The point of transition on the dose‐effect curve as a reference point in the evaluation of in vitro toxicity data

Dose‐effect evaluation is an increasingly important step of health risk assessment. The foreseen increase of in vitro methods argues for the development and evaluation of a clearly defined reference points for dose‐effect modelling of in vitro data. In the present study, the traditional use of a concentration corresponding to 10% or 50% of the maximal effect (EC₁₀ or EC₅₀) is compared with a strategy, under which, a reference point (Benchmark dose, BMD_T) is calculated that represents the dose where the slope of the dose‐effect curve changes the most (per unit log‐dose) in the low dose region. To illustrate the importance of the reference point, dose‐effect data on CYP1A1 enzyme activity for 30 polychlorinated biphenyl (PCB) congeners were evaluated in order to calculate relative potencies, in relation to 2,3,7,8‐TCDD, with confidence intervals (CIs). The present study shows that the interpretation of the results as potency and rank orders potentially depends on the choice and definition of the reference point (BMD_T, EC₁₀ or EC₅₀). This is important as potency ranking may be used as a method for screening and prioritization, in research, in policy development or in pharmaceutical development. The use of the BMD_T implies a focus on the change of structure in the parameter's dose–response rather than a particular percentage change in the response in such a parameter. In conclusion, the BMD_T may be used as an alternative base for evaluation of dose‐effect relationships in vitro. It offers an objective geometrical definition of a reference point in the low‐dose region of the dose‐effect curve. Copyright © 2012 John Wiley & Sons, Ltd.     

Assessment of PCBs exposure in human hair using double focusing high resolution mass spectrometry and single quadrupole mass spectrometry

Polychlorinated biphenyls (PCBs) levels were assessed in human hair samples, originating from two main agricultural regions of Greece. The analysis was performed by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-double focusing high resolution mass spectrometry (GC-DFHRMS). The main analytical procedure involved hair decontamination, solid-liquid extraction and cleanup steps. The recoveries of PCBs ranged from 71.2% to 101.6%, with accuracies greater than 87.5% and the between-run precisions (%RSD) lower than 25% for all analytes. Differences in the frequencies of detection and the median values of PCBs were detected between the examined regions and between the applied analytical techniques. All Peloponnesus' hair examined samples were found positive for each examined PCB, while the percentage of the total positive samples ranged from 86.1% (for PCB 138) to 94.4% (for PCB 28 and 153 congeners) using GC-DFHRMS. The Cretan hair samples were less contaminated (SUM PCBs=0.61 and 1.47pg/mg) unlike the Peloponnesus' samples (SUM PCBs=24.68 and 38.74pg/mg) measured by GC-DFHRMS and GC-MS, respectively. PCBs with high chlorination gave lower concentration values compared to low chlorination PCBs in both populations. No significant differences were observed between women and men. The GC-DFHRMS technique provided higher percentage of positive samples and low PCBs median values, due to higher sensitivity and interferences from isobaric ions in the GC-MS technique and is therefore considered as a powerful tool for such assessments in hair specimens.     

Excitatory and inhibitory synaptic transmission is differentially influenced by two ortho-substituted polychlorinated biphenyls in the hippocampal slice preparation

Exposure to polychlorinated biphenyls impairs cognition and behavior in children. Two environmental PCBs 2,2',3,3',4,4',5-heptachlorobiphenyl (PCB170) and 2,2',3,5',6-pentachlorobiphenyl (PCB95) were examined in vitro for influences on synaptic transmission in rat hippocampal slices. Field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 region using a multi-electrode array. Perfusion with PCB170 (10 nM) had no effect on fEPSP slope relative to baseline period, whereas (100 nM) initially enhanced then depressed fEPSP slope. Perfusion of PCB95 (10 or 100 nM) persistently enhanced fEPSP slope > 200%, an effect that could be inhibited by dantrolene, a drug that attenuates ryanodine receptor signaling. Perfusion with picrotoxin (PTX) to block GABA neurotransmission resulted in a modest increase in fEPSP slope, whereas PTX + PCB170 (1-100 nM) persistently enhanced fEPSP slope in a dose dependent manner. fEPSP slope reached > 250% of baseline period in the presence of PTX + 100 nM PCB170, conditions that evoked marked epileptiform after-potential discharges. PCB95 and PCB170 were found to differentially influence the Ca²⁺-dependence of [³H]ryanodine-binding to hippocampal ryanodine receptors. Non-coplanar PCB congeners can differentially alter neurotransmission in a manner suggesting they can elicit imbalances between inhibitory and excitatory circuits within the hippocampus. Differential sensitization of ryanodine receptors by Ca²⁺ appears to mediate, at least in part, hippocampal excitotoxicity by non-coplanar PCBs.     

Chronic treatment with polychlorinated biphenyls (PCB) during pregnancy and lactation in the rat: Part 1: Effects on somatic growth, growth hormone-axis activity and bone mass in the offspring

Polychlorinated biphenyls (PCBs) are pollutants detected in animal tissues and breast milk. The experiments described in the present paper were aimed at evaluating whether the four PCB congeners most abundant in animal tissues (PCB-138, -153, -180 and -126), administered since fetal life till weaning, can induce long-term alterations of GH-axis activity and bone mass in the adult rat. We measured PCB accumulation in rat brain and liver, somatic growth, pituitary GH expression and plasma hormone concentrations at different ages. Finally, we studied hypothalamic somatostatin expression and bone structure in adulthood, following long-term PCB exposure.Dams were treated during pregnancy from GD15 to GD19 and during breast-feeding. A constant reduction of the growth rate in both male and female offspring from weaning to adulthood was observed in exposed animals. Long-lasting alterations on hypothalamic-pituitary GH axis were indeed observed in PCB-exposed rats in adulthood: increased somatostatin expression in hypothalamic periventricular nucleus (both males and females) and lateral arcuate nucleus (males, only) and decreased GH mRNA levels in the pituitary of male rats. Plasma IGF-1 levels were higher in PCB-exposed male and female animals as compared with controls at weaning and tended to be higher at PN60. Plasma testosterone and thyroid hormone concentrations were not significantly affected by exposure to PCBs. In adulthood, PCBs caused a significant reduction of bone mineral content and cortical bone thickness of tibiae in male rat joint to increased width of the epiphyseal cartilage disk. In conclusion, the developmental exposure to the four selected PCB compounds used in the present study induced far-reaching effects in the adult offspring, the male rats appearing more sensitive than females.     

PCB-induced endothelial cell dysfunction: Role of poly(ADP-ribose) polymerase

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants implicated in the development of pro-inflammatory events critical in the pathology of atherosclerosis and cardiovascular disease. PCB exposure of endothelial cells results in increased cellular oxidative stress, activation of stress and inflammatory pathways leading to increased expression of cytokines and adhesion molecules and ultimately cell death, all of which can lead to development of atherosclerosis. To date no studies have been performed to examine the direct effects of PCB exposure on the vasculature relaxant response which if impaired may predispose individuals to hypertension, an additional risk factor for atherosclerosis. Overactivation of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) following oxidative/nitrosative stress in endothelial cells and subsequent depletion of NADPH has been identified as a central mediator of cellular dysfunction. The aim therefore was to investigate whether 2,2′,4,6,6′-pentachlorobiphenyl (PCB 104) directly causes endothelial cell dysfunction via increased oxidative stress and subsequent overactivation of PARP. Exposure of ex vivo rat aortic rings to PCB 104 impaired the acetylcholine-mediated relaxant response, an effect that was dependent on both concentration and exposure time. In vitro exposure of mouse endothelial cells to PCB 104 resulted in increased cellular oxidative stress through activation of the cytochrome p450 enzyme CYP1A1 with subsequent overactivation of PARP and NADPH depletion. Pharmacological inhibition of CYP1A1 or PARP protected against the PCB 104-mediated endothelial cell dysfunction. In conclusion, the environmental contaminants, PCBs, can activate PARP directly impairing endothelial cell function that may predispose exposed individuals to development of hypertension and cardiovascular disease.     

Predictors of Serum Dioxins and PCBs among Peripubertal Russian Boys

Background

Although sources and routes of exposure to dioxins and polychlorinated biphenyls (PCBs) have been studied, information regarding exposure among children is limited. Breast-feeding and diet are two important contributors to early life exposure. To further understand other significant contributors to childhood exposure, we studied a cohort of children from a city with high environmental dioxin levels.

Objectives

We investigated predictors of serum concentrations of polychlorinated dibenzo-p-dioxins (PCDDs)/polychlorinated dibenzofurans (PCDFs)/co-planar PCBs (C-PCBs), toxic equivalents (TEQs), and PCBs among 8- to 9-year-old boys in Chapaevsk, Russia.

Methods

We used general linear regression models to explore associations of log₁₀-transformed serum concentrations of PCDDs/PCDFs/C-PCBs, TEQs, and PCBs at study entry with anthropometric, demographic, geographic, and dietary factors in 482 boys in Chapaevsk, Russia.

Results

The median (25th, 75th percentile) concentration for total 2005 TEQs was 21.1 pg/g lipid (14.4, 33.2). Boys who were older, consumed local foods, were breast-fed longer, and whose mothers were employed at the Khimprom chemical plant (where chlorinated chemicals were produced) or gardened locally had significantly higher serum dioxins and PCBs, whereas boys with higher body mass index or more educated parents had significantly lower serum dioxins and PCBs. Boys who lived < 2 km from Khimprom had higher total TEQs (picograms per gram lipid) [adjusted mean = 30.6; 95% confidence interval (CI), 26.8–35.0] than boys who lived > 5 km away (adjusted mean = 18.8; 95% CI, 17.2–20.6).

Conclusions

Our findings suggest that there are specific local sources of dioxin and PCB exposure among children in Chapaevsk including maternal gardening, consumption of locally grown food, and residential proximity to the Khimprom plant.     

Maternal Pregnancy Levels of Polychlorinated Biphenyls and Risk of Hypospadias and Cryptorchidism in Male Offspring

Background

The etiologies of the male urogenital anomalies cryptorchidism and hypospadias are poorly understood. It has been suggested, however, that in utero hormone levels may be related to risk. Endocrine-disrupting chemicals, including polychlorinated biphenyl (PCB) compounds, may alter hormone levels and thereby affect the fetus.

Objectives

To examine whether in utero PCB exposure is related to cryptorchidism and hypospadias, we examined PCB levels among pregnant women enrolled in the Collaborative Perinatal Project (CPP).

Methods

The CPP enrolled pregnant women at 12 U.S. medical centers between 1959 and 1965. For the present research, we analyzed third-trimester serum samples from the mothers of 230 sons with cryptorchidism, 201 sons with hypospadias, and 593 sons with neither condition. We estimated adjusted odds ratios (ORs) and 95% confidence intervals (CIs) using logistic regression and examined the associations of each anomaly with individual PCB congener levels, sum of PCBs, and several functional groupings of PCBs.

Results

In general, the ORs for cryptorchidism or hypospadias showed no notable associations with individual PCB congener levels or functional groupings of PCBs. However, the ORs and 95% CIs for the sum of PCBs associated with hypospadias were as follows: 0–1.9 μg/L, reference group; 2–2.9 μg/L, OR = 1.57, 95% CI, 1.05–2.34; 3–3.9 μg/L, OR = 1.45, 95% CI, 0.90–2.34; and ≥ 4.0 μg/L, OR = 1.69, 95% CI, 1.06–2.68; p-value for trend = 0.08.

Conclusions

Given the large number of associations examined, these findings do not strongly support the hypothesis that PCBs are associated with cryptorchidism or hypospadias. Because population serum PCB levels at the time of sample collection were considerably higher than levels at present, it is unlikely that current PCB exposure is related to the development of either anomaly.     

多氯联苯的肝毒性研究进展

多氯联苯(PCBs)是一类广泛存在于环境中的持久性有机污染物。笔者综述了PCBs的特性及其对动物和人类肝毒性影响的研究进展。已有的研究结果表明,PCBs对动物肝脏有致损害及致癌作用;但对人类而言,除在重大污染事件中发现PCBs可导致人类肝疾病,直至死亡外,职业暴露和环境中长期低剂量暴露实验均未证实PCBs暴露与人类肝脏的损伤及其致癌作用有关。但某些地区人群高肝癌发病率及其环境中PCBs的检出预示着其中有一定的关系,值得深入研究。     

The effect of dietary glycine on the hepatic tumor promoting activity of polychlorinated biphenyls (PCBs) in rats

Polychlorinated biphenyls (PCBs) are ubiquitious lipophilic environmental pollutants. Some of the PCB congeners and mixtures of congeners have tumor promoting activity in rat liver. The mechanism of their activity is not fully understood and is likely to be multifactorial. The aim of this study was to investigate if the resident liver macrophages, Kupffer cells, are important in the promoting activity of PCBs. The hypothesis of this study was that the inhibition of Kupffer cell activity would inhibit hepatic tumor promotion by PCBs in rats. To test our hypothesis, we studied the effects of Kupffer cell inhibition by dietary glycine (an inhibitor of Kupffer cell secretory activity) in a rat two-stage hepatocarcinogenesis model using 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153, a non-dioxin-like PCB) or 3,3',4,4'-tetrachlorobiphenyl (PCB-77, a dioxin-like PCB) as promoters. Diethylnitrosamine (DEN, 150 mg/kg) was administered to female Sprague-Dawley rats, which were then placed on an unrefined diet containing 5% glycine (or casein as nitrogen control) starting two weeks after DEN administration. On the third day after starting the diets, rats received PCB-77 (300 micromol/kg), PCB-153 (300 micromol/kg), or corn oil by i.p. injection. The rats received a total of 4 PCB injections, administered every 14 days. The rats were euthanized on the 10th day after the last PCB injection, and the formation of altered hepatic foci expressing placental glutathione S-transferase (PGST) and the rate of DNA synthesis in these foci and in the normal liver tissue were determined. Glycine did not significantly affect foci number or volume. PCB-153 did not significantly increase the focal volume, but increased the number of foci per liver, but only in the rats not fed glycine; PCB-77 increased both the foci number and their volume in both glycine-fed and control rats. Glycine did not alter the PCB content of the liver, but did increase the activity of 7-benzyloxyresorufin O-dealkylase (BROD) in liver microsomes from PCB-153 treated rats. However, glycine did not affect the induction of ethoxyresorufin O-dealkylase activity by PCB-77 in liver microsomes. Glycine diminished hepatocyte proliferation in PGST-positive foci, but not in normal tissue. Overall these results do not support the hypothesis that dietary glycine inhibits the promoting activities of PCBs. The observations that PCB-153 increased the number of foci per liver in control rats but not glycine-fed rats and that dietary glycine reduced cell proliferation in PGST-positive foci, however, do not allow us to completely rule out a role for dietary glycine. But the data overall indicate that Kupffer cells likely do not contribute to the tumor promoting activities of PCB-77 and PCB-153.     

Protective effects of insulin on polychlorinated biphenyls-induced disruption of actin cytoskeleton in hippocampal neurons

Insulin receptors are widely distributed in the brain, and insulin improves learning and memory in some brain injury. Insulin elevates LIM kinase 1 (LIMK-1) activity and induces actin polymerization in some cells, while actin cytoskeleton dynamics mediated via LIMK-1/cofilin signal pathway is considered important to learning and memory formation. Our previous studies have shown that polychlorinated biphenyls (PCBs) disrupt the actin cytoskeleton by inhibiting LIMK-1/cofilin signaling pathway in the cultured hippocampal neurons. To determine potential neuronal protective effects by insulin, we administered insulin to the cultured hippocampal neurons after exposure to PCBs mixture Aroclor 1254 (A 1254). We found that insulin antagonized a loss of filamentous actin and the cytotoxicity induced by A 1254. Similarly, insulin restored the decrease of LIMK-1 and cofilin phosphorylation induced by A 1254. We concluded that insulin could protect neurons, probably partly by ameliorating filamentous actin cytoskeleton disruption mediated via the activation of LIMK-1/cofilin signal pathway in cultured hippocampal neurons after exposure to A 1254. The above protective effects in hippocampal neuron may have important implications in the treatment of PCBs-induced neurotoxicity and the mechanism by which insulin improves learning and memory. (c) 2007 Wiley Periodicals, Inc. Environ Toxicol 22: 152-158, 2007.