Comparison of Chlorantraniliprole and Flubendiamide Activity Toward Wild-Type and Malignant Hyperthermia-Susceptible Ryanodine Receptors and Heat Stress Intolerance

Abstract

Chlorantraniliprole (CP) and flubendiamide (FD) are widely used in agriculture globally to control lepidopteran pests. Both insecticides target ryanodine receptors (RyRs) and promote Ca 2þ leak from sarcoplasmic reticulum (SR) within insect skeletal muscle yet are purportedly devoid of activity toward mammalian RyR1 and muscle. RyRs are ion channels that regulate intracellular Ca 2þ release from SR during physiological excitation-contraction coupling. Mutations in RYR1 genes confer malignant hyperthermia susceptibility (MHS), a potentially lethal pharmacogenetic disorder in humans and animals. Compared with vehicle control, CP (10 mM) triggers a 65-fold higher rate of Ca 2þ efflux from Ca 2þ-loaded mammalian WT-RyR1 SR vesicles, whereas FD (10 mM) produces negligible influence on Ca 2þ leak. We, therefore, compared whether CP or FD differentially influence patterns of high-affinity [ 3 H]ryanodine ([ 3 H]Ry) binding to RyR1 isolated from muscle SR membranes prepared from adult C57BL/6J mice expressing WT, homozygous C-terminal MHS mutation T4826I, or heterozygous Nterminal MHS mutation R163C. Basal [ 3 H]Ry binding differed among genotypes with rank order T4826I)R163C$WT, regardless of [Ca 2þ ] in the assay medium. Both CP and FD (0.01-100 mM) elicited concentration-dependent increase in [ 3 H]Ry binding, although CP showed greater efficacy regardless of genotype or [Ca 2þ ]. Exposure to CP (500 mg/kg; p.o) failed to shift intolerance to heat stress (38 C) characteristic of R163C and T4826I MHS mice, nor cause lethality in WT mice. Although nM-mM of either diamide is capable of differentially altering WT and MHS RyR1 conformation in vitro, human RyR1 mutations within putative diamide N-and C-terminal interaction domains do not alter heat stress intolerance (HSI) in vivo.

Interactions of Dichlorodiphenyltrichloroethane (DDT) and Dichlorodiphenyldichloroethylene (DDE) With Skeletal Muscle Ryanodine Receptor Type 1

“…1A-D) were tested to determine their ability to enhance binding of [ 3 H]Ry to RyR1-enriched JSR membrane preparations in the presence of low nanomolar concentration of [ 3 H]Ry. Allosteric modulation of RyR conformation mediated by pharmacological or toxicological agents has been widely investigated with the use of low nanomolar [ 3 H]Ry due to its ability to bind to the open state of RyR (Morisseau et al, 2009;Truong and Pessah, 2018). All 4 DDx compounds stimulated [ 3 H]Ry binding in a concentration-dependent manner with o,p 0 -DDE exhibiting greatest apparent potency (EC 50 ) and efficacy (maximum binding) ( Figure 2).…”

“…Preparation of RyR1-enriched junctional sarcoplasmic reticulum membrane from rabbit muscles. RyR1-enriched junctional sarcoplasmic reticulum (JSR) membrane fractions were isolated from the back and hind limb skeletal muscles of wild-type male (1.5 kg) New Zealand White rabbits (Charles River, Hollister, California) as previously described in Pessah et al (1986), Saito et al (1984), and Truong and Pessah (2018) to investigate whether o,p 0 -DDT, p,p 0 -DDT, o,p 0 -DDE, or p,p 0 -DDE (DDx, collectively) trigger Ca 2þ release from RyR1. Approximately 200 g of skeletal muscle tissue was flash frozen in liquid nitrogen, grounded into a fine powder with a mortar and pestle, and subsequently homogenized in a Waring blender for 1 min in aqueous homogenization buffer (pH 7.4) containing 300 mM sucrose, 5 mM imidazole, 100 mM PMSF, and 10 mg/ml leupeptin hydrochloride.…”

“…Membrane fraction preparations from mouse skeletal muscle. Crude membrane fraction from the skeletal muscle of 4-to 5-monthold individual male and female wild-type C57BL/6J mice (n ¼ 8 per sex) was prepared as previously described in Truong and Pessah (2018). Limb skeletal muscles were flash frozen, grounded, and placed into ice-cold aqueous buffer (pH 7.4) containing (in mM) 300 sucrose, 10 HEPES, 10 mg/ml leupeptin hydrochloride, 0.1 PMSF, 10 sodium fluoride (NaF), 2 bglycerophosphate, 0.5 mM sodium orthovanadate (Na 3 VO 4 ), and 1.5 ethylene glycol tetraacetic acid (EGTA).…”

“…Macroscopic Ca 21 flux measurements. Net Ca 2þ flux across JSR membrane vesicles were assessed in real time using metallochromic Ca 2þ indicator dye arsenazo III (AsIII) as previously described in Feng et al (2017) and Truong and Pessah (2018). JSR membrane vesicles at 100 mg/ml were added into 35 C cuvettes containing aqueous medium (pH 7.4) consisting of (in mM) 100 KCl, 6 sodium pyrophosphate, 0.25 AsIII, and 20 4-morpholinepropanesulfonic acid (MOPS), 2 Mg 2þ ATP, 10 phosphocreatine, and 20 mg/ml creatine phosphokinase.…”

“…[ 3 H]Ry-binding analysis. Specific [ 3 H]Ry binding to either rabbit JSR membrane preparations or mouse skeletal muscle homogenates were measured as previously described in Pessah and Zimanyi (1991) and Truong and Pessah (2018). Rabbit JSR RyR1enriched membrane preparations were used to establish concentration-effect curves for o,p 0 -DDT, p,p 0 -DDT, o,p 0 -DDE, and p,p 0 -DDE (DDx, collectively) to determine both efficacy and potency; whereas, mouse skeletal muscle microsomal preparations were used to increase power and to investigate sex differences in DDx potency toward RyR1.…”