Polychlorinated biphenyl exposure alters the expression profile of microRNAs associated with vascular diseases

Exposure to persistent organic pollutants, including polychlorinated biphenyls (PCBs) is correlated with multiple vascular complications including endothelial cell dysfunction and atherosclerosis. PCB-induced activation of the vasculature subsequently leads to oxidative stress and induction of pro-inflammatory cytokines and adhesion proteins. Gene expression of these cytokines/proteins is known to be regulated by small, endogenous oligonucleotides known as microRNAs that interact with messenger RNA. MicroRNAs are an acknowledged component of the epigenome, but the role of environmentally-driven epigenetic changes such as toxicant-induced changes in microRNA profiles is currently understudied. The objective of this study was to determine the effects of PCB exposure on microRNA expression profile in primary human endothelial cells using the commercial PCB mixture Aroclor 1260. Samples were analyzed using Affymetrix GeneChip® miRNA 4.0 arrays for high throughput detection and selected microRNA gene expression was validated (RT-PCR). Microarray analysis identified 557 out of 6658 microRNAs that were changed with PCB exposure (p < 0.05). In-silico analysis using MetaCore database identified 21 of these microRNAs to be associated with vascular diseases. Further validation showed that Aroclor 1260 increased miR-21, miR-31, miR-126, miR-221 and miR-222 expression levels. Upregulated miR-21 has been reported in cardiac injury while miR-126 and miR-31 modulate inflammation. Our results demonstrated evidence of altered microRNA expression with PCB exposure, thus providing novel insights into mechanisms of PCB toxicity.     

Acute exposure to aroclor 1016 or 1260 differentially affects dopamine transporter and vesicular monoamine transporter 2 levels

Polychlorinated biphenyls (PCBs) have been shown to specifically target the dopaminergic nervous system, resulting in long-term reduction of striatal dopamine (DA) levels. However, the mechanism(s) by which PCBs exert this effect is not known. Here we report that decreased striatal dopamine levels are observed 1, 7, and 14 days after acute exposure to the common PCB mixtures Aroclor 1016 or 1260. Dopamine transporter (DAT) levels were decreased at all time points in Aroclor 1016 treated animals, and on Days 1 and 7 in Aroclor 1260 treated animals. Vesicular monoamine transporter 2 (VMAT2) levels were not affected by Aroclor 1016, but were significantly decreased 14 days after exposure to Aroclor 1260. Tyrosine hydroxylase expression, a marker of dopamine neuron integrity, was not significantly affected by PCB exposure at any time. These data suggest that PCB-induced reductions in striatal dopamine may be mediated by alterations in DAT and VMAT2 expression.     

Polychlorinated biphenyls disrupt hepatic epidermal growth factor receptor signaling

1.?Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that disrupt hepatic xenobiotic and intermediary metabolism, leading to metabolic syndrome and nonalcoholic steatohepatitis (NASH).

2.?Since phenobarbital indirectly activates Constitutive Androstane Receptor (CAR) by antagonizing growth factor binding to the epidermal growth factor receptor (EGFR), we hypothesized that PCBs may also diminish EGFR signaling.

3.?The effects of the PCB mixture Aroclor 1260 on the protein phosphorylation cascade triggered by EGFR activation were determined in murine (in vitro and in vivo) and human models (in vitro). EGFR tyrosine residue phosphorylation was decreased by PCBs in all models tested.

4.?The IC₅₀ values for Aroclor 1260 concentrations that decreased Y1173 phosphorylation of EGFR were similar in murine AML-12 and human HepG2 cells (～2–4?μg/mL). Both dioxin and non-dioxin-like PCB congeners decreased EGFR phosphorylation in cell culture.

5.?PCB treatment reduced phosphorylation of downstream EGFR effectors including Akt and mTOR, as well as other phosphoprotein targets including STAT3 and c-RAF in vivo.

6.?PCBs diminish EGFR signaling in human and murine hepatocyte models and may dysregulate critical phosphoprotein regulators of energy metabolism and nutrition, providing a new mechanism of action in environmental diseases.     

Delayed effects of pre- and early-life time exposure to polychlorinated biphenyls on tadpoles of two amphibian species (Xenopus laevis and Rana temporaria)

This study examined the effects of polychlorinated biphenyls (PCBs) on the development of amphibians using Xenopus laevis and Rana temporaria as experimental animals. Amphibians were exposed at different life stages and via different routes to the technical mixtures Clophen A50 and Aroclor 1254 or to a non-ortho PCB congener (PCB 126). The effects of PCB exposure in amphibians, such as mortality, number and pattern of malformations, or body weight at the end of successful metamorphosis of tadpoles, depends on the route, the point of time of exposure during the complex life cycle of amphibians, and the length of the observation period. Retinoid concentrations were significantly altered in PCB dosed embryos. Presently used early-life time test systems such as the FETAX assay may underestimate toxic effects of compounds with long term response such as PCBs on amphibians.     

Dosimetry considerations in the enhanced sensitivity of male Wistar rats to chronic ethylene glycol-induced nephrotoxicity

The developmental consequences of exposure to the polychlorinated biphenyls (PCBs) have been widely studied, making PCBs a unique model to understand issues related to environmental mixture of persistent chemicals. PCB exposure in humans adversely affects neurocognitive development, causes psychomotor difficulties, and contributes to attention deficits in children, all of which seem to be associated with altered patterns of neuronal connectivity. In the present study, we examined gene expression profiles in the rat nervous system following PCB developmental exposure. Pregnant rats (Long-Evans) were dosed perinatally with 0 or 6 mg/kg/day of Aroclor 1254 from gestation day 6 through postnatal day (PND) 21. Gene expression in cerebellum and hippocampus from PND7 and PND14 animals was analyzed with an emphasis on developmental aspects. Changes in gene expression (> or =1.5 fold) in control animals identified normal developmental changes. These basal levels of expression were compared to data from Aroclor 1254-treated animals to determine the impact of gestational PCB exposure on developmental parameters. The results indicate that the expression of a number of developmental genes related to cell cycle, synaptic function, cell maintenance, and neurogenesis is significantly altered from PND7 to PND14. Aroclor 1254 treatment appears to dampen the overall growth-related gene expression levels in both regions with the effect being more pronounced in the cerebellum. Functional analysis suggests that Aroclor 1254 delays maturation of the developing nervous system, with the consequences dependent on the ontological state of the brain area and the functional role of the individual gene. Such changes may underlie learning and memory deficits observed in PCB exposed animals and humans.     

Prenatal Aroclor 1254 exposure and brain sexual differentiation: effect on the expression of testosterone metabolizing enzymes and androgen receptors in the hypothalamus of male and female rats

Polychlorinated biphenyls (PCBs) are industrial pollutants detected in human milk, serum and tissues. They readily cross the placenta to accumulate in fetal tissues, particularly the brain. These compounds affect normal brain sexual differentiation by mechanisms that are incompletely understood. The aim of this study was to verify whether a technical mixture of PCBs (Aroclor 1254) would interfere with the normal pattern of expression of hypothalamic aromatase and 5-alpha reductase(s), the two main enzymatic pathways involved in testosterone activation and of androgen receptor (AR). Aroclor 1254 was administered to pregnant rats at a daily dose of 25 mg/kg by gavage from days 15 to 19 of gestation (GD15–19). At GD20 the expression of aromatase, 5-alpha reductase types 1 and 2 and androgen receptor (AR) and aromatase activity were evaluated in the hypothalamus of male and female embryos. The direct effect of Aroclor was also evaluated on aromatase activity adding the PCB mixture to hypothalamic homogenates or to primary hypothalamic neuronal cultures. The data indicate that aromatase expression and activity is not altered by prenatal PCB treatment; 5-alpha reductase type 1 is similarly unaffected while 5-alpha reductase type 2 is markedly stimulated by the PCB exposure in females. Aroclor also decreases the expression of the AR in females. The observed in vivo effects are indicative of a possible adverse effect of PCBs on the important metabolic pathways by which testosterone produces its brain effects. In particular the changes of 5-alpha reductase type 2 and AR in females might be one of the mechanisms by which Aroclor exposure during fetal development affects adult sexual behavior in female rats.     

PCB126 induces differential changes in androgen, cortisol, and aldosterone biosynthesis in human adrenocortical H295R cells.

Dioxins and polychlorinated biphenyls (PCBs) have been shown to accumulate in the adrenal glands when incorporated into the body. However, the impacts of exposure on adrenal steroidogenesis have not been thoroughly investigated. In this study, we demonstrated that dioxin-like PCB126 altered androgen, cortisol, and aldosterone biosynthesis differentially in human adrenocortical H295R cells. PCB126 diminished basal and cAMP-induced androstenedione production as well as CYP17 mRNA expression in a dose-dependent and time-dependent manner. The CYP17 repression was accompanied with decreases in the encoded 17 alpha-hydroxylase and 17,20-lyase activities, particularly the latter. In contrast, high concentrations of PCB126 stimulated basal cortisol and aldosterone biosynthesis, including induction of CYP21B, CYP11B1, and CYP11B2 mRNA expression and elevation of the conversion of cortisol from 17-OH-progesterone and aldosterone from progesterone. cAMP abolished the positive effect of PCB126 on cortisol synthesis, while it synergistically enhanced PCB126 stimulation on CYP11B2 expression and aldosterone production. It seemed likely that the downregulation of CYP21B caused by the combination of PCB126 and cAMP counteracted the CYP11B1 induction stimulated by the co-treatment. In addition, high concentrations of PCB126 might sensitize the regulation of adrenocorticotropin (ACTH) on the adrenocortical cells by increasing ACTH receptor levels. Because adrenal steroids have profound influences on glucose tolerance, insulin sensitivity, lipid metabolism, obesity, vascular function, and cardiac remodeling, this article also discusses the potential association of the detected adrenocortical alterations with increased diabetic and cardiovascular risk found among highly exposed people.     

DNA adducts in cultures of polychlorinated biphenyl-treated human hepatocytes

Polychlorinated biphenyls (PCBs) are persistent environmental chemicals that accumulate at the apex of food chains. Several scientific committees support its designation as a human carcinogen, even though the precise mechanism of carcinogenesis remains controversial. In view of its uncertain ability to cause DNA damage in human liver, we investigated the effects of Aroclor 1254, Aroclor 1016, and 4-chlorobiphenyl (4-CB) on DNA adduct formation in cultures of primary human hepatocytes from five donors. Based on (32)P-postlabeling assays, we detected DNA adducts in native human liver as well as untreated, i.e., control cultures of human hepatocytes. Treatment of human hepatocytes with Aroclor 1016 and Aroclor 1254 resulted in four-fold (NP1) and seven-fold (butanol) increases in DNA adduct formation. Further, two and six new adduct spots were detected by multidirectional thin-layer chromatography after nuclease P1 and butanol enrichment. Treatment of human hepatocyte cultures with 4-CB led to 209 adducts per 10(9) nucleotides at the 60 microM concentration, and we show metabolically activated PCBs to be more efficient in the production of DNA-binding species compared with higher chlorinated PCB mixtures. Our study is therefore highly suggestive for a link between PCB exposure and DNA insult in human hepatocytes.     

Reproductive abnormalities, teratogenicity, and developmental problems in American kestrels (Falco sparverius) exposed to polychlorinated biphenyls

This study found abnormalities in multiple reproductive stages in captive American kestrels (Falco sparverius) when exposed to polychlorinated biphenyls (PCBs) through dietary and in ovo exposure. American kestrels laid eggs with environmentally relevant total PCB levels (34.1 micrograms/g whole egg wet weight) when consuming PCB-spiked (Aroclor 1248:1254:1260) food (5-7 micrograms/g body weight per day) for 100 d only in 1998. In 1999, the same adults laid eggs with estimated total PCBs of 23 micrograms/g. Effects of maternal (only female exposed) and paternal (only male exposed) in ovo PCB exposure were investigated. Maternal F1 eggs contained predicted total PCB concentrations of 0.34 microgram/g. Specific abnormalities occurred more frequently during dietary F0 exposure, particularly aggressive courtship interactions, clutch abandonment, occurrences of cracked eggs, and developmental effects. Multiple developmental effects were more pronounced during than after dietary PCB exposure of adults, and although these effects were limited, nevertheless they occurred in the F1 maternal and F1 paternal pairs. However, the incidence of multiple deformities throughout the breeding season increased dramatically from 1998 (13%) to 1999 (56%) in F0 PCB-exposed pairs. Developmental abnormalities were unlikely to be attributed to the extrinsic factors of disease, genetics, or nutritional (vitamin D3) deficiencies, but rather to adverse changes in parental behavior and intrinsic factors involving altered genetic material and PCB exposure. Readily cleared PCB congeners may induce specific types of behavioral and developmental abnormalities, but persistent congeners and metabolites are likely producing (1) odd laying patterns, (2) odd laying patterns, (2) developmental effects including embryonic underdevelopment and edema, and (3) increased incidences of multiple deformities within a clutch.     

The degree of PCB chlorination determines whether the rise in urinary homovanillic acid production in rats is peripheral or central in origin

Commercial mixtures of polychlorinated biphenyls (PCBs, Aroclor 1016, 1254, and 1260) differing in their degree of chlorination and their accumulation in the brain were employed along with a peripheral monoamine oxidase inhibitor, debrisoquin sulfate (Declinax, DS) to determine whether the rise in urinary homovanillic acid (UHVA) following exposure to these PCBs is derived from the peripheral or central nervous system. Rats were gavaged with either corn oil or corn oil containing Aroclor 1016 or a mixture of Aroclors 1254 and 1260 and 24-hr UHVA production was determined by high-performance liquid chromatography with electrochemical detection. All animals also received ip injections of DS to inhibit peripheral production of HVA. Analysis of variance indicated that, following DS treatment, 24-hr UHVA production remained significantly elevated in the Aroclor 1254/1260-exposed animals; while no significant differences between Aroclor 1016-exposed animals and controls were noted. The rise in UHVA in the Aroclor 1254/1260 group involves HVA of central origin whereas the rise in the Aroclor 1016-treated animals is only peripheral. Thus, PCBs that differ in their degree of chlorination alter dopaminergic functions in anatomically different locations.     

Low-frequency hearing loss following perinatal exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB 126) in rats.

Previous research has demonstrated the sensitivity of the developing rat to the ototoxic effects of exposure to Aroclor 1254. In this study we assessed the effects of developmental exposure to an individual PCB congener (3,3',4,4',5-pentachlorobiphenyl; PCB 126) on auditory function. Nulliparous Long Evans rats received either 0, 0.25, or 1.0 microg/kg/day (5 days/week) for 35 days prior to breeding and throughout gestation and lactation. Auditory thresholds for 0.5-, 1-, 4-, 8-, 16-, 32-, and 40-kHz tones were assessed in offspring on postnatal days (PND) 76-90. Perinatal maternal PCB 126 exposure caused low-frequency hearing deficits. Elevated auditory thresholds occurred in the 1.0 microg/kg/day treated group for 0.5- and 1-kHz tones, whereas thresholds were not significantly affected at any higher frequencies. These results are important in that the data implicate, at least partially, the coplanar PCBs in the developmental ototoxicity induced by Aroclor 1254.     

Comparison of pharmacokinetic interactions and physiologically based pharmacokinetic modeling of PCB 153 and PCB 126 in nonpregnant mice, lactating mice, and suckling pups.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that can induce neurological defects in infants and children via placental and lactational transfer. To investigate the lactational transfer of PCBs and compare pharmacokinetic interactions among nonpregnant, lactating mice and suckling pups, quantitative time-course measurements of PCB accumulation in tissues were performed. On postnatal day 1, nonpregnant and lactating C57BL/6 mice were exposed to PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl, 20 mg/kg) alone or a mixture of PCB 153 (20 mg/kg) and PCB 126 (3,3',4,4',5-pentachlorobiphenyl, 0.2 mg/kg) by oral gavage. At 1, 3, 6, and 13 days after treatment, PCB 153 and PCB 126 were determined in nonpregnant and maternal tissues as well as in neonatal tissues by gas chromatography (GC). Coadministration of PCB 153 and PCB 126 increased PCB 153 retention in the liver and decreased PCB 153 accumulation in the fat of nonpregnant mice. Lactational transfer was confirmed to be an efficient elimination mechanism for the lactating mice but a major source of exposure in the pups. However, little or no significant pharmacokinetic interactions were observed in lactating mice and suckling pups. To describe pharmacokinetic interactions between PCB 153 and PCB 126, a physiologically based pharmacokinetic model for PCB 153 disposition was developed. The effects of PCB 126 on the fat content in liver and a diffusion permeation constant in fat were incorporated into the physiologically based pharmacokinetic (PBPK) model. This model successfully describes PCB 153 disposition altered by PCB 126 in nonpregnant mice.     

Comparison of effects of Aroclors 1016 and 1260 on non-human primate catecholamine function

Adult male non-human primates, Macaca nemestrina, were orally-exposed to corn oil or corn oil containing either Aroclor 1016 or 1260 at doses of 0.8, 1.6 or 3.2 mg/(kg.day) for 20 weeks. Brain concentrations of biogenic amines and individual PCB congeners were determined following exposure. Aroclor 1016 significantly decreased concentrations of dopamine and its metabolites in the caudate, putamen, substantia nigra and hypothalamus but did not alter neurotransmitter or metabolite concentrations in the globus pallidus and hippocampus. Total PCB concentrations ranged from 1 to 5 ppm with only three congeners detected (2,4,4'; 2,4,2',4' and 2,5,2',5') making up, on average, 72%, 18% and 7% respectively of the total residue in brain. There were no discernible differences in the congener make-up between brain regions. Aroclor 1260 reduced dopamine concentrations in the caudate, putamen and hypothalamus but produced no effects in the substantia nigra, globus pallidus or hippocampus. Aroclor 1260 concentrations ranged from 18 to 28 ppm with the highest levels found in the hippocampus. Of the congeners that made up more than 5% of the total residue in brain, all were hexa- and heptachlorinated di-ortho-substituted congeners. There were no discernible differences in congener make-up between brain regions. We conclude that: (1) ortho-substituted non-planar congeners are responsible for the observed changes in neurochemical function; (2) both Aroclor 1016 and Aroclor 1260 decrease dopamine concentrations by similar mechanisms; and (3) based on differences in brain concentrations of Aroclor 1260 congeners compared to Aroclor 1016 congeners, lightly-chlorinated congeners are more effective in reducing central dopamine concentrations than are the more highly chlorinated congeners.     

Polychlorinated biphenyl exposure and CYP19 gene regulation in testicular and adrenocortical cell lines.

Exposure to polychlorinated biphenyls (PCBs) disturbs many estrogen-mediated biochemical processes. PCBs may cause these abnormalities by altering expression of the aromatase gene CYP19. This study demonstrated that high concentrations of PCB126 increased basal CYP19 mRNA abundance in mouse testicular Leydig I-10 cells and human adrenocortical H295R cells. Stimulating the cells with chorionic gonadotropin or 8-Br-cAMP concealed the estrogenic effect of PCB126. PCB126 is a powerful ligand for nuclear receptor AhR. Antagonizing the AhR activity of H295R by an inhibitor abolished PCB126-elicited CYP19 induction. However, PCB126 elevated basal CYP19 expression and aromatase activity in a slow progressive manner contrary to the sharp induction of the classic AhR target gene CYP1A1. Exposure of H295R to PCBs with different AhR activation abilities also varied CYP19 and CYP1A1 expression in dissimilar patterns, although the CYP19 mRNA levels were in line with the AhR activation abilities of the congeners. In contrast to PCB126, PCB39, which could not activate AhR and lacked effect on CYP1A1, significantly reduced CYP19 mRNA expression. AhR apparently played an important role in CYP19 gene regulation, but it might regulate CYP19 differently from CYP1A1 in the adrenocortical cells. Regardless of the action mechanism, PCB exposure increases risk for CYP19 dysregulation.     

Effects of subchronic exposure to complex mixtures of dioxin-like and non-dioxin-like polyhalogenated aromatic compounds on thyroid hormone and vitamin A levels in female Sprague-Dawley rats.

The aim of this study was to determine the effects of subchronic exposure to complex mixtures of polyhalogenated aromatic hydrocarbons (PHAHs) on the thyroid hormone and retinoid status in female Sprague-Dawley rats and to investigate the predictability of these effects by the toxic equivalency factor (TEF) concept. In the first experiment, the focus was on a complex dioxin-like PHAH mixture, which covered > 90% of the total toxic equivalents (TEQ) present in Baltic herring. In the second experiment, the contribution of non-dioxin-like polychlorinated biphenyls (PCBs) was investigated by testing the commercial PCB mixture Aroclor 1260, its 0-1 ortho and 2-4 ortho fractions and the reconstituted 0-4 ortho fraction. Hepatic retinoid levels were severely decreased ( approximately 70%) after treatment with the dioxin-like PHAH mixture, similar to the effect of a TEQ equivalent dose of 1 microg 2,3,7,8-TCDD/kg bw/week. However, the TEF concept failed to predict the effect on plasma retinol; a decrease (21%) was observed after treatment with the PHAH mixture, whereas an increase (21%) was found after treatment with TCDD. A more severe decrease of total thyroid hormone in plasma was observed after exposure to the PHAH mixture compared to treatment with TCDD ( approximately 60% vs. 38%). The discrepancy found between the predicted and observed effects for plasma retinol and thyroid hormone is possibly due to an additional effect of hydroxylated PCBs, formed from metabolizable PCBs present in the PHAH mixture. Aroclor 1260 and its fractions did not significantly alter the retinoid and thyroid hormone status at the dose levels tested, indicating that in case of exposure to complex PCB mixtures at environmental levels, no effects, or at best, only marginal effects can be expected on the retinoid and thyroid hormone status.     

Dose-response immunotoxicities of commercial polychlorinated biphenyls (PCBs) and their interaction with 2,3,7,8-tetrachlorodibenzo-p-dioxin

The relative potencies of the commercial polychlorinated biphenyl (PCB) mixtures Aroclors 1260, 1254, 1248, 1242, 1016 and 1232 to inhibit the murine splenic plaque-forming cell response to sheep red blood cells was determined by dose-response treatment of C57BL/6 mice followed by logit plot analysis of the data. The ED50 values obtained for Aroclors 1260, 1254, 1248, 1242, 1016 and 1232 were 104, 118, 190, 391, 408 and 464 mg/kg or 0.28, 0.35, 0.66, 1.5, 1.5 and 2.0 mmol/kg, respectively. It was apparent that the higher chlorinated PCB preparations (Aroclors 1260, 1254 and 1248) were more potent than the lower chlorinated preparations (Aroclors 1242, 1016 and 1232). Previous studies have shown that the interaction of a subeffective dose of Aroclor 1254 (25 mg/kg) with an immunotoxic dose (3.7 nmol/kg) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in significant antagonism of the toxicity of the latter compound. Co-treatment of mice with a 25 mg/kg dose of all the commercial Aroclors and with a 50 mg/kg dose of a reconstituted PCB mixture (resembling a PCB extract from human milk) with TCDD (3.7 nmol/kg) showed that, with the exception of Aroclor 1232, all of the commercial PCBs and the reconstituted PCB mixture significantly antagonized the TCDD-mediated inhibition of the splenic plaque-forming cell response in C57BL/6 mice. The identities of the PCB congeners responsible for this antagonism and the mechanism of this process are unknown and are currently being investigated.