Glutathione-Deficient Mice Are Susceptible to TCDD-Induced

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Glutathione-Deficient Mice Are Susceptible to TCDD-Induced Hepatocellular Toxicity but Resistant to Steatosis Ying Chen,† Mansi Krishan,‡ Daniel W. Nebert,‡ and Howard G. Shertzer*,‡ †

Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, Aurora, Colorado 80045, United States ‡ Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati, Ohio 45267, United States S Supporting Information *

ABSTRACT: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) generates both hepatocellular injury and steatosis, processes that involve oxidative stress. Herein, we evaluated the role of the antioxidant glutathione (GSH) in TCDD-induced hepatotoxicity. Glutamate-cysteine ligase (GCL), comprising catalytic (GCLC) and modifier (GCLM) subunits, is rate limiting in de novo GSH biosynthesis; GCLM maintains GSH homeostasis by optimizing the catalytic efficiency of GCL holoenzyme. Gclm(−/−) transgenic mice exhibit 10−20% of normal tissue GSH levels. Gclm(−/−) and Gclm(+/+) wild-type (WT) female mice received TCDD for 3 consecutive days and were then examined 21 days later. As compared with WT littermates, Gclm(−/−) mice were more sensitive to TCDD-induced hepatocellular toxicity, exhibiting lower reduction potentials for GSH, lower ATP levels, and elevated levels of plasma glutamic oxaloacetic transaminase (GOT) and γ-glutamyl transferase (GGT). However, the histopathology showed that TCDD-mediated steatosis, which occurs in WT mice, was absent in Gclm(−/−) mice. This finding was consistent with cDNA microarray expression analysis, revealing striking deficiencies in lipid biosynthesis pathways in Gclm(−/−) mice; qrt-PCR analysis confirmed that Gclm(−/−) mice are deficient in expression of several lipid metabolism genes including Srebp2, Elovl6, Fasn, Scd1/2, Ppargc1a, and Ppara. We suggest that whereas GSH protects against TCDD-mediated hepatocellular damage, GSH deficiency confers resistance to TCDD-induced steatosis due to impaired lipid metabolism.

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INTRODUCTION Oxidative stress occurs when oxygen free radicals are produced at levels exceeding those capable of being sequestered by normal cellular antioxidant defenses. Glutathione (GSH) is the most abundant cellular nonprotein thiol, attaining a concentration in the high millimolar range in the liver.1 GSH acts as an antioxidant directly by scavenging free radicals or indirectly by serving as a cofactor for antioxidant enzymes such as glutathione peroxidases and glutathione S-transferases.2 Because of its abundance, GSH plays a key role in cellular defense mechanisms against toxicity and oxidative stress resulting from exposures to xenobiotics, pharmaceuticals, or dietary formulations.3 GSH is a tripeptide composed of glutamate, cysteine, and glycine. It is synthesized in most types of cells by two successive enzymatic reactions.4 Glutamate-cysteine ligase (GCL) catalyzes the first and rate-limiting reaction, which couples L-glutamate and L-cysteine to form γ-glutamylcysteine (γ-GC). Glutathione synthetase then couples γ-GC with L-glycine to form GSH. In higher eukaryotes, GCL is a heterodimer composed of a 72.8 kDa catalytic (GCLC) and a 30.8 kDa modifier (GCLM) subunit. While GCLC possesses all of the catalytic activity, GCLM optimizes the kinetic efficiency of the holoenzyme, thereby regulating tissue GSH levels.4 © 2011 American Chemical Society

The essential role of GSH in hepatic functioning is evidenced in a lethal phenotype of hepatocyte-specific Gclc knockout mice,5 which develop severe steatosis and die of liver failure within 1 month postpartum due to ∼95% depletion of hepatic GSH. Mitochondria are the major affected organelles, displaying abnormal ultrastructure and impaired functioning. The Gclm(−/−) knockout mice,6 on the other hand, have 10− 30% of normal GSH levels in liver, lung, pancreas, erythrocytes, and plasma. When unchallenged, these animals exhibit no overt phenotype, making them a useful model of chronic GSH deficiency. Cells derived from Gclm(−/−) mice are highly sensitive to cell death induced by H2O2,6 arsenite,7 and neurotoxicants.8 Gclm(−/−) mouse fetal fibroblasts senesce prematurely in culture.9 The Gclm(−/−) mice, however, are surprisingly resistant to ozone-induced lung injury,10 likely due to augmented antioxidant response to ozone exposure in these animals. An independently developed Gclm null mouse line is highly sensitive to acetaminophen-induced liver injury;11 in contrast, these mice fed a methionine/choline deficient diet show attenuated progression of steatohepatitis when compared to methionine/choline deficient-fed wild-type mice.12 Results from these studies suggest that GSH may either exacerbate or Received: June 10, 2011 Published: November 14, 2011 94

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CYP1A2,21 and ATP levels22 were quantified as described. Protein was measured by the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL), according to the manufacturer's brochure. RNA Isolation and Microarray Analysis. Liver was removed, quickly rinsed in ice-cold RNase-free PBS, flash-frozen in liquid nitrogen, and stored at −80 °C. Frozen tissue (∼50 mg) was homogenized in Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH), and RNA was isolated from the homogenate per the manufacturer's protocol (catalog no. TR 118). RNA samples (0.1 μg/μL) were submitted to the University of Cincinnati Microarray Core for Agilent Bioanalyzer/Nanodrop analysis (Agilent 2100 Bioanalyzer). Total RNA quantity and quality were assessed by optical density at 260 nm and optical density ratios of 260/280 and 260/230 nm ratios, respectively. OD 260/280 ratios ≥2.0 were used for microarrays and qrt-PCR. RNA samples were amplified, and competitive hybridization to microarrays of labeled cDNA targets, generated from four separate samples per group with two dye flips per group, was performed by the Microarray Core exactly as described.23 Microarray data were analyzed online (http://david.abcc.ncifcrf.gov/) using the NIH National Institute for Allergy and Infectious Diseases program DAVID v6.7 (Database for Annotation, Visualization and Integrated Discovery).24,25 cDNA Synthesis and qrt-PCR. cDNA synthesis was performed using an RNA-to-cDNA kit (Applied Biosystems) according to the manufacturer's instructions. Synthesis of cDNA was carried out using 0.5 μg of total RNA, and optical density was used to determine quantity and quality (as described above); qrt-PCR was conducted with a Stratagene MX 3000P instrument using a SYBR Green 2X qrtPCR master mix (Applied Biosystems) with a total reaction volume of 25 μL. Thermocycling parameters were as follows: 95 °C for 3 min followed by 40 cycles of 95 °C for 30 s, 55 °C for 60 s, 72 °C for 60 s, followed by data collection at the end of each cycle. Primer sequences (Table 1) were confirmed to be specific for the target gene by using a basic local alignment search tool (PRIMER-BLAST; http://blast.ncbi. nlm.nih.gov/Blast.cgi). All reactions were performed in duplicate on each plate with a minimum of three independent replicates, and gene expression values were calculated using the difference in target gene expression relative to 18S mRNA by means of the 2−ΔΔCT method.26 Statistics. Statistical significance of the differences between group sample mean values was determined by two-way ANOVA (treatment and genotype), followed by the Student−Newman−Keuls test for pairwise comparisons of means. There were no significant interactions between treatment (vehicle or TCDD) and genotype [WT or Gclm(−/−)] for any of the data sets. Statistics were performed using SigmaStat Statistical Analysis software (SPSS Inc., Chicago, IL). Hazardous Materials. TCDD is highly toxic and a possible human carcinogen. All personnel were instructed as to safe handling procedures. Lab coats, gloves, and masks were worn at all times, and contaminated materials were collected separately for disposal by the Environmental Health & Safety Office of the University of Cincinnati. TCDD-treated mice were housed separately, and their carcasses were treated as contaminated biological materials.

mitigate toxicity, depending on the nature of the toxicant and the biological exposure target. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is hepatotoxic, generating nonalcoholic fatty liver disease (NAFLD) manifested by both hepatocellular injury and steatosis, and involving oxidative stress.13−15 The major objective of this study, therefore, was to determine whether this specific antioxidant, GSH, is important in protecting liver tissue from TCDDmediated toxicity using the Gclm(−/−) knockout mouse.

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EXPERIMENTAL PROCEDURES

Chemicals. TCDD was purchased from Accustandard (New Haven, CT). All other chemicals and reagents were obtained from SigmaAldrich Chemical Co. (St. Louis, MO) as the highest available grades. Animals and Treatment. Experiments involving mice were conducted in accordance with the National Institutes of Health standards for care and use of experimental animals as stated in Principles of Laboratory Animal Care (NIH Publication No. 85-23, revised 1985; http://grants1.nih.gov/grants/olaw/references/phspol.htm) and the University of Cincinnati Institutional Animal Care and Use Committee. Animals were group-housed, maintained on a 12 h light/ dark cycle, and had access to standard rodent chow and water ad libitum. C57BL/6J mice (10 weeks of age) were purchased from The Jackson Laboratory (Bar Harbor, ME). Gclm(−/−) mice were generated as described,6 and the Gclm(−) allele has been backcrossed into the C57BL/6J background for more than 10 generations. Body weights and food and water consumption were measured twice weekly. Female WT and Gclm(−/−) mice (12 weeks of age) were matched for body weights and then treated intraperitoneally with TCDD (15 μg/kg body weight) in corn oil for 3 consecutive days; vehicle controls were given equivalent volumes of corn oil. At 21 days following treatment, the mice were killed by CO2 asphyxiation. Immediately, blood was collected in heparinized microvettes. The liver was quickly weighed, and aliquots of 0.1 g of liver from each animal were frozen in liquid nitrogen for tissue ATP determinations and gene expression analyses. An additional aliquot was taken for gravimetric lipid analysis. For measuring GSH and glutathione disulfide (GSSG) levels, 0.1 g of liver was placed in 5% ultrapure-grade trichloroacetic acid in an icecold redox-quenching buffer, consisting of 20 mM HCl, 5 mM diethylenetriamine pentaacetic acid, and 1 mM 1,10-phenanthroline. The remaining liver was excised and placed in 10% buffered formalin for histology. GSH, GSSG, and Redox Potential. GSH and GSSG were determined spectrofluorometrically using the o-phthalaldehyde procedure.16 The reduction potential (ΔE) for the GSSG/2GSH half-reaction, GSSG + 2H+ → 2GSH, was calculated as: ΔE = {ΔE0 + (RT/nF) × (actual pH − standard pH)} − {RT/nF ln ([GSH]2/[GSSG])}, where n = number of electrons transferred, R = the universal gas constant (8.31 J K−1 mol−1), T = degrees Kelvin, and F = the Faraday constant (9.65 Coulombs × 104 mol−1). At 37 °C, and assuming a cytosolic pH of 7.2, ΔE = {−240 mV + (−61.5 mV/2e−) × (0.2)} − {(61.5 mV/ 2e−) × log ([GSH]2/[GSSG])]}.3 Quantification of Hepatic Lipid. The number and size of lipid droplets were evaluated histologically. A 100 mg piece of liver was placed in 10% formalin, dehydrated through graded alcohols, embedded in paraffin, sectioned (5 μm), mounted on slides, and stained with hematoxylin and eosin. Slides and a stage micrometer were photographed at 100× magnification and saved as TIFF images. The size and number of lipid droplets per unit area were calculated from 10 randomly selected fields per mouse, utilizing Image J version 1.44 software (http://rsb.info.nih.gov/ij/). Nonpolar lipids were also estimated by gravimetric analysis. For this procedure, total nonpolar lipids were extracted (2 h, dark, 4 °C) from a portion of the liver with CHCl3 :MeOH (2:1, v/v), dried, and weighed, as described previously.17 Other Assays. Activities of hepatic γ-glutamyl transferase (GGT),18 glutamic oxaloacetic transaminase (GOT),19 CYP1A1,20

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RESULTS TCDD-Mediated Hepatotoxicity in WT and Gclm(−/−) Mice. Both WT and Gclm(−/−) mice displayed TCDDmediated increases in plasma GOT (Figure 1A) and GGT (Figure 1B) levels. However, as compared with WT litter mates, Gclm(−/−) mice were more sensitive to TCDDinduced hepatocellular toxicity, exhibiting significantly higher levels of plasma GOT and GGT. Although hepatic ATP levels were not lower in knockout mice, TCDD treatment produced about twice as much depletion of liver ATP in Gclm(−/−) mice (79% decrease) than WT mice (41% decrease) (Figure 1C). As compared with WT, Gclm(−/−) mice exhibited a thiol oxidative stress response, with a large decrease in cytosolic GSH (Figure 1D) and a small but significant decrease in cytosolic GSSG (Figure 1E). These changes resulted in a 24% 95

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Table 1. Gene Expression Data in Gclm(−/−) Micea % expression in Gclm(−/−), relative to WT = 100% gene name

gene symbol (Amplicon, bp)

fatty acid synthase

Fasn (122)

elongation of long chain fatty acids-6

Elovl (76)

steroyl CoA desaturase

Scd1/2 (59)

sterol regulatory element binding protein-1c sterol regulatory element binding protein-2 peroxisome proliferator-activated receptor γ, coactivator 1-α peroxisome proliferator activator receptor-α cytochrome P450 1a1

Srebf1 (72)

cytochrome P450 1a2

Srebf 2 (80) Ppargc1a (134) Ppara (99) Cyp1a1 (156) Cyp1a2 (201)

forward primer (5′-) reverse primer (5′-) F 5′-CCTGGATAGCATTCCGAACCT R 5′-AGCACATCTCGAAGGCTACACA F 5′-GCGCTGTACGCTGCCTTTAT R 5′-GCGGCTTCCGAAGTTCAAA F 5′-CAACACCATGGCGTTCCA R 5′-GGTGGGCGCGGTGAT F 5′-CCAGAGGGTGAGCCTGACAA R 5′-AGCCTCTGCAATTTCCAGATCT F 5′-GCGGACAACACACAATATCATTG R 5′-TGACTAAGTCCTTCAACTCTATGATTTTG F 5′-TATGGAGTGACATAGAGTGTGCT R 5′-CCACTTCAATCCACCCAGAAAG F 5′-TGGTTCCTGGTGCCGATTTA R 5′-ACTAGCATCCCACTTAATTATGTATCTGAA F 5′-CCTCATGTACCTGGTAACCA R 5′-AAGGATGAATGCCGGAAGGT F 5′-AAGACAATGGCGGTCTCATC R 5′-GACGGTCAGAAAGCCGTGGT

microarray (average intensity)

qrt-PCR mean ± SE (n = 4)

31.6 (1629)

2.6 ± 0.7

21.1 (570)

5.5 ± 1.0

32.4 (11431/250)

16.9 ± 1.4

39.2 (299)

14.3 ± 4.9

38.8 (298)

8.5 ± 1.4

208 (88)

29.0 ± 21.7

39.2 (327)

16.4 ± 3.5

127 (27.9)

83 ± 14

116 (299)

102 ± 11

a

Genes that were used in this study to evaluate lipid metabolism and AHR activation. The right hand pair of columns shows the correlation of gene expression data obtained by cDNA microarray and qrt-PCR. The data are shown as the relative gene expression in Gclm(−/−) mice with respect to WT mice, expressed as a percentage.

Morphometric analysis of liver histology supported the gravimetric data, in that TCDD-mediated steatosis occurred in WT mice but not in Gclm(−/−) mice (Figure 2F). The steatosis in WT mice treated with TCDD was the result of a 40% increase in lipid vacuole size (Figure 2C) and a 4.6-fold increase in the number of lipid droplets (Figure 2D), which resulted in an 8.4-fold increase in hepatic lipid (Figure 2E). TCDD did not cause increases in liver weight or histologyevaluated lipid accumulation in Gclm(−/−) mice. Gclm(−/−) Mice Are Deficient in Lipid Metabolism. To determine why TCDD did not produce lipid accumulation in Gclm(−/−) liver, we evaluated gene expression via microarray and qrt-PCR analyses. Microarray gene expression data showed that, in Gclm(−/−) mice, the major pathways affected (relative to WT mouse liver) were associated with immune response, inflammation, and lipid metabolism. Pathways related to the immune response and inflammation (GO:6955, GO:2526, GO:6952, GO:9611, GO:2684, GO:50778, and GO:2252) were at low levels (data not shown). More pertinent to the current study was the strikingly low levels of expression of genes involved in a broad range of pathways involving lipid biosynthesis (Figure 3). Of the many specific genes that appear from the microarray to be poorly expressed in Gclm(−/−) liver, we confirmed by qrt-PCR several key regulatory lipid synthesis genes (Table 1). In every case, the degree of decreased gene expression is underestimated in the microarray, with respect to qrt-PCR. For Ppargc1a, the microarray was unable to correctly predict the diminished level of gene expression, due to the very low levels of fluorescence intensity (expression) for this gene in WT untreated mice. Analysis by qrt-PCR confirmed that Gclm(−/−) mice are dramatically deficient in the expression of several genes controlling the rates of lipid biosynthesis, including fatty acid synthase (Fasn), elongation of long chain fatty acids-6 (Elovl6), steroyl-CoA desaturases-1 and −2 (Scd1/2), and sterol regulatory element-binding protein-1 and -2 (Srebf1 and Srebf 2). The expression of the important lipid regulatory genes, peroxisome proliferator-activated receptor α (Ppara) and

Figure 1. TCDD-mediated hepatotoxicity. Wild-type (wt) and Gclm(−/−) knockout (ko) mice were treated with TCDD (T) or corn oil vehicle (V). Plasma was assayed for GOT (A) and GGT (B). Liver tissue samples were assayed for ATP (C), and GSH (D), and GSSG (E), from which the GSSG/2GSH half-cell redox potentials (F) were calculated. Data are shown as mean values ± SEs (n = 4). (a) Different from vehicle-treated mice of the same genotype (p < 0.05). (b) Different from WT mouse with similar treatment (p < 0.05).

decrease in the magnitude of the GSSG/2GSH reduction potential in the Gclm(−/−) mouse (Figure 1F). Moreover, TCDD treatment produced an increase in GSH content of WT, but not Gclm(−/−) mice, and a greater increase in cytosolic GSSG in Gclm(−/−) than in WT mice. TCDD thus produced an additional 23% decrease in the magnitude of the GSSG/ 2GSH reduction potential in Gclm(−/−) mice, as compared to only 10% in WT mice. TCDD-Mediated Hepatic Lipid Accumulation in WT and Gclm(−/−) Mice. Liver size and lipid content were greatly affected by TCDD. In WT mice, TCDD caused a 36% increase in liver weight (Figure 2A), attributable in part to lipid accumulation. Whereas TCDD produced a 6-fold increase in nonpolar lipid content (primarily triglycerides) in WT mice, only a doubling was observed in Gclm(−/−) mice (Figure 2B). 96

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Gclm(−/−) mice treated with TCDD could not be explained by induction of Cyp1 genes via the aromatic hydrocarbon receptor. The TCDD transcriptional activation of Cyp1a1 (Figure 4A) and Cyp1a2 (Figure 4B) and the inducibility of

Figure 4. TCDD induction of Cyp1 genes. WT (wt) and Gclm(−/−) knockout (ko) mice were treated with TCDD (T) or corn oil vehicle (V). qrt-PCR gene expression relative to 18S mRNA for Cyp1a1 (A) and Cyp1a2 (B) and enzyme activities for CYP1A1 (C) and CYP1A2 (D) are shown. Data are shown as mean values ± SEs (n = 4). (a) Different from vehicle-treated mice of the same genotype (p < 0.05).

Figure 2. TCDD-mediated steatosis. WT (wt) and Gclm(−/−) knockout (ko) mice were treated with TCDD (T) or corn oil vehicle (V). Liver weights (A) and lipid contents (B−E) are shown. Gravimetric determination of nonpolar lipids is shown in panel B. For histological analysis, lipid droplet size (C) and lipid droplet number (D) were used to calculate the percentage of tissue area containing lipid droplets (E). Representative images of HE-stained liver sections from which the lipid data (C−E) were calculated are shown in panel F. Data (A−E) are shown as mean values ± SEs (n = 4). (a) Different from vehicle-treated mice of the same genotype (p < 0.05). (b) Different from WT mouse with similar treatment (p < 0.05).

enzyme activities of CYP1A1 (Figure 4C) and CYP1A2 (Figure 4D) were the same in WT and Gclm(−/−) mice.

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DISCUSSION

Diseases associated with exposure to TCDD include NAFLD, cancer, cardiovascular disease, birth defects, and type 2 diabetes.27 The mechanisms associated with pathogenesis are both transcriptional28 and cell signaling29 in nature. As a ligand, TCDD activates the heterodimeric transcription factor, aromatic hydrocarbon receptor (AHR), leading to downstream pathways that result in oxidative stress.30 Antioxidants have often been used to show that oxidative stress is intimately involved in TCDD-mediated pathogenesis. We recently showed that the potent antioxidant, 4b,5,9b,10-tetrahydroindeno[1,2b]indole, prevents TCDD-mediated decrease in hepatic ATP levels in mice.31 Similarly, the phenolic antioxidant resveratrol, found at high levels in red grapes and peanuts, prevents TCDD-induced hepatic steatosis, oxidative stress, and loss of body weight in C57BL/6J mice.32 It is likely that multiple etiologies are responsible for the chemoprotective effects of various antioxidants, including the cysteinyl thiol GSH, which could alter many different transcriptional and signaling pathways. Hepatic GSH is crucial in maintaining intracellular redox homeostasis and the reduced state of protein thiol moieties. GSH also functions to detoxify xenobiotics and their metabolites and protects against oxidative damage caused by reactive oxygen species. In the current study, an important role of GSH in cellular protection against TCDD toxicity is supported by the observation that mice deficient in GSH are

Figure 3. Hepatic lipid metabolism gene ontologies down-regulated in Gclm(−/−) mice. Gene ontologies were derived from microarray results using the online program DAVID (Database for Annotation, Visualization and Integrated Discovery).

peroxisome proliferator-activated receptor γ coactivator-1α (Ppargc1a), were also acutely decreased in Gclm(−/−) liver. Gclm(−/−) Mice Have Unaltered Espression of Cyp1a1 and Cyp1a2. The TCDD response phenotype described for 97

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Gclm(−/−) mice from that study versus the present paper were derived independently, but reasons for the differences in gene expression profile are not known. There is some indication that NRF2 (NFE2L2; nuclear factor erythroid 2-related factor) may be involved in TCDD-mediated fatty liver, but it is not clear if this is a positive or negative effect. In mice, hepatic triglyceride accumulation is enhanced by schisandrin B, a dibenzocyclooctene component from the fruit of the Chinese herb, Schisandra chinensis.44 Schisandrin B activates mitogen-activated protein kinase (MAPK) signaling through NRF2, thereby transcriptionally activating antioxidant genes such as Gclm.45 In contrast, Nrf 2 knockout mice are acutely sensitive to TCDD steatosis, associated with increased hepatic lipid synthesis and decreased hepatic lipid efflux, resulting from lower levels of such proteins as apolipoprotein B (ApoB).46 Thus, activation of the NRF2-signaling pathway could mitigate TCDD-mediated steatosis by decreasing hepatic lipid synthesis and increasing hepatic lipid efflux. While NRF2 signaling is likely to be involved in the resistance to TCDD-induced steatosis in the Gclm(−/−) mouse, it seems not to involve transcriptional regulation, since the levels of Nfe2l2 and Keap1 transcripts are not different in Gclm(−/−) mouse liver (Supporting Information; filename: Chen cDNA micrarray.pdf). A recent paper reported an interesting association between the steatosis and the TCDD-AHR-mediated transcriptional activation of CD36/fatty acyl translocase.47 TCDD-mediated steatosis depends in part on the transcriptional activation of CD36 and fatty acid transport proteins. We know from the present and prior15 studies that TCDD also produces an increase in hepatic cytosolic and mitochondrial GSH and GSSG concentrations. It would be attractive to hypothesize that upregulation of CD36 by TCDD is mediated by GSH; it would follow that in the Gclm(−/−) mouse, such up-regulation is circumvented. However, microarray results do not support the hypothesis, since neither hepatic gene expression of CD36 nor Ahr is changed in the Gclm(−/−) mouse (Supporting Information; filename: Chen cDNA micrarray.pdf). Furthermore, induction of CYP1A1 and CYP1A2 through the AHR is unchanged in Gclm(−/−) mice. In part, the role of GSH in suceptibility to hepatic steatosis remains unclear because of the complex relationship between GSH concentration and lipid metabolism and deposition. We speculate that the dose−response issue can be explained in part by proposing that 95−99% depletion of GSH levels [Gclc(−/−) mouse] promotes steatosis, 70−90% depletion [Gclm(−/−) mouse] prevents steatosis, and normal or high GSH levels neither promotes nor prevents steatosis, the development of which depends on the nature of the study. However, interpreting the complex literature will require an understanding of the mechanisms by which GSH regulates steatosis. Such mechanisms include not only transcriptional regulation of gene expression but, most importantly, cell signaling pathways initiated by phosphorylation or formation of Michael reaction products between cysteine and electrophiles.

more susceptible to hepatocellular damage following TCDD exposure. A less expected finding from our study is the association between GSH depletion (10−25% of normal) and the strikingly low levels of expression of genes encoding lipogenic enzymes (FASN, ELOVL6, and SCD1/2) and their key regulatory proteins (PPARα, PPARGC1α, SREBF1, and SREBF2). SREBFs are important positive regulators of lipogenic genes, whose induction promotes hepatic de novo lipogenesis.33 PPARs are also important regulators of lipid metabolism in liver.34 PPARGC1A is a member of the PGC1α family of coactivators that regulate lipid metabolism by binding to transcription factors such as PPARα, PPARγ, and SREBP1C (7193}. Activation of either PPARα or PPARγ by agonists, such as peroxisome-proliferating fibrates or thiazolidinediones, respectively, protect against steatosis by decreasing SREBP activity, which results in lower levels of the lipogenic enzymes fatty acid synthase and glycerol 3-phosphate acyltransferase.35 Conversely, knockdown or knockout of the Ppara gene in mice leads to hypertriglyceridemia.36 In humans, low levels of PPARα are associated with diminished rates of β-oxidation of fatty acids through SREBP1, which in turn are associated with greater steatosis.37 It would, therefore, appear that protection against steatosis in Gclm(−/−) mice in the present study is not regulated by PPARα, PPARγ, or SREBP1C, since activation of these transcriptional mediators would up-regulate lipolysis and down-regulate lipogenesis. Rather, expression of these genes is markedly lessened in Gclm(−/−) mice, as are their downstream lipogenic target genes. Several studies suggest a relationship between cellular GSH levels and susceptibility to hepatic steatosis. In cultured HepG2 cells, L-Cys treatment increases intracellular GSH levels, which accompanies a transient down-regulation of SREBF1C and its downstream lipid-metabolizing target genes, including FASN and SCD1.38 In mice, the L-Cys congeners N-acetylcysteine, S-ethylcysteine, and S-propylcysteine elevate hepatic GSH and decrease the expression of lipogenic genes, resulting in protection from high-fat diet-induced steatosis.39 GSH levels are not only associated with hepatic lipogenesis but lipolysis as well. In a rat study, a sucrose-rich diet lowered hepatic GSH levels as well as activities of the lipolytic enzymes, citrate synthase, and β-hydroxyacyl-CoA dehydrogenase; N-acetylcysteine treatment reversed these effects.40 This was confirmed in a mouse model with severe hepatic GSH depletion by virtue of hepatocyte-specific ablation of the Gclc gene; steatosis occurred rapidly5 and was diminished by N-acetylcysteine treatment.41 In contrast to these reports, HepG2 cells incubated with fatty acids showed a positive relationship between cellular lipid accumulation and GSH concentration.42 In rats fed a high-fat diet, there was an association between the elevated GSH levels and the severity of steatosis.43 Thus, the relationship between the GSH levels and the development of hepatic steatosis, and the mechanisms underlying that association, are complex and remain to be defined. We found that Gclm(−/−) mice, with moderate depletion of hepatic GSH levels, were resistant to steatosis. This phenotype is similar to that reported recently in which Gclm(−/−) mice received a nonalcoholic steatohepatitis (NASH)-inducing diet, deficient in methionine and choline.12 In that study, Gclm(−/−) mice were found to be resistant to the development of NASH, including steatosis, fibrosis, and inflammation. In those mice receiving a standard diet, hepatic SCD1 mRNA was diminished, whereas FASN mRNA was unchanged and PPARA mRNA levels were increased in Gclm(−/−) mice.12 The two lines of

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CONCLUSIONS Our study suggests that low hepatic GSH levels in the Gclm(−/−) mouse are a susceptibility factor for TCDD-induced hepatocellular damage, most likely due to exacerbation of TCDD-mediated oxidative stress. Gclm(−/−) mice exhibit exceedingly low levels of expresion of lipid-metabolizing genes, which contributes to preventing the development of TCDD-induced hepatosteatosis. 98

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ASSOCIATED CONTENT S Supporting Information * cDNA microarray data (filename: Chen cDNA micrarray.pdf). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION Corresponding Author *Tel: 513-558-0522. Fax: 513-558-0925. E-mail: shertzhg@ ucmail.uc.edu. Author Contributions H.G.S. and Y.C. designed the study, Y.C. and M.K. conducted the research, H.G.S. and D.W.N. wrote the paper, and H.G.S. had primary responsibility for the final content. All authors edited and approved the final manuscript. Funding This work was funded, in part, by the Center for Environmental Genetics (NIH Grant P30 ES06096).

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ACKNOWLEDGMENTS We thank Dr. Giovanni Coppola, Department of Neurology, UCLA School of Medicine ([email protected]), for the primer sequences for Ppargc1a. We also thank Dr. Mary Beth Genter, Ph.D., D.A.B.T., University of Cincinnati College of Medicine ([email protected]), for her generous assistance in reading the liver histology slides.

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ABBREVIATIONS AHR, aromatic hydrocarbon receptor; GCL, glutamate-cysteine ligase; GCLC, glutamate-cysteine ligase catalytic subunit; GCLM, glutamate-cysteine ligase modifier subunit; GGT, γ-glutamyl transferase; GOT, glutamic oxaloacetic transaminase; GSH, reduced glutathione; GSSG, glutathione disulfide; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

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