Egr1 Gene Expression as a Potential Biomarker for In Vitro Prediction of Ocular Toxicity.

Da-Bin Hwang,Young-Jin Chun,Shin-Young Kim,Kyung-Min Lim,Jong-Hwan Park,Changuk Kim,Dong-Hoon Won,Jun-Won Yun,Yoo-Sub Shin

doi:10.3390/pharmaceutics13101584

Abstract

Animal models are used for preclinical toxicity studies, and the need for in vitro alternative methods has been strongly raised. Our study aims to elucidate the potential mechanism of change in EGR1 expression under situations of toxic injury and to develop an Egr1 promoter–luciferase gene reporter assay for an in vitro alternative method for toxicity prediction in drug discovery. We first found an increase in early growth response-1 (EGR1) mRNA/protein expressions in the liver and kidney of cisplatin-treated injured rats. Additionally, the EGR1 protein level was also elevated under situations of ocular injury after sodium lauryl sulfate (SLS) eye drops. These in vivo observations on injury-related EGR1 induction were confirmed by in vitro studies, where human corneal epithelial cells were treated with representative irritants (SLS and benzalkonium chloride) and 17 chemicals having different UN GHS irritant categories. Additionally, our results suggest the involvement of ERK, JNK, p38 MAPK pathways in EGR1 elevation in response to gamma-butyrolactone-induced injury. As EGR1 is considered to be a pivotal factor in proliferation and regeneration, siRNA-mediated knockdown of Egr1 promoted cytotoxic potential through a delay of injury-related recovery. More importantly, the elevation of promoter activities was observed by various irritants in cells transfected with Egr1 promoter-reporter vector. In conclusion, Egr1 can be a potential biomarker in a promoter-reporter system to improve the accuracy of in vitro predictions for ocular irritation.

Highlights

As animal models share similar biological pathways with humans, preclinical pharmacology and toxicology testing must be performed inevitably on these models before the clinical phase on humans for approval by regulatory agencies [1]
(0.625 × 10−3 %) and GB (1.92%) showing almost normal viability, Small Interfering RNA (siRNA) did not influence the cytotoxicity. These results indicate that the early growth response-1 (Egr1) knockdown by siRNA increases the which is produced by the Jun N-terminal kinases (JNK) signaling pathway and inhibits thethe pathway by susceptibility of eye irritation; the change in early growth response-1 (EGR1) caused by irritants is likely dephosphorylating pERK [36,37,38], was reduced by GB treatment in human corneal epitheto be a compensatory upregulation for the tissue repair process
Gene expression screening for ocular irritation potential can enhance the prediction rate of a prediction model based on cell viability [23]

Summary

Introduction

As animal models share similar biological pathways with humans, preclinical pharmacology and toxicology testing must be performed inevitably on these models before the clinical phase on humans for approval by regulatory agencies [1]. As an essential part of a comprehensive toxicity program in the processes of drug discovery and development, the degree of eye and skin irritation for new chemicals, drugs, and cosmetics should be measured before exposure to humans [2]. The Draize rabbit eye irritation test has been commonly used, in which chemicals are applied directly into the rabbit’s eyes to observe changes in eye state [3,4]. One of the most important in vitro alternative tests is the hen’s egg test–chorioallantoic membrane test. This test evaluates the blood vessel reaction of the chorioallantoic membrane after exposure to liquid or solid test materials using fertilized chicken eggs [7,8]

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