Heat stress induces ROS production and histone phosphorylation in celomocytes of Eisenia hortensis
Keywords:reactive oxygen species (ROS), γH2AX, histone phosphorylation, heat stress, in vitro cell culture
The effect of heat stress on celomocytes (leukocytes) from Eisenia hortensis was investigated by measuring the production of reactive oxygen species (ROS). After culturing celomocytes at
temperatures ranging from 4 °C (control) to 44 °C for 3-16 h, ROS levels were measured using a flow cytometric method employing dihydrorhodamine 123 (DHR123) for ROS detection and 7-
aminoactinomycin D (7-AAD) as a viability stain. Reproducibly we observed significant (p < 0.05)
increases in ROS production and decreases in cell viability at temperatures of 28 °C and above. We then examined the effect of heat stress on histone phosphorylation employing antibodies specific for γH2AX as an indicator of histone modification. Celomocytes were incubated at temperatures ranging between 20 °C to 35 °C for 16 h and antibodies specific for phosphorylated serines in H2AX histones were employed through flow cytometric analysis. Comparing controls to heat-stressed samples using three separate assays reproducibly confirmed significant H2AX phosphorylation (p < 0.05). Collectively, these results emphasize the importance of selecting appropriate temperatures for rearing invertebrates in laboratory-based habitats and for culturing invertebrate cells when conducting in vitro assays in order to minimize oxidative stress. The possible cellular effects of heat stress in soil ecosystems associated with global warming events is also considered.