Study On Oxidative Stress And Apoptosis Induced By Cigarette Smoke Solution Combined With Arsenite And Their Mechanisms

ObjectiveArsenic is a ubiquitous metalloid element detected in virtually all environmental media. Excessive chronic intake of arsenic through food, drinking water and air does great harm to health, which is a severe public health problem to the whole world. It has been estimated that 200 million people worldwide are at risk from health effects associated with high concentrations of arsenic in their drinking water. In China, Thailand, Bengal, Chile and other countries, many populations who drink arsenic contaminated water have been found. Endemic arsenic poisoning is also a severe public health problem in our country. In the mainland , there are about 3000 thousand people who drink arsenic contaminated water. Exposure to arsenic occurs occupationally in industries and agriculture, including mining, pesticide, pharmaceutical, glass and microelectronics. Many people exposed to arsenic are smokers. Smoking and arsenic exposure are both severe public health problems. Epidemiologic investigations showed incidence rate of lung cancer, bladder cancer and cardiovascular disease significantly increased in the population exposed to them both. However, few experiments on their interaction ( synergy ) in laboratories have been carried out. So in the present study, oxidative stress and apoptosis in rat lymphocytes exposed to arsenite and cigarette smoke solution (CSS) together or separately were evaluated in our study. And 2×2 factorial design was used to evaluate the interaction ( synergy). Nuclear expression of Nrf2 proteins and DNA binding activity of NF — kB were also determined.Methods1. Cells preparationLymphocytes were prepared by a standard Ficoll - Hypaque method from freshly obtained heparinized blood from Male Wistar rats, by centrifugation on lymphocytes separation medium. Lymphocytes were diluted with PRMI1640 to a cell concentration corresponding to 3 x 10 cells/ml.2. Cigarette smoke solution (CSS) preparationSmoke - bubbled PBS and subsequent cells exposure were performed as others described with some modifications, and final concentrations expressed as cig/ml. PBS in two flasks were used to generate cigarette smoke solution from mainstream smoke of one cigarette through a puffing mechanism related to the human smoking pattern (1 pufl/min;1 puff/35 ml;each puff of 2 s duration). Exposure of cells was started immediately thereafter.3. Cells treatmentsAccording to 2 x 2 factorial design, lymphocytes were divided into 4 groups: arsenite treatment group ( CSS 0 cig/ml, arsenite 20 fjimol/L ) , CSS treatment group (CSS 8 x 10 " cig/ml, arsenite 0 jxmol/ L ) , arsenite and CSS treatment group (CSS 8 x 10" cig/ml, arsenite 20 jxmol/ L ), and control group (CSS 0 cig/ml, arsenite 0 jxmol/ L). Incubation was performed in PR-MI1640 at 37 °C in a CO2 incubator for 2 hours.4. Determination of intracellular ROSAfter the indicated treatments, cells were washed with PBS twice. Cells were pretreated with 25 |xM DCFH - DA for 30 min at 37 °C. They were then washed with PBS for immediate analysis on a Becton Dickinson FACSCalibur flow cytometer. Fluorescence of 20000 cells was detected. The average intensity of fluorescence stood for contents of intracellular ROS.5. Determination of malondialdehydeThe malondialdehyde levels were determined by the thiobarbituric acid method. After the indicated treatments, cells were washed with PBS twice. Then cells were broken up by ultrasonic in ice bath. The supernatant was obtained bycentrifugation. Thiobarbituric acid was used to detect malondialdehyde by flu-orometry. The proteins concentration was used to eliminate the effect on malondialdehyde of the different number of cells in different tubes.6. Comet assayDNA damage was detected by comet assay. After the indicated treatments, cells were washed with PBS twice. Sandwich layers were made before slides were immersed in lysis solution. The slides were then placed in a horizontal electrophoretic tank in cold buffer to allow DNA unwinding. The electrophoresis was carried out in the same solution for 20 min (at 25V) . After electrophoresis, slides were immersed in neutralization buffer and stained with ethidium bromide. Slides were examined with a fluorescence microscope. The damage of DNA was assessed by the tail length of the comets, which was randomly measured with an optical micrometer, 100 cells on each slide at least.7. Reduction rate of Alamar BlueAfter the indicated treatments, cells were washed with PBS twice. After being added 10% Alamar Blue, cells suspensions were plated in 96 -well plates. Cells were incubated at 31% , 5% CO2. After 8 hours, absorbance at 540nm and 620nm were detected by Labsystem Ascent plate reader, and Alamar Blue reduction rate of cells was then computed.8. Determination of cell apoptosisAccording to the manual of apoptosis detection kit, determination of cell apoptosis was carried out. After the indicated treatments, cells were washed with PBS twice. Cells were resuspended in binding buffer and stained with Annexin - V and PI. Flow cytometer was used to collect 10 thousand cells. The software, Cell Quest, was used to analyze apoptosis.9. Detection of proteins expression of Bel -2, Bax, iKBa and nuclear Nrf2 Western blot was used to detect proteins expression. Cells were harvestedand lysed. Proteins of whole cells were extracted. Extraction of nuclear proteins referred to the manual of the kit. Proteins concentration of whole cells or nuclear fractions was detected. The proteins were subjected to SDS - PAGE, transferred to PVDF membranes, and incubated with first and second antibodies. Bound antibody was visualised using the enhanced chemiluminescence reagents and ex-posed to X - ray film. Densitometric analysis was performed on a computer using the public domain NIH Image program (ImageJ).10. Detection of levels of NF - kB DNA bindingElectrophoretic mobility shift assays (EMSA) was used to detect levels of NF - kB DNA binding. Extraction of nuclear proteins referred to the manual of the kit. Proteins concentration of nuclear fractions was also detected. The NF -kB consensus oligonucleotide was radio - labelled with 7 - 32P. Nuclear proteins were pre - incubated. The reaction mixture was then incubated with radio - labelled NF - kB. Samples were subjected to non - denaturing polyacrylamide gel electrophoresis. Gels were dried and exposed to X — ray film. Densitometric a-nalysis was performed on a computer using the public domain NIH Image program (ImageJ ).11. Statistical analysisFollowing ANOVA, multiple comparisons between individual groups were performed using P < 0.05 as the critical limit. The factorial design of the experiment (2x2 design) allowed statistical analysis of the data to be performed using SPSS software. In addition to simple comparisons between treatment means, the interaction term was also assessed for statistical significance. The statistical analysis of the coefficient of the interaction term allowed determination of synergistic or antagonistic interactions between arsenite treatment and CSS treatment.Results1. Intracellular ROSIntracellular ROS increased when arsenite treatment and CSS treatment were used together or separately (P <0.05). Intracellular ROS levels were: arsenite and CSS treatment group > CSS treatment group > arsenite treatment group > control group.2. Malondialdehyde levelsMalondialdehyde levels increased when arsenite treatment and CSS treatment were used together or separately ( P < 0. 05 ). Malondialdehyde levelswere: arsenite and CSS treatment group > CSS treatment group > arsenite treatment group > control group.3. Results of comet assay:The length of the comet tails revealed the DNA of lymphocytes was significantly damaged after addition of arsenite and CSS together or separately (P <0. 05). The length of the comet tails was: arsenite and CSS treatment group > CSS treatment group > arsenite treatment group > control group.4. Reduction rate of Alamar BlueReduction rate of Alamar Blue in CSS treatment group, arsenite treatment group , arsenite and CSS treatment group was significantly lower than control group (P<0. 05). Reduction rate of Alamar Blue was: control group > arsenite treatment group > CSS treatment group > arsenite and CSS treatment group.5. Apoptosis in rat lymphocytesThe number of apoptotic cells increased when arsenite treatment and CSS treatment were used together or separately ( P < 0.05 ). The number of apoptotic cells was;arsenite and CSS treatment group > CSS treatment group > arsenite treatment group > control group.6. Proteins expression of Bel -2Proteins expression of Bel - 2 decreased when arsenite treatment and CSS treatment were used together or separately (P <0. 05). Proteins expression of Bel - 2 was: control group > arsenite treatment group > CSS treatment group > arsenite and CSS treatment group.7. Proteins expression of BaxProteins expression of Bax increased when arsenite treatment and CSS treatment were used together or separately (P < 0. 05 ). Proteins expression of Bax was: arsenite and CSS treatment group > CSS treatment group > arsenite treatment group > control group.8. Proteins expression of nuclear Nrf2Proteins expression of nuclear Nr￡2 in CSS treatment group, arsenite treatment group and arsenite and CSS treatment group increased than control group (P <0.05). But proteins expression of nuclear Nr￡2 in arsenite and CSS treat-ment group was less than both CSS treatment group and arsenite treatment group (P<0.05).9. Levels of NF - kB DNA bindingLevels of NF - kB DNA binding in CSS treatment group, arsenite treatment group increased than control group ( P < 0. 05 ). But levels of NF - kB DNA binding in arsenite and CSS treatment group was less than both CSS treatment group and arsenite treatment group (P<0.05).10. Proteins expression of IkBo:Proteins expression of IkBo: in CSS treatment group, arsenite treatment group decreased than control group (P < 0. 05). But proteins expression of iKBa in arsenite and CSS treatment group was more than both CSS treatment group and arsenite treatment group (P<0.05).11. Results of 2 x 2 factorial design statistical analysisResults of 2 x 2 factorial design statistical analysis were showed: Combination treatment of arsenite and CSS had interactions on ROS levels, malondialde-hyde levels, and the length of the comet tails. The interactions were synergistic. Combination treatment of arsenite and CSS had interactions on the number of ap-optotic cells, proteins expression of Bel - 2 and Bax. The interactions were also synergistic.Conclusions? 1. Intracellular ROS, Malondialdehyde levels and DNA damage increased when arsenite treatment and CSS treatment were used together or separately. And combination treatment of arsenite and CSS had interactions on them. The interactions were synergistic.2. The number of apoptotic cells, proteins expression of Bax increased when arsenite treatment and CSS treatment were used together or separately. On the contrary, proteins expression of Bel - 2 decreased. And combination treatment of arsenite and CSS had interactions on them. The interactions were synergistic.3. Proteins expression of nuclear Nrf2 in arsenite and CSS treatment groupwas less than both CSS treatment group and arsenite treatment group.4. Proteins expression of IKBa in arsenite and CSS treatment group was more than both CSS treatment group and arsenite treatment group. On the contrary , levels of NF - kB DNA binding were less.