Effects of environmental pollutants on calcium release and uptake by rat cortical microsomes

Abstract

Dysregulation of neuronal intracellular Ca²⁺ homeostasis can play a crucial role in many neurotoxic effects, including impaired brain development and behavioral dysfunctions. This study examined 40 suspected neurotoxicants from different chemical classes for their capacity to alter Ca²⁺ release and uptake from rat cortical microsomes. First, ten suspected neurotoxicants have been tested using a well-established cuvette-based Ca²⁺ flux assay. Five out of ten compounds (TOCP, endosulfan, PCB-95, chlorpyrifos and BDE-49) showed a significant, concentration-dependent alteration of Ca²⁺ release and uptake in adult rat cortical microsomes. The original cuvette assay was downscaled and customized to a fast, higher throughput microplate method and the 40 suspected neurotoxicants were screened for their effects on intracellular Ca²⁺homeostasis. In decreasing order of potency, the 15 test compounds that showed the strongest alteration of Ca²⁺ levels in adult rat microsomes were TOCP, endosulfan, BDE-49, 6-OH-BDE-47, PCB-95, permethrin, alpha-cypermethrin, chlorpyrifos, bioallethrin, cypermethrin, RDP, DEHP, DBP, BDE-47, and PFOS. Results from co-exposure experiments with selective inhibitors suggested that for some compounds Ca²⁺ releasing effects could be attributed to RyR activation (PFOS, DBP, and DEHP) or to SERCA inhibition (a potential novel mechanism of action for all four tested pyrethroid insecticides). The effects of the two most potent compounds, endosulfan and TOCP, were not blocked by any of the inhibitors tested, indicating other possible mechanism of action. For all other potent test compounds, a combined effect on RyR, IP₃R, and/or SERCA has been observed. PFOS and 6-OH-BDE-47 caused increased Ca²⁺ release from adult but not from neonatal rat brain microsomes, indicating age-dependent difference in susceptibility to these test compounds. The current study suggests that the neurotoxic potential of compounds belonging to different chemical classes could partly be attributed to the effects on intracellular Ca²⁺ release and uptake. Although further validation is required, the downscaled method developed in this study presents technical advance that could be used for the future screening of suspected intracellular Ca²⁺ disruptors.

Keywords: Calcium homeostasis; Environmental pollutants; IP(3) receptor; Microsomes; Neurotoxicity; Ryanodine receptor; SERCA.