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. 2017 Aug;32(8):2004-2020.
doi: 10.1002/tox.22374. Epub 2016 Oct 24.

Protection of Nrf2 against arsenite-induced oxidative damage is regulated by the cyclic guanosine monophosphate-protein kinase G signaling pathway

Chengzhi Chen  1   2 , Xuejun Jiang  1   2 , Shiyan Gu  1 , Yanhao Lai  3 , Yuan Liu  3   4   5 , Zunzhen Zhang  1
Affiliations

Protection of Nrf2 against arsenite-induced oxidative damage is regulated by the cyclic guanosine monophosphate-protein kinase G signaling pathway

Chengzhi Chen et al. Environ Toxicol. 2017 Aug.

Abstract

Arsenite has been shown to induce a variety of oxidative damage in mammalian cells. However, the mechanisms underlying cellular responses to its adverse effects remain unknown. We previously showed that the level of Nrf2, a nuclear transcription factor significantly increased in arsenite-treated human bronchial epithelial (HBE) cells suggesting that Nrf2 is involved in responding to arsenite-induced oxidative damage. To explore how Nrf2 can impact arsenite-induced oxidative damage, in this study, we examined Nrf2 activation and its regulation upon cellular arsenite exposure as well as its effects on arsenite-induced oxidative damage in HBE cells. We found that Nrf2 mRNA and protein levels were significantly increased by arsenite in a dose- and time-dependent manner. Furthermore, we showed that over-expression of Nrf2 significantly reduced the level of arsenite-induced oxidative damage in HBE cells including DNA damage, chromosomal breakage, lipid peroxidation and depletion of antioxidants. This indicates a protective role of Nrf2 against arsenite toxicity. This was further supported by the fact that activation of Nrf2 by its agonists, tertiary butylhydroquinone (t-BHQ) and sulforaphane (SFN) resulted in the same protective effects against arsenite toxicity. Moreover, we demonstrated that arsenite-induced activation of Nrf2 was mediated by the cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) signaling pathway. This is the first evidence showing that Nrf2 protects against arsenite-induced oxidative damage through the cGMP-PKG pathway. Our study suggests that activation of Nrf2 through the cGMP-PKG signaling pathway in HBE cells may be developed as a new strategy for prevention of arsenite toxicity. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 2004-2020, 2017.

Keywords: Nrf2; arsenite; cGMP-PKG signaling pathway; oxidative damage.

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Figures

Figure 1
Figure 1. Arsenite reduced cell viability and induced oxidative damage in HBE cells
(a) Cells were treated with 1 μM-60 μM arsenite for 24 h and then subjected to Alama Blue assay for obtaining AR570 value. (b) Cells were treated with 0, 5 μM, 10 μM and 20 μM arsenite for 6, 12 and 24 h, respectively, and LDH release rate was measured as described in the Materials and Methods. (c) Cells were treated with 5 μM, 10 μM, 20 μM arsenite for 24 h or pretreated with 10 mM NAC for 2 h and subsequently treated with 10 μM arsenite for 24 h. The level of ROS was detected with DCFH-DA probe through flow cytometry. The Gm value was obtained from Windows Multiple Document Interface for Flow Cytometry (Version 2.8). Representative images of ROS determination were showed in (d). The levels of MDA (e), SOD (f) and GSH (g) in untreated HBE cells and cells treated with various concentrations of arsenite were determined as described in Materials and Methods. “*” denotes a significant difference (P<0.05) was detected between treated cells and untreated cells, whereas “**” denotes a significant difference (P<0.05) was detected between cells pretreated with NAC and cells treated with 10 μM arsenite alone.
Figure 1
Figure 1. Arsenite reduced cell viability and induced oxidative damage in HBE cells
(a) Cells were treated with 1 μM-60 μM arsenite for 24 h and then subjected to Alama Blue assay for obtaining AR570 value. (b) Cells were treated with 0, 5 μM, 10 μM and 20 μM arsenite for 6, 12 and 24 h, respectively, and LDH release rate was measured as described in the Materials and Methods. (c) Cells were treated with 5 μM, 10 μM, 20 μM arsenite for 24 h or pretreated with 10 mM NAC for 2 h and subsequently treated with 10 μM arsenite for 24 h. The level of ROS was detected with DCFH-DA probe through flow cytometry. The Gm value was obtained from Windows Multiple Document Interface for Flow Cytometry (Version 2.8). Representative images of ROS determination were showed in (d). The levels of MDA (e), SOD (f) and GSH (g) in untreated HBE cells and cells treated with various concentrations of arsenite were determined as described in Materials and Methods. “*” denotes a significant difference (P<0.05) was detected between treated cells and untreated cells, whereas “**” denotes a significant difference (P<0.05) was detected between cells pretreated with NAC and cells treated with 10 μM arsenite alone.
Figure 1
Figure 1. Arsenite reduced cell viability and induced oxidative damage in HBE cells
(a) Cells were treated with 1 μM-60 μM arsenite for 24 h and then subjected to Alama Blue assay for obtaining AR570 value. (b) Cells were treated with 0, 5 μM, 10 μM and 20 μM arsenite for 6, 12 and 24 h, respectively, and LDH release rate was measured as described in the Materials and Methods. (c) Cells were treated with 5 μM, 10 μM, 20 μM arsenite for 24 h or pretreated with 10 mM NAC for 2 h and subsequently treated with 10 μM arsenite for 24 h. The level of ROS was detected with DCFH-DA probe through flow cytometry. The Gm value was obtained from Windows Multiple Document Interface for Flow Cytometry (Version 2.8). Representative images of ROS determination were showed in (d). The levels of MDA (e), SOD (f) and GSH (g) in untreated HBE cells and cells treated with various concentrations of arsenite were determined as described in Materials and Methods. “*” denotes a significant difference (P<0.05) was detected between treated cells and untreated cells, whereas “**” denotes a significant difference (P<0.05) was detected between cells pretreated with NAC and cells treated with 10 μM arsenite alone.
Figure 1
Figure 1. Arsenite reduced cell viability and induced oxidative damage in HBE cells
(a) Cells were treated with 1 μM-60 μM arsenite for 24 h and then subjected to Alama Blue assay for obtaining AR570 value. (b) Cells were treated with 0, 5 μM, 10 μM and 20 μM arsenite for 6, 12 and 24 h, respectively, and LDH release rate was measured as described in the Materials and Methods. (c) Cells were treated with 5 μM, 10 μM, 20 μM arsenite for 24 h or pretreated with 10 mM NAC for 2 h and subsequently treated with 10 μM arsenite for 24 h. The level of ROS was detected with DCFH-DA probe through flow cytometry. The Gm value was obtained from Windows Multiple Document Interface for Flow Cytometry (Version 2.8). Representative images of ROS determination were showed in (d). The levels of MDA (e), SOD (f) and GSH (g) in untreated HBE cells and cells treated with various concentrations of arsenite were determined as described in Materials and Methods. “*” denotes a significant difference (P<0.05) was detected between treated cells and untreated cells, whereas “**” denotes a significant difference (P<0.05) was detected between cells pretreated with NAC and cells treated with 10 μM arsenite alone.
Figure 1
Figure 1. Arsenite reduced cell viability and induced oxidative damage in HBE cells
(a) Cells were treated with 1 μM-60 μM arsenite for 24 h and then subjected to Alama Blue assay for obtaining AR570 value. (b) Cells were treated with 0, 5 μM, 10 μM and 20 μM arsenite for 6, 12 and 24 h, respectively, and LDH release rate was measured as described in the Materials and Methods. (c) Cells were treated with 5 μM, 10 μM, 20 μM arsenite for 24 h or pretreated with 10 mM NAC for 2 h and subsequently treated with 10 μM arsenite for 24 h. The level of ROS was detected with DCFH-DA probe through flow cytometry. The Gm value was obtained from Windows Multiple Document Interface for Flow Cytometry (Version 2.8). Representative images of ROS determination were showed in (d). The levels of MDA (e), SOD (f) and GSH (g) in untreated HBE cells and cells treated with various concentrations of arsenite were determined as described in Materials and Methods. “*” denotes a significant difference (P<0.05) was detected between treated cells and untreated cells, whereas “**” denotes a significant difference (P<0.05) was detected between cells pretreated with NAC and cells treated with 10 μM arsenite alone.
Figure 1
Figure 1. Arsenite reduced cell viability and induced oxidative damage in HBE cells
(a) Cells were treated with 1 μM-60 μM arsenite for 24 h and then subjected to Alama Blue assay for obtaining AR570 value. (b) Cells were treated with 0, 5 μM, 10 μM and 20 μM arsenite for 6, 12 and 24 h, respectively, and LDH release rate was measured as described in the Materials and Methods. (c) Cells were treated with 5 μM, 10 μM, 20 μM arsenite for 24 h or pretreated with 10 mM NAC for 2 h and subsequently treated with 10 μM arsenite for 24 h. The level of ROS was detected with DCFH-DA probe through flow cytometry. The Gm value was obtained from Windows Multiple Document Interface for Flow Cytometry (Version 2.8). Representative images of ROS determination were showed in (d). The levels of MDA (e), SOD (f) and GSH (g) in untreated HBE cells and cells treated with various concentrations of arsenite were determined as described in Materials and Methods. “*” denotes a significant difference (P<0.05) was detected between treated cells and untreated cells, whereas “**” denotes a significant difference (P<0.05) was detected between cells pretreated with NAC and cells treated with 10 μM arsenite alone.
Figure 1
Figure 1. Arsenite reduced cell viability and induced oxidative damage in HBE cells
(a) Cells were treated with 1 μM-60 μM arsenite for 24 h and then subjected to Alama Blue assay for obtaining AR570 value. (b) Cells were treated with 0, 5 μM, 10 μM and 20 μM arsenite for 6, 12 and 24 h, respectively, and LDH release rate was measured as described in the Materials and Methods. (c) Cells were treated with 5 μM, 10 μM, 20 μM arsenite for 24 h or pretreated with 10 mM NAC for 2 h and subsequently treated with 10 μM arsenite for 24 h. The level of ROS was detected with DCFH-DA probe through flow cytometry. The Gm value was obtained from Windows Multiple Document Interface for Flow Cytometry (Version 2.8). Representative images of ROS determination were showed in (d). The levels of MDA (e), SOD (f) and GSH (g) in untreated HBE cells and cells treated with various concentrations of arsenite were determined as described in Materials and Methods. “*” denotes a significant difference (P<0.05) was detected between treated cells and untreated cells, whereas “**” denotes a significant difference (P<0.05) was detected between cells pretreated with NAC and cells treated with 10 μM arsenite alone.
Figure 2
Figure 2. Arsenite induced DNA damage and chromosomal breakage in HBE cells
Cells were treated with 5 μM, 10 μM, 20 μM arsenite for 24 h or pretreated with 10 mM NAC for 2 h and subsequently treated with 10μM arsenite. Arsenite-induced DNA damage and chromosomal breakage were determined by comet assay (a)-(d) and micronucleus assay (e), respectively. Representative images in comet assay were showed (200×) and illustrated in (a). The effects of arsenite on tail DNA (%), tail length and OTM were illustrated in (b)-(d). “*” denotes a significant difference (P<0.05) detected between treated cells and untreated cells, whereas “**” denotes a significant difference (P<0.05) detected between cells pretreated with NAC and cells treated with 10 μM arsenite.
Figure 2
Figure 2. Arsenite induced DNA damage and chromosomal breakage in HBE cells
Cells were treated with 5 μM, 10 μM, 20 μM arsenite for 24 h or pretreated with 10 mM NAC for 2 h and subsequently treated with 10μM arsenite. Arsenite-induced DNA damage and chromosomal breakage were determined by comet assay (a)-(d) and micronucleus assay (e), respectively. Representative images in comet assay were showed (200×) and illustrated in (a). The effects of arsenite on tail DNA (%), tail length and OTM were illustrated in (b)-(d). “*” denotes a significant difference (P<0.05) detected between treated cells and untreated cells, whereas “**” denotes a significant difference (P<0.05) detected between cells pretreated with NAC and cells treated with 10 μM arsenite.
Figure 2
Figure 2. Arsenite induced DNA damage and chromosomal breakage in HBE cells
Cells were treated with 5 μM, 10 μM, 20 μM arsenite for 24 h or pretreated with 10 mM NAC for 2 h and subsequently treated with 10μM arsenite. Arsenite-induced DNA damage and chromosomal breakage were determined by comet assay (a)-(d) and micronucleus assay (e), respectively. Representative images in comet assay were showed (200×) and illustrated in (a). The effects of arsenite on tail DNA (%), tail length and OTM were illustrated in (b)-(d). “*” denotes a significant difference (P<0.05) detected between treated cells and untreated cells, whereas “**” denotes a significant difference (P<0.05) detected between cells pretreated with NAC and cells treated with 10 μM arsenite.
Figure 2
Figure 2. Arsenite induced DNA damage and chromosomal breakage in HBE cells
Cells were treated with 5 μM, 10 μM, 20 μM arsenite for 24 h or pretreated with 10 mM NAC for 2 h and subsequently treated with 10μM arsenite. Arsenite-induced DNA damage and chromosomal breakage were determined by comet assay (a)-(d) and micronucleus assay (e), respectively. Representative images in comet assay were showed (200×) and illustrated in (a). The effects of arsenite on tail DNA (%), tail length and OTM were illustrated in (b)-(d). “*” denotes a significant difference (P<0.05) detected between treated cells and untreated cells, whereas “**” denotes a significant difference (P<0.05) detected between cells pretreated with NAC and cells treated with 10 μM arsenite.
Figure 2
Figure 2. Arsenite induced DNA damage and chromosomal breakage in HBE cells
Cells were treated with 5 μM, 10 μM, 20 μM arsenite for 24 h or pretreated with 10 mM NAC for 2 h and subsequently treated with 10μM arsenite. Arsenite-induced DNA damage and chromosomal breakage were determined by comet assay (a)-(d) and micronucleus assay (e), respectively. Representative images in comet assay were showed (200×) and illustrated in (a). The effects of arsenite on tail DNA (%), tail length and OTM were illustrated in (b)-(d). “*” denotes a significant difference (P<0.05) detected between treated cells and untreated cells, whereas “**” denotes a significant difference (P<0.05) detected between cells pretreated with NAC and cells treated with 10 μM arsenite.
Figure 3
Figure 3. Arsenite activated Nrf2 in HBE cells
Cells were treated with 5 μM, 10 μM and 20 μM arsenite for 24 h or 10 μM arsenite for 6 h, 12 h and 24 h, respectively. The effects of arsenite on Nrf2 protein level at different time points and doses were shown in (a)-(c). The level of Nrf2 in the nucleus of HBE cells treated with arsenite was determined by immunofluorescence (d) and (e). Representative images (400×) of immunofluorescence results were showed in (d). Fluorescence intensity was obtained from Image-Pro Plus software. The mRNA level of Nrf2 in HBE cells was illustrated in (f) and (g). "*" denotes a significant difference (P<0.05) detected between treated and untreated cells.
Figure 3
Figure 3. Arsenite activated Nrf2 in HBE cells
Cells were treated with 5 μM, 10 μM and 20 μM arsenite for 24 h or 10 μM arsenite for 6 h, 12 h and 24 h, respectively. The effects of arsenite on Nrf2 protein level at different time points and doses were shown in (a)-(c). The level of Nrf2 in the nucleus of HBE cells treated with arsenite was determined by immunofluorescence (d) and (e). Representative images (400×) of immunofluorescence results were showed in (d). Fluorescence intensity was obtained from Image-Pro Plus software. The mRNA level of Nrf2 in HBE cells was illustrated in (f) and (g). "*" denotes a significant difference (P<0.05) detected between treated and untreated cells.
Figure 3
Figure 3. Arsenite activated Nrf2 in HBE cells
Cells were treated with 5 μM, 10 μM and 20 μM arsenite for 24 h or 10 μM arsenite for 6 h, 12 h and 24 h, respectively. The effects of arsenite on Nrf2 protein level at different time points and doses were shown in (a)-(c). The level of Nrf2 in the nucleus of HBE cells treated with arsenite was determined by immunofluorescence (d) and (e). Representative images (400×) of immunofluorescence results were showed in (d). Fluorescence intensity was obtained from Image-Pro Plus software. The mRNA level of Nrf2 in HBE cells was illustrated in (f) and (g). "*" denotes a significant difference (P<0.05) detected between treated and untreated cells.
Figure 3
Figure 3. Arsenite activated Nrf2 in HBE cells
Cells were treated with 5 μM, 10 μM and 20 μM arsenite for 24 h or 10 μM arsenite for 6 h, 12 h and 24 h, respectively. The effects of arsenite on Nrf2 protein level at different time points and doses were shown in (a)-(c). The level of Nrf2 in the nucleus of HBE cells treated with arsenite was determined by immunofluorescence (d) and (e). Representative images (400×) of immunofluorescence results were showed in (d). Fluorescence intensity was obtained from Image-Pro Plus software. The mRNA level of Nrf2 in HBE cells was illustrated in (f) and (g). "*" denotes a significant difference (P<0.05) detected between treated and untreated cells.
Figure 3
Figure 3. Arsenite activated Nrf2 in HBE cells
Cells were treated with 5 μM, 10 μM and 20 μM arsenite for 24 h or 10 μM arsenite for 6 h, 12 h and 24 h, respectively. The effects of arsenite on Nrf2 protein level at different time points and doses were shown in (a)-(c). The level of Nrf2 in the nucleus of HBE cells treated with arsenite was determined by immunofluorescence (d) and (e). Representative images (400×) of immunofluorescence results were showed in (d). Fluorescence intensity was obtained from Image-Pro Plus software. The mRNA level of Nrf2 in HBE cells was illustrated in (f) and (g). "*" denotes a significant difference (P<0.05) detected between treated and untreated cells.
Figure 3
Figure 3. Arsenite activated Nrf2 in HBE cells
Cells were treated with 5 μM, 10 μM and 20 μM arsenite for 24 h or 10 μM arsenite for 6 h, 12 h and 24 h, respectively. The effects of arsenite on Nrf2 protein level at different time points and doses were shown in (a)-(c). The level of Nrf2 in the nucleus of HBE cells treated with arsenite was determined by immunofluorescence (d) and (e). Representative images (400×) of immunofluorescence results were showed in (d). Fluorescence intensity was obtained from Image-Pro Plus software. The mRNA level of Nrf2 in HBE cells was illustrated in (f) and (g). "*" denotes a significant difference (P<0.05) detected between treated and untreated cells.
Figure 3
Figure 3. Arsenite activated Nrf2 in HBE cells
Cells were treated with 5 μM, 10 μM and 20 μM arsenite for 24 h or 10 μM arsenite for 6 h, 12 h and 24 h, respectively. The effects of arsenite on Nrf2 protein level at different time points and doses were shown in (a)-(c). The level of Nrf2 in the nucleus of HBE cells treated with arsenite was determined by immunofluorescence (d) and (e). Representative images (400×) of immunofluorescence results were showed in (d). Fluorescence intensity was obtained from Image-Pro Plus software. The mRNA level of Nrf2 in HBE cells was illustrated in (f) and (g). "*" denotes a significant difference (P<0.05) detected between treated and untreated cells.
Figure 4
Figure 4. The effects of Nrf2 on arsenite-induced oxidative damage in HBE cells
The effects of Nrf2 on DNA damage induced by arsenite were examined under Nrf2 gene downregulation and overexpression (a) and (b) with treatment of 10 μM arsenite for 24 h. Representative images of comet assay were shown in (a) (200×). The effect of Nrf2 on arsenite-induced chromosomal breakage was determined under the same condition (c), whereas the effects of the protein on the level of GSH, SOD, MDA (d) as well as LDH release (e) were also determined. "*" denotes a significant difference (P<0.05), detected between HBE cells with Nrf2 gene knockdown or Nrf2 overexpression and normal HBE cells.
Figure 4
Figure 4. The effects of Nrf2 on arsenite-induced oxidative damage in HBE cells
The effects of Nrf2 on DNA damage induced by arsenite were examined under Nrf2 gene downregulation and overexpression (a) and (b) with treatment of 10 μM arsenite for 24 h. Representative images of comet assay were shown in (a) (200×). The effect of Nrf2 on arsenite-induced chromosomal breakage was determined under the same condition (c), whereas the effects of the protein on the level of GSH, SOD, MDA (d) as well as LDH release (e) were also determined. "*" denotes a significant difference (P<0.05), detected between HBE cells with Nrf2 gene knockdown or Nrf2 overexpression and normal HBE cells.
Figure 4
Figure 4. The effects of Nrf2 on arsenite-induced oxidative damage in HBE cells
The effects of Nrf2 on DNA damage induced by arsenite were examined under Nrf2 gene downregulation and overexpression (a) and (b) with treatment of 10 μM arsenite for 24 h. Representative images of comet assay were shown in (a) (200×). The effect of Nrf2 on arsenite-induced chromosomal breakage was determined under the same condition (c), whereas the effects of the protein on the level of GSH, SOD, MDA (d) as well as LDH release (e) were also determined. "*" denotes a significant difference (P<0.05), detected between HBE cells with Nrf2 gene knockdown or Nrf2 overexpression and normal HBE cells.
Figure 4
Figure 4. The effects of Nrf2 on arsenite-induced oxidative damage in HBE cells
The effects of Nrf2 on DNA damage induced by arsenite were examined under Nrf2 gene downregulation and overexpression (a) and (b) with treatment of 10 μM arsenite for 24 h. Representative images of comet assay were shown in (a) (200×). The effect of Nrf2 on arsenite-induced chromosomal breakage was determined under the same condition (c), whereas the effects of the protein on the level of GSH, SOD, MDA (d) as well as LDH release (e) were also determined. "*" denotes a significant difference (P<0.05), detected between HBE cells with Nrf2 gene knockdown or Nrf2 overexpression and normal HBE cells.
Figure 4
Figure 4. The effects of Nrf2 on arsenite-induced oxidative damage in HBE cells
The effects of Nrf2 on DNA damage induced by arsenite were examined under Nrf2 gene downregulation and overexpression (a) and (b) with treatment of 10 μM arsenite for 24 h. Representative images of comet assay were shown in (a) (200×). The effect of Nrf2 on arsenite-induced chromosomal breakage was determined under the same condition (c), whereas the effects of the protein on the level of GSH, SOD, MDA (d) as well as LDH release (e) were also determined. "*" denotes a significant difference (P<0.05), detected between HBE cells with Nrf2 gene knockdown or Nrf2 overexpression and normal HBE cells.
Figure 5
Figure 5. The effects of agonists of Nrf2 on arsenite-induced oxidative damage in HBE cells
The effects of agonists of Nrf2, t-BHQ and SFN on arsenite-induced oxidative damage were determined as described in Materials and Methods. The effects of t-BHQ and SFN on arsenite-induced production of ROS were illustrated in (a) and (b). The effects of t-BHQ and SFN on arsenite-induced DNA damage determined by comet assay were shown in (c) and (d). The effects of t-BHQ and SFN on arsenite-induced chromosomal breakage was determined by micronucleus assay (e), whereas their effects on the level of GSH and MDA as well as the activity of SOD in HBE cells treated by 10 μM arsenite were illustrated in (f). "*" denotes a significant difference (P<0.05) detected between HBE cells treated with arsenite alone and cells treated with arsenite along with t-BHQ or SFN.
Figure 5
Figure 5. The effects of agonists of Nrf2 on arsenite-induced oxidative damage in HBE cells
The effects of agonists of Nrf2, t-BHQ and SFN on arsenite-induced oxidative damage were determined as described in Materials and Methods. The effects of t-BHQ and SFN on arsenite-induced production of ROS were illustrated in (a) and (b). The effects of t-BHQ and SFN on arsenite-induced DNA damage determined by comet assay were shown in (c) and (d). The effects of t-BHQ and SFN on arsenite-induced chromosomal breakage was determined by micronucleus assay (e), whereas their effects on the level of GSH and MDA as well as the activity of SOD in HBE cells treated by 10 μM arsenite were illustrated in (f). "*" denotes a significant difference (P<0.05) detected between HBE cells treated with arsenite alone and cells treated with arsenite along with t-BHQ or SFN.
Figure 5
Figure 5. The effects of agonists of Nrf2 on arsenite-induced oxidative damage in HBE cells
The effects of agonists of Nrf2, t-BHQ and SFN on arsenite-induced oxidative damage were determined as described in Materials and Methods. The effects of t-BHQ and SFN on arsenite-induced production of ROS were illustrated in (a) and (b). The effects of t-BHQ and SFN on arsenite-induced DNA damage determined by comet assay were shown in (c) and (d). The effects of t-BHQ and SFN on arsenite-induced chromosomal breakage was determined by micronucleus assay (e), whereas their effects on the level of GSH and MDA as well as the activity of SOD in HBE cells treated by 10 μM arsenite were illustrated in (f). "*" denotes a significant difference (P<0.05) detected between HBE cells treated with arsenite alone and cells treated with arsenite along with t-BHQ or SFN.
Figure 5
Figure 5. The effects of agonists of Nrf2 on arsenite-induced oxidative damage in HBE cells
The effects of agonists of Nrf2, t-BHQ and SFN on arsenite-induced oxidative damage were determined as described in Materials and Methods. The effects of t-BHQ and SFN on arsenite-induced production of ROS were illustrated in (a) and (b). The effects of t-BHQ and SFN on arsenite-induced DNA damage determined by comet assay were shown in (c) and (d). The effects of t-BHQ and SFN on arsenite-induced chromosomal breakage was determined by micronucleus assay (e), whereas their effects on the level of GSH and MDA as well as the activity of SOD in HBE cells treated by 10 μM arsenite were illustrated in (f). "*" denotes a significant difference (P<0.05) detected between HBE cells treated with arsenite alone and cells treated with arsenite along with t-BHQ or SFN.
Figure 5
Figure 5. The effects of agonists of Nrf2 on arsenite-induced oxidative damage in HBE cells
The effects of agonists of Nrf2, t-BHQ and SFN on arsenite-induced oxidative damage were determined as described in Materials and Methods. The effects of t-BHQ and SFN on arsenite-induced production of ROS were illustrated in (a) and (b). The effects of t-BHQ and SFN on arsenite-induced DNA damage determined by comet assay were shown in (c) and (d). The effects of t-BHQ and SFN on arsenite-induced chromosomal breakage was determined by micronucleus assay (e), whereas their effects on the level of GSH and MDA as well as the activity of SOD in HBE cells treated by 10 μM arsenite were illustrated in (f). "*" denotes a significant difference (P<0.05) detected between HBE cells treated with arsenite alone and cells treated with arsenite along with t-BHQ or SFN.
Figure 5
Figure 5. The effects of agonists of Nrf2 on arsenite-induced oxidative damage in HBE cells
The effects of agonists of Nrf2, t-BHQ and SFN on arsenite-induced oxidative damage were determined as described in Materials and Methods. The effects of t-BHQ and SFN on arsenite-induced production of ROS were illustrated in (a) and (b). The effects of t-BHQ and SFN on arsenite-induced DNA damage determined by comet assay were shown in (c) and (d). The effects of t-BHQ and SFN on arsenite-induced chromosomal breakage was determined by micronucleus assay (e), whereas their effects on the level of GSH and MDA as well as the activity of SOD in HBE cells treated by 10 μM arsenite were illustrated in (f). "*" denotes a significant difference (P<0.05) detected between HBE cells treated with arsenite alone and cells treated with arsenite along with t-BHQ or SFN.
Figure 6
Figure 6. Arsenite increased the levels of PKG and cGMP in HBE cells independent of NO
The effects on arsenite on the levels of NO, tNOS and iNOS in HBE cells were determined as described in Materials and Methods. (a). Cells were treated with 5 μM, 10 μM and 20 μM arsenite for 24 h, respectively. "*" denotes a significant difference (P<0.05) detected in untreated HBE cells and cells treated with various concentrations of arsenite. The levels of PKG and cGMP in HBE cells treated with various concentrations of arsenite (5 μM-20 μM) for 6 h-24 h were determined as described in Materials and Methods. The effect of arsenite on PKG level in HBE cells was illustrated in (b), whereas its effect on cellular cGMP level was shown in (c). "*" denotes a significant difference detected between untreated cells and cells treated with various concentrations of arsenite for 6 h-24 h.
Figure 6
Figure 6. Arsenite increased the levels of PKG and cGMP in HBE cells independent of NO
The effects on arsenite on the levels of NO, tNOS and iNOS in HBE cells were determined as described in Materials and Methods. (a). Cells were treated with 5 μM, 10 μM and 20 μM arsenite for 24 h, respectively. "*" denotes a significant difference (P<0.05) detected in untreated HBE cells and cells treated with various concentrations of arsenite. The levels of PKG and cGMP in HBE cells treated with various concentrations of arsenite (5 μM-20 μM) for 6 h-24 h were determined as described in Materials and Methods. The effect of arsenite on PKG level in HBE cells was illustrated in (b), whereas its effect on cellular cGMP level was shown in (c). "*" denotes a significant difference detected between untreated cells and cells treated with various concentrations of arsenite for 6 h-24 h.
Figure 6
Figure 6. Arsenite increased the levels of PKG and cGMP in HBE cells independent of NO
The effects on arsenite on the levels of NO, tNOS and iNOS in HBE cells were determined as described in Materials and Methods. (a). Cells were treated with 5 μM, 10 μM and 20 μM arsenite for 24 h, respectively. "*" denotes a significant difference (P<0.05) detected in untreated HBE cells and cells treated with various concentrations of arsenite. The levels of PKG and cGMP in HBE cells treated with various concentrations of arsenite (5 μM-20 μM) for 6 h-24 h were determined as described in Materials and Methods. The effect of arsenite on PKG level in HBE cells was illustrated in (b), whereas its effect on cellular cGMP level was shown in (c). "*" denotes a significant difference detected between untreated cells and cells treated with various concentrations of arsenite for 6 h-24 h.
Figure 7
Figure 7. Inhibition of cGMP-PKG pathway suppressed arsenite-induced Nrf2 activation in HBE cells
Cells were treated with 10 μM arsenite for 24 h or pretreated with KT5823 for 2 h followed by treatment of 10 μM arsenite for additional 24 h. The effect of KT5823 on the mRNA level of Nrf2 was determined by quantitative real-time PCR (a). The effect of KT5823 on Nrf2 protein level was determined with Western blot (b) and (c). The effect of KT5823 on the protein level of Nrf2 in nucleus was determined by immunofluorescence (d) and (e). The immunofluorescence intensity was obtained from Image-Pro Plus software. “*” denotes a significant difference (P<0.05) detected between cells treated with arsenite alone and cells treated with arsenite along with KT5823.
Figure 7
Figure 7. Inhibition of cGMP-PKG pathway suppressed arsenite-induced Nrf2 activation in HBE cells
Cells were treated with 10 μM arsenite for 24 h or pretreated with KT5823 for 2 h followed by treatment of 10 μM arsenite for additional 24 h. The effect of KT5823 on the mRNA level of Nrf2 was determined by quantitative real-time PCR (a). The effect of KT5823 on Nrf2 protein level was determined with Western blot (b) and (c). The effect of KT5823 on the protein level of Nrf2 in nucleus was determined by immunofluorescence (d) and (e). The immunofluorescence intensity was obtained from Image-Pro Plus software. “*” denotes a significant difference (P<0.05) detected between cells treated with arsenite alone and cells treated with arsenite along with KT5823.
Figure 7
Figure 7. Inhibition of cGMP-PKG pathway suppressed arsenite-induced Nrf2 activation in HBE cells
Cells were treated with 10 μM arsenite for 24 h or pretreated with KT5823 for 2 h followed by treatment of 10 μM arsenite for additional 24 h. The effect of KT5823 on the mRNA level of Nrf2 was determined by quantitative real-time PCR (a). The effect of KT5823 on Nrf2 protein level was determined with Western blot (b) and (c). The effect of KT5823 on the protein level of Nrf2 in nucleus was determined by immunofluorescence (d) and (e). The immunofluorescence intensity was obtained from Image-Pro Plus software. “*” denotes a significant difference (P<0.05) detected between cells treated with arsenite alone and cells treated with arsenite along with KT5823.
Figure 7
Figure 7. Inhibition of cGMP-PKG pathway suppressed arsenite-induced Nrf2 activation in HBE cells
Cells were treated with 10 μM arsenite for 24 h or pretreated with KT5823 for 2 h followed by treatment of 10 μM arsenite for additional 24 h. The effect of KT5823 on the mRNA level of Nrf2 was determined by quantitative real-time PCR (a). The effect of KT5823 on Nrf2 protein level was determined with Western blot (b) and (c). The effect of KT5823 on the protein level of Nrf2 in nucleus was determined by immunofluorescence (d) and (e). The immunofluorescence intensity was obtained from Image-Pro Plus software. “*” denotes a significant difference (P<0.05) detected between cells treated with arsenite alone and cells treated with arsenite along with KT5823.
Figure 7
Figure 7. Inhibition of cGMP-PKG pathway suppressed arsenite-induced Nrf2 activation in HBE cells
Cells were treated with 10 μM arsenite for 24 h or pretreated with KT5823 for 2 h followed by treatment of 10 μM arsenite for additional 24 h. The effect of KT5823 on the mRNA level of Nrf2 was determined by quantitative real-time PCR (a). The effect of KT5823 on Nrf2 protein level was determined with Western blot (b) and (c). The effect of KT5823 on the protein level of Nrf2 in nucleus was determined by immunofluorescence (d) and (e). The immunofluorescence intensity was obtained from Image-Pro Plus software. “*” denotes a significant difference (P<0.05) detected between cells treated with arsenite alone and cells treated with arsenite along with KT5823.
Figure 8
Figure 8. A model for arsenite-induced Nrf2 activation to protect against oxidative damage via the cGMP-PKG signaling pathway in HBE cells
Arsenite induces oxidative damage while it activates cGMP-PKG signaling pathway by increasing cellular level of cGMP and PKG in HBE cells. This in turn increases the level of Nrf2 which subsequently increases cellular level of GSH and SOD activity, thereby reducing the elevated level of ROS induced by arsenite. This then significantly reduces oxidative DNA damage, chromosomal breakage and lipid peroxidation induced by arsenite.
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