Species-Specific Unbound Fraction Differences in Highly Bound PFAS: A Comparative Study across Human, Rat, and Mouse Plasma and Albumin.

Per- and polyfluoroalkyl substances (PFAS) are a diverse group of fluorinated compounds which have yet to undergo comprehensive investigation regarding potential adverse health effects and bioaccumulative properties. With long half-lives and accumulative properties, PFAS have been linked to several toxic effects in both non-clinical species such as rat and mouse as well as human. Although biological impacts and specific protein binding of PFAS have been examined, there is no study focusing on the species-specific fraction unbound (fu) in plasma and related toxicokinetics. Herein, a presaturation equilibrium dialysis method was used to measure and validate the binding of 14 individual PFAS with carbon chains containing 4 to 12 perfluorinated carbon atoms and several functional head-groups to albumin and plasma of mouse (C57BL/6 and CD-1), rat, and human. Equivalence testing between each species-matrix combination showed positive correlation between rat and human when comparing fu in plasma and binding to albumin. Similar trends in binding were also observed for mouse plasma and albumin. Relatively high Spearman correlations for all combinations indicate high concordance of PFAS binding regardless of matrix. Physiochemical properties of PFAS such as molecular weight, chain length, and lipophilicity were found to have important roles in plasma protein binding of PFAS.

Perinatal exposure to PFOS and sustained high-fat diet promote neurodevelopmental disorders via genomic reprogramming of pathways associated with neuromotor development.

Perfluorooctanesulfonic acid (PFOS) is a neurotoxic widespread organic contaminant which affects several brain functions including memory, motor coordination and social activity. PFOS has the ability to traverse the placenta and the blood brain barrier (BBB) and cause weight gain in female mice. It's also known that obesity and consumption of a high fat diet have negative effects on the brain, impairs cognition and increases the risk for the development of dementia. The combination effect of developmental exposure to PFOS and the intake of a high-fat diet (HFD) has not been explored. This study investigates the effect of PFOS and /or HFD on weight gain, behavior and transcriptomic and proteomic analysis of adult brain mice. We found that female mice exposed to PFOS alone showed an increase in weight, while HFD expectedly increased body weight. The combination of HFD and PFOS exacerbated generalized behavior such as time spent in the center and rearing, while PFOS alone impacted the distance travelled. These results suggest that PFOS exposure may promote hyperactivity. The combination of PFOS and HFD alter social behavior such as rearing and withdrawal. Although HFD interfered with memory retrieval, biomarkers of dementia did not change except for total Tau and phosphorylated Tau. Tau was impacted by either or both PFOS exposure and HFD. Consistent with behavioral observations, global cerebral transcriptomic analysis showed that PFOS exposure affects calcium signaling, MAPK pathways, ion transmembrane transport, and developmental processes. The combination of HFD with PFOS enhances the effect of PFOS in the brain and affects pathways related to ER stress, axon guidance and extension, and neural migration. Proteomic analysis showed that HFD enhances the impact of PFOS on inflammatory pathways, regulation of cell migration and proliferation, and MAPK signaling pathways. Overall, these data show that PFOS combined with HFD may reprogram the genome and modulate neuromotor development and may promote symptoms linked to attention deficit-hyperactivity disorders (ADHD) and autism spectrum disorders (ASD). Future work will be needed to confirm these connections.

Per- and polyfluoroalkyl substances (PFAS) augment adipogenesis and shift the proteome in murine 3T3-L1 adipocytes.

The Per- and polyfluoroalkyl substances (PFAS) are a wide group of fluorinated compounds, which the health effects of many of them have not been investigated. Perfluorinated sulfonates, such as perfluorooctane sulfonate (PFOS) and perfluorinated carboxylates, such as perfluorooctanoic acid (PFOA) are members of this broad group of PFAS, and previous studies have shown a correlation between the body accumulation of PFOS and PFOA and increased adipogenesis. PFOA and PFOS have been withdrawn from the market and use is limited because of their persistence, toxicity, and bioaccumulative properties. Instead, short chain PFAS have been created to replace PFOA and PFOS, but the health effects of other short chain PFAS are largely unknown. Therefore, herein we aimed to comprehensively determined the potential adipogenesis of ten different PFAS (PFBS, PFHxS, PFOS, PFBA, PFHxA, PFHA, PFOA, PFNA, PFDA, and HFPO-DA) and investigated the differences in protein expression of 3T3-L1 cells upon exposure to each PFAS. 3T3-L1 cells were differentiated with or without each PFAS for 4-days, and cellular lipid was quantified using Nile Red staining. Analysis of the adipocyte proteome was performed to identify the pathways related to adipogenesis and quantify proteins significantly affected by each PFAS. The results showed that in general, every PFAS investigated in our study has the potential to induce the 3T3-L1 differentiation to adipocytes in the presence of rosiglitazone, with the concentrations that range between 0.25 and 25 µM. Proteomics analysis revealed specific markers regarding to adipogenesis upregulated upon exposure to each of the ten PFAS.

Per- and polyfluoroalkyl substances, epigenetic age and DNA methylation: a cross-sectional study of firefighters.

Background: Per- and polyfluoroalkyl substances (PFASs) are persistent chemicals that firefighters encounter. Epigenetic modifications, including DNA methylation, could serve as PFASs toxicity biomarkers. Methods: With a sample size of 197 firefighters, we quantified the serum concentrations of nine PFASs, blood leukocyte DNA methylation and epigenetic age indicators via the EPIC array. We examined the associations between PFASs with epigenetic age, site- and region-specific DNA methylation, adjusting for confounders. Results: Perfluorohexane sulfonate, perfluorooctanoate (PFOA) and the sum of branched isomers of perfluorooctane sulfonate (Sm-PFOS) were associated with accelerated epigenetic age. Branched PFOA, linear PFOS, perfluorononanoate, perfluorodecanoate and perfluoroundecanoate were associated with differentially methylated loci and regions. Conclusion: PFASs concentrations are associated with accelerated epigenetic age and locus-specific DNA methylation. The implications for PFASs toxicity merit further investigation.

The role of maternal high fat diet on mouse pup metabolic endpoints following perinatal PFAS and PFAS mixture exposure.

Per- and polyfluoroalkyl substances (PFAS) are a family of chemicals that are ubiquitous in the environment. Some of these chemicals, such as perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonate (PFHxS) and perfluorooctanoic acid (PFOA), are found in human sera and have been shown to cause liver steatosis and reduce postnatal survival and growth in rodents. The purpose of this work is to evaluate the impact of diet and PFAS exposure to mouse dam (mus musculus) on the risk to pup liver and metabolism endpoints later in life, as well as evaluate PFAS partitioning to pups. Timed-pregnant dams were fed a standard chow diet or 60 % kcal high fat diet (HFD). Dams were administered either vehicle, 1 mg/kg PFOA, 1 mg/kg PFOS, 1 mg/kg PFHxS, or a PFAS mixture (1 mg/kg of each PFOA, PFOS, and PFHxS) daily via oral gavage from gestation day 1 until postnatal day (PND) 20. At PND 21, livers of dams and 2 pups of each sex were evaluated for lipid changes while remaining pups were weaned to the same diet as the dam for an additional 10 weeks. Dam and pup serum at PND 21 and PND 90 were also evaluated for PFAS concentration, alanine aminotransferase (ALT), leptin and adiponectin, and glycosylated hemoglobin A1c. Perinatal exposure to a HFD, as expected, increased pup body weight, maternal liver weight, pup liver triglycerides, pup serum ALT, and pup serum leptin. PFOA and the PFAS mixture increased liver weights, and. treatment with all three compounds increased liver triglycerides. The maternal HFD increased dam and pup serum PFAS levels, however, was protective against PFOA-induced increase in serum ALT and observed increases in liver triglycerides. The PFAS mixture had very distinct effects when compared to single compound treatment, suggesting some cumulative effects, particularly when evaluating PFAS transfer from dam to pup. This data highlights the importance of diet and mixtures when evaluating liver effect of PFAS and PFAS partitioning.

Developmental Perfluorooctanesulfonic acid (PFOS) exposure as a potential risk factor for late-onset Alzheimer's disease in CD-1 mice and SH-SY5Y cells.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that accounts for approximately 60-80% of dementia cases worldwide and is characterized by an accumulation of extracellular senile plaques composed of ß-amyloid (Aß) peptide and intracellular neurofibrillary tangles (NFTs) containing hyperphosphorylated tau protein. Sporadic or late-onset AD (LOAD) represents 95 % of the AD cases and its etiology does not appear to follow Mendelian laws of inheritance, thus, implicating the role of epigenetic programming and environmental factors. Apolipoprotein allele 4 (ApoE4), the only established genetic risk factor for LOAD, is suggested to accelerate the pathogenesis of AD by increasing tau hyperphosphorylation, inhibiting the clearance of amyloid-ß (Aß), and promoting Aß aggregation. Perfluorooctanesulfonic acid (PFOS) is a persistent organic pollutant, with potential neurotoxic effects, that poses a major threat to the ecosystem and human health. By employing in vivo and in vitro models, the present study investigated PFOS as a potential risk factor for LOAD by assessing its impact on amyloidogenesis, tau pathology, and rodent behavior. Our behavioral analysis revealed that developmentally exposed male and female mice exhibited a strong trend of increased rearing and significantly increased distance traveled in the open field test. Biochemically, GSK3ß and total ApoE were increased following developmental exposure, in vivo. Furthermore, in vitro, low concentrations of PFOS elevated protein levels of APP, tau, and its site-specific phosphorylation. Differentiated SH-SY5Y cells exposed to a series of PFOS concentrations, also, had elevated protein expression of GSK3ß. These data suggest that total ApoE is inducible by environmental exposure to PFOS.

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.

Perfluorooctanesulfonic Acid (PFOS) Thwarts the Beneficial Effects of Calorie Restriction and Metformin.

A combination of calorie restriction (CR), dietary modification, and exercise is the recommended therapy to reverse obesity and nonalcoholic fatty liver disease. In the liver, CR shifts hepatic metabolism from lipid storage to lipid utilization pathways, such as AMP-activated protein kinase (AMPK). Perfluorooctanesulfonic acid (PFOS), a fluorosurfactant previously used in stain repellents and anti-stick materials, can increase hepatic lipids in mice following relatively low-dose exposures. To test the hypothesis that PFOS administration interferes with CR, adult male C57BL/6N mice were fed ad libitum or a 25% reduced calorie diet concomitant with either vehicle (water) or 100 µg PFOS/kg/day via oral gavage for 6 weeks. CR alone improved hepatic lipids and glucose tolerance. PFOS did not significantly alter CR-induced weight loss, white adipose tissue mass, or liver weight over 6 weeks. However, PFOS increased hepatic triglyceride accumulation, in both mice fed ad libitum and subjected to CR. This was associated with decreased phosphorylated AMPK expression in liver. Glucagon (100 nM) treatment induced glucose production in hepatocytes, which was further upregulated with PFOS (2.5 µM) co-treatment. Next, to explore whether the observed changes were related to AMPK signaling, HepG2 cells were treated with metformin or AICAR alone or in combination with PFOS (25 µM). PFOS interfered with glucose-lowering effects of metformin, and AICAR treatment partially impaired PFOS-induced increase in glucose production. In 3T3-L1 adipocytes, metformin was less effective with PFOS co-treatment. Overall, PFOS administration disrupted hepatic lipid and glucose homeostasis and interfered with beneficial glucose-lowering effects of CR and metformin.

In vitro metabolism of HMTD and blood stability and toxicity of peroxide explosives (TATP and HMTD) in canines and humans.

Triacetone triperoxide (TATP) and hexamethylene triperoxide diamine (HMTD) are prominent explosive threats. Mitigation of peroxide explosives is a priority among the law enforcement community, with canine (K9) units being trained to recognise the scent of peroxide explosives. Herein, the metabolism, blood distribution, and toxicity of peroxide explosives are investigated.HMTD metabolism studies in liver microsomes identified two potential metabolites, tetramethylene diperoxide diamine alcohol aldehyde (TMDDAA) and tetramethylene peroxide diamine dialcohol dialdehyde (TMPDDD).Blood stability studies in dogs and humans showed that HMTD was rapidly degraded, whereas TATP remained for at least one week.Toxicity studies in dog and human hepatocytes indicated minimum cell death for both TATP and HMTD.

An 'Omics Approach to Unraveling the Paradoxical Effect of Diet on Perfluorooctanesulfonic Acid (PFOS) and Perfluorononanoic Acid (PFNA)-Induced Hepatic Steatosis.

Perfluoroalkyl substances (PFAS) are a family of toxicants universally detected in human serum and known to cause dyslipidemia in animals and humans. Hepatic steatosis, which is defined as lipid deposition in the liver, is known to be a consequence of poor diet. Similarly, PFAS are known to induce hepatic steatosis in animals on a low-fat chow. This study explored diet-PFAS interactions in the liver and their potential to modulate hepatic steatosis. Male C57BL/6J mice were fed with either a low-fat diet (10% kcal from fat, LFD) or a moderately high-fat diet (45% kcal from fat, HFD) with or without perfluorooctanesulfonic acid (3 ppm, PFOS) or perfluorononanoic acid (3 ppm, PFNA) in feed for 12 weeks. Livers were excised for histology and quantification of PFAS and lipids. The PFOS and PFNA coadministration with HFD reduced the hepatic accumulation of lipid and PFAS relative to the LFD treatment groups. Furthermore, transcriptomic analysis revealed that PFAS administration in the presence of an HFD significantly reduces expression of known hepatic PFAS uptake transporters, organic anion transporter proteins. Transcriptomics and proteomics further revealed several pathways related to lipid metabolism, synthesis, transport, and storage that were modulated by PFAS exposure and further impacted by the presence of dietary fat. Both dietary fat content and the chemical functional head group exerted significant influence on hepatic PFAS accumulation and the resulting biochemical signature, suggesting that diet and structure should be considered in the design and interpretation of research on PFAS induced hepatic steatosis.

Hepatoprotective and anti-inflammatory effects of a standardized pomegranate (Punica granatum) fruit extract in high fat diet-induced obese C57BL/6 mice.

Diets rich in fats are linked to elevated systemic inflammation, which augments the progression of inflammatory-related disorders including non-alcoholic fatty liver disease (NAFLD) and neurodegenerative diseases. A phenolic-enriched pomegranate fruit extract (PE) was investigated for its hepatoprotective and anti-inflammatory effects in male C57BL/6 mice fed either a high-fat diet or a standard rodent diet with or without 1% of PE for 12 weeks. Mouse livers and hippocampi were evaluated for the expression of genes associated with NAFLD and inflammation by multiplexed gene analysis. PE alleviated diet-induced fatty liver and suppressed hepatic lipid regulating genes including Cd36, Fas, Acot2 and Slc27a1. In addition, PE suppressed gene expression of pro-inflammatory cytokines including Il-1α, Il-7, Il-11, Ifnα, Tnfα and Lepr in the hippocampi. Our findings support the protective effects of PE against high-fat diet-induced hepatic and neurological disease.

Perfluorooctanesulfonic Acid and Perfluorohexanesulfonic Acid Alter the Blood Lipidome and the Hepatic Proteome in a Murine Model of Diet-Induced Obesity.

Perfluoroalkyl substances (PFAS) represent a family of environmental toxicants that have infiltrated the living world. This study explores diet-PFAS interactions and the impact of perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic (PFHxS) on the hepatic proteome and blood lipidomic profiles. Male C57BL/6J mice were fed with either a low-fat diet (10.5% kcal from fat) or a high fat (58% kcal from fat) high carbohydrate (42 g/l) diet with or without PFOS or PFHxS in feed (0.0003% wt/wt) for 29 weeks. Lipidomic, proteomic, and gene expression profiles were determined to explore lipid outcomes and hepatic mechanistic pathways. With administration of a high-fat high-carbohydrate diet, PFOS and PFHxS increased hepatic expression of targets involved in lipid metabolism and oxidative stress. In the blood, PFOS and PFHxS altered serum phosphatidylcholines, phosphatidylethanolamines, plasmogens, sphingomyelins, and triglycerides. Furthermore, oxidized lipid species were enriched in the blood lipidome of PFOS and PFHxS treated mice. These data support the hypothesis that PFOS and PFHxS increase the risk of metabolic and inflammatory disease induced by diet, possibly by inducing dysregulated lipid metabolism and oxidative stress.

Perfluorooctanesulfonic acid (PFOS) administration shifts the hepatic proteome and augments dietary outcomes related to hepatic steatosis in mice.

Hepatic steatosis increases risk of fatty liver and cardiovascular disease. Perfluorooctanesulfonic acid (PFOS) is a persistent, bio-accumulative pollutant that has been used in industrial and commercial applications. PFOS administration induces hepatic steatosis in rodents and increases lipogenic gene expression signatures in cultured hepatocytes. We hypothesized that PFOS treatment interferes with lipid loss when switching from a high fat diet (HFD) to a standard diet (SD), and augments HFD-induced hepatic steatosis. Male C57BL/6 N mice were fed standard chow diet or 60% kCal high-fat diet (HFD) for 4 weeks to increase body weight. Then, some HFD mice were switched to SD and mice were further divided to diet only or diet containing 0.0003% PFOS, for six treatment groups: SD, HFD to SD (H-SD), HFD, SD + PFOS, H-SD + PFOS, or HFD + PFOS. After 10 weeks on study, blood and livers were collected. HFD for 14 weeks increased body weight and hepatic steatosis, whereas H-SD mice returned to SD measures. PFOS administration reduced body weight in mice fed a SD, but not H-SD or HFD. PFOS administration increased liver weight in H-SD + PFOS and HFD + PFOS mice. PFOS increased hepatic steatosis in H-SD and HFD groups. Hepatic mRNA expression and SWATH-MS proteomic analysis revealed that PFOS induced lipid and xenobiotic transporters, as well as metabolism pathways. Overall, the findings herein suggest that PFOS treatment did interfere with lipid loss associated with switch to a SD and similarly augmented hepatic lipid accumulation in mice established on an HFD.

Inhibitory Effect of Cannabidiol on the Activation of NLRP3 Inflammasome Is Associated with Its Modulation of the P2X7 Receptor in Human Monocytes.

Cannabidiol (CBD), a phytocannabinoid, has been reported to have anti-inflammatory effects associated with NLRP3 inflammasome activation, but its mechanism of anti-inflammasome action remains unclear. Herein, we report CBD's effect on NLRP3 inflammasome activation and its modulation of P2X7, an inflammasome activation-related receptor, in human THP-1 monocytes. CBD (0.1, 1, and 10 µM) exerted anti-inflammasome activity in LPS-nigericin-stimulated THP-1 monocytes by reducing media IL-1ß concentration (by 63.9%, 64.1%, and 83.1%, respectively), which was similar to the known NLRP3 inflammasome inhibitors oridonin and MCC950 (16.9% vs 20.8% and 17.4%, respectively; at 10 µM). CBD (10 µM) decreased nigericin-alone- and nigericin-lipopolysaccharide-induced potassium efflux by 13.7% and 13.0%, respectively, in THP-1 monocytes, strongly suggesting P2X7 receptor modulation. Computational docking data supported the potential for CBD binding to the P2X7 receptor via interaction with GLU 172 and VAL 173 residues. Overall, the observed CBD suppressive effect on NLRP3 inflammasome activation in THP-1 monocytes was associated with decreased potassium efflux, as well as in silico prediction of P2X7 receptor binding. CBD inhibitory effects on the NLRP3 inflammasome may contribute to the overall anti-inflammatory effects reported for this phytocannabinoid.

Lack of Multidrug Resistance-associated Protein 4 Prolongs Partial Hepatectomy-induced Hepatic Steatosis.

Multidrug resistance-associated protein 4 (Mrp4) is an efflux transporter involved in the active transport of several endogenous and exogenous chemicals. Previously, we have shown that hepatic Mrp4 expression increases following acetaminophen overdose. In mice, these increases in Mrp4 expression are observed specifically in hepatocytes undergoing active proliferation. From this, we hypothesized that Mrp4 plays a key role in hepatocyte proliferation and that lack of Mrp4 impedes liver regeneration following liver injury and/or tissue loss. To evaluate the role of Mrp4 in these processes, we employed two-third partial hepatectomy (PH) as an experimental liver regeneration model. In this study, we performed PH-surgery on male wildtype (C57BL/6J) and Mrp4 knockout mice. Plasma and liver tissues were collected at 24, 48, and 72 h postsurgery and evaluated for liver injury and liver regeneration endpoints, and for PH-induced hepatic lipid accumulation. Our results show that lack of Mrp4 did not alter hepatocyte proliferation and liver injury following PH as evaluated by Ki-67 antigen staining and plasma alanine aminotransferase levels. To our surprise, Mrp4 knockout mice exhibited increased hepatic lipid content, in particular, di- and triglyceride levels. Gene expression analysis showed that lack of Mrp4 upregulated hepatic lipin1 and diacylglycerol O-acyltransferase 1 and 2 gene expression, which are involved in the synthesis of di- and triglycerides. Our observations indicate that lack of Mrp4 prolonged PH-induced hepatic steatosis in mice and suggest that Mrp4 may be a novel genetic factor in the development of hepatic steatosis.

2,2',4,4',5-Pentabromodiphenyl ether induces lipid accumulation throughout differentiation in 3T3-L1 and human preadipocytes in vitro.

Flame retardants, specifically polybrominated diphenyl ethers (PBDEs), are chemical compounds widely used for industrial purposes and household materials. NHANES data indicate that nearly all Americans have trace amounts of PBDEs in serum, with even higher levels associated with occupational exposure. PBDEs are known to bioaccumulate in the environment due to their lipophilicity and stability, and more importantly, they have been detected in human adipose tissue. The present study examined whether the PBDE congener, BDE-99 (2,2',4,4',5-pentabromodiphenyl ether; 0.2-20 µM), enhances the adipogenesis of mouse and human preadipocyte cell models in vitro via induced lipid accumulation. 3T3-L1 mouse preadipocytes and human visceral preadipocytes demonstrated enhanced hormone-induced lipid accumulation upon BDE-99 treatment. In addition, BDE-99 (20 µM) induced preadipocyte differentiation and lipid development in nondifferentiated human preadipocytes. BDE-99, the second most abundant congener in human adipose tissue, increased total lipids in differentiating adipocytes and therefore showed a potential role in the regulation of adipogenesis. This warrants more research to further understand the impact of lipophilic persistent pollutants on adipose tissue homeostasis.

Pomegranate ellagitannin-gut microbial-derived metabolites, urolithins, inhibit neuroinflammation in vitro.

OBJECTIVES: Urolithins, ellagitannin-gut microbial-derived metabolites, have been reported to mediate pomegranate's neuroprotective effects against Alzheimer's disease (AD), but there are limited data on their effects against neuroinflammation. Herein, we: (1) evaluated whether urolithins (urolithins A and B and their methylated derivatives) attenuate neuroinflammation in murine BV-2 microglia and human SH-SY5Y neurons, and (2) evaluated hippocampus of transgenic AD (R1.40) mice administered a pomegranate extract (PE; 100 or 200 mg/kg/day for 3 weeks) for inflammatory biomarkers. METHODS: Effects of urolithins (10 µM) on inflammatory biomarkers were evaluated in lipopolysaccharide (LPS)-stimulated BV-2 microglia. In a non-contact co-culture cell model, SH-SY5Y cell viability was assessed after exposure to media collected from LPS-BV-2 cells treated with or without urolithins. Effects of urolithins on apoptosis and caspase 3/7 and 9 release from H2O2-induced oxidative stress of BV-2 and SH-SY5Y cells were assessed. Hippocampal tissues of vehicle and PE-treated transgenic R1.40 mice were evaluated for gene expression of inflammatory biomarkers by qRT-PCR. RESULTS: Urolithins decreased media levels of nitric oxide, interleukin 6 (IL-6), prostaglandin E2, and tumor necrosis factor alpha from LPS-BV-2 microglia. In the co-culture cell model, media from LPS-BV-2 cells treated with urolithins preserved SH-SY5Y cell viability greater than media from cells treated without urolithins. Urolithins mitigated apoptosis and caspase 3/7 and 9 release from H2O2-induced oxidative stress of BV-2 and SH-SY5Y cells. While not statistically significant, inflammatory biomarkers (TNF-α, COX-2, IL-1, and IL-6) appeared to follow a decreasing trend in the hippocampus of high-dose PE-treated animals compared to controls. DISCUSSION: The attenuation of neuroinflammation by urolithins may contribute, in part, toward pomegranate's neuroprotective effects against AD.

Physiological Regulation of Drug Metabolism and Transport: Pregnancy, Microbiome, Inflammation, Infection, and Fasting.

This article is a report on a symposium entitled "Physiological Regulation of Drug Metabolism and Transport" sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2017 meeting in Chicago, IL. The contributions of physiologic and pathophysiological regulation of drug-metabolizing enzymes and transporters to interindividual variability in drug metabolism are increasingly recognized but in many cases are not well understood. The presentations herein discuss the phenomenology, consequences, and mechanism of such regulation. CYP2D6 transgenic mice were used to provide insights into the mechanism of regulation of this enzyme in pregnancy, via hepatocyte nuclear factor 4α, small heterodimer partner, and retinoids. Regulation of intestinal and hepatic drug-processing enzymes by the intestinal microbiota via tryptophan and its metabolites was investigated. The potential impact of parasitic infections on human drug metabolism and clearance was assessed in mice infected with Schistosoma mansoni or Plasmodium chabaudi chabaudi AS, both of which produced widespread and profound effects on murine hepatic drug-metabolizing enzymes. Finally, the induction of Abcc drug efflux transporters by fasting was investigated. This was demonstrated to occur via a cAMP, protein kinase A/nuclear factor-E2-related factor 2/Sirtuin 1 pathway via antioxidant response elements on the Abcc genes.

Thymoquinone ameliorates diabetic phenotype in Diet-Induced Obesity mice via activation of SIRT-1-dependent pathways.

Thymoquinone, a natural occurring quinone and the main bioactive component of plant Nigella sativa, undergoes intracellular redox cycling and re-oxidizes NADH to NAD+. TQ administration (20 mg/kg/bw/day) to the Diet-Induced Obesity (DIO) mice reduced their diabetic phenotype by decreasing fasting blood glucose and fasting insulin levels, and improved glucose tolerance and insulin sensitivity as evaluated by oral glucose and insulin tolerance tests (OGTT and ITT). Furthermore, TQ decreased serum cholesterol levels and liver triglycerides, increased protein expression of phosphorylated Akt, decreased serum levels of inflammatory markers resistin and MCP-1, and decreased NADH/NAD+ ratio. These changes were paralleled by an increase in phosphorylated SIRT-1 and AMPKα in liver and phosphorylated SIRT-1 in skeletal muscle. TQ also increased insulin sensitivity in insulin-resistant HepG2 cells via a SIRT-1-dependent mechanism. These findings are consistent with the TQ-dependent re-oxidation of NADH to NAD+, which stimulates glucose and fatty acid oxidation and activation of SIRT-1-dependent pathways. Taken together, these results demonstrate that TQ ameliorates the diabetic phenotype in the DIO mouse model of type 2 diabetes.

Hepatic Lipid Accumulation and Nrf2 Expression following Perinatal and Peripubertal Exposure to Bisphenol A in a Mouse Model of Nonalcoholic Liver Disease.

BACKGROUND: Exposure to chemicals during critical windows of development may re-program liver for increased risk of nonalcoholic fatty liver disease (NAFLD). Bisphenol A (BPA), a plastics component, has been described to impart adverse effects during gestational and lactational exposure. Our work has pointed to nuclear factor E2-related factor 2 (Nrf2) being a modulator of hepatic lipid accumulation in models of NAFLD. OBJECTIVES: To determine if chemical exposure can prime liver for steatosis via modulation of NRF2 and epigenetic mechanisms. METHODS: Utilizing BPA as a model exposure, pregnant CD-1 mice were administered 25µg/kg/day BPA via osmotic minipumps from gestational day 8 through postnatal day (PND)16. The offspring were weaned on PND21 and exposed to same dose of BPA via their drinking water through PND35. Tissues were collected from pups at week 5 (W5), and their littermates at week 39 (W39). RESULTS: BPA increased hepatic lipid content concomitant with increased Nrf2 and pro-lipogenic enzyme expression at W5 and W39 in female offspring. BPA exposure increased Nrf2 binding to a putative antioxidant response element consensus sequence in the sterol regulatory-element binding protein-1c (Srebp-1c) promoter. Known Nrf2 activators increased SREBP-1C promoter reporter activity in HepG2 cells. Methylated DNA immunoprecipitation-PCR and pyrosequencing revealed that developmental BPA exposure induced hypomethylation of the Nrf2 and Srebp-1c promoters in livers of W5 mice, which was more prominent in W39 mice than in others. CONCLUSION: Exposure to a xenobiotic during early development induced persistent fat accumulation via hypomethylation of lipogenic genes. Moreover, increased Nrf2 recruitment to the Srebp-1c promoter in livers of BPA-exposed mice was observed. Overall, the underlying mechanisms described a broader impact beyond BPA exposure and can be applied to understand other models of NAFLD. https://doi.org/10.1289/EHP664.