Studies on the molecular mechanisms of genotoxicity of nitric oxide (NO)

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Studies on the molecular mechanisms of genotoxicity of nitric oxide (NO)

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Abstract

In this study, the amount of S-nitrosoglutathione (GSNO) was measured spectrophotometrically at 334 nm. Spontaneous decrease of absorbency at 334 nm was detected when GSNO was exposed to 37 °C and a high pH (pH 8.0). We investigated the catalytic roles of various metal ions on the decomposition of GSNO. The degradation of GSNO (0.5 mM) was enhanced by the presence of Cu²⁺and Ni²⁺ions. The amount of NO release from GSNO degradation was estimated by the Griess reaction based on nitrite accumulation. The results indicate that nitrite production was elevated by at least 2-fold in the presence of Cu²⁺. Our study further indicates that Cu²⁺

enhance GSNO-induced apoptosis in human colon adenocarcinoma (HT 29) cells. We also found that copper ions

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modulate the expression of bad, bax, and bcl-2 in GSNO-treated HT 29 cells. The levels of bax and bad proteins were significantly elevated by about 4- to 6-fold when compared with mock-treated cells at 24 h after combined treatment of GSNO plus Cu²⁺ or Ni²⁺. On the other hand, significant inhibition of bcl-2 occurred in HT 29 cells with simultaneous treatment of GSNO with Cu²⁺ (or Ni²⁺). It seemed that Cu²⁺ (Ni²⁺) could enhance the decomposition of GSNO that liberated NO to activate the pathways. Our results demonstrated that the apoptotic effects induced by GSNO was promoted by Ni²⁺ and Cu²⁺ through two different mechanisms: by depletion of intracellular GSH level and by triggered of NO release from GSNO which then promoted the NO-induced apoptotic cell death in human cells.

Keywords: Apoptosis, Nitric Oxide, Air pollutant.

Introduction

Nitric oxide (NO) is produced from macrophages or macrophage-like cells by inflammatory stimulation, and has various effects on the immune system. Previous studies have demonstrated that the induction of nitric oxide synthase by LPS plus interferon-γ (INF-γ) or interleukin-1 (IL-1) causes apoptosis in various types of cells [1-4]. Other studies including ours have indicated that high concentrations of NO induce wild-type p53 protein accumulation

and apoptosis [5-9]. Recent studies have demonstrated that p53 performs an essential role in response to NO generated either from an NO donor or from over expression of NOS. Over expression of wild-type p53 in human tumor cell lines results in down-regulation of NOS gene expression, as well as enzymatic activity [10]. Such an observation implies that an increased level of NOS expression in human tumor samples may be a loss of p53-mediated NOS gene regulation due to functional inactivation of wild-type p53. Little is known about the pathways leading to formation of RSNO in biological tissues. NO does not react with sulfhydryl groups directly, but a potent nitrosating species (NO_x) is formed upon reaction of NO with oxygen. There is general agreement that the biologic activity of nitrosothiols, e.g., smooth muscle relaxation or inhibition of platelet aggregation, is due to release of free NO, but the mechanisms involved in the decomposition of these compounds are unclear.

Remarkable variations have been reported in the stability of different nitrosothiols in aqueous solution at physiological pH levels, with their half lives ranging from a few seconds (S-nitroso-L-cysteine) to hours (GSNO). However, more recent studies indicate that nitrosothiols are stable in aqueous solutions and only decompose and release NO in the presence of trace metals. Thus, Cu²⁺

ions have been shown to trigger decomposition of S-nitroso-N-acetyl penicillamine (SNAP) and GSNO, and

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the effect of copper ions was found to be potentiated by reductants such as ascorbate. We further assessed the effects of Cu²⁺ and Ni²⁺ ions on the modulation of bax, bcl-2, and bad gene expression in response to GSNO treatment. The described reactions may have important implications for the biochemistry and physiology of NO–mediated biological processes.

Result and discussion

÷ ÷ As shown in figure 1A, GSNO was stable at room temperature (25 °C) for at least 96 h.

In contrast, the absorbance of 334 nm apparently decreased at 48 h when GSNO was exposed to 37 °C. Such results imply that NO may be released from GSNO at 37 °C in a cell culture system. GSNO (0.5 mM) solution was then prepared with potassium phosphate buffer (50 mM) to different pH values and then exposed to 37 °C or 25 °C at the indicated times (Figure 1B). This experiment demonstrated that GSNO degradation was more rapid at a higher pH (pH 8.0) when exposed to 37 °C. Our results indicate that decomposition of GSNO (0.5 mM) was more rapid in the presence of Cu²⁺ and Ni²⁺ (Figure 2) when compared to other metal ions (data not shown).

The cytotoxicity of Cu²⁺ after 24-h treatment was evaluated in HT 29 cells. We found that a concentration of Cu²⁺ of less than 10 µM was non-toxic to HT 29 cells (Figure 3A). As described above, Cu²⁺ (10 µM) strongly enhances GSNO decomposition; the level of

NO production in HT 29 cells was further estimated with the Griess reaction by determination of nitrite accumulation (Figure 3B). Our results indicate that nitrite production was elevated by at least 2-fold in the presence of Cu²⁺(Figure 3B). In order to investigate whether Cu²⁺ could modulate NO-induced apoptosis, the percentage of apoptotic cells was calculated by TUNEL assay, which further demonstrated that Cu²⁺

enhances GSNO-induced apoptosis (Figure 3C). In order to further demonstrate the critical role of Cu²⁺

in intracellular GSNO decomposition, HT 29 (Figure 4) cells were pretreated with GSNO (1 mM) for 6 h, washed twice with PBS, and then treated with Cu²⁺ (2.5 µM) at the indicated times. Other groups were treated with either GSNO (pretreated for 6 h then washed with PBS), or Cu²⁺ (treated consistently) as a comparison group. We found that exogenous added Cu²⁺ significantly augments the apoptotic effect induced by GSNO (Figure 4).

In our recent studies, we demonstrated that bcl-2 protein expression was down-regulated and bax protein expression was induced in NO-treated human cancer cells. Figure 5 shows that the expression of bcl-2 was down-regulated in a time-dependent manner when cells were treated with GSNO (2mM). On the other hand, bax and bad proteins in HT 29 and COLO 205 cells were significantly elevated by GSNO treatment (Figure 5). By comparison, the alterations of bax and bcl-2 protein expression were more

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significant in the COLO 205 than HT 29 cell by GSNO treatment (Figure 5).

We have gained further insight into the role of Cu²⁺ and Ni²⁺ involved in alteration of gene expression inGSNO-induced apoptosis. The levels of bax and bad proteins were significantly elevated by about 4- to 6-fold when compared with mock-treated cells at 24 h after treatment with GSNO plus Cu²⁺ or Ni²⁺ (Figure 6A and B). On the other hand, significant inhibition of bcl-2 occurred in HT 29 cells when simultaneously treated with GSNO and Cu²⁺ (or Ni²⁺) (Figure 6C). In this figure, regulation of the bax, bad, and bcl-2 levels in only GSNO-treated HT 29 cells was less significantly affected (Figure 6A, B, and C). It seems that Cu²⁺ (Ni²⁺) plays a critical role in decomposition of GSNO which clearly augments the effects on regulation of gene expression in apoptotic cells.

In our previous results, we demonstrated that antioxidants (such as L-N-acetyl-cysteine, LNAC) attenuate NO-induced apoptosis in human colon cancer cells. To test the

protective mechanisms of antioxidants, we demonstrated that the intracellular level of glutathione (GSH) was elevated in cells after exposure to LNAC. Our results suggest that the protective effect of LNAC might be linked to its inducement of increases in cellular glutathione and bcl-2 protein levels and to its suppression of cellular bax protein in treated cells. In this study, we considered the possibility that metals modulating apoptosis in

NO-treated cells might arise from several mechanisms. In order to evaluate the roles of intracellular antioxidants (GSH) in cells exposed to NO and (or) combined treatment with metals, we therefore compared the levels of DNA fragmentation in cells after exposure to LNAC, GSNO, metals, and combined treatment. As shown in figure 7, DNA fragmentation was observed in GSNO (lane 4) and combined treatment of GSNO and metals (lanes 5 and 6). By contrast, LNAC protects apoptosis in cells exposed to GSNO (or combined treatment with GSNO plus metals) (lanes 7 to 9).

Our previous report shown that the intracellular level of GSH was elevated in cells after exposure to LNAC. We found that elevation of intracellular GSH level could attenuate NO-induced apoptosis in human cancer cells. Another study also demonstrated that apoptosis could be induced by GSH depleting agent (BSO) in human cholangiocytes.

In this study, we further investigate whether promotion of NO-induced apoptosis by Cu²⁺ and Ni²⁺ were modulated through depletion of intracellular GSH levels. COLO 205 cells were treated either with BSO, GSNO, ions, or by different combination as indicated in the figure 8. The intracellular GSH levels were extremely depleted in cells treated with BSO (50 µM) (Figure 8A, lane 2) when compared to the control group (Figure 8A, lane 1). Interestingly, significant decreased of GSH levels were also observed in cells by combine treatment of GSNO either

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with Cu²⁺ or Ni²⁺ (Figure 8A, lanes 5 and 7). We further demonstrated that apoptosis induction and intracellular GSH depletion were occurred simultaneously in cells by combine treatment of ions and GSNO (Figure 8A and B, lanes 5 and 7). However, less extent (15%) of cells undergoing apoptosis were observed in the BSO-treated group although the GSH levels were extremely depleted (Figure 8A and B, lane 2). Such results implied that Cu²⁺ (or Ni²⁺) mediated apoptotic effects induced by GSNO were partially through depletion of intracellular GSH level.

To further clarify such observations, the level of nitrite production was then measured in cells treated either by BSO plus GSNO (Figure 8C, lane 3) or ions plus GSNO (Figure 8C, lanes 5 and 7). We demonstrated that the level of nitrite production was consistently with the apoptotic effects induced by combine treatment of GSNO and ions (Figure 8B and C, lanes 5 and 7) whereas cells combine treated with GSNO and BSO do not (Figure 8B and C, lane 3).

Collectively, our results suggest that the apoptotic effects induced by GSNO was promoted by Ni²⁺ and Cu²⁺ through two different mechanisms: by depletion of intracellular GSH level and by triggered of NO release from GSNO which then promoted the apoptotic cell death in human cells.

References

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Molecular Carcinogenesis (in press).

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19:101-113.

Studies on the molecular mechanisms of genotoxicity of nitric oxide (NO)

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