Effects Of Sulfur Dioxide On Gene Expression Profiles, Apoptosis And Cancer-related Genes From Rat

Recent years, many studies in the effect of sulfur dioxide (SO₂) on the genetic toxicity, oxidative damage, DNA damage, signal transduction, membrane damage etc, have been detected in our laboratory, and the results showed that the toxicological effect of SO₂ was abroad and complex. Conventional toxicology research investigates the toxicological effect only from one or two aspect, and it couldn't reflect the response of whole genome to one toxicant. So in order to study the molecular mechanism of toxicological effects of SO₂ from the genome response, Affymetrix GeneChip (RAE230A) analysis was carried to analyze the gene expression profiles in the lungs of Wistar rats exposed to SO₂ (14mg/m³, lh/day, for 30 days), this work was cooperated with Qin Guohua in our laboratory. The results found that 173 genes, containing 79 known genes and 94 novel genes, were up-regulated, and 85 genes, containing 46 known genes and 39 novel genes, were down-regulated in rats long-term exposed to SO₂ compared with control rats. It is suggested that discriminating genes in lungs of rats exposed to SO₂ included those involved in immune, inflammation, oxidative stress, oncogene,tumor suppresses fatty acid and glucose metabolism, extracellular matrix and others. The mechanism of long-term exposed to SO2 is complex. These results lead to a conclusion that exposure to SO2 could cause inordinance of fatty metabolism, effect the phospholipids membrane fluidity, signal transduction and super oxidative damage of membrane, and could have some relations with occurrence of tumor, cause changes in cell growth, transfer, differentiation, proliferation, synthesis of extracellular matrix, and histological structure. SO2 could cause expression changes of many genes and it indicated that expression of many genes was variable and unstable, and there might be a SCVrelated unstable expressional genome in lungs of rats exposed to SO2.Many previous studies showed that SO2 could cause oxidative stress, reduction of glutathione (GSH)—one of the antioxidant, DNA damage, increase of tumor necrosis factor (TNF), elevation of concentration of cytoplasmic free calcium, and increase of membranous permeability of mitochondria, and these results suggested that SO2 might have some relations with apoptosis. In order to study the molecular mechanism of occurrence of apoptosis exposure to SO2, the mRNA and protein levels of p53, bax, bcl-2, caspase-3, caspase-8 were analyzed in livers and lungs of rats after exposed to different concentrations of SO2 ((K 14> 28> 56 mg/m3, 6 h/day, for 7 days) using a real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) assay and immunohistochemistry method, and activities ofcaspase-3 were detected, histological structure were observed using hematoxylin and eosin staining (HE staining), apoptotic cells were detected by the TdT-dUTP terminal nick-end labeling (TUNEL) method, respectively. The results showed that mRNA and protein levels of p53, bax, caspase-3 and caspase-8 were increased in a dose-dependent manner with the increase of SO2 concentrations, while mRNA and protein levels of bcl-2 were decreased in livers and lungs of rats exposed to SC^and it had a dose-effect relationship with concentrations of SO2, and caspase-3 activities were increased. Analysis on histological structure observed several kinds of necrosis of liver including spot necrosis and focal necrosis, and livers were infiltrated with lymphocytes, monocytes, few neutrophil, eosinophils, congestion and inflammation happened in lungs. The results of TUNEL showed that there were some apoptotic cells in livers and lungs of rats after exposure to SO2. These results lead to a conclusion that exposure to SO2 could change the expression of apoptosis-related genes, and it suggested that SO2 could induce apoptosis in liver and lung of rat and it might be related with some apoptosis-related diseases, and it indicated that precise mechanism of regulation of apoptosis was very important to organism. Elucidating the expression patterns of those factors after SO2 inhalation might be critical to our understanding mechanisms of SO2 toxicity and helpful for the therapeutic intervention. Increase of mRNA and protein expression of caspase-8 and elevation of TNF levels in previous study indicated that death-receptor pathway took part in theoccurrence of apoptosis, and expression changes of bax and bcl-2 suggested that mitochondria pathway might play a role in the process of apoptosis, and up-regulation of p53 mRNA and protein levels showed that p53-dependent pathway was concerned with the appearance of apoptosis, and all of this illuminated that inducement of SO2 on apoptosis was regulated by many signal pathway and it was a very complicated biological process.Apoptosis is a responsive reaction to special factor in the environment such as high temperature, radiation, chemical toxicant and many cytokines etc. Many studies considered that trigger action of apoptosis is a chain-model result of many genes taking part in, including oncogenes and tumor suppressor genes such as c-myc, p53, ras etc. Oncogenes and tumor suppressor genes exert important and precise regulatory function in normal biological process, and not only participate in the regulation of apoptosis, but also take part in the regulation of cell cycle, and play important regulative role in the process of tumor occurrence. In order to study the toxicological mechanism of the correlation between occurrence of apoptosis, cancer and exposure to SO2, the mRNA and protein levels of some oncogenes ( c-fos, c-jun, c-myc, K-ras) and tumor suppressor genes (p53, Rb, pi6) were analyzed in livers and lungs of rats after exposed to different concentrations ofSO2((K 14^ 28> 56 mg/m3, 6 h/day, for 7 days) using a real-time RT-PCR assay and western blot method, respectively. The results showed that mRNA and protein levels of oncogenes such as c-fos, c-jun, c-myc, K-ras and tumorsuppressor gene p53 were increased in a dose-dependent manner with the increase of SO2 concentrations, while mRNA and protein levels of tumor suppressor genes Rb and pl6 were decreased in livers and lungs of rats exposed to SO2 and it had a dose-effect relationship with concentration of SO2. Increased expression of c-jun mRNA and protein in livers and lungs of rats exposed to SO2 could be considered as a early sign of activation of inflammatory cells. Abundant expression of oncogenes c-myc, c-fos, c-jun might be one of the signal pathway of the inducement of apoptosis in livers and lungs in rats after inhalation of SO2, and it validated that the occurrence of apoptosis had some relations with abnormal expression of these oncogenes. Increased expression of p53 mRNA and protein and DNA damage effect of SO2 in previous studies indicated that increase of p53 expression played a very important role in the process of DNA damage mediated by hyperoxia. The coadjustment of pi6 and Rb is one of the key factors in cell cycle from Gi phase to S phase, while SO2 inhalation destroyed the balance status of the feedback regulative pathway. One hand exposure to SO2 caused decrease in pi6 mRNA and protein expression levels, and it indicated that regulative function of Rb in cell cycle was weakened by SO2 inhalation. On the other hand, decreased expression of Rb caused by SO2 inhalation could result in abnormity of G] check-point and elevate possibility of cell proliferation immortalization. At the same time of decreased expression of Rb caused by SO2 inhalation, the expression of pi6 did not increased but decreased, and itsuggested that the suppressive function on tumor of Rb pathway was suppressed by SO2 inhalation.In summary, studies on apoptosis-related genes, oncogenes and tumor suppressor genes indicated that promoter genes of apoptosis and tumor suppressor genes could produce anti-apoptosis signal to antagonize the growth signal arise from oncogenes. Since apoptosis is an active and programmed cell death, and it can be considered as an active antagonistic mechanism to tumor in organism, and these results suggested that apoptosis and cell proliferation were coinstantaneous pathological and physiological phenomenon. The results of genechip indicated that toxicological mechanism of SO2 was very complex and it was a biological process that expression of many genes was variable and unstable, all of the toxicological mechanism could form a very complex and enormous network to regulate the effect of SO2 on organism, and further studies should be critical to our understanding mechanisms of SO2 toxicity and helpful for the therapeutic intervention.