No abstract

Targeted mutant models are common in mechanistic toxicology experiments investigating the absorption, metabolism, distribution, or elimination (ADME) of chemicals from individuals. Key models include those for xenosensing transcription factors and cytochrome P450s (CYP). Here we investigated changes in transcript levels, protein expression, and steroid hydroxylation of several xenobiotic detoxifying CYPs in constitutive androstane receptor (CAR)-null and two CYP-null mouse models that have subfamily members regulated by CAR; the Cyp3a-null and a newly described Cyp2b9/10/13-null mouse model. Compensatory changes in CYP expression that occur in these models may also occur in polymorphic humans, or may complicate interpretation of ADME studies performed using these models. The loss of CAR causes significant changes in several CYPs probably due to loss of CAR-mediated constitutive regulation of these CYPs. Expression and activity changes include significant repression of Cyp2a and Cyp2b members with corresponding drops in 6α- and 16β-testosterone hydroxylase activity. Further, the ratio of 6α-/15α-hydroxylase activity, a biomarker of sexual dimorphism in the liver, indicates masculinization of female CAR-null mice, suggesting a role for CAR in the regulation of sexually dimorphic liver CYP profiles. The loss of Cyp3a causes fewer changes than CAR. Nevertheless, there are compensatory changes including gender-specific increases in Cyp2a and Cyp2b. Cyp2a and Cyp2b were down-regulated in CAR-null mice, suggesting activation of CAR and potentially PXR following loss of the Cyp3a members. However, the loss of Cyp2b causes few changes in hepatic CYP transcript levels and almost no significant compensatory changes in protein expression or activity with the possible exception of 6α-hydroxylase activity. This lack of a compensatory response in the Cyp2b9/10/13-null mice is probably due to low CYP2B hepatic expression, especially in male mice. Overall, compensatory and regulatory CYP changes followed the order CAR-null > Cyp3a-null > Cyp2b-null mice.

Exposure to environmentally-relevant chemicals that activate the xenobiotic receptors aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), and peroxisome proliferator-activated receptor alpha (PPARα) in rodent test systems often leads to increases in oxidative stress (OS) that contributes to liver cancer induction. We hypothesized that activation of the oxidant-induced transcription factor Nrf2 could be used as a surrogate endpoint for increases in OS. We examined the relationships between activation of xenobiotic receptors and Nrf2 using previously characterized gene expression biomarkers that accurately predict modulation. Using a correlation approach (Running Fisher Test), the biomarkers were compared to microarray profiles in a mouse liver gene expression compendium. Out of the 163 chemicals examined, 47% from 53 studies activated Nrf2. We found consistent coupling between CAR and Nrf2 activation. Out of the 41 chemicals from 32 studies that activated CAR, 90% also activated Nrf2. CAR was activated earlier and at lower doses than Nrf2, indicating CAR activation preceded Nrf2 activation. Nrf2 activation by two CAR activators was abolished in CAR-null mice. We hypothesized that Nrf2 is activated by ROS from the increased activity of enzymes encoded by Cyp2b family members. However, Nrf2 was similarly activated in the livers of both TCPOBOP-treated wild-type and Cyp2b9/10/13-null mice. This study provides evidence that Nrf2 activation 1) often occurs after exposure to xenobiotic chemicals, 2) is tightly linked to activation of CAR, and 3) does not require induction of three Cyp2b genes secondary to CAR activation.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease; however, progression to nonalcoholic steatohepatitis (NASH) is associated with most adverse outcomes. CYP2B metabolizes multiple xeno-and endobiotics, and male Cyp2b-null mice are dietinduced obese (DIO) with increased NAFLD. However, the DIO study was not performed long enough to assess progression to NASH. Therefore, to assess the role of Cyp2b in fatty liver disease progression from NAFLD to NASH, we treated wildtype (WT) and Cyp2b-null mice with a normal diet (ND) or choline-deficient, L-amino acid-defined high fat diet (CDAHFD) for 8 weeks and determined metabolic and molecular changes. CDAHFD-fed WT female mice gained more weight and had greater liver and white adipose tissue mass than their Cyp2b-null counterparts; males experienced diet-induced weight loss regardless of genotype. Serum biomarkers of liver injury increased in both CDAHFD-fed female and male mice; however CDAHFD-fed Cyp2b-null females exhibited significantly lower serum ALT, AST, and ASP concentrations compared to WT mice, indicating Cyp2b-null females were protected from liver injury. In both genders, hierarchical clustering of RNA-seq data demonstrates several gene ontologies responded differently in CDAHFD-fed Cyp2b-null mice compared to WT mice (lipid metabolism > fibrosis > inflammation). Oil Red O staining and direct triglycerides measurements confirmed that CDAHFD-fed Cyp2b-null females were protected from NAFLD. CDAHFD-fed Cyp2b-null mice showed equivocal changes in fibrosis with transcriptomic and serum markers suggesting less inflammation due to glucocorticoid-mediated repression of immune responses. In contrast to females, CDAHFD-fed Cyp2b-null males had higher triglyceride levels. Results indicate that female Cyp2b-null mice are protected from NAFLD while male Cyp2b-null mice are more susceptible to NAFLD, with few significant changes in NASH development. This study confirms that increased NAFLD development does not necessarily lead to progressive NASH. Furthermore, it indicates a role for Cyp2b in fatty liver disease that differs based on gender.

Recent studies indicate a role for the constitutive androstane receptor (CAR), pregnane X-receptor (PXR), and hepatic xenobiotic detoxifying CYPs in fatty liver disease or obesity. Therefore, we examined whether Cyp3a-null mice show increased obesity and fatty liver disease following 8-weeks of exposure to a 60% high-fat diet (HFD). Surprisingly, HFD-fed Cyp3a-null females fed a HFD gained 50% less weight than wild-type (WT; B6) females fed a HFD. In contrast, Cyp3a-null males gained more weight than WT males, primarily during the first few weeks of HFD-treatment. Cyp3a-null females also recovered faster than WT females from a glucose tolerance test; males showed no difference in glucose tolerance between the groups. Serum concentrations of the anti-obesity hormone, adiponectin are 60% higher and β-hydroxybutyrate levels are nearly 50% lower in Cyp3a-null females than WT females, in agreement with reduced weight gain, faster glucose response, and reduced ketogenesis. In contrast, Cyp3a-null males have higher liver triglyceride concentrations and lipidomic analysis indicates an increase in phosphatidylinositol, phosphatidylserine and sphingomyelin. None of these changes were observed in females. Last, Pxr, Cyp2b, and IL-6 expression increased in Cyp3a-null females following HFD-treatment. Cyp2b and Fatp1 increased, while Pxr, Cpt1a, Srebp1 and Fasn decreased in Cyp3a-null males following a HFD, indicating compensatory biochemical responses in male (and to a lesser extent) female mice fed a HFD. In conclusion, lack of Cyp3a has a positive effect on acclimation to a HFD in females as it improves weight gain, glucose response and ketosis.