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Recent in vitro transcriptomic analyses for the short-chain polyfluoroalkyl substance, HFPO-DA (ammonium, 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate), support conclusions from in vivo data that HFPO-DA-mediated liver effects in mice are part of the early key events of the peroxisome proliferator-activated receptor alpha (PPARα) activator-induced rodent hepatocarcinogenesis mode of action (MOA). Transcriptomic responses in HFPO-DA-treated rodent hepatocytes have high concordance with those treated with a PPARα agonist and lack concordance with those treated with PPARγ agonists or cytotoxic agents. To elucidate whether HFPO-DA-mediated transcriptomic responses in mouse liver are PPARα-dependent, additional transcriptomic analyses were conducted on samples from primary PPARα knockout (KO) and wild-type (WT) mouse hepatocytes exposed for 12, 24, or 72 h with various concentrations of HFPO-DA, or well-established agonists of PPARα (GW7647) and PPARγ (rosiglitazone), or cytotoxic agents (acetaminophen or d-galactosamine). Pathway and predicted upstream regulator-level responses were highly concordant between HFPO-DA and GW7647 in WT hepatocytes. A similar pattern was observed in PPARα KO hepatocytes, albeit with a distinct temporal and concentration-dependent delay potentially mediated by compensatory responses. This delay was not observed in PPARα KO hepatocytes exposed to rosiglitazone, acetaminophen, d-galactosamine. The similarity in transcriptomic signaling between HFPO-DA and GW7647 in both the presence and absence of PPARα in vitro indicates these compounds share a common MOA. [ABSTRACT FROM AUTHOR]

Like many per- or polyfluorinated alkyl substances (PFAS), toxicity studies with HFPO-DA (ammonium, 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate), a short-chain PFAS used in the manufacture of some types of fluorinated polymers, indicate that the liver is the primary target of toxicity in rodents following oral exposure. Although the current weight of evidence supports the PPARα mode of action (MOA) for liver effects in HFPO-DA-exposed mice, alternate MOAs have also been hypothesized including PPARγ or cytotoxicity. To further evaluate the MOA for HFPO-DA in rodent liver, transcriptomic analyses were conducted on samples from primary mouse, rat, and pooled human hepatocytes treated for 12, 24, or 72 h with various concentrations of HFPO-DA, or agonists of PPARα (GW7647), PPARγ (rosiglitazone), or cytotoxic agents (ie, acetaminophen or d -galactosamine). Concordance analyses of enriched pathways across chemicals within each species demonstrated the greatest concordance between HFPO-DA and PPARα agonist GW7647-treated hepatocytes compared with the other chemicals evaluated. These findings were supported by benchmark concentration modeling and predicted upstream regulator results. In addition, transcriptomic analyses across species demonstrated a greater transcriptomic response in rodent hepatocytes treated with HFPO-DA or agonists of PPARα or PPARγ, indicating rodent hepatocytes are more sensitive to HFPO-DA or PPARα/γ agonist treatment. These results are consistent with previously published transcriptomic analyses and further support that liver effects in HFPO-DA-exposed rodents are mediated through rodent-specific PPARα signaling mechanisms as part of the MOA for PPARα activator-induced rodent hepatocarcinogenesis. Thus, effects observed in mouse liver are not appropriate endpoints for toxicity value development for HFPO-DA in human health risk assessment. [ABSTRACT FROM AUTHOR]

HFPO-DA (ammonium, 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate) is a short-chain polyfluorinated alkyl substance (PFAS) used in the manufacture of some types of fluorinated polymers. Like many PFAS, toxicity studies with HFPO-DA indicate the liver is the primary target of toxicity in rodents following oral exposure. Due to the structural diversity of PFAS, the mode of action (MOA) can differ between PFAS for the same target tissue. There is significant evidence for involvement of peroxisome proliferator-activated receptor alpha (PPARα) activation based on molecular and histopathological responses in the liver following HFPO-DA exposure, but other MOAs have also been hypothesized based on limited evidence. The MOA underlying the liver effects in mice exposed to HFPO-DA was assessed in the context of the Key Events (KEs) outlined in the MOA framework for PPARα activator-induced rodent hepatocarcinogenesis. The first 3 KEs (ie, PPARα activation, alteration of cell growth pathways, and perturbation of cell growth/survival) are supported by several lines of evidence from both in vitro and in vivo data available for HFPO-DA. In contrast, alternate MOAs, including cytotoxicity, PPARγ and mitochondrial dysfunction are generally not supported by the scientific literature. HFPO-DA-mediated liver effects in mice are not expected in humans as only KE 1, PPARα activation, is shared across species. PPARα-mediated gene expression in humans produces only a subset (ie, lipid modulating effects) of the responses observed in rodents. As such, the adverse effects observed in rodent livers should not be used as the basis of toxicity values for HFPO-DA for purposes of human health risk assessment. [ABSTRACT FROM AUTHOR]

Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate (HFPO-DA) is a short chain member of per- and polyfluoroalkyl substances (PFAS). To better understand the relevance of histopathological effects seen in livers of mice exposed to HFPO-DA for human health risk assessment, histopathological effects were summarized from hematoxylin and eosin (H&E)-stained sections in several repeat-dose toxicity studies in mice. Findings across studies revealed histopathological changes consistent with peroxisomal proliferation, whereas two reports of steatosis could not be confirmed in the published figures. In addition, mechanisms of hepatocellular death were assessed in H&E sections as well as with the apoptotic marker cleaved caspase-3 (CCasp3) in newly cut sections from archived liver blocks from select studies. A comparison of serially CCasp3 immunolabeled and H&E-stained sections revealed that mechanisms of hepatocellular death cannot be clearly discerned in H&E-stained liver sections alone as several examples of putatively necrotic cells were positive for CCasp3. Published whole genome transcriptomic data were also reevaluated for enrichment of various forms of hepatocellular death in response to HFPO-DA, which revealed enrichment of apoptosis and autophagy, but not ferroptosis, pyroptosis, or necroptosis. These morphological and molecular findings are consistent with transcriptomic evidence for peroxisome proliferator-activated receptor alpha (PPARα) signaling in HFPO-DA exposed mice. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Under the Food Safety Modernization Act (FSMA) and preventive controls (PCs) regulations, food manufacturers must consider whether PCs are needed for potential hazards present in food. The mycotoxin ochratoxin A (OTA) is considered a chemical hazard under FSMA. It is produced by several fungal species and can be present in various agricultural commodities, including coffee. OTA presents a unique scenario in food safety, because it is known to be a potential risk; because heating may destroy it, but not completely; and because the hazard profile suggests it is not acutely toxic at the occurrence levels in coffee, although at high exposure levels, it is potentially nephrotoxic and carcinogenic in animal models. In the absence of US compliance levels, it is important for the risk assessor and risk manager to determine whether PCs are warranted. To address this complex situation in the coffee industry, we combined food safety and toxicology risk assessment principles to examine the available information on OTA hazard and risk in coffee. Exposure and health-based benchmarks for OTA in coffee, established by reviewing peer-reviewed literature, food recall databases, and authoritative reviews, resulted in large margins-of-exposure for both single and repeated exposure scenarios. Furthermore, no evidence was identified from historical data to suggest OTA is acutely toxic in humans from coffee consumption or other exposure sources. Therefore, findings from this assessment indicate that no PC is warranted for US coffee manufactures, based on the low severity and likelihood of risk according to margin-of-exposure estimates and historical data. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Endocrine disrupting compounds ( EDCs) enter aquatic habitats from a variety of anthropogenic sources and can mimic, block, or modulate the synthesis of natural hormones. EDCs affect both reproductive and non-reproductive behaviors because hormones mediate responses associated with aggression and fear. We examined the effects of two EDCs on risk-taking behaviors in guppies ( Poecilia reticulata). We quantified risk-taking in terms of propensity to forage in a risky location and tendency to join groups in the presence of a predator. We found that male and female guppies responded oppositely to environmentally relevant concentrations of an estrogenic EDC, 17α-ethinylestradiol ( EE2), or an androgenic EDC, 17β-trenbolone ( TB). Males decreased risk-taking with increasing EE2 concentration (as predicted), but females increased risk-taking (contrary to prediction). In contrast, females increased risk-taking with increasing TB concentrations (as predicted), but males decreased risk-taking (contrary to prediction). These results did not match our expectation that EE2 would reduce risk-taking and TB would increase risk-taking in both sexes. We suspect EE2 and TB produced these counterintuitive effects by downregulating their corresponding hormone receptors and thus reducing levels of circulating endogenous hormones in females and males, respectively. These results show that EDCs can alter fish behavior and potentially reduce fitness in unexpected ways. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Human CYP2B6 is inducible by PFOS in an in vivo model. • hCYP2B6-Tg mice retain more PFOS than Cyp2b-nulls and in turn show greater toxicity. • A high-fat diet (HFD) reduced PFOS-mediated hepatotoxicity, but increased steatosis. • HFD has opposing effects on PFOS retention in Cyp2b-null and hCYP2B6 mice. • CYP2B6 is protective from PFOS-mediated steatosis but not HFD-mediated steatosis. [ABSTRACT FROM AUTHOR]

What's in Our Waters Jr. (WOW Jr.) is an environmental outreach program designed to teach fourth grade elementary students about watershed protection. This program is unique because of the partnership that brings together students, teachers, scientists, and water resource professionals to promote science education and environmental stewardship in the classroom through mentorship and hands-on learning activities. WOW Jr. is co-led by the Clemson University Cooperative Extension Service (4-H Youth Development and Water Resources Programs) and Clemson University Environmental Toxicology Graduate Program. The major themes covered in WOW Jr. are watersheds, water pollution, and water conservation and protection. The curriculum is designed to help teachers meet several South Carolina academic standards for fourth grade science. Pre- and post-surveys administered to students to assess program effectiveness and progress over time show that engagement with science professionals increases achievement of state standards and knowledge of watershed protection by environmental stewardship, and it alters students' perceptions of scientists. [ABSTRACT FROM AUTHOR]

Assessment of genotoxicity is a critical component of mode of action (MOA) analysis and carcinogen risk assessment due to its influence on quantitative risk extrapolation approaches. To date, clear guidance and expert consensus on the determination of a mutagenic MOA remains elusive, resulting in different estimates of carcinogenic risk for the same chemical among different stakeholders. Oral toxicity criteria for hexavalent chromium [Cr(VI)], for example, differ by orders of magnitude due largely to the interpretation of in vivo genotoxicity data. Herein, we review in vivo genotoxicity studies for Cr(VI) to inform the MOA for Cr(VI)-induced tumors observed in a two-year cancer bioassay in mice and rats exposed via drinking water. Overall, genotoxicity results in carcinogenic target tissues (viz., oral cavity and duodenum) are negative. Results in the intestine are consistent with imaging data indicating little to no chromium present in the crypt compartment following oral exposure. Positive genotoxicity results in nontarget tissues have been reported at high doses mostly following nonphysiological routes of exposure. Given the negative genotoxicity results in carcinogenic target organs from oral exposure to Cr(VI), there is scientific justification to support the use of nonlinear low-dose extrapolation methods in the derivation of oral toxicity criteria for Cr(VI). These results highlight important differences between genotoxicity testing for hazard identification purposes and quantitative risk assessment. [ABSTRACT FROM AUTHOR]

Use of cannabidiol (CBD) in humans has increased considerably in recent years. While currently available studies suggest that CBD is relatively safe for human consumption, data from publicly available studies on CBD conducted according to modern testing guidelines are lacking. In the current study, the potential for toxicity following repeated oral exposure to hemp-derived CBD isolate was evaluated in male and female Sprague Dawley rats. No adverse treatment-related effects were observed following administration of CBD via oral gavage for 14 and 90 days at concentrations up to 150 and 140 mg/kg-bw/d, respectively. Microscopic liver and adrenal gland changes observed in the 90-day study were determined to be resolved after a 28-day recovery period. CBD was well tolerated at these dose levels, and the results of this study are comparable to findings reported in unpublished studies conducted with other CBD isolates. The current studies were conducted as part of a broader research program to examine the safety of CBD. • OECD guideline-compliant subchronic oral toxicity study of CBD isolate conducted in rats. • No adverse treatment-related effects up to the highest doses tested. • Adaptive microscopic liver and adrenal gland changes resolved after a 28-day recovery period. • Oral NOAEL identified as 150 and 140 mg/kg-bw/d in 14- and 90-day toxicity studies, respectively. [ABSTRACT FROM AUTHOR]