The Effects Of Hypoxia On The Antioxidant Response Element-Mediated Gene Expression Of NAD(P)H: Quinone Oxidoreductasel And The Toxicities Of Bioreductive Drugs In Human Hepatoma Cells

[Objective] We take the present study with the goal of whether hypxoia can induce enzyme activity of NADP(H): Quinone Oxidoreductasel(NQOl) and proliferation of human hepatoma cells SMMC-7721, and whether Antioxidant Response Element(ARE) can mediate gene expression in response to hypoxia as antioxidant. On the meantime, we investigate the cytotoxicity and DNA damage of SMMC-7721 cells after bioreductive drugs" (AZQ/DZQ) treatment under different oxygen exposure.[Methods] Human hepatoma cells SMMC-7721 are planted in 96-well plates, after 24h growth, cells were grown under hypoxic exposure(pC>2<0.1%) for different periods. Cell proliferation was determined by MTT assay, NQO1 enzyme activity was detected by spectrophotometric assay using direct measurement of NQO1 fromcells cultured in 96-well plates. Electrophoretic Mobility Shift Assay(EMSA) was employed to assess nuclear proteins binding to the ARE under hypoxic exposure.AZQ/DZQ treatment of cells was also under hypoxia. Cytotoxicity was measured by MTT assay, DNA strand break and cross link was determined by Alkaline Single Cell Gel Electrophoresis (ASCGE) and modified SCGE separately.[Results] The effect of hypoxia on 7721 cells demonstrates a time-related change. A 6h hypoxic exposure can stimulate cell proliferation and inhibit NQO1 enzyme activity in cells, and exposure beyond this time course (9h, 12h, 24h) can inhibit cell proliferation and induce NQO1 enzyme activity. The differences between cells under hypoxia and air were statistically significant at each time point (P<0.01). There are nuclear proteins binding to ARE probe from cells under hypoxia and air all the time, but no proteins were observed binding to the mutated ARE probe under the same conditions .AP-1 probe(at the same concentration of ARE probe) can't compete the specific binding between nuclear extracts and ARE probe, though NF-K B probe(100 times concentration) can compete with ARE probe binding to nuclear proteins from normoxic cells, but can't compete the interation between ARE probe and nuclear extracts from hypoxic cells. The cytotoxicities of AZQ/DZQ were all enhanced under hypoxic exposure, and DZQ has a more selective toxicity towards hypoxic cells. Treatment in air of AZQ can cause DNA strand break and DZQ can cause DNA strand cross link in stead. Under hypoxic exposure theDZQ caused DNA strand cross link was more severe, and the major DNA damage caused by AZQ changed from strand break to strand cross link. There were dosage-dependent effects in the SCGE experiment.[Conclusions] Hypoxia can inhibit cell proliferation and induce NQOI enzyme activity of hepatoma cells SMMC-7721 after a certain period. There are excessive NF- K B binding to NQO1ARE or some transcriptional factors other than NF- K. B after hypoxic exposure. ARE participate in the induction of NQOI gene. The cytotoxicities of AZQ/DZQ were all enhanced under hypoxic exposure, and DZQ has a more selective toxicity towards hypoxic cells. The DNA damage mechanisms of the two bioreductive drugs (AZQ/DZQ) vary under different oxygen exposure. DZQ cause DNA damage by rendering DNA strand cross link and hypoxia can strengthen this effect. AZQ cause DNA damage mainly by DNA strand break in air but DNA strand cross link under hypoxia.