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.

Most Nuclear Receptors (NRs) are ligand-dependent transcription factors crucial in homeostatic physiological responses or environmental responses. We annotated the D. magna NRs and compared them to D. pulex and other species, primarily through phylogenetic analysis. Daphnia species contain 26 NRs spanning all seven gene subfamilies. Thirteen of the 26 receptors found in Daphnia species phylogenetically segregate into the NR1 subfamily, primarily involved in energy metabolism and resource allocation. Some of the Daphnia NRs, such as RXR, HR96, and E75 show strong conservation between D. magna and D. pulex. Other receptors, such as EcRb, THRL-11 and RARL-10 have diverged considerably and therefore may show different functions in the two species. Curiously, there is an inverse association between the number of NR splice variants and conservation of the LBD. Overall, D. pulex and D. magna possess the same NRs; however not all of the NRs demonstrate high conservation indicating the potential for a divergence of function.

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.

HR96 is a CAR/PXR/VDR ortholog in invertebrates, and a promiscuous endo- and xenobiotic nuclear receptor involved in acclimation to toxicants. Daphnia HR96 is activated by chemicals such as atrazine and linoleic acid (LA) (n-6 fatty acid), and inhibited by triclosan and docosahexaenoic acid (DHA)(n-3 fatty acid). We hypothesized that inhibitors of HR96 may block the protective responses of HR96 based on previously performed luciferase assays. Therefore, we performed acute toxicity tests with two-chemical mixtures containing a HR96 inhibitor (DHA or triclosan) and a HR96 activator (LA or atrazine). Surprisingly, results demonstrate that triclosan and DHA are less toxic when co-treated with 20–80 μM atrazine. Atrazine provides concentration-dependent protection as lower concentrations have no effect and higher concentrations cause toxicity. LA, a weaker HR96 activator, did not provide protection from triclosan or DHA. Atrazine’s protective effects are presumably due to its ability to activate HR96 or other toxicologically relevant transcription factors and induce protective enzymes. Atrazine did not significantly induce glucosyltransferase, a crucial enzyme in triclosan detoxification. However, atrazine did increase antioxidant activities, crucial pathways in triclosan’s toxicity, as measured through GST activity and the TROLOX equivalence assay. The increase in antioxidant capacity is consistent with atrazine providing protection from a wide range of toxicants that induce ROS, including triclosan and unsaturated fatty acids predisposed to lipid peroxidation.

The nuclear receptors (NRs) are ligand-dependent transcription factors that respond to various internal as well as external cues such as nutrients, pheromones, and steroid hormones that play crucial roles in regulation and maintenance of homeostasis and orchestrating the physiological and stress responses of an organism. We annotated the Fundulus heteroclitus (mummichog; Atlantic killifish) nuclear receptors. Mummichog are a non-migratory, estuarine fish with a limited home range often used in environmental research as a field model for studying ecological and evolutionary responses to variable environmental conditions such as salinity, oxygen, temperature, pH, and toxic compounds because of their hardiness. F. heteroclitus have at least 74 NRs spanning all seven gene subfamilies. F. heteroclitus is unique in that no RXRα member was found within the genome. Interestingly, some of the NRs are highly conserved between species, while others show a higher degree of divergence such as PXR, SF1, and ARα. Fundulus like other fish species show expansion of the RAR (NR1B), Rev-erb (NR1D), ROR (NR1F), COUPTF (NR2F), ERR (NR3B), RXR (NR2B), and to a lesser extent the NGF (NR4A), and NR3C steroid receptors (GR/AR). Of particular interest is the co-expansion of opposing NRs, Reverb-ROR, and RAR/RXR-COUPTF.