SPERTUS, M.S.*; JULIAN, D.; University of Florida; University of Florida: Cellular oxidative stress in polychaete coelomocytes during in vitro exposure to multiple abiotic stressors
In coastal environments, variations in temperature, salinity, pH and H2O2 may occur alone or in combination. The impact of these abiotic stressors on cellular oxidative stress (OS) in marine invertebrates is still poorly understood, and whether simultaneous exposure to multiple stressors causes interaction effects on OS is even less clear. We examined the effect of temperature, both alone and in combination with other abiotic stressors, on OS in coelomocytes from the marine polychaete Glycera dibranchiata. Coelomocytes were isolated, diluted in buffer and exposed for 3 h to a range of temperatures (5, 15, 25, 35, and 45 C) with gradations of salinity (100, 80, 60, 55, 50, 45, 40, and 20% seawater), pH (6.4-8.4 in 0.2 pH unit increments) and H2O2 (0.01, 0.1 and 1 mM), each with independent replicates of 4-5 worms. After exposure, cell survival was quantified by cell count, overall cellular OS was quantified fluorometrically with carboxy-H2DCFDA, and mitochondrial superoxide (O2–) production was quantified fluorometrically with MitoSOX Red. When present alone, elevated temperature increased OS 1.5-fold and O2– production 1.7-fold. Salinity alone had no effect on OS or O2– production and there was no interaction between salinity and temperature. When applied alone, elevated pH had a very small effect on OS and O2– (less than 1.1 fold), but elevated pH in combination with elevated temperature increased OS and O2– production by 1.7- and 2.4-fold, respectively. H2O2 alone increased OS and O2– production by 1.7-fold, but in combination with elevated temperature, the increase was 2.5-fold and 4.0-fold, respectively. These data suggest that elevated temperature has a strong direct effect and a strong interaction effect on free radical production and OS in vitro, and point to the importance of evaluating abiotic stressors in combination.
