Selective Protein Covalent Binding and Target Organ Toxicity

TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.

143, 1–12 (1997)

TO968074

CONTEMPORARY ISSUES IN TOXICOLOGY Selective Protein Covalent Binding and Target Organ Toxicity1,2 STEVEN D. COHEN,*,3 NEIL R. PUMFORD,† EDWARD A. KHAIRALLAH,‡ KIM BOEKELHEIDE,§ LANCE R. POHL,Ø H. R. AMOUZADEH,Ø AND JACK A. HINSON† Departments of *Pharmaceutical Sciences and ‡Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut; †Division of Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas; §Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island; and ØMolecular and Cellular Toxicology Section, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland Received June 27, 1996; accepted November 12, 1996

Hinson et al., 1994). For many xenobiotics, conjugation reactions of the intermediates with soluble cell constituents, e.g., glutathione (GSH), produces products which may be more readily excreted and are often less toxic than the parent compound. For others similar reactions may produce metabolites (conjugates) which are themselves more toxic than the parent compound (Dekant and Vamvakas, 1993; Dekant et al., 1993; Monks et al., 1990). In addition, reactive intermediates have the potential of forming covalent bonds with larger cellular constituents, e.g., nucleic acids, proteins. Extensive study of the covalent interactions of xenobiotics with nucleic acids has led to greater understanding of the role of such binding in chemical mutagenesis and carcinogenesis (for review see Miller, 1994). Similarly, target organ toxicity has been well correlated with the generation of reactive intermediates and their covalent binding to cellular proteins. The elucidation of mechanisms of toxicant action continues as a major goal of toxicology research, and numerous studies continue to test and generally support the association between reactive intermediate covalent binding to proteins and target organ toxicity. Such research has emphasized the identification of (1) toxic electrophilic metabolites of xenobiotics, (2) bound proteins in target cells, and (3) cellular consequences (protective vs destructive) which result from binding. The general association of protein binding with toxicity has led to the covalent binding hypothesis that binding to critical cellular proteins may be an initiating event in some target organ toxicities. Alternatively, protein binding may also play a protective role by sequestering and/or inactivating reactive electrophile, or by signaling the cell to ‘‘warn’’ of the presence of excess electrophile. Throughout the 1970s and into the 1980s studies of protein covalent binding were based primarily on radioisotopic detection of bound metabolites and these generally supported the covalent binding hypothesis. However, some studies demonstrated diminution of toxicity by intervening treatments without a corresponding change in covalent binding and, on that

Selective Protein Covalent Binding and Target Organ Toxicity. COHEN, S. D., PUMFORD, N. R., KHAIRALLAH, E. A., BOEKELHEIDE, K., POHL, L. R., AMOUZADEH, H. R., AND HINSON, J. A. (1997). Toxicol. Appl. Pharmacol. 143, 1–12. Protein covalent binding by xenobiotic metabolites has long been associated with target organ toxicity but mechanistic involvement of such binding has not been widely demonstrated. Modern biochemical, molecular, and immunochemical approaches have facilitated identification of specific protein targets of xenobiotic covalent binding. Such studies have revealed that protein covalent binding is not random, but rather selective with respect to the proteins targeted. Selective binding to specific cellular target proteins may better correlate with toxicity than total protein covalent binding. Current research is directed at characterizing and identifying the targeted proteins and clarifying the effect of such binding on their structure, function, and potential roles in target organ toxicity. The approaches employed to detect and identify the targeted proteins are described. Metabolites of acetaminophen, halothane, and 2,5-hexanedione form covalently bound adducts to recently identified protein targets. The selective binding may influence homeostatic or other cellular responses which in turn contribute to drug toxicity, hypersensitivity, or autoimmunity. q 1997 Academic Press

Many chemically dissimilar xenobiotics are converted to highly reactive intermediates in living systems where they subsequently interact with cell constituents. This has been widely implicated as a critical event in target organ toxicity induced by therapeutic and environmental chemicals (Nelson and Pearson, 1990; Vermeulen et al., 1993; Boelsterli, 1993; 1

Sponsored by the Mechanisms Specialty Section of the Society of Toxicology. 2 Summary of the symposium presented at the 34th Annual Meeting of the Society of Toxicology, Baltimore, MD, March, 1995. 3 To whom correspondence should be addressed at Department of Pharmaceutical Sciences, University of Connecticut, 372 Fairfield Rd, Storrs, CT 06269-2092. 1

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basis, refuted the hypothesis. Yet, for the toxicants employed, cell death without initial covalent binding to proteins has not been documented, which remains consistent with the hypothesis. For some toxicants, e.g., acetaminophen (APAP) and halothane, more recent immunochemical studies have facilitated the detection and identification of unique proteins bound by electrophiles, in both experimental animals and humans. Mechanistic studies of protein covalent binding have suggested that for some toxicants, e.g., n-hexane and methyl nbutyl ketone, binding may result in alteration of the function of critical target proteins and lead directly to toxicity. For others, e.g., halothane, the binding may result in altered immune recognition of the target proteins. For still others, e.g., APAP, binding results in altered intracellular distribution of some targeted proteins and inhibition of enzymatic activity, the importance of which remains to be clarified. The following sections summarize current findings. IMMUNOCHEMICAL DETECTION AND IDENTIFICATION OF XENOBIOTIC–PROTEIN ADDUCTS

Recently, immunochemical methods have been developed to detect and identify xenobiotics covalently bound to proteins (Pohl, 1993). These methods have been utilized to investigate the mechanisms of toxicity of halothane, diclofenac, trichloroethylene, APAP, and a variety of other chemicals. In general, highly selective polyclonal antibodies have been produced. A very simple and successful method of immunogen preparation is to couple the carboxyl groups on the chemical of interest to the e amino group of lysine on an immunogenic protein such as keyhole limpet hemocyanin (KLH). This method has been used successfully for the APAP–cysteine conjugate (Roberts et al., 1987) and diclofenac (Pumford et al., 1993b). Alternatively, APAP has been linked by diazo coupling with p-aminobenzoic acid followed by mixed anhydride activation and binding to KLH (Bartolone et al., 1987). Effective immunogens for halothane (Satoh et al., 1985) and trichloroethylene (Halmes et al., 1996) have also been produced more directly by use of a reactive chemical that can covalently link to the carrier protein in a manner similar to reaction of the chemical of interest. The species of choice for immunization has been the rabbit because it can produce large quantities of serum relatively inexpensively. When sufficient titers are produced, the antisera are characterized in a competitive ELISA to determine the polyclonal antibody specificity (Potter et al., 1989; Pumford et al., 1993b; Satoh et al., 1985). Thereafter, the protein covalent binding of the chemical or its metabolite can be determined both quantitatively and qualitatively. Western blots permit detection of individual proteins targeted by the toxicant (Bartolone et al., 1987, 1988) and ELISA tech-

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TABLE 1 Identified Halothane Target Proteins Molecular mass

Protein

Reference

100 82 80 63 59 58 57

GRP94/ERp99/endoplasmin GRP78/BiP ERp72 (a heat shock protein) Calreticulin Carboxylesterase Unknown (isomerase or protease) Protein Disulfide Isomerase (PDI)

Thomassen et al. (1990) Davila et al. (1991) Pumford et al. (1993a) Butler et al. (1992) Satoh et al. (1989) Martin et al. (1991) Martin et al. (1993b)

niques permit quantitation of protein adducts in tissues and subcellular fractions (Pumford et al., 1989, 1990b). In addition, immunohistochemical analysis permits assessment of distribution of covalent adducts among tissues and localization within individual cell types (Bartolone et al., 1989; Hargus et al., 1994; Roberts et al., 1991; Satoh et al., 1985; Emeigh Hart et al., 1995). Examples of how this approach has resulted in the identification of selectively targeted proteins for a variety of toxicants are provided below. The inhalation anesthetic halothane causes a rare hepatitis in susceptible individuals and the idiosyncratic reaction is believed to occur by an immunological mechanism. Sera from patients with halothane hepatitis have been shown to contain antibodies that recognize specific liver microsomal proteins from humans (Kenna et al., 1988) and animals (Kenna et al., 1987, 1988) treated with halothane. These proteins may be involved in the initiation and/or may be targets of a hypersensitivity or autoimmune type reaction. Halothane is metabolized to the reactive trifluoroacetyl chloride intermediate which is believed to acetylate lysine groups on proteins forming N e-trifluoroacetyl (TFA) groups. The major proteins recognized by sera from halothane hepatitis patients have been shown to contain the TFA group. These proteins, which have been purified and identified (Table 1), are found in the lumen of the endoplasmic reticulum and many are involved in the maturation of newly synthesized proteins. Cellular processing of TFA antigens is discussed below. The nonsteroidal anti-inflammatory drug diclofenac produces a mild hepatotoxicity in 15% of patients and, in rare instances, causes a fulminant hepatic necrosis. The mechanism of the toxicity is unclear, with some studies suggesting an immune-mediated hypersensitivity (Breen et al., 1986; Sallie et al., 1991; Schapira et al., 1986) and others implicating direct cytotoxicity (Dunk et al., 1982; Helfgott et al., 1990; Iveson et al., 1990; Purcell et al., 1991). Either mechanism could be initiated through covalent modification of endogenous proteins by a reactive metabolite of diclofenac. Therefore, polyclonal antisera were produced in rabbits immunized with antigen prepared by coupling diclofenac to

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KLH using carbodiimide (Pumford et al., 1993b). Competitive ELISA revealed that N e-diclofenac-N a-acetyl-L-lysine was the most potent inhibitor of the resulting antisera. Western blot analysis of liver homogenates from diclofenactreated mice revealed a dose-dependent formation of adducts at approximately 50, 70, 110, and 140 kDa (Pumford et al., 1993b). The 50-kDa microsomal protein was the primary target after 200 mg diclofenac/kg and binding was dependent on cytochrome P450 activation of diclofenac (Hargus et al., 1994). In contrast, the 110- and 140-kDa adducts were localized to the plasma membrane and their formation followed UDP-glucuronosyltransferase metabolic activation of diclofenac (Hargus et al., 1994). Immunofluorescence studies indicated that diclofenac adducts were associated with the surface of the hepatocytes, and immunohistochemistry detected the majority of adducts in the bile canalicular region (Hargus et al., 1994). Recently, the 110-kDa bile canalicular membrane protein has been identified as dipeptidyl peptidase IV (CD 26) (Hargus et al., 1995). Purification and determination of the identity and function of the other major target proteins is a necessary step in determining the role of such diclofenac–protein adducts in diclofenac-induced hepatotoxicity. Trichloroethylene (TCE) has been implicated in the causation of a life-threatening autoimmune disorder known as systemic sclerosis or scleroderma-like syndrome (Haustein and Ziegler, 1985; Lockey et al., 1987; Saihan et al., 1978). TCE-induced scleroderma-like disease is not only associated with occupational exposure but also with environmental contamination of ground water (Kilburn and Warshaw, 1992). To investigate whether formation of TCE–protein conjugates may be involved in a sclerosis-like syndrome, polyclonal rabbit antisera was produced by derivitizing KLH with dichloroacetic anhydride (Halmes and Pumford, 1995). ELISA studies revealed high titer antibodies which reacted with the solid-phase antigen, dichloroacetic anhydridetreated rabbit serum albumin (RSA), but not with RSA alone, indicating specificity for the dichloroacetyl group. Competitive ELISA indicated that the antiserum primarily recognizes chloroacetylated groups on lysine, with greatest inhibition by N e-(dichloroacetyl)-L-lysine, while L-lysine alone inhibited less than 10% at 500 mM. The antiserum was affinitypurified on a column prepared by conjugating dichloroacetic acid to diaminodipropylamine gel with carbodiimide. Western blot analysis was conducted with the resultant antiserum using proteins from mice killed 1, 3, 6, or 9 hr following ip treatment with 1000 mg TCE/kg. The most intensive staining was in the microsomal fractions from both the liver and the lungs, where the primary adducts of 50 and 100 kDa were detected. These increased in a time-responsive manner from 1 to 6 hr, with less intensity of staining seen by 9 hr. The 50- and 100-kDa adducts increased as the dose of TCE was increased from 250 to 2000 mg/kg. Purification and determination of the identity and function of these major target

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proteins is a necessary step in determining the role of such TCE–protein adducts in systemic sclerosis. Following a hepatotoxic dose, the analgesic APAP covalently modifies a select group of proteins and selective protein binding has been better correlated with toxicity than total binding (Bartolone et al., 1987; Pumford et al., 1990a; Beierschmitt et al., 1989). Shortly after toxic APAP treatments, binding to cytosolic proteins of approximately 44, 55–58, and 100 kDa is detected (Bartolone et al., 1987; Pumford et al., 1990a), and it has been postulated that these early (e.g., by 30 min) adducts may be more important than adducts that occur later in the time course. Mitochondrial proteins of 50, 54, and 67 kDa are also early targets for APAP binding (Pumford et al., 1990a; Landin et al., 1996) and may play a critical role in the initiation of events associated with the early decrease in mitochondrial function and subsequent elevation of mitochondrial calcium following a hepatotoxic dose of APAP (Burcham and Harman, 1988, 1990; Meyers et al., 1988; Nazareth et al., 1991). As noted above for other toxicants, the identity of the major protein adducts is also an important step to better understanding the role of covalent modifications in APAP hepatotoxicity, and the immunochemical methods prove very useful for following the purification of individual target proteins. The 55- to 58-kDa protein was the most intensively stained target protein detected with anti-APAP antisera in mouse liver cytosol following a hepatotoxic dose of APAP; therefore, this protein was the first to be purified independently by two laboratories (Bartolone et al., 1992; Pumford et al., 1992). A combination of standard separation/purification techniques such as differential centrifugation, ion exchange chromatography, and hydroxyapatite chromatography was employed and the resulting protein fractions were screened for the 55- to 58-kDa APAP protein adduct by Western blot using anti-APAP antiserum. The purified protein was then digested with trypsin and automated Edman degradation was performed on peptides separated by reverse-phase HPLC. The amino acid sequences (Pumford et al., 1992; Bartolone et al., 1992) of several analyzed peptides exhibited 97– 100% homology with the deduced amino acid sequence from a selenium binding protein whose function is not known (Bansal et al., 1990), but which has been implicated in the cell growth inhibitory effect of selenites (Morrison et al., 1988) (Table 2). Subsequently, two very similar proteins were cloned and one, termed the APAP binding protein (ABP), was found to have 100% homology with the amino acid sequence of all the peptides from the 55- to 58-kDa APAP adduct (Table 2; Lanfear et al., 1993). The other, termed the selenium binding protein (SBP), showed slightly less homology to two of the nine peptides analyzed. Further information about this and other APAP-targeted proteins is presented in the following section. CELLULAR CONSEQUENCES OF PROTEIN ADDUCT FORMATION

The preceding section documents the value of using an immunochemical approach in the study of covalently ad-

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TABLE 2 Major Cytosolic Acetaminophen Binding Protein Peptides Sequenced % Homologya

Peptides from 55-kDa APAP adductb LHK GTWEKPGGASPMGY HNVMVSTEWAAPNVFKDGFNPAHVE IFVWDWQRHEIIQTL VIQVPSK QYDISNPQKP LYATTSLYSD Peptides from 58-kDa APAP adductc GYDFWYQPR HEIIQTLQMTDGLIPLEI

SBP

ABP

100 87 100 100 100 100 90

100 100 100 100 100 100 100

100 100

100 100

a

Lanfear et al. (1993). Pumford et al. (1992). c Bartolone et al. (1992). b

ducted proteins. However, the possibility remains that some adducts may go undetected, perhaps as a result of unique differences in antigenicity. For APAP this has recently been addressed and there is excellent agreement between Western blots utilizing anti-APAP antibodies and the phosphorimage detection of radioactive APAP adducts (Myers et al., 1995). Thus immunochemical approaches appear to detect most APAP targets. To date seven proteins, including the 55- to 58-kDa target, have been identified as major APAP binding proteins (Table 3). Five of the identified targets have known enzymatic activity which is decreased in association with APAP binding prior to any overt liver damage. It is possible that the collective loss of enzyme activities may play a critical role in the onset of APAP toxicity. Thus, glutamine synthetase, glutamate dehydrogenase, and carbamyl phosphate synthetase are involved in trapping ammonia in the liver, and their inhibition may mediate the accumulation of plasma ammonia after APAP (Gupta et al., 1995). Likewise, diminution of mitochondrial aldehyde and glutamate dehy-

drogenase activities may influence the NAD/NADH ratio, disruption of which could adversely affect oxidative phosphorylation (Williamson et al., 1967) and contribute to diminished mitochondrial function after APAP administration (Burcham and Harman, 1988, 1990; Meyers et al., 1988; Nazareth et al., 1991). Mitochondrial binding may be of special importance in toxicity since it is minimal after treatment with the nontoxic APAP analogue, 3*-hydroxyacetanilide (AMAP) which covalently binds to other hepatic proteins (Meyers et al., 1995). The significance of the arylation of tetrahydrofolate dehydrogenase, a major enzyme involved in transmethylation reactions, has not been established. Overall, there is to date no direct evidence to provide a causal link between diminished activity of these arylated enzymes to APAP-induced hepatotoxicity. APAP also arylates a major nuclear structural protein, lamin A (Hong et al., 1994, Khairallah et al., 1995). Lamin A is one of three intermediate filaments that form the nuclear lamina, a discontinuous network anchoring the interphase

TABLE 3 Identification of Acetaminophen Binding Proteins Molecular mass (kDa)

Fraction

Protein

Reference

44 50 54 55–58

Microsomes Mitochondria Mitochondria Cytosol

74–75 100 130

Nucleus Cytosol Mitochondria

Glutamine synthetase Glutamate dehydrogenase Aldehyde dehydrogenase Selenium binding protein or acetaminophen binding protein Lamin-A N10-Formyl tetrahydrofolate dehydrogenase Carbamyl phosphate synthetase I

Bulera et al. (1995) Halmes et al. (1996) Landin et al. (1996) Bartolone et al. (1992) Pumford et al. (1992) Hong et al. (1994) Pumford et al. (1994) Gupta et al. (1995)

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chromatin to the inner nuclear membrane (Spector, 1993). Recent electron microscopic evidence is consistent with APAP-induced disruption of the nuclear lamina in association with lamin A arylation (Hong et al., 1994). Chromatin attachment to the inner nuclear membrane was diminished by 2 hr, and was barely detectable 8–12 hr after APAP. Blebbing of the outer nuclear membrane was observed by 6 hr after APAP. Thus, covalent binding to lamin A may mediate the disruption of nuclear organization, which, in turn, may contribute to the changes of nuclear function that have been observed after APAP (Hongslo et al., 1990, 1994). The cytosolic 55- to 58-kDa APAP binding protein (58ABP), as the most prominent target detected after APAP (Bartolone et al., 1992; Pumford et al., 1992), was the first arylated protein to be identified. A polyclonal antibody raised against the mouse liver 58-ABP has enabled detection of the protein in liver from humans (Bartolone et al., 1992) and most mammalian species but not guinea pig (unpublished observations). Analysis with anti-APAP antibody suggests that 58-ABP is also targeted during human APAP overdose (Birge et al., 1990). Gel permeation chromatography, using nondenaturing conditions, indicated that 58-ABP is a monomer, while 2-D gel electrophoresis revealed a cluster of four 58-ABP isoforms with pI values ranging from 6.2 to 6.6 (Bartolone et al., 1992). The relative degree of early arylation varied among the isoforms and did not appear to depend upon their relative abundance (Hong et al., 1994, Khairallah et al., 1995). Arylation of the 58 ABP is detected within 30–60 min after APAP administration and is closely correlated with APAP toxicity (Bartolone et al., 1987, 1989, 1992; Beierschimtt et al., 1989; Birge et al., 1989, 1990; Brady et al., 1990; Pumford et al., 1990). Recent evidence suggests that 58-ABP may be a preferential target relative to other cytosolic proteins (Hoivik et al., 1996). However, there is no direct evidence of a critical initiating role for 58-ABP arylation in APAP toxicity, and the mere presence of adducted protein is not the sole determinant of cell injury (Bartolone et al., 1987; Tveit et al., 1992). Alternatively, the 58ABP may serve a protective role as an electrophile scavenger. It possesses eight cysteine residues which could provide nucleophilic sites for arylation by NAPQI or other electrophiles. It has also been suggested that APAP electrophile may react with 58-ABP through protein-associated selenium (Pumford et al., 1992). Since the protein can readily form mixed disulfides with glutathione under oxidative conditions (Birge et al., 1991), reaction of sulfhydryl residues with NAPQI would be favored in APAP overdose when GSH reserves are low, thereby permitting the 58-ABP to act as a scavenger of reactive electrophiles. In support of this, indirect evidence suggests that electrophilic metabolites of 2,6dimethyl APAP (Birge et al., 1990), 3-OH acetanilide (Myers et al., 1995), 3-methyl indole (Kaster and Yost, 1996),

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and bromobenzene (Manautou et al., 1996) also appear to target a similar cytosolic protein. Recent studies on the subcellular distribution of APAP targets revealed that within 1 hr after APAP administration in vivo, an arylated 58-kDa protein target was detected in the nuclear fraction (Hong et al., 1994). Immunogold electron microscopy, with anti-58-ABP, detected nuclear 58-ABP in livers from APAP-dosed mice but not in controls. Furthermore, analysis of Western blots from 2-D gels of subnuclear fractions from APAP-treated mice also demonstrated cross reactivity with anti-58-ABP. Since 58-ABP was not detectable in similar hepatic nuclear fractions from control mice, this suggests that arylation of cytosolic 58-ABP during APAP exposure may initiate its translocation into the nucleus. In support of this the earliest arylated, rather than the most abundant, of the four isoforms of 58-ABP were the first two isoforms detected in the nucleus. Selective washing, extraction, and digestion procedures indicated that the translocated protein is chromatin-associated (Hong et al., 1994). The toxicologic significance of the nuclear translocation of 58-ABP remains to be determined. However, it is unlikely to be due to an APAP-induced ‘‘leakiness’’ of nuclear membranes since neither cytosolic lactate dehydrogenase nor the 44-kDa APAP binding protein can be detected in the purified nuclear extracts. In addition, buthionine sulfoximine and Nacetyl cysteine, which accelerate or prevent APAP toxicity, respectively, caused corresponding changes in 58-ABP translocation, and translocation was not detected after AMAP (Hong et al., 1994), which arylates 58-ABP without toxicity. Finally, 58-ABP was evident in the nucleus after a toxic dose of bromobenzene (unpublished observations), which may also target 58-ABP (Manautou et al., 1995). Collectively, these observations on the rapidity and extent of arylation of the 58-ABP suggest a novel role for the 58-ABP as a ‘‘sensor’’ for the presence of electrophiles (Khairallah et al., 1995). Although highly speculative at this point, the translocation subsequent to arylation may be a form of intracellular signal for transmitting information to the nucleus. Once in the nucleus, the arylated 58-ABP can interact with chromatin and alter transcriptional and/or replicative events appropriate to the homeostatic needs of the cell. These nuclear events may enable cells to resist electrophile insult, e.g., via induction of stress proteins which occurs after APAP (Bruno et al., 1992), initiate events which facilitate destruction of APAP-injured cells (Ray et al., 1993), and/or signal replicative responses to neighboring cells for promotion of tissue repair (Mehendale et al., 1994). 2,5-HEXANEDIONE TARGETS MICROTUBULES AND DISRUPTS SEMINIFEROUS TUBULE FLUID SECRETION

2,5-Hexanedione (2,5-HD) exposure produces a unique pattern of nervous system and testicular injury with primary

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FIG. 1. In the working hypothesis which guides this research, these pathogenetic steps have been investigated as links between the covalent binding of 2,5-HD and its induction of testicular injury.

involvement of microtubule-rich axons and Sertoli cells. Given this apparent targeting of microtubule-enriched cells, studying the mechanism of 2,5-HD-induced male reproductive toxicity has involved the elucidation of the role of microtubules in Sertoli cell function and the effects of 2,5-HD on Sertoli cell microtubules. This section briefly summarizes the background information necessary for understanding the mechanism of action of 2,5-HD on the testis, leading to the development of the current working hypothesis (Fig. 1) which guides this ongoing investigation. 2,5-HD is the toxic g-diketone metabolite of n-hexane and methyl n-butyl ketone, solvents responsible for a number of outbreaks of clinical polyneuropathy in humans. In rats, morphological evidence of testicular injury precedes symptomatic neurotoxicity and occurs 3–4 weeks after initiating exposure to 1% 2,5-HD in the drinking water (Boekelheide, 1988a; Chapin et al., 1983). The first morphological change seen in the testis is Sertoli cell vacuolization, suggesting that this cell is the target of 2,5-HD-induced testicular toxicity. After about 5 weeks of 2,5-HD exposure, there is a dramatic loss of germ cells resulting in a persistent testicular atrophy (Boekelheide and Hall, 1991). Sertoli cells are polarized epithelial cells which extend from the base to the lumen of the seminiferous tubule. They form basal tight junctions (the ‘‘blood–testis barrier’’), creating an adluminal space in which germ cells are bathed by a Sertoli cell-derived seminiferous tubule fluid. Interestingly, Sertoli cells share common features of microtubule structure and organization with axons, the target of 2,5-HD-induced neurotoxicity (Neely and Boekelheide, 1988), explaining the focus on this organelle in mechanistic studies of 2,5-HDinduced testicular injury. Microtubules are polymers of the core protein tubulin, a 100-kDa a-b heterodimer, with attached microtubule-associated proteins. Microtubules are abundant in the apical Sertoli cell cytoplasm with a presumptive role of maintaining structure and function in the adluminal compartment (Neely and Boekelheide, 1988; Allard et al., 1993). The chemical basis of 2,5-HD-induced injury is complex. As a g-diketone, 2,5-HD first undergoes an irreversible con-

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densation with a primary amine, such as a protein lysyl eamine, to form a heterocyclic aromatic pyrrole; since pyrroles are unstable, they may react further by oxidation and cross linking (Fig. 2). 2,5-HD is widely distributed in vivo with a propensity for ubiquitous pyrrole formation with any appropriate amine. The implication of this nonspecific chemical behavior is that 2,5-HD-induced toxicity, which is highly tissue selective, must be due to covalent binding to proteins which have unique features of structure or function within the target organs (thus the focus on microtubules, as noted above). Exposure of rats to 1% 2,5-HD in the drinking water produces an alteration in the assembly of tubulin purified from brain or testis within 2 weeks of initiating exposure, before the appearance of morphological or functional abnormalities (Boekelheide, 1988a,b). 2,5-HD-treated rat testis protein has an increased pyrrole content, indicating covalent binding of the 2,5-HD to this target tissue (Boekelheide, 1987a, 1988b). An intramolecular cross link which modifies the structure of the a-tubulin subunit may explain the alteration in microtubule assembly produced by 2,5-HD (Boekelheide et al., 1991; Sioussat and Boekelheide, 1989). The 2,5-HD-induced microtubule assembly alteration is ‘‘taxol-like’’ and characterized by precocious nucleation and abnormally rapid assembly (Boekelheide, 1987a,b, 1988b). Microtubules treated with 2,5-HD in vitro display the same assembly alterations as those derived from the testes of 2,5HD-treated rats (Boekelheide, 1987a,b). The modified tubulin demonstrates altered control responses to the usual modulators of assembly (Boekelheide, 1987b). Using sea urchin zygotes as a model system, mitotic spindle abnormalities have been observed after microinjection of 2,5-HDtreated tubulin (Sioussat et al., 1990). Three separate in vivo approaches have consistently supported an association between altered testicular microtubule assembly and testicular

FIG. 2. The g-diketone, 2,5-HD, reacts with protein-bound primary amines to form unstable heterocyclic aromatic pyrroles which may react further to form intra- and intermolecular protein crosslinks.

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injury: (1) the use of congeners with differential testicular and microtubule effects (Boekelheide, 1987a), (2) the determination of microtubule alterations throughout a time course of 2,5-HD exposure and recovery (Boekelheide, 1988b; Johnson et al., 1991), and (3) examination of the relationship between dose rate, testicular effects, and microtubule assembly alterations (Boekelheide and Eveleth, 1988). Microtubules facilitate the transport of proteins within cells by serving as ‘‘tracks’’ for the movement of vesicles from donor to acceptor membranes. Microtubule motors, such as kinesin and cytoplasmic dynein, bind both their cargo and microtubules at distinct sites and couple the energy of ATP hydrolysis to their movement along microtubules. Both kinesin and cytoplasmic dynein are found in the Sertoli cell in locations compatible with their involvement in intracellular microtubule-dependent transport events (Hall et al., 1992). 2,5-HD exposure has been shown to alter the distribution of cytoplasmic dynein within Sertoli cells (Hall et al., 1995). In vitro studies using purified kinesin as the motor have shown that transport along 2,5-HD-treated microtubules is slower than that along control microtubules (Redenbach et al., 1994). The slowed kinesin-dependent microtubule transport resulting from 2,5-HD exposure requires a cross linking event following pyrrole formation, since: (1) the noncrosslinking 2,5-HD congener 3-acetyl-2,5-hexanedione does not slow microtubule transport, and (2) glutaraldehyde, a cross linking g-dialdehyde, produces a similar slowing of microtubule transport as 2,5-HD (Redenbach et al., 1994). Microtubules are likely critical to the appropriate formation and secretion of the seminiferous tubule fluid. In a time course study of 2,5-HD exposure in the rat, seminiferous tubule fluid secretion, as measured by efferent duct ligation, was shown to be inhibited about 2 days after the appearance of Sertoli cell vacuolation but before bulk germ cell loss (Johnson et al., 1991). Using a more refined technique for measuring seminiferous tubule fluid secretion, the luminal oil drop method applied to isolated seminiferous tubules, seminiferous tubule fluid secretion was decreased after 3 weeks of exposure to 1% 2,5-HD in the drinking water, before the appearance of any morphological changes in the testis (Richburg et al., 1994). During normal spermatogenesis, germ cell production is regulated by spermatogonial apoptosis, or ‘‘programmed cell death.’’ In model systems, apoptosis may be triggered by growth factor withdrawal. Reasoning that one potential consequence of the 2,5-HD-induced inhibition of seminiferous tubule fluid secretion would be a relative deficiency of Sertoli cell-derived growth factors required by germ cells, the incidence of apoptosis was examined during a time course of exposure to 1% 2,5-HD in the drinking water. Apoptosis was assessed by morphology on plastic-embedded sections and by detection of DNA fragmentation both on tissue sec-

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tions and by DNA electrophoresis. A peak in the incidence of apoptosis occurred at 5 weeks after beginning exposure, indicating that germ cell death in this toxicant-induced testicular injury occurred predominantly by apoptosis rather than necrosis. In searching for the connection between covalent binding of 2,5-HD and testicular toxicity, research has focused on microtubules because of their unique structure and organization in the target organs of this toxicant. Since the viability of adluminal germ cells is thought to depend upon the provision of nutritional, hormonal, and growth factors in the seminiferous tubule fluid, disruption of the secretory process is one mechanism by which Sertoli cell dysfunction could result in testicular atrophy. In this working hypothesis, dysfunctional microtubules result in progressive failure of the secretory pathway leading to decreased seminiferous tubule fluid secretion, a relative growth factor deficiency in the adluminal compartment, and germ cell apoptosis. PROCESSING OF TRIFLUOROACETYLATED LIVER ANTIGENS ASSOCIATED WITH HALOTHANE HEPATITIS

The inhalation anesthetic halothane (CF3CHBrCl) is metabolized to the reactive trifluoroacetyl chloride (CF3COCl) metabolite by cytochromes P450 in the endoplasmic reticulum (ER) of the liver (Kenna et al., 1990) (Fig. 3). Several ER proteins, including cytochromes P450, are trifluoroacetylated (TFA) by this reactive metabolite. Many of these TFA-proteins have been purified from rat liver microsomes and have been identified as luminal ER proteins (see Table 1). Most patients diagnosed with halothane hepatitis have serum antibodies that react with one or more of these TFAantigens. In addition, some halothane hepatitis patients have antibodies in their sera that react with the native unaltered forms of these proteins (Pumford et al., 1993a; Martin et al., 1993a,b). Results from other studies indicate that similar proteins are present in the livers of humans (Smith et al., 1993) and they appear to become covalently modified by the TFA moiety after patients have been administered halothane (Pohl et al., 1989). Therefore, it is possible that human liver orthologues of the rat TFA-proteins have an immunopathological role in those patients that develop halothane hepatitis. However, the mechanism by which these ER luminal proteins interact with the immune system is not known because the fate of GRP94, GRP78, ERp72, calreticulin, 59-kDa carboxylesterase, the 58-kDa protein, and protein disulfide isomerase (PDI) or their respective TFA-adducts in the liver have not been studied. In order to address this question, we have recently investigated the mechanisms by which these proteins are processed in primary cultures of rat hepatocytes (Amouzadeh and Pohl, 1995). Parenchymal cells (PCs) and nonparenchymal cells

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FIG. 3. Possible processing pathways of TFA-proteins in the liver. Oxidative metabolism of halothane (CF3CHBrCl) by cytochromes P450 leads to the formation of the trifluoroacetyl chloride (CF3COCl) reactive metabolite. This metabolite can covalently alter cytochromes P450 and several luminal endoplasmic reticulum (ER) proteins of 100, 82, 80, 63, 59, 58, and 57 kDa. Turnover studies in primary cultures of rat hepatocytes indicated that all of the luminal TFA-proteins except for the TFA-100-kDa protein are extensively degraded in the lysosomes (L) or in other acidic compartments (AC) (pathway A). The TFA-100-kDa protein may be degraded in an unknown compartment (pathway B) or in the ER (pathway C).

(NPCs) were isolated from rats 16 hr after treatment with halothane to locate the source of TFA-proteins among liver cells. Immunoblots of PCs lysates with TFA antiserum showed the presence of substantial amounts of previously identified major TFA-proteins at 100, 82, 80, 63, 59, 58, and 57 kDa. However, no TFA-proteins were detected in endothelial cells and Kupffer cells that were isolated by centrifugal elutriation from livers of halothane-treated rats. Consequently, all of the remaining protein processing studies were done in primary cultures of PCs. To help elucidate how both the native carrier proteins of the TFA hapten and the covalently altered TFA-proteins were processed in PCs, the half-lives of these proteins were determined. PCs, isolated from halothane-treated rats, were pulsed with 35S-methionine for approximately 12 hr and chased for 4 days. The radiolabeled 100-, 82-, 80-, 63-, 59-, 58-, and 57-kDa carrier proteins of the TFA hapten were immunoprecipitated from cell lysates with specific protein

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antisera and were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and fluorography. TFA-proteins in cell lysates were detected by immunoblotting with TFA antiserum. The half-lives of the native carrier proteins of the TFA hapten and the TFA-proteins were then determined from the band intensities of fluorograms and immunoblots, respectively, and were all found to be approximately 1 day. These results are similar to the apparent rates of the turnover of the corresponding TFA-proteins in rat liver in vivo, supporting the use of primary cultures of hepatic PCs as a model system for studying the processing of the antigens associated with halothane hepatitis. The findings also suggested that these proteins may have been processed by a similar pathway that was not influenced by the TFA hapten. To learn more about the mechanisms of processing of the TFA-proteins in rat liver, the effect of several protease inhibitors on the degradation of the TFA-proteins in primary cultures of PCs was studied. For example, treatment of the cells with ammonium chloride completely blocked the turnover of all of the TFA-proteins studied except for that of the TFA-100-kDa protein, indicating that the major site of degradation of TFA-proteins in liver PCs was within lysosomes or other acidic vesicles (Fig. 3). Since the rates of the turnover of the native carrier proteins of the TFA hapten and the TFA-proteins were similar, these proteins may be transported to acidic degradative vesicles by a common pathway. Partial inhibition of the turnover of TFA-proteins by 3-methyladenine suggested that one possible mechanism for this process is autophagy. Studies done in rat liver cells indicate that autophagy occurs stepwise and involves the initial formation of double-membrane-bound nascent autophagic vacuoles or autophagosomes from the ER. These vesicles are subsequently transformed into single-membrane-bound acidic vacuoles or autolysosomes that acquire hydrolases by fusing with preexisting lysosomes and/or possibly with Golgi-derived vesicles (Fig. 3). Indeed, PDI has been detected immunohistochemically in the autophagosomes and autolysosomes of rat liver. The labeling density of these vesicles with anti-PDI antibody was increased when the autodegradation of PDI was slowed down by treatment of rats with leupeptin, a known inhibitor of lysosomal cysteine hydrolases. Similarly, we found that leupeptin substantially blocked the turnover of the TFA-proteins, except for that of the TFA-100-kDa protein. The serine protease inhibitor 4(2-aminoethyl)-benzenesulfonyl fluoride produced similar results. Although it is not clear where in the PCs the TFA100-kDa protein is degraded, it may be processed at least partially in the ER. These studies suggest that the degradation of the TFAproteins within the PCs of the liver could be a detoxifying mechanism that prevents the interaction of the intact TFAproteins with B cells and the subsequent formation of specific antibodies directed against them. Perhaps patients who develop halothane hepatitis have a defect in this mechanism.

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DISCUSSION AND CONCLUSION

REFERENCES

New insight into the role of protein covalent binding in target organ toxicity has been presented. The ability to identify individual proteins which become modified by reactive intermediates will greatly increase our understanding of the role of such binding. One should still heed the caution that (some) covalent binding may merely reflect the fleeting presence of electrophiles rather than being mechanistically involved in toxicity (Gillette, 1974). Yet, it is now clear that covalent binding may directly interfere with the functions of some proteins and alter the antigenicity of others. In some cases conclusive evidence is still lacking regarding the means by which bound proteins may disrupt cellular homeostasis. However, recent studies provide new insights on the possibilities that not all binding is damaging and that some may play a role as cellular scavenger and/or signal for the presence of reactive electrophilic intermediates. In spite of the advances which have been made there is still a need for additional research to determine dosimetry relationships between binding to specific target proteins and toxicity. Immunochemical analysis of APAP covalent binding to specific proteins suggests that protein arylation occurs after subtoxic doses with some proteins becoming more prominently targeted as the dose increases from nontoxic to toxic (Bartolone et al., 1987). Such findings would be consistent with there being a threshold for binding to specific proteins which becomes exceeded in association with toxicity, but quantitative assessment of such putative thresholds is lacking. Factors other than adduct accumulation may also contribute to outcome. The nontoxic APAP isomer AMAP also appears to bind to 58-ABP and other APAP target proteins, but the binding of nontoxic AMAP was less stable than the binding of toxic APAP (Myers et al., 1995), suggesting that the nature of the binding may also be a determinant of toxicity. Thus, as the role of protein covalent binding in target organ toxicity continues to be explored and additional protein targets become identified, it will become increasingly important to identify specific binding sites on the target proteins and to characterize the nature of the binding. Advantage must also be taken of evolving quantitative methods to better clarify the threshold question with respect to binding to specific target proteins. This will be important to the ultimate elucidation of the mechanistic importance of covalent binding in target organ toxicity.

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ACKNOWLEDGMENTS N.R.P. and J.A.H. were supported in part by a grant from the National Institute of General Medical Sciences (1 R01 GM48749). E.A.K. and S.D.C. were supported in part by Grant R01 GM31460 from the National Institute of General Medical Sciences. K.B. is a Burroughs Wellcome Fund Scholar in Toxicology, and was supported in part by a grant from the Burroughs Wellcome Fund and in part by Grant R01 ES05033 from the National Institute of Environmental Health Sciences, NIH.

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Thomassen, D., Martin, B. M., Martin, J. L., Pumford, N. R., and Pohl, L. R. (1990). Characterization of a halothane induced trifluoroacetylated 100 kDa neoantigen that is related to a glucose-regulated protein. FASEB J. 4, A599. Thomassen, D., Martin, B. M., Martin, J. L., Pumford, N. R., and Pohl, L. R. (1990). The role of a stress protein in the development of a druginduced allergic response. Eur. J. Pharmacol. 183, 1138–1139. Tveit, A., Leung, C. Y., Emeigh Hart, S. G., Wynad, D. S., Khairallah, E. A., and Cohen, S. D. (1992). Repeated acetaminophen (APAP) dosing results in selective arylation to hepatic and renal proteins without toxicity in the CD-1 mouse. Toxicologist 12, 254. Vermeulen, N. P., Bessens, J. G. M., and Van de Straat, R. (1992). Molecular aspects of paracetamol-induced hepatotoxicity and its mechanismbased prevention. Drug Metab. Rev. 24, 367–407.

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