Human CYP2B6 produces oxylipins from polyunsaturated fatty acids and reduces diet-induced obesity.

Multiple factors in addition to over consumption lead to obesity and non-alcoholic fatty liver disease (NAFLD) in the United States and worldwide. CYP2B6 is the only human detoxification CYP whose loss is associated with obesity, and Cyp2b-null mice show greater diet-induced obesity with increased steatosis than wildtype mice. However, a putative mechanism has not been determined. LC-MS/MS revealed that CYP2B6 metabolizes PUFAs, with a preference for metabolism of ALA to 9-HOTrE and to a lesser extent 13-HOTrE with a preference for metabolism of PUFAs at the 9- and 13-positions. To further study the role of CYP2B6 in vivo, humanized-CYP2B6-transgenic (hCYP2B6-Tg) and Cyp2b-null mice were fed a 60% high-fat diet for 16 weeks. Compared to Cyp2b-null mice, hCYP2B6-Tg mice showed reduced weight gain and metabolic disease as measured by glucose tolerance tests, however hCYP2B6-Tg male mice showed increased liver triglycerides. Serum and liver oxylipin metabolite concentrations increased in male hCYP2B6-Tg mice, while only serum oxylipins increased in female hCYP2B6-Tg mice with the greatest increases in LA oxylipins metabolized at the 9 and 13-positions. Several of these oxylipins, specifically 9-HODE, 9-HOTrE, and 13-oxoODE, are PPAR agonists. RNA-seq data also demonstrated sexually dimorphic changes in gene expression related to nuclear receptor signaling, especially CAR > PPAR with qPCR suggesting PPARγ signaling is more likely than PPARα signaling in male mice. Overall, our data indicates that CYP2B6 is an anti-obesity enzyme, but probably to a lesser extent than murine Cyp2b's. Therefore, the inhibition of CYP2B6 by xenobiotics or dietary fats can exacerbate obesity and metabolic disease potentially through disrupted PUFA metabolism and the production of key lipid metabolites.

Cyp2b-Knockdown Mice Poorly Metabolize Corn Oil and Are Age-Dependent Obese.

We previously made a RNAi-based cytochrome P450 2b (Cyp2b)-knockdown (Cyp2b-KD) mouse to determine the in vivo role of the Cyp2b subfamily in xenobiotic detoxification. Further studies reported here indicate a role for Cyp2b in unsaturated fatty-acid (UFA) metabolism and in turn obesity. Mice were treated intraperitoneally (i.p.) with 100 µL corn oil as a carrier or the potent Cyp2b-inducer 3,3',5,5'-Tetrachloro-1,4-bis(pyridyloxy)benzene (TCPOBOP (TC)) dissolved in corn oil. Surprisingly, female Cyp2b-KD mice but not male mice showed increased liver lipid accumulation. Male Cyp2b-KD mice had higher serum triacylglycerols, cholesterol, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) than wildtype (WT) mice; females had higher cholesterol, LDL, and HDL. Thus, Cyp2b-KD mice are unable to clear a high bolus dose of corn oil, potentially because the Cyp2b-KD mice were unable to metabolize the UFA in the corn oil. Therefore, WT and Cyp2b-KD mice were housed for 35 weeks and necropsies performed to test whether Cyp2b-KD mice develop age onset obesity. Cyp2b-KD mice exhibited a significant increase in body weight caused by an increase in white adipose tissue deposition relative to WT mice. Serum cholesterol, triacylglycerol, LDL, and VLDL were significantly greater in 35-week-old Cyp2b-KD males compared to WT males; only serum triacylglycerol and LDL were higher in females. In conclusion, changes in Cyp2b expression led to perturbation in lipid metabolism and depuration in Cyp2b-KD mice. This suggests that Cyp2b is more than a detoxification enzyme, but also involved in the metabolism of UFA, as Cyp2b-KD mice have increased the body weight, fat deposition, and serum lipids.

Exchange of polar lipids from adults to neonates in Daphnia magna: Perturbations in sphingomyelin allocation by dietary lipids and environmental toxicants.

Because xenosensing nuclear receptors are also lipid sensors that regulate lipid allocation, we hypothesized that toxicant-induced modulation of HR96 activity would alter lipid profiles and the balance between adult survival and neonate production following exposure in Daphnia magna. Adult daphnids were exposed to unsaturated fatty acid- and toxicant- activators or inhibitors of HR96 and later starved to test whether chemical exposure altered allocation toward survival or reproduction. The HR96 activators, linoleic acid and atrazine, decreased reproduction as expected with concomitant changes in the expression of HR96 regulated genes such as magro. The HR96 inhibitors, docosahexaenoic acid (DHA) and triclosan, increased reproduction or neonate starvation survival, respectively. However, pre-exposure to triclosan increased in neonate survival at the expense of reproductive maturation. Lipidomic analysis revealed that sphingomyelins (SM) are predominantly found in neonates and therefore we propose are important in development. DHA and triclosan increased neonatal SM, consistent with HR96's regulation of Niemann-Pick genes. While DHA altered expression of magro, Niemann-Pick 1b, mannosidase, and other HR96-regulated genes as expected, triclosan primarily perturbed sphingomyelinase and mannosidase expression indicating different but potentially overlapping mechanisms for perturbing SM. Overall, SM appears to be a key lipid in Daphnia maturation and further support was provided by carmofur, which inhibits sphingomyelin/ceramide metabolism and in turn severely represses Daphnia maturation and initial brood production. In conclusion, toxicants can perturb lipid allocation and in turn impair development and reproduction.

Arachidonic acid enhances reproduction in Daphnia magna and mitigates changes in sex ratios induced by pyriproxyfen.

Arachidonic acid is 1 of only 2 unsaturated fatty acids retained in the ovaries of crustaceans and an inhibitor of HR97g, a nuclear receptor expressed in adult ovaries. The authors hypothesized that, as a key fatty acid, arachidonic acid may be associated with reproduction and potentially environmental sex determination in Daphnia. Reproduction assays with arachidonic acid indicate that it alters female:male sex ratios by increasing female production. This reproductive effect only occurred during a restricted Pseudokirchneriella subcapitata diet. Next, the authors tested whether enriching a poorer algal diet (Chlorella vulgaris) with arachidonic acid enhances overall reproduction and sex ratios. Arachidonic acid enrichment of a C. vulgaris diet also enhances fecundity at 1.0 µM and 4.0 µM by 30% to 40% in the presence and absence of pyriproxyfen. This indicates that arachidonic acid is crucial in reproduction regardless of environmental sex determination. Furthermore, the data indicate that P. subcapitata may provide a threshold concentration of arachidonic acid needed for reproduction. Diet-switch experiments from P. subcapitata to C. vulgaris mitigate some, but not all, of arachidonic acid's effects when compared with a C. vulgaris-only diet, suggesting that some arachidonic acid provided by P. subcapitata is retained. In summary, arachidonic acid supplementation increases reproduction and represses pyriproxyfen-induced environmental sex determination in D. magna in restricted diets. A diet rich in arachidonic acid may provide protection from some reproductive toxicants such as the juvenile hormone agonist pyriproxyfen. Environ Toxicol Chem 2015;34:527-535. © 2014 SETAC.

Using mummichog (Fundulus heteroclitus) arrays to monitor the effectiveness of remediation at a superfund site in Charleston, South Carolina, U.S.A.

We previously developed a cDNA array for mummichogs (Fundulus heteroclitus), an estuarine minnow, that is targeted for identifying differentially expressed genes from exposure to polycyclic aromatic hydrocarbons and several metals, including chromium. A chromium-contaminated Superfund site at Shipyard Creek in Charleston, South Carolina, USA, is undergoing remediation, providing us a unique opportunity to study the utility of arrays for monitoring the effectiveness of site remediation. Mummichogs were captured in Shipyard Creek in Charleston prior to remediation (2000) and after remediation began (2003 and 2005). Simultaneously, mummichogs were collected from a reference site at the Winyah Bay National Estuarine Research Reserve (NERR) in Georgetown, South Carolina, USA. The hepatic gene expression pattern of fish captured at Shipyard Creek in 2000 showed wide differences from the fish captured at NERR in 2000. Interestingly, as remediation progressed the gene expression pattern of mummichogs captured at Shipyard Creek became increasingly similar to those captured at NERR. The arrays acted as multidimensional biomarkers as the number of differentially expressed genes dropped from 22 in 2000 to four in 2003, and the magnitude of differential expression dropped from 3.2-fold in 2000 to no gene demonstrating a difference over 1.5-fold in 2003. Furthermore, the arrays indicated changes in the bioavailability of chromium caused by hydraulic dredging in the summer of 2005. This research is, to our knowledge, the first report using arrays as biomarkers for a weight-of-evidence hazard assessment and demonstrates that arrays can be used as multidimensional biomarkers to monitor site mitigation because the gene expression profile is associated with chromium bioavailability and body burden.

Alterations in hepatic gene expression by trivalent chromium in Fundulus heteroclitus.

Cr(III) is the dominant toxicant at some Superfund sites within the United States and therefore we are interested in its effects. Cr(III)s mechanisms are not well studied or understood because of its low bioavailability. We have attempted to characterize the effects of Cr(III) on gene expression in Fundulus heteroclitus (mummichog) liver. The NOEC and LOEC were determined at 32 and 64 mg/L, respectively, by measuring growth and mortality after exposing juveniles for 30 days. Secondary adult male exposures were performed at 32 mg/L, livers excised, and RNA extracted. Arrays were probed with cDNA from untreated or Cr(III)-exposed adult fish and gene expression was quantified. Cr(III) at 32 mg/L altered the expression of five genes, including GSTtau, GSTalpha, and ALDH4. Ultimately, we anticipate using this gene expression information to ascertain whether Cr(III) is bioavailable at potentially adverse concentrations in contaminated sites.

Effects of nonylphenol on hepatic testosterone metabolism and the expression of acute phase proteins in winter flounder (Pleuronectes americanus): comparison to the effects of Saint John's Wort.

4-Nonylphenol (4-NP), a major by-product of alkylphenol ethoxylates, is used in several industries and as a consequence is quite common in rivers, estuaries and other aquatic environments that receive sewage discharges or are near offshore oil platforms. 4-NP is an environmental estrogen that also binds human and rodent Pregnane X-receptor (PXR), the orphan nuclear receptor that controls the expression of several detoxication genes in mammals, including several CYP3A and CYP2B family members. These P450s preferentially hydroxylate testosterone in the 6beta- and 16beta-positions, respectively. In this study, the effects of 4-NP on testosterone metabolism and hepatic CYP3A induction were compared to the effects of St. John's Wort (SJW), a well established mammalian PXR agonist, in winter flounder. Male winter flounder (Pleuronectes americanus) were injected with 100 mg/kg/day 4-NP or 500 mg/kg/day SJW or both (S and N) every 24 h. Forty-eight hours after the initial injections, flounder were euthanized. Western blots and testosterone 6beta-hydroxylation indicated that CYP3A was increased 50% by 4-NP, but was not affected by SJW. Testosterone 16beta-hydroxylase activity was also significantly increased in flounder treated with 4-NP (2.8 x), but not with SJW. This is not consistent with our hypothesis that both SJW and 4-NP would induce CYP3A. Subtractive hybridization was performed between control and 4-NP treated hepatic mRNA samples to isolate differentially expressed genes. Subtractive hybridization indicated that several acute phase proteins were altered by 4-NP. Quantitative real-time PCR (Q-PCR) confirmed 4-NP altered the expression of complement components C8b, cathepsin L, C-type lectin domain, FK506 binding protein 2 precursor (FKBP2) and an EST (expressed sequence tag). SJW and 4-NP treated flounder demonstrated similar induction profiles for the EST, cathepsin L and FKBP2, suggesting that SJW was at a sufficient dose to alter gene expression but not induce P450s. In conclusion, testosterone hydroxylase activity and Western blots indicate that SJW did not activate detoxication pathways in a similar manner to 4-NP.