Hugh A. Tilson

Differential effects of polychlorinated biphenyl congeners on phosphoinositide hydrolysis and protein kinase C translocation in rat cerebellar granule cells

Previous reports from our laboratory have suggested that the neuroactivity of some polychlorinated biphenyl (PCB) congeners is associated with perturbations in cellular Ca(2+)-homeostasis. We have characterized further the neurochemical effects of PCBs on signal transduction in primary cultures of cerebellar granule cells. The present experiments found that neither 2,2-dichlorobiphenyl (DCBP), an ortho-substituted congener, nor 3,3,4,4,5-pentachlorobiphenyl (PCBP), a non-ortho-substituted congener, affected basal phosphoinositide (PI) hydrolysis in cerebellar granule cells. However, at concentrations up to 50 microM, DCBP potentiated carbachol-stimulated PI hydrolysis, while decreasing it at 100 microM. PCBP, on the other hand, had no effect on carbachol-stimulated PI hydrolysis in concentrations up to 100 microM. [3H]Phorbol ester ([3H]PDBu) binding was used to determine protein kinase C (PKC) translocation. DCBP increased [3H]PDBu binding in a concentration-dependent manner and a twofold increase was observed at 100 microM in cerebellar granule cells. PCBP had no effect on [3H]PDBu binding at concentrations up to 100 microM. The effect of DCBP on [3H]PDBu binding was time-dependent and was also dependent on the presence of external Ca2+ in the medium. To test the hypothesis that DCBP increases [3H]PDBu binding by acting on receptor-activated calcium channels, the effects of DCBP were compared to those of L-glutamate. The effects of DCBP (50 microM) and glutamate (20 microM) were additive.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of microsomal and mitochondrial Ca2+-sequestration in rat cerebellum by polychlorinated biphenyl mixtures and congeners. Structure-activity relationships.

Recent studies from our laboratory indicate that polychlorinated biphenyl (PCB) congeners in vitro perturbed signal transduction mechanisms including cellular Ca2+-homeostasis and protein kinase C translocation. We have now investigated the structure-activity relationship (SAR) of three PCB mixtures, 24 PCB congeners and one dibenzofuran for their effects on microsomal and mitochondrial Ca2+-sequestration in rat cerebellum. Ca2+-sequestration by these intracellular organelles was determined using radioactive 45CaCl2. All three mixtures studied, Aroclor 1016, Aroclor 1254 and Aroclor 1260, were equally potent in inhibiting microsomal and mitochondrial Ca2+-sequestration with IC50 values of 6–8 μM. 1,2,3,7,8-Pentachlorodibenzofuran had no effect on Ca2+-sequestration by these organelles. The SAR among the congeners revealed: (1) congeners with ortho-/meta- or ortho-, para-chlorine substitutions were the most potent in inhibiting microsomal and mitochondrial Ca2+-sequestration (IC50=2.4–22.3 μM); (2) congeners with only para- but without ortho-substitutions were not effective in inhibiting Ca2+-sequestration by microsomes and mitochondria; (3) increased chlorination was not related to the effectiveness of these congeners. The present SAR studies indicate that the effects of most PCB congeners in vitro may be related to an interaction at specific sites having preference for low lateral substitution or lateral content (meta- or para) in the presence of ortho-substitution.

Neurochemical Effects of Environmental Chemicals: In Vitro and In Vivo Correlations on Second Messenger Pathwaysa

Abstract: Polychlorinated biphenyls (PCBs) are persistent, bioaccumulative, toxic, and widely distributed environmental chemicals. There is now both epidemiological and experimental evidence that PCBs cause cognitive deficits; however, the underlying cellular or molecular mechanism(s) is not known. We have hypothesized that altered signal transduction/second messenger homeostasis by PCBs may be associated with these effects since second messengers in signal transduction pathways, such as calcium, inositol phosphates (IP), and protein kinase C (PKC), play key roles in neuronal development and their function. In vitro studies using cerebellar granule neurons and isolated organelle preparations indicate that ortho‐PCBs increase intracellular free Ca2+ levels by inhibiting microsomal and mitochondrial Ca2+ buffering and the Ca2+ extrusion process. Ortho‐PCBs also increase agonist‐stimulated IP accumulation and cause PKC translocation at low micromolar concentrations where no cytotoxicity is observed. On the other hand, non‐ortho‐PCBs are not effective in altering these events. Further SAR studies indicate that congeners with chlorine substitutions favoring non‐coplanarity are active in vitro, while congeners favoring coplanarity are relatively inactive. Subsequent in vivo studies have shown that repeated exposure to a PCB mixture, Aroclor 1254, increases PKC translocation and decreases Ca2+ buffering in the brain, similar to in vitro studies. These changes in vivo are associated with elevated levels of non‐coplanar ortho‐PCB congeners at levels equivalent to 40–50 μM in brain, the concentrations that significantly inhibited second messenger systems in neuronal cultures in vitro. Current research is focusing on PCB‐induced alterations in second messenger systems following developmental exposure.

Gestational exposure to methylmercury alters the developmental pattern of trk-like immunoreactivity in the rat brain and results in cortical dysmorphology

Nerve growth factor signal transduction mediated through the trk receptor has been implicated in neuronal growth, differentiation, and survival. In this study, we examined the effects of gestational exposure to the developmental neurotoxicant methylmercury (CH3Hg) on the ontogeny of trk-immunoreactivity (IR). Long-Evans dams were dosed on gestational days 6-15 (p.o.) with 0, 1, or 2 mg/kg CH3Hg dissolved in saline. Pups were sacrificed and perfused with buffered paraformaldehyde on postnatal days (PND) 1, 4, 10, 21 and 85. The brains were sectioned sagitally, Nissl-stained or stained immunohistochemically for trk receptors or glial fibrillary acidic protein (GFAP), and examined throughout the medial to lateral extent of the brain. The greatest density of IR in neural cell bodies was seen in the olfactory bulb, hippocampus, cerebral, and cerebellar cortex, striatum, septum, nucleus basalis, inferior colliculus, pons, and brain stem nuclei. trk IR was not limited to nerve cell bodies, with prominent axonal and dendritic staining in the brainstem, neocortex, hippocampus, cerebellum, and olfactory tract. The regional pattern of trk IR varied in an age-dependent manner. In controls, trk-like IR appeared to peak in most regions between PND4-10 and decreased dramatically after PND21. This age-related difference in trk IR was supported by western blot analysis of PND10 and adult neocortex. This reduced and more adult-like pattern of trk IR was apparent on PND21 with some persistent trk-like IR in the olfactory bulb, hippocampus, neocortex, cerebellum and basal forebrain. In contrast to the normal regional patterns of trk IR, CH3Hg produced a dose-related decrease in trk-like IR in the absence of overt maternal toxicity or neonatal toxicity. CH3Hg-induced decreases in trk-like IR were especially apparent during the early postnatal period when trk IR was the greatest. The effects of CH3Hg exposure were restricted regionally, with the largest decrease in trk-like IR apparent in cortical regions, basal forebrain nuclei, and brain stem nuclei. Subsequent to the effects of CH3Hg on cortical trk-like IR were alterations in the development of cortical laminae on PND10 and 21 of neocortex. These alterations were characterized by quantifiable decreases in cell density, cell size and the widths of the layers of posterior neocortex. Not all of the CH3Hg-induced effects were characterized by decreased trk-like IR. Robust increases in trk IR in glial cells in the corpus callosum and brain stem were observed coincident with increased GFAP IR in cells of similar morphology. The present results localize the cellular and regional ontogeny of trk and suggest that developmental exposure to CH3Hg alters the normal ontogeny of this trophic factor receptor which may be associated with the developmental neurotoxicity of this chemical.

Ontogenetic alterations in molecular and structural correlates of dendritic growth after developmental exposure to polychlorinated biphenyls

Objective Perinatal exposure to polychlorinated biphenyls (PCBs) is associated with decreased IQ scores, impaired learning and memory, psychomotor difficulties, and attentional deficits in children. It is postulated that these neuropsychological deficits reflect altered patterns of neuronal connectivity. To test this hypothesis, we examined the effects of developmental PCB exposure on dendritic growth. Methods Rat dams were gavaged from gestational day 6 through postnatal day (PND) 21 with vehicle (corn oil) or the commercial PCB mixture Aroclor 1254 (6 mg/kg/day). Dendritic growth and molecular markers were examined in pups during development. Results Golgi analyses of CA1 hippocampal pyramidal neurons and cerebellar Purkinge cells indicated that developmental exposure to PCBs caused a pronounced age-related increase in dendritic growth. Thus, even though dendritic lengths were significantly attenuated in PCB-treated animals at PND22, the rate of growth was accelerated at later ages such that by PND60, dendritic growth was comparable to or even exceeded that observed in vehicle controls. Quantitative reverse transcriptase polymerase chain reaction analyses demonstrated that from PND4 through PND21, PCBs generally increased expression of both spinophilin and RC3/neurogranin mRNA in the hippocampus, cerebellum, and cortex with the most significant increases observed in the cortex. Conclusions This study demonstrates that developmental PCB exposure alters the ontogenetic profile of dendritogenesis in critical brain regions, supporting the hypothesis that disruption of neuronal connectivity contributes to neuropsychological deficits seen in exposed children.

Extracellular calcium is required for the polychlorinated biphenyl-induced increase of intracellular free calcium levels in cerebellar granule cell culture.

Recent studies from the laboratory indicate that polychlorinated biphenyl (PCB) congeners can alter signal transduction and calcium homeostasis in neuronal preparations. These effects were more pronounced for the ortho-substituted, non-coplanar congeners, although the mechanisms underlying these effects are not clear. In the present study the time-course and concentration-dependent effects of coplanar and non-coplanar PCBs on intracellular free calcium concentration ([Ca2+]i) in cerebellar granule cell cultures were compared using the fluorescent probe fura-2. The ortho-substituted congeners 2,2-dichlorobiphenyl (DCB) and 2,2,4,6,6-pentachlorobiphenyl (PeCB) caused a gradual increase of [Ca2+]i while the non-ortho-substituted congeners 4,4-DCB and 3,3,4,4,5-PeCB had no effect. The increase of [Ca2+]i produced by 2,2-DCB was time- and concentration-dependent. Further studies examined possible mechanisms for this rise in [Ca2+]i. In contrast to the muscarinic agonist carbachol, the effects of 2,2-DCB on [Ca2+]i were not blocked by thapsigargin and required the presence of extracellular calcium. The effects of ortho-substituted PCBs may depend on their ability to inhibit calcium sequestration as 2,2-DCB significantly inhibited 45Ca2+-uptake by microsomes and mitochondria while 3,3,4,4,5-PeCB had no effect. In addition, 2,2-DCB significantly increased the binding of [3H]inositol 1,4,5-trisphosphate to receptors on cerebellar microsomes, suggesting another possible mechanism by which ortho-substituted PCBs can mobilize [Ca2+]i. These results show that PCBs increase [Ca2+]i in vitro via a mechanism that requires extracelluar calcium, and support previous structure-activity studies indicating that ortho-substituted PCBs are more potent than non-ortho-substituted PCBs.

Effects of selected neuroactive chemicals on calcium transporting systems in rat cerebellum and on survival of cerebellar granule cells.

This investigation examined the relationship between alteration of Ca(2+)-transport systems and cytotoxicity in vitro for a number of neuroactive chemicals including environmental pollutants. 45Ca2+ uptake as a measure of Ca2+ sequestration was determined in mitochondria and microsomes isolated from cerebella of adult male Long-Evans hooded rats by differential centrifugation. Ca2+ extrusion, measured as Ca(2+)-ATPase activity, was determined in synaptosomes prepared by sucrose density gradient. Cytotoxicity (lactate dehydrogenase leakage) was assessed in primary cultures of cerebellar granule cells from 6- to 8-day-old Long-Evans rats. N-Methyl-D-aspartic acid (NMDA) did not alter synaptosomal Ca(2+)-ATPase activity or 45Ca2+ uptake in mitochondria and microsomes. However, chlorpromazine (CPZ), aluminum (Al), permethrin (PER), and deltamethrin (DEL) inhibited Ca2+ sequestration by mitochondria and microsomes. The IC50 values (microM) for CPZ, Al, PER, and DEL were 67.8, 671, 4.2, and 91.2 for mitochondrial 45Ca2+ uptake, and 129.9, 1384, > 50, and > 200 for microsomal 45Ca2+ uptake, respectively. CPZ, PER, and DEL also inhibited synaptosomal Ca(2+)-ATPase activity in a concentration-dependent manner with IC50 values of 62.5, > 400, and 246.9 microM, indicating an effect on the Ca(2+)-extrusion process. Al increased Ca(2+)-ATPase activity (EC50 = 833 microM). Although NMDA did not inhibit Ca(2+)-transport systems, it was cytotoxic at 250 microM and higher concentrations after 2 hr of exposure. Similarly, CPZ was cytotoxic at concentrations of 25 and 10 microM after 4 hr exposure. However, PER, DEL, and Al were not cytotoxic at any concentration up to 500 microM. Of all the chemicals tested, CPZ was the most potent in inhibiting Ca(2+)-transporting systems and was also cytotoxic.(ABSTRACT TRUNCATED AT 250 WORDS)

Aluminum decreases muscarinic, adrenergic, and metabotropic receptor-stimulated phosphoinositide hydrolysis in hippocampal and cortical slices from rat brain **

Effects of aluminum chloride (AlCl3) (0.1 to 1000 microM) on inositol phosphate (IP) accumulation stimulated by carbachol (CARB), norepinephrine (NE) or quisqualate (QUIS) were examined in rat hippocampal and cortical slices. In the absence of agonist, only 1000 microM AlCl3 significantly reduced basal accumulation of IPs. For CARB-stimulated IP accumulation, 100 microM and greater AlCl3 significantly inhibited IP accumulation. In cortical slices, 1000 microM AlCl3 reduced CARB-stimulated IP accumulation by 55% and in hippocampal slices 1000 microM AlCl3 inhibited IP accumulation by 40%. Similar effects of AlCl3 were observed for NE-stimulated IP accumulation. In cortical slices, the concentration-response for AlCl3 effects on agonist-stimulated IP accumulation was significantly different from that in hippocampal slices. For QUIS-stimulated accumulation of IPs, 1000 microM AlCl3 significantly inhibited IP accumulation in hippocampal slices. However, in cortical slices a biphasic effect of AlCl3 was observed. 500 and 1000 microM AlCl3 significantly inhibited IP accumulation, whereas 10 and 50 microM AlCl3 significantly enhanced QUIS-stimulated IP accumulation. In both hippocampal and cortical slices, 500 microM AlCl3 significantly inhibited CARB-, NE- or QUIS-stimulated IP accumulation at all agonist concentrations (0.1 to 10000 microM) tested, indicating a post-receptor effect on agonist-mediated IP accumulation. Stimulation of G-proteins with NaF (5-30 mM) resulted in accumulation of IPs in hippocampal and cortical slices in the absence of added agonists. NaF (5-30 mM) plus 1 mM CARB produced increased accumulation of IPs over CARB or NaF alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurobehavioral methods used in neurotoxicological research

Exposure to chemicals in the environment and workplace can have adverse effects on the nervous system. Behavioral endpoints are being used with greater frequency in the hazard identification phase of neurotoxicology risk assessment. One reason behavioral procedures are used in animal neurotoxicology studies is that they evaluate neurobiological functions known to be affected in humans exposed to neurotoxic agents, including alterations in sensory, motor, autonomic, and cognitive function. In hazard identification, behavioral tests are used in a tiered-testing context. Tests in the first tier are designed to determine the presence of neurotoxicity. Examples of first-tier behavioral tests include functional observational batteries and motor activity. Second-tier tests are used to characterize neurotoxicant-induced effects on sensory, motor, and cognitive function. Second-tier tests are usually more complex and costly to perform. Reliance on behavioral endpoints in neurotoxicology risk assessment will likely increase in the future.

Effects of acute and repeated exposures to Aroclor 1254 in adult rats : Motor activity and flavor aversion conditioning

While considerable research has focused on the neurotoxicity of developmental exposures to polychlorinated biphenyls, including Aroclor 1254, relatively little is known about exposures in adult animals. This study investigated the behavioral effects of acute and repeated Aroclor 1254 exposures to adult rats on motor activity and flavor aversion conditioning. Male Long-Evans rats (60 days old) were tested for motor activity in a photocell device after acute (0, 100, 300, or 1000 mg/kg, p.o.) or repeated (0, 1, 3, 10, 30 or 100 mg/kg/day, po, 5 days/week for 4 to 6 weeks exposure to Aroclor 1254. Motor activity was decreased dose-dependently at doses of 300 mg/kg or more after acute exposure. Severe body weight loss and deaths occurred at 1000 mg/kg. Recovery of activity occurred over 9 weeks but was incomplete. After repeated exposure, motor activity was decreased dose-dependently at doses of 30 mg/kg or more, and severe weight loss and deaths occurred at 100 mg/kg. In contrast to acute exposure, complete recovery of activity occurred 3 weeks after exposure. Additional rats were water deprived (30 min/day) and received acute po administration of Aroclor 1254 (0, 10, 15, 25, 30, 100, or 300 mg/kg) shortly after consuming a saccharin solution. Three days later they were given the choice between consuming saccharin or water, and saccharin preferences were recorded. Saccharin preference was decreased at doses of 25 mg/kg or more. Additional experiments determined the effect of repeated saccharin-Aroclor 1254 pairings (0, 3.75, 7.5, or 15 mg/kg/day, 14 days) followed by a choice test 1 day after the last dose. Repeated exposure to 15 mg/kg produced robust flavor aversion conditioning. Repeated exposure to 7.5 mg/kg produced flavor aversion conditioning in four of 12 rats. These results demonstrate that Aroclor 1254 causes hypoactivity and flavor aversions in adult rats; the no observable effect level (NOEL) for motor activity was 100 mg/kg for acute exposure and 10 mg/kg for repeated exposure for a period of up to 6 weeks. The acute NOEL for flavor aversion conditioning was 15 mg/kg while the repeated NOEL was 7.5 mg/kg.