Identification of proteins adducted by reactive naphthalene metabolites in vitro

Metabolic activation of inert chemicals to electrophilic intermediates has been correlated with the incidence and severity of cytotoxicity. The current studies have identified several proteins adducted by reactive metabolites of the lung toxicant, naphthalene. Proteins isolated from microsomal incubations of (14)C-naphthalene were separated by 2-DE, proteins were blotted to PVDF membranes and radioactive proteins were localized by storage phosphor analysis. Adducted proteins were isolated from complimentary gels and identified by peptide mass mapping. A total of 18 adducted proteins were identified including: protein disulfide isomerase precursor, ER-60 protease, alpha actin, mouse urinary proteins, and cytochrome b5 reductase. In supernatant fractions, protein disulfide isomerase, heat shock protein 70, and alpha-actin were key proteins to which reactive naphthalene metabolites were bound. All of the proteins adducted, with the exception of cytochrome b5 reductase were sulfhydryl rich. Although several of the proteins found to be adducted in these studies have also been shown to be adducted by other electrophiles, several others have not been reported as common targets of reactive metabolites. These studies provide a basis for both in situ and in vivo work designed to follow the fate and formation of reactive metabolite protein adducts.     

Characterization of model Peptide adducts with reactive metabolites of naphthalene by mass spectrometry

Naphthalene is a volatile polycyclic aromatic hydrocarbon generated during combustion and is a ubiquitous chemical in the environment. Short term exposures of rodents to air concentrations less than the current OSHA standard yielded necrotic lesions in the airways and nasal epithelium of the mouse, and in the nasal epithelium of the rat. The cytotoxic effects of naphthalene have been correlated with the formation of covalent protein adducts after the generation of reactive metabolites, but there is little information about the specific sites of adduction or on the amino acid targets of these metabolites. To better understand the chemical species produced when naphthalene metabolites react with proteins and peptides, we studied the formation and structure of the resulting adducts from the incubation of model peptides with naphthalene epoxide, naphthalene diol epoxide, 1,2-naphthoquinone, and 1,4-naphthoquinone using high resolution mass spectrometry. Identification of the binding sites, relative rates of depletion of the unadducted peptide, and selectivity of binding to amino acid residues were determined. Adduction occurred on the cysteine, lysine, and histidine residues, and on the N-terminus. Monoadduct formation occurred in 39 of the 48 reactions. In reactions with the naphthoquinones, diadducts were observed, and in one case, a triadduct was detected. The results from this model peptide study will assist in data interpretation from ongoing work to detect peptide adducts in vivo as markers of biologic effect.     

Evidence that 1-naphthol is not an obligate intermediate in the covalent binding and the pulmonary bronchiolar necrosis by naphthalene

Recent studies of a number of volatile aromatic hydrocarbons have suggested that the formation of covalently bound metabolites arises solely through the intermediate formation of phenols. This study further examines the involvement of 1-naphthol in the in vivo and in vitro formation of covalently bound metabolites and pulmonary bronchiolar necrosis by naphthalene. Marked differences were observed in the rate of 1-naphthol formation in lung and liver microsomal incubations without correspondingly large differences between the rates of formation of covalently bound metabolites from naphthalene and 1-naphthol. Glutathione decreased covalent binding in hepatic microsomal incubations containing 14[C]1-naphthol but did not result in the formation of any of the glutathione adducts isolated from identical incubations containing 14[C]naphthalene. Tissue levels of covalently bound radioactivity in mice treated with 14[C]1-naphthol or 14[C]naphthalene were similar; however, in contrast to studies with naphthalene, 1-naphthol administration did not deplete tissue glutathione nor result in detectable tissue injury. These studies indicate that 1-naphthol is not an obligate intermediate in the formation of covalently bound metabolites from naphthalene nor does it appear to be a more proximate lung toxic metabolite.     

Evidence for cellular protein covalent binding derived from styrene metabolite

Styrene is one of the most important industrial intermediates consumed in the world. Human exposure to styrene occurs mainly in the reinforced plastics industry, particularly in developing countries. Styrene has been found to be hepatotoxic and pneumotoxic in humans and animals. The biochemical mechanisms of styrene-induced toxicities remain unknown. Albumin and hemoglobin adduction derived from styrene oxide, a major reactive metabolite of styrene, has been reported in blood samples obtained from styrene-exposed workers. The objectives of the current study focused on cellular protein covalent binding of styrene metabolite and its correlation with cytotoxicity induced by styrene. We found that radioactivity was bound to cellular proteins obtained from mouse airway trees after incubation with ¹⁴C-styrene. Microsomal incubation studies showed that the observed protein covalent binding required the metabolic activation of styrene. The observed radioactivity binding in protein samples obtained from the cultured airways and microsomal incubations was significantly suppressed by co-incubation with disulfiram, a CYP2E1 inhibitor, although disulfiram apparently did not show a protective effect against the cytotoxicity of styrene. A 2-fold increase in radioactivity bound to cellular proteins was detected in cells stably transfected with CYP2E1 compared to the wild-type cells after ¹⁴C-styrene exposure. With the polyclonal antibody developed in our lab, we detected cellular protein adduction derived from styrene oxide at cysteinyl residues in cells treated with styrene. Competitive immunoblot studies confirmed the modification of cysteine residues by styrene oxide. Cell culture studies showed that the styrene-induced protein modification and cell death increased with the increasing concentration of styrene exposure. In conclusion, we detected cellular protein covalent modification by styrene oxide in microsomal incubations, cultured cells, and mouse airways after exposure to styrene and found a good correlation between styrene-induced cytotoxicity and styrene oxide-derived cellular protein adduction.     

Identification of proteins adducted by reactive metabolites of naphthalene and 1-nitronaphthalene in dissected airways of rhesus macaques

Naphthalene and 1-nitronaphthalene are ambient air pollutants, which undergo P450-dependent bioactivation in the lung. Reactive metabolites of naphthalene and 1-nitronaphthalene covalently bind to proteins, and the formation of covalent adducts correlates with airway epithelial cell injury in rodent models. These studies were designed to identify protein adducts generated from these reactive metabolites within distal respiratory airways. Distal bronchioles and parenchyma from rhesus monkeys were incubated with [(14)C]naphthalene or [(14)C]1-nitronaphthalene. Proteins were separated by 2-DE, blotted to PVDF membranes, and adducted proteins imaged by storage phosphor analysis. MS of in-gel tryptic digests identified numerous adducted proteins including: eight cytoskeletal proteins, two chaperone proteins, seven metabolic enzymes, one redox protein, two proteins involved in ion balance and cell signaling, and two extracellular proteins. While many proteins are adducted by both naphthalene and 1-nitronaphthalene, some are unique to the individual toxicant and airway subcompartment. Although the role which adduction of these proteins plays in cytotoxicity was not evaluated, these studies provide candidate proteins for future work designed to determine the importance of protein adducts in the mechanisms of toxicity and for developing biomarkers useful in determining the relevance of findings in animal models to exposed human populations.     

The microsomal metabolism of pentachlorophenol and its covalent binding to protein and DNA

The microsomal metabolism of pentachlorophenol (PCP) was investigated, with special attention to the conversion dependent covalent binding to protein and DNA. The two metabolites detected were tetrachloro-1,2- and tetrachloro-1,4-hydroquinone. Microsomes from isosafrole (ISF)-induced rats were by far the most effective in catalyzing the reaction: the rate of conversion was increased 7-fold over control microsomes. All other inducers tested (hexachlorobenzene (HCB), phenobarbital (PB) and 3-methylcholanthrene (3MC) gave 2--3-fold increases over control. There are indications that the 1,2- and 1,4-isomers are produced in different ratio's by various cytochrome P-450 isoenzymes: Microsomes from PB- and HCB-treated rats produced the tetrachloro-1,4- and tetrachloro-1,2-hydroquinone in a ratio of about 2, while microsomes from rats induced with 3 MC and ISF showed a ratio of about 1.3. When PCP was incubated with microsomes from rats treated with HCB, a mixed type inducer of P-450, the ratio between formation of the 1,4- and 1,2-isomers decreased with increasing concentration of PCP, suggesting the involvement of at least two P-450 isoenzymes with different Km-values. The overall apparent Km-value for HCB-microsomes was 13 microM both for the formation of the soluble metabolites and the covalent binding to microsomal protein, suggesting both stem from the same reaction. The covalent binding could be inhibited by ascorbic acid and this inhibition was accompanied by an increase in formation of tetrachlorohydroquinones (TCHQ). Although a large variation was observed in rates of conversion between microsomes treated with different (or no) inducers, the rate of covalent binding to microsomal protein was remarkably constant. A conversion-dependent covalent binding to DNA was observed in incubations with added DNA which was 0.2 times the amount of binding to protein (37 pmol/mg DNA).     

Actin Cys374 as a nucleophilic target of alpha,beta-unsaturated aldehydes

We have recently shown that actin can be modified by the Michael addition of 4-hydroxynonenal to Cys374. Here, we have exposed purified actin at increasing acrolein concentrations and have identified the sites of acrolein addition using LC-ESI-MS/MS. Acrolein reacted with Cys374, His87, His173, and, minimally, His40. Cys374 adduction by both 4-hydroxynonenal and acrolein negligibly affected the polymerization of aldehyde-modified (carbonylated) actin, as shown by fluorescence measurements. Differently, acrolein binding at histidine residues, when Cys374 was completely saturated, inhibited polymerization in a dose-dependent manner. Molecular modeling analyses indicated that structural distortions of the ATP-binding site, induced by four acrolein-Michael adducts, could explain the changes in the polymerization process. Aldehyde binding to Cys374 does not alter significantly actin polymerization because this residue is located in a very flexible region, whose covalent modifications do not alter the protein folding. These data demonstrate that Cys374 represents the primary target site of alpha,beta-unsaturated aldehyde addition to actin in vitro. As Cys374 is a preferential target for various oxidative/nitrosative modifications, and actin is one of the main carbonylated proteins in vivo, these findings also suggest that the highly reactive Cys374 could serve as a carbonyl scavenger of reactive alpha,beta-unsaturated aldehydes and other electrophilic lipids.     

Drug residue formation from ronidazole, a 5-nitroimidazole. III. Studies on the mechanism of protein alkylation in vitro

Ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reductively metabolized by liver microsomal and purified NADPH-cytochrome P-450 reductase preparations to reactive metabolites that covalently bind to tissue proteins. Kinetic experiments and studies employing immobilized cysteine or blocked cysteine thiols have shown that the principal targets of protein alkylation ara cysteine thiols. Furthermore, ronidazole specifically radiolabelled with 14C in the 4,5-ring, N-methyl or 2-methylene positions give rise to equivalent apparent covalent binding suggesting that the imidazole nucleus is retained in the bound residue. In contrast, the carbonyl-14C-labeled ronidazole gives approx. 6--15-fold less apparent covalent binding indicating that the carbamoyl group is lost during the reaction leading to the covalently bound metabolite. The conversion of ronidazole to reactive metabolite(s) is quantitative and reflects the amazing efficiency by which this compound is activated by microsomal enzymes. However, only about 5% of this metabolite can be accounted for as protein-bound products under the conditions employed in these studies. Consequently, approx. 95% of the reactive ronidazole metabolite(s) can react with other constituents in the reaction media such as other thiols or water. Based on these results, a mechanism is proposed for the metabolic activation of ronidazole.     

Estrogen metabolism in rat liver microsomal and isolated hepatocyte preparations--II. Inhibition studies

The abilities of various inhibitors and metabolism modifiers to alter the metabolism of estradiol and the irreversible binding of estradiol to proteins were examined in subcellular microsomal incubations and in intact hepatocyte preparations. In studies with rat liver microsomal preparations containing estradiol and an NADPH-generating system, the irreversible binding of radiolabeled steroid metabolite(s) to the microsomal proteins was 77.59 pmol/mg/min (SD 6.1; 7.6% of total steroid). 2-Bromoestradiol and 4-bromoestradiol, inhibitors of estrogen 2-hydroxylase, effectively decreased this irreversible binding of radiolabeled estradiol metabolite(s) to microsomal proteins to 17 pmol mg-1 min-1 (2.1% of total estradiol). These haloestrogens were also effective inhibitors in the intact hepatocyte cells, decreasing the amounts of organic metabolites, aqueous-soluble conjugates, and protein-bound materials. The HPLC radiochromatograms of the organic-extracted fractions from the 2 h hepatocyte incubations demonstrate that the catechol estrogen products, i.e. 2-hydroxyestrogens and 2-methoxyestrogens, were present in lower amounts in the incubations containing the bromoestrogens than in control incubations containing no inhibitor. Ascorbic acid and cysteine, general modifiers of oxidative pathways of metabolism, also affected estradiol metabolism in microsomal and hepatocyte preparations. Both these agents were able to decrease the irreversible binding of estradiol to proteins in the microsomal assays. Ascorbic acid decreased the general metabolism of estradiol in the hepatocyte incubations but did not decrease irreversible binding to proteins. The addition of cysteine to the hepatocyte incubation resulted in an increased metabolism of estradiol and the production of more aqueous-soluble radiolabeled metabolites than the control incubations; however, cysteine did not decrease the amounts of estradiol metabolite(s) irreversibly bound to proteins. Investigations of steroid metabolism in the isolated hepatocytes thus provide an effective in vitro technique for examining the overall oxidative, reductive, and conjugative pathways that are functional in the liver and enables one to investigate the abilities of inhibitors, regulators, and modifiers to affect the metabolic processes. Also, these hepatocyte studies demonstrate that the inhibitors of estrogen 2-hydroxylase, 2-bromoestradiol and 4-bromoestradiol, can enter and act in the intact cells. Consequently, these agents may be useful pharmacological probes for examining the functions of catechol estrogens in other tissues.     

Synthesis of vitamin K1 2,3-epoxide in rat liver mitochondria

Metabolism of vitamin K1 in rat liver mitochondria has been studied with succinate as the source of reducing equivalents. A metabolite was isolated that comigrated with vitamin K1 epoxide using four different chromatographic systems. The purified metabolite had an ultraviolet spectrum (200-330 nm) that was identical to that of synthetic vitamin K1 epoxide. The mass spectrum of the purified metabolite was identical to that of synthetic vitamin K1 epoxide. A comparison of production of vitamin K1 epoxide by mitochondrial and microsomal preparations indicates that the mitochondrial production of vitamin K1 epoxide was about 50% of that of the microsomes. Since the mitochondrial preparation was found to have only 3.4% of the glucose-6-phosphatase activity of the microsomal preparation, it can be concluded that the vitamin K1 epoxide isolated from the mitochondrial incubations was due primarily to mitochondrial synthesis. Epoxidation of vitamin K1 in mitochondria suggests that mitochondria might be sites for vitamin K-dependent carboxylation of protein(s).     

The covalent binding of acetaminophen to protein. Evidence for cysteine residues as major sites of arylation in vitro

Covalent binding of the reactive metabolite of acetaminophen has been investigated in hepatic microsomal preparations from phenobarbital-pretreated mice. Low molecular weight thiols (cysteine and glutathione) were found to inhibit this binding, whereas several other amino acids which were tested did not. Bovine serum albumin (BSA), which contains a single free sulfhydryl group per molecule and which thus represents a macromolecular thiol compound, inhibited covalent binding of the reactive acetaminophen metabolite to microsomal protein in a concentration-dependent manner. The acetaminophen metabolite also became irreversibly bound to BSA in these experiments, although this binding was reduced by approx. 47% when the thiol function of BSA was selectively blocked prior to incubation. Covalent binding of the acetaminophen metabolite to bovine alpha s1-casein, a soluble protein which does not contain any cysteine residues, was found to occur to an extent of 37% of that which became bound to native BSA. These results were taken to indicate that protein thiol groups are major sites of covalent binding of the reactive metabolite of acetaminophen in vitro. The covalent binding characteristics of synthetic N-acetyl-p-benzoquinoneimine (NAPQI), the putative electrophilic intermediate produced during oxidative metabolism of acetaminophen, paralleled closely those of the reactive species generated metabolically. These findings support the contention that NAPQI is indeed the reactive arylating metabolite of acetaminophen which binds irreversibly to protein.     

Estrogen 1,2-epoxides or estrogen quinones/semiquinones

Metabolic activation of estradiol leading to the formation of catechol estrogens is a prerequisite for its genotoxic activity. Both estrogen-o-quinones/semiquinones and estrogen 1,2-epoxides have been proposed to be responsible for this activity. Incubations of [3H]estradiol and [3H]1 alpha,2 alpha-epoxy-4-estrene-3-one-17 beta-ol (ketotautomer of estradiol 1,2-epoxide) with rat liver microsomal and cytosol preparations were carried out in the presence of SKF 525A, ascorbic acid, glutathione and cysteine. Ascorbic acid decreased binding to proteins and aqueous-soluble fraction with both [3H] estradiol and [3H]epoxyestrenolone in incubations with microsomes but no effect with cytosol fraction. Incubations of microsomes with thiols gave water-soluble metabolites which were characterized as 1(4)-thioether derivatives of 2-hydroxyestradiol and incubations of [3H]epoxyestrenolone with cytosol and thiols gave estradiol-2-thioether. Incubations with ascorbic acid and thiols resulted in decreased formation of water-soluble metabolites in microsomal incubations but not in cytosol incubations. These studies indicate that the major pathway for irreversible binding of estrogens to macromolecules involves estrogen-o-quinones/semiquinones and not estrogen 1, 2-epoxide.     

Novel non-labile covalent binding of sulfamethoxazole reactive metabolites to cultured human lymphoid cells

Sulfamethoxazole (SMX) causes rare hypersensitivity syndrome reactions characterized by fever and multi-organ toxicity. Covalent binding of SMX reactive metabolites to cellular proteins has been demonstrated but the link between cytotoxicity and targets of covalent binding has not been explored. We therefore investigated the relationship between covalent binding of the reactive SMX-hydroxylamine (SMX-HA) metabolite, and its cytotoxicity to a hystiocytic lymphoma (U937) cell line. Incubation of U937 cells with 0-1 mM SMX-HA for 3 h resulted in dose-dependent cytotoxicity, as assessed by tetrazolium dye conversion at 24 h. SMX-HA caused dose-dependent covalent binding to cellular proteins as assessed by immunoblotting with SMX antisera at 3 and 24 h. Covalent binding was predominantly to proteins of approximately 45, 59 and 75 kDa, but other targets were also observed. The relative extent of binding to proteins was significantly different from the relative cytotoxicity at 24 h. Further, cells surviving at 24 h also had extensive covalent binding. Covalent binding was observed under reducing (beta-mercaptoethanol) and non-reducing conditions to plasma membrane and microsomal but not cytosolic proteins. This non-labile covalent binding has not been previously reported. These observations suggest that extensive covalent binding does not necessarily lead to cell death, allowing the accumulation of potentially immunogenic drug-protein conjugates. These observations in whole cells may be relevant to the immunopathogenesis of SMX hypersensitivity syndrome reactions.     

Selective inhibition of cytosolic epoxide hydrolase activity in vitro by compounds that inhibit catalase

The ability of a number of known inhibitors of catalase activity to affect cytosolic and microsomal epoxide hydrolase activities in vitro, measured as enzymatic trans-stilbene oxide hydrolysis and styrene oxide hydrolysis, respectively, was investigated. Catalase and cytosolic epoxide hydrolase activities are inhibited by hydroxylated metabolites of 2-amino-4,5-diphenylthiazole (DPT). The metabolite hydroxylated on the 4-phenyl ring (4OH-DPT) and the metabolite hydroxylated on both phenyl rings (4,5-DIOH-DPT) are potent inhibitors of both enzymes; the metabolite hydroxylated on the 5-phenyl ring (5OH-DPT) is less potent. Unmetabolized DPT has no effect on either enzyme. 4OH-DPT inhibits, but 5OH-DPT enhances, microsomal epoxide hydrolase activity. 4,5-DIOH-DPT and DPT have no effect on this enzyme. Other compounds that inhibit both catalase and cytosolic epoxide hydrolase activities, but do not inhibit microsomal epoxide hydrolase activity, are nordihydroguaiaretic acid and 2-aminothiazole. Microsomal epoxide hydrolase activity is enhanced by 2-aminothiazole and levamisole in vitro. Thus these inhibitors of catalase are selective epoxide hydrolase inhibitors in that they inhibit cytosolic epoxide hydrolase activity in vitro, but have either no effect on, or increase the activity of, microsomal epoxide hydrolase in vitro. Conversely, the selective cytosolic epoxide hydrolase inhibitors 4-phenylchalcone oxide and 4'-phenylchalcone oxide do not inhibit catalase activity, nor does trichloropropene oxide, a selective microsomal epoxide hydrolase inhibitor.     

Structural and functional consequences of inactivation of human glutathione S-transferase P1-1 mediated by the catechol metabolite of equine estrogens, 4-hydroxyequilenin

The inactivation mechanism(s) of human glutathione S-transferase P1-1 (hGST P1-1) by the catechol metabolite of Premarin estrogens, 4-hydroxyequilenin (4-OHEN), was (were) studied by means of site-directed mutagenesis, electrospray ionization mass spectrometric analysis, titration of free thiol groups, kinetic studies of irreversible inhibition, and analysis of band patterns on nonreducing sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The four cysteines (Cys 14, Cys 47, Cys 101, and Cys 169 in the primary sequence) in hGST P1-1 are susceptible to electrophilic attack and/or oxidative damage leading to loss of enzymatic activity. To investigate the role of cysteine residues in the 4-OHEN-mediated inactivation of this enzyme, one or a combination of cysteine residues was replaced by alanine residues (C47A, C101A, C47A/C101A, C14A/C47A/C101A, and C47A/C101A/C169A mutants). Mutation of Cys 47 decreased the affinity for the substrate GSH but not for the cosubstrate 1-chloro-2,4-dinitrobenzene (CDNB). However, the Cys 47 mutation did not significantly affect the rate of catalysis since V(max) values of the mutants were similar or higher compared to that of wild type. Electrospray ionization mass spectrometric analyses of wild-type and mutant enzymes treated with 4-OHEN showed that a single molecule of 4-OHEN-o-quinone attached to the proteins, with the exception of the C14A/C47A/C101A mutant where no covalent adduct was detected. 4-OHEN also caused oxidative damage as demonstrated by the appearance of disulfide-bonded species on nonreducing SDS--PAGE and protection of 4-OHEN-mediated enzyme inhibition by free radical scavengers. The studies of thiol group titration and irreversible kinetic experiments indicated that the different cysteines have distinct reactivity for 4-OHEN; Cys 47 was the most reactive thiol group whereas Cys 169 was resistant to modification. These results demonstrate that hGST P1-1 is inactivated by 4-OHEN through two possible mechanisms: (1) covalent modification of cysteine residues and (2) oxidative damage leading to proteins inactivated by disulfide bond formation.     

The reactive metabolite target protein database (TPDB) – a web-accessible resource

Background  

The toxic effects of many simple organic compounds stem from their biotransformation to chemically reactive metabolites which bind covalently to cellular proteins. To understand the mechanisms of cytotoxic responses it may be important to know which proteins become adducted and whether some may be common targets of multiple toxins. The literature of this field is widely scattered but expanding rapidly, suggesting the need for a comprehensive, searchable database of reactive metabolite target proteins.     

Tissue factor coagulant function is enhanced by protein-disulfide isomerase independent of oxidoreductase activity

Protein-disulfide isomerase (PDI) switches tissue factor (TF) from coagulation to signaling by targeting the allosteric Cys186-Cys209 disulfide. Here, we further characterize the interaction of purified PDI with TF. We find that PDI enhances factor VIIa-dependent substrate factor X activation 5-10-fold in the presence of wild-type, oxidized soluble TF but not TF mutants that contain an unpaired Cys186 or Cys209. PDI-accelerated factor Xa generation was blocked by bacitracin but not influenced by inhibition of vicinal thiols, reduction of PDI, changes in redox gradients, or covalent thiol modification of reduced PDI by N-ethylmaleimide or methyl-methanethiosulfonate, which abolished PDI oxidoreductase but not chaperone activity. PDI had no effect on fully active TF on either negatively charged phospholipids or in activating detergent, indicating that PDI selectively acts upon cryptic TF to facilitate ternary complex formation and macromolecular substrate turnover. PDI activation was reduced upon mutation of TF residues in proximity to the macromolecular substrate binding site, consistent with a primary interaction of PDI with TF. PDI enhanced TF coagulant activity on microvesicles shed from cells, suggesting that PDI plays a role as an activating chaperone for circulating cryptic TF.     

The formation of chlorobenzene and benzene by the reductive metabolism of lindane in rat liver microsomes

The major metabolite produced by incubating [14C]lindane with rat liver microsomes under anaerobic conditions was determined to be chlorobenzene, with lesser amounts of benzene also being formed. Using relatively high lindane concentrations (250 microM), four nonvolatile metabolites of lindane were also produced anaerobically, the predominant one being identified by mass spectrometry as tetrachlorocyclohexene (TCCH). TCCH, likewise, was reduced to chlorobenzene and benzene in microsomes under anaerobic conditions. Binding of [14C]lindane to microsomal protein occurred under aerobic as well as anaerobic incubation conditions; however, lindane protein binding was greatest in anaerobic incubations compared to those containing an atmosphere of air or 100% oxygen. Hemin reduced by dithionite also readily produced chlorobenzene and benzene from lindane. These results indicate that lindane interacts readily with heme and heme proteins, including cytochrome P-450, in the absence of oxygen to undergo multiple chloride eliminations forming chlorobenzene and benzene as end products.     

Identification of extremely reactive gamma-ketoaldehydes (isolevuglandins) as products of the isoprostane pathway and characterization of their lysyl protein adducts

Isoprostanes are prostaglandin-like compounds produced by non-enzymatic peroxidation of arachidonic acid. The cyclooxygenase-derived endoperoxide, prostaglandin H2, can undergo rearrangement to highly reactive gamma-ketoaldehyde secoprostanoids (levuglandin E2 and D2). We explored whether isoprostane endoperoxide intermediates also rearrange to levuglandin-like compounds (isolevuglandins). Formation of a series of isolevuglandins during oxidation of arachidonic acid in vitro was established utilizing a number of mass spectrometric analyses. However, these compounds could not be detected in free form in protein-containing biological systems, which we hypothesized was due to extremely rapid adduction to amines. This was supported by the finding that >60% of levuglandin E2 adducted to albumin within 20 s, whereas approximately 50% of 4-hydroxynonenal still remained unadducted after 1 h. By utilizing electrospray tandem mass spectrometry, we established that these compounds form oxidized pyrrole adducts (lactams and hydroxylactams) with lysine. Formation of isolevuglandin-lysine adducts on apolipoprotein B was readily detected during oxidation of low density lipoprotein following enzymatic digestion of the protein to single amino acids. These studies identify a novel series of extremely reactive products of the isoprostane pathway that rapidly form covalent adducts with lysine residues on proteins. This provides the basis to explore the formation of isolevuglandins in vivo to investigate the potential biological ramifications of their formation in settings of oxidant injury.