Introduction Hydrogen sulphide (H2S) is thought to have a biphasic effect on cells leading to cellular proliferation at “low” doses (micromolar) and cell death in “high” doses (millimolar). It is unclear how this is related to colonic physiology and in-vivo concentrations. H2S is cytotoxic by inhibiting cytochrome c, while genotoxicity may be mediated by free radicals of uncertain origin. We tested the effect of sub-physiological and physiological concentration of H2S on epithelial cell lines to identify proliferative/cytotoxic effects and whether a free radical scavenger could be protective.
Method Cell lines were seeded at a concentration of 2.5 × 104per well and cultured until 70–80% confluency. NaHS was dissolved in distilled water at 0, 100 μM, 500 μM, 1 mM, 2.5 mM and 5 mM and added to the culture. The number of surviving cells was counted after 24 h. In the protection experiments 1mM Tiron, a free radical scavenger, dissolved in distilled water was added concurrently.
Results H2S proved considerably cytotoxic to CACO2 cells at even physiological doses (100 μM, 76.7% death), while at the higher end of the claimed physiological range (2.5 mM) 98.8% cell death was noted). Similar findings occurred with HCT116, though increased survival was noted with HT29 cells compared to the other colorectal cell lines (100 µM 43.9% death). Tiron was protective in all cell lines, especially in the CACO2 cells, with 100 μM + 1 mM Tiron mitigating cell death at 20% and 2.5 mM at 68.8%. P-values were significant at p < 0.001 for all the above results. HELA and A549 cells were more resilient (100 μM, 7.6%, 2.5 mM, 37.6%) or A549 cells (100 μM, 5.2%, 2.5 mM, 22.6%), though these data were non-significant.
Conclusion H2S is cytotoxic to cell lines, especially colonic lines, at estimated physiological doses. Tiron, a free-radical scavenger, increases cell survival after H2S exposure. This implicates excess free radicals in cytotoxicity. HT29 cells, with intact mismatch repair, were more resilient than other colorectal lines with mismatch deficiency. Contrary to some published research, this experiment suggests that H2S is cytotoxic, even at physiological levels, via a free radical mechanism, rather than protective against such insults. Colonic H2S may potentiate the risk of colorectal cancer. Steps to reduce its production or inhibit its action may reduce the risk of progression to cancer and reduce the chance of recurrence after surgery.
Disclosure of interest None Declared.
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