Jamie Nakai

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 p16INK4a. 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.

Penetration of chloroform, trichloroethylene, and tetrachloroethylene through human skin.

In vitro dermal absorption was measured for three volatile organic compounds in dilute aqueous solution through freshly prepared and previously frozen human skin. The permeability coefficients at 26 degrees C for chloroform (0.14 cm/h) and trichloroethylene (0.12 cm/h) were similar but much larger than that for tetrachloroethylene (0.018 cm/h). Storage of the skin at -20 degrees C did not significantly affect the penetration of these chemicals. The dermal absorption of chloroform through freshly prepared human skin was not changed significantly by pretreatment of the skin with commonly used consumer products (moisturizer, baby oil, insect repellent, sunscreen); however, the permeability coefficient was found to increase from 0.071 cm/h at 11 degrees C to 0.19 cm/h at 50 degrees C. These data suggest that exposure estimates for chloroform and other contaminants in water should consider the appropriate exposure scenario to properly assess the dermal dose.

Effect of environmental conditions on the penetration of benzene through human skin

The in vitro penetration of [14C]benzene through freshly prepared human skin was examined under a variety of skin conditions associated with swimming and bathing. The experimental system utilized a recirculating donor solution and a flow-through receiver solution, and was modified to accommodate the analysis of volatiles. The permeability coefficient of 0.14 cm/h under standard conditions at 26 degrees C was found to increase to 0.26 cm/h at 50 degrees C and decrease to 0.10 cm/h at 15 degrees C. Storage of the skin at- 20 degrees C did not affect the penetration of benzene. Application of baby oil, moisturizer, or insect repellant to the skin before exposure under standard conditions did not affect the flux of benzene, but a significant increase was observed when the skin was pretreated with sunscreen (permeability coefficient 0.24 cm/h). These results suggest that risk assessment or exposure modeling for benzene and other environmental contaminants should account for appropriate changes in the environmental conditions when considering the dermal route of exposure.

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.

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.

Behavioral and thyroid effects of in utero and lactational exposure of Sprague–Dawley rats to the polybrominated diphenyl ether mixture DE71

Exposure of rodents during gestation and lactation to polybrominated diphenyl ethers (PBDEs) has been reported to disrupt neurobehavioral function in offspring, as well as to disrupt thyroid function. To assess this we evaluated development and behavior after gestational and lactational exposure to the technical PBDE mixture DE71. Pregnant Sprague-Dawley rats were exposed to 0, 0.3, 3.0 or 30 mg/kg/day of DE71 from gestation day 1 to postnatal day (PND) 21 and were assessed on a wide range of behavioral functions from early postnatal period until old age (PND 450). DE71 exposure decreased thyroid hormone levels (T3 and T4) in mothers and offspring with offspring being more sensitive that mothers. Developmental landmarks, neuromotor function, anxiety, learning and memory were not affected by DE71 at any age. DE71 produced small changes in motor activity rearing only at PND 110 but not at any other age and no other activity measure was altered by DE71. Cholinergic sensitivity measured by nicotine-stimulated motor activity was not affected by perinatal DE71 exposure. Acoustic startle responses were potentiated by DE71 at PND 90 indicating delayed effects on sensory reactivity. Habituation was measured in motor activity tests at five ages but was not altered by DE71 at any age. Habituation measured in startle tests was also not affected by exposure to DE71. For thyroid hormone levels at PND 21, the lowest adverse effect level was 3.0 mg/kg. Few behavioral effects were observed and the lowest adverse effect level was 30 mg/kg. Our results confirm that DE71 produces transient effects on thyroid hormone levels but does not result in learning or motor impairment and does not alter non-associative learning (habituation).

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 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.

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.

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.