Increased Perfluorooctanesulfonate (PFOS) Toxicity and Accumulation Is Associated with Perturbed Prostaglandin Metabolism and Increased Organic Anion Transport Protein (OATP) Expression.

Perfluorooctanesulfonate (PFOS) is a widespread environmental pollutant with a long half-life and clearly negative outcomes on metabolic diseases such as fatty liver disease and diabetes. Male and female Cyp2b-null and humanized CYP2B6-transgenic (hCYP2B6-Tg) mice were treated with 0, 1, or 10 mg/kg/day PFOS for 21 days, and surprisingly it was found that PFOS was retained at greater concentrations in the serum and liver of hCYP2B6-Tg mice than those of Cyp2b-null mice, with greater differences in the females. Thus, Cyp2b-null and hCYP2B6-Tg mice provide new models for investigating individual mechanisms for PFOS bioaccumulation and toxicity. Overt toxicity was greater in hCYP2B6-Tg mice (especially females) as measured by mortality; however, steatosis occurred more readily in Cyp2b-null mice despite the lower PFOS liver concentrations. Targeted lipidomics and transcriptomics from PFOS-treated Cyp2b-null and hCYP2B6-Tg mouse livers were performed and compared to PFOS retention and serum markers of toxicity using PCA. Several oxylipins, including prostaglandins, thromboxanes, and docosahexaenoic acid metabolites, are associated or inversely associated with PFOS toxicity. Both lipidomics and transcriptomics indicate PFOS toxicity is associated with PPAR activity in all models. GO terms associated with reduced steatosis were sexually dimorphic with lipid metabolism and transport increased in females and circadian rhythm associated genes increased in males. However, we cannot rule out that steatosis was initially protective from PFOS toxicity. Moreover, several transporters are associated with increased retention, probably due to increased uptake. The strongest associations are the organic anion transport proteins (Oatp1a4-6) genes and a long-chain fatty acid transport protein (fatp1), enriched in female hCYP2B6-Tg mice. PFOS uptake was also reduced in cultured murine hepatocytes by OATP inhibitors. The role of OATP1A6 and FATP1 in PFOS transport has not been tested. In summary, Cyp2b-null and hCYP2B6-Tg mice provided unique models for estimating the importance of novel mechanisms in PFOS retention and toxicity.

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.

The reproductive effects of the cancer chemotherapy agent, Carmofur, on Daphnia magna are mediated by its metabolite, 5-Fluorouracil.

Carmofur is an antineoplastic agent that inhibits ceramidase, a key enzyme in the sphingolipid pathway. Previous research suggests carmofur represses reproductive maturity in Daphnia magna. The purpose of this experiment was to confirm carmofur's effects on fecundity and reproductive maturity over two generations. A chronic toxicity test found reproductive maturity was delayed from 9 to 19 days by 0.80 µM carmofur with a 99.7% drop in reproduction, probably caused by delayed ovarian development. Second generation effects were even greater with 0% reproductive success at 0.40 µM. To our surprise, carmofur was not measured in the media by HPLC 24 h after exposure. Previous research indicated that carmofur is unstable in water and hydrolyzed into 5-fluorouracil (5-FU). Therefore, the chronic toxicity study was repeated with 5-FU and similar effects on reproductive maturity were observed at similar concentrations despite very different acute toxicities (48 h carmofur LC50 = 1.93 µM; 5-FU LC50 = 207 µM). 5-FU delayed reproductive maturity from 9 to 21 days with a 71.12% drop in reproduction at 0.80 µM and greater effects in the 2nd generation similar to carmofur. 5-FU was found stable in aquatic media and HPLC confirmed 5-FU was hydrolyzed from carmofur within 24 h. In conclusion, carmofur and 5-FU reduce fecundity because they delay reproductive maturity and ovarian development in Daphnia magna. We conclude that the reproductive effects observed after carmofur treatment are primarily mediated by its breakdown product, 5-FU. This further underscores the importance of measuring chemical concentrations and evaluating chemical metabolism and decomposition when determining toxicity, especially of chemotherapeutic agents.Clinical trials registration Not applicable.

Age- and Diet-Dependent Changes in Hepatic Lipidomic Profiles of Phospholipids in Male Mice: Age Acceleration in Cyp2b-Null Mice.

Increases in traditional serum lipid profiles are associated with obesity, cancer, and cardiovascular disease. Recent lipidomic analysis has indicated changes in serum lipidome profiles, especially in regard to specific phosphatidylcholines, associated with obesity. However, little work has evaluated murine hepatic liver lipidomic profiles nor compared these profiles across age, high-fat diet, or specific genotypes, in this case the lack of hepatic Cyp2b enzymes. In this study, the effects of age (9 months old), high-fat diet (4.5 months old), and the loss of three primarily hepatic xeno- and endobiotic metabolizing cytochrome P450 (Cyp) enzymes, Cyp2b9, Cyp2b10, and Cyp2b13 (Cyp2b-null mice), on the male murine hepatic lipidome were compared. Hierarchical clustering and principal component analysis show that age perturbs hepatic phospholipid profiles and serum lipid markers the most compared to young mice, followed by a high-fat diet and then loss of Cyp2b. Several lipid biomarkers such as PC/PE ratios, PE 38 : 6, and LPC concentrations indicate greater potential for NAFLD and hypertension with mixed effects in Cyp2b-null mice(less NAFLD and greater hypertension-associated markers). Lipid profiles from older mice contain greater total and n-6 fatty acids than normal diet (ND)-fed young mice; however, surprisingly, young Cyp2b-null mice contain high n-6 : n-3 ratios. Overall, the lack of Cyp2b typically enhanced adverse physiological parameters observed in the older (9 mo) mice with increased weight gain combined with a deteriorating cholesterol profile, but not necessarily all phospholipid profiles were adversely perturbed.

Dataset of endo- and xenobiotic inhibition of CYP2B6: Comparison to CYP3A4.

Cytochrome P450 2B6 (CYP2B6) is a human enzyme important in chemical detoxification, steroid and fatty acid metabolism that is primarily hepatic. Therefore, induction or inhibition of CYP2B6 may perturb endo- and xenobiotic metabolism and cause adverse reactions. Recent research indicates that mice lacking Cyp2b enzymes are obese with liver steatosis [1] (Heintz et al., J Nutr Biochem, 70:125-137, 2019). Current work is underway to determine the role of CYP2B6 in obesity and fatty acid metabolism, and CYP2B6 fluorescent inhibition assays were used to determine the IC50s of multiple industrial chemicals, pesticides, bile acids, steroids, and fatty acids. In many cases, inhibition of CYP3A4 was also performed in comparison because CYP3A4 is the most abundant hepatic detoxification CYP and therefore by abundance alone may also play a key role in the chemical's metabolism. Further, using the ratio of comparative potency of these compounds for CYP2B6 and CYP3A4, specificity can be estimated for these CYP2B6 inhibitors. These results indicate strong preferential inhibition (greater than 10-fold) of CYP2B6 and include lithocholic acid, arachidonic acid, atrazine, chlorpyrifos, endosulfan, parathion, and nonylphenol. Estradiol was a strong preferential inhibitor of CYP3A4. Other screened CYP2B6 inhibitors include triclosan, ticlopidine, jet fuel, docosahexaenoic acid, linoleic acid, linolenic acid, oleic acid, lithocholic acid, butylate, hexachlorocyclohexane, vinclozolin, pentachlorophenol, metalachlor, butylate, diazinon, avermectin, tribufos, ticlopidine, and bisphenol A. Documentation of xenobiotic and endobiotic inhibition by these CYPs is necessary for proper modeling of the effects of diet, chemical exposure or even mixtures on drug metabolism and potential adverse reactions.

Detoxification Cytochrome P450s (CYPs) in Families 1-3 Produce Functional Oxylipins from Polyunsaturated Fatty Acids.

This manuscript reviews the CYP-mediated production of oxylipins and the current known function of these diverse set of oxylipins with emphasis on the detoxification CYPs in families 1-3. Our knowledge of oxylipin function has greatly increased over the past 3-7 years with new theories on stability and function. This includes a significant amount of new information on oxylipins produced from linoleic acid (LA) and the omega-3 PUFA-derived oxylipins such as α-linolenic acid (ALA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA). However, there is still a lack of knowledge regarding the primary CYP responsible for producing specific oxylipins, and a lack of mechanistic insight for some clinical associations between outcomes and oxylipin levels. In addition, the role of CYPs in the production of oxylipins as signaling molecules for obesity, energy utilization, and development have increased greatly with potential interactions between diet, endocrinology, and pharmacology/toxicology due to nuclear receptor mediated CYP induction, CYP inhibition, and receptor interactions/crosstalk. The potential for diet-diet and diet-drug/chemical interactions is high given that these promiscuous CYPs metabolize a plethora of different endogenous and exogenous chemicals.

Increased toxicity and retention of perflourooctane sulfonate (PFOS) in humanized CYP2B6-Transgenic mice compared to Cyp2b-null mice is relieved by a high-fat diet (HFD).

PFOS is a persistent, fluorosurfactant used in multiple products. Murine Cyp2b's are induced by PFOS and high-fat diets (HFD) and therefore we hypothesized that human CYP2B6 may alleviate PFOS-induced steatosis. Cyp2b-null and hCYP2B6-Tg mice were treated with 0, 1, or 10 mg/kg/day PFOS by oral gavage for 21-days while provided a chow diet (ND) or HFD. Similar to murine Cyp2b10, CYP2B6 is inducible by PFOS. Furthermore, three ND-fed hCYP2B6-Tg females treated with 10 mg/kg/day PFOS died during the exposure period; neither Cyp2b-null nor HFD-fed mice died. hCYP2B6-Tg mice retained more PFOS in serum and liver than Cyp2b-null mice presumably causing the observed toxicity. In contrast, serum PFOS retention was reduced in the HFD-fed hCYP2B6-Tg mice; the opposite trend observed in HFD-fed Cyp2b-null mice. Hepatotoxicity biomarkers, ALT and ALP, were higher in PFOS-treated mice and repressed by a HFD. However, PFOS combined with a HFD exacerbated steatosis in all mice, especially in the hCYP2B6-Tg mice with significant disruption of key lipid metabolism genes such as Srebp1, Pparg, and Hmgcr. In conclusion, CYP2B6 is induced by PFOS but does not alleviate PFOS toxicity presumably due to increased retention. CYP2B6 protects from PFOS-mediated steatosis in ND-fed mice, but increases steatosis when co-treated with a HFD.

Evaluation of oral citrulline administration as a mitigation strategy for fescue toxicosis in sheep.

Gestating ewes consuming ergot alkaloids, from endophyte-infected (E+) tall fescue seed, suffer from intrauterine growth restriction and produce smaller lambs. Arginine (Arg) supplementation has been shown to increase birth weight and oral citrulline (Cit) administration is reported to increase arginine concentrations. Two experiments were conducted to: 1) evaluate if oral supplementation with Cit or water, to ewes consuming E+ fescue seed, increases lamb birth weight and 2) determine the effectiveness of Cit and citrulline:malate as an oral drench and elevating circulating levels of Cit to determine levels and dose frequency. In experiment 1, gestating Suffolk ewes (n = 10) were assigned to one of two treatments [oral drench of citrulline-malate 2:1 (CITM; 81 mg/kg/d of citrulline) or water (TOX)] to start on d 86 of gestation and continued until parturition. Ewes on CITM treatment had decreased (P < 0.05) plasma Arg and Cit concentrations during gestation. At birth, lambs from CITM ewes had reduced (P < 0.05) crude fat and total fat but did not differ (P > 0.05) in birth weight from lambs born to TOX ewes. In experiment 2, nonpregnant Suffolk ewes (n = 3) were assigned to either oral citrulline (CIT; 81 mg/kg/d), citrulline-malate 2:1 (CITM; 81 mg/kg/d of citrulline), or water (CON) drench in a Latin Square design for a treatment period of 4 d with a washout period of 3 d. On d 4, blood samples were collected at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 18 h post drench. Oral drenching of CIT and CITM increased (P < 0.0001) Cit concentrations within 2 h and levels remained elevated for 6 h. Apparent half-life of elimination for CIT and CITM were 8.484 and 10.392 h, respectively. Our results show that lamb birth weight was not altered with a single oral drench of citrulline-malate; however, lamb body composition was altered. The level and frequency of citrulline dosing may need to be greater in order to observe consistent elevation of Cit/Arg concentrations to determine its effectiveness in mitigating fescue toxicosis.

20th Pollutant Responses in Marine Organisms (PRIMO 20): Global issues and fundamental mechanisms caused by pollutant stress in marine and freshwater organisms.

The 20th Pollutant Responses in Marine Organisms (PRIMO 20) conference provided a forum for scientists from around the world to communicate novel toxicological research findings specifically focused on aquatic organisms, by combining applied and basic research at the intersection of environmental and mechanistic toxicology. The work highlighted in this special issue of Aquatic Toxicology, a special issue of Marine Environmental Research, and presented through posters and presentations, encompass important and emerging topics in freshwater and marine toxicology. This includes multiple types of emerging contaminants including microplastics and UV filtering chemicals. Other studies aimed to further our understanding of the effects of endocrine disrupting chemicals, pharmaceuticals, and personal care products. Further research presented in this virtual issue examined the interactive effects of chemicals and pathogens, while the final set of manuscripts demonstrates continuing efforts to combine traditional biomonitoring, data from -omic technologies, and modeling for use in risk assessment and management. An additional goal of PRIMO meetings is to address the link between environmental and human health. Several articles in this issue of Aquatic Toxicology describe the appropriateness of using aquatic organisms as models for human health, while the keynote speakers, as described in the editorial below, presented research that highlighted bioaccumulation of contaminants such as PFOS and mercury from fish to marine mammals and coastal human populations such as the Gullah/GeeChee near Charleston, South Carolina, USA.

Gender differences in diet-induced steatotic disease in Cyp2b-null mice.

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 diet-induced 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.

Phase 0 of the Xenobiotic Response: Nuclear Receptors and Other Transcription Factors as a First Step in Protection from Xenobiotics.

This mini-review examines the crucial importance of transcription factors as a first line of defense in the detoxication of xenobiotics. Key transcription factors that recognize xenobiotics or xenobiotic-induced stress such as reactive oxygen species (ROS), include AhR, PXR, CAR, MTF, Nrf2, NF-κB, and AP-1. These transcription factors constitute a significant portion of the pathways induced by toxicants as they regulate phase I-III detoxication enzymes and transporters as well as other protective proteins such as heat shock proteins, chaperones, and anti-oxidants. Because they are often the first line of defense and induce phase I-III metabolism, could these transcription factors be considered the phase 0 of xenobiotic response?

Cyp2b-null male mice are susceptible to diet-induced obesity and perturbations in lipid homeostasis.

Obesity is an endemic problem in the United States and elsewhere, and data indicate that in addition to overconsumption, exposure to specific chemicals enhances obesity. CYP2B metabolizes multiple endo- and xenobiotics, and recent data suggests that repression of Cyp2b activity increases dyslipidemia and age-onset obesity, especially in males. To investigate the role played by Cyp2b in lipid homeostasis and obesity, we treated wildtype and Cyp2b-null mice with a normal (ND) or 60% high-fat diet (HFD) for 10 weeks and determined metabolic and molecular changes. Male HFD-fed Cyp2b-null mice weigh 15% more than HFD-fed wildtype mice, primarily due to an increase in white adipose tissue (WAT); however, Cyp2b-null female mice did not demonstrate greater body mass or WAT. Serum parameters indicate increased ketosis, leptin and cholesterol in HFD-fed Cyp2b-null male mice compared to HFD-fed wildtype mice. Liver triglycerides and liver:serum triglyceride ratios were higher than their similarly treated wildtype counterparts in Cyp2b-null male mice, indicating a role for Cyp2b in fatty acid metabolism regardless of diet. Furthermore, RNAseq demonstrates that hepatic gene expression in ND-fed Cyp2b-null male mice is similar to HFD-fed WT male mice, suggestive of fatty liver disease progression and a role for Cyp2b in lipid homeostasis. Females did not show as demonstrative changes in liver health, and significantly fewer changes in gene expression, as well as gene expression associated with liver disease. Overall our data indicates that the repression or inhibition of CYP2B may exacerbate metabolic disorders and cause obesity by perturbing fatty acid metabolism, especially in males.

Chemical Activation of the Constitutive Androstane Receptor Leads to Activation of Oxidant-Induced Nrf2.

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 with 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 2 CAR activators was abolished in CAR-null mice. We hypothesized that Nrf2 is activated by reactive oxygen species 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 3 Cyp2b genes secondary to CAR activation.

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.

High fat diet induced obesity is mitigated in Cyp3a-null female mice.

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 ß-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.

RNA sequencing indicates that atrazine induces multiple detoxification genes in Daphnia magna and this is a potential source of its mixture interactions with other chemicals.

Atrazine is an herbicide with several known toxicologically relevant effects, including interactions with other chemicals. Atrazine increases the toxicity of several organophosphates and has been shown to reduce the toxicity of triclosan to D. magna in a concentration dependent manner. Atrazine is a potent activator in vitro of the xenobiotic-sensing nuclear receptor, HR96, related to vertebrate constitutive androstane receptor (CAR) and pregnane X-receptor (PXR). RNA sequencing (RNAseq) was performed to determine if atrazine is inducing phase I-III detoxification enzymes in vivo, and estimate its potential for mixture interactions. RNAseq analysis demonstrates induction of glutathione S-transferases (GSTs), cytochrome P450s (CYPs), glucosyltransferases (UDPGTs), and xenobiotic transporters, of which several are verified by qPCR. Pathway analysis demonstrates changes in drug, glutathione, and sphingolipid metabolism, indicative of HR96 activation. Based on our RNAseq data, we hypothesized as to which environmentally relevant chemicals may show altered toxicity with co-exposure to atrazine. Acute toxicity tests were performed to determine individual LC50 and Hillslope values as were toxicity tests with binary mixtures containing atrazine. The observed mixture toxicity was compared with modeled mixture toxicity using the Computational Approach to the Toxicity Assessment of Mixtures (CATAM) to assess whether atrazine is exerting antagonism, additivity, or synergistic toxicity in accordance with our hypothesis. Atrazine-triclosan mixtures showed decreased toxicity as expected; atrazine-parathion, atrazine-endosulfan, and to a lesser extent atrazine-p-nonylphenol mixtures showed increased toxicity. In summary, exposure to atrazine activates HR96, and induces phase I-III detoxification genes that are likely responsible for mixture interactions.

Annotation of the Nuclear Receptors in an Estuarine Fish species, Fundulus heteroclitus.

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.

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.