Methoxychlor and Vinclozolin Induce Rapid Changes in Intercellular and Intracellular Signaling in Liver Progenitor Cells

Abstract

Methoxychlor (MXC) and vinclozolin (VIN) are well-recognized endocrine disrupting chemicals known to alter epigenetic regulations and transgenerational inheritance; however, non-endocrine disruption endpoints are also important. Thus, we determined the effects of MXC and VIN on the dysregulation of gap junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells. Both chemicals induced a rapid dysregulation of GJIC at non-cytotoxic doses, with 30 min EC 50 values for GJIC inhibition being 10 mM for MXC and 126 mM for VIN. MXC inhibited GJIC for at least 24 h, while VIN effects were transient and GJIC recovered after 4 h. VIN induced rapid hyperphosphorylation and internalization of gap junction protein connexin43, and both chemicals also activated MAPK ERK1/2 and p38. Effects on GJIC were not prevented by MEK1/2 inhibitor, but by an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), resveratrol, and in the case of VIN, also, by a p38 inhibitor. Estrogen (ER) and androgen receptor (AR) modulators (estradiol, ICI 182,780, HPTE, testosterone, flutamide, VIN M2) did not attenuate MXC or VIN effects on GJIC. Our data also indicate that the effects were elicited by the parental compounds of MXC and VIN. Our study provides new evidence that MXC and VIN dysregulate GJIC via mechanisms involving rapid activation of PC-PLC occurring independently of ER-or AR-dependent genomic signaling. Such alterations of rapid intercellular and intracellular signaling events involved in regulations of gene expression, tissue development, function and homeostasis, could also contribute to transgenerational epigenetic effects of endocrine disruptors.

Improved multiparametric scrape loading-dye transfer assay for a simultaneous high-throughput analysis of gap junctional intercellular communication, cell density and viability

“…Importantly, we demonstrated applicability of our innovative multiparametric SL-DT assay for variety of in vitro cell types such as liver cells (WB-F344), lung cells (BEAS-2B, HBE1) or testicular cells (TM4 and TM3 cells), which all are GJIC-competent cells commonly used for the assessment of GJIC in response to different chemicals [50][51][52][53][54] or physical treatments 55 . All these cell types express connexin 43 51,56,57 , which is widely expressed in many cell types (especially in the epithelial cells) and whose gap junction channels are diffusible for Lucifer Yellow fluorescent dye 58 .…”

Structure-Dependent Effects of Phthalates on Intercellular and Intracellular Communication in Liver Oval Cells

“…Since MAPKs and their pathways are crucial for healthy liver function [ 49 ] and implicated in the control of GJIC [ 45 ], the changes in the activity of MAPK-Erk1/2 were investigated in response to phthalates in rat liver oval WB-F344 cells. After 0.5 h exposure of WB-F344 cells to 10 nM TPA (12-O-tetradecanoylphorbol 13-acetate), which was used as a positive control and known GJIC inhibitor and tumor promoter, rapidly and significantly increased (~30-fold increase) the levels of phosphorylated, i.e., activated, MAPK-Erk1 and 2 ( Figure 4 ; original, uncropped and unadjusted images in Figures S6–S9 ).…”

“…Connexin 43 is the most abundant and studied connexin type, whose activity and function can be regulated by the direct phosphorylation caused by many kinases, including MAPK-Erk-1/2 [ 39 , 41 , 75 ]. While the activation of MAPK kinases is considered to be one of the mechanisms of GJIC dysregulation [ 41 , 76 , 77 ], it has been reported that various environmental toxicants and endocrine disrupters can dysregulate GJIC via mechanisms independent from the MAPK-Erk1/2 pathway, even when MAPK-Erk1/2 activation is associated with GJIC dysregulation [ 41 , 43 , 44 , 45 ]. Although GJIC-inhibiting phthalates also activated the MAPK-Erk1/2 pathways in our study, the rapid MAPK-Erk1/2 activation caused by phthalates from Groups E and F was not necessarily associated with the rapid inhibition of GJIC.…”

“…The MAPK-Erk-1/2 pathway is known to regulate GJIC in different cell types, including liver oval cells [ 41 ]. Via the disturbance of liver GJIC, toxicants can cause their disruptive effects on epigenetic regulations, homeostatic processes, and healthy liver functions [ 35 , 45 ]. Previously, there was a structure-dependent relationship of phthalates shown to inhibit intercellular communication in mouse hepatocytes [ 46 ].…”

Testimony for veterinary apitherapy

“…Subsequently, animals contract MXC toxicity result several toxicological implications as ovarian toxicity, 77 where it causes impairment of ovarian follicular development and other ovarian function 77,86 leading to reproductive tract malformations in the male offspring. 87 MXC hepto-toxicity results in significant reduction in total serum protein rather than marked elevation in hepatic marker enzymes(ALT, AST & ALP) 76 as well as changes in inter and intra cellular signaling. 88 Histopathologically, there is changes in hepatocytes 76,89 owing to induction changes in mRNA abundance of genes in the liver and testes, 90 where alterations involved in regulations of gene expression, tissue development, function and homeostasis, could also contribute to transgenerational epigenetic effects of endocrine disruptors.…”