Polychlorinated Biphenyls Mixture Research Articles

Levels of polychlorinated biphenyls and organochlorine pesticides in serum samples of Egyptian Vulture ( Neophron percnopterus) from Spain

Concentrations of 23 polychlorinated biphenyls (PCBs), p, p ′-DDT and two of its metabolites, p, p ′-DDE and p, p ′-TDE have been measured in serum samples of up to 1 ml of Egyptian Vulture ( Neophron percnopterus) gathered from five populations in Spain. ∑PCB concentrations were found to be in the range 3.2–97 ng/ml, while those of ∑DDTs ranged from 0.93 to 38 ng/ml. p, p ′-DDT/ p, p ′-DDE ratios higher than one were only found in the Segovia population, which could be an indication of recent use of p, p ′-DDT in the area. In all cases, PCB profiles were dominated by congeners 52, 132 + 105, 138, 153 and 180. However, some differences among the five populations studied became evident when their profiles were compared with those of technical PCB mixtures by principal components analysis. The DDT and PCB levels detected in the serums analysed were lower than those previously reported for similar avian species and those reported to have deleterious effects on survival or reproduction of birds.

Effects of a mixture of polychlorinated biphenyls (Aroclor 1254) on the transcriptional activity of thyroid hormone receptor.

Polychlorinated biphenyls (PCBs) are environmental contaminants which may affect thyroid function. PCBs may reduce serum thyroid hormone (TH) concentrations by either displacing T4 from TH transport proteins or increasing its hepatic metabolism. The reduced serum T4 causes neurological and growth defects in animals exposed to PCBs during the perinatal period, which can partially be reverted by T4 administration. In addition to a hypothyroid-like syndrome, a direct action of PCBs on TH-sensitive genes has been postulated. In the present study the effects of Aroclor 1254 (ARO), a mixture of PCBs, on transcription of TH-dependent genes were investigated. A reporter plasmid containing the TH-responsive element (TRE) of malic enzyme (ME) gene was used in transient transfections to assess the responsiveness to ARO. ARO (10 microM) reduced the CAT activity by about 50% and competed with T3 to reduce the induction of transcription. Cotransfection of TH receptor (TR) and a wild type TRE was required to reveal ARO inhibitiry effect, which was abolished by a mock reaction not containing TR or by a mutated TRE. ARO reduced the 125I-T3 binding to TR by 30%, but did not affect the interaction of TR with a 32P-labeled TRE in gel shift assay. ARO is likely to produce a conformational change in in vitro translated TR, leading to its increased proteolysis by trypsin. These results demonstrate that ARO interacts with TR, thereby affecting the transcription of TH-sensitive genes, and provide a molecular basis to further explain the complex effects of PCBs on TH disruption.

Occurrence of polychlorinated biphenyls and polybrominated diphenyl ethers in green mussels (Perna viridis) from Singapore, Southeast Asia.

The green mussel, Perna viridis, was used in this study to measure levels of polychlorinated biphenyls (PCBs) and, for the first time, polybrominated diphenyl ethers (PBDE) in the marine environment. Samples were collected from eight different locations in the coastal waters of Singapore between April and May 2002. Forty-one PCB and 21 PBDE congeners were quantified by gas chromatography-mass spectrometry and were all positively detected in the mussel tissues. Total concentrations in green mussel tissues ranged from 6.1 to 82 ng/g and 2.0 to 38 ng/g on a dry-weight basis for PCBs and PBDEs, respectively. Such levels reflect the ubiquity of these persistent organic pollutants in a tropical marine environment. Principal component analysis was applied to the PCB data and revealed similarities in the congener composition of mussel tissues to that of the commercial PCB mixture, Aroclor 1254. The PBDE levels, to date, were approximately one order of magnitude greater than the upper concentrations reported for blue mussel (Mytilus edulis) tissues in Europe. At some sampling sites, the congener composition of PBDEs in P. viridis tissues indicated recent exposure to a commercial pentabrominated flame retardant.

Modulation of ovarian follicle maturation in Long–Evans rats exposed to polychlorinated biphenyls (PCBs) in-utero and lactationally

Polychlorinated biphenyls (PCBs) are ubiquitous man-made toxicants capable of endocrine disruption. Studies in several species have shown that exposure to PCBs and their hydroxylated metabolites reduces fecundity and decreases circulating concentrations of thyroid hormones, causing serious reproductive and developmental defects. Thyroid hormones modulate both follicular development and steroidogenesis, and affect estrogen metabolism and the regulation of estrogen receptor. This study was designed (1) to determine whether exposure to a commercially prepared PCB mixture (Aroclor 1016) exerts detrimental effects on follicle maturation in the Long–Evans hooded rat; and (2) to determine whether the modulatory effects of Aroclor can be attenuated by levo-thyroxine sodium (T 4) supplementation. Animals were treated on gestation days 7–13 with a single daily intraperitoneal injection (2.5 mg/kg per day) of Aroclor. Half of the Aroclor-treated dams were also given T 4 supplements (2.89 μg/kg per day) via drinking water. Female pups were sacrificed on postnatal days 24/25, and the ovaries were excised, fixed for histology and analyzed. The analysis included a count, measurement and classification of healthy and atretic preantral and antral follicles in the greatest cross-sectional area. The results indicated that treatment with Aroclor significantly reduced the number of preantral follicles <50,000 μm 2 and the total number of antral follicles in the 50–100,000 and >100,000 μm 2 size classes. T 4 circumvented the Aroclor effect on the number of preantral follicles <50,000 μm 2; however, a significant reduction in the antral follicle number persisted in the 50–100,000 and >100,000 μm 2 size classes. In addition, we observed a significant increase in atresia in the Aroclor-treated ovaries in the antral <50,000 μm 2 size class, which was not present in ovaries exposed to both Aroclor and T 4. These data support the hypothesis that Aroclor reduces the number of preantral and antral follicles of certain size classes in rats exposed during the critical period of development, and that supplementation with T 4 can attenuate the effects of Aroclor on small, but not medium or large antral follicles. Atresia of small, antral follicles may constitute one of the underlying mechanisms by which folliculogenesis is modulated by Aroclor 1016.

Effects of polychlorinated biphenyls on estrogen receptor-beta expression in the anteroventral periventricular nucleus.

Polychlorinated biphenyls (PCBs) can disrupt the reproductive axis, particularly when the exposure occurs during the vulnerable developmental periods. Some effects of environmental endocrine disruptors such as PCBs may be exerted through binding to estrogen receptors (ERs). In this study we examined the endocrine-disrupting effects of Aroclor 1221 (a commercial PCB mixture), focusing on its actions on the ER-ss, which has been implicated in mediating effects of endocrine-disrupting chemicals. A low, ecologically relevant dose of Aroclor 1221 or vehicle (ethanol) was administered three times each to rat dams, on gestational day 16 and on postpartum days 1 and 4, a developmental period during which steroid hormones have permanent effects on adult brain structure and function. Effects on ER-ss cell number in the anteroventral periventricular nucleus (AVPV) were quantified; this sexually dimorphic nucleus of the brain is essential to female reproductive function. For comparison, we quantified ER-ss cell number in another hypothalamic region, the supraoptic nucleus (SON). Using a stereologic approach, we found that Aroclor 1221 caused a highly significant down-regulation of the number of ER-ss-expressing cells in the AVPV, but had no effect in the SON. Thus, PCB exposure has consequences for neural ER expression, and these findings have implications for wildlife and humans that have been exposed to environmental estrogens, particularly during the susceptible periods of early development.

Effect of a single dose of polychlorinated biphenyls on hepatic cell proliferation and the DNA binding activity of NF-kappaB and AP-1 in rats.

Polychlorinated biphenyls (PCBs) are environmental pollutants that, because of their persistence and biomagnification, raise concerns about the health consequences of long-term exposure. PCB mixtures induce hepatocellular carcinomas in rodents, but the mechanism of their promoting activity is not clear. Previous studies have shown that oxidative stress occurs after PCB administration, with the induction of lipid peroxidation and oxidative DNA damage, which may contribute to their promoting activity. In this study, we examined whether the oxidative stress-sensitive transcription factors NF-kappaB or AP-1 were activated by PCBs in the liver. Male Sprague-Dawley rats were injected i.p. with corn oil, 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153, 30, 150, or 300 micromol/kg), 3,3',4,4'-tetrachlorobiphenyl (PCB-77, 30, 150, or 300 micromol/kg), or both PCBs (each 30 or 150 micromol/kg). Rats were euthanized 2, 6, or 24 h, or 2, 6, and 10 d after the PCB injection. Electrophoretic mobility shift assays (EMSAs) were performed to determine NF-kappaB and AP-1 DNA binding activities. The highest NF-kappaB DNA binding activity was observed in rats receiving higher doses of PCB-153 (150 and 300 micromol/kg), with peak activation occurring 2 d after injection. AP-1 activation was not detected at any timepoint. Hepatocyte proliferation, as measured by the labeling index, was increased only in groups receiving the highest dose of PCB-153 or the combination of two PCBs (150 micromol/kg each) at day 2, and not by any other PCB treatment at any timepoint. These results show that PCB-153, but not PCB-77, can induce hepatocyte proliferation and hepatic NF-kappaB activation after a single dose.

Accumulation of polychlorinated biphenyls in sediments of the Venice Lagoon and the industrial area of Porto Marghera

Sediment cores, collected at seven sites in the Venice Lagoon and within the canals of the industrial area were analyzed for polychlorinated biphenyls (PCBs) in order to assess the chronology of pollution and its present trends. The surficial concentration of PCBs is very high (more than 2049 μg kg −1) only in the Brentella Canal, probably due to a very recent contaminating episode. Very high values downcore (up to 41 639 μg kg −1) can be found in different parts of the industrial area, especially in the canals Lusore-Brentelle and Salso. Lagoon samples are much less contaminated (2.7–123 μg kg −1), being influenced only occasionally by polluted sediments resuspended from the canals. Sediment chronology shows that the delivery of contaminants peaked in the 1970s–early 1980s, decreasing since at most sites. Congener profiles distinguish PCBs in two main categories: heavy congeners characterize a baseline pollution, probably due to a large variety of sources within the lagoon system, whereas a mixture of light PCBs was discharged into the canals Brentella and Salso.

Perinatal Exposure to Polychlorinated Biphenyls Alters Excitatory Synaptic Transmission and Short-term Plasticity in the Hippocampus of the Adult Rat

Developmental exposure to polychlorinated biphenyls (PCBs) has been associated with cognitive deficits in humans and laboratory animals. Previous work has demonstrated a reduced capacity to support long-term potentiation (LTP) in animals exposed to a PCB mixture, Aroclor 1254 (A1254) via the dam in utero and throughout the preweaning period [Brain Res. 850;1999:87–95; Toxicol. Sci. 57;2000:102–11]. Assessment of normalized input/output (I/O) functions collected prior to LTP induction failed to reveal consistent differences in baseline synaptic transmission between control and PCB-exposed groups. The present study was designed to systematically evaluate excitatory and inhibitory synaptic transmission using a more extensive I/O analysis and paired pulse functions to assess short-term plasticity. Pregnant Long-Evans rats were administered either corn oil (control) or 6 mg/kg per day of A1254 by gavage from gestational day (GD) 6 until pups were weaned on postnatal day (PND) 21. In adult male offspring (5–11 months of age), field potentials evoked by perforant path stimulation were recorded in the dentate gyrus under urethane anesthesia. Detailed I/O functions were assessed by averaging the responses evoked in the dentate gyrus to stimulus pulses delivered to the perforant path in an extensive ascending intensity series. Population spike (PS) and postsynaptic potential (PSP) amplitudes recorded in the dentate gyrus were significantly enhanced in PCB-exposed animals relative to controls at midrange intensities. No group differences were observed in EPSP slope amplitudes. Short-term plasticity was assessed by delivering pairs of stimulus pulses at interpulse intervals (IPIs) ranging from 10 to 70 ms. In the dentate gyrus this range of intervals activates both inhibitory and excitatory mechanisms leading to a pattern of depression at brief intervals (<30 ms) followed by facilitation as the interval between pulses is extended. Paired pulse depression was decreased at an intermediate IPI (30 ms) with submaximal stimulus intensities. These data augment previous work demonstrating persistent changes in hippocampal plasticity as a result of exposure to PCBs during development. Furthermore, as increases in field potential amplitudes were observed, these findings support previous conclusions that A1254-induced LTP deficits are not readily attributable to reductions in synaptic excitability. Thus, in addition to impairment in use-dependent synaptic plasticity reported previously, the present report reveals that basic components of information processing within the hippocampus are permanently altered as a result of perinatal exposure to PCBs.

Inhibition of LPS-induced splenocyte proliferation by ortho-substituted polychlorinated biphenyl congeners

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants, and their ubiquitous nature has prompted studies of their potential health hazards. As a result of their lipophilic nature, PCBs accumulate in breast milk and subsequently affect the health of offspring of exposed individuals. Biological effects of PCBs in animals have mostly been attributed to coplanar congeners, although effects of ortho congeners also have been demonstrated. To investigate the relationship of immunotoxicity and chlorine substitution pattern, the effects of PCB congeners and mixtures of ortho and non- ortho-substituted constituents of Aroclor 1242 on splenocytes from C57B1/6 mice were examined. The immunotoxic endpoints investigated included splenocyte viability, lipopolysaccharide (LPS)-induced splenocyte proliferation, and LPS-induced antibody secretion. Congeners with multiple ortho chlorines preferentially inhibited splenocyte proliferation as compared with non- or mono- ortho-substituted congeners. However, mixtures of non- and mono- ortho-substituted congeners and multi- ortho-substituted congeners inhibited LPS-induced splenocyte proliferation and antibody secretion at similar concentrations. Exposure of splenocytes to these mixtures did not activate the aryl hydrocarbon receptor (AhR) signal transduction pathway. These results suggest individual multi- ortho-substituted congeners preferentially inhibit LPS-induced splenocyte proliferation, while congeners not exhibiting an effect individually may have additive effects in a mixture to produce an immunotoxic response through an AhR-independent pathway.

A mixture of dioxins, furans, and non-ortho PCBs based upon consensus toxic equivalency factors produces dioxin-like reproductive effects.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin) and related polyhalogenated aromatic hydrocarbons (PHAHs) alter the reproductive development of laboratory animals. Therefore, we exposed animals to a mixture of dioxins, furans, and polychlorinated biphenyls (PCBs) that included TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzofuran (1-PeCDF), 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), octachlorodibenzofuran (OCDF), 3,3′,4,4′-tetrachlorobiphenyl (PCB77), 3,3′,4,4′,5-pentachlorobiphenyl (PCB126), and 3,3′,4,4′,5,5′-hexachlorobiphenyl (PCB169). The mixture composition approximated the relative abundance of these compounds in foodstuff (L. S. Birnbaum and M. J. DeVito, 1995, Toxicology Vol. 105, pp. 391–401). Following the work of Gray et al. with TCDD (1997, Toxicology and Applied Pharmacology Vol. 146, pp. 11–20), we exposed time-pregnant dams on gestation day (GD) 15 at doses up to 1.0 μg TCDD toxic equivalency (TEQ)/kg and the development of offspring was monitored. This mixture significantly increased the time to puberty in both male and female offspring. At postnatal day (PND) 32 seminal vesicle weights were decreased; however, only ventral prostate weight was affected at PND 49 and no effects were seen at PND 63. In female offspring, the mixture caused dose-dependent increases in the incidence of vaginal thread. Ethoxyresorufin-O-deethylase (EROD) activity was higher than with TCDD the comparable TEQ exposure. Based on the slightly lowered responsiveness to the mixture, we used 2.0 μg TEQ/kg to examine reproductive effects. This dose elicited the responses observed with 1.0 μg TCDD/kg. Results indicate that the mixture causes a similar spectrum of effects seen with TCDD and the slightly lowered degree of response based on administered dose appears to be due to decreased transfer of mixture components to the offspring. Thus, the use of the WHO consensus TEFs (M. Van den Berg et al., 1998, Environ. Health Perspec. 106, 775–792) reasonably predicts the developmental toxicity of this mixture of dioxin-like PHAHs.

PCB Muscle Content and Liver EROD Activity in the European EEL (Anguilla Anguilla) Treated with Aroclor 1254

The European eel (Anguilla anguilla) is a euryaline fish suitable for investigating exposure, accumulation patterns and biological effects of lipophilic pollutants such as polychlorinated biphenyls (PCBs). Eels were collected from the Orbetello Lagoon (Tyrrhenian coast of Italy) and injected with increasing doses of Aroclor 1254 to better characterise EROD activity as biomarker in eels from Mediterranean brackish environments. Fish muscle was analysed for PCB content and EROD activity was measured in liver microsomiaI fraction. Significant differences of PCB levels were observed in exposed fish (p < 0.05) while EROD activity increased significantly reaching the highest values at the maximum dose of 50mg/Kg (p < 0.001). Highly positive correlation (r = 0.82; p < 0.01) was found between PCB content and EROD activity. Isomer profiles showed decreases in hexa-CBs. The European eel is confirmed to be a useful sentinel species for assessing pollution in brackish environments whereas EROD activity is characterised as a sensitive biomarker of exposure for PCB mixtures.

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 32P-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 heptocyte cultures with 4-CB led to 209 adducts per 10 9 nucleotides at the 60 μM 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.

Effects of PCB exposure on neuropsychological function in children.

In the last decade advances in the analytic methods for quantification of polychlorinated biphenyls (PCBs) have resulted in widespread availability of congener-specific analysis procedures, and large amounts of data on PCB congener profiles in soil, air, water, sediments, foodstuffs, and human tissues have become available. These data have revealed that the PCB residues in environmental media and human tissues may not closely resemble any of the commercial PCB mixtures, depending on source of exposure, bioaccumulation through the food chain, and weathering of PCBs in the environment. At the same time, toxicological research has led to a growing awareness that different classes of PCB congeners have different profiles of toxicity. These advances in analytic techniques and toxicological knowledge are beginning to influence the risk assessment process. As the data from ongoing PCB studies assessing the mediators of neurobehavioral outcomes in children are published, the weight of evidence for PCB effects on neurodevelopment is growing. Studies in Taiwan, Michigan (USA), New York (USA), Holland, Germany, and the Faroe Islands have all reported negative associations between prenatal PCB exposure and measures of cognitive functioning in infancy or childhood. The German study also reported a negative association between postnatal PCB exposure and cognitive function in early childhood--a result that had not been found in previous studies. Only one published study in North Carolina (USA) has failed to find an association between PCB exposure and cognitive outcomes. Despite the fact that several more recent studies have used congener-specific analytic techniques, there have been only limited attempts to assess the role of specific PCB congeners or classes of congeners in mediating neurodevelopmental outcomes. From a statistical standpoint, attempts to determine the role of individual congeners in mediating outcomes are hampered by the fact that concentrations of most individual congeners are highly correlated with each other and with total PCBs. From a toxicological standpoint, these efforts are hampered by the fact that many of the PCB congeners present in human tissues have never been studied in the laboratory, and their relative potency to produce nervous system effects is unknown. More complete information on the health effects of various congeners or congener classes would allow more informed scientific and risk assessment decisions.

Cumulative Effects of Natural and Anthropogenic Stress on Immune Function and Disease Resistance in Juvenile Chinook Salmon

Abstract Previous studies have shown that juvenile chinook salmon Oncorhynchus tshawytscha exposed in the field or the laboratory to polychlorinated biphenyls (PCBs), an anthropogenic stressor, are immunosuppressed. It is not known whether simultaneous exposure to natural stressors can increase this immunosuppression. To examine the effects of natural and anthropogenic stressors on immune function, we infected juvenile chinook salmon with metacercariae of the trematode Nanophyetus salmincola by exposing the fish to infected freshwater snails Juga plicifera. Infected (>300 metacercariae per fish) and noninfected salmon were then injected with either the commercial PCB mixture Aroclor 1254 or an acetone–emulphor carrier. B cell function was examined by in vitro hemolytic plaque-forming cell (PFC) assay. Nanophyetus salmincola infection resulted in significantly lower anterior kidney primary PFCs and lower splenic secondary PFCs. The combination of N. salmincola infection and Aroclor 1254 exposure caused a l...

Exposure to the polychlorinated biphenyl mixture Aroclor® 1254 alters melanocyte and tail muscle morphology in developing Xenopus laevis tadpoles

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that have damaging effects on both ecosystem and human health. Numerous studies have shown that exposure to PCBs can alter growth and development of aquatic organisms, including frogs. In this report, developing Xenopus laevis tadpoles were exposed to the PCB mixture Aroclor 1254. Tadpoles were exposed from 5 through 9 d postfertilization to either 0, 1, 10, 50, or 100 ppm Aroclor 1254. Exposure to an acute, high concentration of Aroclor 1254 (10, 50, and 100 ppm) caused statistically significant reductions in survival and body size. In addition, tadpoles exposed to these higher concentrations showed histological abnormalities, including aberrant tail tip, myotomal, and melanocyte morphologies. Described adverse health effects associated with PCB exposure of developing frogs will serve as useful health endpoints in ongoing and future molecular-based studies that correlate health effects with changes in gene expression.

Alterations in brain protein kinase C isoforms following developmental exposure to a polychlorinated biphenyl mixture

PCBs have been shown to alter several neurochemical end-points and are implicated in the etiology of some neurological diseases. Recent in vivo studies from our laboratory indicated that developmental exposure to a commercial PCB mixture, Aroclor 1254 ®, caused perturbations in calcium homeostasis and changes in protein kinase C (PKC) activities in rat brain. However, it is not known which molecular substances are targets for PCB-induced developmental neurotoxicity. Since the PKC signaling pathway has been implicated in the modulation of motor behavior as well as learning and memory, and the roles of PKC are subspecies specific, the present study attempted to analyze the effects on selected PKC isozymes in the cerebellum and the hippocampus following developmental exposure (gestational day 6 through postnatal day 21) to a PCB mixture, Aroclor 1254. The results indicated that the developmental exposure to PCBs caused significant hypothyroxinemia and age-dependent alterations in the translocation of PKC isozymes; the effects were greatly significant at postnatal day (PND) 14. Immunoblot analysis of PKC-alpha (α) from both cerebellum and hippocampus revealed that developmental exposure to Aroclor 1254 caused a significant decrease in cytosolic fraction and an increase in particulate fraction. There was no significant difference between these two brain regions on the level of fractional changes. However, the ratio between the fractions (particulate/cytosol) from cerebellum only was increased in a dose-dependent manner. Analysis of PKC-gamma (γ) in cerebellum on PND14 showed a decrease in cytosolic fraction in both dose groups and an increase in particulate fraction at high dose (6 mg/kg) only. The ratio between the two fractions was increased in a dose-dependent manner. In the hippocampus, there was a significant decrease in PKC-γ in cytosolic fraction of the high-dose group and a significant increase in particulate fraction of the low-dose group. But, the ratio between the fractions showed a significant increase (2.6-fold increase in high dose on PND14). Analysis of PKC-epsilon (ε) in cerebellum showed a significant decrease in cytosolic fraction at PND14, while particulate PKC-ε was unchanged. But in hippocampus, there was a significant decrease in cytosolic PKC-ε and an increase in ratio between fractions at 6 mg/kg on PND14. The results from this study indicate that the patterns of subcellular distributions of PKC isoforms following a developmental PCB exposure were PKC isozyme- and developmental stage-specific. Considering the significant role of PKC signaling in motor behavior, learning and memory, it is suggested that altered subcellular distribution of PKC isoforms at critical periods of brain development may be a possible mechanism of PCB-induced neurotoxic effects and that PKC-α, γ, and ε may be among the target molecules implicated with PCB-induced neurological impairments during developmental exposure. It is believed that this is the first report successfully identifying PKC isoforms responding to PCBs during developmental exposure.

Development and validation of a herring gull embryo toxicokinetic model for PCBs.

A toxicokinetic model was developed to describe polychlorinated biphenyl (PCB) accumulation by herring gull (Larus argentatus) embryos during development. The model consists of two components, a bioenergetics model that predicts the lipid mass balance of embryo and yolk compartments with time and an empirical toxicokinetic model that describes PCB partitioning between lipid compartments in the egg. The model was calibrated using data on PCB and lipid partitioning between embryo and yolk + albumen at four time points during incubation in herring gull eggs injected with a PCB mixture, combined with data sets on herring gull embryo growth rates and bioenergetic demands with time. The model was validated using independent data consisting of maternally exposed, field-incubated Lake Superior herring gull eggs that varied in incubation ages over the range of 8.5 d to pipping age (26-28 days). PCB concentrations in 6-9 d embryos were nearly an order of magnitude less than predicted by equilibrium lipid partitioning between the embryo and yolk + albumen compartments of the eggs. PCB concentrations in embryos were adequately predicted by equilibrium partitioning, however, for eggs incubated for 23-28 d. An empirical relationship was developed to account for the apparent nonequilibrium behaviour of PCBs during early development. The model was sensitive to the mass of yolk lipids and the mass of PCBs deposited to fresh eggs and much of the variability in embryo PCB concentrations could by explained by accounting for variability in these input parameters. Consistent with experimental data for other avian species, the model predicts that the highest PCB concentrations in the embryo/chick occur during pipping or soon after when yolk lipids have been completely resorbed by the embryo.

Gene expression patterns predict exposure to PCBs in developing Xenopus laevis tadpoles.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that pose global ecological and human health problems. Although it is well established that PCBs are associated with a variety of adverse health effects in wildlife and in humans, it is often difficult to determine direct cause-and-effect relationships between exposure and specific health outcomes. In this study, gene expression signatures were used to relate exposure to PCBs with altered physiological responses and/or specific health effects. Real-time PCR was used to measure gene expression levels for 10 genes in Xenopus laevis tadpoles (18 days postfertilization, PF) after acute exposure (2 days) to the PCB mixture Aroclor 1254. Specific gene expression signatures correlated with exposure and were predictive of adverse health effects. Exposure to low levels of Aroclor 1254 (5-50 ppb) significantly increased expression of six genes, independent of any health effects; exposure to midlevel concentrations (300-400 ppb) significantly decreased expression levels of two genes, NGF and beta-actin, prior to the onset of observable health effects; exposure to higher doses (500-700 ppb) significantly decreased NGF and beta-actin expression concomitant with the appearance of gross morphological abnormalities, behavioral deficits, and a statistically significant decrease in survival. This study expands upon our previous work that demonstrated an age-dependent susceptibility to Aroclor 1254 in Xenopus laevis tadpoles and that defined specific gene expression signatures as useful bioindicators of exposure and as predictors of overt or impending health effects.

Identification of calcium-dependent and -independent signaling pathways involved in polychlorinated biphenyl-induced cyclic AMP-responsive element-binding protein phosphorylation in developing cortical neurons

Cyclic AMP (cAMP)-responsive element-binding protein (CREB) is a transcription factor important in developing nervous system cells and is activated by a variety of signaling molecules. Aroclor 1254 (A1254), a polychlorinated biphenyl mixture, perturbs Ca 2+ homeostasis and increases CREB phosphorylation in rat neonatal cortical cell cultures in a time- and concentration-dependent manner. The present experiments determined that the cell type responding to A1254 with Ca 2+ increases and phosphorylated CREB (phospho-CREB) was predominantly of neuronal morphology and microtubule-associated protein (MAP2)-positive phenotype. Similarly, glutamate (100 μM) increased phospho-CREB immunoreactivity selectively in MAP2-immunopositive cells. Using Western blotting and immunocytochemical techniques, we identified key signal transduction pathways operative in phosphorylating CREB in cortical cell cultures and examined their participation in 3 ppm A1254-induced CREB activation. Cortical cultures treated with glutamate, forskolin or the phorbol ester phorbol 12-myristate 13-acetate exhibited robust increases in phospho-CREB. Tetrodotoxin (1 μM) completely inhibited CREB phosphorylation by A1254, suggesting that synaptic activity is involved in A1254-induced CREB activation. Buffering [Ca 2+] i with bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid tetrakis(acetoxymethyl) ester in the absence of extracellular Ca 2+ partially inhibited A1254-induced CREB phosphorylation. Inhibition of mitogen-activated protein kinase (10 μM U0126) or protein kinase C (PKC; bisindoylmaleimide, 5 μM) activation did not inhibit A1254-induced CREB phosphorylation. By contrast, inhibition of protein kinase A (PKA) with 100 μM PKA inhibitor peptide, PKI, blocked A1254-induced CREB phosphorylation. Thus, we examined whether A1254 activates PKA by increasing cAMP; 10 μM forskolin, but not A1254, elevated intracellular cAMP levels. These results indicate that in neocortical cells in culture, CREB phosphorylation occurs via Ca 2+-, PKA-, and PKC-dependent pathways. Furthermore, A1254-induced CREB phosphorylation occurs predominantly in neurons, is dependent on synaptic activity and mediated by Ca 2+- and PKA-dependent pathways.

A novel mechanism for endocrine-disrupting effects of polychlorinated biphenyls: direct effects on gonadotropin-releasing hormone neurones.

Polychlorinated biphenyls (PCBs) cause abnormal development and physiology of the reproductive system. We hypothesized that these effects may be mediated, at least in part, by neuroendocrine cells in the hypothalamus that integrate inputs to and outputs from the central nervous system and reproductive systems. The effects of two PCB mixtures, Aroclor 1221 and Aroclor 1254, were tested on the hypothalamic GT1-7 cells, which synthesize and secrete the key hypothalamic hormone, gonadotropin-releasing hormone (GnRH). GT1-7 cells were treated for 24 h in dose-response experiments and GnRH gene expression and release were quantified. Aroclor 1221 was stimulatory to GnRH gene expression, particularly at post-transcriptional levels (GnRH cytoplasmic mRNA), and increased GnRH peptide levels, suggesting a post-translational regulation of GnRH biosynthesis. It also caused a qualitative increase in GT1-7 neurite outgrowth and cell confluency. Aroclor 1254 had very different effects from Aroclor 1221. It inhibited GnRH nuclear mRNA levels at high dosages, and stimulated GnRH mRNA at low doses, suggesting a post-transcriptional mechanism of regulation. Aroclor 1254 did not alter GnRH peptide levels. Qualitatively, Aroclor 1254 caused a retraction of GT1-7 cell processes and neurotoxicity at high dosages. In order to gauge the involvement of the oestrogen receptor in these responses, the oestrogen receptor antagonist, ICI 182,780 (ICI) was coadministered in other studies with the PCBs. While effects of Aroclor 1221 on GnRH gene expression were not blocked by ICI, its effects on GnRH peptide levels were blocked by ICI, indicating that some but not all of the effects of Aroclor 1221 are mediated by the classical oestrogen receptor alpha and/or beta. The inhibitory effects of Aroclor 1254 on GnRH gene expression were not prevented by ICI, although ICI itself had stimulatory effects on GnRH gene expression that were blocked by cotreatment with Aroclor 1254. These results demonstrate a novel mechanism for effects of the two PCBs directly on GnRH gene expression, and indicate a hypothalamic level for endocrine disruption by these environmental toxicants.