Responsiveness of hepatic and cerebral cytochrome P450 in rat offspring prenatally and lactationally exposed to a reconstituted PCB mixture

Perinatal polychlorinated biphenyl (PCB) exposures still remain a serious health concern because offspring receive PCB burden from mother during vulnerable processes of development. Since cytochrome P450 (CYP) represents a toxicological endpoint, in the present study, representing an extended investigation of a previous multitasked one, we explored the long‐term responsiveness of CYP1A and CYP2B isoforms by Western blot analysis in liver and whole brain of lactating (PN12), weaning (PN21), and adult offspring (PN60) rats prenatally and lactationally exposed to a reconstituted PCB mixture (RM) of noncoplanar PCB138, 153, 180, and coplanar PCB126 congeners. We chose highly chlorinated PCBs instead of lower chlorinated one, because their recalcitrance to biotransformation makes easy their accumulation/persistence in tissues and breast milk. Dioxin‐like congener PCB126 binding aryl hydrocarbon receptor (AHR) is responsible of many toxic effects. Pregnant Sprague–Dawley dams with high affinity AHR received subcutaneous injection of RM (10 mg/kg body weight) daily during gestation (days 15–19) and twice a week during breast‐feeding. The results evidenced a transfer of PCBs to neonates through milk and a significant responsiveness of hepatic CYP in both mothers and offspring. In liver of exposed progeny, CYP isoforms exhibited a significant increment at PN12 (70% over control) and at PN21 (270% over control). Contrary to dams, in adult PCB offspring CYP levels showed a decline up to values similar to those of control. This transient developmental responsiveness of CYP isoforms in offspring liver reflects roughly the time course of hepatic PCB levels previously reported. Even if congeners were detected in brain, we failed in evidencing a responsiveness of CYP isoforms probably because of region‐specific CYP expression in this organ. In conclusion, induction of offspring hepatic CYP is index of liver PCB burden, and despite the insensitivity of whole brain CYP we cannot exclude brain vulnerability toward PCB. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 856–866, 2014.     

Learning and memory in rats gestationally and lactationally exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Recently we reported that in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or coplanar polychlorinated biphenyls (PCBs) resulted in a reduction of errors on a radial arm maze (RAM) working memory task. The effect was more pronounced in males than in females. In this study, we further investigated the effects of in utero and lactational exposure to TCDD on learning and memory by testing male and female TCDD-exposed rats on three different spatial learning and memory tasks: the RAM, the Morris water maze (MWM), and spatial discrimination-reversal learning (RL), as well as on a nonspatial learning task, visual RL. Time-mated Sprague-Dawley rats were gavaged with either TCDD (0.1 microg/kg/day) or corn oil vehicle on gestation days 10-16. Litters were culled to eight on day 2 and weaned on day 21. Beginning on day 80, one male and one female from each litter were tested on the same RAM working memory task used in the previous study. Again, the TCDD-exposed male rats displayed a pronounced decrease in errors relative to control males. Following the RAM testing, the same animals were tested on the MWM, but no differences between the exposed and control rats were observed. Another male and female from each litter were tested on spatial RL on a T-maze. There were no differences between the exposed and control rats on this task. Following spatial RL, the same rats were tested on visual RL on the same maze. The exposed animals did not differ from controls on original learning, but took more trials to reach criterion on the first and second reversals. These results demonstrate a reliable, but task-specific, facilitation of spatial learning and memory in male rats exposed to TCDD during gestation and lactation. In contrast, both male and female TCDD-exposed rats showed a deficit in learning on the visual RL task. This pattern is consistent with that seen in earlier monkey studies. Perinatally TCDD-exposed monkeys were facilitated on certain spatial tasks, but impaired on visual RL tasks.     

Distribution of cytochrome P450 1A1 and NADPH-cytochrome P450 reductase in lungs of rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin: ultrastructural immunolocalization and in situ hybridization.

Induction of cytochrome P450 1A1 (P450 1A1) in a variety of tissues is a well established consequence of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although localization of the induced protein within the lung has been described, the precise intracellular distribution of the enzyme is not clear. Analysis of tissue sections, microsomal proteins, and mRNA from lungs of treated and untreated rabbits established that P450 1A1 had been induced by treatment with TCDD. Rabbit lungs from animals treated with TCDD were examined with immunocytochemistry and in situ hybridization, to identify the cell types that contain P450 1A1 and those that contain mRNA encoding P450 1A1. Endothelial cells of the entire vascular bed of rabbit lung reacted markedly with anti-P450 1A1. Likewise, cells lining both arteries and veins, as well as capillary endothelial cells, reacted strongly with the cDNA probe for mRNA encoding P450 1A1. Clara cells at all levels of airway labeled prominently for both P-450 1A1 and P450 1A1 mRNA. In addition, type 2 cells, alveolar macrophages, and to a lesser degree, ciliated cells reacted with the cDNA probe. P450 reductase, which is required for P450 activity, has previously been identified in Clara cells, type 2 cells, and alveolar macrophages, but not in endothelium of rabbit lung. We have now obtained similar results for the localization of mRNA encoding P-450 reductase. This finding brings into question the function of P450 1A1 in endothelium.     

Diminished thymosin alpha-1 levels in persons exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

There is evidence from animal studies that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) impairs immune responses, with the thymus being a principal target organ. The purpose of this study was to evaluate thymic function, through measurement of thymic hormone levels, in persons exposed to TCDD. We examined thymosin alpha-1 (Thya-1) levels in sera from a group of 94 persons who were presumed to be exposed to TCDD from living, working, or recreating in a contaminated residential area. We compared these results, along with results from in vitro and in vivo tests of immune function, with those from a group of 105 unexposed persons who were similar with regard to age, sex, and race. The exposed group had a significantly lower mean Thya-1 serum level (977.3 +/- 304.1 pg/ml vs. 1148.7 +/- 482.1 pg/ml, p less than .01 by t-test). We also found a statistically significant trend of decreasing Thya-1 levels with increasing number of years of residence in the TCDD-contaminated area. However, Thya-1 levels were not associated with other measures of immune function in the TCDD-exposed group. Thus, while the principal findings suggest that long-term TCDD exposure may be associated with diminished secretion of Thya-1, the lack of an association with an increased prevalence of clinically diagnosed immune suppression in these TCDD-exposed persons makes the biologic significance of the findings unclear. Further studies are needed to more fully evaluate possible long-term TCDD-induced effects on the thymus and human immune function.     

Sex ratio of the offspring of Sprague-Dawley rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in utero and lactationally in a three-generation study

Reports of a decreased male/female sex ratio in children born to males exposed to TCDD in Seveso, Italy, at a young age have sparked examinations of this endpoint in other populations exposed to TCDD or related compounds. Overall, the male/female sex ratio results reported in these studies, with slightly different age-exposed male populations, have shown mixed results. Experimental studies of the effects of in utero exposure to TCDD in laboratory animals have reported no effect on the f(1) sex ratio and mixed results for the sex ratio of the f(2) generation. In order to better understand the potential effects of TCDD on second generation sex ratio, we retrieved archived data from a comprehensive three-generation feeding study of TCDD in rats that was conducted and published in the 1970s, but which did not publish data on sex ratio of the offspring [Murray, F.J., Smith, F.A., Nitschke, K.D., Humiston, C.G., Kociba, R.J., Schwetz, B.A., 1979. Three-generation reproduction study of rats given 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the diet. Toxicol. Appl. Pharmacol. 50, 241-252]. A re-examination of the original Murray et al. data found no statistically significant treatment-related changes in postnatal day 1 sex ratio in any generation of treated animals, consistent with one other relatively large study reporting on this endpoint. We discuss mechanistic data underlying a potential effect of TCDD on this endpoint. We conclude that the inconsistency in findings on sex ratio of the offspring of male rats exposed to TCDD in utero is likely due to random variation associated with a relatively small sample size, although differences between studies in strain of rat, dose regimen, and day of ascertainment of sex ratio cannot be ruled out.     

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the expression of cytochrome P450 1A1, the aryl hydrocarbon receptor, and the aryl hydrocarbon receptor nuclear translocator in rat brain and pituitary

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related substances are ubiquitous environmental pollutants causing a wide variety of pathological alterations, with the most severe being progressive anorexia and body weight loss. These features suggest a possible involvement of the nervous system and neuroendocrine-related organs including the pituitary gland. However, so far there is little evidence for direct effects of TCDD on these areas. In the present study, male Sprague-Dawley rats were treated with a single oral dose of TCDD (10 microg/kg) and euthanized 1, 3, or 28 days after treatment. The expression of cytochrome P450 1A1 (CYP1A1), the aryl hydrocarbon receptor (AHR), and the aryl hydrocarbon receptor nuclear translocator (ARNT) were analyzed in different brain regions and pituitaries using semiquantitative RT-PCR and Western blotting. Relative levels of CYP1A1 mRNA and protein were dramatically increased in the pituitary. A significant increase in CYP1A1 mRNA was also detected in all the brain regions examined including olfactory bulb, striatum-caudate, hypothalamus, hippocampus, cortex, cerebellum, and substantia nigra. The increase in the expression was time-dependent with the highest level observed 1 day after TCDD treatment. The AHR and ARNT mRNAs were detected in the same areas but in contrast to CYP1A1 the changes in AHR and ARNT mRNA expression were limited to the 28-day time point. The present results provide evidence for the presence of CYP1A1, AHR, and ARNT in the central nervous system and in the pituitary, suggesting that TCDD may exert a direct effect on these regions.     

Cross-species comparisons of transcriptomic alterations in human and rat primary hepatocytes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

A toxicogenomics approach was used to qualitatively and quantitatively compare the gene expression changes in human and rat primary hepatocytes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Hepatocytes from five individual rats and five individual humans were exposed for 24 h to 11 concentrations of TCDD ranging from 0.00001 to 100nM and a vehicle control. Gene expression changes were analyzed using whole-genome microarrays containing 13,002 orthologs. Significant changes in expression of individual orthologs at any concentration (fold change [FC] ± 1.5 and false discovery rate < 0.05) were higher in the rat (1547) compared with human hepatocytes (475). Only 158 differentially expressed orthologs were common between rats and humans. Enrichment analysis was performed on the differentially expressed orthologs in each species with 49 and 34 enriched human and rat pathways, respectively. Only 12 enriched pathways were shared between the two species. The results demonstrate significant cross-species differences in expression at both the gene and pathway level. Benchmark dose analysis of gene expression changes showed an average 18-fold cross-species difference in potency among differentially expressed orthologs with the rat more sensitive than the human. Similar cross-species differences in potency were observed for signaling pathways. Using the maximum FC in gene expression as a measure of efficacy, the human hepatocytes showed on average a 20% lower efficacy among the individual orthologs showing differential expression. The results provide evidence for divergent cross-species gene expression changes in response to TCDD and are consistent with epidemiological and clinical evidence showing humans to be less sensitive to TCDD-induced hepatotoxicity.     

Localization of rat cytochrome P450 in various tissues and comparison of arachidonic acid metabolism by rat P450 with that by human P450 orthologs

Metabolites of arachidonic acid produced by P450 are interesting substances with prominent physiological functions. To elucidate the physiological function of P450, it is necessary to identify a specific P450 in a particular tissue or organ and to characterize its catalytic activities. In this study, the expression of CYP2A1, 2B1, 2C23, 2J3, and 4F1 was investigated in liver, lung, kidney, spleen, heart, brain, and testis of rats by RT-PCR. Furthermore, arachidonic acid metabolism was investigated using the rat P450s described above and human CYP2A6, 2B6, 2C9, 2C18, 2C19, 2J2, and 4F2. Among the rat P450s, CYP2B1 and 2C23 efficiently produced EETs and CYP4F1 produced 19/20-HETE in abundace. CYP2B1 was specifically expressed in the lung. CYP2C23 was detected in all tissues used in this study. CYP4F1 was expressed in the kidney as well as in the liver. Among the human P450s, CYP2C9 and 2C19 efficiently produced EETs. CYP4F2 produced 19/20-HETE. The catalytic properties of rat CYP2C23 were similar to those of human CYP2C9 and 2C19. The catalytic properties of CYP4F isoforms were also similar between humans and rats. A systematic analysis of P450 expression in various tissues and of its catalytic property may provide valuable information on the physiological roles of P450s in each tissue.     

Responsiveness of cerebral and hepatic cytochrome P450s in rat offspring prenatally exposed to lindane

Prenatal exposure to low doses of lindane has been shown to affect the ontogeny of xenobiotic metabolizing cytochrome P450s (CYPs), involved in the metabolism and neurobehavioral toxicity of lindane. Attempts were made in the present study to investigate the responsiveness of CYPs in offspring prenatally exposed to lindane (0.25 mg/kg b. wt.; 1/350th of LD(50); p. o. to mother) when challenged with 3-methylcholanthrene (MC) or phenobarbital (PB), inducers of CYP1A and 2B families or a sub-convulsant dose of lindane (30 mg/kg b. wt., p. o.) later in life. Prenatal exposure to lindane was found to produce an increase in the mRNA and protein expression of CYP1A1, 1A2, 2B1, 2B2 isoforms in brain and liver of the offspring at postnatal day 50. The increased expression of the CYPs in the offspring suggests the sensitivity of the CYPs during postnatal development, possibly, to low levels of lindane, which may partition into mother's milk. A higher increase in expression of CYP1A and 2B isoenzymes and their catalytic activity was observed in animals pretreated prenatally with lindane and challenged with MC (30 mg/kg, i. p. x 5 days) or PB (80 mg/kg, i. p. x 5 days) when young at age (approx. 7 weeks) compared to animals exposed to MC or PB alone. Further, challenge of the control and prenatally exposed offspring with a single sub-convulsant dose of lindane resulted in an earlier onset and increased incidence of convulsions in the offspring prenatally exposed to lindane have demonstrated sensitivity of the CYPs in the prenatally exposed offspring. Our data assume significance as the subtle changes in the expression profiles of hepatic and cerebral CYPs in rat offspring during postnatal development could modify the adult response to a later exposure to xenobiotics.     

Antiteratogenic effect of resveratrol in mice exposed in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin

The effect of resveratrol, an aryl hydrocarbon receptor antagonist, on the teratogenicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated. Pregnant C57BL/6J mice were orally administered resveratrol (50 mg/kg) for 6 consecutive days, from gestational day (GD) 8 to GD13, followed by an oral challenge with TCDD (14 mug/kg) on GD12. TCDD caused severe fetal malformations including cleft palate (40.7%), renal pelvic dilatation (100%, mean score 3.060), and ureteric dilatation (100%, mean score 3.210) and tortuosity (95.1%). Resveratrol significantly reduced both the incidence of TCDD-induced cleft palate to 18.4% and the degrees of renal pelvic and ureteric dilatations caused by TCDD. The results suggest that pretreatment with resveratrol might bring a beneficial outcome for reducing the incidence and severity of fetal malformations caused by TCDD exposure in utero.     

Interactions of indoles with specific binding sites for 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver

In order to identify some of the structural requirements for binding of indoles to the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we have investigated the capacity of various indoles to inhibit specific [1,6-3H]TCDD binding in rat liver cytosol, as analyzed by electrofocusing in polyacrylamide gel. Of these indoles, indolo[3,2-b]carbazole was the most active. The IC50 value for receptor binding of indolo[3,2-b]carbazole as well as for 2,3,7,8-tetrachlorodibenzofuran was 3.6 nM, whereas that of 5,6-benzoflavone was 26 nM. Both indolo[3,2-b]carbazole and 2,3,7,8-tetrachlorodibenzofuran competitively inhibited the binding of [3H]TCDD to the receptor. The well-known microsomal enzyme inducer 3,3'-diindolymethane did not interact significantly with the TCDD receptor. Previous concepts of structure-activity relationships for binding of chlorinated dioxins to the TCDD receptor fail to account for the receptor binding of unhalogenated aryl hydrocarbon hydroxylase inducers such as 5,6-benzoflavone. We have instead considered the true three-dimensional space occupied by some receptor ligands by means of a computer using crystallographic data as inputs. When the atomic van der Waals radii were included, all potent receptor ligands studied could be fitted into a rectangle of 6.8 X 13.7 A.     

Organ specific induction of drug metabolizing enzymes by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied on the activities of arylhydrocarbon hydroxylase, ethoxycoumarin deethylase, cytochrome c reductase, epoxide hydratase, UDP glucuronosyltransferase, and glutathione S-transferase in the liver, kidney, lung, small intestinal mucosa, and testis of male Wistar rats. There was a severalfold increase in the activity of monooxygenase in the liver, kidney, and lung, whereas virtually no effect could be detected in the intestine or testes. The proportion of 3- and 9-hydroxylation of the total hydroxylation of benzo(a)pyrene decreased in the liver, but increased in the kidney. TCDD had no significant effect on epoxide hydratase or glutathione S-transferase activities in any tissues. UDP glucuronosyltransferase exhibited a sevenfold increase in the liver, less than twofold in the kidney, and none in other tissues. Treatment of the microsomes with digitonin, trypsin, or phospholipase A did not reveal additional induction UDP glucurnosyltransferase, although all were able to increase measurable enzymatic activity in control and TCDD-treated animals. TCDD seems to be different from phenobarbital and polycyclic hydrocarbons as an effector of not only monooxygenase but also epoxide hydratase, UDP glucuronosyltransferase, and glutathione S-transferase.     

Sensitivity of bald eagle (Haliaeetus leucocephalus) hepatocyte cultures to induction of cytochrome P4501A by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Graded doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were added to primary hepatocyte cultures of bald eagle (Haliaeetus leucocephalus) embryos to determine their sensitivity to induction of cytochrome P4501A (CYP1A) and porphyrin accumulation. No porphyrin accumulation was observed, but both CYP1A catalytic activity (using the ethoxyresorufin-O-deethylase (EROD) assay) and immunodetectable CYP1A were induced by relatively high concentrations of TCDD. Bald eagle hepatocytes were less sensitive to CYP1A induction than hepatocytes from any other avian species that we have studied to date. These in vitro results are in general agreement with recent assessments of field data, which indicate that bald eagles are relatively insensitive to some of the effects of TCDD and related compounds. Preparation of bald eagle hepatocytes was challenging because existing methods did not yield monolayers of cells. Here we describe details of a new method that was successful for bald eagle hepatocytes. This new method is used routinely in our laboratory to prepare hepatocyte cultures from birds for examination of various biochemical responses to environmental contaminants.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits steroidogenesis in the rat testis by inhibiting the mobilization of cholesterol to cytochrome P450scc.

Testosterone synthesis in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats is decreased because pregnenolone production by the testis is inhibited. This inhibition can only be caused by a reduction in the activity of the mitochondrial enzyme which converts cholesterol into pregnenolone (cytochrome P450scc), and/or by an impairment in the multistep process by which luteinizing hormone (LH) stimulates the mobilization of cholesterol to this enzyme. Seven days after rats were treated with 100 micrograms TCDD/kg, testicular cytochrome P450scc activity (assayed with 20 alpha-hydroxycholesterol as substrate) was decreased to 45% of control. If this decrease were responsible for the inhibition of testicular steroidogenesis in vivo, substrate pools for cytochrome P450scc in the testis would be increased. Yet TCDD decreased the amount of cholesterol that was readily available to cytochrome P450scc in isolated testis mitochondria (the reactive cholesterol pool), even when steroidogenesis was maximally stimulated in vivo with the LH analogue human chorionic gonadotropin (hCG). These decreases in substrate pools were not due to a reduction in mitochondrial capacity for reactive cholesterol. We conclude that the 55% decrease in cytochrome P450scc activity is not severe enough to inhibit testicular steroidogenesis in vivo. Instead, TCDD must act by inhibiting the LH-stimulated mobilization of cholesterol to cytochrome P450scc. This conclusion is supported by two observations. First, when pregnenolone formation was blocked by treating rats with the cytochrome P450scc inhibitor aminoglutethimide, TCDD greatly reduced the rate at which hCG caused reactive cholesterol to accumulate in testis mitochondria in vivo. Second, TCDD inhibited both testosterone synthesis and the mobilization of cholesterol to cytochrome P450scc within 1 day. The steroidogenic inhibition does not appear to be due to an LH receptor defect, because TCDD inhibited dibutyryl cAMP- and hCG-stimulated steroid secretion by isolated perfused testes to comparable extents. We conclude that TCDD inhibits testicular steroidogenesis predominantly if not exclusively by inhibiting the mobilization of cholesterol to cytochrome P450scc, and that this inhibition occurs subsequent to cAMP formation.