Effects of co‐exposure to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin and polychlorinated biphenyls on nonalcoholic fatty liver disease in mice

2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin (TCDD) and polychlorinated biphenyls (PCBs) are persistent organic pollutants which coexist in environment, and human are co‐exposed to these chemicals. Our present study was aimed to investigate the possible enhanced nonalcoholic fatty liver disease (NAFLD) in ApoE^?/? mice co‐exposed to TCDD and PCBs and to reveal the potential mechanisms involved in. Male ApoE^?/? mice were exposed to TCDD (15 μg/kg) and Aroclor1254 (55 mg/kg, a representative mixture of PCBs) alone or in combination by intraperitoneal injection four times over a 6‐week period. Those mice co‐exposed to PCBs and TCDD developed serious liver steatosis, necrosis, and inflammatory stimuli. Interestingly, all treatment induced hepatic cytochrome P450 1A1 (CYP1A1) expression, but the maximal level of CYP1A1 was not observed in the co‐exposure group. Furthermore, microarray analysis by ingenuity pathway analysis software showed that the nuclear factor‐erythroid 2‐related factor 2 (Nrf2)‐mediated oxidative stress response pathway was significantly activated following co‐exposure to TCDD and PCBs. Our data demonstrated that co‐exposure to TCDD and PCBs markedly worsen NAFLD in ApoE^?/? mice. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1364–1374, 2015.     

Exposure to Aroclor‐1254 impairs spindle assembly during mouse oocyte maturation

Polychlorinated biphenyls (PCBs), as typical environmental estrogen disruptors, are a structurally‐related group of halogenated aromatic hydrocarbons that are composed of 209 isomers and present as a mixture in the environment. PCBs congener with different numbers and positions of chlorine atoms substituted on the biphenyl moiety. Aroclor‐1254 is a mixture of more than 60 PCB congeners. Previous studies have provided the evidence that PCBs have severe negative effects on reproductive functions, but the effects of PCBs on spindle assembly during mouse oocyte maturation in vitro have not been reported. In the present study, female ICR mouse immature oocytes were cultured in M2 medium with 1 and 10 μg mL^?1 Aroclor‐1254 separately in vitro. The percentage of germinal vesicle breakdown (GVBD) and the first polar body extrusion were recorded. The results showed no significant difference in the percentage of GVBD or the first polar body extrusion between control oocytes and Aroclor‐1254‐treated oocytes. Further studies showed that the normal localization of γ‐tubulin and Aurora‐A kinase was interfered and α‐tubulin assembling into spindle was affected when mouse oocytes were exposed to Aroclor‐1254. The length of spindle from 10 μg mL^?1 Aroclor‐1254‐treated oocytes was longer than that from control oocytes, and the spindle area in the Aroclor‐1254‐treated groups were decreased. Furthermore, the percentage of DNA damage in cumulus cells revealed an increase after exposed to Aroclor‐1254. These results will provide the important reference for the prevention of reproductive disorders caused by PCBs. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1652–1662, 2016.     

Age‐dependent electrocardiographic changes in Pgc‐1β deficient murine hearts

Increasing evidence implicates chronic energetic dysfunction in human cardiac arrhythmias. Mitochondrial impairment through Pgc‐1β knockout is known to produce a murine arrhythmic phenotype. However, the cumulative effect of this with advancing age and its electrocardiographic basis have not been previously studied. Young (12‐16 weeks) and aged (>52 weeks), wild type (WT) (n = 5 and 8) and Pgc‐1β^?/? (n = 9 and 6), mice were anaesthetised and used for electrocardiographic (ECG) recordings. Time intervals separating successive ECG deflections were analysed for differences between groups before and after β1‐adrenergic (intraperitoneal dobutamine 3 mg/kg) challenge. Heart rates before dobutamine challenge were indistinguishable between groups. The Pgc‐1β^?/? genotype however displayed compromised nodal function in response to adrenergic challenge. This manifested as an impaired heart rate response suggesting a functional defect at the level of the sino‐atrial node, and a negative dromotropic response suggesting an atrioventricular conduction defect. Incidences of the latter were most pronounced in the aged Pgc‐1β^?/? mice. Moreover, Pgc‐1β^?/? mice displayed electrocardiographic features consistent with the existence of a pro‐arrhythmic substrate. Firstly, ventricular activation was prolonged in these mice consistent with slowed action potential conduction and is reported here for the first time. Additionally, Pgc‐1β^?/? mice had shorter repolarisation intervals. These were likely attributable to altered K⁺ conductance properties, ultimately resulting in a shortened QT_c interval, which is also known to be associated with increased arrhythmic risk. ECG analysis thus yielded electrophysiological findings bearing on potential arrhythmogenicity in intact Pgc‐1β^?/? systems in widespread cardiac regions.     

Regulation of cardiac angiotensin‐converting enzyme and angiotensin AT1 receptor gene expression in Npr1 gene‐disrupted mice

1. Understanding of the regulatory mechanisms of gene expression in the control of blood pressure and fluid volume is a key issue in cardiovascular medicine. Guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) signalling antagonizes the physiological and pathophysiological effects mediated by the renin–angiotensin–aldosterone system (RAAS) in the regulation of cardiovascular homeostasis. 2. The targeted‐disruption of the Npr1 gene (coding for GC‐A/PRA) leads to activation of the cardiac RAAS involved in the hypertrophic remodelling process, which influences cardiac size, expression of pro‐inflammatory cytokine genes and the behaviour of various hypertrophy marker genes. The Npr1 gene‐knockout (Npr1^?/?) mice exhibit 35–40 mmHg higher systolic blood pressure and a significantly greater heart weight to bodyweight ratio than wild‐type (Npr1^+/+) mice. 3. The expression of both angiotensin‐converting enzyme (ACE) and angiotensin II AT_1a receptors are significantly increased in hearts from Npr1^?/? mice compared with hearts from Npr1^+/+ mice. In parallel, the expression of interleukin‐6 and tumour necrosis factor‐α is also markedly increased in hearts from Npr1^?/? mice. 4. These findings indicate that disruption of NPRA/cGMP signalling leads to augmented expression of the cardiac RAAS in conjunction with pro‐inflammatory cytokines in Npr1‐null mutant mice, which promotes the development of cardiac hypertrophy and remodelling.     

Age‐related changes in hepatic expression and activity of drug metabolizing enzymes in male wild‐type and breast cancer resistance protein knockout mice

This study aimed to reveal age‐related changes in the expression and activity of seven hepatic drug metabolizing enzymes (DMEs) in male wild‐type and breast cancer resistance protein knockout (Bcrp1^?/?) FVB mice. The protein expression of four cytochrome P450 (Cyps) (Cyp3a11, 2d22, 2e1, and 1a2), and three UDP‐glucuronosyltransferases (Ugts) (Ugt1a1, 1a6a, and 1a9) in liver microsomes of wild‐type and Bcrp1^?/? FVB mice at different ages were determined using a validated ultra high performance liquid chromatography with tandem mass spectrometry (UHPLC–MS/MS) method. The activities and mRNA levels of these DMEs were measured using the probe substrates method and real‐time PCR, respectively. In the liver of wild‐type FVB mice, Cyp3a11, 2d22, 2e1, 1a2, Ugt1a1, and 1a6a displayed maximum protein levels at 6–9 weeks of age. Cyp1a2, Ugt1a1, 1a6a, and 1a9 showed maximum activities at 6–9 weeks of age, whereas Cyp3a11, 2d22, and 2e1 showed maximum activities in 1–3‐week‐old mice. Additionally, most of the DMEs showed maximum mRNA levels in 17‐week‐old mice liver. Compared with wild‐type FVB mice, the protein levels of these DMEs showed no significant changes in Bcrp1^?/? FVB mice liver. However, the activity of Cyp2e1 was increased and that of Cyp2d22 was decreased. In conclusion, t he seven hepatic DMEs in FVB mice liver showed significant alterations in an isoform‐specific manner with increased age. Although the protein levels of these DMEs showed no significant changes, the activities of Cyp2e1 and 2d22 were changed in Bcrp1^?/? mice.     

Role of N-methyl-D-aspartate receptors in polychlorinated biphenyl mediated neurotoxicity

Polychlorinated biphenyls (PCBs) are widespread persistent environmental pollutants. Chronic human and animal exposure to PCBs results in various harmful effects including neurotoxicity. This study investigates the effects of the PCB mixture Aroclor 1254 (A1254) and two PCB congeners (coplanar, non-ortho PCB 126, and non coplanar PCB 99) on the expression of N-methyl-D-aspartate receptors (NMDARs) and the subsequent toxic effects using a human SHS5-SY neuroblastoma cell line. NMDAR was measured using a radiolabeled phencyclidine receptor ligand [³H]-MK801, apoptosis was quantified using fluorogenic substrates specific for caspase-3 (DEVD-AFC) and cell death using lactate dehydrogenase (LDH) release. After treatment, a positive dose–response relationship of increasing NMDARS, increasing caspase-3 activity and cell death was observed in all PCB compounds. The non-coplanar PCB compounds were found to be significantly more toxic than the coplanar congener and the PCB mixture A1254. PCB-mediated cell death was attenuated with 10 μM NMDAR antagonists: 1-amino-3,5-dimethyladamantane hydrochloride (memantine) and (+)-5-methyl-10,11-dihydro-5H-debenzocyclhepten-5,10-imine maleate ((+)-MK-801), thus demonstrating the importance of NMDAR in PCB neurotoxicity. Intracellular calcium [Ca²⁺]_i chelator BAPTA-AM (1 μM) partially attenuated the neurotoxic effect of the PCBs suggesting a role of calcium homeostasis disruption in the neurotoxicity of PCBs. These results suggest that the neurotoxicity of PCBs can be mediated through activation of NMDARs.     

Role of cyclooxygenase‐1 and ‐2 in endothelium‐dependent contraction of atherosclerotic mouse abdominal aortas

The objective of this study was to determine the role of cyclooxygenase (COX)‐1 or ‐2 in endothelium‐dependent contraction under atherosclerotic conditions. Atherosclerosis was induced in apoE knockout (apoE^?/?) mice and those with COX‐1^?/? (apoE^?/?‐COX‐1^?/?) by feeding with high fat and cholesterol food. Aortas (abdominal or the whole section) were isolated for functional and/or biochemical analyses. As in non‐atherosclerotic conditions, the muscarinic receptor agonist acetylcholine (ACh) evoked an endothelium‐dependent, COX‐mediated contraction following NO synthase (NOS) inhibition in abdominal aortic rings from atherosclerotic apoE^?/? mice. Interestingly, COX‐1 inhibition not only abolished such a contraction in rings showing normal appearance, but also diminished that in rings with plaques. Accordingly, only a minor contraction (<30% that of apoE^?/? counterparts) was evoked by ACh (following NOS inhibition) in abdominal aortic rings of atherosclerotic apoE^?/?‐COX‐1^?/? mice with plaques, and none was evoked in those showing normal appearance. Also, the contraction evoked by ACh in apoE^?/?‐COX‐1^?/? abdominal aortic rings with plaques was abolished by non‐selective COX inhibition, thromboxane‐prostanoid (TP) receptor antagonism, or endothelial denudation. Moreover, it was noted that ACh evoked a predominant production of the prostacyclin (PGI₂, which mediates abdominal aortic contraction via TP receptors in mice) metabolite 6‐keto‐PGF_1α, which was again sensitive to COX‐1 inhibition or COX‐1^?/?. Therefore, in atherosclerotic mouse abdominal aortas, COX‐1 can still be the major isoform mediating endothelium‐dependent contraction, which probably results largely from PGI₂ synthesis as in non‐atherosclerotic conditions. In contrast, COX‐2 may have only a minor role in such response limited to areas of plaques under the same pathological condition.     

Resistance to Cyp3a induction by polychlorinated biphenyls,including non-dioxin-like PCB153, in gills of killifish (Fundulus heteroclitus) from New Bedford Harbor

Previous reports suggested that non-dioxin-like (NDL) PCB153 effects on cytochrome P450 3A (Cyp3a) expression in Atlantic killifish (Fundulus heteroclitus) gills differed between F0 generation fish from a PCB site (New Bedford Harbor; NBH) and a reference site (Scorton Creek; SC). Here, we examined effects of PCB153, dioxin-like (DL) PCB126, or a mixture of both, on Cyp3a56 mRNA in killifish generations removed from the wild, without environmental PCB exposures. PCB126 effects in liver and gills differed between populations, as expected. Gill Cyp3a56 was not affected by either congener in NBH F2 generation fish, but was induced by PCB153 in SC F1 fish, with females showing a greater response. PCB153 did not affect Cyp3a56 in liver of either population. Results suggest a heritable resistance to NDL-PCBs in killifish from NBH, in addition to that reported for DL PCBs. Induction of Cyp3a56 in gills may be a biomarker of exposure to NDL PCBs in fish populations that are not resistant to PCBs.     

Comparative effects of 1α‐hydroxyvitamin D3 and 1,25‐dihydroxyvitamin D3 on transporters and enzymes in fxr(+/+) and fxr(‐/‐) mice

Previous studies have shown that 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] treatment in mice resulted in induction of intestinal and renal Cyp24a1 and Trpv6 expression, increased hepatic Cyp7a1 expression and activity, as well as higher renal Mdr1/P‐gp expression. The present study compared the equimolar efficacies of 1α‐hydroxyvitamin D₃ [1α(OH)D₃] (6 nmol/kg i.p. q2d × 4), a lipophilic precursor with a longer plasma half‐life that is converted to 1,25(OH)₂D₃, and 1,25(OH)₂D₃ on vitamin D receptor (VDR) target genes. To clarify whether changes in VDR genes was due to VDR and not secondary, farnesoid X receptor (FXR)‐directed effects, namely, lower Cyp7a1 expression in rat liver due to increased bile acid absorption, wildtype [fxr(+/+)] and FXR knockout [fxr(‐/‐)] mice were used to distinguish between VDR and FXR effects. With the exception that hepatic Sult2a1 mRNA was increased equally well by 1α(OH)D₃ and 1,25(OH)₂D₃, 1α(OH)D₃ treatment led to higher increases in hepatic Cyp7a1, renal Cyp24a1, VDR, Mdr1 and Mrp4, and intestinal Cyp24a1 and Trpv6 mRNA expression in both fxr(+/+) and fxr(‐/‐) mice compared to 1,25(OH)₂D₃ treatment. A similar induction in protein expression and microsomal activity of hepatic Cyp7a1 and renal P‐gp and Mrp4 protein expression was noted for both compounds. A higher intestinal induction of Trpv6 was observed, resulting in greater hypercalcemic effect following 1α(OH)D₃ treatment. The higher activity of 1α(OH)D₃ was explained by its rapid conversion to 1,25(OH)₂D₃ in tissue sites, furnishing higher plasma and tissue 1,25(OH)₂D₃ levels compared to following 1,25(OH)₂D₃‐treatment. In conclusion, 1α(OH)D₃ exerts a greater effect on VDR gene induction than equimolar doses of 1,25(OH)₂D₃ in mice. Copyright © 2013 John Wiley & Sons, Ltd.     

Interactive effects of hypoxia and PCB co‐exposure on expression of CYP1A and its potential regulators in Atlantic croaker liver

Although marine and coastal environments which are contaminated with xenobiotic organic compounds often become hypoxic during the summer, the interactive effects of hypoxia and xenobiotic exposure on marine species such as teleost fishes remain poorly understood. The expression and activity of monooxygenase enzyme cytochrome P450‐1A (CYP1A) in fishes are upregulated by exposure to polychlorinated biphenyls (PCBs), whereas they are down‐regulated during hypoxia exposure. We investigated the interactive effects of hypoxia and PCB co‐exposure on hepatic CYP1A expression in Atlantic croaker and on potential regulators of CYP1A. Croaker were exposed to hypoxia (1.7 mg/L dissolved oxygen), 3,3′,4,4′‐tetrachlorobiphenyl (PCB 77, dose: 2 and 8 µg/g body weight), and Aroclor 1254 (a common PCB mixture, dose: 0.5 and 1 µg/g body weight), alone and in combination for 4 weeks. PCB 77 exposure markedly increased hepatic CYP1A mRNA and protein expression, and ethoxyresorufin‐O‐deethylase (EROD, an indicator of CYP1A enzyme) activity and increased endothelial nitric oxide synthase (eNOS) protein expression. PCB 77 treatment also increased interleukin‐1β (IL‐1β, a cytokine) mRNA levels and protein carbonyl (PC, an indicator of reactive oxygen species, ROS) contents. These marked PCB 77‐ and Aroclor 1254‐induced increases in CYP1A mRNA levels and EROD activity were significantly attenuated by co‐exposure to hypoxia, whereas the increases in hepatic eNOS protein and IL‐1β mRNA expression, and PC contents were augmented by hypoxia co‐exposure. The results suggest that biotransformation of organic xenobiotics by CYP1A is reduced in fish during co‐exposure to hypoxia and is accompanied by alterations in eNOS, ROS, and IL‐1β levels.     

Effects of embryonic exposure to polychlorinated biphenyls on zebrafish (Danio rerio) retinal development

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that affect embryonic development. The purpose of this study was to examine the effects of embryonic exposure to PCBs on early retinal development in zebrafish, Danio rerio. Zebrafish embryos were immediately exposed to different concentrations (0, 0.125, 0.25, 0.5, 1.0 and 2.0 mg) of PCBs per liter of medium at 28.5 °C. Embryos were assessed at 30, 48, 72 and 96 h post‐fertilization (hpf) for changes in embryonic survival rate, development, larval retinal morphology and ultrastructure of the retina. The results show that PCB exposure decreased the survival rate of embryos in a time‐ and dose‐dependent manner. Embryos exposed to the higher concentrations of PCBs (0.5, 1.0 and 2.0 mg l^?1) displayed obvious gross morphological deformities. At 72 hpf, the retinal layer development of zebrafish was delayed at higher PCB concentrations (1.0 mg l^?1). At 96 hpf, irregularity of photoreceptor cells arrangement and thickening of photoreceptor and ganglionic layers were observed in PCB‐treated larvae at concentrations of 0.25–1 mg l^?1. Ultrastructural examination showed signs of growth inhibition of the photoreceptor outer segment at 0.25–1 mg l^?1 PCB exposure at 72 hpf, as well as the appearance of massive vacuoles and holes inside the outer segments in the PCB exposure group at 96 hpf. These results suggest that embryonic exposure to moderate and high levels of PCBs induced developmental deficits in zebrafish retinas, particularly in photoreceptor cells. Copyright © 2011 John Wiley & Sons, Ltd.     

The toll like receptor 4‐myeloid differentiation factor 88 pathway is essential for particulate matter‐induced activation of CD4‐positive cells

Asian sand dust (ASD), a type of particulate matter (PM) found in Asia, can be transported to East Asia. We recently found that acute splenic inflammation is induced by ASD in mouse models. In this study, we examined the effect of sub‐chronic ASD exposure on mouse immune cells. Mice were intratracheally administered ASD once every 2 weeks for 8 weeks and killed 24 hours after the final administration. Wild‐type (WT) mice showed increased cell viability after ASD administration. In contrast, ASD administration induced splenocyte activation in toll‐like receptor (TLR)2^?/?, but not TLR4^?/? mice. Furthermore, concanavalin A‐induced interleukin‐2 production increased after ASD administration in WT and TLR2^?/? mice, but not in TLR4^?/? or myeloid differentiation factor (MyD)88^?/? mice. Immunoblotting demonstrated that nuclear factor κB (NF‐κB) was activated in WT mice, but not in TLR4^?/? or MyD88^?/? mice. The NF‐κB‐dependent gene products CDK2 and intercellular cell adhesion molecule‐1 were upregulated upon ASD administration in WT mice, but not in TLR4^?/? or MyD88^?/? mice. Furthermore, the particles themselves, rather than particle constituents, activated NF‐κB in CD4‐positive cells through the TLR4 or MyD88 pathway. Taken together, these results indicate that particle‐induced splenic inflammation occurs via TLR4‐MyD88 signaling.     

PCB Concentrations and Dioxin‐like Activity in Blood Samples from Danish School Children and Their Mothers living in Urban and Rural Areas

Human exposure to persistent organic pollutants (POPs) is of major concern due to a diversity of adverse effects from prolonged exposure and bioaccumulation. Manufacturing of polychlorinated biphenyls (PCBs), a subgroup of POPs, has been prohibited for many decades; however, human exposure still occurs due to the persistent nature of the chemicals. The concentrations of the dioxin‐like PCB congeners 105, 118 and 156 and the non‐dioxin‐like PCB congeners 28, 52, 101, 138, 153 and 180, p,p′‐DDE, p,p′‐DDT, o,p′‐DDE, o,p′‐DDT, HCB and β‐HCH as well as the dioxin‐like activity using the AhR transactivity assay were analysed in blood samples from Danish schoolchildren and their mothers in the European framework of the DEMOCOPHES/COPHES projects. The participants were selected from an urban and a rural area, respectively. The PCB concentrations and the AhR‐TEQ (TCDD toxic equivalent) were significantly higher in schoolchildren living in the urban area compared with the rural, and for AhR‐TEQ, a strong correlation between the mothers and children was observed. We found a significant negative correlation between BMI and PCB concentrations in the children. Finally, in the mothers, there was a positive association between age and PCB concentration. These results show that both PCBs and dioxin‐like activity can be measured as biomarkers of exposure and effects in blood samples from children and women. The results indicate that people living in urban areas may be exposed to higher concentrations of PCBs, dioxins and dioxin‐like chemicals, which may lead to a greater risk of adverse effects for urban populations.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin treatment alters eicosanoid levels in several organs of the mouse in an aryl hydrocarbon receptor-dependent fashion

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) adversely affects many mammalian organs and tissues. These effects are mediated by the aryl hydrocarbon receptor (AHR). CYP1A1, CYP1A2 and CYP1B1 are upregulated by the liganded AHR. These (and other) cytochromes P450 can metabolize arachidonic acid into a variety of bioactive eicosanoids. Towards investigating a potential role of eicosanoids in TCDD toxicity, arachidonic acid, two other unsaturated long-chain fatty acids, and up to twenty-five eicosanoids were measured in five organs/tissues of male and female wild-type and Ahr null mice treated or untreated with TCDD. TCDD generally increased the levels of the four dihydroxyeicosatrienoic acids (DHETs) and (where measured) 5,6-epoxyeicosatrienoic acid and 18-, 19- and 20-hydroxyeicosatrienoic acids (HETEs) in the serum, liver, spleen and lungs, but not the heart, of both sexes, and increased the levels in the serum, liver and spleen of several metabolites that are usually considered products of lipoxygenase activity, but which may also be generated by cytochromes P450. TCDD also increased the levels of the esterified forms of these eicosanoids in the liver in parallel with the corresponding free forms. The levels of prostanoids were generally not affected by TCDD. The above changes did not occur in Ahr null mice, and are therefore mediated by the AHR. TCDD increased the mRNA levels of Cyp1a1, Cyp1a2, Cyp1b1 and the Pla2g12a form of phospholipase A₂ to varying degrees in the different organs, and these increases correlated with some but not all the changes in eicosanoids levels in the organs, suggesting that other enzymes may also be involved.     

Effects of structurally different noncoplanar and coplanar PCBs on HELF cell proliferation,cell cycle,and potential molecular mechanisms

Polychlorinated biphenyls (PCBs) are a group of chemicals that persist in the environment, indoors, and humans. Lung exposure to airborne and food contaminants, such as PCBs, may cause possible lung disorders, such as cancer. In the present study, we investigated the effects of structurally different lower chlorinated (≤4Cl), noncoplanar PCB40, and coplanar PCB77 on human lung fibroblast cell line (HELF) cell proliferation, cell cycle progression, and possible molecular mechanisms. Noncoplanar PCB40 and coplanar PCB77 exhibited concentration‐ and time‐dependent biphasic dose–response effects on HELF cell proliferation. Noncoplanar PCB40 and coplanar PCB77 induced 23 and 45% cytotoxicity at higher concentrations than the control. The flow cytometry analysis showed that exposure to PCB40 caused a significant increase in time spent in the G1 phase but decreased length of the S phase in a concentration‐ and time‐dependent manner, whereas PCB77 exposure decreased time spent in the G1 and S phases but increased time spent in the G2 phase. Western blot analysis indicated that PCB77 increased the expression of cyclin E, CDK2, p21, and caspase‐9, while PCB40 decreased the expression of these proteins (except CDK2 and p21). An increase in CDK expression after exposure to PCB77 suggests that it may cause carcinogenic effects on HELF cells at higher doses. Our results also demonstrate that the different cytotoxic effects induced by coplanar and nonplanar PCBs were correlated with their structural characteristics; the coplanar congener was more cytotoxic than the nonplanar congener. The study elaborates threshold levels for these chemicals and suggests that the cytotoxicity mechanisms by which PCB congeners act on HELF cells depend on their planarity and chemical structures. Furthermore, the study will be important for developing antidotes to the adverse effects and risk assessment practices for PCBs. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1183–1190, 2017.     

Synthesis and evaluation of a 18F‐labeled 4‐phenylpiperidine‐4‐carbonitrile radioligand for σ1 receptor imaging

We report the design and synthesis of several 4‐phenylpiperidine‐4‐carbonitrile derivatives as σ₁ receptor ligands. In vitro radioligand competition binding assays showed that all the ligands exhibited low nanomolar affinity for σ₁ receptors (K_i(σ₁) = 1.22–2.14 nM) and extremely high subtype selectivity (K_i(σ₂) = 830–1710 nM; K_i(σ₂)/K_i(σ₁) = 680–887). [¹⁸F]9 was prepared in 42–46% isolated radiochemical yield, with a radiochemical purity of >99% by HPLC analysis after purification, via nucleophilic ¹⁸F^‐ substitution of the corresponding tosylate precursor. Biodistribution studies in mice demonstrated high initial brain uptakes and high brain‐to‐blood ratios. Administration of SA4503 or haloperidol 5 min prior to injection of [¹⁸F]9 significantly reduced the accumulation of radiotracers in organs known to contain σ₁ receptors. Two radioactive metabolites were observed in the brain at 30 min after radiotracer injection. [¹⁸F]9 may serve as a lead compound to develop suitable radiotracers for σ₁ receptor imaging with positron emission tomography.     

Toxaphene‐induced mouse liver tumorigenesis is mediated by the constitutive androstane receptor

Toxaphene was shown to increase liver tumor incidence in B6C3F1 mice following chronic dietary exposure. Preliminary evidence supported a role for the constitutive androstane receptor (CAR) in the mode of action of toxaphene‐induced mouse liver tumors. However, these results could not rule out a role for the pregnane X receptor (PXR) in liver tumor formation. To define further the nuclear receptors involved in this study, we utilized CAR, PXR and PXR/CAR knockout mice (CAR^−/−, PXR^−/− and PXR^−/−/CAR^−/−) along with the wild‐type C57BL/6. In this study CAR‐responsive genes Cyp3a11 and Cyp2b10 were induced in the liver of C57BL/6 (wild‐type) mice by toxaphene (30–570‐fold) (at the carcinogenic dose 320 ppm) and phenobarbital (positive control) (16–420‐fold) following 14 days' dietary treatment. In contrast, in CAR^−/− mice, no induction of these genes was seen following treatment with either chemical. Cyp3a11 and Cyp2b10 were also induced in PXR^−/− mice with toxaphene and phenobarbital but were not changed in treated PXR^−/−/CAR^−/− mice. Similarly, induction of liver pentoxyresorufin‐O‐deethylase (CAR activation) activity by toxaphene and phenobarbital was absent in CAR^−/− and PXR^−/−/CAR^−/− mice treated with phenobarbital or toxaphene. Ethoxyresorufin‐O‐deethylase (EROD, represents aryl hydrocarbon receptor activation) activity in CAR^−/− mice treated with toxaphene or phenobarbital was increased compared with untreated control, but lower overall in activity in comparison to the wild‐type mouse. Liver EROD activity was also induced by both phenobarbital and toxaphene in the PXR^−/− mice but not in the PXR^−/−/CAR^−/− mice. Toxaphene treatment increased 7‐benzyloxyquinoline activity (a marker for PXR activation) in a similar pattern to that seen with pentoxyresorufin‐O‐deethylase. These observations indicate that EROD and PXR activation are evidence, as expected, of secondary overlap to primary CAR receptor activation. Together, these results definitively show that activation of the CAR nuclear receptor is the mode of action of toxaphene‐induced mouse liver tumors. Copyright © 2017 John Wiley & Sons, Ltd.