Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that activate the aryl hydrocarbon receptor, triggering many biological effects typified by induction of cytochrome P450 1A1 (CYP1A1). Interestingly, little is known about their effects on human CYP3A4, despite this enzymeâ s clinical relevance. Controversially, studies have reported both inductive and suppressive effects of PAHs on CYP3A expression. The current study used 3-methylcholanthrene (3-MC) to study the effects of PAHs on CYP3A4 activity and expression in humanized PXR-CAR-CYP3A4/3A7 mice. Adult mice were treated by intraperitoneal injection with 3-MC (80 mg/kg), or corn oil vehicle, and euthanized 24 or 72 hours later. Basal hepatic CYP3A4 activity and expression was significantly higher in female vs. male mice. 3-MC decreased CYP3A4 activity and expression in female mice at 72 hours, whilemales showed similar, but non-statistically significant, trends. This mouse model will facilitate further in vivo studies of the mechanisms and consequences of CYP3A4 suppression by PAHs.

M.Sc.

The aryl hydrocarbon receptor (AHR) nuclear translocator (ARNT), a partner in the hypoxia and AHR signaling pathways, is induced in rat liver by dexamethasone (DEX), a steroid that activates the glucocorticoid (GR) and pregnane X (PXR) receptors. I conducted in vivo rat studies to examine the roles of GR and PXR in ARNT regulation. A selective GR agonist or a low DEX dose activating GR but not PXR increased hepatic levels of ARNT mRNA and an unidentified ARNT antibody-reactive protein (UAARP). The trend for increased ARNT protein levels required high DEX doses activating GR and PXR. A GR antagonist prevented the induction of ARNT mRNA and UAARP levels by low-dose DEX and the increased ARNT protein levels by high-dose DEX. DEX-induced ARNT mRNA and protein levels did not differ between wild-type and PXR-knockout rats. These results support a role for GR in the DEX induction of rat hepatic ARNT expression.

M.Sc.

3-Methylcholanthrene (MC) is a model polycyclic aromatic hydrocarbon that induces cytochrome P450 1A1 (CYP1A1) expression. This laboratory has shown previously that aromatic hydrocarbons, which are important environmental toxicants, down-regulate the expression of rat liver CYP2C11. Recent observations also suggested that CYP2C8, a human enzyme that metabolizes antineoplastic and antidiabetic drugs, among others, is down-regulated in response to aromatic hydrocarbon exposure in primary human hepatocytes. I examined the regulation of CYP2C8 at the mRNA level by MC in two human hepatocellular carcinoma cell lines, HepG2 and HepaRG. MC down-regulated CYP2C8 mRNA levels in HepG2 cells at 24 hours and in HepaRG cells at 48 hours. CYP1A1 mRNA was induced by MC in both cell lines and HepaRG cells appeared to be more sensitive than HepG2 cells to MC-induced cytotoxicity. Further studies are warranted to define the mechanisms and functional impacts of the modulation of this important human CYP by environmental toxicants.

MAST

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene (MC); the prototypical response is induction of drug-metabolizing enzymes. Factors that regulate AHR levels in vivo are poorly understood. It is also not clear how AHR levels affect MC responsiveness. I hypothesize that glucocorticoids enhance hepatic AHR based on previous findings of decreased hepatic AHR protein in hypophysectomised rats and increased AHR levels following glucocorticoid treatment in rodent hepatoma cells. To study this, adrenalectomized (ADX) or SHAM-ADX rats were treated with dexamethasone or vehicle. AHR protein was decreased by 50–60% at 4 days after ADX, but was not altered by dexamethasone. Dexamethasone induced hepatic AHR nuclear translocator (ARNT) mRNA by up to 9-fold, with no corresponding change in ARNT protein. AHR target gene expression was measured in MC-treated ADX rats to assess MC responsiveness given the decrease in AHR protein following ADX. MC-induced hepatic CYP1B1 mRNA was reduced by 50% in ADX rats relative to SHAM. AHR mRNA was increased 4-fold, 6 h after MC in SHAM rats, but no induction was observed in ADX rats. MC-induced 7-ethoxyresorufin O-deethylation activity in ADX rats was 35% of the activity in the MC-treated SHAM group at 6 h. To assess the capacity for hepatic P450-mediated metabolism, NADPH-cytochrome P450 oxidoreductase (POR) was measured. POR activity was decreased by 50-65% following ADX. DEX induced hepatic POR mRNA by up to 7-fold, 6 h after treatment in SHAM, ADX and intact rats. Putative glucocorticoid responsive elements in the rat Por gene were identified, but recruitment of the glucocorticoid receptor to these elements was not detected using chromatin immunoprecipitation. In rat H-4-II-E hepatoma cells, dexamethasone induced POR, but not ARNT, mRNA. I have shown that ADX decreases hepatic AHR protein and subsequently, MC responsiveness is suppressed for some AHR-mediated responses. Decreased POR activity following ADX might contribute to a decreased capacity for P450-dependent metabolism. The novel findings with respect to glucocorticoid regulation of ARNT and POR demonstrate the complexity of AHR-glucocorticoid cross-talk and the need for further study.

PhD