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A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23-24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling.

Low-grade inflammation is a characteristic of the obese state, and adipose tissue releases many inflammatory mediators. The source of these mediators within adipose tissue is not clear, but infiltrating macrophages seem to be especially important, although adipocytes themselves play a role. Obese people have higher circulating concentrations of many inflammatory markers than lean people do, and these are believed to play a role in causing insulin resistance and other metabolic disturbances. Blood concentrations of inflammatory markers are lowered following weight loss. In the hours following the consumption of a meal, there is an elevation in the concentrations of inflammatory mediators in the bloodstream, which is exaggerated in obese subjects and in type 2 diabetics. Both high-glucose and high-fat meals may induce postprandial inflammation, and this is exaggerated by a high meal content of advanced glycation end products (AGE) and partly ablated by inclusion of certain antioxidants or antioxidant-containing foods within the meal. Healthy eating patterns are associated with lower circulating concentrations of inflammatory markers. Among the components of a healthy diet, whole grains, vegetables and fruits, and fish are all associated with lower inflammation. AGE are associated with enhanced oxidative stress and inflammation. SFA and trans-MUFA are pro-inflammatory, while PUFA, especially long-chain n-3 PUFA, are anti-inflammatory. Hyperglycaemia induces both postprandial and chronic low-grade inflammation. Vitamin C, vitamin E and carotenoids decrease the circulating concentrations of inflammatory markers. Potential mechanisms are described and research gaps, which limit our understanding of the interaction between diet and postprandial and chronic low-grade inflammation, are identified.

A fully validated multiple-transition recording isotope dilution liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitative determination of N(epsilon)-carboxymethyllysine (CML) and lysine in dairy products is described. Internal standards were [N-1',2'-(13)C(2)]CML and [1,2,3,4,5,6-(13)C(6)-2,6-(15)N(2)]lysine, and the method was validated by evaluating the selectivity, linearity, precision (repeatability and reproducibility) and trueness, using both powder and liquid products. For liquid dairy products, the repeatability and reproducibility was 2.79% and 11.0%, while 4.85% and 4.92% were determined for powder dairy products, respectively. The trueness of the method ranged from -9.6% to -3.6% for powder and from -0.99% to 6.8% for liquid dairy products. The limit of detection for CML was estimated to be 8 ng CML per mg protein while the limit of quantification was 27 ng CML per mg protein. The method encompasses a proteolytic cleavage mediated by enzymatic digestion to reach a complete release of the amino acids prior to a sample cleanup based on solid phase extraction, and followed by LC-MS/MS analysis of CML and lysine residues. To ensure a suitable performance of the enzymatic digestion, CML measurements were compared to values obtained with an acid hydrolysis-mediated proteolysis. Finally, the method was employed for the analysis of CML in various dairy products. The values compare well to the data available in the literature when similar methods were used, even if some discrepancies were observed upon comparison with the results obtained by other techniques such as enzyme-linked immunosorbent assay and GC-MS.

There is now strong evidence that probiotic bacteria can regulate inflammatory immune responses. Here, we analyzed whether oral supplementation with the probiotic bacterial strain Lactobacillus johnsonii (La1) could interfere with skin immune status following UV exposure. A randomized, double-blind, placebo controlled clinical trial was conducted with 54 healthy volunteers receiving either La1 or placebo, during six weeks prior to solar-simulated UV irradiation. Blister roofs and skin biopsies were recovered 1, 4 and 10 days after UV exposure from un-irradiated and irradiated skin and used for immunohistochemical analysis and mixed epidermal cell lymphocyte reaction (MECLR), respectively. La1 supplementation did not prevent the UV-induced phenotypic maturation of Langerhans cells (LCs) or the decrease in MECLR in irradiated skin samples, one day post-irradiation. On day 4, MECLR was still decreased in the placebo group, with a parallel reduction in the CD1a LC marker in irradiated epidermis. In contrast, the allostimulatory capacity of epidermal cells was totally recovered in the La1 group correlating with the normalization of CD1a expression within the epidermis. For the first time, the results provide evidence that ingested probiotic bacteria accelerate the recovery of skin immune homeostasis after UV-induced immunosuppression.

Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N(epsilon)-fructoselysine (FL), N(epsilon)-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34+/-3.81 nmol CML per micromol of free Lys (Lys(free)) and 81.5+/-87.8 nmol Pyr micromol(-1) Lys(free)(-1) vs. 3.72+/-1.29 nmol FL micromol(-1) Lys(free)(-1). In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47+/-0.08 nmol FL micromol(-1) of protein-bound Lys (Lys(p-b)), 0.04+/-0.03 nmol CML micromol(-1) Lys(p-b)(-1) and 0.06+/-0.02 nmol Pyr micromol(-1)Lys(p-b)(-1). It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products.

A common approach for the quantification of 3-nitrotyrosine (NY) in routine analyses relies on the cleavage of peptide bonds in order to release the free amino acids from proteins in tissues or fluids. NY is usually monitored by either GC-MS(/MS) or LC-MS/MS techniques. Various proteolysis methods have been employed to combine digestion efficiency with prevention of artifactual nitration of tyrosine. However, so far, no study was designed to compare the HCl-based hydrolysis method with enzymatic digestion in terms of reliability for the measurement of NY. The present work addresses the digestion efficiency of BSA using either 6M HCl, pronase E or a cocktail of enzymes (pepsin, pronase E, aminopeptidase, prolidase) developed in our laboratory. The HCl-based hydrolysis leads to a digestion yield of 95%, while 25 and 75% are achieved with pronase E and the cocktail of enzymes, respectively. These methods were compared in terms of NY measurement and the results indicate that a prior reduction of the disulfide bonds ensures a reliable quantification of NY. We additionally show that the enzyme efficacy is not altered when the digestion is carried out in the presence of BSA with a high content of NY.

1. The major side effects of the immunosuppressive drug cyclosporin A (CsA) are hypertension and nephrotoxicity. It is likely that both are caused by local vasoconstriction. 2. We have shown previously that 20 h treatment of rat vascular smooth muscle cells (VSMC) with therapeutically relevant CsA concentrations increased the cellular response to [Arg8]vasopressin (AVP) by increasing about 2 fold the number of vasopressin receptors. 3. Displacement experiments using a specific antagonist of the vasopressin V1A receptor (V1AR) showed that the vasopressin binding sites present in VSMC were exclusively receptors of the V1A subtype. 4. Receptor internalization studies revealed that CsA (10(-6) M) did not significantly alter AVP receptor trafficking. 5. V1AR mRNA was increased by CsA, as measured by quantitative polymerase chain reaction. Time-course studies indicated that the increase in mRNA preceded cell surface expression of the receptor, as measured by hormone binding. 6. A direct effect of CsA on the V1AR promoter was investigated using VSMC transfected with a V1AR promoter-luciferase reporter construct. Surprisingly, CsA did not increase, but rather slightly reduced V1AR promoter activity. This effect was independent of the cyclophilin-calcineurin pathway. 7. Measurement of V1AR mRNA decay in the presence of the transcription inhibitor actinomycin D revealed that CsA increased the half-life of V1AR mRNA about 2 fold. 8. In conclusion, CsA increased the response of VSMC to AVP by upregulating V1AR expression through stabilization of its mRNA. This could be a key mechanism in enhanced vascular responsiveness induced by CsA, causing both hypertension and, via renal vasoconstriction, reduced glomerular filtration.

The clinical use of the immunosuppressive drug cyclosporin A (CsA) is limited by its side effects, namely hypertension and nephrotoxicity. It has been proposed that reactive oxygen species (ROS) could be involved as mediators of the toxic effects of CsA. Here, we have studied the possible interrelationship between CsA metabolism and production of ROS. Using cultures of rat aortic smooth muscle cells (RASMC), CsA (1 microM) produced a rapid (within 10 min) increase in reactive oxygen species, detected by oxidation of the fluorescent probes 2,7-dichlorofluorescin and dihydrorhodamine-123. DNA synthesis was increased in the presence of CsA as assessed by [3H]thymidine incorporation. The superoxide dismutase inhibitor diethyldithiocarbamate (1 mM) and the iron chelator desferal (5 microM), as well as ketoconazole (1 microM) and troleandomycin (10 microM), inhibitors of the cytochrome P-450 3A, were able to block both effects. High-performance liquid chromatography analysis revealed that RASMC were capable to metabolize CsA to its primary metabolites (AM1, AM9 and AM4N), and that their formation was inhibited by ketoconazole and troleandomycin. Furthermore, mRNAs encoding cytochrome P-450 3A1 and 3A2 were detected in RASMC by reverse transcriptase-polymerase chain reaction. Our data suggest that CsA is metabolized by cytochrome P-450 3A in RASMC producing reactive oxygen species, most likely superoxide and the hydroxyl radical, known to damage lipids and DNA.

Because acute infection and inflammation affect drug metabolism and drug-metabolizing enzymes, the effect of the acute-phase response on the expression of glutathione S-transferase (GST) isoenzymes, glutathione synthesis, and several antioxidant enzymes was investigated. Hepatic expression of GST isozymes, positive and negative acute-phase reactants, and antioxidant enzymes were determined by Northern blotting and hybridization with gene-specific oligonucleotide probes after lipopolysaccharide treatment of rats. Lipopolysaccharide caused the expected acute-phase response as judged by the increased expression of positive and decreased expression of negative acute-phase proteins. The messenger RNA (mRNA) expression of the major hepatic rat GST isozymes A1, A2, A3, M1, and M2 was decreased 50% to 90%. Total hepatic GST activity toward 1-chloro-2,4-dinitrobenzene was also significantly decreased. mRNA expression of gamma-glutamylcysteine synthetase (GCS) large subunit and catalase was reduced by approximately 60%. GCS enzyme activity was also decreased, resulting in a 35% decrease in the hepatic content of reduced glutathione 4 days after lipopolysaccharide challenge. Mn-Superoxide dismutase expression was increased 13-fold, and thioredoxin level was elevated 3-fold after lipopolysaccharide challenge. The expression of all parameters determined returned to near control levels 7 days after treatment. Together, these data show that GSTs and GCS are negative acute-phase proteins and that decreased GCS activity results in a decrease in hepatic glutathione content. Thus, in addition to the phase I drug-metabolizing enzymes known to be decreased during the acute-phase response, some phase II enzymes involved in the elimination of xenobiotics and carcinogens are also decreased.

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.

Various natural and synthetic compounds are known to protect against cancer by elevating phase II detoxification enzymes. Generally classified as monofunctional, these inducers are believed to trigger cellular signal(s) that activate gene transcription through an antioxidant or electrophile response element (ARE/EpRE) in responsive genes. In contrast, the phase I enzymes of drug metabolism (cytochrome P450s) are not believed to be induced by monofunctional inducers and P450 genes have not been found to contain functional ARE/EpREs. In this study, rats were treated with the monofunctional inducers tert-butylated hydroxyanisole, ethoxyquin, and oltipraz to study the inducibility of individual glutathione S-transferase isozymes, NADP(H):quinone oxidoreductase, gamma-glutamylcysteine synthetase, UDP-glucuronosyl transferase, and cytochrome P450 enzymes. Hepatic mRNAs were analyzed on Northern blots using gene-specific oligonucleotide probes for GST Ya1, Ya2, Yc1, Yc2, Yb1, Yb2, and Yf, for UGT 1*06, and for P450 1A1, 1A2, 2B1, 2C11, 3A2, and 4A1. NADP(H):quinone oxidoreductase and gamma-glutamylcysteine synthetase mRNAs were detected using cDNA probes. All the phase II detoxification enzymes analyzed, except GST Yf, were induced by the three monofunctional inducers, suggesting that these genes may be regulated by a mechanism involving an ARE/EpRE element in their promoter region. Interestingly, it was found that ethoxyquin was a particularly good inducer for both members of the P450 2B family, 2B1 and 2B2, and both ethoxyquin and oltipraz were also capable of modestly inducing P450 1A2 and 3A2. Oltipraz was found to slightly induce P450 2B2, but not 2B1, at the dose and time analyzed. Induction of mRNA generally correlated well with induction of protein levels determined by Western blot and/or enzyme activity measurements for selected enzymes. The results of this study suggest that many phase II enzymes may contain ARE/EpRE elements in addition to those confirmed to be regulated by a mechanism involving ARE/EpRE elements. In addition, it was found that several P450 enzymes were induced by monofunctional inducers, suggesting a possibility that some phase I enzymes may also be regulated by a mechanism involving ARE/EpRE elements.

Although the mechanisms responsible for chemically induced oxidative stress are under intense investigation, little is known about the effects of prooxidant chemicals on the expression of drug-metabolizing enzymes. We examined the effects of diquat (0.1 mmol/kg, ip) and ciprofibrate (0.025% w/w, diet), chemicals which induce oxidative stress via different biochemical mechanisms, on the steady-state messenger RNA (mRNA) levels of six cytochrome P450 enzymes, seven glutathione S-transferase (GST) isoenzymes, UDP-glucuronosyl transferase 1-06 (UGT1*06), gamma-glutamylcysteine synthetase (gamma GCS), NADP(H):quinone oxidoreductase (quinone reductase), Cu/Zn superoxide dismutase (SOD), catalase, and 18S ribosomal RNA in the livers of male Sprague-Dawley rats. Effects of chemical treatments on mRNA levels were compared to changes in catalytic activities for selected enzymes. Ciprofibrate treatment selectively decreased CYP1A2 mRNA expression, whereas both chemicals suppressed CYP3A2 mRNA expression. CYP4A1 mRNA expression and lauric acid hydroxylase activities were induced by ciprofibrate treatment, whereas diquat treatment moderately increased CYP4A1 mRNA levels without affecting lauric acid hydroxylase activities. The steady-state mRNA levels encoding constitutively expressed GST isozymes (Ya1, Ya2, Yb1, Yb2, and Yc1) were decreased by diquat exposure, and the mRNA encoding four of the five constitutively expressed GSTs (Ya1, Ya2, Yb1, and Yc1) were also decreased by ciprofibrate treatment. Nonconstitutively expressed or low constitutively expressed genes (CYP1A1, CYP2B1, CYP2B2, GST Yc2, GST Yf, and UGT1*06) were not induced by exposure to the prooxidants. Changes in isozyme-specific catalytic activities were more consistent with the observed changes in mRNA expression for the GSTs than for the P450s. Both treatments had inhibitory effects on hepatic GSH biosynthesis by decreasing gamma GCS large-subunit mRNA expression, gamma GCS catalytic activities, and hepatic GSH concentrations. Cu/Zn SOD and quinone reductase mRNA levels were increased after ciprofibrate exposure, whereas Cu/Zn SOD mRNA expression was decreased in the diquat-treated animals. The results of this study indicate that diquat and ciprofibrate can decrease the expression profile of a number of phase I, phase II, and antioxidant enzymes and inhibit GSH biosynthesis. These effects may involve the pretranslational loss of hepatic mRNAs, possibly due to accelerated production of reactive oxygen species.

Mice are resistant to aflatoxin carcinogenicity primarily due to expression of a glutathione S-transferase (mYc) with high catalytic activity toward aflatoxin B1-8,9-epoxide (AFBO). In contrast, rats are more sensitive to aflatoxin carcinogenicity due to the constitutive expression of a glutathione S-transferase with relatively low catalytic activity toward AFBO (rYc1). To identify the contribution of different regions of the mYc protein that confer high catalytic activity toward AFBO, six chimeric mYc/rYc1 GST enzymes were generated utilizing full and partial restriction enzyme digestions at two conserved StyI sites in the mYc and rYc1 complementary DNAs (between amino acid residues 56-57 and 142-143). Recombinant wild-type and chimeric glutathione S-transferases were bacterially expressed, affinity purified, and their catalytic activities measured toward AFBO, delta 5-androstene-3,17-dione, 1-chloro-2,4-dinitrobenzene, and ethacrynic acid. The set of chimeras displayed a wide range of catalytic activities toward the substrates assayed. The chimeras with the greatest activity toward AFBO were 1:56rat-57: 221mouse and 1:56mouse-57:142rat-143:221mouse, with AFBO conjugating activities 200 and 8 times greater than wild-type rYc1, respectively. These results demonstrate that the residues that confer high AFBO conjugation activity in mYc are located in the region spanning residues 57-221.

Dietary 2(3)-tert-butyl-4-hydroxyanisole (BHA) treatment has been shown to increase hepatic glutathione (GSH) content in rats and mice. Subsequent studies in our laboratory have demonstrated that hepatic gamma-glutamylcysteine synthetase (GCS) activity is increased in mice treated with dietary BHA. To test whether this increase in GCS activity follows an increase in hepatic messenger RNA for the large subunit of GCS (GCS-LS mRNA), a 390-base pair fragment corresponding to a region near the 5' end of the rat GCS-LS cDNA sequence was amplified using the PCR reaction and used to detect GCS-LS mRNA on Northern blots. Hepatic GSH, GCS activity, and GCS-LS mRNA levels were determined either in mice treated with BHA in the diet for 12 days or mice injected with diethyl maleate (DEM), phorone, and/or DL-buthionine-[S,R]-sulfoximine (BSO) over a 24 hr period. BHA caused a 1.5-fold increase in GSH levels, a 1.7-fold increase in hepatic GCS activity by Day 12, and a rapid 5-fold increase in hepatic GCS mRNA levels reaching maximal levels after 2-3 days. Partial depletion of GSH with either phorone (70%) or DEM (50%) resulted in a 4- to 5-fold increase in hepatic GCS-LS mRNA levels by 9 hr and a 1.5- to 2-fold increase in hepatic GSH and GCS activity by 24 hr. Depletion of GSH with the GCS enzyme inhibitor BSO had no effect on GCS mRNA expression, even though GSH was depleted to 30%. When BSO was combined with the phorone treatment GSH levels were depleted to < 10%, but the large increase in GCS-LS mRNA seen with phorone alone was greatly attenuated. These data suggest that depletion of GSH per se, is not sufficient to induce elevation of GCS-LS mRNA levels, but that the formation of GSH conjugates may be required to trigger GCS-LS mRNA induction. The increase in GCS-LS mRNA levels may account for the increase in GCS activity and elevation of GSH observed following BHA treatment, as well as the "rebound" of GSH above control levels observed 18-24 hr following depletion of GSH by other chemicals. These results are consistent with the Michael acceptor, hypothesis by Talalay.

The complementary DNAs of rat glutathione S-transferase (GST, EC 2.5.1.18) Yc1 and of mouse Yc were expressed from a prokaryotic expression vector in E. coli. The purified proteins were analyzed for their activity toward aflatoxin B1-8,9-epoxide (AFBO), the reactive intermediate of the fungal mycotoxin aflatoxin B1 (AFB). The mouse Yc isozyme had about 50-fold higher conjugating activity toward AFBO than the rat Yc1 isozyme (144 nmol/mg/min versus 3.3 nmol/mg/min). The rat Yc1 isozyme had specific activities toward 1-chloro-2,4-dinitrobenzene, cumene hydroperoxide and ethacrynic acid of 10.7, 0.98 and 0.92 mumol/mg/min, respectively, whereas the mouse Yc isozyme had specific activities of 5.7, 2.1 and 0.1 mumol/mg/min for these substrates, respectively. These data provide further support for the hypothesis that the constitutive presence of the alpha class GST Yc isozyme in mouse liver protects mice from the hepatocarcinogenic effects of aflatoxin B1.

Glutathione S-transferases (EC 2.5.1.18) are a multigene family of related proteins divided into four classes. Each class has multiple isoforms that exhibit tissue-specific expression, which may be an important determinant of susceptibility of that tissue to toxic injury or cancer. Recent studies have suggested that alpha-class glutathione S-transferase isoforms may play an important role in the development of cancers. Several alpha-class glutathione S-transferase isozymes have been characterized, purified, and cloned from a number of species, including rats, mice, and humans. Here we report on the cloning, sequencing, and mRNA expression of two alpha-class glutathione S-transferases from mouse liver, termed mYa and mYc. While mYa was shown to be identical to the known alpha-class glutathione S-transferase complementary DNA clone pGT41 (W. R. Pearson et al., J. Biol. Chem., 263: 13324-13332, 1988), the other clone, mYc, was demonstrated to be a novel complementary DNA clone encoding a glutathione S-transferase homologous to rat Yc (subunit 2). The mRNA for this novel complementary DNA is expressed constitutively in mouse liver. It also is the major alpha-class glutathione S-transferase isoform expressed in lung. The levels of expression of the butylated hydroxyanisole-inducible form (mYa) are highest in kidney and intestine. Treatment of mice with butylated hydroxyanisole had little effect on the expression levels of mYc but strongly induced mYa expression in liver. Butylated hydroxyanisole treatment increased expression levels for both mYa and mYc to varying degrees in kidney, lung, and intestine. The importance of the novel mouse liver alpha-class glutathione S-transferase isoform (mYc) in the metabolism of aflatoxin B1 and other carcinogens is discussed.