Introduction

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Recent studies have shown that levels of certain hsps are increased in the liver in response to hepatotoxicants. The livers of rats treated with halothane have elevated levels of hsp70i (also termed hsp72) (VanDyke et al., 1992), and cadmium treatment in rats results in increased synthesis of hsp70i, grp94 and a 110-kDa protein (Goering et al., 1993). Mice treated with hepatotoxic doses of cocaine have elevated hepatic levels of hsp25 and hsp70i (Salminen et al., 1997). The significance of increased de novo synthesis of these proteins in response to hepatotoxicants is unclear, although hsp induction has been correlated with protection from some hepatotoxicants (Salminen et al., 1996). These observations, when taken together with the fact that hsps function as chaperones (for reviews, see Jaattela and Wissing, 1992; Lindquist and Craig, 1988; Welch, 1992), suggest that hsp induction may constitute an important cell defense mechanism against proteotoxic chemicals.

Activation of HSF, the factor responsible for the activation of hsp genes during stress (Abravaya et al., 1992; Baler et al., 1993), appears to result from the presence within the cell of non-native proteins (Ananthan et al., 1986; Zuo et al., 1995). It is reasonable to postulate that adduction of proteins by reactive metabolites of hepatotoxicants may render them sufficiently "non-native" to enable them to trigger the activation of HSF, resulting in upregulation of hsp synthesis. Indirect evidence is provided by recent studies comparing the intralobular localization of hsp accumulation and reactive metabolite binding from cocaine (Salminen et al., 1997). In mice treated with an hepatotoxic dose of cocaine, elevated hsp levels were observed only in cells with detectable cocaine reactive metabolite binding, and shifting the location within the lobule of metabolite binding through pretreatment with phenobarbital or -naphthoflavone produced a corresponding shift in cells expressing the hsps. However, because reactive metabolite binding was also correlated with cytotoxicity, the possibility could not be ruled out that increased hsp expression occurred in response to some manifestation of toxicity rather than as a direct consequence of protein adduction.

In an effort to discriminate between protein adduction vs. some unidentified facet of cytotoxicity as a stimulus for hsp synthesis, additional studies were conducted using APAP and its regioisomer AMAP. APAP is a commonly used analgesic/antipyretic agent that can cause liver necrosis in overdose situations (Ambre and Alexander, 1977; Boyer and Rouff, 1971; Hinson, 1980). Cytochrome P450-mediated oxidation of APAP results in the formation of an electrophilic intermediate, NAPQI, that is detoxified by conjugation with GSH (Corcoran et al., 1980; Dahlin et al., 1984; Plaa, 1993). After hepatotoxic doses of APAP, hepatocellular GSH becomes depleted, permitting unconjugated NAPQI to bind cellular macromolecules (Manautou et al., 1996; Rashed et al., 1990). Although many possible nucleophilic targets exist in the cell, certain proteins are preferentially bound by NAPQI, and their adduction has been proposed to play a role in causing cell death (Bartolone et al., 1992; Bulera et al., 1995).

AMAP also undergoes oxidation to produce electrophilic intermediates which bind to proteins. Based on GSH conjugates identified after AMAP administration to mice, at least three AMAP metabolites (viz., 2-acetamido-p-benzoquinone, 4-acetamido-o-benzoquinone and N-acetyl-3-methoxy-p-benzoquinone imine) have been postulated to account for this binding (Rashed and Nelson, 1989). Although equimolar doses of APAP and AMAP produce similar levels of covalently bound metabolites, AMAP is not hepatotoxic (Roberts et al., 1990). Even when the AMAP dose was increased to its highest nonlethal amount, and peak reactive metabolite binding was nearly twice that of an hepatotoxic dose of APAP, no liver injury from AMAP was observed, and biochemical effects associated with APAP were absent (i.e., perturbation of calcium homeostasis, inhibition of mitochondrial function and inhibition of glutathione peroxidase and thioltransferase activity) (Rashed et al., 1990; Tirmenstein and Nelson, 1990).

In the study reported here, APAP and AMAP were compared with respect to toxicity, reactive metabolite binding and effects on hepatic hsp levels. The difference between these two structurally related compounds in terms of hepatotoxic effect was verified. Total hepatic hsp responses were evaluated using SDS-PAGE and Western blots, and potential differences in responses among cells within the hepatic lobule were examined using immunohistochemistry. Also, the distribution of cells expressing hsps was compared with the intralobular distribution of reactive metabolite binding.

	Materials and Methods

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Animals and treatments. Adult B6C3F1 male mice (Harlan Sprague-Dawley, Indianapolis, IN) weighing 22 to 25 g were used. Mice were housed on corn cob bedding in temperature- and humidity-controlled animal quarters with a 12-hr light/dark cycle and allowed free access to water before and during the experiments. Mice were fasted for 16 hr before APAP or AMAP (Sigma Chemical, St. Louis, MO) doses and then given food for the duration of the experiments. Mice were administered a single i.p. dose of 200 mg/kg APAP or 1000 mg/kg AMAP in warm saline. The dose for each compound was determined in preliminary experiments to be the maximum tolerated dose that allowed survival for the duration of the experiments. Some mice were pretreated 1 hr before the AMAP dose with BSO (222 mg/kg i.p.) dissolved in saline. APAP and BSO were given with an injection volume of 10 ml/kg b.wt. AMAP was given with an injection volume of 20 ml/kg because its limited solubility precluded the use of a smaller injection volume. Mice were killed by carbon dioxide asphyxiation.

PAGE, protein blotting and immunostaining. The hsp levels in total liver protein were detected by SDS-PAGE and Western blotting as previously described (Salminen et al., 1996) with the following modifications. Four hundred milligrams of liver was homogenized in sample buffer (0.05 M Tris, 2% SDS, 10 mM dithiothreitol, 10% glycerol and 1 mM phenylmethylsulfonyl fluoride, pH 6.8), boiled for 5 min, passed through a 22-gauge needle three times to shear DNA and stored at 80°C until use. Fifty micrograms of protein from each sample was aliquoted to separate tubes, and bromphenol blue was added to a final concentration of 0.0025%. Each aliquot was boiled for 5 min, loaded onto separate lanes of a 12.5% SDS-PAGE gel and resolved by electrophoresis. Proteins separated by SDS-PAGE were immediately blotted to Hybond-ECL Western membrane (Amersham Life Science, Arlington Heights, IL). On completion, the membrane was blocked in TBS (20 mM Tris, 500 mM sodium chloride, pH 7.5) containing 3% gelatin and then probed with one of the following antibodies: anti-hsp25 (rabbit polyclonal), anti-hsp60 (mouse monoclonal), anti-hsc70 (mouse monoclonal), anti-hsp70i (mouse monoclonal) or anti-hsp90 (mouse monoclonal). Each of these antibodies was obtained from Stressgen (Victoria, B.C., Canada) and used at a 1:1000 dilution in TTBS (TBS containing 0.05% polyoxyethylenesorbitan monolaurate) containing 1% gelatin. Incubation was for 18 hr at 24°C with continuous shaking. Primary antibody binding was detected using a sheep anti-mouse or donkey anti-rabbit (depending on the primary antibody used) horseradish peroxidase-conjugated antibody (Amersham) at a 1:3000 dilution in TTBS containing 1% gelatin. The chemiluminescent horseradish peroxidase substrate Luminol (Amersham) was added to the membrane, and the membrane was exposed to standard X-ray film to localize antibody binding.

Immunohistochemical detection of hsp25, hsp70i and APAP adducts in mouse liver. Five-millimeter-thick sections from several lobes of each liver were placed in tissue cassettes and fixed in neutral buffered formalin for 3 hr. The livers were rinsed and stored in saline, processed routinely and embedded in paraffin. Four sequential sections that were 4- to 6-µm thick were cut from the same block to facilitate comparison of localization of hsp induction, APAP or AMAP reactive metabolite binding and morphological changes. One section was stained with hematoxylin and eosin and examined for histopathology by light microscopy, and the remaining sections were immunohistochemically stained with an anti-hsp25 antibody, an anti-hsp70i monoclonal antibody, or an anti-APAP antibody as follows. Paraffin-embedded sections were deparaffinized by passing through three changes of xylene for 5 min each. The sections were passed through 100% ethanol two times for 1 min each, 95% ethanol for 1 min and double-distilled water (ddH₂O) two times for 2 min each. Endogenous peroxidase activity was quenched by submerging the slides in 3% hydrogen peroxide containing 0.1% sodium azide for 10 min. The slides were then washed in ddH₂O three times for 2 min each and equilibrated in TBS for 2 min. All the following incubations were performed in a humidified chamber. Blocking solution [TBS containing 25% (v/v) bovine serum plus 3% (w/v) purified bovine serum albumin] was placed on each section and incubated at 37°C for 1 hr. Fab fragment goat anti-mouse IgG (H+L; Jackson Immuno Research Laboratories, West Grove, PA) was added to the blocking solution (10 µg/ml final concentration) before blocking slides subsequently probed for hsp70i induction. The latter addition blocked any endogenous mouse IgG that was present in the sections and prevented false-positive signals when probing with the biotinylated anti-mouse IgG secondary antibody. The slides were washed two times in TBS for 2 min each. Anti-hsp25 antibody (rabbit polyclonal; Stressgen) was diluted 1:100 in blocking solution and placed on the appropriate slides, whereas anti-hsp70i (mouse monoclonal; Stressgen) was diluted 1:100 in blocking solution devoid of the Fab fragment goat anti-mouse IgG antibody and placed on the appropriate slides. Anti-APAP antibody was diluted 1:100 in blocking solution and placed on the appropriate slides. The antibodies were incubated with the sections at 37°C for 1 hr and then at 24°C for 18 hr. The sections were washed three times in TBS for 2 min each. Biotinylated goat anti-mouse or goat anti-rabbit (Southern Biotechnology Associates, Birmingham, AL), depending on the primary antibody used, was diluted 1:500 in TBS containing 3% BSA, placed on the slides and incubated at 37°C for 30 min. The sections were washed three times in TBS for 2 min each. Streptavidin-linked horseradish peroxidase (Southern Biotechnology Associates) was diluted 1:200 in TBS containing 3% BSA, placed on the slides and incubated at 37°C for 30 min. The sections were washed three times in TBS for 2 min each. The horseradish peroxidase colorimetric substrate 3,3-diaminobenzidine (DAB; Sigma Chemical, St. Louis, MO) supplemented with 0.03% NiCl₂ (w/v) was incubated with each section for 15 min at 24°C to provide a permanent location of antibody binding. The sections were then counterstained with hematoxylin and dehydrated by passing through graded alcohols and xylene in the reverse order as for deparaffinizing the sections. The sections were mounted using Permount (Fisher Scientific, Orlando, FL) and a glass coverslip. With this procedure, no binding of normal rabbit serum or mouse IgG, used as negative controls for the anti-hsp25 and anti-APAP, or anti-hsp70i antibodies, respectively, was observed. In addition, secondary antibody-only-treated slides exhibited no binding.

Liver NPSH depletion. Liver NPSH levels were measured as the total acid-soluble thiols according to the method of Ellman (1959). Each liver was homogenized in 5 ml of 6% trichloroacetic acid (w/v) and 1 mM [ethylenedinitrilo]tetraacetic acid and then centrifuged at 1000 × g at 4°C for 15 min. Eighty microliters of supernatant solution was added to 2 ml of phosphate buffer (0.1 M, pH 8.0). After the addition of 40 µl of 5,5-dithiobis-(2-nitrobenzoic acid) [(DTNB) 4 mg/ml in 95% ethanol], the solution was vortex-mixed and allowed to stand at 24°C for 5 min. The absorbance at 412 nm was then measured, and the corresponding NPSH concentrations were determined using a standard curve derived from reduced glutathione.

Protein determination. Protein was measured with the Micro Protein Determination assay (Sigma Chemical) using bovine serum albumin as standard.

Statistical analysis. NPSH data were analyzed by a one-way analysis of variance followed by a Student-Neuman-Keuls post hoc test. The level of significant difference was defined as the.05 level of probability.

	Results

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Western blot analysis was used to measure the induction of hsp accumulation in mouse liver by APAP or AMAP. Representative Western blots in figure 1 show the hepatic levels of hsp25, hsp60, hsc70, hsp70i and hsp90 at 0 (control), 3, 6 and 24 hr after treatment of naive, male B6C3F1 mice with APAP (200 mg/kg i.p.). The doses of APAP and AMAP used for this study were chosen because they allowed survival for 72 hr, by which time the APAP-induced lesions were being repaired (not shown). Lower doses of APAP were not used because they failed to cause toxicity and hsp induction, whereas higher doses of both compounds often resulted in mortality by 24 hr. Control levels of all the hsps screened were consistent among animals, and control levels in a representative animal are included in figure 1. The levels of hsp60, hsc70 and hsp90 in the liver were unaffected by APAP treatment, whereas hsp25 levels were increased at 6 and 24 hr, and hsp70i levels were increased at each time point. Maximal accumulation was observed at 24 hr for both hsps. No increase in any of the hsps was observed in response to AMAP (1000 mg/kg i.p.) (not shown).

View larger version (62K): [in this window] [in a new window]   Fig. 1.   HSP (hsp) induction in mouse liver 0, 3, 6 or 24 hr after treatment with 200 mg/kg APAP. Liver protein was resolved on a 12.5% SDS-PAGE gel, and the level of various hsps determined by Western blotting using an antibody specific for the indicated hsp. The Western blots are from a single experiment, with each lane containing a liver sample from a single mouse. Control levels of the various hsps were consistent between animals and are represented by the single animal shown in this figure.

With information gained from Western blots identifying the hsps increased by APAP treatment, subsequent experiments used immunohistochemistry to determine the distribution of hsp25 and hsp70i accumulation within the lobule. In addition, the intralobular location of covalently bound APAP or AMAP was determined immunohistochemically using an anti-APAP antibody. APAP treatment caused visible hepatocellular swelling by 3 hr with single-cell necrosis occurring by 6 hr followed by extensive centrilobular necrosis at 24 hr. Visible increases in hsp25 and hsp70i immunostaining coincided with the hepatocellular swelling observed at the earliest observation time, 3 hr after the dose (not shown). The detection of hsp25 accumulation at 3 hr by immunostaining but not by Western blotting was not surprising because Western blotting was probably less sensitive in that it measured whole-tissue hsp levels in which many hepatocytes did not exhibit hsp induction. Consistent with the Western blot experiments, hsp25 and hsp70i levels were greatly increased at 6 and 24 hr. Both hsp25 and hsp70i accumulation at 6 hr was evident throughout the centrilobular region, and the distribution of cells with increased hsps, covalently bound APAP and mild morphological changes (i.e., cell swelling) were essentially superimposable (not shown). By 24 hr, the pattern of hsp25 induction had changed dramatically. At that time, hsp70i accumulation was similar to the pattern of hsp25 and hsp70i accumulation observed at 3 and 6 hr and was uniform throughout the centrilobular region and restricted to necrotic hepatocytes (fig. 2). In contrast, hsp25 accumulation was minimal within the most affected centrilobular hepatocytes and strongest in hepatocytes on the periphery and surrounding the lesions. Similar to observations at 6 hr, hsp25 and hsp70i accumulation was restricted to hepatocytes that had covalently bound APAP.

View larger version (K): [in this window] [in a new window]   Fig. 2.   Immunohistochemical detection of hsp25 and hsp70i accumulation and APAP adduction of cellular macromolecules in mouse liver 24 hr after treatment of naive mice with 200 mg/kg APAP. Four sequential sections were cut to facilitate comparison of hsp induction with APAP adduction and morphological changes. The slides were treated as follows: a, hematoxylin and eosin stain; b, immunohistochemical stain using an anti-hsp25 antibody; c, immunohistochemical stain using an anti-hsp70i antibody; and d, immunohistochemical stain using an anti-APAP antibody. All immunostained slides were counterstained with hematoxylin. Arrow, portal triad; arrowhead, central vein. Extensive centrilobular (zone 3) necrosis is evident. Original field magnification, 200×.

In contrast to the hepatocellular swelling and necrosis produced by APAP, no significant morphological changes were observed in mice treated with AMAP (1000 mg/kg. i.p.), up to 24 hr after the dose (fig. 3). To confirm reactive metabolite binding from AMAP, liver sections from AMAP-treated mice were immunostained. As has been observed previously,³ the pattern of binding was panlobular rather than localized in the centrilobular region as is the case with APAP. Despite the panlobular binding, the greatest level of AMAP immunostaining was observed in the single layer of cells immediately surrounding the centrilobular veins. As reflected by the intensity of immunostaining, AMAP binding was greatest 1 hr after the dose and slightly reduced at 3 and 6 hr post-treatment. AMAP binding was barely detectable 24 hr after the dose. It is possible that Western blotting (as shown for APAP in figure 1) could have been insufficiently sensitive to detect overexpression of hsps in a small subset of cells. For this reason, liver sections from AMAP-treated mice were also immunostained for hsp25 and hsp70i. No immunostaining for hsp25 or hsp70i was detected anywhere in the lobule at any of the time points (fig. 3).

View larger version (K): [in this window] [in a new window]   Fig. 3.   Immunohistochemical detection of hsp25 and hsp70i levels and AMAP adduction of cellular macromolecules in mouse liver 3 hr after treatment of naive mice with 1000 mg/kg AMAP. Four sequential sections were cut to facilitate comparison of hsp induction with AMAP adduction and morphological changes. The slides were treated as follows: a, hematoxylin and eosin stain; b, immunohistochemical stain using an anti-hsp25 antibody; c, immunohistochemical stain using an anti-hsp70i antibody; and d, immunohistochemical stain using an anti-APAP antibody. All immunostained slides were counterstained with hematoxylin. Arrow, portal triad; arrowhead, central vein. Histopathological analysis was similar to that of control-treated animals with no obvious signs of morphological changes evident. Original field magnification, 200×.

Previous reports that cellular thiol status may be important in hsp gene activation (Chen et al., 1992; Huang et al., 1994; Liu et al., 1996) prompted an additional experiment in which the effect of APAP and AMAP on hepatic NPSHs was measured. APAP caused a rapid decline in NPSH to 21% of control values by 1 hr after administration (fig. 4). NPSH levels began rising by 3 hr and exceeded control levels at 24 hr. The depression in hepatic NPSH produced by AMAP was much less pronounced, with a nadir at 46% of control observed 6 hr after the dose. The depression of hepatic NPSH was more protracted than after APAP, however. These data suggested that the lower efficacy of AMAP in depleting NPSH levels might be a possible explanation of its inability to trigger hsp induction.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   The effects of APAP, AMAP and BSO and AMAP on the liver levels of NPSH. Mice were administered 200 mg/kg APAP or 1000 mg/kg AMAP, and the level of NPSH in the liver measured 0, 1, 3, 6 or 24 hr after the dose as described in Materials and Methods. Some mice were pretreated with BSO (222 mg/kg) 1 hr before the AMAP dose. The AMAP dose was lowered to 600 mg/kg for the combined BSO and AMAP treatment because the 1000 mg/kg AMAP dose produced an unacceptably high level of mortality. Results are expressed as percentage of the mean NPSH concentration in concurrently euthanized, saline-treated controls. Data are displayed as the mean ± S.E.M. (three or four mice). *, Statistically significant decrease/increase in NPSH caused by the APAP or BSO and AMAP treatments compared with control levels; **, statistically significant decrease in NPSH caused by all three treatments compared with control levels (P < .05).

To produce a hepatic NPSH depression in AMAP-treated mice similar to that observed in animals treated with APAP, mice were pretreated with the glutathione synthesis inhibitor BSO 1 hr before the AMAP dose. The dose of AMAP was lowered to 600 mg/kg for these experiments because >50% mortality was observed in mice administered BSO and 1000 mg/kg AMAP. Despite the use of a lower AMAP dose, the BSO and AMAP treatment resulted in only a slightly lower level of binding of AMAP to liver protein compared with the 1000 mg/kg AMAP dose, with no change in the pattern of binding (i.e., binding was still panlobular, with the greatest binding in the single layer of hepatocytes surrounding the central veins), as measured by immunostaining (not shown). Unlike the results reported by Tirmenstein and Nelson (1991), we did not observe any indications of hepatotoxicity (i.e., serum ALT levels and liver morphology were the same as those measured in vehicle-treated mice) after the BSO and AMAP treatment. This can perhaps be explained in that the dose of BSO used in our study was ~4-fold lower than that used by Tirmenstein and Nelson (1991). The BSO and AMAP treatment produced a similar decrease in NPSH as APAP at 1 hr (26% for BSO and AMAP vs. 21% for APAP) with a nadir of 14% at 3 hr (fig. 4). BSO alone caused a maximal decline of NPSH of 52% and 46% of control levels 1 and 3 hr after the dose, respectively (not shown). Similar to AMAP alone, BSO and AMAP failed to cause hepatotoxicity detectable by light microscopy at any of the observation times (fig. 5). Western blot analysis of hsp25 and hsp70i induction by BSO and AMAP was inconclusive but suggested that a slight induction of hsp25 might have occurred by 24 hr (not shown). Immunohistochemical detection of hsp25 accumulation confirmed that BSO and AMAP did produce hsp25 accumulation by 24 hr in a small fraction of cells surrounding the central veins of the liver with no concurrent induction of hsp70i (fig. 5). BSO treatment alone failed to cause induction of either hsp as measured by Western blotting and immunohistochemistry (not shown).

View larger version (K): [in this window] [in a new window]   Fig. 5.   Immunohistochemical detection of hsp25 and hsp70i accumulation in mouse liver 24 hr after treatment of naive mice with BSO and AMAP. Mice were pretreated with BSO (222 mg/kg) 1 hr before the AMAP dose (600 mg/kg). Three sequential sections were cut to facilitate comparison of hsp induction with morphological changes. The slides were treated as follows: a, hematoxylin and eosin stain; b, immunohistochemical stain using an anti-hsp25 antibody; and c, immunohistochemical stain using an anti-hsp70i antibody. All immunostained slides were counterstained with hematoxylin. Arrow, portal triad; arrowhead, central vein. Histopathological analysis was similar to that of control treated animals with no obvious signs of morphological changes evident. Original field magnification, 200×.

	Discussion

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The results of this study show that toxicity from APAP, like that from a number of other hepatotoxicants, is accompanied by increased hepatic concentrations of hsps. Consistent with previous observations with cocaine, bromobenzene and carbon tetrachloride (Roberts et al., 1996; Salminen et al., 1997), induction of hsps selectively involved hsp25 and hsp70i; no changes in hsp60, hsc70 and hsp90 were detected. Western blot analysis appeared to suggest that accumulation of hsp70i may precede that of hsp25 (see fig. 1). This distinction is probably an artifact resulting from the limited sensitivity of measuring whole-tissue hsps with this method. Using immunohistochemical staining, both hsp25 and hsp70i concentrations were increased at the earliest time point examined (i.e., 3 hr after APAP administration). Observations in this study suggest that hsp25 may, in fact, be a more sensitive indicator of cytotoxicity. At 24 hr after the APAP dose, cells at the margin of the lesions had detectable reactive metabolite binding and appeared to be only minimally affected. Within these cells, hsp25 but not hsp70i was elevated (see fig. 2). Additional evidence is provided by the observation that combined BSO and AMAP treatment caused induction of only hsp25 (see fig. 5).

The strong correlation in intralobular distribution of APAP reactive metabolite binding and increased levels of hsp25 and hsp70i is consistent with protein adduction as a stimulus for hsp synthesis. On the other hand, the absence of increased levels of hsps in response to AMAP, despite extensive reactive metabolite binding, argues that protein adduction alone may not be an effective trigger for hsp upregulation. The latter observation must be interpreted cautiously because the data are merely correlative and there are a number of potential explanations for the lack of an hsp response. One is that it may not be protein adduction per se but an adduction of certain critical proteins that leads to increased hsp synthesis. There is ample evidence that the reactive metabolites of APAP and AMAP bind cell organelles to different extents (Myers et al., 1995; Tirmenstein and Nelson, 1989), and it has been postulated that the greater reactivity of AMAP metabolites leads to binding with protein targets more proximal to the site of metabolism (i.e., microsomal protein), and less critical, than is the case with APAP (Ramsay et al., 1989; Rashed et al., 1990). Differences in hsp induction between APAP and AMAP may reflect differences in the intracellular site(s) of the adducted proteins or perhaps the nature of the adducted proteins themselves. Tirmenstein and Nelson (1991) showed that pretreatment with BSO caused an increase in binding of AMAP to mitochondrial proteins. Even though we did not explicitly show increased binding of AMAP to mitochondrial proteins by BSO pretreatment in our study, the ability of the combined BSO and AMAP treatment to trigger hsp25 induction might be representative of the increased binding of AMAP to critical cellular targets that were not accessible without BSO pretreatment.

Another possibility relates to the amount or concentration of adducted protein that may be required to trigger increased hsp synthesis. Previous studies have shown the overall extent of binding of AMAP and APAP metabolites to hepatic protein to be similar (Rashed et al., 1990; Roberts et al., 1990). Immunohistochemical staining shows that AMAP reactive metabolite binding occurs in hepatocytes throughout the liver, however, whereas binding of APAP metabolites is confined to the centrilobular region (see figs. 2 and 3). The more diffuse binding of AMAP metabolites may result in a lower amount or concentration of adducted protein per cell, perhaps less than the threshold required to trigger hsp synthesis. Increasing adducted protein concentrations by increasing the AMAP dose to test this hypothesis is unfortunately precluded by the respiratory depression that occurs at higher dosages of AMAP (Rashed et al., 1990). The ability of BSO and AMAP to trigger hsp25 induction around the central veins is compatible with the notion that the concentration of adducted protein may play a critical role in triggering hsp induction. Although AMAP binding occurs panlobularly, the greatest binding occurs in the single layer of hepatocytes surrounding the central veins (see fig. 3), which is where hsp25 induction occurs after BSO and AMAP treatment.

Another possible explanation for the absence of hsp induction after AMAP treatment is that protein adduction alone is an insufficient stimulus. Chen et al. (1992) found that binding of an electrophilic metabolite of nephrotoxic cysteine conjugates to protein in LLC-PK1 cells was associated with induction of hsp70 mRNA and increased hsp70 synthesis. Treatment of cells with the thiol-reducing agent dithiothreitol did not affect protein adduction but nevertheless inhibited induction of hsp70 mRNA. Based on these observations, the authors proposed that a combination of protein adduction and alterations in cellular nonprotein thiols may be needed to activate hsp transcription factor. Huang et al. (1994) found that dithiothreitol prevented induction of hsps by hyperthermia, further suggesting that oxidation of cellular thiols is an important trigger of hsp induction.

In the present study, both APAP and AMAP diminished hepatocellular NPSH content, but the effect of AMAP was relatively modest compared with that of APAP. When NPSH depletion from AMAP was enhanced by BSO pretreatment to produce declines in NPSH levels comparable to those caused by APAP, accumulation of hsp25 was observed, albeit only in cells surrounding the central vein in which AMAP binding was greatest. These results are consistent with a requirement for both protein adduction and a substantial loss of reduced thiols in triggering hsp25 induction. It is unclear why the hsp25 response in BSO- and AMAP-treated mice was restricted to cells surrounding the central vein, but there are several possible explanations. As discussed above, the level of protein adduction in other cells may have been below some threshold requirement, the nature of the protein adducts may be different in different regions of the liver, the extent of NPSH depletion in other cells may have been lesser and insufficient to permit the hsp25 response, the instability of the AMAP-protein adducts (Myers et al., 1995) may have prevented permanent damage of the target proteins or there may be a combination of these factors. The absence of an hsp70i response in BSO- and AMAP-treated animals suggests that there may be somewhat different requirements in terms of cellular events for hsp70i vs. hsp25 induction. This hypothesis is supported by the observed differences in localization of hsp25 and hsp70i within the hepatic lobule 24 hr after hepatotoxic doses of APAP (see fig. 2).

All of the observations presented here suggest that a combination of APAP-arylation of protein and sulfhydryl oxidation may trigger hsp induction; however, they do not address whether hsp induction may play a defensive role during APAP exposure. Overexpression of hsps has been shown to be cytoprotective against a handful of insults (Huot et al., 1991; Jaattela et al., 1992; Li et al., 1991) and elevated levels of hsps, produced by a mild hyperthermic treatment, have been correlated with protection against a wide array of stressors (Feige and Mollenhauer, 1992; Salminen et al., 1996), suggesting that hsps do play a protective role during stress. It has been proposed that hsps bind nonnative proteins and help them either refold or enhance their degradation, thereby aiding cell survival (Parsell and Linquist, 1994). This is consistent with the ability of hsps to bind nascent proteins and fully mature, but denatured, proteins and aid their refolding. Therefore, hsps may play a protective role during APAP exposure by binding APAP-arylated proteins and aiding their refolding or degradation, thereby aiding cell survival.

In conclusion, hepatotoxic doses of APAP in mice lead to increased levels of hsp25 and hsp70i in the liver. As has been observed previously with cocaine, there is a close temporal and spatial correlation within the lobule of reactive metabolite binding, cytotoxicity and accumulation of these hsps. The absence of similar increases in hsps in response to AMAP, despite reactive metabolite binding similar in extent to that from APAP (at least on a whole-organ basis), implies that the simple presence of adducted protein may not be a sufficient stimulus for increased hsp gene expression. The data presented, even though they are correlative, suggest that at least for hsp25, a reduction in NPSH levels may have to occur simultaneously with protein adduction to trigger hsp induction.