Genomic indicators in the blood predict drug-induced liver injury.

Genomic biomarkers for the detection of drug-induced liver injury (DILI) from blood are urgently needed for monitoring drug safety. We used a unique data set as part of the Food and Drug Administration led MicroArray Quality Control Phase-II (MAQC-II) project consisting of gene expression data from the two tissues (blood and liver) to test cross-tissue predictability of genomic indicators to a form of chemically induced liver injury. We then use the genomic indicators from the blood as biomarkers for prediction of acetaminophen-induced liver injury and show that the cross-tissue predictability of a response to the pharmaceutical agent (accuracy as high as 92.1%) is better than, or at least comparable to, that of non-therapeutic compounds. We provide a database of gene expression for the highly informative predictors, which brings biological context to the possible mechanisms involved in DILI. Pathway-based predictors were associated with inflammation, angiogenesis, Toll-like receptor signaling, apoptosis, and mitochondrial damage. The results show for the first time and support the hypothesis that genomic indicators in the blood can serve as potential diagnostic biomarkers predictive of DILI.

Functional analysis of multiple genomic signatures demonstrates that classification algorithms choose phenotype-related genes.

Gene expression signatures of toxicity and clinical response benefit both safety assessment and clinical practice; however, difficulties in connecting signature genes with the predicted end points have limited their application. The Microarray Quality Control Consortium II (MAQCII) project generated 262 signatures for ten clinical and three toxicological end points from six gene expression data sets, an unprecedented collection of diverse signatures that has permitted a wide-ranging analysis on the nature of such predictive models. A comprehensive analysis of the genes of these signatures and their nonredundant unions using ontology enrichment, biological network building and interactome connectivity analyses demonstrated the link between gene signatures and the biological basis of their predictive power. Different signatures for a given end point were more similar at the level of biological properties and transcriptional control than at the gene level. Signatures tended to be enriched in function and pathway in an end point and model-specific manner, and showed a topological bias for incoming interactions. Importantly, the level of biological similarity between different signatures for a given end point correlated positively with the accuracy of the signature predictions. These findings will aid the understanding, and application of predictive genomic signatures, and support their broader application in predictive medicine.

Hepatocellular proliferation in response to a peroxisome proliferator does not require TNFalpha signaling.

Rodents exposed to peroxisome proliferator xenobiotics respond with marked increases in hepatocellular replication and growth that results in tumor formation. Recently, tumor necrosis factor-alpha (TNFalpha) was proposed as the central mediator of this maladaptive response. To define the role of TNFalpha signaling in hepatocellular growth induced by peroxisome proliferators we administered three daily gavage doses of the potent peroxisome proliferator, Wy-14 643, to mice nullizygous for TNF-receptor I (TNFR1), TNFR2, or both receptors. We demonstrate here that regardless of genotype the mice responded with almost identical increases in liver to body weight ratios and hepatocyte proliferation. Lacking evidence that TNFalpha signaling mediates these effects, we then examined the possible contribution of alternative cytokine pathways. Semi-quantitative, reverse transcriptase polymerase chain reaction analysis revealed that wild type mice acutely exposed to Wy-14 643 had increased hepatic expression of Il1beta, Il1r1, Hnf4, and Stat3 genes. Moreover, hepatic adenomas from mice chronically exposed to Wy-14 643 had increased expression of Il1beta, Il1r1, Il6, and Ppargamma1. Expression of Il1alpha, Tnfalpha, Tnfr1, Tnfr2, Pparalpha, or C/ebpalpha was not altered by acute Wy-14 643 exposure or in adenomas induced by Wy-14643. These data suggest that the hepatic mitogenesis and carcinogenesis associated with peroxisome proliferator exposure is not mediated via TNFalpha but instead may involve an alternative pathway requiring IL1beta and IL6.

Altered expression of the carboxylesterases ES-4 and ES-10 by peroxisome proliferator chemicals.

The nonspecific carboxylesterases (EC.3.1.1.1) are a large group of enzymes that play important roles in the metabolism of foreign xenobiotics and endogenous lipids, including activators of the peroxisome proliferator-activated receptor alpha, a nuclear receptor that is the central mediator of peroxisome proliferator (PP) effects in the rodent liver. A number of reports have demonstrated that PP exposure leads to alterations in levels of carboxylesterases in the liver. In this study, we determined by Western blot analysis whether exposure to diverse PP results in alteration of expression of two highly expressed microsomal carboxylesterases. Chronic exposure to the PP WY-14,643 (WY) and gemfibrozil (GEM), but not di-n-butyl phthalate (DBP), led to decreases in ES-4 in male rat livers. ES-4 was increased in female rat livers treated with GEM. WY exposure led to decreases in ES-10 in male and female rat livers. ES-10 was increased in female rats treated with DBP. Compared with other end points that are altered within days after PP exposure, the downregulation of ES-4 and ES-10 by WY was considerably slower, occurring between 1 and 5 weeks of exposure. Decreased expression of ES-4 was observed at doses of WY or GEM as low as 10 or 8000 ppm, respectively, whereas decreased expression of ES-10 was more resistant to changes by any PP occurring only with WY at doses as low as 50 ppm. After chronic exposure to WY or diethylhexyl phthalate in wild-type mice, kidney, but not liver, expression of ES-4 and ES-10 was downregulated. These decreases in kidney ES expression were not observed in PPARalpha-null mice lacking a functional PPARalpha gene, demonstrating the importance of this transcription factor in these changes. These studies demonstrate that ES protein expression is under complex control by PP that is sex- and compound-dependent. These results lend support to the hypothesis that PP exposure leads to a reprogramming of expression of enzymes important in the metabolism of PPARalpha activators.

Role of thyroid hormones in hepatic effects of peroxisome proliferators.

Peroxisome proliferators are endocrine disrupting chemicals that cause liver tumors in rodents but not humans. Although the receptor that mediates key hepatic effects, the peroxisome proliferator-activated receptor alpha (PPAR-alpha), and its endogenous ligands have been identified, the mechanism whereby these commonly used chemicals cause liver tumors in rodents has yet to be elucidated. Species differences in PPAR-alpha and DNA response elements may explain some of the variability in response upon exposure to peroxisome proliferators. The possibility that thyroid-modulating effects of peroxisome proliferators may contribute to the hepatic effects of peroxisome proliferators has yet to be fully explored. When the potent peroxisome proliferator, WY-14,643, was given to hypothyroid rats, there was a blunting of the hepatomegaly and hepatocyte proliferative responses seen in thyroid-intact animals. Acyl-CoA oxidase activity was unaltered by changes in thyroid hormone status. In addition, preliminary evidence indicates that peroxisome proliferators increased hepatic thyroid receptor (TRalpha1) expression, but TRalpha1 levels in liver tumors were similar to those in unexposed animals. Significant differences between humans and rodents with respect to thyroid hormone physiology and metabolism, in conjunction with the results of these studies, may be indicative of yet another mechanism to explain differential sensitivity to hepatic effects of peroxisome proliferators.

Peroxisome proliferator-activated receptor alpha-null mice lack resistance to acetaminophen hepatotoxicity following clofibrate exposure.

The purpose of this study was to investigate whether activation of the nuclear receptor PPARalpha is needed for protection from acetaminophen (APAP) hepatotoxicity produced by repeated administration of the peroxisome proliferator clofibrate (CFB). Female wild-type and PPARalpha-null mice received corn oil vehicle or 500 mg CFB/kg, ip, daily for 10 days. They were then fasted overnight (18 h) and either killed at 4 or 24 h after challenge with 400 mg APAP/kg. Controls received 50% propylene glycol vehicle only. In this model of CFB hepatoprotection, liver injury was assessed by measuring plasma sorbitol dehydrogenase activity and by histopathology at 24 h after APAP challenge. Significant hepatocellular necrosis was evident in both corn oil-pretreated PPARalpha-null and wild-type mice at 24 h after APAP challenge. In agreement with previous studies, CFB-pretreated wild-type mice showed marked protection against APAP toxicity. In contrast, CFB did not provide protection against APAP hepatotoxicity in the PPARalpha-null mice. Similarly, at 4 h after APAP challenge, hepatic glutathione depletion and selective arylation of cytosolic proteins were reduced significantly in CFB-pretreated wild-type mice, but not in PPARalpha-null mice. The lack of changes in APAP binding and NPSH depletion in CFB-pretreated, PPARalpha-null mice is consistent with the presence of significant liver injury at 24 h in this treatment group. These findings demonstrate that the protection against APAP hepatotoxicity by peroxisome proliferator treatment is mediated by the activation of PPARalpha.

Central role of peroxisome proliferator-activated receptors in the actions of peroxisome proliferators.

Peroxisome proliferators (PPs) are a large class of structurally dissimilar chemicals that have diverse effects in rodents and humans. Most, if not all, of the diverse effects of PPs are mediated by three members of the nuclear receptor superfamily called peroxisome proliferator-activated receptors (PPARs). In this review, we define the molecular mechanisms of PPs, including PPAR binding specificity, alteration of gene expression through binding to DNA response elements, and cross talk with other signaling pathways. We discuss the roles of PPARs in growth promotion in rodent hepatocarcinogenesis and potential therapeutic effects, including suppression of cancer growth and inflammation.

Apoptosis, mitosis and cyclophilin-40 expression in regressing peroxisome proliferator-induced adenomas.

Chronic exposure to peroxisome proliferators (PP), including certain industrial and pharmaceutical chemicals, causes liver cancer in rodents. Continuous exposure to PP is needed for tumor development since the frequency of hepatocellular neoplasms is decreased in animals returned to control diet. To determine cellular and molecular events responsible for enhanced growth in PP-induced liver tumors, we evaluated the relationships of WY-14,643 levels, apoptosis, mitosis and cyclophilin-40 (Cyp-40) expression in regressing tumors induced by WY-14,643, a potent PP. Male F344 rats were fed WY-14,643 (0.1%) in the diet for 43 weeks and then switched to control diet for 2, 3, 5 or 36 days. Mean serum and hepatic concentrations of WY-14,643 were decreased as early as 2 days following removal of WY-14,643 as compared with rats continuously fed WY-14,643. Adenomas from rats maintained on WY-14,643 markedly compressed surrounding parenchyma. Evidence of adenoma regression was observed by 3 days of WY-14,643 withdrawal and was characterized by loss of compression. Decreased compression corresponded to increases in the apoptotic index and decreases in the mitotic index in regressing adenomas at 2, 3, and 5 days following the switch to control diet. Cyclophilins are multifunctional receptor proteins involved in numerous signal transduction pathways, including those mediated by cyclosporin, a liver tumor promoter in rats. Cyp-40 expression was markedly increased in adenomas from continuously exposed rats, but expression returned to levels similar to surrounding parenchyma in adenomas after 5 days of WY-14,643 withdrawal. Taken together, these results indicate that WY-14, 643-induced adenomas regress rapidly following withdrawal of the PP in association with declining liver WY-14,643 levels, suggesting that peroxisome proliferator-activated receptor alpha may mediate PP-induced alterations in mitogenic and/or apoptotic regulation in growing tumors, in conjunction with alterations in Cyp-40 signal transduction.

Central role of PPARalpha in the mechanism of action of hepatocarcinogenic peroxisome proliferators.

Peroxisome proliferators (PP) are a large class of structurally dissimilar chemicals. These chemicals have diverse effects in rodents and humans, including regulation of lipid metabolism, growth promotion, and induction of hepatocarcinogenesis. Most, if not all, effects of PP are mediated by three members of the nuclear receptor superfamily called PP-activated receptors (PPAR). In this review, we discuss the evidence that PPARalpha, the predominant PPAR in the, liver is involved in the growth promoting and hepatocarcinogenic effects of PP.

Hepatic expression of acute-phase protein genes during carcinogenesis induced by peroxisome proliferators.

Concern exists regarding peroxisome proliferator (PP) xenobiotic exposure because many PPs are potent hepatocarcinogens in rodents. The mechanism of carcinogenicity induced by PPs is atypical compared with those of other hepatocarcinogens in that the former appears to involve alterations in expression of PP-activated receptor (PPAR) target genes rather than direct mutagenicity. To begin to identify some of these genes, we used differential display to compare mRNA expression between hepatic adenomas and adjacent non-tumor liver from rats fed the potent PP Wy-14643 (WY) for 78 wk. Here, we report increased expression of the acute-phase protein (APP) gene alpha-1 antitrypsin (AT) and decreased expression of alpha2-urinary globulin in the tumors. Similar changes were seen in hepatic adenomas induced by a diethylnitrosamine and phenobarbital protocol, indicating a lack of specificity for PP-induced tumors. Additional APP genes, including ceruloplasmin, haptoglobin, beta-fibrinogen, and alpha1-acid glycoprotein were also upregulated in WY-induced tumors but were downregulated in the livers of rats administered a different PP for 13 wk. Mice treated with either WY or di(2-ethylhexyl) phthalate for 3 wk had decreased hepatic AT expression but increased expression of ceruloplasmin and haptoglobin. PPARalpha-null mice showed no hepatic APP gene alteration after PP treatment but had higher basal expression than did wild-type controls. We conclude that PPARalpha activation by several different PPs leads to dysregulation of hepatic APP gene expression in rats and mice. This dysregulation may indicate alterations in cytokine signaling networks regulating both APP gene expression and hepatocellular proliferation.

Altered bcl-2 family expression during non-genotoxic hepatocarcinogenesis in mice.

Dysregulation of apoptosis is an important component of multistage hepatocarcinogenesis. Members of the bcl-2 protein family are important in the regulation of apoptosis and their expression is altered in several cancers. The objectives of the present study were to determine whether the expression of members of the bcl-2 protein family are altered in mouse liver during acute treatment with non-genotoxic carcinogens and throughout non-genotoxic hepatocarcinogenesis. Acute treatment of B6C3F1 mice with phenobarbital resulted in increased levels of bcl-2 and decreased levels of bax protein, while acute treatment with WY-14,643 resulted in increased bcl-2 and BAG-1 protein in the liver. Following chronic treatment, altered hepatic foci and adenomas were classified as: small-cell, heterogeneous basophilic lesions (spontaneous or tetrachlorodibenzo-p-dioxin-induced); large-cell, homogeneous basophilic lesions (WY-14,643-induced); acidophilic lesions (phenobarbital- or chlordane-induced). Of the small-cell heterogeneous basophilic lesions, 86% of foci (31/36) and 85% of adenomas (35/41) exhibited increased bcl-2 protein levels compared with surrounding normal hepatocytes, whereas only 12.5% of foci (4/36) and 12% of adenomas (5/41) exhibited increased bcl-X(L) levels. Of the large-cell, homogenous, basophilic lesions, 100% of foci (3/3) and 90% of adenomas (9/10) expressed bcl-2 protein, whereas 100% of foci (3/3) and 80% of adenomas (8/10) exhibited increased bcl-X(L) protein levels compared with surrounding normal hepatocytes. Of the acidophilic lesions, the majority of foci (28/32, 88%) and adenomas (47/50, 94%) expressed increased bcl-X(L), whereas increased bcl-2 was observed in only 12.5% of acidophilic preneoplastic foci (4/32) and 14% of acidophilic adenomas (7/50). Of the carcinomas analyzed, 81% expressed increased bcl-2 (54/67), 78% expressed increased bcl-X(L) (52/67) and 69% expressed increased levels of both bcl-2 and bcl-X(L) (46/67). Collectively, only 8% of preneoplastic foci, 3% of adenomas and 1.5% of carcinomas did not express either bcl-2 or bcl-X(L). These results suggest that regulation of apoptotic proteins is altered during non-genotoxic carcinogenesis in mouse liver. Furthermore, there were both chemical- and lesion-specific aspects of expression of apoptotic proteins during hepatocarcinogenesis in mice.

Modulation of testosterone-metabolizing hepatic cytochrome P-450 enzymes in developing Sprague-Dawley rats following in utero exposure to p,p'-DDE.

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) causes sexual developmental aberrations in male rats through a likely mechanism of androgen receptor antagonism. DDE is also known to induce liver cytochrome P-450 (CYP). The expression of CYP enzymes is regulated by steroid hormones, which, in turn, are inactivated in the liver by CYP-catalyzed hydroxylations and subsequent conjugations. This study was undertaken to examine the potential of in utero DDE exposure to affect the developmental expression of the hepatic CYP enzymes that are responsible for testosterone hydroxylations. Pregnant Sprague-Dawley rats were dosed daily by gavage with DDE at 0, 10, or 100 mg/kg body weight or with flutamide at 40 mg/kg body weight from gestation day 14 to 18. Additional adult male rats were given seven daily doses of DDE at 100 mg/kg. Liver samples were collected from the offspring of the dosed dams on postnatal days (PND) 10 and 21 and from the adult rats a day after the last dosing. Assays for regioselective and sterospecific testosterone hydroxylase activities were performed using hepatic microsomal preparations. Specific liver CYP proteins were detected by immunoblotting. While the CYP2B1 and 3A1 and their hydroxylated testosterone products were highly elevated by the DDE treatments in both adult and developing rats, the responses of 2C11 and 2A1 were development-dependent. The flutamide treatment had little effect on CYP enzyme expression. This study demonstrated that developing offspring rats are susceptible to the hepatic CYP enzyme-modulating action of DDE following its administration to the pregnant dams.

Alteration of protein kinase C isoform-specific expression during rat hepatocarcinogenesis after exposure to the peroxisome proliferator WY-14,643.

The role of protein kinase C (PKC) isoforms in mediating peroxisome proliferator chemical- (PPC) induced hepatocarcinogenesis was examined. After an acute gavage exposure to WY-14,643 (WY) membrane-bound PKCdelta and cytosolic PKCbeta decreased, whereas the expression of the other isoforms was not altered. After a 13-week chronic exposure, membrane-bound PKCbeta, delta and zeta levels decreased. In WY-induced hepatocellular adenomas, PKCalpha was increased, and PKCbeta was further decreased in membrane fractions. These results, taken together with previous studies, indicate that alterations in PKCalpha, beta and delta isoforms, which regulate mitogenesis, could play important roles in perpetuating the high cell proliferative rate in PPC-induced hepatocellular adenomas.

Tissue-specific induction of 17 beta-hydroxysteroid dehydrogenase type IV by peroxisome proliferator chemicals is dependent on the peroxisome proliferator-activated receptor alpha.

The 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) family of proteins regulates the levels of the active 17 beta-hydroxy forms of sex steroids. The expression of 17 beta-HSD type IV is induced by peroxisome proliferator chemicals (PPC) in rat liver. In order to characterize more generally the impact of PPC on 17 beta-HSD expression, we determined (1) if expression of other members of the 17 beta-HSD family was coordinately induced by PPC exposure, (2) the tissues in which 17 beta-HSD was induced by PPC, and (3) whether the induction of 17 beta-HSD by PPC was dependent on the peroxisome proliferator-activated receptor alpha (PPAR alpha), the central mediator of PPC effects in the mouse liver. The mRNA levels of 17 beta-HSD I, II, and III were not altered in the liver, kidney, and testis or uterus of rats treated with PPC. The mRNA or 80 kDa a full-length protein levels of 17 beta-HSD IV were strongly induced in liver and kidney, but not induced in adrenals, brown fat, heart, testis, and uterus of rats treated with diverse PPC. In liver and kidneys from treated rats, additional proteins of 66 kDa, 56 kDa, and 32 kDa were also induced which reacted with the anti-17 beta-HSD IV antibodies and were most likely proteolytic fragments of 17 bega-HSD IV. Treatment of mice which lack a functional form of PPAR alpha with PPC, demonstrated that PPC-inducibility of 17 beta-HSD IV mRNA or the 80 kDa protein was dependent on PPAR alpha expression in liver and kidney. Our results demonstrate that 17 beta-HSD IV is induced by PPC through a PPAR alpha-dependent mechanism and support the hypothesis that exposure to PPC leads to alterations in sex steroid metabolism.

Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta.

The rat, mouse and human estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activity of environmental chemicals and phytoestrogens in competition binding assays with ER alpha or ER beta protein, and in a transient gene expression assay using cells in which an acute estrogenic response is created by cotransfecting cultures with recombinant human ER alpha or ER beta complementary DNA (cDNA) in the presence of an estrogen-dependent reporter plasmid. Saturation ligand-binding analysis of human ER alpha and ER beta protein revealed a single binding component for [3H]-17beta-estradiol (E2) with high affinity [dissociation constant (Kd) = 0.05 - 0.1 nM]. All environmental estrogenic chemicals [polychlorinated hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT) and derivatives, alkylphenols, bisphenol A, methoxychlor and chlordecone] compete with E2 for binding to both ER subtypes with a similar preference and degree. In most instances the relative binding affinities (RBA) are at least 1000-fold lower than that of E2. Some phytoestrogens such as coumestrol, genistein, apigenin, naringenin, and kaempferol compete stronger with E2 for binding to ER beta than to ER alpha. Estrogenic chemicals, as for instance nonylphenol, bisphenol A, o, p'-DDT and 2',4',6'-trichloro-4-biphenylol stimulate the transcriptional activity of ER alpha and ER beta at concentrations of 100-1000 nM. Phytoestrogens, including genistein, coumestrol and zearalenone stimulate the transcriptional activity of both ER subtypes at concentrations of 1-10 nM. The ranking of the estrogenic potency of phytoestrogens for both ER subtypes in the transactivation assay is different; that is, E2 >> zearalenone = coumestrol > genistein > daidzein > apigenin = phloretin > biochanin A = kaempferol = naringenin > formononetin = ipriflavone = quercetin = chrysin for ER alpha and E2 >> genistein = coumestrol > zearalenone > daidzein > biochanin A = apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin for ER beta. Antiestrogenic activity of the phytoestrogens could not be detected, except for zearalenone which is a full agonist for ER alpha and a mixed agonist-antagonist for ER beta. In summary, while the estrogenic potency of industrial-derived estrogenic chemicals is very limited, the estrogenic potency of phytoestrogens is significant, especially for ER beta, and they may trigger many of the biological responses that are evoked by the physiological estrogens.

Down-regulation of cytochrome P450 2C family members and positive acute-phase response gene expression by peroxisome proliferator chemicals.

In this study, we show that peroxisome proliferator chemical (PPC) exposure leads to alterations in the expression of genes in rat liver regulated by the sex-specific growth hormone secretory pattern and induced during inflammation. Expression of the male-specific cytochrome P450 (P450) 2C11 and alpha2 urinary globulin (alpha2u) genes and the female-specific P450 2C12 gene was down-regulated by some PPC. Expression of P450 2C13, also under control by the sex-specific growth hormone secretory pattern, was not altered by PPC treatment, indicating that regulation of CYP2C family members does not involve perturbation of the growth hormone secretory pattern. In contrast to the increases in expression observed when inflammation was induced in male rats, two positive acute-phase response genes, alpha1-acid glycoprotein and beta-fibrinogen, were decreased by PPC exposure. The down-regulation of the P450 2C11 by WY-14,643 could be reproduced in cultured rat hepatocytes, indicating the down-regulation is a direct effect. Experiments in wild-type mice and mice that lacked a functional peroxisome proliferator-activated receptor-alpha gene showed that down-regulation by WY of alpha1-acid glycoprotein, beta-fibrinogen, and a mouse homologue of alpha2u was dependent on peroxisome proliferator-activated receptor-alpha expression. Our results demonstrate that PPC exposure leads to down-regulation of diverse liver-specific genes, including CYP2C family members important in hormonal homeostasis and acute-phase response genes important in inflammatory responses.

Alterations in the GAL4 DNA-binding domain can affect transcriptional activation independent of DNA binding.

The GAL4 protein belongs to a large class of fungal transcriptional activator proteins encoding within their DNA-binding domains (DBD) six cysteines that coordinate two atoms of zinc (the Zn2Cys6 domain). In an effort to characterize the interactions between the Zn2Cys6 class transcriptional activator proteins and their DNA-binding sites, we have replaced in the full-length GAL4 protein small regions of the Zn2Cys6 domain with the analogous regions of another Zn2Cys6 protein called PPR1 an activator of pyrimidine biosynthetic genes. Alterations between the first and third cysteines abolished binding to GAL4 (upstream activation sequence of GAL (UASG)) or PPR1 (upstream acitvation sequence of UAS) DNA-binding sites and severely reduced transcriptional activation in yeast. In contrast, alterations between the third and fourth cysteines had only minor effects on binding to UASG but led to substantial decreases in activation in both yeast and a mammalian cell line. In the crystal structure of the GAL4 DBD-UASG complex (Marmorstein, R., Carey, M., Ptashne, M., and Harrison, S. C. (1992) Nature 356, 408-414), this region is facing away from the DNA, making it likely that there exists within the GAL4 DBD an accessible domain important in activation.

Cloning genes responsive to a hepatocarcinogenic peroxisome proliferator chemical reveals novel targets of regulation.

To better understand the molecular basis of the hepatocyte proliferation and induction of hepatocellular adenomas by exposure to peroxisome proliferator chemicals (PPC), a systematic search for genes modulated by a PPC (WY-14643) in rat liver was carried out using the differential display technique. The fragments fell into two classes based on the time of initial and maximal induction by WY-14643. The class I genes (clones 5 and 30) were induced 3 h after a gavage exposure to WY-14643 with maximal expression at 24 h. The class II genes (clones 13 and 16) were induced after 24 h with maximal expression at 78 weeks. Expression of the class II genes was also increased after other treatments that cause cell proliferation. Clone 30 was identified as CYP4A2, previously shown to be regulated by PPC. Clone 13 was homologous to the mouse protein H gene, a component of the heterogeneous nuclear ribonucleoprotein particle important in mRNA splicing. Clone 16 was identified as cyclophilin-A, the receptor for the immunosuppressant drug cyclosporin A. The sequence of clone 5 was unique. These data demonstrate that WY-14643 increases the levels of a number of novel genes that are coordinately regulated with increases in chronic cell proliferation and fatty acid metabolism.