The arylation of microsomal membrane proteins by acetaminophen is associated with the release of a 44 kDa acetaminophen-binding mouse liver protein complex into the cytosol.

When analyzed by Western blotting with affinity purified antibodies against acetaminophen, proteins of molecular weight 44 and 58 kDa appear to be the major macromolecular targets in livers of mice administered hepatotoxic concentrations of acetaminophen. In this study, we have examined the characteristics and biochemical properties of the 44 kDa acetaminophen-binding protein in mouse liver. Data are presented which indicate that the 44-kDa protein is the earliest detectable protein targeted by acetaminophen; 30 min after acetaminophen administration in vivo, the binding to the 44 kDa protein is primarily localized in the microsomal fraction. After 1 hr, the 44 kDa acetaminophen-binding protein can be detected in both the microsomes and the cytosol. Extractions of microsomes with Triton X-114 or 1 M NaCl suggests that the acetaminophen-bound 44-kDa protein behaves as a peripheral membrane protein associated with the endoplasmic reticulum by ionic interactions. The cytosolic and microsomal 44-kDa proteins possess similar biochemical properties; both exist natively as components of a protein complex of greater than 200 kDa and both consist of two major isovariants with isoelectric points of 7.0 and 7.1 on two-dimensional gels. When N-acetyl-p-benzoquinone imine, the reactive metabolite of acetaminophen, is incubated with cytosolic or microsomal fractions from control liver, targeting of a 44-kDa protein is only observed in the microsomes. However, when acetaminophen is activated in an NADPH-regenerating microsomal system in vitro, some of the microsomal 44-kDa protein complex can be solubilized and released into the cytosol. Thus, acetaminophen administration can alter the subcellular distribution of at least one protein target in the cell.     

Binding of brominated diphenyl ethers to male rat carrier proteins

1. Two [14 C]-labelled brominated diphenyl ethers, 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) and decabromodiphenyl ether (BDE-209), were separately administered to the male Sprague-Dawley rat as a single oral dose (2.2?mg kg -1 body weight and 3.0?mg kg -1, respectively). 2. Very low [14 C] urine excretion was observed for both congeners (<1% of the dose), and cumulative biliary excretion was approximately 4% for BDE-99 and 9% for BDE-209. 3. More than 6% of the pooled urine from the BDE-99-treated rat was protein-bound to an 18-kDa protein characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western immunoblot analysis as α 2u -globulin. Eighteen per cent of the radioactivity from the pooled urine from the BDE-209 treated rat was bound to albumin; no binding to α 2u -globulin was detected. 4. In bile, 27-39% of the radioactivity from the BDE-99-dosed rat was bound to an unidentified 79-kDa protein, whereas essentially all (>87%) of the biliary radioactivity from BDE-209 was bound to the 79-kDa protein. Both parent BDE-99 and-209 and their metabolites were detected by thin layer chromatography in the extracted fraction of this bile protein. 5. By differential centrifugation, the subcellular localization of the 14 C derived from each congener in selected tissues was quantified. The cytosolic [14 C] from livers of the BDE-209-treated rat was bound to a 14-kDa protein, which was characterized as a fatty acid-binding protein.     

Immunochemical evidence of trifluoroacetylated cytochrome P-450 in the liver of halothane-treated rats

Four hours after the administration of halothane to phenobarbital-pretreated rats, subcellular fractions of liver were isolated and the proteins in the fractions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets, and immunochemically stained with anti-trifluoroacetylated antibodies. The microsomal fraction contained the highest level of trifluoroacetylated adducts. Its major trifluoroacetylated component was immunochemically identified as a phenobarbital-inducible form of cytochrome P-450 (54 kDa), whereas the other observed trifluoroacetylated protein fraction (59 kDa) was not identified. The plasma membrane fraction also contained a 54-kDa trifluoroacetylated adduct, which was immunochemically related to the 54-kDa cytochrome P-450. Microsomes from untreated rats that were administered halothane contained only the 59-kDa trifluoroacetylated protein fraction. The specificity of the immunochemical staining for the bound oxidative metabolite of halothane was confirmed by the finding that rats treated with deuterated halothane had considerably less stained liver proteins than did those treated with halothane. These results suggest that the CF3COX oxidative metabolite of halothane is so reactive that it binds predominantly to the cytochrome P-450 that produced it.     

Binding of brominated diphenyl ethers to male rat carrier proteins

1. Two [(14)C]-labelled brominated diphenyl ethers, 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) and decabromodiphenyl ether (BDE-209), were separately administered to the male Sprague-Dawley rat as a single oral dose (2.2 mg kg(-1) body weight and 3.0 mg kg(-1), respectively). 2. Very low [(14)C] urine excretion was observed for both congeners (<1% of the dose), and cumulative biliary excretion was approximately 4% for BDE-99 and 9% for BDE-209. 3. More than 6% of the pooled urine from the BDE-99-treated rat was protein-bound to an 18-kDa protein characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western immunoblot analysis as alpha(2u)-globulin. Eighteen per cent of the radioactivity from the pooled urine from the BDE-209 treated rat was bound to albumin; no binding to alpha(2u)-globulin was detected. 4. In bile, 27-39% of the radioactivity from the BDE-99-dosed rat was bound to an unidentified 79-kDa protein, whereas essentially all (>87%) of the biliary radioactivity from BDE-209 was bound to the 79-kDa protein. Both parent BDE-99 and-209 and their metabolites were detected by thin layer chromatography in the extracted fraction of this bile protein. 5. By differential centrifugation, the subcellular localization of the (14)C derived from each congener in selected tissues was quantified. The cytosolic [(14)C] from livers of the BDE-209-treated rat was bound to a 14-kDa protein, which was characterized as a fatty acid-binding protein.     

Acetaminophen hepatotoxicity: correspondence of selective protein arylation in human and mouse liver in vitro, in culture, and in vivo

Human and mouse liver were exposed to an APAP-activating system, in vitro. Subsequent immunochemical analysis of electrophoretically separated proteins with an affinity-purified anti-APAP antibody indicated that when a cytosolic fraction from human liver was incubated with APAP, an NADPH-regenerating system, and mouse microsomes selective APAP binding occurred predominantly to proteins of approximately 38, 58, and 130 kDa. To evaluate whether similar proteins are targeted in situ, primary cultures of human hepatocytes were treated with 10 mM APAP for 4 hr prior to immunochemical analysis. APAP binding was again detected in protein bands of approximately 38, 58, and 130 kDa. In addition, selective binding was also noted to other cytosolic protein bands, e.g., approximately 52 and 62 kDa. For mouse liver, the majority of the binding, in vitro or in culture, was to proteins of approximately 44 and 58 kDa with lesser binding to proteins of approximately 33 and 130 kDa among others. By contrast, at the times monitored, little covalent binding was detected in the 44-kDa region in the human liver experiments. Most noteworthy was the finding that when the protein arylation patterns on liver samples from a human APAP fatality were compared to those from a mouse given a hepatotoxic dose of APAP, the binding patterns were similar to those detected after the in vitro and the culture experiments with mouse and human livers. Furthermore, an immunohistochemical analysis revealed that as with the mouse, APAP covalent binding in the human liver exhibited a distinct zonal pattern consistent with centrilobular binding. That APAP arylation of the 58- and 130-kDa proteins was observed in livers from both mice and humans suggests that the mouse provides a valid model for studying the mechanistic importance of covalent binding. Elucidation of the identities and functions of the common targeted proteins may clarify their toxicological significance.     

Selective acetaminophen metabolite binding to hepatic and extrahepatic proteins: an in vivo and in vitro analysis

Acetaminophen (APAP) administration (600 mg/kg, po) to fasted male CD-1 mice resulted in cellular damage to liver, lung, and kidney. An affinity purified antibody against covalently bound APAP was used to identify APAP-protein adducts in microsomal and cytosolic extracts from these target organs. The proteins were resolved on SDS-PAGE, transblotted to nitrocellulose membranes, and analyzed immunochemically. Covalent binding of APAP to intracellular proteins was only observed in those organs which exhibited cellular damage; no APAP adducts were detected in tissues which did not undergo necrosis. In all target tissues the arylation of proteins was not random but highly selective with two adducts of 44 and 58 kDa accounting for the majority of the total APAP-bound proteins which were detected immunochemically. In addition, a third major APAP-protein adduct of 33 kDa was also observed in kidney cytosol. The severity of tissue damage and the amount of adducts present in these tissues could be significantly reduced when mice were pretreated with the mixed function oxidase inhibitor, piperonyl butoxide, prior to APAP dosing. Immunochemical analysis of plasma from APAP-treated animals indicated the presence of several protein adducts by 4 hr following drug administration. These adducts did not appear to be of plasma origin. Incubation of cytosolic proteins from liver, lung, kidney, spleen, brain, and heart with an APAP metabolite generating liver microsomal system demonstrated that the cytosolic 58-kDa protein target was native to all tissues tested. By contrast, the 58-kDa protein target did not appear to be endogenous to plasma since it was not detected when plasma was incubated in vitro with the liver microsomal system. These studies indicate that, although the 58-kDa proteins appear to be endogenous to both target and nontarget tissues, the 58-kDa APAP-protein adducts are detectable only in tissues which become damaged by APAP.     

Studies on the metabolism of the pneumotoxin O,S,S-trimethyl phosphorodithioate--II. Lung and liver slices

The metabolism of O,S,S-trimethyl phosphorodithioate (OSSMe), a pneumotoxic impurity in some organophosphorus insecticides, was investigated by incubating rat lung and liver slices with 1 mM OSSMe, labelled with 3H or 14C on one of its thiolo-methyl (CH3S-) groups. Protein bound radioactivity was higher in lung slices than in liver slices. In lung slices the predominant diester produced was O,S-dimethyl phosphorothioate (OSMeO-), whereas in liver slices it was S,S-dimethyl phosphorodithioate (SSMeO-). Other studies had shown binding of radioactivity and OSMeO- production to be cytochrome P-450-dependent processes in microsomes and SSMeO- production to result from the action of cytosolic glutathione-S-transferase on OSSMe. Preincubation of slices with 10(-5) M paraoxon did not influence the amount of protein-bound radioactivity, suggesting that binding of radioactivity did not simply result from protein phosphorylation. Pretreatments of the rats with O,O,O-trimethyl phosphorothioate [OOOMe(S) 0.5, 2.5 and 12.5 mg/kg p.o.], with p-xylene (1 g/kg, i.p.) or with bromophos (5.3 mg/kg, i.p.) which all protect against the lung toxicity of OSSMe probably by inhibiting pulmonary mixed-function oxidase, also led to significant decreases in both protein binding of radioactivity and OSMeO- production in lung slices, but not in liver slices. These results show that tissue slices are a convenient system for investigating xenobiotic metabolism in the lung and they suggest that the susceptibility of the lung to OSSMe probably results from a relatively high rate of activation, coupled with a relatively low rate of metabolism by non-toxic pathways and/or removal of reactive metabolites in some lung cells, possibly the alveolar type I cells.     

Halothane metabolism: immunochemical evidence for molecular mimicry of trifluoroacetylated liver protein adducts by constitutive polypeptides.

A monoform antibody [anti-TFA antibody] against TFA-protein adducts (TFA-adducts) was obtained by affinity purification of a polyclonal antiserum, raised in rabbits against TFA-rabbit serum albumin, on a N-epsilon-TFA-L-lysine matrix coupled to Affi-Gel 102. The anti-TFA antibody did recognize TFA-adducts of distinct molecular mass on Western blots of hepatocyte homogenates or microsomal membranes obtained from rats pretreated with halothane. The anti-TFA antibody also recognized cross-reactive polypeptides with apparent molecular masses of 52 kDa and 64 kDa on Western blots of hepatocyte homogenates obtained from rats not treated with halothane or metabolites thereof. The 52-kDa and 64-kDa cross-reactive polypeptides were localized in the 3,000 x g particulate fraction of liver homogenates. Recognition, on Western blots, of TFA-adducts and both the 52-kDa and 64-kDa cross-reactive polypeptides by anti-TFA antibody was sensitive to competition by N-epsilon-TFA-L-lysine (IC50 less than 100 microM) and N-epsilon-acetyl-L-lysine (IC50 approximately 10 mM). Treatment with piperidine (1 M) did abolish the recognition of TFA-adducts but not that of the 52-kDa and the 64-kDa cross-reactive polypeptides by anti-TFA antibody on Western blots. In antibody-exchange experiments, anti-TFA antibody was affinity-adsorbed on Western blots to the 52-kDa or the 64-kDa cross-reactive polypeptides of the rat heart, followed by spontaneous transfer to target TFA-adducts present on Western blots of rat liver microsomal membranes. The majority of these target TFA-adducts were recognized by anti-TFA antibody transferring from the source 52-kDa or 64-kDa cross-reactive polypeptides. When examined up to 10 days after exposure of rats to a single dose of halothane, no influence on the constitutive level of expression, in the liver, of either cross-reactive polypeptide was observed. In contrast, TFA-adducts were persistent for greater than 90 hr but less than 10 days. In addition to the liver, the 52-kDa and the 64-kDa cross-reactive polypeptides were prominently expressed in the heart and the kidney and, to a much lesser degree, in the spleen, the thymus, the lung, and skeletal muscle of the rat. Considerable variation in the level of expression of the 52-kDa and the 64-kDa cross-reactive polypeptides was recognized in livers of the six human individuals tested so far.(ABSTRACT TRUNCATED AT 250 WORDS)     

Halothane: inhibition and activation of rat hepatic glutathione S-transferases

Multiple halothane anesthesias (1.25 MAC for 1 hr on 3 alternate days) of male Long-Evans rats initially decreased by up to 30% and subsequently increased to up to 185% liver cytosolic glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene, 3,4-dichloro-1-nitrobenzene and trans-4-phenyl-3-buten-2-one and glutathione peroxidase activity. Halothane rapidly and reversibly activated hepatic cytosolic glutathione S-transferases and purified isoenzyme 1-2 but not isoenzymes 1-1 and 3-3. At high concentrations of halothane (ca. 22 mM), maximal activation was ca. 25%. Halothane, enflurane, isoflurane and methoxyflurane, but not the halothane metabolite 1-chloro-2,2-difluoroethylene, inhibited a mixture of liver cytosolic glutathione S-transferases with time (ca. 30% inhibition/15 min). The inhibition exhibited pseudo-first order kinetics (kobs = 0.13 min-1) and an I50 for halothane of greater than or equal to 15 mM. Halothane inhibited glutathione S-transferases 3-3, 3-4, and 4-4 by 50-60%, but did not affect isoenzymes 1-1 and 1-2. The ability of halothane to diminish hepatic glutathione S-transferase activity in vivo may in part reflect the time-dependent inhibition of glutathione S-transferase isoenzymes containing the 3- and 4-subunits.     

Halothane induces oxidative stress and NF-kappaB activation in rat liver: protective effect of propofol

We investigated the effects of propofol on markers of oxidative stress, nuclear factor kappa B (NF-kappaB) activation and inducible nitric oxide synthase (iNOS) expression in liver of rats treated with halothane under hypoxic conditions. Male Wistar rats received halothane 1%/oxygen 14%, oxygen 14%/propofol 60 mg kg(-1) i.p., or halothane 1%/oxygen 14%/propofol 60 mg kg(-1) i.p. Morphological examination showed complete loss of architecture with massive necrosis of parenchyma in the halothane group, while only minor histological abnormalities were observed in rats receiving halothane plus propofol. The cytosolic concentration of TBARS and the hydroperoxide-initiated chemiluminescence increased significantly in the liver of animals from the halothane group (+62% and +40% versus controls, respectively), and this increase was abolished by propofol administration. Halothane induced a marked activation of NF-kappaB (+180%), and resulted in a significant decrease of the nonphosphorylated form of the inhibitor IkappaBalpha (-53%), while phosphorylated IkappaBalpha protein level was markedly increased (+146%). Propofol administration lowered these effects to +30% (NF-kappaB), -26% (nonphosphorylated IkappaBalpha), and +56% (phosphorylated IkappaBalpha). The increase of iNOS protein level (+59%) induced by halothane was significantly reduced to +22% by additional administration of propofol. Results obtained show that administration of propofol inhibits oxidative stress, NF-kappaB nuclear traslocation and iNOS overexpression in liver of rats receiving halothane. Propofol treatment, by inhibiting the NF-kappaB signal transduction pathway, might block the production of noxious mediators involved in the development of halothane-induced injury.     

Relationship between stress protein induction in rat kidney by mercuric chloride and nephrotoxicity.

Adverse environmental stimuli increase the synthesis of a class of proteins referred to as stress proteins. The effect of mercuric chloride, a model nephrotoxin, on protein synthesis in male rat kidney has been evaluated. Renal slices from exposed rats were incubated with [35S]methionine for 1 hr and subjected to SDS-PAGE, after which 35S-labeled proteins were detected by autoradiography. Enhanced de novo synthesis of 70- and 90-kDa relative molecular mass (M(r)) proteins were detected 2 hr after exposure to 1 mg Hg/kg, with maximum activity occurring at 4-8 hr. By 16 hr postinjection, synthesis of these two proteins had decreased. Dose-related increases in synthesis of these proteins, and of a 110-kDa protein, were observed 4 hr after i.v. injection of 0.25, 0.5, and 1.0 mg Hg/kg, with concomitant inhibition of synthesis of proteins of M(r) 38 and 68 kDa. At a dose of 1 mg/kg, kidney proximal tubules exhibited progressive degenerative changes from 4 to 24 hr. A functional deficit, decreased uptake of [para-3H]aminohippurate into renal slices, was not observed until 16 hr after i.v. injection of 1 mg/kg. No significant histopathologic changes were observed in kidneys 4 hr after treatment with 0.25 or 0.5 mg Hg/kg, iv. No changes in liver protein synthesis were apparent until 16-24 hr, where an increase in the 70- and 90-kDa proteins was observed. A concomitant increase in plasma sorbitol dehydrogenase activity occurred at 16-24 hr; however, there was no histopathological evidence of liver injury. The 72-kDa inducible member of the 70-kDa stress protein family and the 88-kDa member of the 90-kDa protein family were detected by immunoblotting techniques using monoclonal antibodies. The data demonstrate that Hg induces alterations in the expression of renal gene products in vivo as evidenced by enhanced stress protein synthesis and inhibition of synthesis of constitutive proteins. These changes in renal protein synthesis preceded overt renal injury, occurring in the early stages of nephropathy. Altered patterns of stress protein synthesis appeared to be target organ specific. The data suggest that altered protein synthesis patterns may serve as biomarkers of renal injury.     

Comparative studies of the covalent binding of [14C]-2-chloro-4-acetotoluidide by liver and kidney slices of the starling

In tissue slices of female starlings, binding of [14C]-2-chloro-4-acetotoluidide (CAT) radioactivity to liver proteins was almost five times greater than binding to kidney proteins after 2 h of incubation. Binding to protein of liver slices increased in a log linear fashion with increasing CAT concentrations. Binding to protein of kidney slices also increased with increasing concentrations but not in a log linear fashion. Mixed-function oxidase inhibitors, SKF 525-A and alpha-naphthoflavone, decreased binding to liver slices but did not affect binding to kidney slices. Anaerobic incubation conditions inhibited binding to both tissues. P-Hydroxymercuribenzoate and sodium cyanide did not affect the binding of radioactivity associated with [14C]-CAT to proteins of either liver or kidney slices. Diethyl maleate increased binding of the radioactivity to proteins of the kidney slices but not to liver slices. Cysteine also increased binding in kidney slices. Binding in liver slices did not increase significantly with cysteine. The cysteine-induced increase in protein binding in kidney slices did not appear to depend on the formation of sulfate from the metabolism of cysteine. There was no sex-dependent difference in starlings as to the binding of radioactivity in either liver or kidney slices. Male chicken kidney slices bound a much higher amount of radioactivity associated with [14C]-CAT than male starling kidney slices, while the liver slices bound comparable amounts. Male hamster liver slices bound much more radioactivity than did male starling liver slices. However, hamster kidney slices bound much less than did starling kidney slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoblot analysis of protein containing 3-(cystein-S-yl)acetaminophen adducts in serum and subcellular liver fractions from acetaminophen-treated mice

The hepatotoxicity of acetaminophen is believed to be mediated by the metabolic activation of acetaminophen to N-acetyl-p-benzoquinone imine which covalently binds to cysteinyl residues on proteins as 3-(cystein-S-yl)acetaminophen adducts. The formation of these adducts in hepatic protein correlates with the hepatotoxicity. In this study, the formation of 3-(cystein-S-yl)acetaminophen adducts in specific cellular proteins was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected using affinity-purified antisera specific for 3-(cystein-S-yl)acetaminophen adducts on immunoblots. These techniques were used to investigate the liver 10,000g supernatant and serum from B6C3F1 mice that received hepatotoxic doses of acetaminophen. More than 15 proteins containing 3-(cystein-S-yl)acetaminophen adducts were detected in the liver 10,000g supernatant. The most prominent protein containing 3-(cystein-S-yl)acetaminophen adducts in the hepatic 10,000g supernatant had a relative molecular mass of 55 kDa. Serum proteins containing 3-(cystein-S-yl)acetaminophen adducts had molecular masses similar to those found in the liver 10,000g supernatant (55, 87, and approximately 102 kDa). These data, combined with our previous findings describing the temporal relationship between the appearance of 3-(cystein-S-yl)acetaminophen adducts in protein in the serum and the decrease in the levels of 3-(cystein-S-yl)acetaminophen adducts in protein in the liver, suggested that liver adducts were released into the serum following lysis of hepatocytes. The temporal relationship between the formation of specific adducts and hepatotoxicity in mice following a hepatotoxic dose of acetaminophen was examined using immunoblots of mitochondria, microsomes, cytosol, and plasma membranes. Hepatotoxicity indicated by serum alanine aminotransferase levels was increased at 2 and 4 hr after dosing. The cytosolic fraction contained numerous proteins with 3-(cystein-S-yl)acetaminophen adducts, the most intensely stained of which was a 55-kDa protein. 3-(Cystein-S-yl)acetaminophen adducts were detected in the 55-kDa liver protein 30 min after dosing and prior to the development of significant toxicity. Examination of gels suggested that maximal levels of immunochemically detectable adducts in the 55-kDa protein occurred at 1-2 hr, with a decrease in intensity 4 hr after dosing. The presence of 3-(cystein-S-yl)acetaminophen adducts in proteins prior to hepatotoxicity suggests a threshold for adduct formation in the development of toxicity. Protein in microsomes which contained 3-(cystein-S-yl)acetaminophen adducts ranged in molecular weight from 38 to approximately 106 kDa. The major proteins containing 3-(cystein-S-yl)acetaminophen adducts in the mitochondria had molecular masses of 39, 50, 68, and 79 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)     

Liver function following hypovolemic hypotension in rats anaesthetized with halothane or enflurane

Rats which had approximately 25-30% of their calculated blood volume removed were exposed to halothane (1%) or enflurane (2%) in 33% oxygen for 30 min. Hepatic function was evaluated by determining, at various time intervals, serum activities of glutamic-oxalacetic and glutamic-pyruvic transaminase, acid phosphatase and gamma-glutamyl-transpeptidase. In this model serum enzyme activities and animal mortality were significantly increased when hypovolemic hypotension was induced during halothane anaesthesia. The same events did not occur in bleeding animals anaesthetized with enflurane. The marked disparity in hepatic dysfunction and mortality between halothane and enflurane-anaesthetized rats during hypovolemic hypotension may be explained by the more pronounced decrease of oxygen available for the liver and production of reductive toxic intermediates in animals exposed to halothane.     

The calcium-binding protein calreticulin is covalently modified in rat liver by a reactive metabolite of the inhalation anesthetic halothane.

A general procedure is presented for the isolation of several liver microsomal target proteins of the reactive trifluoroacetyl halide metabolite of halothane. It was found that most of these proteins could be selectively extracted from microsomes with 0.1% sodium deoxycholate and separated into partially purified fractions by DEAE-Sepharose anion-exchange chromatography. Using this method, we describe the isolation and identification of a 63-kDa target protein of halothane in rat liver. Amino acid sequences of the N-terminal and of several internal peptides of the protein, as well as the deduced amino acid sequence of a nearly full-length rat liver cDNA clone of the protein, showed 98% identity with a reported murine cDNA that encodes for calreticulin, a major calcium-binding protein of the lumen of endoplasmic reticulum. Although it remains to be determined what role calreticulin has in the development of halothane hepatitis, this study has shown that calreticulin can be a target of reactive metabolites of xenobiotics.     

Factors influencing halothane hepatotoxicity in the rat hypoxic model

Halothane, a widely used inhalation anesthetic, was shown to be hepatotoxic to male, phenobarbital-pretreated rats, only when administered under hypoxic conditions (fraction of inspired oxygen = 0.14). The degree of hepatotoxicity as determined from morphological alterations and serum glutamic-pyruvic transaminase (SGPT) activities, correlated well with concentrations of hepatic cytochrome P-450 and concentration of inspired halothane. Maximal lesion intensity developed within 12 to 24 hr after exposure to 1% halothane for as little as 30 min. By 4 days after exposure, the liver had repaired, since no morphological alterations were apparent and SGPT activities had returned to normal values. Female rats, when pretreated with phenobarbital and exposed to 1% halothane under hypoxic conditions did not develop liver injury. SKF-525A and metyrapone reduced the severity of liver injury when administered preanesthesia and 4 hr postanesthesia. The free sulhydryl-containing compounds, cysteine, cystamine, and N-acetylcysteine afforded protection when administered at 4 or 8 hr (cystamine) after ending anesthesia. These results support the hypothesis that reductive or noxoxygen-dependent biotransformation of halothane results in toxic intermediates that can initiate halothane-induced liver injury.     

Enhanced proteolysis and changes in membrane-associated calpain following phenylhydrazine insult to human red cells

Phenylhydrazine-mediated protein damage in human red cells has been assessed using HPLC, one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblot analysis of major membrane proteins. The association of the Ca(2+)-activated neutral protease, calpain, with membrane proteins following hydrazine insult was also examined using immunoblot analysis. HPLC amino acid analysis of red cell suspensions was employed to quantify proteolysis. Phenylhydrazine (4 mM) increased the rate of leucine, lysine, and histidine release by approximately 12-, 7-, and 5-fold, respectively. N-acetylcysteine (20 mM), dithiothreitol (50 mM), and dimethylthiourea (50 mM) decreased the rate of phenylhydrazine-stimulated amino acid release by approximately 30-50%; in contrast, the free radical scavengers and antioxidants dimethylfuran (50 mM) and dimethyl sulfoxide (50 mM) were without significant effect. The calcium chelator, EGTA (10 mM) inhibited phenylhydrazine-stimulated proteolysis by approximately 30%. Phenylhydrazine (4 mM) caused attenuation of the major membrane protein bands present in the SDS-PAGE pattern and extensive smearing of a band in the region of approximately 28 kDa. Free radical scavengers and antioxidants failed to ameliorate significantly membrane protein damage in phenylhydrazine-treated cells as judged by SDS-PAGE. Immunoblot analysis of spectrin confirmed these results. Two-dimensional SDS-PAGE of membrane proteins following phenylhydrazine treatment, however, revealed the appearance of new protein spots and a loss of existing protein spots as compared to control. Western blot analysis of membrane-associated calpain (79 kDa (proenzyme), 77- and 75-kDa forms) was also performed. Phenylhydrazine-treated red blood cells exhibited concentration- and time-dependent changes in the level of membrane-associated procalpain relative to control. The inhibitors N-acetylcysteine, dithiothreitol, dimethylthiourea, and dimethyl sulfoxide in the presence of phenylhydrazine appeared to preserve the level of procalpain in association with the membrane proteins, but only N-acetylcysteine and dithiothreitol protected the 77- and 75-kDa forms. In contrast, dimethylfuran in the presence of phenylhydrazine caused a substantial decrease in all three forms of membrane-associated calpain. In phenylhydrazine-treated hemolysate, the level of the 77- and 75-kDa forms of membrane-associated calpain was decreased relative to control. These forms were absent when EGTA (10 mM) was included in the incubation and the level of proenzyme was decreased. These data suggest that calpain is recruited to the membrane following hydrazine insult, undergoes a Ca(2+)-dependent conversion to the active forms, and may be involved in the degradation of damaged cytosolic and membrane protein(s).     

Binding of reactive metabolites of CCl4 to specific microsomal proteins

The covalent binding of [14C]carbon tetrachloride to microsomal proteins in rat liver microsomes under anaerobic conditions was investigated by SDS-polyacrylamide slab gel electrophoresis and fluorography. Most of the labeled proteins were observed in the molecular weight range of 52-61 kDa, indicating that cytochrome P-450 forms (EC 1.14.14.1) were labeled. Protein bands at the position of the NADPH-cytochrome P-450 reductase (78 kDa) (EC 1.6.2.4) and NADH-cytochrome b5 reductase (33 kDa) (EC 1.6.2.2) also showed radioactivity. The fluorographic pattern of the protein labeling was cytochrome P-450-dependent, as was demonstrated by CO and metyrapone inhibition as well as by pretreatment of rats with inducing drugs such as 3-methylcholanthrene, benzo(a)pyrene, phenobarbitone and Aroclor 1254. Immuno-precipitation with a purified anti-P-450 immunoglobulin against cytochrome P-450 PB-B (52 kDa) of rat liver indicated that this protein contained about 10-20% of the total bound radioactivity in an average ratio of 0.8 mol [14C]CCl4-metabolite/mol cytochrome P-450 PB-B.     

Tissue distribution of a major mevalonate pyrophosphate decarboxylase in rats.

The 45- and 35-kDa subunits of mevalonate pyrophosphate decarboxylase (MPD) have been purified from rat liver. In this study, we examined the relationship between 45- and 35-kDa MPD and the tissue distribution of a major MPD in rat liver. When the crude extract of rat liver fed on normal chow was subjected to immunoblot analysis using anti-rat 45-kDa MPD antibody, only the 45-kDa band was detected. In a pulse-chase experiment using anti-rat 45-kDa MPD antibody, there was no precursor-product relationship between the 45- and the 35-kDa MPD. In immunoprecipitation, more than 85% of MPD activity in the rat liver was depleted from the crude extract with an excess of the above antibody. When 45-kDa MPD contents in tissues were analyzed by immunoblotting, a single protein band with an apparent molecular weight of 45 kDa was detected in all tissues. The specific protein content of 45-kDa MPD in liver was markedly higher than in other tissues. The activity/amount ratio varied among brain, liver, and testis, being significantly highest in the liver. From these data, it is suggested that 45-kDa MPD serves as a major enzyme involved in cholesterol biosynthesis in rat liver and that a tissue-specific regulator or isozyme of 45-kDa MPD is present in rat liver.     

Identification of three protein targets for reactive metabolites of bromobenzene in rat liver cytosol

The hepatotoxicity of bromobenzene and many other simple organic chemicals is believed to be associated with covalent binding of chemically reactive metabolites to cellular proteins. Recently, a rat liver microsomal esterase was shown to be targeted by bromobenzene metabolites formed in vitro [Rombach, E. M., and Hanzlik, R. P. (1998) Chem. Res. Toxicol. 11, 178-184]. To identify protein targets for bromobenzene metabolites in cytosol, we incubated liver microsomes and glutathione-depleted liver cytosol from phenobarbital-treated rats with [(14)C]bromobenzene in vitro. In a separate experiment, we intraperitoneally injected a hepatotoxic dose of [(14)C]bromobenzene to phenobarbital-treated rats. The cytosol fractions from both experiments were recovered and analyzed for protein-bound radioactivity. Under the conditions that were used, 2.6 and 3.9 nmolar equiv of bromobenzene/mg of cytosolic protein was bound in vitro and in vivo, respectively. Denaturing polyacrylamide gel electrophoresis of these cytosolic proteins followed by phosphor imaging analysis revealed several radiolabeled protein bands over a broad molecular mass range, the patterns observed in vitro and in vivo being generally similar to each other. Cytosolic proteins labeled in vitro were separated by ion exchange chromatography and electrophoresis, and three major radioactive bands with estimated molecular masses of ca. 14, 25, and 30 kDa were in-gel digested with trypsin, followed by on-line HPLC electrospray ionization mass spectrometry of the resulting peptide mixtures. For the three protein bands, the observed peptide masses were found to match the predicted tryptic fragments of liver fatty acid binding protein, glutathione transferase subunit A1, and carbonic anhydrase isoform III, respectively, with 83, 45, and 59% coverage of the corresponding complete sequences. The possible relationship of the adduction of these proteins to the toxicological outcome is discussed.