Protective Effects of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Against Oxidative Stress in Zebrafish Hair Cells.

Natalia Kasica,Dora Reglodi,Jerzy Kaleczyc,Andrea Tamas,Maria Sundvik,Piotr Podlasz

doi:10.1007/s12640-016-9659-8

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide, with known antiapoptotic functions. Our previous in vitro study has demonstrated the ameliorative role of PACAP-38 in chicken hair cells under oxidative stress conditions, but its effects on living hair cells is now yet known. Therefore, the aim of the present study was to investigate in vivo the protective role of PACAP-38 in hair cells found in zebrafish (Danio rerio) sense organs—neuromasts. To induce oxidative stress the 5-day postfertilization (dpf) zebrafish larvae were exposed to 1.5 mM H2O2 for 15 min or 1 h. This resulted in an increase in caspase-3 and p-38 MAPK level in the hair cells as well as in an impairment of the larvae basic behavior. To investigate the ameliorative role of PACAP-38, the larvae were incubated with a mixture of 1.5 mM H2O2 and 100 nM PACAP-38 following 1 h preincubation with 100 nM PACAP-38 only. PACAP-38 abilities to prevent hair cells from apoptosis were investigated. Whole-mount immunohistochemistry and confocal microscopy analyses revealed that PACAP-38 treatment decreased the cleaved caspase-3 level in the hair cells, but had no influence on p-38 MAPK. The analyses of basic locomotor activity supported the protective role of PACAP-38 by demonstrating the improvement of the fish behavior after PACAP-38 treatment. In summary, our in vivo findings demonstrate that PACAP-38 protects zebrafish hair cells from oxidative stress by attenuating oxidative stress-induced apoptosis.

Highlights

Oxidative stress is a pathological state when reactive oxygen species (ROS), products of normal cellular metabolism, are overproduced and antioxidant defenses are not sufficiently efficient (Valko et al 2007)
The exposure to 2.5 mM H2O2 resulted in the amount of caspase-3 immunoreactive (IR) hair cells within the neuromast comparable with 1.5 mM exposure, the hair cells rosette remained much more disintegrated, resulting in higher amount of hair cells occurring outside the neuromast (Fig. 2e)
Apoptotic events appeared within other cell types, suggesting that 5 mM H2O2 concentration is too strong and not specific for affecting only the neuromast hair cells (Fig. 2g)

Summary

Introduction

Oxidative stress is a pathological state when reactive oxygen species (ROS), products of normal cellular metabolism, are overproduced and antioxidant defenses are not sufficiently efficient (Valko et al 2007). One of the cell death types is apoptosis, initiated by several proapoptotic factors including p-38 MAPK and executive caspase-3. There are many types of insult inducing oxidative stress in hair cells resulting in apoptosis and ototoxicity. They include heavy metals (Olivari et al 2008), aminoglycosides (Ylikoski et al 2002; Jiang et al 2005), cisplatin (Alam et al 2000), as well as noise (Henderson et al 2006). Another ubiquitous ototoxic initiator is hydrogen peroxide (H2O2). The intervention strategy against oxidative stress-induced ototoxicity could be the administration of antioxidant or antiapoptotic drugs (Seidman and Vivek 2004)

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