Ethel C. Derr-Yellin

Alterations in brain protein kinase C isoforms following developmental exposure to a polychlorinated biphenyl mixture.

PCBs have been shown to alter several neurochemical end-points and are implicated in the etiology of some neurological diseases. Recent in vivo studies from our laboratory indicated that developmental exposure to a commercial PCB mixture, Aroclor 1254, caused perturbations in calcium homeostasis and changes in protein kinase C (PKC) activities in rat brain. However, it is not known which molecular substances are targets for PCB-induced developmental neurotoxicity. Since the PKC signaling pathway has been implicated in the modulation of motor behavior as well as learning and memory, and the roles of PKC are subspecies specific, the present study attempted to analyze the effects on selected PKC isozymes in the cerebellum and the hippocampus following developmental exposure (gestational day 6 through postnatal day 21) to a PCB mixture, Aroclor 1254. The results indicated that the developmental exposure to PCBs caused significant hypothyroxinemia and age-dependent alterations in the translocation of PKC isozymes; the effects were greatly significant at postnatal day (PND) 14. Immunoblot analysis of PKC-alpha (alpha) from both cerebellum and hippocampus revealed that developmental exposure to Aroclor 1254 caused a significant decrease in cytosolic fraction and an increase in particulate fraction. There was no significant difference between these two brain regions on the level of fractional changes. However, the ratio between the fractions (particulate/cytosol) from cerebellum only was increased in a dose-dependent manner. Analysis of PKC-gamma (gamma) in cerebellum on PND14 showed a decrease in cytosolic fraction in both dose groups and an increase in particulate fraction at high dose (6 mg/kg) only. The ratio between the two fractions was increased in a dose-dependent manner. In the hippocampus, there was a significant decrease in PKC-gamma in cytosolic fraction of the high-dose group and a significant increase in particulate fraction of the low-dose group. But, the ratio between the fractions showed a significant increase (2.6-fold increase in high dose on PND14). Analysis of PKC-epsilon (epsilon) in cerebellum showed a significant decrease in cytosolic fraction at PND14, while particulate PKand an increase in ratio between fractions at 6 mg/kg on PND14. The results from this study indicate that the patterns of subcellular distributions of PKC isoforms following a developmental PCB exposure were PKC isozyme- and developmental stage-specific. Considering the significant role of PKC signaling in motor behavior, learning and memory, it is suggested that altered subcellular distribution of PKC isoforms at critical periods of brain development may be a possible mechanism of PCB-induced neurotoxic effects and that PKC-alpha, gamma, and epsilon may be among the target molecules implicated with PCB-induced neurological impairments during developmental exposure. It is believed that this is the first report successfully identifying PKC isoforms responding to PCBs during developmental exposure.

Age-dependent effects of Aroclor 1254R on calcium uptake by subcellular organelles in selected brain regions of rats☆

Earlier reports from our laboratory have indicated that polychlorinated biphenyls (PCBs) affect signal transduction mechanisms in brain, including Ca2+ homeostasis, phosphoinositol hydrolysis, and protein kinase C (PKC) translocation in mature neurons and adult brain homogenate preparations. Present studies were designed to investigate whether there were any brain region-, gender-, or age-dependent effects of PCBs on 45Ca2+-uptake by two subcellular organelles, microsomes and mitochondria. We have studied in vitro effects of a widely studied commercial PCB mixture, Aroclor 1254R, on 45Ca2+-uptake by microsomes and mitochondria in cerebellum, frontal cortex and hippocampus of postnatal day (PND) 7, 21, and 90-120 (adult) male and female Long-Evans (LE)-rats. In general, microsomal and mitochondrial 45Ca2+-uptake in selected brain regions increased with age; PND 7<PND 21< or =adults. Among three brain regions, hippocampus had relatively lower microsomal 45Ca2+-uptake than cerebellum and frontal cortex throughout the development. Mitochondrial 45Ca2+-uptake was comparable in three brain regions of PND 7 and adult animals, but in PND 21 rats, the cerebellum had much higher activity than frontal cortex and hippocampus. No gender-related differences were seen in 45Ca2+-uptake by either microsomes or mitochondria in selected brain regions throughout development. Inhibition of 45Ca2+-uptake by Aroclor 1254 in a concentration-dependent manner was observed throughout the study. However, the degree of inhibition of microsomal 45Ca2+-uptake in these brain regions by Aroclor 1254 increased with age, PND 7<PND 21< or =adults (IC50s=21-34, 8-20 and 10-14 microM, respectively). Brain region-specific differential sensitivity to Aroclor 1254 on the inhibition of microsomal 45Ca2+-uptake was not seen in PND 7 and adult animals but in PND 21 rats, hippocampus was more sensitive than the other selected brain regions. There were no age-, gender- or brain region-specific differential effects of Aroclor 1254 on mitochondrial 45Ca2+-uptake. These results indicate that a commercial PCB mixture, Aroclor 1254, inhibited 45Ca2+-uptake by both microsomes and mitochondria uniformly in selected brain regions of males and females during development. However, the inhibition of microsomal 45Ca2+-uptake by Aroclor 1254 increased with age. The age- and gender-related differential sensitivity to Aroclor 1254 may be attributed to the changes in calcium homeostasis in various brain regions during development.

Flash-, somatosensory-, and peripheral nerve-evoked potentials in rats perinatally exposed to Aroclor 1254 ☆

Pregnant Long-Evans rats were exposed to 0, 1 or 6 mg/kg/day of Aroclor 1254 (A1254; Lot no. 124-191), a commercial mixture of polychlorinated biphenyls (PCBs), from gestation day (GD) 6 through postnatal day (PND) 21. At 128-140 days of age, male and female offspring were tested for visual-, somatosensory- and peripheral nerve-evoked potentials. The evoked responses increased in amplitude with larger stimulus intensities, and gender differences were detected for some endpoints. In contrast, developmental exposure to A1254 failed to significantly affect the electrophysiological measures. A subset of the animals were tested for low-frequency hearing dysfunction using reflex modification audiometry (RMA). An elevated threshold for a 1-kHz tone was observed, replicating previous findings of A1254-induced auditory deficits [Hear. Res. 144 (2000) 196; Toxicol. Sci. 45(1) (1998) 94; Toxicol. Appl. Pharmacol. 135(1) (1995) 77.]. These findings indicate no statistically significant changes in visual-, somatosensory- or peripheral nerve-evoked potentials following developmental exposure to doses of A1254 that produce behavioral hearing deficits. However, subtle changes in the function of the visual or somatosensory systems cannot be disproved.