Lindane increases in vitro respiratory burst activity and intracellular calcium levels in rainbow trout ( Oncorhynchus mykiss) head kidney phagocytes

Abstract

Phagocytic cells are the main actors of the fish immune system. They secrete reactive oxygen species (ROS) involved in their bactericidal activity. The effects of lindane on ROS production in rainbow trout phagocytes are contradictory. Here, we study the effects of high concentrations of lindane on ROS production (by chemiluminescence) and on intracellular calcium levels ([Ca 2+] i) (by spectrofluorimetry) in trout phagocytes. In these cells, lindane from 2.5 to 10 μM, increases ROS production and has no effect on [Ca 2+] i. From 25 to 200 μM, lindane leads to a rise in ROS production (maximal value measured: 41 152±6253 RLU for 100 μM lindane) associated with an increase in [Ca 2+] i (+3149±96 nM for 100 μM lindane) and with cytotoxicity which appears 2 min after addition of 100 μM lindane (25.4±3.75%; P<0.05). In the absence of extracellular calcium, ROS production of lindane-treated cells remains significantly higher than in controls (maximal value measured: 1899±254 RLU for 25 μM lindane), a significant decrease in [Ca 2+] i is observed in cells treated with 5 or 10 μM lindane (−54±35 nM for 10 μM lindane), and an increase in [Ca 2+] i in cells treated with 100 μM lindane (330±33 nM). The rise in [Ca 2+] i induced by lindane is inhibited when cells are preincubated with thapsigargin (Thaps). We conclude that lindane induces an increase in [Ca 2+] i by mobilising the endoplasmic reticulum calcium store. High concentrations of lindane (>50 μM) alter Ca 2+ homeostasis in the absence of extracellular Ca 2+, confirming that lindane can affect other intracellular stores of Ca 2+. At low concentrations (<25 μM), lindane stimulates ROS production by Ca 2+-independant mechanisms without inducing cytotoxicity. From 25 μM, lindane increases [Ca 2+] i and maximal cytotoxicity appears from 100 μM lindane. Lindane toxicity in fish phagocytes may be associated with high [Ca 2+] i and high ROS production. Thus, ROS are beneficial in protection of the organism but when ROS are produced in excess, they can be toxic for cells and tissues.