Oral arsenic administration to humanizedUDP-glucuronosyltransferase1 neonatal mice induces UGT1A1 through a dependence on Nrf2 and PXR.

Inorganic arsenic (iAs) is an environmental toxicant that can lead to severe health consequences, which can be exacerbated if exposure occurs early in development. Here, we evaluated the impact of oral iAs treatment on UDP-glucuronosyltransferase 1A1 (UGT1A1) expression and bilirubin metabolism in humanized UGT1 (hUGT1) mice. We found that oral administration of iAs to neonatal hUGT1 mice that display severe neonatal hyperbilirubinemia leads to induction of intestinal UGT1A1 and a reduction in total serum bilirubin values. Oral iAs administration accelerates neonatal intestinal maturation, an event that is directly associated with UGT1A1 induction. As a reactive oxygen species producer, oral iAs treatment activated the Keap-Nrf2 pathway in the intestinal tract and liver. When Nrf2-deficient hUGT1 mice (hUGT1/Nrf2-/-) were treated with iAs, it was shown that activated Nrf2 contributed significantly toward intestinal maturation and UGT1A1 induction. However, hepatic UGT1A1 was not induced upon iAs exposure. We previously demonstrated that the nuclear receptor PXR represses liver UGT1A1 in neonatal hUGT1 mice. When PXR was deleted in hUGT1 mice (hUGT1/Pxr-/-), derepression of UGT1A1 was evident in both liver and intestinal tissue in neonates. Furthermore, when neonatal hUGT1/Pxr-/- mice were treated with iAs, UGT1A1 was superinduced in both tissues, confirming PXR release derepressed key regulatory elements on the gene that could be activated by iAs exposure. With iAs capable of generating reactive oxygen species in both liver and intestinal tissue, we conclude that PXR deficiency in neonatal hUGT1/Pxr-/- mice allows greater access of activated transcriptional modifiers such as Nrf2 leading to superinduction of UGT1A1.

NCoR1 Protects Mice From Dextran Sodium Sulfate-Induced Colitis by Guarding Colonic Crypt Cells From Luminal Insult.

BACKGROUND & AIMS: Colonic stem cells are essential for producing the mucosal lining, which in turn protects stem cells from insult by luminal factors. Discovery of genetic and biochemical events that control stem cell proliferation and differentiation can be leveraged to decipher the causal factors of ulcerative colitis and aid the development of more effective therapy. METHODS: We performed in vivo and in vitro studies from control (nuclear receptor corepressor 1 [NCoR1F/F]) and intestinal epithelial cell-specific NCoR1-deficient mice (NCoR1ΔIEC). Mice were challenged with dextran sodium sulfate to induce experimental ulcerative colitis, followed by colitis examination, barrier permeability analysis, cell proliferation immunostaining assays, and RNA sequencing analysis. By using crypt cultures, the organoid-forming efficiency, cell proliferation, apoptosis, and histone acetylation were analyzed after butyrate and/or tumor necrosis factor α treatments. RESULTS: NCoR1ΔIEC mice showed a dramatic increase in disease severity in this colitis model, with suppression of proliferative cells at the crypt base as an early event and a concomitant increase in barrier permeability. Genome expression patterns showed an important role for NCoR1 in colonic stem cell proliferation and secretory cell differentiation. Colonic organoids cultured from NCoR1ΔIEC mice were more sensitive to butyrate-induced cell growth inhibition and apoptosis, which were exaggerated further by tumor necrosis factor α co-treatment, which was accompanied by increased histone acetylation. CONCLUSIONS: NCoR1 regulates colonic stem cell proliferation and secretory cell differentiation. When NCoR1 is disrupted, barrier protection is weakened, allowing luminal products such as butyrate to penetrate and synergistically damage the colonic crypt cells. Transcript profiling: RNA sequencing data have been deposited in the GEO database, accession number: GSE136153.

NRF2-Independent Regulation of Intestinal Constitutive Androstane Receptor by the Pro-Oxidants Cadmium and Isothiocyanate in hUGT1 Mice.

Environmental toxicants such as heavy metals from contaminated water or soil and isothiocyanates (ITC) from dietary sources act as pro-oxidants by directly generating reactive oxygen species (ROS) or through depleting cellular antioxidants such as glutathione. Toxicants can alter drug metabolism, and it was reported that CYP2B10 and UGT1A1 are induced by phenethyl isothiocyanate (PEITC) through the constitutive androstane receptor (CAR). The possibility that nuclear factor erythroid 2-related factor 2 (NRF2), the master regulator of the antioxidant response, could coactivate CAR was investigated in neonatal hUGT1/Nrf2 -/- mice. Neonatal mice were treated with PEITC or cadmium (Cd2+) by oral gavage for 2 days. Both PEITC and Cd2+ induced UGT1A1 RNA and protein in intestinal tissues in both hUGT1/Nrf2 +/- and hUGT1/Nrf2 -/- neonates, indicating NRF2-independent regulation of UGT1A1. Increases in CYP2B10 RNA in intestinal tissues were observed following PEITC or Cd2+ exposure. Activation of intestinal CAR by Cd2+ exposure was directly assessed by nuclear fractionation and Western blot analyses at 0.5, 1, 2, and 4 hours after treatment in hUGT1 neonates and after 48 hours in hUGT1/Nrf2 +/- and hUGT1/Nrf2 -/- neonates. CAR localized to the nucleus independently of NRF2 48 hours after exposure. Substantial CAR localization to the nucleus occurred at the 2- and 4-hour time points, coinciding with a decrease in phosphorylation of cytoplasmic extracellular signal-regulated kinases 1 and 2 and a nuclear increase in P38/p-P38 content. This suggests that a novel oxidative stress-MAPK-CAR axis exists with phenotypic consequences. SIGNIFICANCE STATEMENT: Pro-oxidant toxicants can alter drug metabolism through activation of CAR, independent of the NRF2-KEAP1 signaling pathway. Changes in proteins associated with drug metabolism and linked to increases in intestinal maturation are mediated through an oxidative stress-MAPK-CAR axis.

Crypt Organoid Culture as an in Vitro Model in Drug Metabolism and Cytotoxicity Studies.

The gastrointestinal tract is enriched with xenobiotic processing proteins that play important roles in xenobiotic bioactivation, metabolism, and detoxification. The application of genetically modified mouse models has been instrumental in characterizing the function of xenobiotic processing genes (XPG) and their proteins in drug metabolism. Here, we report the utilization of three-dimensional crypt organoid cultures from these animal models to study intestinal drug metabolism and toxicity. With the successful culturing of crypt organoids, we profiled the abundance of Phase I and Phase II XPG expression, drug transporter gene expression, and xenobiotic nuclear receptor (XNR) gene expression. Functions of XNRs were examined by treating crypt cells with XNR prototypical agonists. Real-time quantitative polymerase chain reaction demonstrated that the representative downstream target genes were induced. These findings were validated from cultures developed from XNR-null mice. In crypt cultures isolated from Pxr-/- mice, pregnenolone 16α-carbonitrile failed to induce Cyp3a11 gene expression; similarly, WY14643 failed to induce Cyp4a10 in the Pparα-/- crypts. Crypt cultures from control (Ugt1F/F ) and intestinal epithelial cell (IEC) specific Ugt1 null mice (Ugt1ΔIEC ) were treated with camptothecin-11, an anticancer prodrug with severe intestinal toxicity that originates from insufficient UGT1A1-dependent glucuronidation of its active metabolite SN-38. In the absence of Ugt1 gene expression, Ugt1ΔIEC crypt cultures exhibit very limited production of SN-38 glucuronide, concordant with increased apoptosis in comparison with Ugt1F/F crypt cultures. This study suggests crypt organoid cultures as an effective in vitro model for studying intestinal drug metabolism and toxicity.

Isothiocyanates induce UGT1A1 in humanized UGT1 mice in a CAR dependent fashion that is highly dependent upon oxidative stress.

Isothiocyanates, such as phenethyl isothiocyanate (PEITC), are formed following the consumption of cruciferous vegetables and generate reactive oxygen species (ROS) that lead to the induction of cytoprotective genes such as the UDP-glucuronosyltransferases (UGTs). The induction of ROS activates the Nrf2-Keap 1 pathway leading to the induction of genes through antioxidant response elements (AREs). UGT1A1, the sole enzyme responsible for the metabolism of bilirubin, can be induced following activation of Nrf2. When neonatal humanized UGT1 (hUGT1) mice, which exhibit severe levels of total serum bilirubin (TSB) because of a developmental delay in expression of the UGT1A1 gene, were treated with PEITC, TSB levels were reduced. Liver and intestinal UGT1A1 were induced, along with murine CYP2B10, a consensus CAR target gene. In both neonatal and adult hUGT1/Car-/- mice, PEITC was unable to induce CYP2B10. A similar result was observed following analysis of UGT1A1 expression in liver. However, TSB levels were still reduced in hUGT1/Car-/- neonatal mice because of ROS induction of intestinal UGT1A1. When oxidative stress was blocked by exposing mice to N-acetylcysteine, induction of liver UGT1A1 and CYP2B10 by PEITC was prevented. Thus, new findings in this report link an important role in CAR activation that is dependent upon oxidative stress.

Intestinal NCoR1, a regulator of epithelial cell maturation, controls neonatal hyperbilirubinemia.

Severe neonatal hyperbilirubinemia (SNH) and the onset of bilirubin encephalopathy and kernicterus result in part from delayed expression of UDP-glucuronosyltransferase 1A1 (UGT1A1) and the inability to metabolize bilirubin. Although there is a good understanding of the early events after birth that lead to the rapid increase in serum bilirubin, the events that control delayed expression of UGT1A1 during development remain a mystery. Humanized UGT1 (hUGT1) mice develop SNH spontaneously, which is linked to repression of both liver and intestinal UGT1A1. In this study, we report that deletion of intestinal nuclear receptor corepressor 1 (NCoR1) completely diminishes hyperbilirubinemia in hUGT1 neonates because of intestinal UGT1A1 gene derepression. Transcriptomic studies and immunohistochemistry analysis demonstrate that NCoR1 plays a major role in repressing developmental maturation of the intestines. Derepression is marked by accelerated metabolic and oxidative phosphorylation, drug metabolism, fatty acid metabolism, and intestinal maturation, events that are controlled predominantly by H3K27 acetylation. The control of NCoR1 function and derepression is linked to IKKß function, as validated in hUGT1 mice with targeted deletion of intestinal IKKß. Physiological events during neonatal development that target activation of an IKKß/NCoR1 loop in intestinal epithelial cells lead to derepression of genes involved in intestinal maturation and bilirubin detoxification. These findings provide a mechanism of NCoR1 in intestinal homeostasis during development and provide a key link to those events that control developmental repression of UGT1A1 and hyperbilirubinemia.