of TA and TASO. The improved understanding of the metabolism and covalent binding of TA and TASO facilitates the use of hepatocytes to prepare protein adducts for target protein identification

Metabolism and Toxicity of Thioacetamide and Thioacetamide <i>S</i>‑Oxide in Rat Hepatocytes

The hepatotoxicity of thioacetamide (TA) has been known since 1948. In rats, single doses cause centrolobular necrosis accompanied by increases in plasma transaminases and bilirubin. To elicit these effects, TA requires oxidative bioactivation, leading first to its <i>S</i>-oxide (TASO) and then to its chemically reactive <i>S</i>,<i>S</i>-dioxide (TASO₂), which ultimately modifies amine-lipids and proteins. To generate a suite of liver proteins adducted by TA metabolites for proteomic analysis and to reduce the need for both animals and labeled compounds, we treated isolated hepatocytes directly with TA. Surprisingly, TA was not toxic at concentrations up to 50 mM for 40 h. On the other hand, TASO was highly toxic to isolated hepatocytes as indicated by LDH release, cellular morphology, and vital staining with Hoechst 33342/propidium iodide. TASO toxicity was partially blocked by the CYP2E1 inhibitors diallyl sulfide and 4-methylpyrazole and was strongly inhibited by TA. Significantly, we found that hepatocytes produce TA from TASO relatively efficiently by back-reduction. The covalent binding of [¹⁴C]-TASO is inhibited by unlabeled TA, which acts as a “cold-trap” for [¹⁴C]-TA and prevents its reoxidation to [¹⁴C]-TASO. This in turn <i>increases</i> the net consumption of [¹⁴C]-TASO despite the fact that its oxidation to TASO₂ is inhibited. The potent inhibition of TASO oxidation by TA, coupled with the back-reduction of TASO and its futile redox cycling with TA, may help explain phenomena previously interpreted as “saturation toxicokinetics” in the in vivo metabolism and toxicity of TA and TASO. The improved understanding of the metabolism and covalent binding of TA and TASO facilitates the use of hepatocytes to prepare protein adducts for target protein identification

Metabolism and Toxicity of Thioacetamide and Thioacetamide <i>S</i>‑Oxide in Rat Hepatocytes

The hepatotoxicity of thioacetamide (TA) has been known since 1948. In rats, single doses cause centrolobular necrosis accompanied by increases in plasma transaminases and bilirubin. To elicit these effects, TA requires oxidative bioactivation, leading first to its <i>S</i>-oxide (TASO) and then to its chemically reactive <i>S</i>,<i>S</i>-dioxide (TASO₂), which ultimately modifies amine-lipids and proteins. To generate a suite of liver proteins adducted by TA metabolites for proteomic analysis and to reduce the need for both animals and labeled compounds, we treated isolated hepatocytes directly with TA. Surprisingly, TA was not toxic at concentrations up to 50 mM for 40 h. On the other hand, TASO was highly toxic to isolated hepatocytes as indicated by LDH release, cellular morphology, and vital staining with Hoechst 33342/propidium iodide. TASO toxicity was partially blocked by the CYP2E1 inhibitors diallyl sulfide and 4-methylpyrazole and was strongly inhibited by TA. Significantly, we found that hepatocytes produce TA from TASO relatively efficiently by back-reduction. The covalent binding of [¹⁴C]-TASO is inhibited by unlabeled TA, which acts as a “cold-trap” for [¹⁴C]-TA and prevents its reoxidation to [¹⁴C]-TASO. This in turn <i>increases</i> the net consumption of [¹⁴C]-TASO despite the fact that its oxidation to TASO₂ is inhibited. The potent inhibition of TASO oxidation by TA, coupled with the back-reduction of TASO and its futile redox cycling with TA, may help explain phenomena previously interpreted as “saturation toxicokinetics” in the in vivo metabolism and toxicity of TA and TASO. The improved understanding of the metabolism and covalent binding of TA and TASO facilitates the use of hepatocytes to prepare protein adducts for target protein identification