The Aryl hydrocarbon receptor mediates reproductive toxicity of polychlorinated biphenyl congener 126 in rats.

Polychlorinated biphenyls (PCBs) are endocrine disrupting chemicals with documented, though mechanistically ill-defined, reproductive toxicity. The toxicity of dioxin-like PCBs, such as PCB126, is mediated via the aryl hydrocarbon receptor (AHR) in non-ovarian tissues. The goal of this study was to examine the uterine and ovarian effects of PCB126 and test the hypothesis that the AHR is required for PCB126-induced reproductive toxicity. Female Holzman-Sprague Dawley wild type (n = 14; WT) and Ahr knock out (n = 11; AHR-/-) rats received a single intraperitoneal injection of either corn oil vehicle (5 ml/kg: WT_O and AHR-/-_O) or PCB126 (1.63 mg/kg in corn oil: WT_PCB and AHR-/-_PCB) at four weeks of age. The estrous cycle was synchronized and ovary and uterus were collected 28 days after exposure. In WT rats, PCB126 exposure reduced (P < 0.05) body and ovary weight, uterine gland number, uterine area, progesterone, 17ß-estradiol and anti-Müllerian hormone level, secondary and antral follicle and corpora lutea number but follicle stimulating hormone level increased (P < 0.05). In AHR-/- rats, PCB126 exposure increased (P ≤ 0.05) circulating luteinizing hormone level. Ovarian or uterine mRNA abundance of biotransformation, and inflammation genes were altered (P < 0.05) in WT rats due to PCB126 exposure. In AHR-/- rats, the transcriptional effects of PCB126 were restricted to reductions (P < 0.05) in three inflammatory genes. These findings support a functional role for AHR in the female reproductive tract, illustrate AHR's requirement in PCB126-induced reprotoxicity, and highlight the potential risk of dioxin-like compounds on female reproduction.

Comprehensive Subchronic Inhalation Toxicity Assessment of an Indoor School Air Mixture of PCBs.

Few in vivo inhalation studies have explored the toxicity of environmentally relevant mixtures of polychlorinated biphenyls (PCBs). The manufacture of industrial PCBs was banned in 1978, but PCBs continue to be formed in industrial and consumer products. Schools represent a significant source of airborne exposures to legacy and nonlegacy PCBs, placing children at risk. To evaluate the impact of these exposures, we generated an airborne mixture of PCBs, called the School Air Mixture (SAM), to match the profile of an older school from our adolescent cohort study. Female Sprague-Dawley rats were exposed either to SAM or filtered air in nose-only exposure systems, 4 h/day for 4 weeks. Congener-specific air and tissue PCB profiles were assessed using gas chromatography with tandem mass spectrometry (GC-MS/MS). PCB exposures recapitulated the target school air profile with a similarity coefficient, cos θ of 0.83. PCB inhalation yielded µg/g ∑209 PCB levels in tissues. Neurobehavioral testing demonstrated a modest effect on spatial learning and memory in SAM-exposed rats. PCB exposure induced oxidative stress in the liver and lungs, affected the maturational stages of hematopoietic stem cells, reduced telomerase activity in bone marrow cells, and altered the gut microbiota. This is the first study to emulate PCB exposures in a school and comprehensively evaluate toxicity.

Skeletal toxicity resulting from exposure of growing male rats to coplanar PCB 126 is associated with disruption of calcium homeostasis and the GH-IGF-1 axis and direct effects on bone formation.

Skeletal toxicity has been reported following exposure to polychlorinated biphenyl (PCB) mixtures. However, molecular mechanisms remain poorly understood. We exposed groups of male 4-5-week-old Sprague-Dawley rats to 3,3', 4, 4', 5-pentachlorobiphenyl (PCB 126), a dioxin-like coplanar PCB congener by a single i.p. injection of 5 µmol/kg in soy oil vehicle or vehicle alone. After 4 weeks, rats were euthanized. PCB exposure resulted in hypocalcemia (P < 0.05) and significant increases in serum PTH without changes in serum phosphorous. Hyperparathyroidism was accompanied by increased expression of mRNAs of vitamin D3 metabolizing cytochrome P450 enzymes CYP27B1 and CYP24 in the kidney (P < 0.05). PCB exposure also reduced body weight, serum IGF-1, and hepatic expression of mRNAs encoding the male-specific GH-pattern-regulated CYP2C11 and CYP3A2 relative to controls (P < 0.05). PCB exposure reduced long bone length, diameter, and surface area, but increased trabecular thickness and volume (P < 0.05). Serum osteocalcin (P < 0.05), a marker and a regulator of bone formation, was reduced, but PCB exposure had no effect on the bone resorption marker RatLaps. Exposure of human intestinal Caco-2 cells to 10-100 nM PCB 126 in the presence of vitamin D3 resulted in inhibition of mRNAs for the calcium transporters TRPV6 and PMCA1b (P < 0.05). In addition, PCB 126 suppressed osteoblastogenesis in primary bone marrow mesenchymal stem cell cultures which was blunted by the AhR antagonist CH-223191. These data provide novel evidence that skeletal toxicity after exposure to PCB 126 is a result of disruption of calcium homeostasis and the GH-IGF-1 axis, and involves direct AhR-mediated effects on bone formation.

Diminished Phosphorylation of CREB Is a Key Event in the Dysregulation of Gluconeogenesis and Glycogenolysis in PCB126 Hepatotoxicity.

The dioxin-like PCB126 elicits toxicity in various target organs. In rat liver, an alteration in the transcript levels of several genes involved in glucose and fatty acid metabolism provides insights into the origin of its hepatotoxicity. To explore the mechanisms, male Sprague-Dawley rats, fed an AIN-93G diet, were injected with PCB126 (1 or 5 µmol/kg) or corn oil and euthanized after 2 weeks. PCB126 significantly decreased serum glucose levels and the transcript levels of genes of many gluconeogenic and glycogenolytic enzymes under the transcriptional control of a nuclear transcription factor, cAMP response element-binding protein (CREB). As a novel finding, we show that PCB126 significantly decreases CREB phosphorylation, which is important for regulating both gluconeogenesis and fatty acid oxidation in the liver and explains CREB's integrative effects on both carbohydrate and lipid metabolism in PCB126 toxicity.

Assessment of the Mitigative Capacity of Dietary Zinc on PCB126 Hepatotoxicity and the Contribution of Zinc to Toxicity.

Hepatic levels of the essential micronutrient, zinc, are diminished by several hepatotoxicants, and the dietary supplementation of zinc has proven protective in those cases. 3,3',4,4',5-Pentachlorobiphenyl (PCB126), a liver toxicant, alters hepatic nutrient homeostasis and lowers hepatic zinc levels. The current study was designed to determine the mitigative potential of dietary zinc in the toxicity associated with PCB126 and the role of zinc in that toxicity. Male Sprague-Dawley rats were divided into three dietary groups and fed diets deficient in zinc (7 ppm Zn), adequate in zinc (30 ppm Zn), and supplemented in zinc (300 ppm). The animals were maintained for 3 weeks on these diets, then given a single IP injection of vehicle or 1 or 5 µmol/kg PCB126. After 2 weeks, the animals were euthanized. Dietary zinc increased the level of ROS, the activity of CuZnSOD, and the expression of metallothionein but decreased the levels of hepatic manganese. PCB126 exposed rats exhibited classic signs of exposure, including hepatomegaly, increased hepatic lipids, increased ROS and CYP induction. Liver histology suggests some mild ameliorative properties of both zinc deficiency and zinc supplementation. Other metrics of toxicity (relative liver and thymus weights, hepatic lipids, and hepatic ROS) did not support this trend. Interestingly, the zinc supplemented high dose PCB126 group had mildly improved histology and less efficacious induction of investigated genes than did the low dose PCB126 group. Overall, decreases in zinc caused by PCB126 likely contribute little to the ongoing toxicity, and the mitigative/preventive capacity of zinc against PCB126 exposure seems limited.

Sulfation of Lower Chlorinated Polychlorinated Biphenyls Increases Their Affinity for the Major Drug-Binding Sites of Human Serum Albumin.

The disposition of toxicants is often affected by their binding to serum proteins, of which the most abundant in humans is serum albumin (HSA). There is increasing interest in the toxicities of environmentally persistent polychlorinated biphenyls (PCBs) with lower numbers of chlorine atoms (LC-PCBs) due to their presence in both indoor and outdoor air. PCB sulfates derived from metabolic hydroxylation and sulfation of LC-PCBs have been implicated in endocrine disruption due to high affinity-binding to the thyroxine-carrying protein, transthyretin. Interactions of these sulfated metabolites of LC-PCBs with HSA, however, have not been previously explored. We have now determined the relative HSA-binding affinities for a group of LC-PCBs and their hydroxylated and sulfated derivatives by selective displacement of the fluorescent probes 5-dimethylamino-1-naphthalenesulfonamide and dansyl-l-proline from the two major drug-binding sites on HSA (previously designated as Site I and Site II). Values for half-maximal displacement of the probes indicated that the relative binding affinities were generally PCB sulfate ≥ OH-PCB > PCB, although this affinity was site- and congener-selective. Moreover, specificity for Site II increased as the numbers of chlorine atoms increased. Thus, hydroxylation and sulfation of LC-PCBs result in selective interactions with HSA which may affect their overall retention and toxicity.

Metabolism and metabolites of polychlorinated biphenyls.

Abstract The metabolism of polychlorinated biphenyls (PCBs) is complex and has an impact on toxicity, and thereby on the assessment of PCB risks. A large number of reactive and stable metabolites are formed in the processes of biotransformation in biota in general, and in humans in particular. The aim of this document is to provide an overview of PCB metabolism, and to identify the metabolites of concern and their occurrence. Emphasis is given to mammalian metabolism of PCBs and their hydroxyl, methylsulfonyl, and sulfated metabolites, especially those that persist in human blood. Potential intracellular targets and health risks are also discussed.

Tissue Distribution, Metabolism, and Excretion of 3,3'-Dichloro-4'-sulfooxy-biphenyl in the Rat.

Polychlorinated biphenyls (PCBs) with less chlorine atoms exhibit a greater susceptibility to metabolism than their more-chlorinated counterparts. Following initial hydroxylation of these less-chlorinated PCBs, metabolic sulfation to form PCB sulfates is increasingly recognized as an important component of their toxicology. Because procedures for the quantitative analysis of PCB sulfates in tissue samples have not been previously available, we have now developed an efficient, LC-ESI-MS/MS-based protocol for the quantitative analysis of 4-PCB 11 sulfate in biological samples. This procedure was used to determine the distribution of 4-PCB 11 sulfate in liver, kidney, lung, and brain as well as its excretion profile following its intravenous administration to male Sprague-Dawley rats. Following initial uptake of 4-PCB 11 sulfate, its concentration in these tissues and serum declined within the first hour following injection. Although biliary secretion was detected, analysis of 24 h collections of urine and feces revealed recovery of less than 4% of the administered 4-PCB 11 sulfate. High-resolution LC-MS analysis of bile, urine, and feces showed metabolic products derived from 4-PCB 11 sulfate. Thus, 4-PCB 11 sulfate at this dose was not directly excreted in the urine but was instead redistributed to tissues and/or subjected to further metabolism.

Disposition of phenolic and sulfated metabolites after inhalation exposure to 4-chlorobiphenyl (PCB3) in female rats.

PCBs, such as PCB3, are air contaminants in buildings and outdoors. Metabolites of PCB3 are potential endocrine disrupting chemicals and genotoxic agents. We studied the disposition of phenolic and sulfated metabolites after acute nose-only inhalation exposure to airborne PCB3 for 2 h in female rats. Inhalation exposure was carried out in three groups. In the first group, rats exposed to an estimated dose of 26 µg/rat were euthanized at 0, 1, 2, and 4 h after exposure. Highest concentrations of phenols and sulfates were observed at 0 h, and the values were 7 ± 1 and 560 ± 60 ng/mL in serum, 213 ± 120 and 842 ± 80 ng/g in liver, 31 ± 27 and 22 ± 7 ng/g in lung, and 27 ± 6 and 3 ± 0 ng/g in brain, respectively. First-order serum clearance half-lives of 0.5 h for phenols and 1 h for sulfates were estimated. In the second group, rats exposed to an estimated dose of 35 µg/rat were transferred to metabolism cages immediately after exposure for the collection of urine and feces over 24 h. Approximately 45 ± 5% of the dose was recovered from urine and consisted mostly of sulfates; the 18 ± 5% of the dose recovered from feces was exclusively phenols. Unchanged PCB3 was detected in both urine and feces but accounted for only 5 ± 3% of the dose. Peak excretion of metabolites in both urine and feces occurred within 18 h postexposure. In the third group, three bile-cannulated rats exposed to an estimated dose of 277 µg/rat were used for bile collection. Bile was collected for 4 h immediately after 2 h exposure. Biliary metabolites consisted mostly of sulfates, some glucuronides, and lower amounts of the free phenols. Control rats in each group were exposed to clean air. Clinical serum chemistry values, serum T4 level, and urinary 8-hydroxy-2'-deoxyguanosine were similar in treated and control rats. These data show that PCB3 is rapidly metabolized to phenols and conjugated to sulfates after inhalation and that both of these metabolites are distributed to liver, lungs, and brain. The sulfates elaborated into bile are either reabsorbed or hydrolyzed in the intestine and excreted in the feces as phenols.

Sulfate conjugates are urinary markers of inhalation exposure to 4-chlorobiphenyl (PCB3).

PCBs are contaminants in the air of older buildings and cities, which raises the concern of inhalation exposure. No reliable biomarker of such exposure is available. We exposed rats to air containing 2 mg/m(3) PCB3 via nose-only inhalation for 2 h, collected urine, and analyzed it by LC/MS. Each rat inhaled an estimated dose of 35 µg PCB3, and excreted 27 ± 2% of it as sulfates within 24 h. Peak excretion occurred within 6 h. PCB sulfates were stable in urine for at least three days at room temperature without chemical preservatives. These data support the use of PCB sulfate conjugates as suitable urinary biomarkers of PCB3 and other airborne PCBs.

Chlorinated biphenyl quinones and phenyl-2,5-benzoquinone differentially modify the catalytic activity of human hydroxysteroid sulfotransferase hSULT2A1.

Human hydroxysteroid sulfotransferase (hSULT2A1) catalyzes the sulfation of a broad range of environmental chemicals, drugs, and other xenobiotics in addition to endogenous compounds that include hydroxysteroids and bile acids. Polychlorinated biphenyls (PCBs) are persistent environmental contaminants, and oxidized metabolites of PCBs may play significant roles in the etiology of their adverse health effects. Quinones derived from the oxidative metabolism of PCBs (PCB-quinones) react with nucleophilic sites in proteins and also undergo redox cycling to generate reactive oxygen species. This, along with the sensitivity of hSULT2A1 to oxidative modification at cysteine residues, led us to hypothesize that electrophilic PCB-quinones react with hSULT2A1 to alter its catalytic function. Thus, we examined the effects of four phenylbenzoquinones on the ability of hSULT2A1 to catalyze the sulfation of the endogenous substrate, dehydroepiandrosterone (DHEA). The quinones studied were 2'-chlorophenyl-2,5-benzoquinone (2'-Cl-BQ), 4'-chlorophenyl-2,5-benzoquinone (4'-Cl-BQ), 4'-chlorophenyl-3,6-dichloro-2,5-benzoquinone (3,6,4'-triCl-BQ), and phenyl-2,5-benzoquinone (PBQ). At all concentrations examined, pretreatment of hSULT2A1 with the PCB-quinones decreased the catalytic activity of hSULT2A1. Pretreatment with low concentrations of PBQ, however, increased the catalytic activity of the enzyme, while higher concentrations inhibited catalysis. A decrease in substrate inhibition with DHEA was seen following preincubation of hSULT2A1 with all of the quinones. Proteolytic digestion of the enzyme followed by LC/MS analysis indicated PCB-quinone- and PBQ-adducts at Cys55 and Cys199, as well as oxidation products at methionines in the protein. Equilibrium binding experiments and molecular modeling suggested that changes due to these modifications may affect the nucleotide binding site and the entrance to the sulfuryl acceptor binding site of hSULT2A1.

Does dietary copper supplementation enhance or diminish PCB126 toxicity in the rodent liver?

Copper is essential for the function of the mitochondrial electron transport chain and several antioxidant proteins. However, in its free form copper can participate in Fenton-like reactions that produce reactive hydroxyl radicals. Aryl-hydrocarbon receptor (AhR) agonists, including the most potent polychlorinated biphenyl (PCB) congener, 3,3',4,4',5-pentachlorobiphenyl (PCB126), increase copper levels in rodent livers. This is accompanied by biochemical and toxic changes. To assess the involvement of copper in PCB toxicity, male Sprague-Dawley rats were fed an AIN-93G diet with differing dietary copper levels: low (2 ppm), adequate (6 ppm), and high (10 ppm). After three weeks, rats from each group were given a single ip injection of corn oil (control), 1, or 5 µmol/kg body weight PCB126. Two weeks following injections, biochemical and morphological markers of hepatic toxicity, trace metal status, and hepatic gene expression of metalloproteins were evaluated. Increasing dietary copper was associated with elevated tissue levels of copper and ceruloplasmin. In the livers of PCB126-treated rats, the hallmark signs of AhR activation were present, including increased cytochrome P450 and lipid levels and decreased glutathione. In addition, a doubling of hepatic copper levels was seen, and overall metal homeostasis was disturbed, resulting in decreased hepatic selenium, manganese, zinc, and iron. Expression of key metalloproteins was either decreased (cytochrome c oxidase), unchanged (ceruloplasmin and CuZnSOD), or increased (tyrosinase and metallothioneins 1 and 2) with exposure to PCB126. Increases in metallothionein may contribute/reflect the increased copper seen. Alterations in dietary copper did not amplify or abrogate the hepatic toxicity of PCB126. PCB126 toxicity, i.e., oxidative stress and steatosis, is clearly associated with disturbed metal homeostasis. Understanding the mechanisms of this disturbance may provide tools to prevent liver toxicity by other AhR agonists.

Disposition and Metabolomic Effects of 2,2',5,5'-Tetrachlorobiphenyl in Female Rats Following Intraperitoneal Exposure.

The disposition and toxicity of lower chlorinated PCBs (LC-PCBs) with less than five chlorine substituents have received little attention. This study characterizes the distribution and metabolomic effects of PCB 52, an LC-PCB found in indoor and outdoor air, three weeks after intraperitoneal exposure of female Sprague Dawley rats to 0, 1, 10, or 100 mg/kg BW. PCB 52 exposure did not affect overall body weight. Gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis identified PCB 52 in all tissues investigated. Hydroxylated, sulfated, and methylated PCB metabolites, identified using GC-MS/MS and nontarget liquid chromatography-high resolution mass spectrometry (Nt-LCMS), were primarily found in the serum and liver of rats exposed to 100 mg/kg BW. Metabolomic analysis revealed minor effects on L-cysteine, glycine, cytosine, sphingosine, thymine, linoleic acid, orotic acid, L-histidine, and erythrose serum levels. Thus, the metabolism of PCB 52 and its effects on the metabolome must be considered in toxicity studies. Highlights: PCB 52 was present in adipose, brain, liver, and serum 3 weeks after PCB exposureLiver and serum contained hydroxylated, sulfated, and methylated PCB 52 metabolitesMetabolomics analysis revealed minor changes in endogenous serum metabolitesLevels of dopamine and its metabolites in the brain were not affected by PCB 52.

Disposition and metabolomic effects of 2,2',5,5'-tetrachlorobiphenyl in female rats following intraperitoneal exposure.

The disposition and toxicity of lower chlorinated PCBs (LC-PCBs) with less than five chlorine substituents have received little attention. This study characterizes the distribution and metabolomic effects of PCB 52, an LC-PCB found in indoor and outdoor air, three weeks after intraperitoneal exposure of female Sprague Dawley rats to 0, 1, 10, or 100 mg/kg BW. PCB 52 exposure did not affect overall body weight. Gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis identified PCB 52 in all tissues investigated. Hydroxylated, sulfated, and methylated PCB metabolites, identified using GC-MS/MS and nontarget liquid chromatography-high resolution mass spectrometry (Nt-LCMS), were primarily found in the serum and liver of rats exposed to 100 mg/kg BW. Metabolomic analysis revealed minor effects on L-cysteine, glycine, cytosine, sphingosine, thymine, linoleic acid, orotic acid, L-histidine, and erythrose serum levels. Thus, the metabolism of PCB 52 and its effects on the metabolome must be considered in toxicity studies.

Identification of sulfated metabolites of 4-chlorobiphenyl (PCB3) in the serum and urine of male rats.

Polychlorinated biphenyls (PCBs) are legacy pollutants that exert toxicities through various mechanisms. In recent years exposure to PCBs via inhalation has been recognized as a hazard. Those PCBs with lower numbers of chlorine atoms (LC-PCBs) are semivolatile and have been reported in urban air, as well as in the indoor air of older buildings. LC-PCBs are bioactivated to phenols and further to quinone electrophiles with genotoxic/carcinogenic potential. We hypothesized that phenolic LC-PCBs are subject to conjugation and excretion in the urine. PCB3, often present in high concentrations in air, is a prototypical congener for the study of the metabolism and toxicity of LC-PCBs. Our objective was to identify metabolites of PCB3 in urine that could be potentially employed in the estimation of exposure to LC-PCBs. Male Sprague-Dawley rats (150-175 g) were housed in metabolism cages and received a single intraperitoneal injection of 600 µmol/kg body weight of PCB3. Urine was collected every 4 h; rats were euthanized at 36 h; and serum was collected. LC/MS analysis of urine before and after incubation with ß-glucuronidase and sulfatase showed that sulfate conjugates were in higher concentrations than glucuronide conjugates and free phenolic forms. At least two major metabolites and two minor metabolites were identified in urine that could be attributed to mercapturic acid metabolites of PCB3. Quantitation by authentic standards confirmed that approximately 3% of the dose was excreted in the urine as sulfates over 36 h, with peak excretion occurring at 10-20 h after exposure. The major metabolites were 4'PCB3sulfate, 3'PCB3 sulfate, 2'PCB3 sulfate, and presumably a catechol sulfate. The serum concentration of 4'PCB3 sulfate was 6.18 ± 2.16 µg/mL. This is the first report that sulfated metabolites of PCBs are formed in vivo. These findings suggest a prospective approach for exposure assessment of LC-PCBs by analysis of phase II metabolites in urine.

Transcriptome sequencing of 3,3',4,4',5-Pentachlorobiphenyl (PCB126)-treated human preadipocytes demonstrates progressive changes in pathways associated with inflammation and diabetes.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that accumulate in adipose tissue and have been associated with cardiometabolic disease. We have previously demonstrated that exposure of human preadipocytes to the dioxin-like PCB126 disrupts adipogenesis via the aryl hydrocarbon receptor (AhR). To further understand how PCB126 disrupts adipose tissue cells, we performed RNAseq analysis of PCB126-treated human preadipocytes over a 3-day time course. The most significant predicted upstream regulator affected by PCB126 exposure at the early time point of 9 h was the AhR. Progressive changes occurred in the number and magnitude of transcript levels of genes associated with inflammation, most closely fitting the pathways of cytokine-cytokine-receptor signaling and the AGE-RAGE diabetic complications pathway. Transcript levels of genes involved in the IL-17A, IL-1ß, MAP kinase, and NF-κB signaling pathways were increasingly dysregulated by PCB126 over time. Our results illustrate the progressive time-dependent nature of transcriptional changes caused by toxicants such as PCB126, point to important pathways affected by PCB126 exposure, and provide a rich dataset for further studies to address how PCB126 and other AhR agonists disrupt preadipocyte function. These findings have implications for understanding how dioxin-like PCBs and other dioxin-like compounds are involved in the development of obesity and diabetes.

Dataset of transcriptomic changes that occur in human preadipocytes over a 3-day course of exposure to 3,3',4,4',5-Pentachlorobiphenyl (PCB126).

Exposure to polychlorinated biphenyls (PCBs) has been associated with the development of metabolic syndrome, a cluster of diseases that includes obesity, diabetes, liver steatosis, and cardiovascular problems. PCBs accumulate and fat and are known to act on adipocytes and their precursors, termed preadipocytes. The PCB congener, PCB126, has been shown to activate the aryl hydrocarbon receptor (AhR) as well as proinflammatory genes. Here, we used RNAseq to assess gene transcript changes that occur in PCB126-exposed human preadipocytes over a time course. RNA was collected from 4 replicates of PCB126-exposed and control-treated preadipocytes at 9 h, 24 h, and 72 h post-exposure. RNA was processed for RNAseq analysis using a NovaSeq 6000 with an obtained minimum of 25 million paired-end 50 bp reads per sample. Reads were aligned using the salmon aligner and transcript expression values were summarized to the gene level using tximport. Gene transcript level counts comparing treated- versus control-treated cells were used for differential expression analysis using DESeq2. Differential expression Excel tables (one for each time point) were generated displaying average differential expression (log2 fold change) of the 4 replicates of treated versus control samples with cutoffs of 0.3 log2 fold change (increase or decrease) and p-values of less than 0.05. FastQ, raw, and differential expression tables were uploaded to GEO. A heat map of genes that were changed in common across all time points was generated using GraphPrism. The data generated from this analysis provides a full transcriptional profile of changes that occur over time in preadipocytes that have been exposed to PCB126. The rich datasets can be mined by other researchers to understand how PCB126 and other dioxin-like compounds, including other PCB congeners such as PCB77 and PCB118, affect biological pathways in preadipocytes and other cell types to cause disease.

PCB126 induced toxic actions on liver energy metabolism is mediated by AhR in rats.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in the regulation of biological responses to more planar aromatic hydrocarbons, like TCDD. We previously described the sequence of events following exposure of male rats to a dioxin-like polychlorinated biphenyl (PCB) congener, 3,3',4,4',5-pentachlorobiphenyl (PCB126), that binds avidly to the AhR and causes various types of toxicity including metabolic syndrome, fatty liver, and disruption of energy homeostasis. The purpose of this study was, to investigate the role of AhR to mediate those toxic manifestations following sub-acute exposure to PCB126 and to examine possible sex differences in effects. For this goal, we created an AhR knockout (AhR-KO) model using CRISPR/Cas9. Comparison was made to the wild type (WT) male and female Holtzman Sprague Dawley rats. Rats were injected with a single IP dose of corn oil vehicle or 5 µmol/kg PCB126 in corn oil and necropsied after 28 days. PCB126 caused significant weight loss, reduced relative thymus weights, and increased relative liver weights in WT male and female rats, but not in AhR-KO rats. Similarly, significant pathologic changes were visible which included necrosis and regeneration in female rats, micro- and macro-vesicular hepatocellular vacuolation in males, and a paucity of glycogen in livers of both sexes in WT rats only. Hypoglycemia and lower IGF1, and reduced serum non-esterified fatty acids (NEFAs) were found in serum of both sexes of WT rats, low serum cholesterol levels only in the females, and no changes in AhR-KO rats. The expression of genes encoding enzymes related to xenobiotic metabolism (e.g. CYP1A1), gluconeogenesis, glycogenolysis, and fatty acid oxidation were unaffected in the AhR-KO rats following PCB126 exposure as opposed to WT rats where expression was significantly upregulated (PPARα, females only) or downregulated suggesting a disrupted energy homeostasis. Interestingly, Acox2, Hmgcs, G6Pase and Pc were affected in both sexes, the gluconeogenesis and glucose transporter genes Pck1, Glut2, Sds, and Crem only in male WT-PCB rats. These results show the essential role of the AhR in glycogenolysis, gluconeogenesis, and fatty acid oxidation, i.e. in the regulation of energy production and homeostasis, but also demonstrate a significant difference in the effects of PCB126 in males verses females, suggesting higher vulnerability of glucose homeostasis in males and more changes in fatty acid/lipid homeostasis in females. These differences in effects, which may apply to more/all AhR agonists, should be further analyzed to identify health risks to specific groups of highly exposed human populations.

Structure-activity relationships for hydroxylated polychlorinated biphenyls as inhibitors of the sulfation of dehydroepiandrosterone catalyzed by human hydroxysteroid sulfotransferase SULT2A1.

Polychlorinated biphenyls (PCBs) are persistent worldwide pollutants that are of concern due to their bioaccumulation and health effects. Metabolic oxidation of PCBs results in the formation of hydroxylated metabolites (OHPCBs). Among their biological effects, OHPCBs have been shown to alter the metabolism of endocrine hormones, including inhibition of mammalian cytosolic sulfotransferases (SULTs) that are responsible for the inactivation of thyroid hormones and phenolic steroids (i.e., hSULT1A1, hSULT1B1, and hSULT1E1). OHPCBs also interact with a human hydroxysteroid sulfotransferase that plays a role in the sulfation of endogenous alcohol-containing steroid hormones and bile acids (i.e., hSULT2A1). The objectives of our current study were to examine the effects of a series of OHPCB congeners on the activity of hSULT2A1 and to develop a three-dimensional quantitative structure-activity relationship (3D-QSAR) model for OHPCBs as inhibitors of the enzyme. A total of 15 OHPCBs were examined, and the sulfation of 1 µM [(3)H] dehydroepiandrosterone (DHEA) was utilized as a model reaction catalyzed by the enzyme. All 15 OHPCBs inhibited the sulfation of DHEA, with IC(50) values ranging from 0.6 µM to 96 µM, and eight of these OHPCBs were also substrates for the enzyme. Comparative molecular field analysis (CoMFA) provided a predictive 3D-QSAR model with a q(2) value of 0.697 and an r(2) value of 0.949. The OHPCBs that had the highest potency as inhibitors of DHEA sulfation were those with a 3, 5-dichloro-4-hydroxy substitution pattern on the biphenyl ring system, and these congeners were also substrates for sulfation catalyzed by hSULT2A1.

Species difference in the regulation of cytochrome P450 2S1: lack of induction in rats by the aryl hydrocarbon receptor agonist PCB126.

CYP2S1 is an evolutionarily conserved, mainly extra-hepatic member of the CYP2 family and proposed to be regulated by the aryl hydrocarbon receptor (AhR). The present study explores AhR's regulation of CYP2S1 in male Sprague Dawley rats using PCB126 (3,3',4,4',5-pentachlorobiphenyl), the most potent AhR agonist among the PCBs. Additionally, CYP2S1 expression was examined after treatments with the classic CYP-inducers ß-naphthoflavone (ß-NF, AhR activator), phenobarbital (PB, CAR activator) and dexamethasone (Dex, PXR activator). CYP2S1 and CYP1A1/2, CYP1B1, CYP2B and CYP3A mRNAs were measured in liver, lung, spleen, stomach, kidney, and thymus at different time points. Constitutive CYP2S1 was expressed at comparable levels to other CYPs with the highest expression levels in stomach, kidney and lung. CYP2S1 mRNA was only non-significantly elevated by ß-NF in liver tissues. PCB126 did not increase CYP2S1 mRNA in any organ and at any time point examined despite a significant induction of CYP1 genes. PCB126 reduced CYP2S1 mRNA by 40% (not significant) from the 7th post-exposure day in thymus. PB and Dex had no effect on CYP2S1 mRNA levels. These observations show that in this model CYP2S1 is not, or only weakly, regulated by AhR and not induced by CAR or PXR activators.