chain inhibitors did not produce similar effects. Furthermore, pharmacological inhibition of the mitochondrial permeability transition pore failed to prevent sulfideinduced depolarization. Finally, increased oxidation of the free radical indicators H 2 DCFDA and MitoSOX TM in erythrocytes exposed to sulfide suggests that sulfide oxidation increased oxidative stress and superoxide production, respectively. Together, these results indicate that sulfide exposure causes mitochondrial depolarization in cells of a sulfide-tolerant annelid, and that this effect, which differs from the actions of other COX inhibitors, may be via increased free radical damage.

Hydrogen sulfide acts as an environmental toxin across a range of concentrations and as a cellular signaling molecule at very low concentrations. Despite its toxicity, many animals, including the mudflat polychaete Glycera dibranchiata, are periodically or continuously exposed to sulfide in their environment. We tested the hypothesis that a broad range of ecologically relevant sulfide concentrations induces oxidative stress and oxidative damage to RNA and DNA in G. dibranchiata. Coelomocytes exposed in vitro to sulfide (0–3 mmol L−1 for 1 h) showed dose-dependent increases in oxidative stress (as 2′,7′-dichlorofluorescein fluorescence) and superoxide production (as dihydroethidine fluorescence). Coelomocytes exposed in vitro to sulfide (up to 0.73 mmol L−1 for 2 h) also acquired increased oxidative damage to RNA (detected as 8-oxo-7,8-dihydroguanosine) and DNA (detected as 8-oxo-7,8-dihydro-2′-deoxyguanosine). Worms exposed in vivo to sulfide (0–10 mmol L−1 for 24 h) acquired elevated oxidative damage to RNA and DNA in both coelomocytes and body wall tissue. While the consequences of RNA and DNA oxidative damage are poorly understood, oxidatively damaged deoxyguanosine bases preferentially bind thymine, causing G-T transversions and potentially causing heritable point mutations. This suggests that sulfide can be an environmental mutagen in sulfide-tolerant invertebrates.

Marine organisms adapted to H2S exposure utilize a variety of mechanisms, including H2S oxidation, to minimize its toxicity. However, this may lead to increased free radical production. We tested whether H2S exerts at least some of its toxicity via free radical damage in erythrocytes from the sulfide‐tolerant marine polychaete Glycera dibranchiata. We exposed erythrocytes to H2S in vitro and assessed oxidative stress, oxidative damage and cell death, and whether exogenous antioxidants reduced H2S toxicity. Exposure to 0.1–3% H2S for 0.5–2 h significantly increased oxidative stress (carboxy‐H2DCFDA labeling; p<0.0001), mitochondrial superoxide production (dihydroethidium labeling; p=0.0003), and oxidative damage to RNA and DNA (via HPLC‐EC‐UV; p<0.05), consistent with increased free radical production. Exposure to H2S caused a dose‐dependent increase in cell death (via propidium iodide and calcein green AM labeling, p<0.0001), but the antioxidants glutathione ethyl ester, dithiothreitol and N‐acetylcysteine reduced this cell death by up to 70% (p<0.0001), 78% (p=0.041), and 85% (p=0.0006), respectively. Therefore, in erythrocytes of a sulfide‐tolerant marine invertebrate, oxidative damage was increased by H2S, and H2S‐induced cell death was reduced by antioxidants, both of which are consistent with free radical damage as an important mechanism of H2S toxicity.