Additivity of Pyrethroid Actions on Sodium Influx in Cerebrocortical Neurons in Primary Culture

Abstract

Background: Pyrethroid insecticides bind to voltage-gated sodium channels and modify their gating kinetics, thereby disrupting neuronal function. Although previous work has tested the additivity of pyrethroids in vivo, this has not been assessed directly at the primary molecular target using a functional measure.Objectives: We investigated the potency and efficacy of 11 structurally diverse food-use pyrethroids to evoke sodium (Na+) influx in neurons and tested the hypothesis of dose additivity for a mixture of these same 11 compounds.Methods: We determined pyrethroid-induced increases in Na+ influx in primary cultures of cerebrocortical neurons using the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI). Concentration-dependent responses for 11 pyrethroids were determined, and the response to dilutions of a mixture of all 11 compounds at an equimolar mixing ratio was assessed. Additivity was tested assuming a dose-additive model.Results: Seven pyrethroids produced concentration-dependent, tetrodotoxin-sensitive Na+ influx. The rank order of potency was deltamethrin > S-bioallethrin > β-cyfluthrin > λ-cyhalothrin > esfenvalerate > tefluthrin > fenpropathrin. Cypermethrin and bifenthrin produced modest increases in Na+ influx, whereas permethrin and resmethrin were inactive. When all 11 pyrethroids were present at an equimolar mixing ratio, their actions on Na+ influx were consistent with a dose-additive model.Conclusions: These data provide in vitro relative potency and efficacy measurements for 7 pyrethroid compounds in intact mammalian neurons. Despite differences in individual compound potencies, we found the action of a mixture of all 11 pyrethroids to be additive when we used an appropriate statistical model. These results are consistent with a previous report of the additivity of pyrethroids in vivo.

Evidence for dose-additive effects of a type II pyrethroid mixture. In vitro assessment

“…Alterations in NO and MDA production by a mixture of Type II pyrethroids can be predicted using this dose-additive model. The results of the present study are consistent with previous report of the additivity of pyrethroids in vivo (Wolansky et al, 2009) and in vitro (Cao et al 2011b) and suggest that doseadditive approaches should be used when assessing the risk of exposures to chemical mixtures that contain Type II pyrethroids. Further studies are required for the validation of this experimental in vitro model for toxicity testing of those pyrethroid mixtures potentially involved in human exposures from dietary and environmental residues.…”

“…Comprensive data sets from other in vitro studies permiting determination of relative potencies of pyrethroids are generally lacking. It was demonstrated that 11 pyrethroids in a manner concentration-dependent increase in Ca 2 þ influx (Cao et al, 2011a) as well as Na þ influx (Cao et al, 2011b) in cerebrocortical neurons; the order of potency was deltamethrin 4S-bioallethrin 4 β-cyfluthrin4λ-cyhalothrin4esfenvalerate, and four compounds cypermethrin, bifenthrin, permethrin and resmethrin did not produce significant concentration-response profiles. These authors demonstrated that alterations in Na þ influx by a mixture of food-use pyrethoids can be predicted using a dose-additive model, results consistent with a previous report of the additivity of pyrethroids in vivo (Wolansky et al, 2009).…”

“…activation of many neural pathways and processes decrease motor activity). Recently, Cao et al (2011aCao et al ( , 2011b) evaluated directly at the primary molecular target the potency and efficacy of the same 11 pyrethroids determining the sodium (Na þ ) influx in cerebrocortical neuron cultures. The results demonstrated that they stimulate TTX-sensitive Na þ influx in primary cultures of cerebrocortical neurons with differing potencies and efficacies.…”

“…Of the 11 pyrethroids examined, 7 pyrethroids [Type I (S-bioallethrin and tefluthrin) and Type II (β-cyfluthrin, deltamethrin, esfenvalerate, fenpropathrin, and λ-cyhalothrin) pyrethroids] caused concentration-dependent increases in Na þ influx; whereas 2 pyrethroids (bifenthrin and cypermethrin, Type I and Type II pyrethroids, respectively) caused small increases and 2 pyrethroids (permethrin and resmethrin, Type I pyrethroids) were without effect. Despite differences in individual compound potencies, it was demonstrated dose additivity for a mixture of these same 11 compounds (Cao et al, 2011a(Cao et al, , 2011b. In order to obtain complementary data on additivity of pyrethroid actions as well as potential uses of different neurotoxicity in vitro tests, in the present study we investigated in SH-SY5Y, HepG2 and Caco-2 human cells: (1) the relative potency of 6 Type II pyrethroids (α-cypermethrin, cyfluthrin, λ-cyhalothrin, deltamethrin, cyphenothrin and esfenvalerate) to induce cytotoxicity determining dose-response curves for cell viability MTT and LDH assays, and (2) the dose-additive effects of the mixture of the same 6 pyrethroids on levels of nitric oxide (NO) and lipid peroxides measured as malondialdehyde (MDA) on the basis that induction of oxidative stress is a key element in pyrethroid-mediated neurotoxicity (El-Gohary et al, 1999;Kale et al, 1999;Giray et al, 2001;El-Demerdash, 2007;Romero et al, 2012).…”

“…In order to obtain complementary data on additivity of pyrethroid actions as well as potential uses of different neurotoxicity in vitro tests, in the present study we investigated in SH-SY5Y, HepG2 and Caco-2 human cells: (1) the relative potency of 6 Type II pyrethroids (α-cypermethrin, cyfluthrin, λ-cyhalothrin, deltamethrin, cyphenothrin and esfenvalerate) to induce cytotoxicity determining dose-response curves for cell viability MTT and LDH assays, and (2) the dose-additive effects of the mixture of the same 6 pyrethroids on levels of nitric oxide (NO) and lipid peroxides measured as malondialdehyde (MDA) on the basis that induction of oxidative stress is a key element in pyrethroid-mediated neurotoxicity (El-Gohary et al, 1999;Kale et al, 1999;Giray et al, 2001;El-Demerdash, 2007;Romero et al, 2012). The Type II pyrethroids used in the present study were selected on the basis that the same compounds, except cyphenothrin, produced a concentration-dependent elevation in neuronal Na þ influx (Cao et al, 2011a(Cao et al, , 2011b, as well as on biomonitoring data from human exposure (Food and Drug Administration FDA, 2011; Centre for Disease Control and Prevention CDC, 2011).…”