砷及其代谢物对BCL-2基因转录本BCL-2α和BCL-2β表达的影响
Effects of arsenic and its metabolites on expressions of BCL-2α and BCL-2β transcripts
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摘要:背景 砷是一种毒物，会影响细胞抗凋亡基因BCL-2及其蛋白质的表达，但砷对BCL-2不同亚型转录本BCL-2α和BCL-2
\beta 的影响尚无相关报道。
目的 研究砷及其代谢物一甲基胂酸（MMA）和二甲基胂酸（DMA）对人正常支气管上皮样细胞（16HBE）和人肺腺癌细胞（A549）中BCL-2基因转录本BCL-2α、BCL-2\beta 及其总和BCL-2T的影响。
方法 将16HBE细胞和A549细胞经体外培养后随机分成3大组：等浓度的不同砷化合物（MMA、DMA和亚砷酸钠）单独染毒组（16HBE细胞的处理浓度为4.5 μmol·L−1，A549细胞为60 μmol·L−1），不同浓度的亚砷酸钠单独染毒组（16HBE细胞的处理浓度为1.5、3.0、4.5 μmol·L−1，A549细胞为20、40、60 μmol·L−1）以及联合染毒组（即MMA+亚砷酸钠、DMA+亚砷酸钠；16HBE细胞的染毒浓度分别均为1.5 μmol·L−1和均为4.5 μmol·L−1，A549细胞的染毒浓度分别均为20 μmol·L−1及60 μmol·L−1），同时在各染毒组中均设立空白对照组。持续染毒48 h后采用实时荧光定量PCR检测两种细胞中BCL-2α、BCL-2\beta 和BCL-2T的相对表达情况。
结果 不同砷化合物单独染毒：16HBE细胞在4.5 μmol·L−1MMA处理下，BCL-2α和BCL-2T的表达水平均低于对照组（q=3.27、2.93，均P<0.05），在4.5 μmol·L−1亚砷酸钠处理下BCL-2α、BCL-2\beta 和BCL-2T的表达水平均低于各自对照组（q=11.06、3.65、10.70，均P<0.05）。A549细胞在60 μmol·L−1DMA处理下BCL-2T的表达水平低于对照组（q=3.12，P＜0.05），在60 μmol·L−1亚砷酸钠处理下BCL-2α、BCL-2\beta 和BCL-2T的表达水平均低于各自对照组（q=7.59、7.27、8.06，均P<0.05）。亚砷酸钠单独染毒：16HBE细胞在1.5 μmol·L−1亚砷酸钠处理下，BCL-2α的表达水平低于对照组，BCL-2\beta 的表达水平高于对照组（q=6.06、11.92，均P<0.05）；在3.0 μmol·L−1亚砷酸钠处理下，BCL-2α的表达水平低于对照组（q=12.72，P<0.05）；在4.5 μmol·L−1亚砷酸钠处理下，BCL-2α、BCL-2\beta 和BCL-2T的表达水平均低于各自对照组（q=15.72、6.79、6.62，均P<0.05）。BCL-2α的表达水平随亚砷酸钠浓度升高逐渐下降（Fα趋势=144.80，P<0.001），BCL-2\beta 和BCL-2T则在1.5 ~ 4.5 μmol·L−1范围内呈剂量依赖性降低（F_\beta 趋势=135.40，FT趋势=38.24，均P<0.001）。在各浓度亚砷酸钠处理下A549细胞BCL-2α、BCL-2\beta 和BCL-2T的表达水平均低于各自对照组（均P<0.05）；且进一步趋势检验结果显示，BCL-2α、BCL-2\beta 和BCL-2T的表达水平均随亚砷酸钠浓度增加逐渐下降（Fα趋势=31.97，F_\beta 趋势=549.50，FT趋势=252.40，均P<0.001）。联合染毒：在均为60 μmol·L−1的MMA+亚砷酸钠处理下，A549细胞BCL-2α、BCL-2\beta 和BCL-2T的表达水平均高于对照组（q=6.37、14.91、5.33，均P<0.05）；在均为60 μmol·L−1的DMA+亚砷酸钠处理下，A549细胞BCL-2α、BCL-2\beta 和BCL-2T的表达水平也均高于各自对照组（q=8.60、17.29、6.91，均P<0.05）
结论 高浓度（16HBE：4.5 μmol·L−1；A549：60 μmol·L−1）的砷代谢产物单独染毒对16HBE及A549细胞的BCL-2转录本多无影响。低浓度（1.5 μmol·L−1）的亚砷酸钠单独染毒可降低16HBE细胞BCL-2α的表达水平，升高其BCL-2\beta 的表达水平；各浓度亚砷酸钠单独染毒可降低A549细胞中所有转录本的表达水平。高浓度（60 μmol·L−1）的MMA与亚砷酸钠联合染毒以及高浓度（60 μmol·L−1）的DMA与亚砷酸钠联合染毒均会升高A549细胞中BCL-2α、BCL-2\beta 和BCL-2T的表达水平，与单独染毒呈现的效应不同。
Abstract:Background Arsenic is a toxicant that can affect the expressions of the cellular anti-apoptotic gene BCL-2 and its protein, but the effects of arsenic on BCL-2α and BCL-2\beta transcripts have not been reported.
Objective To investigate the potential effects of arsenic and its metabolites, methylarsonic acid (MMA) and dimethylarsonic acid (DMA), on BCL-2α, BCL-2\beta , and BCL-2T (total of α and\beta transcripts) in human bronchial epithelial cells (16HBE) and human lung adenocarcinoma cells (A549).
Methods 16HBE cells and A549 cells were randomly divided into three categories of exposure after in vitro culture: single-selected arsenic compound exposure groups with isoconcentration (16HBE cells were treated with 4.5 μmol·L−1 of MMA, DMA, and sodium arsenite, respectively, while A549 cells were treated with 60 μmol·L−1 of MMA, DMA, and sodium arsenite, respectively), sodium arsenite exposure groups with different concentrations (16HBE cells were treated with 1.5, 3.0, and 4.5 μmol·L−1 of sodium arsenite respectively, while A549 cells were treated with 20, 40, and 60 μmol·L−1 of sodium arsenite respectively), and combined exposure groups (i.e. MMA+sodium arsenite, and DMA+sodium arsenite; the exposure concentrations of 16HBE cells were both 1.5 μmol·L−1 and both 4.5 μmol·L−1 respectively, and those of A549 cells were both 20 μmol·L−1 and both 60 μmol·L−1 respectively). Meanwhile, a blank control group was also set up in each exposure category. After 48 h of continuous exposure, the relative expressions of BCL-2α, BCL-2\beta , and BCL-2T in both cells were detected by real-time PCR.
Results Regarding the single-selected arsenic compound exposure, in 16HBE cells, the expression levels of BCL-2α and BCL-2T under 4.5 μmol·L−1 MMA treatment were lower than those in their control groups (q=3.27, 2.93, both P<0.05), and the expression levels ofBCL-2α, BCL-2\beta , and BCL-2T under 4.5 μmol·L−1 sodium arsenite were lower than those in their respective control groups (q=11.06, 3.65, 10.70, all P<0.05). In A549 cells, the expression level ofBCL-2T treated with 60 μmol·L−1 DMA was lower than that in the control group (q=3.12, P<0.05), and the expression levels ofBCL-2α, BCL-2\beta , and BCL-2T treated with 60 μmol·L−1 sodium arsenite were lower than those in their respective control groups (q=7.59, 7.27, 8.06, all P<0.05). Regarding the sodium arsenite exposure: 16HBE cells treated with 1.5 μmol·L−1 sodium arsenite had a lower expression level of BCL-2α and a higher expression level of BCL-2\beta than those in their respective control groups (q=6.06, 11.92, both P<0.05); the expression level ofBCL-2α under 3.0 μmol·L−1 sodium arsenite was lower than that in the control group (q=12.72, P<0.05); and under 4.5 μmol·L−1 sodium arsenite treatment, the expression levels of BCL-2α, BCL-2\beta , and BCL-2T were lower than those in their respective control groups (q=15.72, 6.79, 6.62, all P<0.05). The expression levels ofBCL-2α gradually decreased with increasing concentrations of sodium arsenite (Fα trend=144.80, P<0.001), whileBCL-2\beta and BCL-2T decreased in a dose-dependent manner in the range of 1.5-4.5 μmol·L−1 (F_\beta trend=135.40, FT trend=38.24, both P<0.001). In A549 cells, the expression levels ofBCL-2α, BCL-2\beta , and BCL-2T under each concentration of sodium arsenite treatments were lower than those in their respective control groups (all P<0.05); the results of further trend tests showed that their expression levels gradually decreased with increasing concentrations of sodium arsenite (Fα trend =31.97, F_\beta trend=549.50, FT trend=252.40, all P<0.001). Regarding the combined exposure, under MMA+sodium arsenite treatment at both 60 μmol·L−1, the expression levels of BCL-2α, BCL-2\beta , and BCL-2T in A549 cells were higher than those in their respective control groups (q=6.37, 14.91, 5.33, all P<0.05); under DMA+sodium arsenite treatment at both 60 μmol·L−1, their expression levels in A549 cells were also higher than those in their respective control group (q=8.60, 17.29, 6.91, all P<0.05).
Conclusion Exposure to a high concentration (16HBE: 4.5 μmol·L−1, A549: 60 μmol·L−1) of a single arsenic metabolite has no effect on BCL-2 transcripts in 16HBE cells and A549 cells. Exposure to a low concentration (1.5 μmol·L−1) of sodium arsenite alone would decrease the expression level of BCL-2α and increase the expression level of BCL-2\beta in 16HBE cells, and exposure to all designed concentrations of sodium arsenite alone would decrease the expressions of all transcripts in A549 cells. The combined exposure to high concentrations (both 60 μmol·L−1) of MMA plus sodium arsenite or high concentrations (both 60 μmol·L−1) of DMA plus sodium arsenite would increase the expressions of BCL-2α, BCL-2\beta , and BCL-2Tin A549 cells, which are different from the effects presented by single exposure.
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