type 1 diabetic mice are protected from acetaminophen hepatotoxicity

Type 1 Diabetic Mice Are Protected fromAcetaminophen Hepatotoxicity

Kartik Shankar,* Vishal S. Vaidya,* Udayan M. Apte,* Jose E. Manautou,† Martin J. J. Ronis,‡ Thomas J. Bucci,§ andHarihara M. Mehendale*,1

*Department of Toxicology, School of Pharmacy, The University of Louisiana at Monroe, Monroe, Louisiana 71209; †Department of PharmaceuticalSciences, College of Pharmacy, University of Connecticut, Storrs, Connecticut 06269; ‡Arkansas Children’s Hospital Research Institute, Department of

Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72202; and §Pathology Associates International,National Center for Toxicological Research, Jefferson, Arkansas 72079

Received November 21, 2002; accepted February 7, 2003

Streptozotocin (STZ)-induced diabetic (DB) mice challengedwith single ordinarily lethal doses of acetaminophen (APAP),carbon tetrachloride (CCl4), or bromobenzene (BB) were resistantto all three hepatotoxicants. Mechanisms of protection againstAPAP hepatotoxicity were investigated. Plasma alanine amino-transferase, aspartate aminotransferase, and liver histopathologyrevealed significantly lower hepatic injury in DB mice after APAPadministration. HPLC analysis of plasma and urine revealedlower plasma t1/2, increased volume of distribution (Vd), and in-creased plasma clearance (CLp) of APAP in the DB mice and nodifference in APAP-glucuronide, a major metabolite in mice. In-terestingly, covalent binding of 14C-labeled APAP to liver targetproteins; arylation of APAP to 58, 56, and 44 kDa acetaminophenbinding proteins (ABPs); and glutathione (GSH) depletion in theliver did not differ between nondiabetic (non-DB) and DB mice inspite of downregulated hepatic microsomal CYP2E1 and 1A2proteins in the DB mice, known to be involved in bioactivation ofAPAP. Compensatory cell division measured via 3H-thymidinepulse labeling and immunohistochemical staining for proliferatingcell nuclear antigen (PCNA) indicated earlier onset of S-phase inthe DB mice after exposure to APAP. Antimitotic intervention ofliver cell division by colchicine (CLC) after administration ofAPAP led to significantly higher mortality in the DB mice sug-gesting a pivotal role of liver cell division and tissue repair in theprotection afforded by diabetes. In conclusion, the resistance ofDB mice against hepatotoxic and lethal effects of APAP appearsto be mediated by a combination of enhanced APAP clearance androbust compensatory tissue repair.

Key Words: covalent binding; CYP2E1; diabetes; species differ-ences; tissue repair.

Acetaminophen (APAP) causes fulminant hepatic necrosisin humans and experimental animals (Jollow et al., 1973).APAP is bioactivated to N-acetyl-p-benzoquinoneimine

(NAPQI), predominantly via hepatic cytochrome P450 2E1(CYP2E1) and to minor extent by cytochrome P450 1A2(CYP1A2; Zaher et al., 1998). There is general agreement thatNAPQI-induced covalent binding and protein arylation, com-bined with depletion of cytosolic and mitochondrial glutathi-one (GSH), are pivotal in triggering the events culminating inloss of cellular homeostasis leading to liver injury (Jollow etal., 1973). At least 23 cytosolic, mitochondrial, and microso-mal target proteins that are covalently modified after APAPtreatment have been identified using matrix-assisted laser de-sorption ionization mass spectrometry (MALDI-MS; Qiu et al.,1998). These include the cytosolic 58, 56, and 44 KDa acet-aminophen binding proteins (ABPs), arylation of which showssubstantial correlation with hepatic necrosis (Bartolone et al.,1988; Birge et al., 1990). Several studies have established thecritical role of liver cell division and compensatory tissuerepair response in determining the ultimate outcome of toxicityviz. survival or death (Chanda and Mehendale, 1996; Dalu andMehendale, 1996; Mehendale, 1995; Rao et al., 1996; Soni andMehendale, 1998).

Diabetic rats are highly sensitive to several hepatotoxicants,including carbon tetrachloride (CCl4), bromobenzene (BB),thioacetamide (TA), and chloroform (CHCl3; El-Hawari andPlaa, 1983; Hanasono et al., 1975; Wang et al., 2000a; Watkinset al., 1988). Earlier mechanistic studies focused on changes inbioactivation and metabolism of toxicants in relation to in-creased sensitivity to hepatotoxicants in diabetes. Compensa-tory tissue repair, which is markedly compromised in diabeticrats, is also a major factor leading to higher sensitivity ofdiabetic (DB) rats to TA (Wang et al., 2000a, 2001). Incontrast, DB mice are in fact resistant to TA hepatotoxicity andsurvive a normally lethal challenge of TA (Shankar et al.,2003), revealing a major species difference. Diabetic C57BL6mice exhibited considerably less liver injury after TA admin-istration over 0 to 120 h, accompanied by an augmented repairresponse.

The objective of the present studies was to characterize theresponse of Type 1 diabetic mice to hepatotoxins other than TA

1 To whom correspondence should be addressed at The University of Lou-isiana at Monroe, School of Pharmacy, Department of Toxicology, Sugar Hall306, Monroe, LA 71209-0495. Fax: (318) 342-1686. E-mail: [email protected].

TOXICOLOGICAL SCIENCES 73, 220–234 (2003)DOI: 10.1093/toxsci/kfg059Copyright © 2003 by the Society of Toxicology

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and to identify mechanisms that render DB mice resistant toAPAP hepatotoxicity. Here we report remarkable resistance ofDB mice to several mechanistically different hepatotoxicants.Since in lethality experiments maximum protection was ob-served against APAP hepatotoxicity, we investigated possiblemechanisms of this protection against APAP hepatotoxicity.Several mechanisms have been shown to protect from APAPhepatotoxicity. Broadly these mechanisms deal with inhibitionof total or specific isozymes of P450s (Mitchell et al., 1973a;Wang et al., 1996; Zaher et al., 1998), enhanced detoxificationvia nontoxic conjugation (APAP-glucuronide or sulfate; Ha-zelton et al., 1986), supplementation with GSH or other sulf-hydryl compounds (Corcoran and Wong, 1986; Corcoran et al.,1985), enhanced antioxidant status (Jaeschke, 1990; Kyle etal., 1987), or preplacement of cell division (Chanda et al.,1995; Shayiq et al., 1999). In our studies we have systemati-cally examined changes in bioactivation and disposition ofAPAP, followed by the compensatory tissue repair response.Diabetes is known to modulate several P450 isozymes, includ-ing those involved in bioactivation of APAP (CYP2E1 and1A2). Induction of these isozymes in Type 1 DB rats is thoughtto contribute to the higher bioactivation mediated liver injuryof several hepatotoxicants. However, it is not clear if a similarmodulation occurs in DB mice. Hence, we have examined thehypothesis that changes in these P450s in DB mice may me-diate the protection from APAP-induced toxicity.

Our findings suggest that protection in the DB mice is notdue to altered bioactivation but is mediated via a combinationof enhanced clearance and augmented compensatory liver tis-sue repair after APAP-induced liver damage.

MATERIALS AND METHODS

Animals and chemicals. Male Swiss Webster mice (27–33 g) were ob-tained from Harlan Sprague-Dawley (Indianapolis, IN). Male Sprague-Dawleyrats (250–300 g) were obtained from our central animal facility. Animals werehoused in our central animal facility over sawdust bedding (Harlan Sani-Chips�, Harlan Sprague-Dawley, Indianapolis, IN) in a controlled environment(temperature 21 � 1°C, humidity 50 � 10%, and 12 h light/12 h dark, lightcycle) and were acclimatized for a week before use in experiments. Animalshad access to unlimited water and rodent chow (Harlan Teklad Rat Chow No.7001, Madison, WI). Animal maintenance and treatments were conducted inaccordance with the National Institute of Health Guide for Animal Welfare, asapproved by Institutional Animal Care and Use Committee (IACUC). Allchemicals and biochemical kits were obtained from Sigma Chemical Co. (St.Louis, MO). Perchloric acid and scintillation fluid were obtained from FisherScientific (Fair Lawn, NJ). [3H-Methyl] thymidine (3H-T; 2 Ci/mmol) waspurchased from Moravek Biochemicals (Brea, CA).

Induction of diabetes. Mice were made diabetic with a single dose ofstreptozotocin (STZ, 200 mg/kg, ip; Gaynes and Watkins, 1989; Jeffery et al.,1991; Rerup and Tarding, 1969); rats were also made diabetic with STZinjection (60 mg/kg, ip; Wang et al., 2000a) in 0.01 M citrate buffer (pH 4.3,10 ml/kg). Vehicle control (nondiabetic group) mice and rats received citratebuffer alone. Blood samples were withdrawn retroorbitally under diethyl etheranesthesia to monitor plasma glucose, and diabetes was confirmed by plasmaglucose values (�250 mg/dl) using Sigma kit no. 315-100. Ten days wereallowed for complete removal of STZ (half-life of STZ is 5 min) beforebeginning of experiments. Glycated hemoglobin levels were determined on

day 10 after STZ (or citrate buffer) treatment as an index of overall glucoselevels using Sigma kit number 441-B.

Assessment of proliferating cell nuclear antigen (PCNA) and apoptosisduring diabetes protocol. At 0, 1, 3, 5, and 10 days after STZ treatmentnondiabetic (non-DB) and DB mice were euthanized and blood and liversamples collected. Liver sections were fixed in formalin and processed asdescribed below for PCNA and apoptosis immunohistochemistry.

Lethality study. For lethality experiments, on day 10 of the diabetesprotocol, non-DB and DB mice were divided into two groups each (n � 10 pergroup). One group of mice was treated with APAP (600 mg/kg, ip, warm basicsaline, pH 8, 20 ml/kg), CCl4 (1 ml/kg, ip, corn oil, 5 ml/kg), or BB (0.5 ml/kg,ip, corn oil, 5 ml/kg). The other group received vehicle control saline (20ml/kg) or corn oil (5 ml/kg). Mice were observed twice daily for 14 days andsurvival/mortality was recorded.

APAP treatment and collection of plasma and liver samples. Assess-ments of plasma alanine aminotransferase (ALT), aspartate aminotransferase(AST), glucose, liver histopathology, 3H-thymidine incorporation and PCNAafter APAP treatment were done in the same experiment. On day 10 afteradministration of STZ or citrate buffer, DB and non-DB mice received a singleinjection of either APAP (600 mg/kg, ip in basic saline, pH 8) or basic saline(20 ml/kg, ip) alone. Two h prior to sacrifice, 10 �Ci of 3H-T (in 0.2 mldistilled water/mouse) was administered ip to each mouse in all groups. At 0,6, 12, 24, 36, 48, 72, and 120 h after APAP treatment, blood samples forbiochemical assays and glucose measurements and liver samples for pulselabeling metrics were collected from DB and non-DB mice under diethyl etheranesthesia. At 0, 6, 12, 24, 36, 48, and 72 h after APAP treatment to bothgroups, liver sections were collected and handled as described below forhistopathology. Four mice were treated per group per time point, except fornon-DB groups treated with APAP, in order to obtain an adequate number ofsurviving mice (n � 15 for 36, 48, 72, and 120 h). A minimum of four micewere surviving in each group at all time points.

In a separate experiment, APAP covalent binding was determined viaimmunochemical analysis. DB and non-DB mice were divided into twogroups. One group of DB mice (n � 4) received APAP (600 mg/kg, ip in basicsaline, pH 8) while the other group of DB mice received saline vehicle. Bothgroups of non-DB mice also received similar treatments. Animals were eutha-nized at 4 h after APAP or saline administration and liver samples were flashfrozen in liquid nitrogen and stored at –800C until needed for preparation ofcytosolic fraction.

Plasma enzymes and glucose estimation. Plasma was separated by addi-tion of heparin followed by centrifugation. Plasma ALT (EC 2.6.1.2) and AST(EC 2.6.1.1) were measured as biochemical markers of liver injury usingSigma kit no. 59 UV and Sigma kit no. DG158-Kb, respectively. Glucose wasmeasured using Sigma kit no. 315-100.

Histopathology. Livers were surgically removed from mice under diethylether anesthesia. Portions of liver were taken from the left lateral lobes andwashed with ice-cold normal saline (0.9% NaCl), cut into small pieces, andfixed immediately in 10% phosphate-buffered formalin for 48 h. The livertissue was then transferred to 70% ethyl alcohol and stored until processed.The liver specimens were processed, embedded in paraffin, sectioned at 5 �m,and stained with hematoxylin and eosin (H&E) for histological examinationunder a light microscope. The extent of liver injury was estimated semiquan-titatively and lesions scored as multifocal necrosis. Scoring was as follows: 0,no necrosis; 1, minimal, defined as only occasional necrotic cells in any lobule;2, mild, defined as less than one third of the lobular structure affected; 3,moderate, defined as between one third and two thirds of the lobular structureaffected; 4, severe, defined as greater than two thirds of the lobular structureaffected; 5, more severe, defined as damage to most of the parenchyma of theliver (Wang et al., 2000a, 2001).

3H-T incorporation into hepatonuclear DNA. DNA synthesis was mea-sured by 3H-T incorporation into hepatonuclear DNA as described by Changand Looney (1965). After a single ip injection of 3H-T (10 �Ci in 0.2 mldistilled water/mouse) 2 h prior to sacrifice under diethyl ether anesthesia, the

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following procedure was used. Liver samples (0.5 g) were homogenized in 10ml of 2.2 M sucrose on ice and centrifuged at 40,000 � g for 1 h to isolatehepatic nuclei. The nuclei were then washed, suspended in 0.25 M sucrose, andmixed with 0.5 ml of 0.6 N perchloric acid (PCA) on ice. The mixture wascentrifuged at 10,000 � g for 20 min. Pellets were washed twice with 0.2 NPCA, resuspended in 3 ml of 0.5 N PCA, and heated at 80°C for 20 min. Thesuspension was centrifuged at 10,000 � g for 20 min. DNA was estimated bydiphenylamine reaction (Burton, 1956) in the supernatant. Radiolabeled 3H-Tin the supernatant was estimated using a liquid scintillation counter (PackardInstrument Co., Meriden, CT).

Pharmacokinetic parameters of APAP. Blood (50 �l) was collected fromnon-DB and DB mice at 0, 2, 5, 10, 15, 30, 45, 60, 90, 120, 150, and 240 minafter APAP administration (600 mg/kg, ip). To complete the entire time courseonce (n � 1), six mice had to be used. Three time points were collected fromeach mouse overlapping one time point between mice. Four such replicate timecourses were performed for non-DB and DB mice. In a separate study (n � 20and 6 for non-DB and DB mice, respectively), urine was collected at 0 and 24 hafter APAP administration to non-DB and DB mice. Levels of APAP and AGwere estimated using a reverse-phase HPLC analysis using 7% acetonitrile, 50mM sodium sulfate, and 50 mM potassium phosphate buffer as the mobilephase (1 ml/min; Kulkarni et al., 2000). A C18 column (5 �m, Alltech,Deerfield, IL) was used to separate the components. APAP and AG (HPLCgrade, Sigma Chemical Co.) were used as standards. AG and APAP weredetected at 254 nm, with retention times of 2.6 and 6.9 min, respectively usinga PDA-100 photodiode array detector (Dionex Systems, Sunnyvale, CA).Plasma half-life (t1/2) for APAP was computed using the elimination constant(�), where t1/2 � 0.693/�. The value of � was estimated using a semi-logarithmic plot of time versus concentration (up to 180 min) using JMP-INstatistical software (SAS Institute, Cary, NC).

In a separate study, plasma APAP levels were quantitated using the above-mentioned HPLC assay after low dose of APAP (300 mg/kg, ip). Bloodsamples were drawn from non-DB and DB mice at 15, 30, 60, 90, and 120 minafter APAP administration.

PCNA assay. The PCNA immunohistochemical analysis was conductedas described by Greenwell et al. (1991). Briefly, the liver sections mounted onglass slides were first blocked with casein (0.5%) for 20 min and then reactedwith monoclonal antibody (1:5000) to PCNA (PC.10, Dako Corporation,Carpentaria, CA) for 60 min. Antigen retrieval was performed by heatingslides in 1% zinc sulfate solution for 6.5 min. The antibody was then linkedwith biotinylated goat anti-mouse IgG antibody (1:500 for 20 min, Boehringer/Mannheim, Indianapolis, IN), which was then labeled with streptavidin-con-jugated peroxidase (1:500 for 20 min, Jackson Immunoresearch, West Grove,PA). Brown color was developed by exposing the peroxidase to diaminoben-zidine (one tablet in 10 ml of PBS, filtered and 3% hydrogen peroxide, SigmaChemical Co.) for 10 min. The sections were counterstained with Gill’shematoxylin. The nuclei of G0 cells were blue. G1 cells had light brown nuclei;S-phase nuclei stained dark brown. G2 cells showed brown cytoplasmic stain-ing with or without brown speckling of the nucleus. M cells were identified bymitotic figures. For histomorphometric analysis, each section was observed forcells in the above gap phases of the cell cycle in 10 high power fields, asreported previously (Wang et al., 2000a).

Assessment of apoptosis. Apoptosis was assessed via two independentmethods. First, apoptotic hepatocytes were identified by morphological crite-ria, which included increased eosinophilic cytoplasm, darkened nucleus, andpyknotic separation of cytoplasmic membrane from neighboring hepatocytes(Herrman et al., 1999). Morphologically determined apoptotic cells wereexpressed as apoptotic index (number of apoptotic cells/total number ofcells � 100). Secondly, apoptosis was confirmed via in situ end labeling offree 3�-hydroxyl ends generated during apoptosis using a commercial kit(ApopTag, Intergen, Purchase, NY). The sections were counterstained withGill’s hematoxylin. Apoptotic bodies and cells appeared brown. At least 2000cells were counted from liver sections mounted on each slide for both methods.

Preparation of microsomes for characterization of P450 isozymes. Livermicrosomes were prepared from livers of non-DB and DB, rats and mice, on

day 10 after STZ (or citrate buffer) treatment by differential ultracentrifugationusing the method of Chipman et al. (1979). Protein concentration of themicrosomes was determined by the Bradford method using the Bio-RadProtein Assay (Bio-Rad Laboratories, CA). Microsomes were stored at –800C.

Analysis of CYP2E1, 1A1/1A2, and 3A proteins. Microsomal protein (10�g) from each animal was separated by SDS–PAGE and transferred to nitro-cellulose membranes. For detecting CYP2E1, 1A1/1A2, and 3A proteins,membranes were incubated with primary antibodies against respective pro-teins. Primary antibodies were goat anti-rat CYP2E1 polyclonal antibody, goatanti-rat CYP1A2 polyclonal antibody (which recognizes both rat and mouse,CYP1A1 and 1A2; Gentest Corporation, Woburn, MA) and rabbit anti-humanCYP3A4 polyclonal antibody (Research Diagnostics Inc., Flanders, NJ). Theantibodies were cross-reactive for the corresponding mouse CYP2E1, 1A1/1A2, and 3A proteins. The blots were then further incubated with anti-goat(Sigma Biochemicals, St. Louis, MO)/anti-rabbit secondary antibody (Amer-sham Life Sciences, Piscataway, NJ) conjugated to horseradish peroxidase andvisualized using a chemiluminescence kit obtained from Pierce Biochemicals(Rockford, IL).

Analysis of CYP2E1, 1A1/1A2, and 3A activities. Activity of hepaticmicrosomal CYP2E1 was determined via formation of malondialdehyde(MDA) as a result of lipid peroxidation initiated by tricholromethyl free radical(•CCl3), a metabolite of CCl4. The formation of MDA was estimated spectro-photometrically by reaction with thiobarbituric acid (Hu et al., 1994). SinceCCl4 is bioactivated only via CYP2E1, formation of MDA is specific forCYP2E1 activity. CYP2E1 activity was corroborated via p-nitrophenol hy-droxylase activity (Reinke and Moyer, 1985). Ethoxyresorufin O-deethylase(EROD) activity is specific for CYP1A1 (Burke et al., 1994) and me-thoxyresorufin O-demethylase (MROD) as a marker of CYP1A2 (Rodriguesand Prough, 1991) were measured by following resorufin formation spec-trofluorimetrically at 536 nm (excitation) and 586 (emission) using RF-5301PC scanning spectroflurometer (Shimadzu Scientific Instruments, Colum-bia, MD) under conditions of linearity of incubation time and protein.Cytochrome P450 3A (CYP3A) activity was measured as erythromycin N-demethylase. Erythromycin N-demethylase was measured according to themethod of Werringloer (1978) with a 45 min incubation containing 12.5mmol/l erythromycin in the presence of 0.5 mmol/l NADPH and 1 mg ofmicrosomal protein. The rate of formaldehyde formation was determinedspectrophotometrically at 412 nm using Nash reagent.

Covalent binding analyses of 14C-APAP. Livers from non-DB and DBmice treated with 14C-APAP (600 mg/kg, ip, 4.3 mCi/mmol, 3 �Ci/mouse,Sigma Chemical Co.) were flash frozen at 2 h after APAP treatment. Co-valently bound 14C-APAP derived 14C label to liver macromolecules wasestimated using procedure described by Jollow et al. (1973). Briefly, livertissue (200 mg) was homogenized in 1 ml of 0.9% saline and total proteinprecipitated using 2 ml of 0.9 M trichloroacetic acid (TCA). Samples werecentrifuged at 1000 � g for 15 min at room temperature. The supernatant wasdiscarded and the protein precipitate was resuspended in 3 ml of 0.6 M TCA,mixed on a Vortex agitating mixer for 1 min and centrifuged at 1000 � g for3 min. After two more washings with 0.6 M TCA (3 ml per wash), pellet waswashed six times with 80% methanol (3 ml per wash). After six methanolwashings radioactivity was no longer detected in the supernatant. The remain-ing pellet was dissolved in 2 ml of 1 M NaOH and an aliquot was used toestimate 14C using a liquid scintillation counter (Packard Instrument Co.,Meriden, CT). Results are expressed as DPM/g of liver.

Assessment of Selective Protein Arylation Due to APAP

Preparation of cytosolic fraction for covalent binding studies. Liverswere homogenized (10% w/v) in a buffer containing 0.25 M sucrose, 10 mMTris–HCl, 1 mM MgCl2, pH 7.4 (STM buffer). Homogenates were centrifugedat 9000 � g. The resultant supernatant was centrifuged at 105,000 � g for 1 h(Manautou et al., 1994, 1996). Cytosolic fractions were collected and stored at–800 C until needed. Total protein concentrations in the cytosol were deter-mined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA).

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Immunochemical assessment of APAP covalent binding and levels of 58ABP. Expression of 58-ABP (an APAP selective target protein) and APAPcovalent binding to 58, 56, and 44 kDa ABPs were determined by Westernanalysis of cytosolic fractions. Assessment of expression of 58-ABP was doneto examine if DB changes the levels of the target protein itself. Essentially themethod of Bartolone et al. (1988) and Birge et al. (1989, 1990) were followed.Details of the method have also been described by Manautou et al. (1994).Briefly, proteins (30 �g per lane) were resolved by SDS–PAGE. Resolvedproteins were transferred to nitrocellulose membranes. APAP-bound proteinsor 58-ABP were analyzed by immunostaining with either affinity-purifiedanti-APAP antibody (Bartolone et al., 1988) or anti-58 antisera (Bartolone etal., 1992), respectively, followed with peroxidase-conjugated anti-rabbit IgG.Proteins were visualized using ECL Western blotting detection reagents (Am-ersham Life Sciences, Piscataway, NJ).

APAP-induced hepatic GSH depletion. Total hepatic GSH was estimatedin livers of non-DB and DB mice challenged with APAP (600 mg/kg, ip) at 0,1, 2, and 12 h after APAP treatment. A commercially available kit (Cat.#703002) from Cayman Chemical Company (Ann Arbor, MI) was used. Thekit is based on enzymatic recycling of GSH using GSH reductase followed byreaction of GSH with Ellman’s reagent. The colored product is read at 405 nmand quantitated using a standard curve.

Colchicine antimitotic intervention. Colchicine (CLC) antimitotic inter-vention has been previously used to examine the role of cell division inultimate outcome of hepatotoxic injury in several models (Chanda et al., 1995;Dalu and Mehendale, 1996; Kulkarni et al., 1997; Mangipudy et al., 1995,1996; Rao and Mehendale, 1991a,b, 1993; Rao et al., 1996). Colchicine isknown to exert its antimitotic effects by microtubule perturbations (Kirshner,1978; Manfredi and Horwitz, 1984). In addition, CLC also inhibits S-phaseDNA synthesis by inhibition of thymidine kinase and thymidylate synthetase(Tsukamoto and Kojo, 1989). At low doses (1 mg/kg) CLC is devoid oftoxicity and does not cause any adverse effects on liver histology or on liverfunction (Rao and Mehendale, 1991b). On day 10 of diabetes two groups ofDB mice were treated with APAP (600 mg/kg, ip, basic saline). One groupreceived CLC (1 mg/kg, ip in saline) at 2 and 30 h after APAP treatment; theother group of diabetic mice received saline as control at the same time points.Another group of DB mice receiving saline (vehicle for APAP) was treatedwith two doses of CLC to control for any effects of CLC itself. Mice wereobserved twice daily for 14 days and survival/mortality was recorded.

Data and statistical analysis. All Western blots were scanned using aGS-700 imaging densitometer (Bio-Rad, Hercules, CA) and quantitated using

Quantity One� software (Bio-Rad, Hercules, CA). Data are expressed asmeans � SE. SPSS software package version 11.0 (SPSS Inc., Chicago, IL)was used to perform all statistical tests. Chi-square test was used to analyzestatistical significance between groups in lethality experiments. In all otherexperiments, statistical significance between DB and non-DB groups at thesame time point was analyzed by Student’s t-test. Equality of variances wastested using Levene’s test for equality of variance. Statistical significancebetween DB and non-DB groups with respective controls (at 0 time point) wasevaluated using one-way ANOVA, followed by Duncan’s multiple range test.The criterion for significance was p � 0.05.

RESULTS

STZ-Induced Diabetic Mice Are Resistant to Mechanisticallyand Structurally Diverse Hepatotoxins

On day 10 after STZ treatment, DB mice showed significantelevation in plasma glucose (148 � 26 in non-DB mice com-pared to 490 � 37 mg/dl in DB mice) and glycated hemoglobinlevels (2.2 � 0.1 in non-DB mice compared to 5.3 � 0.5 in DBmice). All three hepatotoxicants caused substantial mortality inthe non-DB mice. APAP led to 70% mortality while CCl4 andBB caused 50 and 70% mortalities in the non-DB mice, re-spectively (Table 1). In contrast, the DB mice showed lowermortality to all hepatotoxicants. No mortality resulted in DBmice treated with the same doses of APAP and CCl4 that werelethal to non-DB mice. BB treatment to the DB mice resultedin 30% mortality.

Temporal Assessment Revealed Significantly LowerDevelopment of Liver Injury in the DB Mice after APAPChallenge.

Plasma ALT (Fig. 1A) and AST (Fig. 1B) in the non-DBmice exhibited significant increase of both transaminases,which correlated with the high lethality observed in this group.

TABLE 1Effect of Streptozotocin-Induced Diabetes on Acetaminophen, Carbon Tetrachloride, and Bromobenzene-Induced Mortality in Male

Swiss Webster Mice

Treatment groups Plasma glucose (mg/dl) HepatotoxinSingle dose ofhepatotoxina

Number of miceTime of death after hepatotoxin

treatment (h)Per group Mortality

Citrate buffer � APAP 134 � 13 APAP 600 10 7 12–36STZ � APAP 525 � 61* APAP 600 10 0 —

Citrate buffer � CCl4 145 � 34 CCl4 1 10 5 48–72STZ � CCl4 465 � 21* CCl4 1 10 0 —

Citrate buffer � BB 166 � 32 BB 0.5 10 7 48–72STZ � BB 478 � 31* BB 0.5 10 3 48–72

Note. On day 0, male Swiss Webster mice (27–33 g) were injected with either STZ (200 mg/kg, ip in citrate buffer, pH 4.3) or citrate buffer (10 ml/kg) alone.On day 10, both groups (STZ and vehicle treated) were injected with single dose of acetaminophen (APAP, 600 mg/kg, ip in warm basic saline, pH 8), carbontetrachloride (CCl4, 1 ml/kg, ip in corn oil), or bromobenzene (BB, 0.5 ml/kg, ip in corn oil). Mortality was observed every 12 h for 14 days. Number of micedying and time of deaths were recorded. Plasma glucose was estimated on day 10 using Sigma kit no. 315-100.

aip (mg/kg or ml/kg).*Indicates significant difference compared to citrate buffer-treated group (p � 0.05).




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The plasma ALT activity was �6.5-fold lower in the DB miceat 12 h compared to non-DB mice consistent with the lack ofany mortality in the DB mice. The transaminase activities fornon-DB mice from 36 h onwards are representative of the 30%surviving mice. Liver sections stained with H&E were exam-ined by light microscopy for multifocal necrosis, vacuolization,and inflammatory cell infiltration (Fig. 2, Table 2). Non-DBmice treated with APAP showed marked histopathology in theliver as early as 6 h. Intense accumulation of erythrocytes inthe central vein characteristic of APAP-induced hepatotoxicitywas evident. The necrosis and vacuolization spread toward the

periportal area over time. On the other hand, DB mice treatedwith the same dose of APAP showed minimal necrosis andhistological damage (Fig. 2, Table 2). By 72 h the survivingnon-DB mice showed some mitotic figures and regression ofmultifocal necrosis. The diabetic mice staged greater numbersof mitotic figures earlier, consistent with earlier recovery.

Robust DNA Synthesis in the DB Mice after APAPAdministration

S-phase DNA synthesis was higher and occurred muchearlier at 6, 24, and 36 h in the DB mice compared to APAPtreated non-DB cohorts (Fig. 3). The DNA synthesis returnedto basal levels in both groups by 120 h. PCNA immunohisto-chemical staining procedure helps to identify cells in differentphases of the cell cycle. In the non-DB mice livers mosthepatocytes were found in the quiescent G0 phase. After APAPtreatment, hepatocytes entered the cell cycle and progressed tomitosis. However, remarkable differences were observed be-tween the DB and non-DB mice. The DB mice exhibitedsignificantly greater numbers of hepatocytes in S-phase ascompared to the non-DB cohorts, at 36 and 48 h after APAPadministration (0.65 and 2.5% in the non-DB group comparedto 2.9 and 8% in the DB group, at 36 and 48 h after APAPtreatment, respectively; Fig. 4). Significant numbers of hepa-tocytes in S-phase were seen at 72 h in the DB and non-DBmice (Fig. 4). It should be recalled that by 48 h only 30% of thenon-DB mice survive, while none of the DB mice die. The DBmice exhibited earlier onset of the cell proliferation.

Plasma t1/2 of APAP Is Lower in DB Mice Along withHigher Clearance

Plasma t1/2 of APAP in non-DB mice was 61 � 2 mincompared to 40 � 4 min in the DB mice (Fig. 5, top; Table 3).Clearance of APAP from the plasma was also significantlyincreased (�twofold) in the DB mice. Surprisingly, plasmalevels of acetaminophen-glucuronide (AG) were similar inboth non-DB and DB mice (Fig. 5, bottom). The DB miceexhibited significant increases in urine volume (�11.2-fold)and in the fraction of dose excreted as APAP in the urine(�5.5-fold) (Table 3). Therefore, clearance of APAP wassubstantially increased in the DB mice even though glucuro-nide formation, which contributes to over 75% of APAP me-tabolism, was not affected.

To examine if changes in plasma t1/2 of APAP are related tohigher injury in the non-DB mice, we employed a nontoxicdose of APAP (300 mg/kg, ip). At this dose, no liver injury wasobserved as measured by plasma ALT levels in either group(preliminary experiments). In spite of lack of injury, the DBmice showed a greater clearance of APAP from the plasma(Fig. 5A), consistent with the hypothesis that the diabetic stateis responsible for enhanced clearance of APAP.

FIG. 1. (A) Plasma alanine aminotransferase (ALT) and (B) aspartateaminotransferase (AST) activities over a time-course after the administrationof APAP. On day 0, male Swiss Webster mice were treated with either STZ(200 mg/kg ip in citrate buffer, pH 4.3) or citrate buffer (10 ml/kg) alone. Onday 10, both groups were treated with a single administration of APAP (600mg/kg ip, in warm basic saline pH 8). Mice were euthanized at 0, 6, 12, 24, 36,48, 72, and 120 h after APAP administration. For diabetic (DB) and non-diabetic (non-DB) mice treated with saline (vehicle for APAP) alone, sampleswere collected at 0, 12, and 72 h only (n � 4 per group at all time points).Transaminase activities for 36, 48, 72, and 120 h time points are representedby only 30% surviving mice in the non-DB group. Results expressed asmeans � SE. *Significantly different from non-DB control at the same timepoint. #Significantly different from the respective 0 h control. p � 0.05.

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Species Differences in CYP2E1 and 1A1/1A2 Modulation inDiabetic Mice versus Rats

Western blot analysis indicated �fivefold induction ofCYP2E1 protein (Fig. 6A) and a �fourfold induction inCYP2E1 enzyme activity in the DB rats as compared to con-trols (Figs. 6B and 6C). By contrast, CYP2E1 protein de-creased to 50% in the diabetic mice compared to non-DB mice(Fig. 6A). However, no significant change in the activity ofCYP2E1 was evident in the DB mice (Figs. 6B and 6C).Diabetic rats showed a twofold induction in CYP1A1/1A2proteins (Fig. 6D). EROD and MROD activities, specific forCYP1A1 and 1A2, respectively, were also increased in DB ratscompared to non-DB controls (Fig. 6E). Similar to modulationof CYP2E1, DB mice showed 57% lower CYP1A1/1A2 pro-teins along with 35% lesser EROD and 51% lesser MROD

activities, compared with respective non-DB mice (Figs. 6D,6E, and 6F). Diabetic rats showed no change in CYP3A proteinand activity (Figs. 6G and 6H). Activity of CYP3A was mea-sured by N-demethylation of erythromycin. Diabetic miceshowed decreased CYP3A protein as compared to non-DBmice (Fig. 6G). However, there was no change in the enzymeactivity in the DB mice (Fig. 6H).

No Differences in 14C-APAP Covalent Binding betweenNon-DB and DB Mice Livers

Total covalent binding of 14C-APAP has been accepted to besuggestive of reactive intermediate production. Covalent bind-ing sharply increases from 30 min to 2 h after APAP treatmentand reaches a peak by 2 h (Jollow et al., 1973). In our studieswe found no differences in 14C-APAP covalent binding be-

FIG. 2. Liver histopathological anal-ysis of H&E-stained liver sections over atime-course after APAP administrationto DB and non-DB mice. Treatment de-tails as in Figure 1. Samples were col-lected only after 0, 6, 12, 24, 36, 48, and72 h after APAP administration. Bothnon-DB and DB mice treated with saline(vehicle for APAP) alone showed no ne-crosis (data not shown). (A) Non-DBmice 0 h, (B) DB mice 0 h, (C) non-DBmice 6 h, (D) DB mice 6 h, (E) non-DBmice 24 h, (F) DB mice 24 h. See Table2 for necrosis scoring. C, central vein; N,areas of necrosis. Magnification in A–Fx400.




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tween non-DB and DB mice (39,902 � 1069 and 32,676 �3340 DPM/g liver, respectively). These data suggest that bio-activation of APAP is similar in both non-DB and DB mice.

Arylation of APAP Target Proteins and Hepatic GSHDepletion Is Similar in Both Non-DB and DB Mice

Expression of the 58-ABP in the DB mice was similar tonon-DB mice at both 0 and 4 h time points (Fig. 7A). Selectivearylation of these target proteins was similar in both DB andnon-DB mice treated with APAP in spite of significantly lowerliver injury and histological damage in the DB mice (Fig. 7B).GSH depletion occurs subsequent to APAP administration,preceding development of necrosis. Since GSH levels havebeen shown to be affected early after APAP challenge, weestimated hepatic GSH levels 1 and 2 h after APAP treatment(Ruepp et al., 2002). Similar to 14C-APAP covalent bindingand arylation of ABPs, depletion of hepatic GSH was alsosimilar in both groups (Fig. 8). Consistent with earlier reportsGSH depletion preceded 14C-APAP covalent binding (Jollowet al., 1973; Mitchell et al., 1973b).

Colchicine Antimitotic Intervention Results in Diminution ofDB-Induced Resistance

Treatment of DB mice with CLC after APAP administrationresulted in increased mortality in the DB mice (60% vs. 0%, in

CLC treated group vs. CLC untreated group) suggesting apivotal role of cell division in mediating the DB-inducedresistance (Table 4). Diabetic mice treated with saline � CLCalone did not show any mortality.

PCNA and Apoptotic Changes in Diabetes

PCNA in non-DB and DB mice examined at 0, 1, 3, 5, and10 days after STZ treatment revealed that in non-DB mice,most cells were in the quiescent (G0) phase with only 0.075%cells in G2 phase of the cell cycle. On day 3 after induction ofdiabetes by STZ, 0.1% cells were in the G2 phase locatedaround the centrilobular area. The number of G2 cells washigher than non-DB controls but not statistically different (Fig.9). However, on days 5 and 10, significantly higher numbers ofG2 cells (0.45 and 1.26%, respectively) were found in the DBmice (Fig. 9). A minimum of 4000 cells was examined pergroup/time point.

No changes in apoptotic cells were seen at any time pointbetween non-DB and DB mice (apoptotic indices of 0.05 �0.02 and 0.03 � 0.01, in non-DB and DB mice, respectively).Assessment of apoptotic bodies under H&E stained liver sec-tions was corroborated by immunohistochemical assessment ofapoptosis. These results are similar to those reported by Herr-man et al. (1999).

DISCUSSION

In the present work, we report resistance of DB mice to threestructurally and mechanistically dissimilar hepatotoxicants.Only a limited number of studies have employed the mousemodel of diabetes. Previous studies have reported lower liver

FIG. 3. 3H-T incorporation into hepatonuclear DNA after APAP admin-istration to non-DB and DB mice. Treatment details are as under Figure 1 andin Methods section. [3H-CH3]-Thymidine (3H-T, 10 �Ci in 0.2 ml distilledwater/per mouse) was administered 2 h prior to sacrifice. Results expressed asmeans � SE (n � 4). *Significantly different from nondiabetic control at thesame time point. #Significantly different from respective 0 h control. p � 0.05.

TABLE 2Effect of Streptozotocin-Induced Diabetes on Acetaminophen-

Induced Hepatic Necrosis in Male Swiss Webster Mice

Hours after APAPadministration

Liver injury score

Non-DB � APAP(600 mg/kg, ip)

DB � APAP(600 mg/kg, ip)

0 0, 0, 0, 0 0, 0, 0, 06 2, 3, 3, 2 0, 1, 0, 1

12 3, 4, 4, 4 1, 1, 1, 124 4, 5, 5, 5 1, 2, 1, 136 4, 4, 4, 3 1, 1, 1, 248 3, 3, 4, 4 0, 1, 1, 172 2, 2, 2, 1 0, 0, 1, 0

Note. On day 0, male Swiss Webster mice (27–33 g) were injected witheither STZ (200 mg/kg, ip in citrate buffer, pH 4.3) or citrate buffer (10 ml/kg)alone. On day 10, both groups (STZ and vehicle treated) were injected withsingle dose of acetaminophen (APAP, 600 mg/kg, ip in warm basic saline, pH8). Liver samples were collected for histopathology at 0, 6, 12, 24, 36, 48, and72 h after APAP treatment. See under Methods for details. Four H&E-stainedslides from each group, each from a separate animal were scored. The extentof liver necrosis was estimated semiquantitatively and lesions scored as mul-tifocal necrosis. Scoring was as follows: 0, no necrosis; 1, minimal, defined asonly occasional necrotic cells in any lobule; 2, mild, defined as less than onethird of the lobular structure affected; 3, moderate, defined as between onethird and two thirds of the lobular structure affected; 4, severe, defined asgreater than two thirds of the lobular structure affected; 5, more severe, definedas damage to most of the parenchyma of the liver.

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injury after APAP in DB mice (Gaynes and Watkins, 1989;Jeffery et al., 1991). In separate studies, Watkins et al. havepreviously examined modulation of CCl4 hepatotoxicity of inboth DB rats and DB mice and observed a species difference inhepatotoxic outcome (Gaynes and Watkins, 1989; Watkins etal., 1988). However, the mechanisms of this resistance remainto be investigated. No data were available on either temporaldevelopment of liver injury or the ultimate outcome of thetoxic injury viz. survival or death in DB mice. The presentstudy investigated both of these aspects.

APAP clearance and bioactivation were the two potentialmechanisms selected for additional investigation. Firstly, ourstudies demonstrate 34% lower plasma half-life of APAP in theDB mice along with twofold higher clearance of APAP, pre-sumably due to a tenfold increase in urine output in the DB

mice. To test the possibility that the delay in APAP clearancein non-DB mice might be due to higher liver injury of APAP(600 mg/kg, ip), we used low dose of APAP (300 mg/kg) toexamine the effect of diabetes alone on APAP clearance in theabsence of any liver injury. Our data clearly suggest that thediabetic state enhances the clearance of APAP from the plasmairrespective of level of liver injury. Several changes in renalmorphology and function reported in diabetes may account forthis (Sharma et al., 1999). Glomerular hypertrophy, acuteincrease in basement membrane mass, proximal tubularchanges, and microalbuminuria are some changes that occur inearly stages of STZ-induced diabetes. Early stages of diabetesare also associated with increased glomerular area and hyper-filtration (Sharma et al., 1999). Polyuria due to a glucose-induced osmotic diuresis is common in patients with hyper-

FIG. 4. Results of the proliferatingcell nuclear antigen (PCNA) immunohis-tochemistry in non-DB and DB mice af-ter APAP administration. Representativephotomicrographs of liver sections from(A) non-DB mice 36 h, (B) DB mice36 h, (C) non-DB mice 48 h, (D) DBmice 48 h, (E) non-DB mice 72 h, (F) DBmice 72 h. C, central vein; nuclei of G0

cells were blue; G1 cells had light brownnuclei; S-phase cells had dark brown nu-clear staining; G2 cells showed browncytoplasmic staining with or withoutbrown speckling of nucleus; M cellswere visualized as mitotic figures withdiffuse cytoplasmic and deep chromo-somal staining. Magnification in A–Fx400.




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glycemia (Spira et al., 1997). The roles of these mechanisms inprotection from APAP toxicity are worthy of a more thoroughinvestigation. The HPLC analysis in our study only presentsAPAP and the APAP-glucuronide data. Other metabolites such

as the APAP-sulfate and the APAP-mercapturate conjugateswere not measured. The APAP-glucuronide represents forabout 70–75% of metabolism in the mice. The APAP-sulfate isonly a minor metabolite in this species accounting for about5–7% of the metabolism (Hazelton et al., 1986). From our data,the APAP-glucuronide and the parent compound account for�80% of APAP metabolism. Quantitation of these metabolitesmight further elucidate the protection mechanisms in DB mice.

Furthermore, we investigated whether or not diabetes de-creases bioactivation of APAP. Three independent indirectmarkers of actual reactive intermediate argue against this. First,covalent binding of 14C-APAP was not different between DBand non-DB mice. Second, selective protein arylation of cyto-solic proteins has served as an indirect indicator of net avail-ability of reactive electrophile in the liver (Bartolone et al.,1988; Birge et al., 1990; Manautou et al., 1996). Third, thedegree of GSH depletion was also similar between non-DB andDB mice, further strengthening the concept that equal bioacti-vation occurred in both groups. Hence it appears that a de-crease in CYP2E1 and 1A1/1A2-mediated bioactivation is notthe mechanism by which the marked hepatotoxic protectionagainst APAP toxicity is achieved in the DB mice.

Other examples of hepatoprotection in the absence of de-creased covalent binding of APAP are available. Treatmentwith cimetidine (Peterson et al., 1983), dithiothreitol (Tee etal., 1986), disulfiram (Poulsen et al., 1987), and chlorproma-zine (Saville et al., 1988) were shown to protect against APAPhepatotoxicity in the absence of significant reduction in cova-lent binding of APAP metabolite. Pretreatment with a singledose of peroxisome proliferator, clofibrate also protects againstAPAP toxicity without any change in selective protein aryla-tion of target proteins or depletion of nonprotein sulfhydrylgroups (Manautou et al., 1996).

Mitochondrial damage is an early event after APAP admin-istration (Qiu et al., 1998; Ruepp et al., 2002), leading todecreased oxidative phosphorylation and energy production(Meyers et al., 1988) and decreased ATP content (Vendemialeet al., 1996). Liver mitochondria from diabetic animals are

FIG. 5. Effect of diabetes on plasma levels of acetaminophen (APAP) andacetaminophen-glucuronide (AG). See Methods for sample collection anddetailed HPLC procedure. (top) Plasma APAP after either 600 mg/kg or 300mg/kg, ip dose of APAP. (bottom) Plasma AG levels over a time-course of0–240 min after APAP administration (600 mg/kg, ip). (n � 4 per group pertime point). *Indicates significant difference compared to respective non-DBgroup at same time point, p � 0.05.

TABLE 3Effect of Diabetes on Disposition of Acetaminophen and Acetaminophen-Glucuronide in the Plasma and Urine

Parameter Non-DB DB % Increase (�) or decrease (–) in DB mice

APAP plasma t1/2 (min) 61 � 2 40 � 4* –34APAP plasma clearance (l/kg � min) 0.01513 � 0.0002 0.03098 � 0.0016* �105Volume of distribution (l) 1.34 � 0.05 1.78 � 0.11* �33Volume of urine/24 h (ml/day) 1.25 14* �1020Fraction of total dose excreted as APAP by 24 h (%) 3.7 � 0.4 18.9 � 3.8* �410Fraction of total dose excreted as AG by 24 h (%) 75.6 � 5.1 76.4 � 7.7 —

Note. Plasma and urine APAP and AG concentrations were determined using reversed-phase HPLC as described under Methods. Plasma was collected fromnon-DB and DB mice (n � 4 per group per time point) at 0, 2, 5, 10, 15, 30, 45, 60, 90, 120, 150, and 240 min after APAP administration. In a separate study(n � 20 and 6 for non-DB and DB mice, respectively) urine was collected at 0 and 24 h after APAP administration to non-DB and DB mice.

*Indicates significant difference compared to the respective non-DB group, p � 0.05.

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reported to be less susceptible to oxidative damage (Ferreira etal., 1999; Santos et al., 2001; Sukalski et al., 1993) and havea higher antioxidant capacity (Elangovan et al., 2000), criticalin the elimination of mitochondrially generated reactive oxy-gen species. Diabetes also alters the mitochondrial permeabil-ity transition (MPT) due to changes in calcium uniporter func-tions and offers resistance of diabetic tissues to ischemiareperfusion injury (Kristal et al., 1996). Although speculativeat this stage, this resistance of mitochondria from DB micemay significantly decrease initiation and progression of injuryafter APAP challenge without changes in arylation of cytosolictarget proteins as observed in our studies.

In addition to the mechanisms that initiate toxicity, thedynamics of the compensatory tissue repair response influencethe ultimate outcome of toxicity (survival or death) (Chandaand Mehendale, 1996; Mehendale, 1995; Soni and Mehendale,1998). Timely onset of cell division and sustained continuationof the cell proliferative response are of pivotal importance forsurvival. Marked cell cycle changes in the present study ob-served in the DB mice are consistent with the early onset oftissue repair after APAP challenge. The movement of other-wise quiescent hepatocytes to G2 phase may be of criticalimportance to the DB mice. The number of cells in the G2

phase normally is small, yet these cells are an importantdefense against CCl4-induced hepatotoxicity (Calabrese andMehendale, 1996). It appears that the greater number of cells inG2 phase in the DB mice provide the stimulus for early onsetof tissue repair.

Antimitotic intervention has been shown to inhibit tissuerepair and permit progression of liver injury initiated by CCl4

(Rao and Mehendale, 1993), TA (Mangipudy et al., 1996), anddichlorobenzene (Dalu et al., 1998; Kulkarni et al., 1997).Also, antimitotic intervention with CLC abolishes autoprotec-tion by low priming doses of hepatotoxicants, against highdoses of the same toxicant (Mangipudy et al., 1995; Rao andMehendale, 1993) or heteroprotection against a different tox-icant (Chanda et al., 1995). But how does absence of celldivision lead to progression of liver injury, even after thetoxicant has been cleared? Recent evidence suggests that thisprogression may be mediated via the action of lytic enzymes(e.g., calpain, phospholipaseA2, etc.) leaking from cells dyingof cytotoxic injury (Limaye et al., 2002). New cells are resis-tant to the action of these lytic enzymes because they overex-press endogenous inhibitors of these enzymes. Hence, by re-placing necrotic cells with new cells, further progression ofliver injury is curbed (Limaye et al., 2002). In the absence ofnew cells (antimitotic action of colchicine) lytic enzymes leadto accelerated progressive injury (Mangipudy et al., 1996). In

FIG. 6. Effect of diabetes on hepatic CYP2E1, 1A1/1A2, and 3A proteinexpression and enzyme activity. See Methods for sample collection, prepara-tion, and assay procedures. Rats and mice were made diabetic using STZ (n �3). (A) Microsomal CYP2E1 protein expression was estimated by Westernblots and quantitated by densitometry. Graphical form of densitometric scan-ning units. (B) CYP2E1 enzyme activity was measured as malondialdehydeformation and estimated using thiobarbituric acid. (C) CYP2E1 enzyme ac-tivity measured as p-nitrophenol hydroxylase activity. (D) MicrosomalCYP1A1/1A2 protein expression. (E) CYP1A1 enzyme activity was measured

by EROD activity. (F) CYP1A2 enzyme activity was measured by MRODactivity. (G) Microsomal CYP3A proteins. (H) CYP3A enzyme activity wasmeasured by erythromycin N-demethylase activity. *Significantly differentfrom nondiabetic control, p � 0.05.




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the study depicted in Table 4, progressive phase of injury(ALT) was accelerated upon CLC intervention of cell divisionin DB mice (data not shown).

What determines the extent of tissue repair? Two primaryreasons may be considered to explain the observed increase inDNA synthesis in the DB mice. First, the lower liver injury inthe DB mice might be considered as a reason for higher tissue

repair. However, the tissue repair response, although initiatedin response to injury, has been noted to be dependent on thephysiological status of the animal and not on the initial extentof injury. For instance, diet restricted rats on treatment withTA, have markedly higher (2.5-fold higher) liver injury com-pared to ad libitum fed rats, but still survive due to increasedtissue repair response (Apte et al., 2002; Ramaiah et al., 1998).Another illustration where the extent of initial injury does notpredict the extent of tissue repair can be seen in DB rats. DBrats challenged with tenfold lower dose of TA as compared tonon-DB rats attain same level of initial injury as the non-DBrats. In spite of equal initial injury, DB rats show significantlydelayed tissue repair (Wang et al., 2000a).

The second possibility is that mechanisms that regulate celldivision in DB mice are upregulated (Shankar et al., 2002).Although, mechanisms for the increased tissue repair remainunclear, altered hormonal regulation of tissue repair in DBmice is an attractive explanation. Growth hormone is known toaccelerate hepatic regeneration. Hepatocyte growth factor geneexpression and DNA synthesis in partial hepatectomized liverswere both accelerated by treatment with growth hormone (Ek-berg et al., 1992). Similarly, CCl4 hepatotoxicity is potentiateddue to delayed compensatory liver tissue repair in the Mini ratmodel, a Wistar-derived transgenic rat in which the expressionof growth hormone (GH) gene is suppressed by the presence ofan antisense transgene (Shimizu et al., 2001). DB mice haveabout tenfold higher circulating levels of growth hormone(Grønbæk et al., 2002). Although speculative at this point, this

FIG. 7. Effect of diabetes on APAPcovalent binding and expression of58-ABP. Mice were made diabetic withSTZ. After treatment with APAP as de-tailed under Figure 1, mice were sacrificed4 h after challenge. See under Methods fordetails. (A) Effect of diabetes on hepatic58-ABP. Proteins were analyzed immuno-chemically via Western blots. Lane 1 rep-resents positive control. Lanes 2 to 5 rep-resent non-DB mice at 4 h after APAPtreatment; 6, 7 represent non-DB mice at0 h after APAP treatment; 8, 9 representDB mice at 0 h after APAP treatment; 10to 13 represent DB mice at 4 h after APAPtreatment. Results expressed as means �SE (n � 4). (B) Covalent binding of APAPto 58, 56, and 44 kDa proteins in the cy-tosol. Proteins were analyzed immuno-chemically via Western blots. Lanes 1 to 4represent DB mice at 4 h after APAP treat-ment; 5, 6 represent DB mice at 0 h afterAPAP treatment; 7, 8 represent non-DBmice at 0 h after APAP treatment; 9 to 12represent non-DB mice after APAP treat-ment. Lane 13 represents positive control.Results expressed as means � SE (n � 4).

FIG. 8. Effect of diabetes on APAP-induced GSH depletion. Mice weresacrificed at 0, 1, 2, and 12 h after APAP treatment. See Methods for details.Results expressed as mean � SE (n � 4). *Significantly different from non-DBcontrol at the same time point. #Significantly different from respective 0 hcontrol. p � 0.05.

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marked increase in growth hormone may play a role in theincreased repair response in the DB mice.

Another important finding from our studies is the species-specific modulation of P450 isozymes in DB mice versus rats.Type I diabetes induced via STZ or alloxan in rats is known tobe accompanied by induction of a multitude of P450 isozymesincluding hepatic CYP2E1 (Barnett et al., 1994; Shimojo,1994; Wang et al., 2000b). However, very little is known aboutsuch changes in P450 isozymes in DB mice. Similar to ourfindings, Sakuma et al. (2001) have also reported a species

difference in the modulation of hepatic CYP2E1 and 1A2between DB rats and mice. The mechanism of a dichotomousimpact of diabetes on microsomal CYP2E1 in the two rodentspecies (rats vs. mice) is not known. Hypophysectomy inducesCYP2E1 in rats but not in mice (Hong et al., 1990). Ketonebodies, thought to regulate CYP2E1 are increased in DB rats(Miller and Yang, 1984) but are unchanged in DB mice (Po-lotsky et al., 2001). These and other differences may collec-tively be responsible for differential modulation of CYP2E1and other isozymes.

TABLE 4Effect of Colchicine Administration on Acetaminophen-Induced Mortality in Streptozotocin Induced-Diabetic

Male Swiss Webster Mice

Treatment groups Plasma glucose (mg/dl) Hepatotoxin dosea

No. of mice

Time of death after hepatotoxin treatment (h)Total Mortality

STZ � APAP 492 � 43 600 10 0 —STZ � APAP � CLC 465 � 25 600 10 6 24–48STZ � saline � CLC 472 � 61 — 10 0 —

Note. On day 0, male Swiss Webster mice (27–33 g) were injected with either STZ (200 mg/kg, ip in citrate buffer, pH 4.3) or citrate buffer (10 ml/kg) alone.On day 10, DB mice were divided into two groups. Plasma glucose was estimated on day 10. Both groups were injected with single dose of acetaminophen(APAP, 600 mg/kg, ip in warm basic saline, pH 8). At 2 and 30 h after APAP administration, one group of diabetic mice received colchicine (CLC, 1 mg/kg,ip in saline) while the other received saline alone. Another group of DB mice receiving saline (vehicle for APAP) was treated with two doses of CLC to controlfor any effects of CLC itself. Lethality was observed every 12 h for 14 days. Number and time of deaths were recorded.

aSingle dose of hepatotoxin (mg/kg, ip).

FIG. 9. Effect of diabetes on PCNAexpression in hepatocytes. Mice weresacrificed on 0, 1, 3, 5, and 10 days afterSTZ (200 mg/kg, ip, citrate buffer) treat-ment. See procedure for PCNA immuno-histochemistry for details. (A) Non-DBmice, (B) day 3 after STZ, (C) day 5 afterSTZ, (D) day 10 after STZ. Nuclei of G0

cells were blue; G2 cells showed browncytoplasmic staining with or withoutbrown speckling of nucleus (indicated byarrow). Magnification x400.




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In conclusion, we report marked resistance of DB mice toseveral mechanistically and structurally diverse hepatotoxi-cants. Even though hepatoprotection against APAP is accom-panied by downregulation of hepatic CYP2E1 and 1A2 pro-teins, protection against liver injury is not due to decreasedcovalent binding of the APAP reactive intermediate. Instead,protection appears to be due to accelerated clearance of APAPand upregulation of mechanisms that enhance early and sub-stantial tissue repair, averting progressive injury.

ACKNOWLEDGMENTS

These studies were partly supported by the Louisiana Board of RegentsSupport Fund through the University of Louisiana at Monroe, Kitty DeGreeEndowed Chair in Toxicology.

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type 1 diabetic mice are protected from acetaminophen hepatotoxicity

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