Environmental Chemicals Compete for 2‐[125I]‐Iodomelatonin Binding to Melatonin Receptors in Brain Slices from C3H/HeN Mice

Abstract

Melatonin binds to MT1 and MT2 G protein‐coupled receptors to modulate physiological responses. In‐silico molecular modeling and in‐vitro bioassays support that a common carbamate pesticide, carbaryl, acts as a putative melatonin receptor antagonist at MT1 and agonist at MT2 (Popovska‐Gorevski et al., FASEB J 2014; 28: 662.12) while common plasticizers (phthalate esters) act as inverse agonists at MT1 and antagonists at MT2 (Jones et al. EB Abstract 2017). Due to the persistent and widespread use of phthalates (plastic, vinyl, and leather products) and carbaryl (agriculture), humans are exposed daily to low levels of these compounds in household, food, and water products. Evidence indicates that environmental exposure to pesticides or phthalates are linked to metabolic disorders, though it is not clear which biological targets contribute to disease pathology (Montgomery et al., Am J Epidemiol. 2008, 167[10]: 1235–1246; James‐Todd et al., Environ Int. 2016, 96:118–126). This study assessed the ability and specificity of carbaryl and several commonly used phthalate esters to compete for 2‐[125I]‐iodomelatonin binding in adjacent coronal brain slices (20μm) from C3H/HeN mice using quantitative receptor autoradiography following in‐vitro or ex‐vivo treatment. Optical density (OD) values and specific binding were determined from autoradiograms in brain regions of interest (suprachiasmatic nucleus [SCN] and paraventricular nucleus of the thalamus [PVNT]) known to modulate circadian rhythms, metabolic, behavioral, and cardiovascular responses through actions at melatonin receptors. Environmental chemicals (dibutyl phthalate [DBP], carbaryl, diphenyl phthalate [DPP], benzyl butyl phthalate [BzBP], and diethyl phthalate [DEP]; 1, 10, 100 μM) competed in a concentration dependent manner for 2‐[125I]‐iodomelatonin (75pM) binding to melatonin receptors in brain slices in vitro. The concentrations of environmental chemicals inhibiting binding by 50% (IC50) were 1.31, 1.71, 1.82, 3.76, and 42.1 μM in the SCN and 1.52, 3.75, 1.60, 1.99, and 28.8 μM in the PVNT respectively. In‐vivo administration of carbaryl (10mg/kg, ip) significantly reduced specific 2‐[125I]‐iodomelatonin binding (50pM) OD in SCN brain slices processed ex‐vivo at 30 (mean ± SEM = 1.7 ± 0.12, n=5, p<0.01), 60 (1.5 ± 0.10, n=6, p<0.001), and 120 (1.8 ± 0.06, n=4, p<0.05) minutes post administration compared to vehicle treated mice (2.4 ± 0.15, n=6, One‐Way ANOVA [F5,22=10.14], p<0.05; Dunnet's Post Test), which recovered by 240 minutes (2.5 ± 0.16, n=4). Phthalate diesters (DBP, DPP, BzBP, and DEP) competed for melatonin receptor binding in the SCN and PVNT in a concentration dependent manner, however a phthalate monoester (monobenzyl phthalate) did not (IC50 > 100 μM). Carbaryl competed both in‐vitro (SCN and PVNT) and ex‐vivo (SCN) for melatonin receptor binding in brain slices from C3H/HeN mice demonstrating the ability to reach target areas in the brain responsible for the control of circadian rhythms. Environmental exposure to phthalates or carbaryl may disrupt time of day messages conveyed by endogenous melatonin to its receptors in target tissues such as the SCN and PVNT.Support or Funding InformationSupported by ES 023684 and JSMBS funds to MLD and RVR.