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Chromate Reductase YieF from Escherichia coli Enhances Hexavalent Chromium Resistance of Human HepG2 Cells.

Liu X, Wu G, Zhang Y, Wu D, Li X, Liu P - Int J Mol Sci (2015)

Bottom Line: The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI) induction.This result was further supported by the evidence of increased Cr(VI)-removing ability of crude cell extract of HepG2-YieF.Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI) stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology, School of Life Sciences, Lanzhou University, Lanzhou 730000, China. kathyliu0505@gmail.com.

ABSTRACT
Hexavalent chromium (Cr(VI)) is a serious environmental pollutant and human toxicant. Mammalian cells are very sensitive to chromate as they lack efficient chromate detoxifying strategy, e.g., chromate-reducing genes that are widely present in prokaryotes. To test whether introduction of prokaryotic chromate-reducing gene into mammalian cells could render higher chromate resistance, an Escherichia coli chromate-reducing gene yieF was transfected into human HepG2 cells. The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI) induction. In chromate-reducing ability test, HepG2-YieF cells that harbored the reductase showed significantly higher reducing ability of Cr(VI) than HepG2 control cells. This result was further supported by the evidence of increased Cr(VI)-removing ability of crude cell extract of HepG2-YieF. Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI) stress. Subcellular localization of YieF was determined by tracing GFP-YieF fusion protein that was detected in both nucleus and cytoplasm by laser confocal microscopy. Altogether, this study successfully demonstrated that the expression of a prokaryotic Cr(VI)-reducing gene yieF endowed mammalian cell HepG2 with enhanced chromate resistance, which brought new insight of Cr(VI) detoxification in mammalian cells.

No MeSH data available.


Related in: MedlinePlus

Relative mRNA expressions of glutathione reductase and glutathione synthetase were compared between transfectants and non-transfectants. Bars represent HepG2 without (▤) or with (■) Cr(VI) treatment, and HepG2-YieF without (▢) or with (▨) Cr(VI) treatment. The values are the mean of four individual samples. Data were normalized to the expression of the housekeeping gene β-actin. Differences between bar a and b and between A and B are significant (p < 0.05).
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ijms-16-11892-f003: Relative mRNA expressions of glutathione reductase and glutathione synthetase were compared between transfectants and non-transfectants. Bars represent HepG2 without (▤) or with (■) Cr(VI) treatment, and HepG2-YieF without (▢) or with (▨) Cr(VI) treatment. The values are the mean of four individual samples. Data were normalized to the expression of the housekeeping gene β-actin. Differences between bar a and b and between A and B are significant (p < 0.05).

Mentions: Some enzymes in mammalian cells are involved indirectly in the reduction of Cr(VI) to Cr(III), for example, glutathione synthetase (for GSH synthesis) and glutathione reductase (for the convertion of reduced GSH from its oxidized form) [27]. The expression of glutathione reductase gene was up-regulated in HepG2 control but down-regulated in HepG2-YieF cells under Cr(VI). Meanwhile, the expression of glutathione synthetase was downregulated in HepG2-YieF cells even without Cr(VI) treatment (Figure 3). These suggested that both of the two GSH-related enzymes were involved in Cr(VI) resistance in HepG2 cells and yieF expression might reduce the oxidative stress caused by Cr(VI).


Chromate Reductase YieF from Escherichia coli Enhances Hexavalent Chromium Resistance of Human HepG2 Cells.

Liu X, Wu G, Zhang Y, Wu D, Li X, Liu P - Int J Mol Sci (2015)

Relative mRNA expressions of glutathione reductase and glutathione synthetase were compared between transfectants and non-transfectants. Bars represent HepG2 without (▤) or with (■) Cr(VI) treatment, and HepG2-YieF without (▢) or with (▨) Cr(VI) treatment. The values are the mean of four individual samples. Data were normalized to the expression of the housekeeping gene β-actin. Differences between bar a and b and between A and B are significant (p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490421&req=5

ijms-16-11892-f003: Relative mRNA expressions of glutathione reductase and glutathione synthetase were compared between transfectants and non-transfectants. Bars represent HepG2 without (▤) or with (■) Cr(VI) treatment, and HepG2-YieF without (▢) or with (▨) Cr(VI) treatment. The values are the mean of four individual samples. Data were normalized to the expression of the housekeeping gene β-actin. Differences between bar a and b and between A and B are significant (p < 0.05).
Mentions: Some enzymes in mammalian cells are involved indirectly in the reduction of Cr(VI) to Cr(III), for example, glutathione synthetase (for GSH synthesis) and glutathione reductase (for the convertion of reduced GSH from its oxidized form) [27]. The expression of glutathione reductase gene was up-regulated in HepG2 control but down-regulated in HepG2-YieF cells under Cr(VI). Meanwhile, the expression of glutathione synthetase was downregulated in HepG2-YieF cells even without Cr(VI) treatment (Figure 3). These suggested that both of the two GSH-related enzymes were involved in Cr(VI) resistance in HepG2 cells and yieF expression might reduce the oxidative stress caused by Cr(VI).

Bottom Line: The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI) induction.This result was further supported by the evidence of increased Cr(VI)-removing ability of crude cell extract of HepG2-YieF.Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI) stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology, School of Life Sciences, Lanzhou University, Lanzhou 730000, China. kathyliu0505@gmail.com.

ABSTRACT
Hexavalent chromium (Cr(VI)) is a serious environmental pollutant and human toxicant. Mammalian cells are very sensitive to chromate as they lack efficient chromate detoxifying strategy, e.g., chromate-reducing genes that are widely present in prokaryotes. To test whether introduction of prokaryotic chromate-reducing gene into mammalian cells could render higher chromate resistance, an Escherichia coli chromate-reducing gene yieF was transfected into human HepG2 cells. The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI) induction. In chromate-reducing ability test, HepG2-YieF cells that harbored the reductase showed significantly higher reducing ability of Cr(VI) than HepG2 control cells. This result was further supported by the evidence of increased Cr(VI)-removing ability of crude cell extract of HepG2-YieF. Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI) stress. Subcellular localization of YieF was determined by tracing GFP-YieF fusion protein that was detected in both nucleus and cytoplasm by laser confocal microscopy. Altogether, this study successfully demonstrated that the expression of a prokaryotic Cr(VI)-reducing gene yieF endowed mammalian cell HepG2 with enhanced chromate resistance, which brought new insight of Cr(VI) detoxification in mammalian cells.

No MeSH data available.


Related in: MedlinePlus