ToxSci Advance Access originally published online on June 21, 2007
Toxicological Sciences 2007 99(2):628-636; doi:10.1093/toxsci/kfm165
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Glutamate Cysteine Ligase Modifier Subunit Deficiency and Gender as Determinants of Acetaminophen-Induced Hepatotoxicity in Mice
* Department of Environmental and Occupational Health Sciences
Department of Pathology
Department of Comparative Medicine, University of Washington, Seattle, Washington 98195
Schering-Plough Biopharma, Palo Alto, California 94304
2 To whom correspondence should be addressed at Department of Environmental and Occupational Health Sciences, Box 354695, University of Washington, Seattle, WA 98195. Fax: (206) 685-4696. E-mail: tjkav{at}u.washington.edu.
Received June 8, 2007; accepted June 12, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The analgesic and antipyretic drug acetaminophen (APAP) is bioactivated to the reactive intermediate N-acetyl-p-benzoquinoneimine, which is scavenged by glutathione (GSH). APAP overdose can deplete GSH leading to the accumulation of APAP–protein adducts and centrilobular necrosis in the liver. N-acetylcysteine (NAC), a cysteine prodrug and GSH precursor, is often given as a treatment for APAP overdose. The rate-limiting step in GSH biosynthesis is catalyzed by glutamate cysteine ligase (GCL) a heterodimer composed of catalytic and modifier (GCLM) subunits. Previous studies have indicated that GCL activity is likely to be an important determinant of APAP toxicity. In this study, we investigated APAP toxicity, and NAC or GSH ethyl ester (GSHee)–mediated rescue in mice with normal or compromised GCLM expression. Gclm wild-type, heterozygous, and null mice were administered APAP (500 mg/kg) alone, or immediately following NAC (800 mg/kg) or GSHee (168 mg/kg), and assessed for hepatotoxicity 6 h later. APAP caused GSH depletion in all mice. Gclm null and heterozygous mice exhibited more extensive hepatic damage compared to wild-type mice as assessed by serum alanine aminotransferase activity and histopathology. Additionally, male Gclm wild-type mice demonstrated greater APAP-induced hepatotoxicity than female wild-type mice. Cotreatment with either NAC or GSHee mitigated the effects of APAP in Gclm wild-type and heterozygous mice, but not in Gclm null mice. Collectively, these data reassert the importance of GSH in protection against APAP-induced hepatotoxicity, and indicate critical roles for GCL activity and gender in APAP-induced liver damage in mice.
Key Words: glutathione deficiency; acetaminophen; gender differences; transgenic mice.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The popular over-the-counter analgesic and antipyretic drug acetaminophen (APAP) can be hepatotoxic when taken in large doses. At nontoxic therapeutic doses, the principal routes of APAP metabolism are direct sulfation or glucuronidation followed by excretion (Hjelle and Klaassen, 1984
). However, in the case of APAP overdose, these pathways may become saturated leading to increased bioactivation by cytochrome P450s 2E1 (CYP2E1), CYP1A2, CYP2D6, and CYP3A to generate the reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI) (Corcoran et al., 1980; Mitchell et al., 1973). NAPQI is detoxified by glutathione (GSH), but when GSH is limiting, NAPQI forms covalent protein adducts which, in the presence of other downstream events, may lead to hepatocellular necrosis and acute liver failure (Corcoran et al., 1980; James et al., 2003a; Malhi et al., 2006). APAP overdose is a common cause of drug-induced acute liver failure in the United States (Larson et al., 2005). In a recently published clinical trial, APAP administered at the maximum recommended daily dose (4 g/day) was associated with elevated serum alanine aminotransferase (ALT) activity in nearly 40% of study participants (Watkins et al., 2006).
In addition to its role in APAP metabolism, the antioxidant sulfhydryl tripeptide GSH is a cofactor for glutathione S-transferase–mediated xenobiotic conjugation and is an important determinant of cellular redox status (Meister and Anderson, 1983; Schafer and Buettner, 2001). GSH is comprised of glutamate, cysteine, and glycine and is synthesized in a two-step reaction. The first and rate-limiting step is the ligation of glutamate and cysteine to form 
-glutamylcysteine (-GC), which is catalyzed by glutamate cysteine ligase (GCL). The second step is catalyzed by GSH synthetase, whereby glycine is ligated to -GC to form GSH.
GCL is a heterodimer consisting of catalytic and modifier subunits. The catalytic subunit (GCLC, 73 kDa) possesses all of the catalytic activity, while the modifier subunit (GCLM, 31 kDa) functions to increase the catalytic efficiency of GCLC by decreasing the Km for glutamate and adenosine triphosphate [ATP], and attenuating feedback inhibition by GSH (Chen et al., 2005; Griffith and Mulcahy, 1999). Importantly, it appears that human polymorphisms related to both GCLC and GCLM are associated with a number of pathologies including chronic beryllium disease, type-I diabetes, cystic fibrosis lung disease, myocardial infarction, and schizophrenia (Bekris et al., 2006, 2007; Koide et al., 2003; McKone et al., 2006; Nakamura et al., 2002, 2003; Tosic et al., 2006).
Enhancement of liver GSH levels mitigates both the initiation and progression of APAP-induced liver injury (Chen et al., 2000; Corcoran and Wong, 1986; James et al., 2003b; Puri and Meister, 1983; Reid et al., 2005). Clinically, the cysteine prodrug N-acetyl-L-cysteine (NAC) is the long-standing standard therapy for APAP overdose (Prescott et al., 1977). By using buthionine sulfoximine (BSO) to inhibit GCL (Miners et al., 1984; Puri and Meister, 1983; Wong and Corcoran, 1987), or by administering N-acetyl-D-cysteine (Corcoran and Wong, 1986), it has been shown that most of the protective effects of NAC are attributable to the incorporation of cysteine into GSH by GCL.
We recently reported that transgenic mice with enhanced GCL activity are resistant to APAP-induced liver injury (Botta et al., 2006). In this current study, we tested whether Gclm null or heterozygous mice, with impaired GCL activity relative to wild-type, are more susceptible to APAP overdose and if NAC or GSH ethyl ester (GSHee) can mitigate the liver damage. We also determined whether these effects are influenced by gender.
We report that both male and female Gclm null and heterozygous mice are susceptible to APAP-induced liver damage. Cotreatment with either NAC or GSHee mitigated the hepatotoxic effects of APAP in Gclm wild-type and heterozygous mice, but neither was effective in Gclm null mice. Additionally, similar to the findings of Chan et al. (2001) and Dai et al. (2006), and in agreement with our previous work (Botta et al., 2006), male mice on a C57BL/6 background, regardless of Gclm genotype, were more sensitive to APAP-induced liver damage compared to female mice of the same genotype. These findings underscore the importance of GSH synthesis in moderating susceptibility to and rescue from APAP-induced liver damage as well as the potentially dominant influence of gender on susceptibility to xenobiotic-induced toxicity. Furthermore, this study validates the Gclm null mouse as a tool for investigating the role of GSH in chemical–biological interactions and other biological processes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Reagents.
All reagents were obtained from Sigma-Aldrich (St Louis, MO), unless otherwise noted.
Generation of Gclm null mice.
All procedures for animal use were in accordance with the National Institutes of Health Guide for the Use and Care of Laboratory Animals and were approved by the University of Washington Institutional Animal Care and Use Committee. Gclm null mice were derived by homologous recombination techniques in mouse embryonic stem (ES) cells. A ß-galactosidase/neomycin phosphotransferase (ß-geo) fusion gene, a gift from Dr Phil Soriano, Fred Hutchinson Cancer Research Center (Soriano et al., 1991), was flanked with approximately 2 kb of the mouse Gclm gene promoter (left arm) and 1.5 kb of the first intron (right arm). This construct also contained a diphtheria toxin gene driven by a thymidine kinase promoter to select against random integrants. After selection of transfected 129SV strain ES cells (R1) with G418 (Invitrogen, Carlsbad, CA), surviving colonies were assessed for disruption of exon 1 of Gclm using PCR and subsequent restriction digest and Southern blot analysis. ES cells from correctly targeted clones were injected into C57BL/6 mouse blastocysts and transplanted into pseudopregnant mice according to standard techniques (Nagy, 2003). Chimeric male pups born from these mothers were mated to C57BL/6 females. Black agouti offspring were screened for the targeted allele. These heterozygotes were intercrossed to obtain Gclm null mice. Upon generation of the Gclm null mice, they were then crossed onto a C57BL/6 background for at least seven generations prior to APAP treatment experiments.
Genotyping.
Genotyping of pups was carried out by analyzing for the presence of both the native Gclm gene and ß-geo sequences in two separate PCR reactions. The reactions utilized the same Gclm forward primer 5'-GCC CGC TCG CCA TCT CTC-3' (1nM); the ß-geo sequence was detected with the reverse primer 5'-CAG TTT GAG GGG ACG ACG ACA-3' (1.25nM); and the native Gclm sequence was detected with the reverse primer 5'-GTT GAG CAG GTT CCC GGT CT-3' (0.5nM). Reactions (20 µl total volume) contained 0.4mM of each deoxy-nucleotidyl triphosphate (Roche Diagnostics, Indianapolis IN), 1 unit of Taq polymerase, and 1x reaction buffer (Qiagen, Inc., Valencia, CA), and 0.8M dimethylsulfoxide. The cycling conditions were as follows: 94°C for 2 min, followed by 35 cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 2 min, and a final extension at 72°C for 5 min. PCR amplicons were visualized by agarose gel electrophoresis.
Initial model characterization.
For biochemical characterization experiments, mice were euthanized by CO2 narcosis followed by cervical dislocation. Select tissues (kidney, liver, brain, and lung) were either homogenized in TES/SB buffer (20mM Tris, 1mM ethylenediaminetetraacetic acid [EDTA], 250mM sucrose, 20mM sodium borate, 2mM serine) with 1x complete protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN), or frozen in liquid nitrogen and stored at – 80°C. Biochemical analyses were conducted on fresh homogenate or on frozen tissues that were homogenized in TES/SB with protease inhibitors at 4°C.
Western blot for GCLC and GCLM.
GCLC and GCLM proteins were detected with the use of rabbit polyclonal antisera raised against ovalbumin conjugates of peptides specific to each subunit, using previously described procedures (Thompson et al., 1999).
Real-time PCR for messenger RNA expression.
Real-time PCR 5'-fluorogenic nuclease assays were performed to quantify Gclc and Gclm messenger RNA (mRNA) expression as described previously (Diaz et al., 2001). Briefly, 2 µg of total RNA was subjected to reverse transcription using random hexamers as primers. For PCR, sequence-specific fluorogenic probes for Gclc and Gclm were used, in conjunction with two specific PCR primers that amplified a 120-bp region surrounding each probe. Glyceraldehyde phosphate dehydrogenase mRNA levels were used as an internal control for expression normalization.
GCL activity assays.
GCL activity was determined using either high-performance liquid chromatography (HPLC) with fluorescence detection (White et al., 1999), or a fluorometric 96-well microtiter plate assay (White et al., 2003) as previously described. In brief, clarified supernatants of tissue homogenates prepared in TES/SB buffer with protease inhibitors were diluted 1:1 and preincubated for 5 min at 37°C in 50 µl of GCL reaction cocktail (40mM ATP, 20mM L-glutamic acid, 400mM Tris, 2.0mM EDTA, 20mM sodium borate, 2mM serine, and 40mM MgCl2). Reactions were initiated by the addition of 50 µl of 2mM cysteine and terminated at either 10 min (liver and kidney) or 30 min (brain and lung) by the addition of 50 µl of 200mM 5-sulfosalicylic acid. Clarified supernatants of the reaction mixture were derivatized with either 10mM monobromobimane (for HPLC assay) or 10mM 2,3-naphthalenedicarboxaldehyde (NDA; for microtiter plate assay) and the fluorescence intensity of the derivatized products was measured at 
ex375 and em475 (for HPLC assay), or at ex472 and em528 (microtiter plate assay). Baseline GSH was subtracted from newly synthesized -GC and GSH to determine net -GC production and therefore GCL activity.
Total GSH content determination.
Clarified tissue homogenates prepared in TES/SB were standardized to 8–10 mg/ml protein concentration, diluted 1:1 with 10% 5-sulfosalicylic acid, incubated on ice for 10 min, and then centrifuged at 18,200 x 9 rpm in a microcentrifuge for 2 min to obtain nonprotein supernatants. Twenty-five-microliter aliquots of the supernatants were added to a 96-well black microtiter plate in triplicate followed by addition of 100 µl of 0.2M N-ethylmorpholine/0.02M NaOH. Glutathione disulfide (GSSG) was reduced to GSH by the addition of 10 µl of 10mM tris(2-carboxyethyl)phosphine (Pierce, Rockford, IL) and incubation for 15 min at room temperature. Total GSH was derivatized, following the addition 50 µl of 0.5N NaOH, by the addition of 10 µl of 10mM NDA. The reaction was incubated at room temperature for 30 min with the fluorescence intensity of NDA–GSH measured at ex472 and em528, and quantified by interpolation on a standard curve constructed with NDA-conjugated GSH in TES/SB:10% sulfosalicylic acid (1:1).
APAP treatment.
Following a 12-h overnight fast, mice were ip injected with either saline (10 µl/g bw) (Baxter, Deerfield, IL); 500 mg/kg APAP immediately followed by 168 mg/kg GSHee; 500 mg/kg APAP immediately followed by 800 mg/kg NAC prepared in 0.9% saline, pH 7.0; GSHee alone; or NAC alone. Immediately following treatment, food was returned to the mice. The number of animals per treatment group is indicated in Table 1. Mice were euthanized with CO2 narcosis and cervical dislocation 6 h following treatment. Immediately thereafter, blood was collected via cardiac puncture for serum ALT activity measurement according to the manufacturer's protocol (Diagnostic Chemicals Limited (USA) Oxford, CT). Liver homogenates were prepared and total GSH content in the clarified supernatant was determined as described above. Portions of the medial lobe were fixed in 4% paraformaldehyde overnight prior to dehydration, paraffin embedding, and histological preparation of hematoxylin and eosin–stained sections according to standard protocols. These sections were evaluated and assessed for pathological alterations by three independent observers, including a board certified pathologist (R.H.P.), blinded with respect to genotype and treatment. Injury was scored on a scale of 0–5 (0 indicating no damage and 5 indicating severe hepatic alterations).
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Statistical analyses.
Data were analyzed by ANOVA followed by paired two-tailed Student's t-test for direct comparison of normally distributed data. ALT data were analyzed nonparametrically by the Kruskal–Wallis test followed by two-sample Wilcoxon rank-sum test for direct comparisons. Differences yielding a p value of less than 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The DNA construct used to disrupt the Gclm gene in the present study incorporated a promoterless ß-geo fusion protein gene flanked by the upstream Gclm promoter and Intron 1 of the native Gclm sequence (Fig. 1).
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and 
) indicate the locations of the PCR forward and reverse primers, respectively. The * symbol represents the location of the probe used for Southern analysis to confirm correctly targeted integration of the ß-geo insert.Gclm heterozygous mating generated 29% null, 48% heterozygous, and 24% wild-type pups in accordance with Mendelian inheritance, indicating no obvious embryo or fetal loss due to the Gclm null mutation. All pups developed normally and exhibited no visible phenotypic differences relative to wild-type littermates.
Initial Model Characterization
Gclm null mice express no immunodetectable GCLM protein, and heterozygous mice express approximately 50% of that present in wild-type controls by Western blot analysis (Fig. 2A). Expression of GCLC protein in the liver of Gclm null mice was 1.7-fold higher than that found in wild-type mice (Fig. 2B). There were no differences between male and female mice with respect to their relative GCLM or GCLC protein expression within genotypes.
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3) of protein expression relative to wild-type (WT) liver, with representative Western blots. Paired two-tailed t-tests, *p < 0.05, **p < 0.01, and ***p < 0.001, relative to wild-type littermates.Reverse transcription–PCR assessment confirmed Gclm and Gclc mRNA expression to be in agreement with the protein expression (Fig. 3). There was no detectable Gclm mRNA expression in Gclm null mice, and the heterozygous mice expressed approximately 50% that of the wild-type controls (p < 0.005, relative to wild-type). Gclc mRNA was upregulated in Gclm null mice relative to either heterozygous or wild-type.
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Total GSH content in Gclm null mice was markedly diminished in all tissues tested (Fig. 4A). GSH was approximately 23% and 89% of wild-type mice in Gclm null and heterozygous mice, respectively. Thus, GSH levels in Gclm null mice are severely compromised while being only slightly changed in Gclm heterozygous mice. Some differences were noted in GSH content between males and females within genotypes and tissues. Relative to male mice, females showed diminished GSH levels in liver (heterozygous only, with a suggestion in wild-type) and in kidney and brain (all genotypes) (p < 0.05).
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Gclm null and heterozygous mice have markedly diminished GCL activity when compared to wild-type mice (Fig. 4B). Collectively, for all tissues and for both genders combined, Gclm null mice exhibited approximately 30%, and heterozygous mice 75% of the GCL activity found in wild-type mice, under our assay conditions. In contrast to total GSH, regardless of genotype, female mice exhibited significantly higher hepatic GCL activity than their male counterparts. Female heterozygous and wild-type mice had higher renal GCL activity than male mice of the same genotype.
APAP Treatment Experiments—Total Liver GSH
Gclm null, heterozygous, and wild-type mice were administered (ip) 500 mg/kg APAP following 12 h of food withdrawal (8 P.M.–8 A.M.). Mitigation of APAP-induced hepatotoxicity was assessed by administration (ip) of NAC (800 mg/kg) or GSHee (168 mg/kg) immediately prior to APAP dosing. These compounds were also administered in the absence of APAP.
APAP treatment of both male and female mice induced depletion of total GSH at 6-h posttreatment, relative to saline-treated controls (Figs. 5A and 5C, p < 0.01 for each genotype). In male wild-type and heterozygous mice, coadministration of either NAC or GSHee mitigated APAP-induced liver GSH depletion (Fig. 5C, p < 0.05). In contrast, APAP-induced total GSH depletion was unaffected by either NAC or GSHee treatment in female wild-type mice, although mitigation of GSH depletion by NAC was observed in female heterozygous mice (Fig. 5A, p < 0.05).
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In male and female Gclm null mice, APAP treatment induced further depletion of total GSH relative to saline-treated controls (Figs. 5A and 5C, p < 0.001). Furthermore, cotreatment with NAC afforded no protection against APAP-induced total GSH depletion in Gclm null mice of either gender (Figs. 5A and 5C, p < 0.001). GSHee partially mitigated depletion of total GSH in male Gclm null mice (Fig. 5C, p < 0.005), however, this effect was not observed in female null mice.
Regardless of genotype, however, female mice were relatively resistant to APAP-induced GSH depletion (Figs. 5A and 5C). Even though male mice exhibited slightly higher control levels of total GSH in all but null mice, APAP treatment induced greater GSH depletion in male than female mice, an observation which also held for null mice (p < 0.05). Interestingly, the observed gender differences became less pronounced when NAC or GSHee was coadministered with APAP. Lastly, the administration of either NAC or GSHee alone did not result in any significant change in total GSH levels at 6-h posttreatment (data not shown).
APAP Treatment Experiments—Liver Histopathology
APAP-induced histopathological alterations including (in order of increasing severity), glycogen depletion, microvesicular steatosis, and mitochondrial swelling, hemorrhage, endoplasmic reticulum vacuolization, and necrosis, progressed in severity from zone III to zone I in all APAP-treated mice. Injury scores indicated that medial lobe tissue from APAP-treated wild-type mice was moderately damaged in both female and male mice (Figs. 5B and 5D, p < 0.001). It should be noted that while APAP-treated female wild-type mice exhibited injury, the severity of the damage observed was less than that in male mice (p = 0.001), which is consistent with both ALT activity (Fig. 6) and total GSH (Figs. 5A and 5C). Cotreatment with NAC or GSHee afforded partial protection against liver injury in male wild-type mice (Fig. 5D, p < 0.001), with neither NAC nor GSHee significantly mitigating the alterations in female wild-type mice (Fig. 5B).
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APAP treatment of Gclm heterozygous mice resulted in increased histopathological alterations in both female and male mice, relative to saline-treated controls (Figs. 5B and 5D, p < 0.005 and p < 0.001, respectively). NAC and GSHee cotreatments mitigated damage in both genders. However, similar to the findings in wild-type mice, these cotreatments did not afford complete protection as a modest degree of alteration was still present. While there was a suggestion of increased damage in male heterozygous mice as compared to female heterozygotes, this difference was not statistically significant (p = 0.15).
APAP treatment of Gclm null mice resulted in extensive damage to the medial lobe in both genders (Figs. 5B and 5D, p < 0.001). Interestingly, neither NAC nor GSHee cotreatments afforded protection against APAP-induced liver damage in null mice. Similar to the heterozygous group, there were no statistically significant differences observed between female and male Gclm null mice.
APAP Treatment Experiments—Serum ALT Activity
In agreement with histopathology, serum ALT activity, an indicator of hepatocellular necrosis, exhibited both gender- and genotype-specific elevations after APAP treatment. Specifically, APAP treatment in female wild-type mice did not induce elevation of serum ALT activity (Fig. 6A). Male wild-type mice, on the other hand, exhibited a significant ALT elevation with APAP treatment relative to both saline-treated male mice (Fig. 6B, p < 0.005) and to APAP-treated female mice (p < 0.001). APAP-induced hepatocellular necrosis in male wild-type mice was mitigated by NAC or GSHee cotreatment with APAP (Figs. 6A and 6B, p < 0.05).
Similar serum ALT results were observed in both female and male Gclm heterozygous mice, with elevated ALT following APAP treatment (Fig. 6, p < 0.01) and mitigation with NAC and GSHee cotreatment (Fig. 6, p < 0.05).
In Gclm null mice, ALT levels were greatly elevated with APAP treatment in both male and female mice (Fig. 6, p < 0.001). Consistent with histopathology, neither NAC nor GSHee treatment afforded hepatoprotection against APAP in Gclm null mice (Fig. 6).
APAP Treatment Experiments—Survival
Initial dose finding studies demonstrated that APAP at 500 mg/kg was not lethal to any gender or Gclm genotype combination up to 6-h postexposure. However, coadministration of 800 mg/kg NAC with APAP resulted in the death of three of five male Gclm null mice at approximately 4-h posttreatment. A fourth mouse in this group died just prior to the 6-h time point following handling. Monitoring of these mice enabled the collection of biological fluids and tissues soon after death, with their analysis included in the above results. Importantly, no mouse receiving NAC alone demonstrated toxicity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The aim of this study was to investigate the implications of genetic impairment of GCL activity and depleted GSH on susceptibility to and rescue from APAP-induced liver injury in a mouse model. Following preliminary observations, we expanded our focus to include the potential modification of this effect by gender.
Gclm null mice have previously been reported by Yang et al. (2002). Unlike Gclc null mice, Gclm null mice exhibit normal viability. While not reported in our study, the Gclm null/ß-geo mice developed in our lab and utilized for this work permit the assessment of endogenous Gclm promoter activity in various tissues and cell types, and under conditions in which Gclm gene expression is modulated by oxidative stress.
Under basal conditions, our Gclm null mice exhibited approximately 20% of normal total GSH and 30% of normal GCL activity in most tissues. Gclm null mice are, therefore, a model of chronic GSH depletion and compromised GCL activity as has been concluded by Yang et al. (2002). Similar to their report, we found that the loss of the Gclm gene did not overtly affect growth and development. The compromised GCL activity and severely decreased total GSH levels are likely attributable to the inefficiency of the GCLC subunit alone in catalyzing -GC formation under physiological conditions (Chen et al., 2005).
Although Gclm heterozygous mice have total GSH levels approximately equivalent to wild-type littermates, tissue GCL activity was decreased as compared to wild-type mice under our assay conditions. This observation suggests that Gclm heterozygous mice may be a good model for modestly impaired GCL activity, but normal basal GSH levels.
Indeed, the observed APAP-induced liver damage supports prior observations of the importance of GCL activity and GSH in protecting against APAP-induced liver injury (Miners et al., 1984; Mitchell et al., 1973; Puri and Meister, 1983). In female mice in particular, the modest decrease in GCL activity in Gclm heterozygous mice rendered C57BL/6 female mice susceptible to APAP-induced liver damage, with Gclm null mice displaying even greater sensitivity. The effect of impaired GCL activity in male mice was not as apparent; this is likely due to the inherent sensitivity of male C57BL/6 mice to APAP, which apparently minimizes the contribution of GCLM expression to APAP susceptibility.
The inability of NAC to mitigate liver damage in Gclm null mice is in agreement with prior studies of either BSO-inhibited GCL activity or N-acetyl-D-cysteine (Miners et al., 1984; Puri and Meister, 1983; Wong and Corcoran, 1987). However, the lethality associated with APAP and NAC coadministration in male Gclm null mice in the current study was unexpected. This result is not without precedent, as Corcoran and Wong (1986) reported 24-h survival of 14 of 20 male mice treated with 500 mg/kg APAP + 1200 mg/kg N-acetyl-D-cysteine and of only 7 of 12 male mice treated with APAP + 900 mg/kg D-cysteine. Nonetheless, we believe this to be the first observation of NAC enhancing APAP toxicity, and have yet to understand the mechanism. A recent study of NAC metabolism in male BALB/c mice by Zwingmann and Bilodeau (2006) demonstrates the limited capacity of NAC to elevate GSH levels above a dose of 300 mg/kg. Higher doses instead result in a significant elevation in hypotaurine and alteration of mitochondrial metabolic pathways. Once again, the lethality associated with APAP + NAC was only observed in male Gclm null mice, thus accumulation of NAC, its metabolites, or altered mitochondrial energetics due to the impaired incorporation into GSH may have all played a role in this effect. Interestingly, this observation illustrates another gender specific differential response.
As with NAC, GSHee successfully mitigated APAP-induced liver damage in Gclm wild-type and heterozygous mice of both genders, but failed to do so in Gclm null mice. It has been demonstrated that GSHee is able to elevate hepatic GSH levels in the absence of GCL activity (Puri and Meister, 1983). However, the amount of GSHee necessary to elevate GSH in Gclm null mice to within normal range was not determined prior to conducting these experiments. As such, our current conclusion of this observation is that there was insufficient supplementation of hepatic GSH at a dose of 168 mg/kg GSHee to afford protection against 500 mg/kg APAP in Gclm null mice.
The gender differences observed in this study are interesting and subject to further investigation. In agreement with previous studies (Botta et al., 2006; Dai et al., 2006), we observed that female C57BL/6 mice are relatively resistant to APAP-induced hepatotoxicity. There are a number of factors that may simultaneously influence the relative resistance of these female mice to APAP-induced toxicity. The most likely include steroid hormone regulation of genes that protect against APAP-induced liver damage, which include Gclc and therefore may include Gclm (Montano et al., 2004). In this and previous work (Botta et al., 2006), we found that female mice demonstrate slightly higher liver GCL activity than male mice. Additionally, in the prior study no correlation was observed between GCL activity and liver injury (as quantified by serum ALT activity) in female mice (r2 = 0.03), whereas in male mice there was a strong inverse correlation (r2 = 0.87). These data suggested that enhancing GCL activity protected male mice against APAP-mediated injury, whereas basal female GCL activity or inducibility was sufficient for protection. In this study, there was less liver damage and GSH depletion induced by APAP in female wild-type mice than in male wild-type mice, which supports this hypothesis. Importantly, however, decreased GCLM expression predisposed both genders to APAP-induced injury. Thus, in these mice, the mechanisms of resistance of female mice to APAP were rendered insignificant with a modest decrease in GCL activity, suggesting involvement of GCL and GSH production in the resistance of female wild-type mice to APAP.
With the increasing incidence of APAP overdose in the United States (Larson et al., 2005), as well as recent observations of APAP-induced hepatotoxicity in humans at the current approved Food and Drug Administration maximal dosing of 4 g/day (Watkins et al., 2006), the investigation of genetic factors that predispose individuals to APAP-induced hepatotoxicity is necessary and timely. This work has further demonstrated a significant role for GCL activity in susceptibility to APAP-induced liver damage in mice. It also suggests that variation in GCL activity may be an important determinant of APAP-induced liver damage, and NAC-mediated protection from such damage in humans.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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National Institutes of Health (R01ES10849, P42ES004696, T32AG000057, T32ES007032, and P30ES07033).
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NOTES |
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1 L.A.M. and I.M. contributed equally in the preparation of this manuscript.
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ACKNOWLEDGMENTS |
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We thank Ms Dianne Botta, Ms Portia Vliet-Gregg, and Ms Monica McGrath for excellent technical assistance.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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