Extracellular calcium is required for the polychlorinated biphenyl-induced increase of intracellular free calcium levels in cerebellar granule cell culture
Introduction
Accidental exposure to high levels of polychlorinated biphenyls (PCBs) and dibenzofurans in contaminated rice oil in Japan and Taiwan indicated that neurological disorders can result from human exposure to PCBs both as an adult (Kuratsune et al., 1972, Hsu et al., 1985) and during development (Rogan et al., 1988, Chen and Hsu, 1994). Of particular concern are studies indicating that exposure to environmental levels of PCBs are associated with impairment of neurological and cognitive development in infants which may be long lasting (Jacobson et al., 1985, Jacobson et al., 1990, Gladen et al., 1988, Rogan and Gladen, 1992, Jacobson and Jacobson, 1993, Jacobson and Jacobson, 1996). Animal studies have also demonstrated the neurotoxic potential of PCBs. Perinatal exposure of laboratory animals to PCBs result in neurobehavioral effects including motor dysfunction (Tilson et al., 1979, Pantaleoni et al., 1988) and cognitive deficits (Levin et al., 1988, Lilienthal and Winneke, 1991, Schantz et al., 1991, Schantz et al., 1995). Neurochemically, PCBs can decrease neurotransmitter and receptor levels after both adult (Seegal et al., 1985, Seegal et al., 1986, Seegal et al., 1991) and developmental exposure (Agrawal et al., 1981, Eriksson, 1988).
The mechanism and structure-activity relationships which underlie the neurotoxicity of PCBs are not clear; however there is accumulating evidence that the site of action may be distinct from the aryl-hydrocarbon (Ah) receptor which is thought to mediate much of the toxicity of the dioxin-like coplanar PCBs (Safe, 1990, Li and Hansen, 1997). PCB congeners with ortho-substitutions that favor a non-coplanar conformation of the phenyl rings appear to have a greater neurotoxic potential in a number of systems. In vivo, a decrease in brain dopamine levels was related to the degree of exposure to ortho-substituted PCB congeners (Seegal et al., 1990, Seegal et al., 1991). Subsequent in vitro studies in PC12 cells found that ortho-substituted congeners were more potent in decreasing dopamine levels than meta- or para-substituted congeners (Shain et al., 1991). Recent in vitro studies from the laboratory examining calcium-related signaling in primary cultures of cerebellar granule cells support the structure-activity relationships reported by Shain et al. (1991). It has been reported that PCB congeners with ortho- or ortho-lateral (meta, para) chlorine substitutions were more potent than non-ortho congeners in causing calcium-dependent translocation of protein kinase C (PKC; Kodavanti et al., 1994, Kodavanti et al., 1995). A similar structure-activity relationship was found for the inhibition of calcium sequestration by PCB congeners in microsomes and mitochondria isolated from cerebellum (Kodavanti et al., 1993, Kodavanti et al., 1996). It was found that the processes involved in maintaining calcium homeostasis are very sensitive to perturbation by ortho-substituted PCBs. The initial studies indicated that 2,2′-dichlorobiphenyl (DCB) inhibited synaptosomal Ca2+-ATPase activity and 45Ca2+ sequestration by mitochondria and microsomes isolated from rat cerebellum with IC50 values below 10 μM (Kodavanti et al., 1993). The non-ortho-substituted 3,3′,4,4′,5-pentachlorobiphenyl (PeCB) was much less potent. Further work confirmed the structure-activity relationship for the inhibition of 45Ca2+ sequestration in mitochondria and microsomes by PCBs (Kodavanti et al., 1996). Others have reported that ortho-substituted PCBs can perturb calcium homeostasis by acting at the ryanodine receptor (Wong and Pessah, 1996).
The actions of ortho-substituted PCBs on processes involved in maintaining calcium homeostasis would be expected to lead to changes in intracellular free calcium ([Ca2+]i). Using the fluorescent probe Fluo-3 in cerebellar granule cell cultures, it was observed that 2,2′-DCB resulted in a rise in [Ca2+]i which was concentration-dependent and increased with the duration of exposure, while 3,3′,4,4′,5-PeCB produced a smaller increase which was not concentration- or time-dependent (Kodavanti et al., 1993). In the same study prolonged incubation with 2,2′-DCB, but not 3,3′,4,4′,5-PeCB, was cytotoxic. Similar studies in human granulocytes using the fluorescent probe Fura-2 showed that ortho-substituted PCBs increased [Ca2+]i in a concentration-dependent manner (Voie and Fonnum, 1998). In the present study the effects of ortho-substituted PCBs on [Ca2+]i and the relationship of this effect to the inhibition of mechanisms which maintain calcium homeostasis were examined further. The use of the fluorescent probe Fura-2 in the present study allowed us to determine quantitatively the effects of selected PCBs on [Ca2+]i in primary cultures of rat cerebellar granule cells. Cerebellar granule cell cultures provide a homogenous population of cells (>95% neurons) in which intracellular calcium responses have been well characterized.
Section snippets
Chemicals and test solutions
2,2′-DCB (IUPAC #4), 4,4′-DCB (IUPAC #15), 2,2′,4,6,6′-PeCB (IUPAC #104), and 3,3′,4,4′,5-PeCB (IUPAC #126) (purity>99%) were purchased from AccuStandard (New Haven, CT). Stock solutions of PCB congeners were prepared in dimethyl sulfoxide (DMSO) and aliquots (3 μl/3 ml) were added to the appropriate buffer solution to achieve the final concentration. DMSO (3 μl/3 ml) was used as a control and had no effect on any measure.
Animals
Timed pregnant female (16 days gestation) Long–Evans rats were obtained
Effects of PCBs on [Ca2+]i
To examine the time-course and concentration-response to an ortho-substituted PCB, cerebellar granule cells were loaded with fura-2, washed, then exposed to 0–50 μM 2,2′-DCB for 30, 60, or 120 min. At the end of the exposure period the cells were mounted in the recording chamber and [Ca2+]i determined. The DMSO vehicle (0.1%) had no effect on basal [Ca2+]i, which ranged from 20 to 60 nM. As shown in Fig. 1, there was concentration-dependent increase in [Ca2+]i which was significant after
Discussion
There is now evidence from a number of different laboratories indicating that non-coplanar PCBs, which do not effectively compete with dioxin at the Ah receptor, can affect calcium-dependent signal transduction. Kodavanti et al., 1993, Kodavanti et al., 1996 reported that ortho-substituted, non-coplanar PCBs inhibited calcium sequestration in mitochondria and microsomes isolated from rat cerebellum. Other studies using cerebellar granule cell cultures have shown that non-coplanar PCBs increase
Acknowledgements
The authors wish to express their appreciation for the excellent technical assistance of Ms Theresa Freudenrich, Ms Ethel Derr-Yellin, and Ms Sue Willig and thank Thomas R. Ward and Richard Seegal for their comments and suggestions on an earlier version of this paper. This article has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, and is approved for publication. Mention of trade names or commercial products does not
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