Thomas R. Sutter

Isolation and Characterization of the Human Cytochrome P450 CYP1B1 Gene

Previously, we identified a novel human cytochrome P450 cDNA that is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and represents the first member of a new subfamily designated cytochrome P4501B1 (CYP1B1; Sutter, T. R., Tang, Y. M., Hayes, C. L., Wo, Y. P., Jabs, E. W., Li, X., Yin, H., Cody, C. W., and Greenlee, W. F. (1994) J. Biol. Chem. 269, 13092-13099). Here, we report on the isolation and initial characterization of the CYP1B1 gene. The CYP1B1 gene maps to human chromosome 2 at 2p21-22 and contains three exons and two introns. The putative open reading frame starts in the second exon and is 1629 base pairs in length. Southern analysis using DNA probes directed to each of the three exons confirmed that CYP1B1 is a single copy gene. Human CYP1B1 differs from its two most closely related members of the cytochrome P450 superfamily, CYP1A1 and CYP1A2, in the number of exons (3 versus 7) and chromosome location (2 versus 15). A single transcription initiation site was identified by primer extension analysis and S1 nuclease mapping. Based on nucleotide sequence analysis, the CYP1B1 gene lacks a consensus TATA box in the promoter region and contains nine TCDD-responsive enhancer core binding motifs (5′-GCGTG-3′) located within a 2.5-kilobase pair genomic fragment 5′-ward of the transcription initiation start site. Deletion analysis of chloramphenicol acetyltransferase reporter gene constructs containing 5′ CYP1B1 genomic fragments indicates that a region from −1022 to −835 containing three of the nine core binding motifs contributes to the TCDD-inducible expression of CYP1B1.

The effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on estrogen metabolism in MCF-7 breast cancer cells: Evidence for induction of a novel 17β-estradiol 4-hydroxylase

Rates of microsomal 17 beta-estradiol (E2) hydroxylation at the C-2, -4, -6 alpha, and -15 alpha positions are each induced greater than 10-fold by treating MCF-7 breast cancer cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The TCDD-induced activities at the C-2, -6 alpha and -15 alpha positions have been attributed to cytochrome P450 1A1 (CYP1A1); however, the low Km 4-hydroxylase induced by TCDD appears to be a distinct enzyme. We report here that antibodies to cytochrome P450-EF (mouse CYP1B1) selectivity inhibited the C-4 hydroxylation of E2 catalyzed by microsomes from TCDD-treated MCF-7 cells. Western blots probed with anti-CYP1B antibodies showed the induction of a 52 kDa microsomal protein in response to treatment with TCDD in MCF-7 cells. Western blots of microsomes from HepG2 cells did not show the TCDD-induced 52 kDa protein, and microsomes from TCDD-treated HepG2 cells did not catalyze a low Km hydroxylation of E2 at C-4. Cellular metabolism experiments also showed induction of both the C-2 and -4 hydroxylation pathways in TCDD-treated MCF-7 cells as evidenced by elevated 2- and 4-methoxyestradiol (MeOE2) formation. In contrast, TCDD-treated HepG2 cells showed 2-MeOE2 formation predominantly over 4-MeOE2. Northern blots of RNA isolated from untreated and TCDD-treated cells, when probed with the human CYP1B1 cDNA, showed induction of a 5.2 kb RNA in MCF-7 cells but not in HepG2 cells in response to treatment with TCDD. These results provide additional evidence for the induction by TCDD of a novel E2 4-hydroxylase in MCF-7 cells but not in HepG2 cells and indicate possible endocrine regulatory roles for the newly discovered group of enzymes of the CYP1B subfamily.

Genetic versus chemoprotective activation of Nrf2 signaling: overlapping yet distinct gene expression profiles between Keap1 knockout and triterpenoid-treated mice

Loss of NF-E2-related factor 2 (Nrf2) signaling increases susceptibility to acute toxicity, inflammation and carcinogenesis in mice due to the inability to mount adaptive responses. In contrast, disruption of Keap1 (a cytoplasmic modifier of Nrf2 turnover) protects against these stresses in mice, although inactivating mutations in Keap1 have been identified recently in some human cancers. Global characterization of Nrf2 activation is important to exploit this pathway for chemoprevention in healthy, yet at-risk individuals and also to elucidate the consequences of hijacking the pathway in Keap1-mutant human cancers. Liver-targeted conditional Keap1-null, Albumin-Cre:Keap1((flox/-)) (CKO) mice provide a model of genetic activation of Nrf2 signaling. By coupling global gene expression analysis of CKO mice with analysis of pharmacologic activation using the synthetic oleanane triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), we are able to gain insight into pathways affected by Nrf2 activation. CDDO-Im is an extremely potent activator of Nrf2 signaling. CKO mice were used to identify genes modulated by genetic activation of Nrf2 signaling. The CKO response was compared with hepatic global gene expression changes in wild-type mice treated with CDDO-Im at a maximal Nrf2 activating dose. The results show that genetic and pharmacologic activation of Nrf2 signaling modulates pathways beyond detoxication and cytoprotection, with the largest cluster of genes associated with lipid metabolism. Genetic activation of Nrf2 results in much larger numbers of detoxication and lipid metabolism gene changes. Additionally, analysis of pharmacologic activation suggests that Nrf2 is the primary mediator of CDDO-Im activity, though other cell-signaling targets are also modulated following an oral dose of 30 micromol/kg.

Antioxidant-Inducible Genes

Publisher Summary Antioxidants inhibit the propagation of free radical reactions. Because of their widespread use as preservatives in processed foods, their biochemical effects have been investigated. Early feeding studies indicated that butylated hydroxytoluene (BHT) increased liver weight, induced proliferation of smooth endoplasmic reticulum, and elevated several hepatic microsomal mono-oxygenase activities typical of phase 1 metabolism. In addition to phase1, antioxidants also induce phase2 xenobiotic-metabolizing enzymes. Phase2 enzymes detoxify activated electrophilic metabolites of xenobiotics via conjugation of endogenous substrates such as glutathione (GSH). Antioxidants act to directly terminate the propagation of free radical reactions, and increase the activity of enzymes those readily metabolize and aid in the elimination of potential cytotoxic chemicals. Dietary administration of antioxidants induced phase 2 xenobiotic-metabolizing enzymes. Induction of phase 2 enzymes by antioxidants was found in many organs and tissues, such as liver, lung, kidney, small intestine, colon, and spleen, thereby affording protection at many anatomical sites .This chapter presents evidence for several newly identified antioxidant-inducible genes, describes the proposed mechanisms of antioxidant signal transduction leading to enhanced expression of these enzymes, and complements the information presented in related reviews concerning the mechanisms and consequences of induction of phase 2 xenobiotic-metabolizing enzymes by antioxidants. The chapter discusses the genes induced by antioxidants—cytochrome P450s, Glutathione, S-transferases, NAD(P)H: quinone reductase, UDP-glucuronosyltransferases, microsomal epoxide hydrolase, aflatoxin BI-aldehyde reductase, dihydrodiol dehydrogenases, aldehyde dehydrogenases, enzymes of glutathione and reduced nicotinamide metabolism, and other proteins and enzymes. The chapter delves into the mechanisms of gene induction by antioxidants—the antioxidant-response element (ARE), proteins binding and signal transduction through the ARE—and the consequences of antioxidant gene induction. The regulation of antioxidant-inducible genes helps understand the signals leading to cellular transformation and carcinogenesis. Antioxidants can be used to decipher the encrypted signal transduction pathways that prevent cell transformation and carcinogenesis.

Genetics of the Hippocampal Transcriptome in Mouse: A Systematic Survey and Online Neurogenomics Resource

Differences in gene expression in the CNS influence behavior and disease susceptibility. To systematically explore the role of normal variation in expression on hippocampal structure and function, we generated an online microarray database for a diverse panel of strains of mice, including most common inbred strains and numerous recombinant inbred lines (www.genenetwork.org). Using this resource, coexpression networks for families of genes can be generated rapidly to test causal models related to function. The data set is optimized for quantitative trait locus (QTL) mapping and was used to identify over 5500 QTLs that modulate mRNA levels. We describe a wide variety of analyses and novel synthetic approaches that take advantage of this resource, and demonstrate how both the data and associated tools can be applied to the study of gene regulation in the hippocampus and relations to structure and function.

Increased oxidative DNA damage in livers of 2,3,7,8-tetrachlorodibenzo-p-dioxin treated intact but not ovariectomized rats

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a more potent hepatocarcinogen in female than in male or ovariectomized rats. A possible mechanism for this increased sensitivity is through enhanced metabolic activation of estrogens by TCDD-induced enzymes leading to oxidative damage in the cell. As a marker for oxidative DNA damage, 8-oxo-deoxyguanosine (8-oxo-dG) was quantitated in livers of intact and ovariectomized Sprague-Dawley rats chronically treated with TCDD (125 ng/kg per day) with and without diethylnitrosamine as initiator. Elevated levels of 8-oxo-dG were detected in a significantly greater number of the intact compared to ovariectomized TCDD-treated rats. Expression of CYP1B1 mRNA, a newly identified cytochrome P450 with proposed estrogen hydroxylase activity, was highly induced by TCDD. The results are consistent with the hypothesis that increased metabolism of endogenous estrogens to catechols by TCDD-induced enzymes may lead to increased oxidative DNA damage and hence contribute to TCDD-mediated hepatocarcinogenicity in female rats.

Induction of cytochrome P450 1B1 and catechol estrogen metabolism in ACHN human renal adenocarcinoma cells

The catechol estrogen metabolites of 17beta-estradiol (E2), 2-hydroxyestradiol (OHE2) and 4-OHE2, differ in hormonal properties and carcinogenic potential. In Syrian hamster kidney, 4-OHE2 induces clear-cell carcinoma whereas 2-OHE2 does not, and an E2 4-hydroxylase appears to be involved in E2-induced carcinogenesis in these animals. Specific E2 4-hydroxylase activity has been observed in extrahepatic tissues from several species. In humans, cytochrome P450 1B1 (CYP1B1) appears to be an extrahepatic E2 4-hydroxylase under the regulatory control of the aromatic hydrocarbon receptor (AhR). As an initial approach to investigating CYP1B1 expression and E2 4-hydroxylase activity in human kidney, we used the ACHN cell line, derived from a human renal adenocarcinoma. In untreated ACHN cells, a very low level of CYP1B1 mRNA expression was observed and CYP1B1 protein could not be detected; however, in ACHN cells exposed to the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), CYP1B1 mRNA levels were elevated 28-fold, and the CYP1B1 protein was detected by immunoblot analysis. Exposure of ACHN cells to TCDD resulted in minimal induction of the CYP1A1 mRNA, and the CYP1A1 protein was not detectable prior to or after exposure to TCDD. E2 hydroxylase activity could not be detected with microsomes from untreated ACHN cells, although activities at C-4 and, to a lesser extent, at C-2 of E2 were observed with microsomes from TCDD-treated ACHN cells. In experiments with intact ACHN cells, elevated rates of formation of 4-methoxyestradiol (MeOE2) and 2-MeOE2 were observed in response to treatment with TCDD. The EC50 for induction of the CYP1B1 mRNA was 1.5 nM TCDD; EC50s for the stimulation of 2- and 4-MeOE2 formation were 0.68 and 1.1 nM TCDD. These results indicate that the ACHN cell line may be a useful in vitro model system to study the regulation of CYP1B1 expression and the cytotoxic effects associated with E2 4-hydroxylation.

EGF receptor signaling blocks aryl hydrocarbon receptor-mediated transcription and cell differentiation in human epidermal keratinocytes

Dioxin is an extremely potent carcinogen. In highly exposed people, the most commonly observed toxicity is chloracne, a pathological response of the skin. Most of the effects of dioxin are attributed to its activation of the aryl hydrocarbon receptor (AHR), a transcription factor that binds to the Ah receptor nuclear translocator (ARNT) to regulate the transcription of numerous genes, including CYP1A1 and CYP1B1. In cultures of normal human epidermal keratinocytes dioxin accelerates cell differentiation, as measured by the formation of cornified envelopes. We show that this acceleration is mediated by the AHR; also, that dioxin increases the expression of several genes known to be regulated by ARNT, which have critical roles in the cornification and epidermal barrier function of the skin. Importantly, we demonstrate that all of these responses are opposed by ligand-activation of the EGF receptor (R), an important regulator of keratinocyte cell fate. In the CYP1A1 enhancer, EGFR activation prevents recruitment of the p300 coactivator, although not affecting the binding of the AHR or ARNT. The total cellular level of p300 protein does not decrease, and overexpression of p300 relieves EGFR-mediated repression of transcription, indicating that p300 is a critical target for the repression of the AHR complex by EGFR signaling. These results provide a mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin is able to disrupt epidermal homeostasis and identify EGFR signaling as a regulator of the AHR. This signaling may modulate the incidence and severity of chloracne and be of therapeutic relevance to human poisonings by dioxin.

Epithelial to Mesenchymal Transition in Human Breast Epithelial Cells Transformed by 17β-Estradiol

The estrogen dependence of breast cancer has long been recognized; however, the role of 17β-estradiol (E2) in cancer initiation was not known until we showed that it induces complete neoplastic transformation of the human breast epithelial cells MCF-10F. E2 treatment of MCF-10F cells progressively induced high colony efficiency and loss of ductulogenesis in early transformed (trMCF) cells and invasiveness in Matrigel invasion chambers. The cells that crossed the chamber membrane were collected and identified as bsMCF; their subclones were designated bcMCF; and the cells harvested from carcinoma formation in severe combined immunodeficient mice were designated caMCF. These phenotypes correlated with gene dysregulation during the progression of the transformation. The highest number of dysregulated genes was observed in caMCF, being slightly lower in bcMCF, and lowest in trMCF. This order was consistent with the extent of chromosome aberrations (caMCF > bcMCF >>> trMCF). Chromosomal amplifications were found in 1p36.12-pter, 5q21.1-qter, and 13q21.31-qter. Losses of the complete chromosome 4 and 8p11.21-23.1 were found only in tumorigenic cells. In tumor-derived cell lines, additional losses were found in 3p12.1-14.1, 9p22.1-pter, and 18q11.21-qter. Functional profiling of dysregulated genes revealed progressive changes in the integrin signaling pathway, inhibition of apoptosis, acquisition of tumorigenic cell surface markers, and epithelial-mesenchymal transition. In tumorigenic cells, the levels of E-cadherin, epithelial membrane antigen, and various keratins were low and CD44E/CD24 were negative, whereas SNAI2, vimentin, S100A4, FN1, HRAS, transforming growth factor β1, and CD44H were high. The phenotypic and genomic changes triggered by estrogen exposure that lead normal cells to tumorigenesis confirm the role of this steroid hormone in cancer initiation. [Cancer Res 2007;67(23):11147–57]

How replicable are mRNA expression QTL

Applying quantitative trait analysis methods to genome-wide microarray-derived mRNA expression phenotypes in segregating populations is a valuable tool in the attempt to link high-level traits to their molecular causes. The massive multiple-testing issues involved in analyzing these data make the correct level of confidence to place in mRNA abundance quantitative trait loci (QTL) a difficult problem. We use a unique resource to directly test mRNA abundance QTL replicability in mice: paired recombinant inbred (RI) and F2 data sets derived from C57BL/6J (B6) and DBA/2J (D2) inbred strains and phenotyped using the same Affymetrix arrays. We have one forebrain and one striatum data set pair. We describe QTL replication at varying stringencies in these data. For instance, 78% of mRNA expression QTL (eQTL) with genome-wide adjusted p ≤ 0.0001 in RI data replicate at a genome-wide adjusted p < 0.05 or better. Replicated QTL are disproportionately putatively cis-acting, and approximately 75% have higher apparent expression levels associated with B6 genotypes, which may be partly due to probe set generation using B6 sequence. Finally, we note that while trans-acting QTL do not replicate well between data sets in general, at least one cluster of trans-acting QTL on distal Chr 1 is notably preserved between data sets.