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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

(A) Comparison of Cr(VI)-reducing ability of cultured HepG2 (□) and HepG2-YieF (▧) cells at different time points. (■) indicates cell-free control. * p < 0.05, ** p < 0.01; (B) Absolute concentration of remaining Cr(VI) in HepG2 (■) and HepG2-YieF (●) cell crude extract at different timepoints; (C) The lessened amount of Cr(VI) in HepG2 (▢) and HepG2-YieF (▨) cell crude extract by calculation. Mean and SEs were obtained from three separate measurements of a representative experiment. Experiments were replicated at least twice. Error bars were in some cases smaller than the size of the symbol. * p < 0.05, ** p < 0.01.
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ijms-16-11892-f002: (A) Comparison of Cr(VI)-reducing ability of cultured HepG2 (□) and HepG2-YieF (▧) cells at different time points. (■) indicates cell-free control. * p < 0.05, ** p < 0.01; (B) Absolute concentration of remaining Cr(VI) in HepG2 (■) and HepG2-YieF (●) cell crude extract at different timepoints; (C) The lessened amount of Cr(VI) in HepG2 (▢) and HepG2-YieF (▨) cell crude extract by calculation. Mean and SEs were obtained from three separate measurements of a representative experiment. Experiments were replicated at least twice. Error bars were in some cases smaller than the size of the symbol. * p < 0.05, ** p < 0.01.

Mentions: Both HepG2 cells and HepG2-YieF cells are capable of removing Cr(VI) from culture media within 36 h of culturing with K2CrSO4, but the reducing ability of the latter was significantly greater than the former (Figure 2A). In addition, to measure the in vitro reducing activities of reductants within HepG2-YieF cells, crude cell extracts were prepared from cultured cells. As shown in Figure 2B,C, the reduction rate of HepG2-YieF cell extract was consistently higher than that of HepG2. These results indicated that yieF increased the Cr(VI)-reducing ability of HepG2 cells. We also compared the relative cell viabilities of HepG2-YieF and HepG2 and found that the viability of stable HepG2-YieF cells was significantly higher than that of HepG2 cells under Cr stress ( Figure S2).


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)

(A) Comparison of Cr(VI)-reducing ability of cultured HepG2 (□) and HepG2-YieF (▧) cells at different time points. (■) indicates cell-free control. * p < 0.05, ** p < 0.01; (B) Absolute concentration of remaining Cr(VI) in HepG2 (■) and HepG2-YieF (●) cell crude extract at different timepoints; (C) The lessened amount of Cr(VI) in HepG2 (▢) and HepG2-YieF (▨) cell crude extract by calculation. Mean and SEs were obtained from three separate measurements of a representative experiment. Experiments were replicated at least twice. Error bars were in some cases smaller than the size of the symbol. * p < 0.05, ** p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4490421&req=5

ijms-16-11892-f002: (A) Comparison of Cr(VI)-reducing ability of cultured HepG2 (□) and HepG2-YieF (▧) cells at different time points. (■) indicates cell-free control. * p < 0.05, ** p < 0.01; (B) Absolute concentration of remaining Cr(VI) in HepG2 (■) and HepG2-YieF (●) cell crude extract at different timepoints; (C) The lessened amount of Cr(VI) in HepG2 (▢) and HepG2-YieF (▨) cell crude extract by calculation. Mean and SEs were obtained from three separate measurements of a representative experiment. Experiments were replicated at least twice. Error bars were in some cases smaller than the size of the symbol. * p < 0.05, ** p < 0.01.
Mentions: Both HepG2 cells and HepG2-YieF cells are capable of removing Cr(VI) from culture media within 36 h of culturing with K2CrSO4, but the reducing ability of the latter was significantly greater than the former (Figure 2A). In addition, to measure the in vitro reducing activities of reductants within HepG2-YieF cells, crude cell extracts were prepared from cultured cells. As shown in Figure 2B,C, the reduction rate of HepG2-YieF cell extract was consistently higher than that of HepG2. These results indicated that yieF increased the Cr(VI)-reducing ability of HepG2 cells. We also compared the relative cell viabilities of HepG2-YieF and HepG2 and found that the viability of stable HepG2-YieF cells was significantly higher than that of HepG2 cells under Cr stress ( Figure S2).

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