Our previous study has demonstrated that instead of neuromuscular blockage
cartap, an organonitrogen
insecticide, could cause a marked irreversible Ca2+-dependent
contracture in both isolated mouse and rabbit phrenic nerve-diaphragms. We further examined the potential of direct myocytotoxicity of
cartap and the possible roles of
calcium ion and oxidative stress on
cartap-induced muscle cell injury using the mouse myoblast cell line, C2C12.
Cartap exerted a dose- and time-dependent cytotoxic effect in C2C12 cells measured by MTT colorimetric assay and
trypan blue dye exclusion. The extracellular activities of both
creatine kinase (CK) and
lactate dehydrogenase (LDH) were elevated in the
cartap-treated groups at or greater than 100 microM. The isoenzymatic profiles showed that the elevations were mainly due to CK-3,
LDH-3, and
LDH-4. Following the addition of 0.5-2.5mM
EGTA, a Ca2+
chelator, or 30-100 microM
verapamil, an L-type Ca2+ channel blocker, the
cartap-induced reduction in MTT metabolic rate of C2C12 cells was significantly restored in a dose-dependent manner in both
EGTA and
verapamil-treated cells. Furthermore,
EGTA could significantly reduce the
cartap-induced elevation in the levels of total extracellular CK and LDH activities. Additionally,
cartap significantly increased the level of endogenous
reactive oxygen species (ROS) in C2C12 cells in a dose- and time-dependent manner. The
cartap-induced ROS generation could be significantly inhibited by
antioxidants, including
Vitamins C and E,
catalase, and
superoxide dismutase, with
catalase the most effective.
EGTA could significantly inhibit
cartap-induced ROS generation in a dose-dependent manner. The results suggested that
cartap could induce ROS generation in C2C12 cells via a Ca2+-dependent mechanism resulting in subsequent cytotoxicity, at least partially, to C2C12 cells. It is speculated that both Ca2+ and Ca2+-induced ROS may also play the central role on the myogenic
contracture and myofiber injury of the diaphragm leading to
respiratory failure and subsequent death in rabbits exposed ocularly to
cartap.