Gestational and lactational exposure to the polychlorinated biphenyl mixture Aroclor 1254 modulates retinoid homeostasis in rat offspring.

Polychlorinated biphenyls (PCBs) induce a broad spectrum of biochemical and toxic effects in mammals including alterations of the vital retinoid (vitamin A) system. The aim of this study was to characterize alterations of tissue retinoid levels in rat offspring and their dams following gestational and lactational exposure to the PCB mixture Aroclor 1254 (A1254) and to assess the interrelationship of these changes with other established sensitive biochemical and toxicological endpoints. Sprague-Dawley rat dams were exposed orally to 0 or 15 mg/kg body weight/day of A1254 from gestational day 1 to postnatal day (PND) 23. Livers, kidneys and serum were collected from the offspring on PNDs 35, 77 and 350. Tissue and serum retinoid levels, hepatic cytochrome P450 (CYP) enzymes and serum thyroid hormones were analyzed. A multivariate regression between A1254 treatment, hepatic retinoid levels, hepatic CYP enzymes activities, thyroid hormone levels and body/liver weights was performed using an orthogonal partial least-squares (PLS) analysis. The contribution of dioxin-like (DL) components of A1254 to the observed effects was also estimated using the toxic equivalency (TEQ) concept. In both male and female offspring short-term alterations in tissue retinoid levels occurred at PND35, i.e. decreased levels of hepatic retinol and retinoic acid (RA) metabolite 9-cis-4-oxo-13,14-dihydro-RA with concurrent increases in hepatic and renal all-trans-RA levels. Long-term changes consisted of decreased hepatic retinyl palmitate and increased renal retinol levels that were apparent until PND350. Retinoid system alterations were associated with altered CYP enzyme activities and serum thyroid hormone levels as well as body and liver weights in both offspring and dams. The estimated DL activity was within an order of magnitude of the theoretical TEQ for different endpoints, indicating significant involvement of DL congeners in the observed effects. This study shows that tissue retinoid levels are affected both short- and long-term by developmental A1254 exposure and are associated with alterations of other established endpoints of toxicological concern.

In utero and lactational exposure to a mixture of environmental contaminants detected in Canadian Arctic human populations alters retinoid levels in rat offspring with low margins of exposure.

Arctic inhabitants are highly exposed to persistent organic pollutants (POP), which may produce adverse health effects. This study characterized alterations in tissue retinoid (vitamin A) levels in rat offspring and their dams following in utero and lactational exposure to the Northern Contaminant Mixture (NCM), a mixture of 27 contaminants including polychlorinated biphenyls (PCB), organochlorine (OC) pesticides, and methylmercury (MeHg), present in maternal blood of the Canadian Arctic Inuit population. Further, effect levels for retinoid system alterations and other endpoints were compared to the Arctic Inuit population exposure and their interrelationships were assessed. Sprague-Dawley rat dams were dosed with NCM from gestational day 1 to postnatal day (PND) 23. Livers, kidneys and serum were obtained from offspring on PND35, PND77, and PND350 and their dams on PND30 for analysis of tissue retinoid levels, hepatic cytochrome P-450 (CYP) enzymes, and serum thyroid hormones. Benchmark doses were established for all endpoints, and a partial least-squares regression analysis was performed for NCM treatment, hepatic retinoid levels, CYP enzyme induction, and thyroid hormone levels, as well as body and liver weights. Hepatic retinoid levels were sensitive endpoints, with the most pronounced effects at PND35 though still apparent at PND350. The effects on tissue retinoid levels and changes in CYP enzyme activities, body and liver weights, and thyroid hormone levels were associated and likely driven by dioxin-like compounds in the mixture. Low margins of exposure were observed for all retinoid endpoints at PND35. These findings are important for health risk assessment of Canadian Arctic populations and further support the use of retinoid system analyses in testing of endocrine-system-modulating compounds.

Effects of environmentally relevant mixtures of persistent organic pollutants on the developmental neurobiology in rats.

We report the developmental neuropathology for rat pups at postnatal day (PND) 37 and PND 77 and the molecular biomarkers for PND 35, 75, and 350 after perinatal exposure to a reconstituted mixture of persistent organochlorine pollutants (POPs) based on the blood profiles of people living in the Great Lake Basin. The developmental neuropathology included routine histopathology evaluation, quantification of cell proliferation and death in the subventricular zone, linear morphometric measurements, and transcriptional analysis. No histopathological, structural, or stereological changes were observed in animals treated with the POPs or Aroclor 1254, on PND 37 or PND 77. While no transcriptional changes were found in Arcolor-treated animals, significant transcriptional changes were observed on PND 350 in female offspring perinatally exposed to 0.13 mg/kg of the POP mixture. Markers of the cholinergic system including acetylcholinesterase and the muscarinic receptors (subtypes M1-M5) were downregulated 2- to 6-fold. In addition, structural genes including neurofilaments (NFLs) and microtubule-associated protein (MAP-2) were downregulated at least 2-fold or greater. Our results support that in utero and lactational exposure to the chemical mixture of POPs lead to developmental changes in adult rat brains.

Perinatal exposure to environmental contaminants detected in Canadian Arctic human populations changes bone geometry and biomechanical properties in rat offspring.

Arctic inhabitants consume large proportions of fish and marine mammals, and are therefore continuously exposed to levels of environmental toxicants, which may produce adverse health effects. Fetuses and newborns are the most vulnerable groups. The aim of this study was to evaluate changes in bone geometry, mineral density, and biomechanical properties during development following perinatal exposure to a mixture of environmental contaminants corresponding to maternal blood levels in Canadian Arctic human populations. Sprague-Dawley rat dams were dosed with a Northern Contaminant Mixture (NCM) from gestational day 1 to postnatal day (PND) 23. NCM contains 27 contaminants comprising polychlorinated biphenyls, organochlorine pesticides, and methylmercury. Femurs were collected on PND 35, 77 and 350, and diaphysis was analyzed by peripheral quantitative computed tomography and three-point bending test, while femoral neck was assessed in an axial loading experiment. Dose-response modeling was performed to establish the benchmark dose (BMD) for the analyzed bone parameters. Exposure to the high dose of NMC resulted in short and thin femur with reduced mechanical strength in offspring at PND35. BMD of femur length, cortical area, and stiffness were 3.2, 1.6, and 0.8 mg/kg bw/d, respectively. At PND77 femur was still thin, but at PND350 no treatment-related bone differences were detected. This study provides new insights on environmental contaminants present in the maternal blood of Canadian Arctic populations, showing that perinatal exposure induces bone alterations in the young offspring. These findings could be significant from a health risk assessment point of view.

In utero and lactational exposure to Aroclor 1254 affects bone geometry, mineral density and biomechanical properties of rat offspring.

Exposure to polychlorinated biphenyls (PCBs) induce a broad spectrum of toxic effects in various organs including bone. The most susceptible age-groups to the toxic effects of PCBs are foetuses and infants. The aim of the present study was to quantitatively evaluate changes in bone geometry, mineral density and biomechanical properties following perinatal exposure to the PCB mixture, Aroclor 1254 (A1254), and to examine the persistence of observed bone alterations by following the offspring over time. Sprague-Dawley rat offspring were exposed to A1254 from gestational day 1 to post-natal day (PND) 23. Femur and tibia were collected on PNDs 35, 77 and 350 and were analyzed by peripheral quantitative computed tomography and biomechanical testing. At PND35, exposure to A1254 induced short, thin femur and tibia, with reduced mechanical strength of femoral neck. No treatment-related bone changes were detected in offspring at PND77 or PND350. In conclusion, the present investigation suggests that perinatal exposure to A1254 leads to shorter, thinner and weaker bones in juvenile rats at PND35, with these effects being absent at later time-points as exposure is discontinued. The results indicate that the observed bone effects are mainly driven by the dioxin-like congeners, although it cannot exclude the contribution of the non dioxin-like congeners to the exposure outcome.

Effects of anesthetics and terminal procedures on biochemical and hormonal measurements in polychlorinated biphenyl treated rats.

This investigation reports the effects of various terminal procedures, and how they modified the responses to a toxicant (polychlorinated biphenyls [A1254], 130 mg/kg/day × 5 days) administered by gavage to Sprague-Dawley male rats. Terminal procedures included exsanguination via the abdominal aorta under anesthesia (isoflurane inhalation or Equithesin injection), decapitation with or without anesthesia, or narcosis induced by carbon dioxide inhalation. Effects of repeated anesthesia were also tested. Terminal procedures induced confounding stress responses, particularly when Equithesin was used. The terminal procedures modified the conclusions about effects of A1254 on the concentrations of corticosterone, insulin, glucagon, glucose, alkaline phosphatase, lactate dehydrogenase, uric acid, and blood urea nitrogen, from nonstatistically significant to significant changes, and in the case of luteinizing hormone from a statistically significant increase to a significant decrease. Investigations of effects of toxicants should be designed and interpreted considering potential changes induced by the selection of a terminal procedure.

Effects of mixtures of polychlorinated biphenyls, methylmercury, and organochlorine pesticides on hepatic DNA methylation in prepubertal female Sprague-Dawley rats.

DNA methylation is one of the epigenetic mechanisms that regulates gene expression, chromosome structure, and stability. Our objective was to determine whether the DNA methylation system could be a target following in utero and postnatal exposure to human blood contaminants. Pregnant rats were dosed daily from gestation day 1 until postnatal day 21 with 2 dose levels of either organochlorine pesticides (OCP; 0.019 or 1.9 mg/kg/day), methylmercury chloride (MeHg; 0.02 or 2 mg/kg/day), polychlorinated biphenyls (PCBs; 0.011 or 1.1 mg/kg/day), or a mixture (Mix; 0.05, or 5 mg/kg/day) including all 3 groups of chemicals. Livers from 1 female offspring per litter were collected at postnatal day 29. Hepatic analysis revealed that the mRNA abundance for DNA methyltransferase (DNMT)-1, -3a, and -3b were significantly reduced by the high dose of PCB, that the high dose of MeHg also reduced mRNA levels for DNMT-1, and -3b, but that OCP had no significant effects compared with control. The high dose of PCB and Mix reduced the abundance of the universal methyl donor S-adenosylmethionine, and Mix also reduced global genome DNA methylation (5-methyl-deoxycytidine/5-methyl-deoxycytidine + deoxycytidine). The latter is consistent with pyrosequencing methylation analysis, revealing that the high-dose groups (except OCP) generally decreased the methylation of CpG sites (position -63 to -29) in the promoter of the tumor suppressor gene p16(INK4a). Overall, these hepatic results suggest that the DNA methylation system can be affected by exposure to high doses of blood contaminants, and that OCP is the least potent chemical group from the investigated mixtures.

Contribution of methylmercury, polychlorinated biphenyls and organochlorine pesticides to the toxicity of a contaminant mixture based on Canadian Arctic population blood profiles.

Human populations are simultaneously exposed to a variety of anthropogenic contaminants. However, despite extensive literature on animal exposure to single compounds, data on the toxicity of complex mixtures are scarce. The Northern Contaminant Mixture (NCM) was formulated to contain the 27 most abundant contaminants in the same relative proportions found in the blood of Canadian Arctic populations. Sprague-Dawley rat dams were dosed from the first day of gestation until weaning with methylmercury (MeHg), polychlorinated biphenyls (PCBs) or organochlorines pesticides (OCs) administered either separately or together in the NCM. An additional control group for hypothyroxinemia was included by dosing dams with the goitrogen 6-propyl-2-thiouracil (PTU). Offspring growth, survival, serum thyroxine and Thyroid Stimulating Hormone (TSH) levels, thyroid gland morphology, brain taurine content and cerebellum and hippocampus protein expression patterns resulting from such exposures were monitored. Pups' increased mortality rate and impaired growth observed in the NCM treatment group were attributed to MeHg, while decreased circulating thyroxine levels and perturbations of thyroid gland morphology were mostly attributable to PCBs. Interestingly, despite comparable reduction in serum thyroxine levels, PCBs and PTU exposures produced markedly different effects on pup's growth, serum TSH level and brain taurine content. Analysis of cerebellum and hippocampus protein expression patterns corroborated previous cerebellum gene expression data, as contaminant co-exposure in the NCM significantly masked the effects of individual components on protein two-dimensional electrophoresis patterns. Identification by MALDI-TOF/TOF MS of differentially expressed proteins involved notably in neuronal and mitochondrial functions provided clues on the cellular and molecular processes affected by these contaminant mixtures.

Toxicological effects of in utero and lactational exposure of rats to a mixture of environmental contaminants detected in Canadian Arctic human populations.

As part of the program to investigate mixture effects of environmental pollutants, this study describes clinical, biochemical, and histopathological effects in rats perinatally exposed to a mixture of persistent organochlorine pollutants and methylmercury that simulates the blood contaminant profile of humans residing in the Canadian Arctic. Groups of pregnant rats were administered orally 0, 0.05, 0.5, or 5 mg/kg body weight (bw)/d of a reconstituted mixture of organochlorine pollutants (referred to as mixture hereafter) from gestational day (GD) 1 to postnatal day (PND) 23. Positive and vehicle controls were given Aroclor 1254 (Aroclor hereafter, 15 mg/kg bw) and corn oil (vehicle), respectively. After parturition, the pups were colled to 8 per litter on PND 4, and killed on PND 35, 77, or 350, when tissues were collected for analysis. Gestational and lactational exposure of rats to mixture up to 5 mg/kg bw produced adverse effects in the offspring, including growth suppression, decreased spleen and thymic weights, increased serum cholesterol and liver microsomal enzyme activities, lower liver retinoid levels, and histological changes in the liver, thyroid, and spleen. Histological changes in the liver consisted of hepatic inflammation, vacuolation, and hypertrophy, while alterations in the thyroid were characterized by hypertrophy and hyperplasia of follicles. The hepatic and thyroidal effects were mild even at the highest dose. The spleen showed a dose-dependent atrophy in the lymphoid nodules and periarteriolar lymphatic sheath regions. Aroclor produced effects similar to those seen in the highest mixture group. In summary, this study demonstrates that exposure to the reconstituted mixture at 5 mg/kg bw produced growth suppression, changes in organ weights, and biochemical and histopathological changes in liver, thyroid, and spleen. This study also demonstrated that the blood level in rats given the 5-mg/kg dose, where most of the effects were observed, is 100-fold higher than the blood level in the 0.05-mg/kg group, which is comparable to that found in humans living in the Canadian Arctic region.

Toxicological effects of gestational and lactational exposure to a mixture of persistent organochlorines in rats: systemic effects.

A large multi-disciplinary study was conducted to investigate the systemic, neurodevelopmental, neurochemical, endocrine, and molecular pathological effects of a mixture of reconstituted persistent organochlorine pollutants (POP) based on the blood profiles of Canadians residing in the Great Lakes/St. Lawrence region. This report outlines the overall study design and describes the systemic effects in rat offspring perinatally exposed to the POP mixture. Maternal rats were administered orally 0, 0.013, 0.13, 1.3, or 13 mg/kg bw/day of the mixture from gestational day (GD) 1 to postnatal day (PND) 23. Positive and negative controls were given Aroclor 1254 (15 mg/kg bw/day) and corn oil (vehicle), respectively. The rat pups were reared, culled to 8 per litter, and killed on postnatal days 35, 70, and 350, at which time tissues were collected for analysis. Exposure to high doses of the mixture elicited clinical, biochemical, and pathological changes and high mortality rates in rat offspring. Aroclor 1254 produced similar effects but a lower mortality than was seen in POP mixture groups. Biochemical changes consisted of increased liver microsomal activities and elevated serum cholesterol. Hepatomegaly was observed in the highest dose group of the mixture and in the positive control. Liver, thymus, and spleen were the target organs of action. Microscopic changes in the liver consisted of vacuolation and hypertrophy, and those in the thymus were characterized by reduced cortical and medullary volume. The spleen showed a treatment-related reduction in lymphocyte density and lymphoid areas. This study demonstrates that exposure to the POP mixture up to 13 mg/kg/day perinatally produced growth suppression, elevated serum cholesterol, increased liver microsomal enzyme activities, and immunopathological changes in the thymus and spleen, and lethality. Most of the effects were seen at dose levels much higher than expected human exposure.

Effect of anaesthetics[sol ]terminal procedures on neurotransmitters from non-dosed and aroclor 1254-dosed rats.

Subtle effects of low-dose exposure to environmental toxicants may be altered or masked by an inappropriate choice of anaesthesia prior to manipulation or termination of experimental animals. This study was designed to investigate effects of various anaesthetics and terminal procedures on neurotransmitters from male Sprague-Dawley rats. Significant changes in neurotransmitters were observed in the caudate nucleus, nucleus accumbens, hippocampus and substantia nigra but not the frontal cortex upon exposure to isoflurane, an Equithesin-like mixture or carbon dioxide relative to control animals that were decapitated without anaesthesia. Terminal use of any of these three anaesthetics also masked or altered some of the changes induced by exposure to Aroclor 1254. These results suggest that it is critical to avoid anaesthetizing experimental animals and that decapitation is the preferred method for euthanasia when conducting neurochemical studies.

Early developmental neurotoxicity of a PCB/organochlorine mixture in rodents after gestational and lactational exposure.

The developmental and neurobehavioral effects of gestational and lactational exposure to a mixture of 14 polychlorinated biphenyls (PCBs) and 11 organochlorine pesticides was examined and compared against the commercial PCB mixture Aroclor 1254. The mixture was based on blood levels reported in Canadian populations living in the Great Lakes/St. Lawrence basin. Pregnant Sprague-Dawley rats were dosed orally with 0.013, 0.13, 1.3, or 13 mg/kg of the chemical mixture or 15 mg/kg of Aroclor 1254 from gestation day (GD) 1 to postnatal day (PND) 23. The highest mixture dose decreased maternal gestation and lactation body weight, and produced high mortality rates (80% overall) and reductions in offspring weight that persisted to adulthood. Aroclor 1254 produced smaller but persistent decreases in offspring weight without affecting maternal weight or offspring mortality. Aroclor 1254 and 13 mg/kg of the mixture produced comparable decreases in maternal and offspring serum T4 levels and comparable alterations to maternal thyroid morphology. Aroclor 1254 delayed the righting reflex and ear opening, accelerated eye opening, and reduced grip strength at PNDs 10-14. The mixture at 13 mg/kg delayed negative geotaxis in addition to delaying righting reflex and ear opening and reducing grip strength but had no effect on eye opening. Lower mixture doses (0.13 and 1.3 mg/kg) also delayed ear opening but affected no other parameters. Developmental exposure to the chemical mixture was found to be more toxic than exposure to Aroclor 1254 and produced a different profile of effects on early neurodevelopment. This PCB/organochlorine pesticide mixture affects mortality, growth, thyroid function, and neurobehavioral development in rodents.