Joyce E. Royland

A Genomic Analysis of Subclinical Hypothyroidism in Hippocampus and Neocortex of the Developing Rat Brain

Hypothyroidism during pregnancy and the early postnatal period has severe neurological consequences for the developing offspring. The impact of milder degrees of perturbation of the thyroid axis as encompassed in conditions of subclinical hypothyroidism and hypothyroxinemia, however, has not been established. The present investigation examined the effects of graded levels of hypothyroidism, from subclinical to severe, on global gene expression in the developing rodent brain. Thyroid hormone insufficiency was induced by administration of propylthiouracil (PTU) to pregnant rats via drinking water from gestational day 6 until sacrifice of pups prior to weaning. In the first study a specialised microarray, the Affymetrix Rat Neurobiology array RN_U34, was used to contrast gene expression in the hippocampus of animals exposed to 0 or 10 ppm (10 mg/l) PTU, a treatment producing severe hypothyroidism. In the second study, a more complete genome array (Affymetrix Rat 230A) was used to compare gene expression in the neocortex and hippocampus of postnatal day (PN) 14 animals experiencing graded degrees of thyroid hormone insufficiency induced by delivery of 0, 1, 2 or 3 ppm PTU to the dam. Dose‐dependent up‐ and down‐regulation were observed for gene transcripts known to play critical roles in brain development and brain function. Expression levels of a subset of approximately 25 genes in each brain region were altered at a dose of PTU (1 ppm) that induced mild hypothyroxinemia in dams and pups. These data indicate that genes driving important developmental processes are sensitive to relatively modest perturbations of the thyroid axis, and that the level of gene expression is related to the degree of hormone reduction. Altered patterns of gene expression during critical windows of brain development indicate that thyroid disease must be viewed as a continuum and that conditions typically considered ‘subclinical’ may induce structural and functional abnormalities in the developing central nervous system.

Gene expression profiles following exposure to a developmental neurotoxicant, Aroclor 1254 : Pathway analysis for possible mode(s) of action

Epidemiological studies indicate that low levels of polychlorinated biphenyl (PCB) exposure can adversely affect neurocognitive development. In animal models, perturbations in calcium signaling, neurotransmitters, and thyroid hormones have been postulated as potential mechanisms for PCB-induced developmental neurotoxicity. In order to understand the role of these proposed mechanisms and to identify other mechanisms in PCB-induced neurotoxicity, we have chosen a global approach utilizing oligonucleotide microarrays to examine gene expression profiles in the brain following developmental exposure to Aroclor 1254 (0 or 6 mg/kg/day from gestation day 6 through postnatal day (PND) 21) in Long-Evans rats. Gene expression levels in the cerebellum and hippocampus from PNDs 7 and 14 animals were determined on Affymetrix rat 230A_2.0 chips. In the cerebellum, 87 transcripts were altered at PND7 compared to 27 transcripts at PND14 by Aroclor 1254 exposure, with only one transcript affected at both ages. In hippocampus, 175 transcripts and 50 transcripts were altered at PND7 and PND14, respectively, by Aroclor 1254 exposure with five genes commonly affected. Functional analysis suggests that pathways related to calcium homeostasis (Gng3, Ryr2, Trdn, Cacna1a), intracellular signaling (Camk2d, Stk17b, Pacsin2, Ryr2, Trio, Fert2, Ptk2b), axonal guidance (Lum, Mxd3, Akap11, Gucy1b3), aryl hydrocarbon receptor signaling (Nfia, Col1a2), and transcripts involved in cell proliferation (Gspt2, Cdkn1c, Ptk2b) and differentiation (Ifitm31, Hpca, Zfp260, Igsf4a, Hes5) leading to the development of nervous system were significantly altered by Aroclor 1254 exposure. Of the two brain regions examined, Aroclor 1254-induced genomic changes were greater in the hippocampus than the cerebellum. The genomic data suggests that PCB-induced neurotoxic effects were due to disruption of normal ontogenetic pattern of nervous system growth and development by altering intracellular signaling pathways but not by endocrine disruption.

Developmental Exposure to a Commercial PBDE Mixture: Effects on Protein Networks in the Cerebellum and Hippocampus of Rats

Background: Polybrominated diphenyl ethers (PBDEs) are structurally similar to polychlorinated biphenyls (PCBs) and have both central (learning and memory deficits) and peripheral (motor dysfunction) neurotoxic effects at concentrations/doses similar to those of PCBs. The cellular and molecular mechanisms for these neurotoxic effects are not fully understood; however, several studies have shown that PBDEs affect thyroid hormones, cause oxidative stress, and disrupt Ca2+-mediated signal transduction. Changes in these signal transduction pathways can lead to differential gene regulation with subsequent changes in protein expression, which can affect the development and function of the nervous system. Objective: In this study, we examined the protein expression profiles in the rat cerebellum and hippocampus following developmental exposure to a commercial PBDE mixture, DE-71. Methods: Pregnant Long-Evans rats were dosed perinatally with 0 or 30.6 mg/kg/day of DE-71 from gestation day 6 through sampling on postnatal day 14. Proteins from the cerebellum and hippocampus were extracted, expression differences were detected by two-dimensional difference gel electrophoresis, and proteins were identified by tandem mass spectrometry. Protein network interaction analysis was performed using Ingenuity® Pathway Analysis, and the proteins of interest were validated by Western blotting. Results: Four proteins were significantly differentially expressed in the cerebellum following DE-71 exposure, whereas 70 proteins were significantly differentially expressed in the hippocampus. Of these proteins, 4 from the cerebellum and 47 from the hippocampus, identifiable by mass spectrometry, were found to have roles in mitochondrial energy metabolism, oxidative stress, apoptosis, calcium signaling, and growth of the nervous system. Conclusions: Results suggest that changes in energy metabolism and processes related to neuroplasticity and growth may be involved in the developmental neurotoxicity of PBDEs. Citation: Kodavanti PR, Royland JE, Osorio C, Winnik WM, Ortiz P, Lei L, Ramabhadran R, Alzate O. 2015. Developmental exposure to a commercial PBDE mixture: effects on protein networks in the cerebellum and hippocampus of rats. Environ Health Perspect 123:428–436; http://dx.doi.org/10.1289/ehp.1408504

Gene expression profiles in the cerebellum and hippocampus following exposure to a neurotoxicant, Aroclor 1254: Developmental effects

The developmental consequences of exposure to the polychlorinated biphenyls (PCBs) have been widely studied, making PCBs a unique model to understand issues related to environmental mixture of persistent chemicals. PCB exposure in humans adversely affects neurocognitive development, causes psychomotor difficulties, and contributes to attention deficits in children, all of which seem to be associated with altered patterns of neuronal connectivity. In the present study, we examined gene expression profiles in the rat nervous system following PCB developmental exposure. Pregnant rats (Long-Evans) were dosed perinatally with 0 or 6 mg/kg/day of Aroclor 1254 from gestation day 6 through postnatal day (PND) 21. Gene expression in cerebellum and hippocampus from PND7 and PND14 animals was analyzed with an emphasis on developmental aspects. Changes in gene expression (> or =1.5 fold) in control animals identified normal developmental changes. These basal levels of expression were compared to data from Aroclor 1254-treated animals to determine the impact of gestational PCB exposure on developmental parameters. The results indicate that the expression of a number of developmental genes related to cell cycle, synaptic function, cell maintenance, and neurogenesis is significantly altered from PND7 to PND14. Aroclor 1254 treatment appears to dampen the overall growth-related gene expression levels in both regions with the effect being more pronounced in the cerebellum. Functional analysis suggests that Aroclor 1254 delays maturation of the developing nervous system, with the consequences dependent on the ontological state of the brain area and the functional role of the individual gene. Such changes may underlie learning and memory deficits observed in PCB exposed animals and humans.

Toluene effects on oxidative stress in brain regions of young-adult, middle-age, and senescent Brown Norway rats.

The influence of aging on susceptibility to environmental contaminants is not well understood. To extend knowledge in this area, we examined effects in rat brain of the volatile organic compound, toluene. The objective was to test whether oxidative stress (OS) plays a role in the adverse effects caused by toluene exposure, and if so, if effects are age-dependent. OS parameters were selected to measure the production of reactive oxygen species (NADPH Quinone oxidoreductase 1 (NQO1), NADH Ubiquinone reductase (UBIQ-RD)), antioxidant homeostasis (total antioxidant substances (TAS), superoxide dismutase (SOD), γ-glutamylcysteine synthetase (γ-GCS), glutathione transferase (GST), glutathione peroxidase (GPX), glutathione reductase (GRD)), and oxidative damage (total aconitase and protein carbonyls). In this study, Brown Norway rats (4, 12, and 24 months) were dosed orally with toluene (0, 0.65 or 1g/kg) in corn oil. Four hours later, frontal cortex, cerebellum, striatum, and hippocampus were dissected, quick frozen on dry ice, and stored at -80°C until analysis. Some parameters of OS were found to increase with age in select brain regions. Toluene exposure also resulted in increased OS in select brain regions. For example, an increase in NQO1 activity was seen in frontal cortex and cerebellum of 4 and 12 month old rats following toluene exposure, but only in the hippocampus of 24 month old rats. Similarly, age and toluene effects on glutathione enzymes were varied and brain-region specific. Markers of oxidative damage reflected changes in oxidative stress. Total aconitase activity was increased by toluene in frontal cortex and cerebellum at 12 and 24 months, respectively. Protein carbonyls in both brain regions and in all age groups were increased by toluene, but step-down analyses indicated toluene effects were statistically significant only in 12month old rats. These results indicate changes in OS parameters with age and toluene exposure resulted in oxidative damage in frontal cortex and cerebellum of 12 month old rats. Although increases in oxidative damage are associated with increases in horizontal motor activity in older rats, further research is warranted to determine if these changes in OS parameters are related to neurobehavioral and neurophysiological effects of toluene in animal models of aging.

Toluene effects on gene expression in the hippocampus of young adult, middle-age, and senescent Brown Norway Rats.

Differential susceptibility to environmental exposures across life stages is an area of toxicology about which little is known. We examined the effects of toluene on transcriptomic changes and oxidative stress (OS) parameters (e.g., NQO1 and GPX) in the rat brain at different life stages to elucidate key molecular pathways responsible for toluene-induced neurotoxicity, as well as possible age-related interactions. Changes in assessed end points following acute oral toluene (0, 0.65, and 1.0 g/kg) were examined 4 h after exposure in hippocampi of Brown Norway Rats at 4, 12, and 24 months of age. Genomic data were analyzed by two-way ANOVA to identify the effects of age, toluene, and interactions between the two factors. Analysis by one-way ANOVA identified 183 genes whose expression changed ≥ 1.25-fold with age. The majority of the genes were upregulated between life stages (> 79%). Similar analysis for toluene-related genes found only two sequences to vary significantly with dose. Fifty-six genes were identified to have expression changes due to an age-toluene interaction. Expression of genes with roles in immune response, cytoskeleton, protein, and energy metabolism was changed with advancing life stage, indicating changes in basic cellular homeostasis. Toluene affected similar cell functions, enhancing the effects of aging. OS parameters also indicated age-related changes in response mechanisms, evidence of toluene damage, and supported an age-toluene interaction. The data indicate that life stage can alter the toxicity of acute toluene exposure in various and complex ways, highlighting the need for further investigation into the role of aging in susceptibility.

Acute and subchronic toxicity of inhaled toluene in male Long–Evans rats: Oxidative stress markers in brain☆☆☆

The effects of exposure to volatile organic compounds (VOCs), which are of concern to the EPA, are poorly understood, in part because of insufficient characterization of how human exposure duration impacts VOC effects. Two inhalation studies with multiple endpoints, one acute and one subchronic, were conducted to seek effects of the VOC, toluene, in rats and to compare the effects between acute and subchronic exposures. Adult male Long-Evans rats were exposed to toluene vapor (n=6 per group) at a concentration of 0 or 1019 ± 14 ppm for 6h in the acute study and at 0 ± 0, 10 ± 1.4, 97 ± 7, or 995 ± 43 ppm for 6h/d, 5d/week for 13 weeks in the subchronic study. For the acute study, brains were dissected on ice within 30 min of the end of exposure, while for the subchronic study, brains were dissected 18 h after the last exposure. Frontal cortex, hippocampus, cerebellum, and striatum were assayed for a variety of oxidative stress (OS) parameters including total aconitase (TA), protein carbonyls, glutathione peroxidase (GPX), glutathione reductase (GRD), glutathione transferase (GST), γ-glutamylcysteine synthetase (GCS), superoxide dismutase (SOD), total antioxidants (TAS), NADPH quinone oxidoreductase-1 (NQO1), and NADH ubiquinone reductase (UBIQ-RD) activities using commercially available kits. Following acute exposure, UBIQ-RD, GCS and GRD were increased significantly only in the cerebellum, while TAS was increased in frontal cortex. On the other hand, subchronic exposure affected several OS markers including increases in NQO1 and UBIQ-RD. The effect of subchronic toluene exposure on SOD and TAS was greater in the striatum than in the other brain regions. TA activity (involved in maintaining iron homeostasis and an indicator of DNA damage) was inhibited in striatum and cerebellum, increased in hippocampus, and unchanged in frontal cortex. Protein carbonyls increased significantly in both the frontal cortex and cerebellum. In general, the results showed that acute exposure to toluene affected OS parameters to a lesser extent than did subchronic exposure. These results suggest that toluene exposure induces OS in the brain and this may be a component of an adverse outcome pathway for some of the neurotoxic effects reported following toluene exposure.

Atypical microglial response to biodiesel exhaust in healthy and hypertensive rats

&NA; Accumulating evidence suggests a deleterious role for urban air pollution in central nervous system (CNS) diseases and neurodevelopmental disorders. Microglia, the resident innate immune cells and sentinels in the brain, are a common source of neuroinflammation and are implicated in air pollution‐induced CNS effects. While renewable energy, such as soy‐based biofuel, is of increasing public interest, there is little information on how soy biofuel may affect the brain, especially in people with preexisting disease conditions. To address this, male spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats were exposed to 100% Soy‐based Biodiesel Exhaust (100SBDE; 0, 50, 150 and 500 &mgr;g/m3) by inhalation, 4 h/day for 4 weeks (5 days/week). Ionized calcium‐binding adapter molecule‐1 (IBA‐1) staining of microglia in the substantia nigra revealed significant changes in morphology with 100SBDE exposure in rats from both genotypes, where SHR were less sensitive. Aconitase activity was inhibited in the frontal cortex and cerebellum of WKY rats exposed to 100SBDE. No consistent changes occurred in pro‐inflammatory cytokine expression, nitrated protein, or arginase1 expression in brain regions from either rat strain exposed to 100SBDE. However, while IBA‐1 mRNA expression was not modified, CX3CR1 mRNA expression was lower in the striatum of 100SBDE exposed rats regardless of genotype, suggesting a downregulation of the fractalkine receptor on microglia in this brain region. Together, these data indicate that while microglia are detecting and responding to 100SBDE exposure with changes in morphology, there is reduced expression of CX3CR1 regardless of genetic background and the activation response is atypical without traditional inflammatory markers of M1 or M2 activation in the brain. HighlightsMicroglia morphology in the substntia nigra changed to an activated morphology in response to biodesiel exhaust exposure with no evidence of traditional markers of M1 or M2 activation.Biodiesel exhaust perturbed the fractaline axis in the midbrain.Biodiesel exhaust caused minor effects on oxidative stress markers in the brain.