Pharmacogenomics of Chemically Distinct Classes of Keap1-Nrf2 Activators Identify Common and Unique Gene, Protein, and Pathway Responses In Vivo.

The Kelch-like erythroid-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) signaling pathway is the subject of several clinical trials evaluating the effects of Nrf2 activation on the prevention of cancer and diabetes and the treatment of chronic kidney disease and multiple sclerosis. 3H-1,2-dithiole-3-thione (D3T) and 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) are representative members of two distinct series of Nrf2 chemical activators. Previous reports have described activator-specific effects on Nrf2-dependent gene regulation and physiologic outcomes. Here we used a robust chemical genomics approach to characterize expression profiles between D3T and CDDO-Im in livers from wild-type and Nrf2-null mice. At equally efficacious doses in wild-type mice, 406 genes show common RNA responses to both treatments. These genes enriched the Nrf2-regulated pathways of antioxidant defense and xenobiotic metabolism. In addition, 197 and 745 genes were regulated uniquely in response to either D3T or CDDO-Im, respectively. Functional analysis of the D3T-regulated set showed a significant enrichment of Nrf2-regulated enzymes involved in cholesterol biosynthesis. This result was supported by Nrf2-dependent increases in lanosterol synthase and CYP51 protein expression. CDDO-Im had no effect on cholesterol biosynthesis regardless of the dose tested. However, unlike D3T, CDDO-Im resulted in Nrf2-dependent elevation of peroxisome proliferator α and Kruppel-like factor 13, as well as the coactivator peroxisome proliferator γ coactivator 1ß, together indicating regulation of ß-oxidation and lipid metabolic pathways. These findings provide novel insights into the pharmacodynamic action of these two activators of Keap1-Nrf2 signaling. Although both compounds modify Keap1 to affect canonical cytoprotective gene expression, additional unique sets of Nrf2-dependent genes were regulated by each agent with enrichment of selective metabolic pathways.

EGFR regulation of epidermal barrier function.

Keratinocyte terminal differentiation is the process that ultimately forms the epidermal barrier that is essential for mammalian survival. This process is controlled, in part, by signal transduction and gene expression mechanisms, and the epidermal growth factor receptor (EGFR) is known to be an important regulator of multiple epidermal functions. Using microarray analysis of a confluent cell density-induced model of keratinocyte differentiation, we identified 2,676 genes that are regulated by epidermal growth factor (EGF), a ligand of the EGFR. We further discovered, and separately confirmed by functional assays, that EGFR activation abrogates all of the known essential processes of keratinocyte differentiation by 1) decreasing the expression of lipid matrix biosynthetic enzymes, 2) regulating numerous genes forming the cornified envelope, and 3) suppressing the expression of tight junction proteins. In organotypic cultures of skin, EGF acted to impair epidermal barrier integrity, as shown by increased transepidermal water loss. As defective epidermal differentiation and disruption of barrier function are primary features of many human skin diseases, we used bioinformatic analyses to identify genes that are known to be associated with skin diseases. Compared with non-EGF-regulated genes, EGF-regulated genes were significantly enriched for skin disease genes. These results provide a systems-level understanding of the actions of EGFR signaling to inhibit keratinocyte differentiation, providing new insight into the role of EGFR imbalance in skin pathogenesis.

Chemical genomics of cancer chemopreventive dithiolethiones.

3H-1,2-dithiole-3-thione (D3T) and its analogues 4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (OLT) and 5-tert-butyl-3H-1,2-dithiole-3-thione (TBD) are chemopreventive agents that block or diminish early stages of carcinogenesis by inducing activities of detoxication enzymes. While OLT has been used in clinical trials, TBD has been shown to be more efficacious and possibly less toxic than OLT in animals. Here, we utilize a robust and high-resolution chemical genomics procedure to examine the pharmacological structure-activity relationships of these compounds in livers of male rats by microarray analyses. We identified 226 differentially expressed genes that were common to all treatments. Functional analysis identified the relation of these genes to glutathione metabolism and the nuclear factor, erythroid derived 2-related factor 2 pathway (Nrf2) that is known to regulate many of the protective actions of dithiolethiones. OLT and TBD were shown to have similar efficacies and both were weaker than D3T. In addition, we identified 40 genes whose responses were common to OLT and TBD, yet distinct from D3T. As inhibition of cytochrome P450 (CYP) has been associated with the effects of OLT on CYP expression, we determined the half maximal inhibitory concentration (IC(50)) values for inhibition of CYP1A2. The rank order of inhibitor potency was OLT >> TBD >> D3T, with IC(50) values estimated as 0.2, 12.8 and >100 microM, respectively. Functional analysis revealed that OLT and TBD, in addition to their effects on CYP, modulate liver lipid metabolism, especially fatty acids. Together, these findings provide new insight into the actions of clinically relevant and lead dithiolethione analogues.

CYP1B1 is not a major determinant of the disposition of aromatase inhibitors in epithelial cells of invasive ductal carcinoma.

CYP1B1 and CYP19 (aromatase) have been shown to be expressed in breast tumors. Both enzymes are efficient estrogen hydroxylases, indicating the potential for overlapping substrate and inhibitor specificity. We measured the inhibition properties of aromatase inhibitors (AIs) against CYP1B1-catalyzed hydroxylation of 17beta-estradiol (E2) to determine whether CYP1B1 affects the disposition of AIs. In addition, we estimated the frequency of coexpression of these enzymes in breast tumor epithelium. Immunohistochemical analyses of CYP19 and CYP1B1 in a panel of 29 cases of invasive ductal carcinoma of the breast showed epithelial cell staining for CYP19 in 76% and for CYP1B1 in 97% of the samples. Statistical analysis showed no significant correlation (0.33) for positive expression of CYP19 and CYP1B1 (p > 0.07). CYP1B1 inhibition was determined for two steroidal inhibitors: formestane and exemestane and five nonsteroidal inhibitors: aminoglutethimide, fadrozole, anastrozole, letrozole, and vorozole. Of the seven compounds tested, only vorozole exhibited inhibition of CYP1B1 activity with IC(50) values of 17 and 21 microM for 4-hydroxy estradiol and 2-hydroxy estradiol, respectively. The estimated K(i) values of vorozole for E2 4- and 2-hydroxylation were 7.26 and 6.84 microM, respectively. Spectrophotometric studies showed that vorozole was a type II inhibitor of CYP1B1. This study shows that with the exception of vorozole, the aromatase inhibitors are selective for CYP19 relative to CYP1B1. Thus, although both CYP19 and CYP1B1 are expressed in a high percentage of breast cancers, CYP1B1 is not a major determinant of the disposition of AIs.

Effects of in utero exposure of C57BL/6J mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin on epidermal permeability barrier development and function.

BACKGROUND: Development of the epidermal permeability barrier (EPB) is essential for neonatal life. Defects in this barrier are found in many skin diseases such as atopic dermatitis. OBJECTIVE: We investigated the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the development and function of the EPB. METHODS: Timed-pregnant C57BL/6J mice were gavaged with corn oil or TCDD (10 µg/kg body weight) on gestation day 12. Embryos were harvested on embryonic day (E) 15, E16, E17, and postnatal day (PND) 1. RESULTS: A skin permeability assay showed that TCDD accelerated the development of the EPB, beginning at E15. This was accompanied by a significant decrease in transepidermal water loss (TEWL), enhanced stratification, and formation of the stratum corneum (SC). The levels of several ceramides were significantly increased at E15 and E16. PND1 histology revealed TCDD-induced acanthosis and epidermal hyperkeratosis. This was accompanied by disrupted epidermal tight junction (TJ) function, with increased dye leakage at the terminal claudin-1-staining TJs of the stratum granulosum. Because the animals did not have enhanced rates of TEWL, a commonly observed phenotype in animals with TJ defects, we performed tape-stripping. Removal of most of the SC resulted in a significant increase in TEWL in TCDD-exposed PND1 pups compared with their control group. CONCLUSIONS: These findings demonstrate that in utero exposure to TCDD accelerates the formation of an abnormal EPB with leaky TJs, warranting further study of environmental exposures, epithelial TJ integrity, and atopic disease.

Analysis of the CYP1A1 mRNA dose-response in human keratinocytes indicates that relative potencies of dioxins, furans, and PCBs are species and congener specific.

Reports indicate that toxic equivalency factors (TEFs) based primarily on rodent data do not accurately predict in vitro human responsiveness to certain dioxin-like chemicals (DLCs). To investigate this in cells responsive to dioxins and relevant to chloracne, normal human epidermal keratinocytes were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and several DLCs, each with a TEF value of 0.1, representing three classes of congeners. We estimated half maximal effective concentration (EC50)-based donor-specific relative potency (REP) values for cytochrome P450 1A1 (CYP1A1) messenger RNA (mRNA) induction for TCDD, 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (HxCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,6,7,8-hexachlorodibenzofuran (HxCDF), and 3,3',4,4',5-pentachlorobiphenyl (PCB 126). We also determined EC50-based population-level REP values (n = 4) for CYP1A1 mRNA induction for TCDD, HxCDF, and PCB 126. Furthermore, an alternative factor, the relative threshold factor (RTF) based on the low end (threshold) of the dose-response curve, was calculated. Our results demonstrated that HxCDF had a population-based REP value of 0.98, 9.8-fold higher than its assigned TEF value of 0.1. Conversely, PCB 126 had an REP value of 0.0027 and an RTF of 0.0022, 37-fold and 45-fold less than its assigned TEF of 0.1, respectively. The REP values for HxCDD and TCDF were 0.24 and 0.10, respectively, similar to their assigned value of 0.1. Therefore, although the DLCs tested in the current study all possessed the same assigned TEF value of 0.1, congener-specific differences in REPs and RTFs were observed for human keratinocytes. These congener-specific discrepancies are likely because of differences in interspecies factors that have yet to be defined.