The effect of nicotinamide on gene expression in a traumatic brain injury model.

Microarray-based transcriptional profiling was used to determine the effect of nicotinamide on gene expression in an experimental traumatic brain injury (TBI) model. Ingenuity Pathway Analysis (IPA) was used to evaluate the effect on relevant functional categories and canonical pathways. At 24 h, 72 h, and 7 days, respectively, 70, 58, and 76%, of the differentially expressed genes were up-regulated in the vehicle treated compared to the sham animals. At 24 h post-TBI, there were 150 differentially expressed genes in the nicotinamide treated animals compared to vehicle; the majority (82%) down-regulated. IPA analysis identified a significant effect of nicotinamide on the functional categories of cellular movement, cell-to-cell-signaling, antigen presentation and cellular compromise, function, and maintenance and cell death. The canonical pathways identified were signaling pathways primarily involved with the inflammatory process. At 72 h post-cortical contusion injury, there were 119 differentially expressed genes in the nicotinamide treated animals compared to vehicle; the majority (90%) was up-regulated. IPA analysis identified a significant effect of nicotinamide on cell signaling pathways involving neurotransmitters, neuropeptides, growth factors, and ion channels with little to no effect on inflammatory pathways. At 7 days post-TBI, there were only five differentially expressed genes with nicotinamide treatment compared to vehicle. Overall, the effect of nicotinamide on counteracting the effect of TBI resulted in significantly decreased number of genes differentially expressed by TBI. In conclusion, the mechanism of the effect of nicotinamide on secondary injury pathways involves effects on inflammatory response, signaling pathways, and cell death.

Transcriptome correlation analysis identifies two unique craniosynostosis subtypes associated with IRS1 activation.

The discovery of causal mechanisms associated with nonsyndromic craniosynostosis has proven to be a difficult task due to the complex nature of the disease. In this study, differential transcriptome correlation analysis was used to identify two molecularly distinct subtypes of nonsyndromic craniosynostosis, termed subtype A and subtype B. In addition to unique correlation structure, subtype A was also associated with high IGF pathway expression, whereas subtype B was associated with high integrin expression. To identify a pathologic link between altered gene correlation/expression and the disease state, phosphorylation assays were performed on primary osteoblast cell lines derived from cases within subtype A or subtype B, as well as on primary osteoblast cell lines with novel IGF1R variants previously reported by our lab (Cunningham ML, Horst JA, Rieder MJ, Hing AV, Stanaway IB, Park SS, Samudrala R, Speltz ML. Am J Med Genet A 155A: 91-97, 2011). Elevated IRS1 (pan-tyr) and GSK3ß (ser-9) phosphorylation were observed in two novel IGF1R variants with receptor L domain mutations. In subtype A, a hypomineralization phenotype coupled with decreased phosphorylation of IRS1 (ser-312), p38 (thr-180/tyr-182), and p70S6K (thr-412) was observed. In subtype B, decreased phosphorylation of IRS1 (ser-312) as well as increased phosphorylation of Akt (ser-473), GSK3ß (ser-9), IGF1R (tyr-1135/tyr-1136), JNK (thr-183/tyr-187), p70S6K (thr-412), and pRPS6 (ser-235/ser-236) was observed, thus implicating the activation of IRS1-mediated Akt signaling in potentiating craniosynostosis in this subtype. Taken together, these results suggest that despite the stimulation of different pathways, activating phosphorylation patterns for IRS1 were consistent in cell lines from both subtypes and the IGF1R variants, thus implicating a key role for IRS1 in the pathogenesis of nonsyndromic craniosynostosis.

Influence of Matrigel-overlay on constitutive and inducible expression of nine genes encoding drug-metabolizing enzymes in primary human hepatocytes.

1. Previous studies reported that rat hepatocytes overlaid with extracellular matrix components (Matrigel) maintain the expression and responsiveness of drug-metabolizing enzymes. However, whether Matrigel provides similar advantages in human hepatocytes remains largely uncertain.2. The influence of Matrigel-overlay on the constitutive and phenobarbital- and oltipraz-inducible expression of nine biotransformation enzymes, cytochrome P450s 1A1, 1A2, 2B6, 3A4, and glutathione S-transferases A1, A2, M1, T1, P1, in primary human hepatocytes was evaluated.3. Hepatocytes from five livers were maintained on a rigid collagen substratum with or without Matrigel overlay and treated for 48?h with two doses of each inducer. Quantitative RT-PCR, and for selected genes, immunoblot and enzyme activity analyses, demonstrated that human hepatocytes overlaid with Matrigel showed consistently higher constitutive and inducible expression of biotransformation genes. 4. Phenobarbital-mediated responsiveness of cytochrome P450 2B6, a potential indicator of hepatocyte differentiation status, was markedly higher in overlaid relative to non-overlaid hepatocytes. 5. It is concluded that an Matrigel overlay facilitates the maintenance and induction of xenobiotic metabolizing enzymes in primary cultures of human hepatocytes.

Phytochemical-induced changes in gene expression of carcinogen-metabolizing enzymes in cultured human primary hepatocytes.

1. The naturally occurring compounds curcumin (CUR), 3,3'-diindolylmethane (DIM), isoxanthohumol (IXN), 8-prenylnaringenin (8PN), phenethyl isothiocyanate (PEITC) and sulforaphane (SFN) protect animals against chemically induced tumours. Putative chemoprotective mechanisms include modulated expression of hepatic biotransformation enzymes. However, few, if any, studies have used human primary cells as test models. 2. The present study investigated the effects of these phytochemicals on the expression of four carcinogenesis-relevant enzymes--cytochrome P450 (CYP)1A1 and 1A2, NAD(P)H:quinone oxidoreductase (NQO1) and glutathione S-transferase A1 (GSTA1)--in primary cultures of freshly isolated human hepatocytes. 3. Quantitative RT-PCR analyses demonstrated that CYP1A1 was up-regulated by PEITC and DIM in a dose-dependent manner. CYP1A2 transcription was significantly activated following DIM, IXN, 8PN and PEITC treatments. DIM exhibited a remarkably effective induction response of CYP1A1 (474-, 239- and 87-fold at 50, 25 and 10 microM, respectively) and CYP1A2 (113-, 70- and 31-fold at 50, 25 and 10 microM, respectively), that was semiquantitatively reflected in protein levels. NQO1 expression responded to PEITC (11 x at 25 microM), DIM (4.5 x at 50 microM) and SFN (5 x at 10 microM) treatments. No significant effects on GSTA1 transcription were seen. 4. The findings show novel and unexpected effects of these phytochemicals on the expression of human hepatic biotransformation enzymes that play key roles in chemical-induced carcinogenesis.

Interindividual differences in response to chemoprotection against aflatoxin-induced hepatocarcinogenesis: implications for human biotransformation enzyme polymorphisms.

It is now evident that most, if not all, of the remarkable species differences in susceptibility to AFB hepatocarcinogenesis is due in large part, if not exclusively, to differences in biotransformation. Certainly the relative rate of oxidative formation of the proximate carcinogen, AFB-8,9-exo-epoxide, is an important determinant of species and interindividual differences in susceptibility to AFB. However, mice produce relatively large amounts of exo-AFBO, yet are highly resistant to AFB-hepatocarcinogenesis because they express a particular form of GST with remarkably high catalytic activity toward the exo-epoxide of AFB. Rats, which are highly susceptible to AFB hepatocarcinogenesis,can be made resistant through dietary induction of an orthologous form of GST that is normally expressed in only very small amounts. Based on these findings in laboratory animal models, there is great interest in identifying chemicals and/or specific dietary constituents that could offer protection against AFB-hepatocarcinogenesis to humans. Current experimental strategies have focused on the antiparasitic drug, oltipraz, which induces protection in rats and has also shown some promise in humans. The mechanism of protection in rats appears to be via induction of an alpha class GST with high catalytic activity toward AFBO (rGSTA5-5). vet human alpha class GST proteins that are constitutively expressed in the liver (hGSTA1 and hGSTA2) have little, if any activity toward AFBO. Rather, it appears that mu class GSTs may be responsible for the very low, but potentially significant, detoxification activity toward AFBO. Oltipraz and certain dietary constituents may induce mu class GSTs in human liver, and this could afford some protection against the genotoxic effects of AFBO. However, it also appears that oltipraz, and perhaps certain dietary constituents, act as competitive inhibitors of human CYP1A2. As CYP1A2 appears to mediate most of the activation of AFB to exo-AFBO in human liver at low dietary concentrations of AFB encountered in the human diet, much of the putative protective effects of oltipraz could be mediated via inhibition of CYP1A2 rather than induction of GSTs. There is now evidence that human microsomal epoxide hydrolase (mEH) could play a role in protecting human DNA from the genotoxic effects of AFB, although the importance of this detoxification pathway, relative to mu class GSTs, remains to be elucidated. Oltipraz is an effective inducer of mEH in rats (Lamb Franklin, 2000), and thus induction of this pathway in humans could also potentially contribute to the protective effects of this drug toward AFB genotoxicity. Because the dihydrodiol of AFB may contribute indirectly to the carcinogenic effects of AFB via protein adduction and subsequent hepatotoxicity, the recently characterized human aflatoxin aldehyde reductase (AFAR) may also offer some protection against AFB-induced carcinogenicity in humans. Current and future dietary and/or chemointervention strategies aimed at reducing the carcinogenic effects of AFB in humans should consider all of the possible mechanistic approaches for modifying AFB-induced genotoxicity.

Mu-class GSTs are responsible for aflatoxin B(1)-8, 9-epoxide-conjugating activity in the nonhuman primate macaca fascicularis liver.

Mice are resistant to the carcinogenic effects of the mycotoxin aflatoxin B(1) (AFB(1)) because they constitutively express an alpha-class glutathione S-transferase (mGSTA3-3) that has high (approximately 200,000 pmol/min/mg) activity toward aflatoxin B(1)-8, 9-epoxide (AFBO). Rats do not constitutively express a GST with high AFBO-conjugating activity and are sensitive to AFB(1)-induced hepatocarcinogenesis. Constitutively expressed human hepatic alpha-class GSTs (hGSTA1-1 and hGSTA2-2) possess little or no AFBO-detoxifying activity (<2 pmol/min/mg). Recently, we found that the nonhuman primate, Macaca fascicularis (Mf), exhibits significant (approximately 300 pmol/min/mg) constitutive hepatic GST activity towards AFBO. To determine which specific GST isoenzyme(s) is (are) responsible for this activity, MF: GSTs were purified from liver tissue and characterized and, Mf mu-class GST cDNAs were cloned by reverse transcriptase-coupled polymerase chain reaction (RT-PCR). Purification by glutathione agarose (GSHA) affinity chromatography yielded a protein, GSHA-GST, that exhibited relatively high AFBO-conjugating activity (239 pmol/min/mg) compared to other GST-containing peaks. Western blotting and enzymatic activity analyses revealed that GSHA-GST belongs to the mu class. Two distinct mu-class GST cDNAs, mfaGSTM1 (GenBank accession # AF200709) and mfaGSTM2 (GenBank accession # AF200710), were generated by RT-PCR. CDNA-derived amino acid sequence analysis revealed that mfaGSTM1 and mfaGSTM2 share 97% and 96% homology with the human mu-class GSTs hGSTM4 and hGSTM2, respectively. In contrast to recombinant mfaGSTM1-1, which had no detectable AFBO-conjugating activity, mfaGSTM2-2 exhibited this activity at 333 pmol/min/mg. Activity profiles for the stereoisomers exo- and endo-AFBO, and of 1-chloro-2,4-dinitrobenzene of the purified protein GSHA-GST and recombinant mfaGSTM2-2, suggested that they are two distinct enzymes. Our results indicate that, in contrast to rodents, mu-class GSTs are responsible for the majority of AFBO-conjugating activity in the liver of Macaca fascicularis.

Effects of dietary oltipraz and ethoxyquin on aflatoxin B1 biotransformation in non-human primates.

Following aflatoxin B1 (AFB) exposure, rats readily develop liver tumors. However, treatment of rats with a variety of compounds, including the synthetic dithiolthione oltipraz and the antioxidant ethoxyquin, protects these rodents from AFB-induced hepatocarcinogenesis. Several epidemiological studies strongly suggest that AFB is also a causative agent of liver cancer in humans. However, relatively little is known about the efficacy of cancer chemoprevention in human and non-human primates. To this end, we examined the effects of chemopreventive agents on AFB metabolism in non-human primates. Hepatic aflatoxin B1 metabolism profiles of macaque (Macaca nemestrina) and marmoset (Callithrix jacchus) monkeys were determined and compared to humans. Quantitatively, the oxidative metabolism of this mycotoxin was similar in the three primate species. In contrast to macaques, both humans and marmosets lacked AFB-glutathione conjugating activity. It was concluded that marmosets resembled human AFB metabolism more closely than the macaques, and therefore, marmoset monkeys were chosen for this study. Eleven adult male marmosets were randomly assigned to three groups. Animals received the synthetic dithiolthione oltipraz, the antioxidant ethoxyquin, or vehicle only. In addition, two single doses of AFB were also administered orally before and after animals were treated with aforementioned compounds. Both oltipraz and ethoxyquin induced aflatoxin B1-glutathione conjugating activity in the livers of some but not all marmosets. In addition, 10 microM oltipraz inhibited cytochrome P450-mediated activation of AFB to the ultimate carcinogenic metabolite, aflatoxin B1-8,9-epoxide, in vitro, up to 51%. Furthermore, animals treated in vivo with oltipraz, but not ethoxyquin, exhibited a significant reduction (53% average) in AFB-DNA adduct formation relative to the control animals (p < 0.05). Together, our data suggest that chemoprevention is also effective in primates; however, most likely to a lesser degree than in rodents.

Identification of amino acid residues essential for high aflatoxin B1-8,9-epoxide conjugation activity in alpha class glutathione S-transferases through site-directed mutagenesis.

Mice constitutively express glutathione S-transferase mGSTA3-3 in liver. This isoform possesses uniquely high conjugating activity toward aflatoxin B1-8,9-epoxide (AFBO), thereby protecting mice from aflatoxin B1-induced hepatocarcinogenicity. In contrast, rats constitutively express a closely related GST isoenzyme, rGSTA3-3, with low AFBO activity and, therefore, are sensitive to aflatoxin B1 exposure. Although the two GSTs share 86% sequence identity and have similar catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB), they have an approximately 1000-fold difference in catalytic activity toward AFBO. To identify amino acids that confer high activity toward AFBO, non-conserved rGSTA3-3 residues were replaced with mGSTA3-3 residues in two regions believed to form the substrate binding site. Twenty-one mutant rGSTA3-3 enzymes were generated by site-directed mutagenesis using combinations of nine different residues. Except for the E208D mutant, single mutations of rGSTA3-3 produced enzymes with no detectable AFBO activity. Generally, AFBO conjugation activity increased in additive fashion as mGSTA3-3 residues were introduced into the rGSTA3-3 enzyme with the six site mutant E104I/H108Y/Y111H/L207F/E208D/V217K displaying the highest AFBO activity (40 nmol/mg/min) of all the mutant enzymes. When this mutant enzyme was further modified by three additional substitutions (D103E/I105M/V106I) AFBO conjugation activity decreased 14-fold to 2. 8 nmol/mg/min. Although wild-type mGSTA3-3 AFBO conjugation activity (265 nmol/mg/min) could not be obtained by our rGSTA3-3 mutants, we were able to identify six mGSTA3-3 residues; Ile104, Tyr108, His111, Phe207, Asp208, and Lys217 that, when collectively substituted into rGSTA3-3, substantially increased (>200-fold) glutathione conjugation activity toward AFBO.

Oltipraz-mediated changes in aflatoxin B(1) biotransformation in rat liver: implications for human chemointervention.

Oltipraz (OPZ) is currently being considered for human use to protect against aflatoxin B1 (AFB)-induced hepatocarcinogenesis based on its proven protective effect in rats. The effectiveness of this treatment presumes that orthologous cytochrome P450 and glutathione S-transferase (GST) isozymes metabolize AFB in humans as they do in rats. In this study, alterations in the expression of multiple forms of cytochrome P450 and GST were evaluated after treatment with OPZ, as well as other known P450 inducers, including 3-methylcholanthrene, pregnenolone-16alpha-carbonitrile, and ciprofibrate. Evidence is presented that the male-specific rat CYP 3A2, an orthologue of human CYP 3A4, may be primarily responsible for AFB activation in rat liver at both high and low AFB substrate concentrations. The CYP 1A2 enzyme does not appear to play a role in AFB activation in rat liver at any substrate concentration, whereas the major human P450 enzyme capable of activating AFB at a low substrate concentration has been identified as CYP 1A2. Surprisingly, we found that the CYP 1A2 steady-state mRNA level and the CYP 1A2-associated methoxyresorufin-O-demethylase activity were induced approximately 3- and 2-fold, respectively, by OPZ in rat liver. However, because CYP 1A2 does not appear to participate in AFB activation, induction of CYP 1A2 may be insignificant for AFB-induced hepatocarcinogenesis in rat models. In the rat, a heterodimeric alpha class GST enzyme containing the Yc2 subunit is the only polypeptide characterized to date in this species with high catalytic activity for the conjugation of activated AFB with glutathione. The GST Yc2 steady-state mRNA level was induced 5-fold by OPZ treatment. This induction was mirrored by significant increases in both the corresponding protein level and AFB-8,9-epoxide-conjugating enzyme activity, which may contribute significantly to protection against AFB-induced carcinogenesis in the rat. Investigations from this and other laboratories have not revealed any evidence for a Yc2-like GST isozyme with high AFB-8,9-epoxide-conjugating activity in human liver. We have also been unable to demonstrate that the two major human alpha class GST isozymes, A1-1 and A2-2, purified from bacteria expressing the corresponding cDNAs, exhibit any significant AFB-8,9-epoxide-conjugating activity. Our results suggest that humans may not be protected to the same extent as rats against AFB-induced hepatocarcinogenesis by treatment with OPZ and that further investigations are needed to establish the usefulness of OPZ for protection against human exposure to AFB.

A comparative study of hepatic and colonic metabolic enzymes in inbred mouse lines before and after treatment with the colon carcinogen, 1,2-dimethylhydrazine.

1,2-Dimethylhydrazine (DMH) is an organotropic colon carcinogen that undergoes metabolic activation to DNA-reactive metabolites. Twenty hours after parenteral treatment of AKR/J (colon tumor resistant) and SWR/J (susceptible) mice with DMH.2HCl (70 mg/kg), functional levels of Cyp1a1 and Cyp2e1 were examined by measuring O-deethylation of ethoxyresorufin (EROD) and hydroxylation of p-nitrophenol, respectively. In control animals, SWR/J mice exhibited higher hepatic EROD activity (1.4-fold) when compared with AKR/J mice. In carcinogen-treated animals, EROD activity was decreased 20-30% in both mouse lines. Hepatic p-nitrophenol hydroxylase activity, similar in control animals of both strains, was reduced comparably (45-50% of control) after DMH administration. In liver, a decrease in immunoreactive Cyp2e1 protein paralleled the decline in enzyme activity, whereas in the colon, no significant treatment-related differences were detected in either strain. In liver and colon cytosols, alcohol dehydrogenase activity was not significantly different in either mouse line, both in control and DMH-treated animals. Glutathione levels were elevated (1.7-fold) in livers of AKR/J mice after DMH administration. Total glutathione-S-transferase (GST) activity was significantly increased (1.8-fold) in the colons of SWR/J mice and in the livers (1.4-fold) of AKR/J mice. Furthermore, the GST isoform, GST-Yp, was reduced 40% in the SWR/J colon. These data demonstrate the importance of metabolic capacity as a factor in conferring differential tumor susceptibility in a murine cancer model to the indirect-acting colon carcinogen, DMH.

Differential single- versus double-strand DNA breakage produced by doxorubicin and its morpholinyl analogues.

The morpholinyl analogues of doxorubicin (DOX) have previously been reported to be non-cross-resistant in multidrug resistant (MDR) cells due to a lower affinity for P-glycoprotein relative to the parent compound. In order to further investigate the mechanisms of action of these morpholinyl anthracyclines, we examined their ability to cause DNA single- and double-strand breaks (SSB, DSB) and their interactions with topoisomerases. Alkaline elution curves were determined after 2-h drug treatment at 0.5, 2 and 5 microM, while neutral elution was conducted at 5, 10 and 25 microM in a human ovarian cell line, ES-2. A pulse-field gel electrophoresis assay was used to confirm the neutral elution data under the same conditions. Further, K-SDS precipitation and topoisomerase drug inhibition assays were used to determine the effects of DOX and the morpholinyl analogues on topoisomerase (Topo) I and II. Under deproteinated elution conditions (pH 12.1), DOX, morpholinyl DOX (MRA), methoxy-morpholinyl DOX (MMDX) and morpholinyl oxaunomycin (MX2) were equipotent at causing SSB in the human ovarian carcinoma cell line, ES-2. However, neutral elution (pH 9.6) under deproteinated conditions revealed marked differences in the degree of DNA DSB. After 2-h drug exposures at 10 microM, DSBs were 3300 rad equivalents for MX2, 1500 for DOX and 400 for both MRA and MMDX in the ES-2 cell line. Pulse-field data substantiated these differences in DSBs, with breaks easily detected after MX2 and DOX treatment, but not with MRA and MMDX. DOX and MX2 thus cause DNA strand breaks selectively through interaction with Topo II, but not Topo I. In contrast, MRA and MMDX cause DNA breaks through interactions with both topoisomerases with a predominant inhibition of Topo I.

Role of cytochrome P4501A2 in chemical carcinogenesis: implications for human variability in expression and enzyme activity.

Cytochrome P4501A2 (CYP1A2) has been identified as a key factor in the metabolic activation of numerous chemical carcinogens, including aflatoxin B1, various heterocyclic and aromatic amines, and certain nitroaromatic compounds. In addition, CYP1A2 contributes to the inactivation of several common drugs and dietary constituents, including acetaminophen and caffeine. Two xenobiotic-responsive-element (XRE)-like sequences and an antioxidant response element (ARE) have been identified in the regulatory region of the CYP1A2 gene; however, the functionality of the ARE remains to be demonstrated. Based on in vivo phenotyping assays, substantial interindividual variability in CYP1A2 activity has been reported. Some population-based studies have reported either bi- or tri-modal distributions in CYP1A2 phenotype, suggesting a genetic basis for the large interindividual differences in CYP1A2 activity. However, despite the polymodal distributions reported for CYP1A2 activity, a distinct functional genetic polymorphism in the gene has not been identified. Potential mechanisms contributing to the large interindividual variability in CYP1A2 activity are discussed. A thorough understanding of the functions and regulation of the CYP1A2 gene may ultimately lead to new methods for preventing or intervening in the development of certain chemically-related human cancers.

Amino acid differences at positions 10, 11, and 104 explain the profound catalytic differences between two murine pi-class glutathione S-transferases.

The glutathione S-transferases play a pivotal role in the detoxification of toxic and carcinogenic electrophiles. We have previously reported the isolation of two actively transcribed murine pi-class glutathione S-transferase genes. In this study the two proteins encoded by these genes, Gst p-1 and Gst p-2, were expressed in Escherichia coli and found to exhibit profoundly different catalytic activities, the activity of Gst p-2 toward a panel of electrophilic substrates being 1-3 orders of magnitude lower than that of Gst p-1. In order to establish the basis for the difference between these highly homologous proteins, mutants were generated where specific amino acids had been exchanged. Kinetic analysis of the wild-type and mutant enzymes revealed that the amino acid differences occurring at positions 10 (Val/Ser), 11 (Arg/Pro), and 104 (Val/Gly) are responsible for the reduced enzymatic activity of Gst p-2. This analysis together with computer graphics modeling for Gst p-2 indicated that these changes affected both substrate and glutathione binding to the enzyme.

Isolation and characterization of two mouse Pi-class glutathione S-transferase genes.

Pi-class glutathione S-transferases (GSTs) play an important role in the detoxification of chemical toxins and mutagens and are implicated in neoplastic development and drug resistance. In all species characterized to date, only one functional Pi-class GST gene has been described. In this report we have identified two actively transcribed murine Pi-class GST genes, Gst p-1 and Gst p-2. The coding regions of Gst p-1 and the mouse Pi-class GST cDNA (GST-II) reported by Hatayama, Satoh and Satoh (1990) (Nucleic Acids Res. 18, 4606) are identical, whereas Gst p-2 encodes a protein that has not been described previously. The two genes are approximately 3 kb long and contain seven exons interrupted by six introns. In addition to a TATA box and a sequence motif matching the phorbol-ester-responsive element, the promoters of Gst p-1 and Gst p-2 exhibit one and two G+C boxes (GGGCGG) respectively. The cDNAs of the two genes were isolated from total liver RNA using reverse PCR. The peptide sequence deduced from the cDNAs share 97% identity and differ in six amino acids. Both genes are transcribed at significantly higher levels in male mouse liver than in female, and Gst p-1 mRNA is more abundant in both sexes than Gst p-2.