In vivo covalent binding of [14C]trinitrotoluene to proteins in the rat.

When a single dose of [14C]trinitrotoluene was administered intraperitoneally (i.p.) to rats at 1, 10 or 50 mg/kg of body weight, covalently bound radioactivity was detected in globin, plasma proteins and proteins in the liver and kidney. The extent of covalent binding was dose dependent and was highest in plasma and renal proteins at all times up to 4 h after dosing. Covalent adduct levels in globin, however, decline slower than others. At a dose of 50 mg/kg of body weight, globin covalent adduct levels peaked at 1 h after dosing at 182 pmol/mg protein and subsequently decreased to approximately 50 pmol/mg protein between days 1 and 8. Of the covalent adduct levels in liver and kidney, those in the 10,000 x g and microsomal fractions were found to be higher than that in the cytosolic fraction. Radioactivity covalently bound to globin and the hepatic proteins was susceptible to dilute acid hydrolysis from which 2-amino-4,6-dinitrotoluene (2A) and 4-amino 2,6-dinitrotoluene (4A) were the major products recovered by solvent extraction. Upon acetylation, the hydrolysate gave rise to derivatives identified as the acetates of 2A and 4A on the basis of mass spectrometry and HPLC cochromatography with authentic samples. Four hours after an i.p. dose of [14C]TNT at 50 mg/kg of body weight about 0.4% of the dose was found as bound adducts to hemoglobin, of which approximately 48% was recovered as solvent extractable radioactivity after acid hydrolysis. About 2% of the radioactive dose was in the liver, of which approximately 30% was covalently bound to hepatic proteins, and approximately 49% of that was convertible to solvent extractable radioactivity upon acid hydrolysis. In vitro incubation of [14C]TNT with blood showed that there was a linear increase of covalent adducts in globin during the first 2 h of incubation; the concentration of covalent adducts was slightly higher than that with plasma proteins. The major compounds recovered from the hydrolysate of the globin adducts were also 2A and 4A as obtained from globin in the in vivo studies. On the basis of the in vitro and in vivo study results, we have confirmed the formation of protein adducts following a single i.p. administration of [14C]TNT at 1, 10 or 50 mg/kg of body weight to the rat or by in vitro incubation with blood.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

SIRT3 deacetylase: the Jekyll and Hyde sirtuin

Mitochondrial deacetylase SIRT3 protects against oxidative damage. In an article published online this month in EMBO reports, it is shown to also aggravate paracetamol-induced liver toxicity, calling for caution in trying to pharmacologically enhance SIRT3 activity.EMBO Rep (2011) advance online publication. doi:10.1038/embor.2011.121Post-translational modifications have crucial roles in regulating the functions of many eukaryotic proteins. Among them, lysine acetylation has been traditionally studied in the context of nuclear histone modifications, and was one of the first to be described as part of the ‘histone code'' hypothesis (Kim et al, 2006). More recently, work from several groups has demonstrated that lysine acetylation also modulates the activity of several non-histone proteins. In this context, this modification seems particularly abundant on mitochondrial proteins (Schwer et al, 2009). However, the way in which acetylation influences enzyme function and metabolic reprogramming in pathological states remains unknown. In an article published online this month in EMBO reports, Sack and colleagues shed new light on the role of mitochondrial SIRT3 deacetylase during paracetamol-induced toxicity, describing the mitochondrial protein aldehyde dehydrogenase 2 (ALDH2) as a new target of SIRT3, and a protective role for protein acetylation in this context (Lu et al, 2011).The sirtuin family of NAD⁺-dependent deacetylases comprises seven mammalian homologues (SIRT1–SIRT7) that have diverse functions and cellular localizations (Finkel et al, 2009). Among them, mitochondrial SIRT3 is the main deacetylase involved in the modulation of mitochondrial metabolic and oxidative-stress regulatory pathways (Schwer et al, 2009). SIRT3 seems to mediate protection against oxidative damage under caloric restriction (Someya et al, 2010), as well as promoting enhanced protection against redox and nutrient-excess stress (Zhong & Mostoslavsky, 2011).…these results raise the tantalizing possibility that—at least in the context of [paracetamol] toxicity—the less SIRT3 the betterAcetaminophen (APAP)—commonly known as paracetamol—is a widely used analgesic and anti-pyretic drug that is safe at therapeutic-dose levels. However, APAP overdose has been linked to liver injury in both humans and mice (Jaeschke & Bajt, 2006), with a high mortality rate due to acute liver failure. Remarkably, this hepatotoxic effect seems to be enhanced by fasting (Whitcomb & Block, 1994), a phenomenon that was poorly understood. In the initial phases of cell injury, a product of APAP oxidation—the highly reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI)—binds to protein cysteine and lysine residues (Zhou et al, 1996), eventually depleting hepatic glutathione and leading to the concomitant hepatotoxicity. Although there has been extensive research, the underlying molecular mechanisms of liver injury have not been fully elucidated.In this new study, Lu and colleagues aimed to decipher the way in which fasting or caloric-restriction exacerbate the redox-stress-dependent toxicity of APAP (Lu et al, 2011). Given the known increase in SIRT3 activity on nutrient deprivation, they proposed that, if protein acetylation inhibits NAPQI binding, SIRT3-mediated deacetylation might aggravate acetaminophen-induced liver injury (AILI).First, they tested whether lack of SIRT3 protects against AILI, by analysing susceptibility to liver injury in SIRT3^+/+ and SIRT3^−/− mice treated with a single toxic dose of APAP under fed and fasted conditions. Strikingly, they found that fasted SIRT3^−/− mice showed less hepatotoxicity than the SIRT3-competent mice. By using two-dimensional gel and immunoblot analyses, they then compared hepatic mitochondrial-protein acetylation profiles between fasted SIRT3^−/− and SIRT3^+/+ mice. In these experiments they identified, among several candidates, ALDH2—a known target of NAPQI, binding to which is known to reduce ALDH2 activity (Landin et al, 1996). This dehydrogenase oxidizes and detoxifies aldehydes—including lipid peroxidation products such as trans-4-hydroxy-2-nonenal (4-HNE; Doorn et al, 2006)—and thus buffers these highly reactive metabolites.…SIRT3 might act as a double-edged sword [raising] a word of caution regarding therapeutic strategies aimed at potentiating SIRT3 activityLu and colleagues then focused on ALDH2. In a series of elegant studies, they demonstrated that ALDH2 is a direct target of SIRT3, and deacetylation of ALDH2 modifies NAPQI binding. Liver mitochondria from SIRT3-deficient mice had increased ALDH2 acetylation, indicating a direct interaction between SIRT3 and ALDH2. ALDH2 was then shown to be a direct target of SIRT3 by using in vitro deacetylation assays. Despite these differences, basal ALDH2 activity remained the same in both genotypes; enzymatic activity was therefore evaluated in response to APAP treatment in fasted mice. Remarkably, SIRT3-deficient mitochondria exhibited approximately 40% higher levels of ALDH2 activity after APAP administration and, consequently, significantly lower levels of 4-HNE adducts were detected, in comparison to SIRT3^+/+ mice. SIRT3 is a known protective factor against oxidative stress; however, these results raise the tantalizing possibility that—at least in the context of APAP toxicity—the less SIRT3 the better.Logically, the next step was to show that the protective effect of SIRT3 deficiency is directly dependent on sustained ALDH2 activity. A marked increased in liver injury in the SIRT3-deficient animals was observed after knockdown of ALDH2 by using a lentiviral short-hairpin RNA approach, supporting their argument. To gain further molecular insight, the authors followed previous observations indicating that binding of NAPQI to ALDH2 diminishes ALDH2 activity (Landin et al, 1996). They hypothesized that SIRT3 might deacetylate ALDH2, in turn increasing its binding to NAPQI and leading to the concomitant inactivation of the protein. Indeed, through elegant SIRT3 gain- and loss-of-function experiments, they demonstrated that SIRT3-dependent deacetylation of ALDH2 enhances binding of the enzyme to NAPQI, whereas SIRT3 inactivation decreases NAPQI binding to ALDH2.The Sack group went one step further and used mass spectrometry to identify ALDH2 Lys 377 as the residue deacetylated by SIRT3. They showed that acetylation of Lys 377 is increased in SIRT3-deficient mice, and a mutant ALDH2 with an acetyl-mimicking mutation (K377Q) exhibited significantly less binding to NAPQI, giving a detailed molecular explanation for the protective effect observed in the absence of this sirtuin.These findings demonstrate that SIRT3-mediated deacetylation of mitochondrial proteins modulates susceptibility to AILI. Furthermore, the identification of ALDH2 as the substrate for SIRT3 deacetylation in this process provides a molecular framework in which to understand the apparent paradox of enhanced APAP toxicity under conditions of fasting or caloric restriction. Fasting induces SIRT3-mediated deacetylation of ALDH2, leading to increased NAPQI binding, which in turn reduces ALDH2 activity. This causes an accumulation of highly reactive adducts, probably contributing to the exacerbated hepatotoxicity observed after APAP treatment under nutrient restriction (Fig 1).Open in a separate windowFigure 1SIRT3-mediated exacerbation of acetaminophen-induced liver injury. SIRT3 deacetylates Lys 377 of ALDH2, making it available for NAPQI binding, which de-activates it. The concomitant reduction in the aldehyde-detoxifying activity of ALDH2 aggravates liver injury. AILI, acetaminophen-induced liver injury; ALDH2, aldehyde dehydrogenase 2; NAPQI, N-acetyl-p-benzoquinoneimine.The toxic effects of AILI have been traditionally addressed by using anti-oxidant therapies based on NAPQI binding to cysteine residues. Surprisingly, the functional outcome of NAPQI binding to lysine residues has not been explored so far, although it was described almost 15 years ago (Zhou et al, 1996). The Sack laboratory approached this issue, providing clear, supportive data for an interesting and provocative hypothesis: although it is widely accepted that SIRT3 has protective, anti-oxidant effects, ALDH2 deacetylation by SIRT3 exacerbates APAP-induced hepatotoxicity. This indicates that SIRT3 might act as a double-edged sword, and raises a word of caution regarding therapeutic strategies aimed at potentiating SIRT3 activity. Although this study provides support for this paradoxical effect, some questions remain. First, is ALDH2 the only SIRT3 substrate involved in this phenotype? The authors show that several other proteins were identified in their study, but their roles remain to be explored. Second, what is the physiological role of SIRT3-mediated ALDH2 deacetylation? Does this modification alter ALDH2 activity under conditions of nutrient stress? If so, how? Third, how general is this phenomenon? Does protein deacetylation modulate the binding of other toxic metabolites to proteins in detoxifying organs, such as the liver? Although answers to these questions await future investigation, one thing is certain: we need to exercise caution when evaluating the therapeutic potential of sirtuin modulators.     

Structural Basis of Inactivation of Thiol Protease by N-Acetyl-p-benzoquinone Imine (NAPQI). A Knowledge-Based Molecular Modeling of the Adduct of NAPQI with Thiol Protease of the Papain Family

Inhibition of hepatic cysteine proteases by non-steroidal anti-inflammatory drug (NSAID) metabolites is implicated in several pathological conditions. It has been reported in the literature that N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen (APAP) can quickly arylate and oxidize thiol (cysteine) protease of the papain family to form an adduct in the pathogenesis of acetaminophen-induced hepatotoxicity. It was also clarified by earlier NMR studies that the 3-position of the aromatic ring (C-3) is the only site of conjugation with cysteinyl thioethers for protein arylation. In a recent study, the adduct of NAPQI has been identified and characterized by LC/MS/MS, LC/NMR and UV spectroscopy, and two possible covalent binding modes corresponding to the 2-position (model-1) and the 3 -position (model-2) of the aromatic ring of NAPQI have been proposed. The work presented here has been initiated to check the structural viability of inhibition for the two proposed adducts at the atomic level. Results of our investigation by computer-assisted molecular modeling structurally demonstrate why model-2 would be more applicable to the static x-ray structure of the complex at physiological pH. This coordinated computational and molecular biology experiment can be used for metabolic screening of NSAIDs. A combinatorial approach of this kind alleviates the doubts in interpreting the results of metabolic function and enhances our insights obtained from either computational or experimental studies alone.     

Macromolecular covalent binding of [14C]nitrobenzene in the erythrocyte and spleen of rats and mice

Nitrobenzene exposure is known to produce red blood cell damage as well as engorgement and sinusoidal congestion of the spleen in male Fischer-344 (F-344) rats but not in male B6C3F1 mice. These studies were conducted to investigate the species differences in the covalent binding of [¹⁴C]nitrobenzene in the erythrocyte and spleen and to assess the contribution of nitrobenzene-induced erythrocytic damage to the splenic effects. Total and covalently bound ¹⁴C concentrations in erythrocytes of rats were 6–13 times greater than those of mice following a single oral dose of 75, 150, 200 or 300 mg/kg [¹⁴C]nitrobenzene, suggesting that species differences in nitrobenzene-induced red blood cell toxicity may be related to differences in erythrocytic accumulation of nitrobenzene and its metabolites. Covalently bound ¹⁴C in erythrocytes of rats peaked 24 h following administration of 200 mg [¹⁴C]nitrobenzene/kg; in contrast, bound radio-label in erythrocytes from mice plateaued at 10 h. Splenic engorgement increased in a time-related manner in treated rats but not in mice. Species specificity was also observed in the accumulation of bound radiolabel in the spleen. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of lysed, dialyzed erythrocytes from treated rats revealed that hemoglobin was the primary, if not the exclusive, site of macromolecular covalent binding following nitrobenzene treatment. SDS-PAGE of dialyzed rat spleens revealed that 82% of total bound ¹⁴C migrated identically to hemoglobin. These data indicate that covalent binding of [¹⁴C]nitrobenzene and its metabolites in the spleen is primarily derived from bound ¹⁴C from scavenged erythrocytes. Therefore, the species differences in splenic engorgement and accumulation of [¹⁴C]nitrobenzene may be related to differences in susceptibility to nitrobenzene-induced red blood cell damage.     

Identification of S-(2,5-dihydroxyphenyl)-cysteine and S-(2,5-dihydroxyphenyl)-N-acetyl-cysteine as urinary metabolites of acetaminophen in the mouse. Evidence for p-benzoquinone as a reactive intermediate in acetaminophen metabolism

S-(2,5-Dihydroxyphenyl)-cysteine and S-(2,5-dihydroxyphenyl)-N-acetyl-cysteine [the cysteine- and N-acetyl-cysteine adducts, respectively, of hydroquinone (HQ)] were identified and quantified in the urine of mice administered [ring-U-14C]acetaminophen [14C]APAP, 200 mg kg-1, i.p.). Urine was collected for 24 h and fractionated by HPLC to isolate the above adducts. These conjugates were then converted to a common derivative, viz. O,O',S-tris-acetyl-3-thio-hydroquinone, which was characterized by GC/MS. Neither of the HQ adducts was detected in the urine of control mice which had not received APAP. Quantification of urinary HQ-cysteine and HQ-N-acetyl-cysteine was performed by HPLC techniques, which indicated that these conjugates accounted for approx. 1.5% of the administered dose of APAP after 24 h, a figure which is equivalent to 6.3% of the corresponding APAP-thiol conjugates in the urine. These findings provide strong indirect evidence that p-benzoquinone is formed as a reactive, but apparently non-hepatotoxic, metabolite of APAP in vivo.     

Binding of chloroform to mouse and rat hemoglobin

The covalent binding of chemical carcinogens and mutagens to hemoglobin has been proposed as a dose monitor for environmental exposure. The binding of chloroform to hemoglobin in rats was demonstrated to result from the formation of addition adducts to amino acids in the globin. The altered amino acids were isolated with an amino acid analyzer employing ion exchange chromatography. The covalent binding of orally-administered [¹⁴C]chloroform to rat hemoglobin reached a peak within 10 h. Afterwards, the amount of chloroform bound decreased slowly with half remaining at 7 weeks. The extent of chloroform binding was linearly dependent upon dose between 0.1 and 100 μmol/kg. Above 100 μmol/kg chloroform the binding to hemoglobin increased at a reduced rate. The extent of [¹⁴C]chloroform binding resulting from either 10 daily doses of 0.1 μmol/kg or a single 1.0 μmol/kg was similar. The binding to hemoglobin in three strains of mice and rats varied from 85 ± 7 μmol/g Hb for Swiss (CFN) mice to 152 ± 14 for Fisher-344 rats. We have demonstrated that the binding of chloroform to hemoglobin appears to have the following attributes of a dose monitor: (1) easily accessible from laboratory animals (and humans); (2) dose dependent; (3) stability, so that exposure can be determined weeks after it has ceased; (4) integration of low exposures which individually would be undetectable.     

Structural characterization of the major covalent adduct formed in vitro between acetaminophen and bovine serum albumin

The structure of the covalent adduct formed in vitro between [14C]-acetaminophen ([14C]APAP) and bovine serum albumin (BSA) has been investigated with the aid of new analytical methodology. The APAP-BSA adduct, isolated from mouse liver microsomal incubations to which the radiolabeled drug and BSA had been added, was cleaved using a combination of specific (cyanogen bromide) and non-specific (acid hydrolysis) procedures, following which the mixture of amino acids obtained was derivatized, in aqueous solution, with ethyl chloroformate. The resulting ethoxycarbonyl derivatives were recovered by extraction into ethylacetate, methylated and subjected to profile analysis using both reverse-phase and normal-phase HPLC techniques. In each HPLC step, one major radioactive amino acid adduct was detected and was identified by mass spectrometry as the derivative of 3-cystein-S-yl-4-hydroxyaniline. Based on this finding, and with a knowledge of the behavior under acidic hydrolysis conditions of the 3-cysteinyl conjugate of APAP, it could be concluded that the major APAP-BSA adduct is one in which the drug is bound, via a thioether linkage at the C-3 position, to a sulfhydryl group on the protein. Furthermore, it could be established that this -SH function almost certainly is that associated with the cys-34 residue of BSA.     

Evidence for cytochrome P4501A2-mediated protein covalent binding of thiabendazole and for its passive intestinal transport: use of human and rabbit derived cells

Thiabendazole (TBZ), an anthelmintic and fungicide benzimidazole, was recently demonstrated to be extensively metabolized by cytochrome P450 (CYP) 1A2 in man and rabbit, yielding 5-hydroxythiabendazole (5OH-TBZ), the major metabolite furtherly conjugated, and two minor unidentified metabolites (M1 and M2). In this study, exposure of rabbit and human cells to 14C-TBZ was also shown to be associated with the appearance of radioactivity irreversibly bound to proteins. The nature of CYP isoforms involved in this covalent binding was investigated by using cultured rabbit hepatocytes treated or not with various CYP inducers (CYP1A1/2 by beta-naphthoflavone, CYP2B4 by phenobarbital, CYP3A6 by rifampicine, CYP4A by clofibrate) and human liver and bronchial CYP-expressing cells. The covalent binding to proteins was particularly increased in beta-naphthoflavone-treated rabbit cells (2- to 4-fold over control) and human cells expressing CYP1A2 (22- to 42-fold over control). Thus, CYP1A2 is a major isoenzyme involved in the formation of TBZ-derived residues bound to protein. Furthermore, according to the good correlation between covalent binding and M1 or 5OH-TBZ production, TBZ would be firstly metabolized to 5OH-TBZ and subsequently converted to a chemically reactive metabolic intermediate binding to proteins. This metabolic activation could take place preferentially in liver and lung, the main biotransformation organs, rather than in intestines where TBZ was shown to be not metabolized. Moreover, TBZ was rapidly transported by passive diffusion through the human intestinal cells by comparison with the protein-bound residues which were not able to cross the intestinal barrier. Consequently, the absence of toxicity measured in intestines could be related to the low degree of TBZ metabolism and the lack of absorption of protein adducts. Nevertheless, caution is necessary in the use of TBZ concurrently with other drugs able to regulate CYP1A2, particularly in respect to liver and lung tissues, recognised as sites of covalent-binding.     

The metabolism of pentachlorobenzene by rat liver microsomes: the nature of the reactive intermediates formed

Metabolism of [14C]-pentachlorobenzene by liver microsomes from dexamethasone-induced rats results in the formation of pentachlorophenol and 2,3,4,6-tetrachlorophenol as major primary metabolites in a ratio of 4:1, with 2,3,4,5- and 2,3,5,6-tetrachlorophenols as minor metabolites. The unsubstituted carbon atom is thus the favourite site of oxidative attack, but the chlorine substituted positions still play a sizable role. As secondary metabolites both para- and ortho-tetrachlorohydroquinone are formed (1.4 and 0.9% of total metabolites respectively). During this cytochrome P450-dependent conversion of pentachlorobenzene, 5-15% of the total amount of metabolites becomes covalently bound to microsomal protein. Ascorbic acid inhibits this binding to a considerable extent, indicating that quinone metabolites play an important role in the binding. However, complete inhibition was never reached by ascorbic acid, nor by glutathione, suggesting that other reactive intermediates, presumably epoxides, are also responsible for covalent binding.     

SIRT3-dependent deacetylation exacerbates acetaminophen hepatotoxicity

Acetaminophen/paracetamol-induced liver failure--which is induced by the binding of reactive metabolites to mitochondrial proteins and their disruption--is exacerbated by fasting. As fasting promotes SIRT3-mediated mitochondrial-protein deacetylation and acetaminophen metabolites bind to lysine residues, we investigated whether deacetylation predisposes mice to toxic metabolite-mediated disruption of mitochondrial proteins. We show that mitochondrial deacetylase SIRT3(-/-) mice are protected from acetaminophen hepatotoxicity, that mitochondrial aldehyde dehydrogenase 2 is a direct SIRT3 substrate, and that its deacetylation increases acetaminophen toxic-metabolite binding and enzyme inactivation. Thus, protein deacetylation enhances xenobiotic liver injury by modulating the binding of a toxic metabolite to mitochondrial proteins.     

Evidence that covalent binding of metabolically activated phenol to microsomal proteins is caused by oxidised products of hydroquinone and catechol

The metabolic activation of [14C]phenol resulting in covalent binding to proteins has been studied in rat liver microsomes. The covalent binding was dependent on microsomal enzymes and NADPH and showed saturation kinetics for phenol with a Km-value of 0.04 mM. The metabolites hydroquinone and catechol were formed at rates which were 10 or 0.7 times that of the binding rate of metabolically activated phenol. The effects of cytochrome P-450 inhibitors and cytochrome P-450 inducers on the metabolism and binding of phenol to microsomal proteins, suggest that cytochrome P-450 isoenzyme(s) other than P-450 PB-B or P-450 beta NF-B catalyses the metabolic activation of phenol. Furthermore, reconstituted mixed-function oxidase systems containing cytochrome P-450 PB-B and P-450 beta NF-B were (on basis of cytochrome P-450 content) 6 and 11 times less active in catalysing the formation of hydroquinone than microsomes. The isolated metabolites hydroquinone and catechol bound more extensively to microsomal proteins than phenol and the binding of these was not stimulated by NADPH. The binding occurring during the metabolism of phenol could be predicted by the rates of formation of hydroquinone and catechol and the rates by which the isolated metabolites were bound to proteins.     

Analysis of naphthalene adduct binding sites in model proteins by tandem mass spectrometry

The electrophilic metabolites of the polyaromatic hydrocarbon naphthalene have been shown to bind covalently to proteins and covalent adduct formation correlates with the cytotoxic effects of the chemical in the respiratory system. Although 1,2-naphthalene epoxide, naphthalene diol epoxide, 1,2-naphthoquinone, and 1,4-napthoquinone have been identified as reactive metabolites of interest, the role of each metabolite in total covalent protein adduction and subsequent cytotoxicity remains to be established. To better understand the target residues associated with the reaction of these metabolites with proteins, mass spectrometry was used to identify adducted residues following (1) incubation of metabolites with actin and protein disulfide isomerase (PDI), and (2) activation of naphthalene in microsomal incubations containing supplemental actin or PDI. All four reactive metabolites bound to Cys, Lys or His residues in actin and PDI. Cys(17) of actin was the only residue adducted by all metabolites; there was substantial metabolite selectivity for the majority of adducted residues. Modifications of actin and PDI, following microsomal incubations containing (14)C-naphthalene, were detected readily by 2D gel electrophoresis and phosphor imaging. However, target modifications on tryptic peptides from these isolated proteins could not be readily detected by MALDI/TOF/TOF and only three modified peptides were detected using high resolution-selective ion monitoring (HR-SIM). All the reactive metabolites investigated have the potential to modify several residues in a single protein, but even in tissues with very high rates of naphthalene activation, the extent of modification was too low to allow unambiguous identification of a significant number of modified residues in the isolated proteins.     

Activation of microsomal glutathione transferase activity by reactive intermediates formed during the metabolism of phenol

The activity of microsomal glutathione transferase was increased 1.7-fold in rat liver microsomes which carried out NADPH dependent metabolism of phenol. Known phenol metabolites were therefore tested for their ability to activate the microsomal glutathione transferase. The phenol metabolites benzoquinone and 1,2,4-benzenetriol both activated the glutathione transferase in microsomes 2-fold independently of added NADPH. However, NADPH was required to activate the enzyme in the presence of hydroquinone. Catechol did not activate the enzyme in microsomes. The purified enzyme was activated 6-fold and 8-fold by 5 mM benzenetriol and benzoquinone respectively. Phenol, catechol or hydroquinone had no effect on the purified enzyme. When microsomal proteins that had metabolized [14C]phenol were examined by SDS polyacrylamide gel electrophoresis and fluorography it was found that metabolites had bound covalently to a protein which comigrated with the microsomal glutathione transferase enzyme. We therefore suggest that reactive metabolites of phenol activate the enzyme by covalent modification. It is discussed whether the binding and activation has general implications in the regulation of microsomal glutathione transferase and, since some reactive metabolites might be substrates for the enzyme, their elimination through conjugation.     

The regioselective binding of CHCl3 reactive intermediates to microsomal phospholipids.

Microsomal phospholipids (PL) are a good target for the reactive intermediates produced by either the oxidative or the reductive biotransformation of CHCl3 (Testai et al. (1990), Toxicol. Appl. Pharmacol. 104, 496-503). In order to preliminarily characterize the different PL with CHCl3 reactive intermediates, two common methods of PL breakdown have been exploited: the acid-catalyzed transmethylation and the enzymatic hydrolysis with phospholipase C. The results indicated that radioactivity derived from the adducts of PL with the oxidation metabolite, phosgene, partitioned preferentially in the aqueous phase (the ratio of aqueous to organic phase radioactivity contents was about 10); the opposite occurred (ratio about 0.1) when the PL adducts were produced by the reductive process metabolites (dichloromethyl radicals). Therefore, the two methods of PL adduct breakdown can be used to detect and quantitate selectively the two reactive intermediates of CHCl3 biotransformation. The use of phospholipase C, which specifically cleaves the bond between the glyceryl-oxygen and the phosphor atom of PL also gave some structural information. Indeed, the radioactivity partitioning in the aqueous phase after enzymatic hydrolysis of CHCl3 oxidation-associated PL adducts, indicated the selective covalent binding of phosgene residues with the PL polar heads. The clear-cut different partition of radioactivity observed after hydrolysis of PL adducts with CHCl3 reduction intermediates, analogously indicated that dichloromethyl radicals were selectively bound to the PL fatty acyl chains. Using this method we could confirm that in in vitro experimental conditions resembling the physiological status of the liver, both metabolic pathways were concurrently active in hepatic microsomes of B6C3F1 mice. Extents of reactive metabolites similar to those found in B6C3F1 mouse liver microsomes, could be measured in Sprague-Dawley rat liver microsomes only after pretreatment of the animals with PB and incubation with higher CHCl3 concentrations. The toxicological implications of these findings are discussed.     

Evidence for binding of metabolically activated 1,2-dibromoethane to chromatin of forestomach and liver

The covalent binding of metabolically activated 1,2-dibromoethane (DBE), a potent carcinogen, to chromatin constituents of forestomach and liver was examined $\text{in vitro}$. Chromatin was prepared from forestomach and liver of B6C3F1 mice and characterized. In order to activate DBE, microsomes and cytosol were isolated from mouse forestomach and liver and incubated with [¹⁴C]-DBE in the presence of a NADPH regenerating system. Results demonstrate that DBE bound covalently to the same extent to protein of microsomes and chromatin isolated from forestomach and liver. On the contrary, DBE bound significantly more to chromatin DNA of forestomach or liver than it did to salmon sperm DNA. It appears from these results that the metabolically activated DBE is more reactive to homologous DNA than exogenous DNA. Fractionation of DBE-bound chromatin protein into histone and nonhistone proteins resulted in higher binding of DBE to non-histone than to histone proteins isolated from forestomach and liver.     

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.     

Chloroacetates of 2- and 3-demethylthiocolchicine: specific covalent interactions with tubulin with preferential labeling of the beta-subunit.

We synthesized two chemically reactive A ring modified analogs of colchicine, 2-chloroacetyl-2-demethylthiocolchicine (2-CTC) and 3-chloroacetyl-3-demethylthiocolchicine (3-CTC). Both are similar to colchicine as inhibitors of tubulin polymerization and act as competitive inhibitors of colchicine binding (apparent Ki values, 3 microM). [14C]-labeled 2-CTC and 3-CTC bound to tubulin at 37 degrees C but not at 0 degree C, and bound drug formed covalent bond(s) with tubulin. The binding and covalent reactions were inhibited by podophyllotoxin. About 60% of the bound 3-CTC rapidly formed a covalent bond with tubulin. With 2-CTC the covalent reaction was slower than the binding reaction, and only one-third of the bound 2-CTC reacted covalently with tubulin. The ratio of radiolabel in beta-tubulin to that in alpha-tubulin was about 4:1 with both 2-CTC and 3-CTC.     

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.     

The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes

3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N′-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1 mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5–3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5–3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5–3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT.