Guillermo Elizondo

CYP3A4 allelic variants with amino acid substitutions in exons 7 and 12 : Evidence for an allelic variant with altered catalytic activity

To determine the existence of mutant and variant CYP3A4 alleles in three racial groups and to assess functions of the variant alleles by complementary deoxyribonucleic acid (cDNA) expression.

Over‐production of IFN‐γ and IL‐12 in AhR‐null mice

The aryl hydrocarbon receptor (AhR) is a ligand‐activated transcription factor that mediates toxicity of environmental pollutants such as 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin. The exposure to AhR agonists results in profound suppression of cellular and humoral immune responses and compromises host to infectious disease. Therefore, to define the role of AhR in the immune response, spleen cells from ovalbumin (OVA)‐immunized and naïve mice were removed and stimulated in vitro with either OVA or mitogen concanavalin‐A (Con A), respectively. Proliferation, CD19+, F4/80+, CD4+ and CD8+ T cells expansion and cytokines production were measured in C57BL/6‐AhR−/− mice (AhR−/−) and compared with immune response in similarly immunized age‐matched wild type (AhR+/+) mice. In response to OVA immunization, AhR−/− mice had similar levels of serum OVA‐specific IgG2a, IgG1, and IgG2b compared with AhR+/+ animals. However, AhR−/− mice showed splenomegalia and an increase in B cells. No changes were observed on proliferation and IL‐4 secretion, although AhR−/− cells produced more IFN‐γ and IL‐12 than AhR+/+ cells. Similar results were observed with Con A stimulation, a decrease on IL‐5 and no change on IL‐2 secretion were observed on AhR−/− cells compared with AhR+/+ cells in response to Con A stimulation. High levels of IFN‐γ mRNA were detected in AhR−/− lymphocytes, but IL‐4 mRNA levels in AhR−/− cells were similar to those in AhR+/+ mice. These data suggest that AhR may play an important role in the normal development and function of immune system by down‐regulating IFN‐γ and IL‐12 expression.

The unexpected role for the aryl hydrocarbon receptor on susceptibility to experimental toxoplasmosis.

The aryl hydrocarbon receptor (AhR) is part of a signaling system that is mainly triggered by xenobiotic agents. Increasing evidence suggests that AhR may regulate immunity to infections. To determine the role of AhR in the outcome of toxoplasmosis, we used AhR−/− and wild-type (WT) mice. Following an intraperitoneal infection with Toxoplasma gondii (T. gondii), AhR−/− mice succumbed significantly faster than WT mice and displayed greater liver damage as well as higher serum levels of tumor necrosis factor (TNF)-α, nitric oxide (NO), and IgE but lower IL-10 secretion. Interestingly, lower numbers of cysts were found in their brains. Increased mortality was associated with reduced expression of GATA-3, IL-10, and 5-LOX mRNA in spleen cells but higher expression of IFN-γ mRNA. Additionally, peritoneal exudate cells from AhR−/− mice produced higher levels of IL-12 and IFN-γ but lower TLR2 expression than WT mice. These findings suggest a role for AhR in limiting the inflammatory response during toxoplasmosis.

Retinoic acid modulates retinaldehyde dehydrogenase 1 gene expression through the induction of GADD153–C/EBPβ interaction

Mammalian class I aldehyde dehydrogenase (ALDH) plays an important role in the biosynthesis of the hormone retinoic acid (RA), which modulates gene expression and cell differentiation. RA has been shown to mediate control of human ALDH1 gene expression through modulation of the retinoic acid receptor alpha (RARalpha) and the CCAAT/enhancer binding protein beta (C/EBPbeta). The positive activation of these transcription factors on the ALDH1 promoter is inhibited by RA through a decrease of C/EBPbeta binding to the ALDH1 CCAAT box response element. However, the mechanism of this effect remains unknown. Here we report that the RARalpha/retinoid X receptor beta (RXRbeta) complex binds to the mouse retinaldehyde dehydrogenase 1 (Raldh1) promoter at a non-consensus RA response element (RARE) with similar affinity to that of the consensus RARE. We found that C/EBPbeta binds to a Raldh1 CCAAT box located at -82/-58bp, adjacent to the RARE. Treatment with RA increases GADD153 and GADD153-C/EBPbeta interaction resulting in a decreased cellular availability of C/EBPbeta for binding to the Raldh1 CCAAT box. These data support a model in which high RA levels inhibit Raldh1 gene expression by sequestering C/EBPbeta through its interaction to GADD153.

Reversal of liver fibrosis in aryl hydrocarbon receptor null mice by dietary vitamin A depletion

Aryl hydrocarbon receptor (AHR)‐null mice display a liver fibrosis phenotype that is associated with a concomitant increase in liver retinoid concentration, tissue transglutaminase type II (TGaseII) activity, transforming growth factorβ (TGFβ) overexpression, and accumulation of collagen. To test the hypothesis that this phenotype might be triggered by the observed increase in liver retinoid content, we induced the condition of retinoid depletion by feeding AHR‐null mice a vitamin A‐ deficient diet with the purpose to reverse the phenotype. Liver retinoid content decreased sharply within the first few weeks on the retinoid‐deficient diet. Analysis of TGFβ1, TGFβ2, and TGFβ3 expression revealed a reduction to control levels in the AHR ‐/‐ mice accompanied by parallel changes in TGaseII protein levels. In addition, we observed an increase in the TGFβ receptors, TGFβ RI and TGFβ RII, as well as in Smad4, and their reduction to wild‐type mouse liver levels in AHR ‐/‐ mice fed the retinoid‐deficient diet. Reduction of peroxisomal proliferator‐activated receptor γ (PPARγ) messenger RNA (mRNA) and protein levels in AHR ‐/‐ mice was consistent with the presence of hepatic stellate cell (HSC) activation and liver fibrosis. Vitamin A deficiency normalized PPARγ expression in AHR ‐/‐ mice. In conclusion, livers from AHR ‐/‐ mice fed the vitamin A‐deficient diet showed a decrease in collagen deposition, consistent with the absence of liver fibrosis. (HEPATOLOGY 2004;39:157–166.)

Ube2l3 gene expression is modulated by activation of the aryl hydrocarbon receptor: implications for p53 ubiquitination.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a halogenated aromatic hydrocarbon and environmental contaminant, results in several deleterious effects, including fetal malformation and cancer. These effects are mediated by the aryl hydrocarbon receptor (AhR), a ligand-activated receptor that regulates the expression of genes encoding xenobiotic-metabolizing enzymes. Several reports suggest that AhR function is beyond the adaptive chemical response. In the present study, we analyzed and compared gene expression profiles of C57BL/6N wild-type (WT) and Ahr-null mice. DNA microarray and quantitative RT-PCR analyses revealed changes in the expression of genes involved in the ubiquitin-proteasome system (UPS). UPS has an important role in cellular homeostasis control and dysfunction of this pathway has been implicated in the development of several human pathologies. Protein ubiquitination is a multi-step enzymatic process that regulates the stability, function, and/or localization of the modified proteins. This system is highly regulated post-translationally by covalent modifications. However, little information regarding the transcriptional regulation of the genes encoding ubiquitin (Ub) proteins is available. Therefore, we investigated the role of the AhR in modulation of the UPS and regulation of Ube2l3 transcription, an E2 ubiquitin-conjugating enzyme, as well as the effects on p53 degradation. Our results indicate that AhR inactivation decreases on liver proteasome activity, probably due to a down-regulation on the expression of several proteasome subunits. On the other hand, AhR activation increases Ube2l3 mRNA and protein levels by controlling Ube2l3 gene expression, resulting in increased p53 ubiquitination and degradation. In agreement with this, induction of apoptosis was attenuated by the AhR activation.

CYP2D6 genotype and phenotype in Amerindians of Tepehuano origin and Mestizos of Durango, Mexico.

Although the drug‐metabolizing enzyme CYP2D6 has been studied extensively in subjects of differing ethnicities, limited CYP2D6 pharmacogenetic data are available for the Amerindian population and Mestizos of Mexico. Dextromethorphan hydroxylation phenotype was studied in Tepehuano Amerindian (n = 58) and Mestizo (n = 88) subjects, and 195 individuals (85 Tepehuano Amerindians and 110 Mestizos) were genotyped by polymerase chain reaction‐restriction fragment length polymorphism methods to identify the frequencies of the CYP2D6*3, *4, *6, and *10 alleles. Tepehuano Amerindian subjects lacked the poor metabolizer (PM) phenotype, whereas in Mestizos the PM phenotype frequency was 6.8%. The CYP2D6*3, *6, and *10 alleles were not found in Tepehuano Amerindians. The CYP2D6*4 allele had a low frequency (0.006) in this Amerindian group. In the Mestizo group, the CYP2D6*3, *4, and *10 alleles had frequencies of 0.009, 0.131, and 0.023, respectively. The CYP2D6*6 allele was not found in Mestizos. The genotype‐phenotype association was strongly statistically significant (r2 = .45; P = .005) in Mestizos. The Tepehuano population was found to have a low phenotypic and genotypic CYP2D6 diversity and differed from other Amerindian groups. On the other hand, the frequencies of the CYP2D6 variant alleles in Mestizos were similar to those reported for whites.

Induction of CYP3A4 by 1α,25-dyhydroxyvitamin D3 in HepG2 cells

Abstract CYP3A4, the predominant cytochrome P450 (CYP) expressed in human liver, contributes to the metabolism of approximately half the drugs in use today. In general, human-derived cell lines fail to express CYPs. It was previously shown that CYP3A4 mRNA and CYP3A immunoreactive protein are induced by 1α,25-dyhydroxyvitamin D 3 (1α,25-(OH) 2 D 3 ) in the human colon carcinoma cell line Caco-2. The aim of the present study was to examine whether 1α,25-(OH) 2 D 3 regulates CYP3A4 gene expression in HepG2 cells, a human hepatocarcinoma cell line. Treatment with 1α,25-(OH) 2 D 3 resulted in an induction of CYP3A4 mRNA and CYP3A4 immunoreactive protein, 1.5-fold and 4.0-fold respectively, when compared to control cultures, in a time-dependent fashion. These observations are in agreement with previous reports suggesting a role of 1α,25-(OH) 2 D 3 on CYP3A4 transcription regulation, and demonstrate that this hormone, as in Caco-2 cells, increase CYP3A4 levels in HepG2 cells. In conclusion, HepG2 cell cultures treated with 1α,25-(OH) 2 D 3 , provides a useful model to study the function of CYP3A4 and its role in drug liver metabolism.

Pregnane X Receptor-Dependent Induction of the CYP3A4 Gene by o,p′-1,1,1,-Trichloro-2,2-Bis (p-Chlorophenyl)ethane

CYP3A4, the predominant cytochrome P450 (P450) expressed in human liver and intestine, contributes to the metabolism of approximately half the drugs in clinical use today. CYP3A4 catalyzes the 6β-hydroxylation of a number of steroid hormones and is involved in the bioactivation of environmental procarcinogens. The expression of CYP3A4 is affected by several stimuli, including environmental factors such as insecticides and pesticides. The o,p′-1,1,1,-trichloro-2,2-bis (p-chlorophenyl)ethane (DDT) isomer of DDT comprises approximately 20% of technical grade DDT, which is an organochloride pesticide. We have recently shown that o,p′-DDT exposure increases CYP3A4 mRNA levels in HepG2 cells. To determine the mechanism by which o,p′-DDT induces CYP3A4 expression, transactivation and electrophoretic mobility shift assays were carried out, revealing that o,p′-DDT activates the CYP3A4 gene promoter through the pregnane X receptor (PXR). CYP3A4 gene promoter activation resulted in both an increase in CYP3A4 mRNA levels and an increase in the total CYP3A4 activity in HepG2 cells. We also observed induction of CYP3A4 and mouse Cyp3a11 mRNA in the intestine of CYP3A4-transgenic mice after exposure to 1 mg/kg o,p′-DDT. At higher doses, a decrease of CYP3A4 inducibility was observed together with an increase in levels of interleukin 6 mRNA, a proinflammatory cytokine that strongly represses CYP3A4 transcription. The present study indicates that regulation of other genes under PXR control may be altered by o,p′-DDT exposure.

Aryl hydrocarbon receptor influences nitric oxide and arginine production and alters M1/M2 macrophage polarization.

AIMS The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxicity of environmental pollutants. It is also implicated in the regulation of the immune system. Ahr-null macrophages overproduce several proinflammatory cytokines following LPS-mediated stimulation, suggesting that AHR affects the balance between the inflammatory M1 and anti-inflammatory M2 phenotypes. Therefore, the present study aimed to examine whether the loss of AHR modifies macrophage polarization. MATERIALS AND METHODS Peritoneal macrophages from wild-type and Ahr-null mice were differentiated into M1 or M2 phenotype by treatment with LPS/IFNγ or IL-4, and several M1 and M2 markers were determined by qPCR and ELISA assays. Macrophage phagocytic capacity was determined through phagocytosis of yeast and Leishmania major infection assays. Nitric oxide (NO) and urea production, and arginase activity were also determined. KEY FINDINGS When macrophages were polarized to the M1 phenotype, Ahr-null cells presented a mixed response; higher levels of IL-1β, IL-6, IL-12, and TNFα were observed after IFNγ- and LPS-mediated activation. However, Ahr-null cells also exhibited decreased NO production and phagocytic capacity. When macrophage was polarized to the M2 phenotype, Ahr-null cells exhibited lower levels of Fizz1, Ym1, and IL-10. In contrast, arginase activity was increased when compared to wild-type macrophages. In addition, macrophages from Ahr-null mice were more susceptible to L. major infection. SIGNIFICANCE Disruption of the Ahr gene alters macrophage polarization when compared to WT macrophage. These changes may affect the development and resolution of several diseases such as bacterial or parasitic infections.