Study Of Sulfur Dioxide On Gene Expression Profiles, Cytochrome P450, And Cancer Related Gene Expressions In Vivo And Vitro

Sulfur dioxide (SO₂) is a ubiquitous air pollutant, present in low concentrations in the urban air, and in higher concentrations in the working environment. Few data are available on the effects of exposed to this pollutant on the molecular mechanism, although some biochemical changes, genetic toxicity, oxidative stress, and DNA damage have been detected. Affymetrix GeneChip (RAE230A) analysis was carried to analyze the gene expression profiles in the lungs of Wistar rats exposed to SO₂ for short-term (56 mg/m³, 6h/day, for 7 days) and long-term (14 mg/m³, 1h/day, for 30 days). Study of long-term inhalation SO₂ was cooperated with my classmate Bai Juli under directing by our tutor. It was found that 31 genes, containing 18 known genes and 13 novel genes, were up-regulated, and 30 genes, containing 19 known genes and 11 novel genes, were down-regulated in rats short-term exposed to SO₂ compared with control rats. While there were 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. These results lead to the conclusions: (1) A notable feature of the gene expression profiles was the changed expressions of genes related to oxidative phosphorylation in lungs of rats short-term exposed to SO₂, which showshigh-dose short-term exposed to SO₂ may cause the deterioration of mitochondrial functions;(2) Discriminating genes in lungs of rats long-term exposed to SO₂ included those involved in fatty acid metabolism, immune, inflammatory, oxidative stress, oncogene, tumor suppressor genes, extracellular matrix, and so on. The mechanism of low-dose long-term exposed to SO₂ is more complex. (3) SO₂ exerts its effects by different mechanisms in vivo at high-dose short-term inhalation and at low-dose long-term inhalation. Because low-dose long-term inhalation is the SO₂ exposures main approach of human beings. It indicates that SO₂ exerts its toxicological effects in human beings by regulating the expressions of a series of genes.In order to investigate the effects of SO₂ on cytochrome P450s, male Wistar rats were housed in exposure chambers and treated with 14, 28 and 56 mg /m³ SO₂ for 6 h/day for 7 days, while control rats were exposed to filtered air in the same condition. The contents of hepatic cytochrome P450 contents and the activities of CYP2E1 of rats were measured by spectrophotometry. Fluorescence spectrophotometry was used to study the activities of CYP1 Al, CYP1A2 and CYP2B1/2. The mRNA levels of CYP1A1, CYP1A2, CYP2B1/2, and CYP2E1 were analyzed in livers and lungs by using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) and RT-PCR assay, respectively. It showed that the hepatic total CYPs contents, the activities and mRNA levels of CYP1A1, CYP1A2, CYP2B1/2 in lungs and livers, the activities and mRNA levels of CYP2E1 in lungs were decreased in rats exposed to SO₂. Furthermore, the decreases of total CYPs contents, activities and mRNA levels of these P450 enzymes caused by SO₂ at different concentrations in lungs and livers of rats followed lineardose-response curves. However, the hepatic CYP2E1-dependent p-nitrophenol hydroxylases (p-NP) and mRNA levels were unaltered by SO2 at all detected concentrations. It indicates that SO2 exposure can suppress the total CYPs contents, the activities and mRNA levels of hepatic and pulmonary CYP1A1/2, CYP2B1/2 and 2E1 of rats except for hepatic CYP 2E1 and ROS and/or cytokines might act as mediators of this effect. Reduction of CYP450s expression during SO2 exposure may be part of an adaptive response by the liver and lung to minimize cell damage. The capacity of the liver and the lung to metabolize xenobiotics can be compromised in both animals and humans.Benzo(a)pyrene (B(a)P), a polycyclic aromatic hydrocarbon, is a ubiquitous environmental contaminant with diverse toxicological effects. In order to investigate the interactions between SO2 and B(a)P, male Wistar rats were exposed to intratracheally instilled with benzo(a)pyrene (B(a)P;3 mg) or SO2 (56 mg/m3) inhalation alone or together. The mRNA of CYP1A1/2, proto-oncogenes (c-fos, c-jun, c-myc, and K-ras), tumor suppressor genes (p53, pi6, and Rb) and protein levels of those proto-oncogenes and tumor suppressor genes were analyzed in livers and lungs using real-time RT-PCR assay and Western blot analysis, respectively. And 7-ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities were detected. In livers and lungs of rats exposed to SO2 alone, the mRNA expressions and activities of CYP1A1 and CYP1A2, the mRNA and protein expressions of p 16 and Rb were decreased. Meanwhile, the mRNA and protein levels of c-jun, c-fos, c-myc, K-ras, and p53 were increased significantly. Exposure to B(a)P alone induced the mRNA expressions and activities of CYP1A1 and CYP1A2, the mRNA and protein expressions ofc-jun, c-myc, K-ras, and p53 in livers and lungs except for c-jun mRNA levels. However, exposure to B(a)P plus inhaled SO2 neither increased nor decreased the mRNA expressions and activities of CYP1A1/2, c-myc mRNA expressions, and K-ras mRNA and protein expressions in livers and lungs, pi6 and Rb mRNA expressions in lungs, versus exposed to B(a)P or SO2 alone. Nevertheless, exposure to B(a)P plus inhaled SO2 increased the mRNA and protein levels of c-jun, c-fos, and p53 in both livers and lungs, decreased the mRNA and protein levels of p 16 and Rb in livers compared with rats exposed to SO2 or B(a)P alone. The synergistic inductive effects were observed in the mRNA levels of c-myc but not in the protein levels in rats exposure to B(a)P plus inhaled SO2. The synergistic inhibitive effects were observed in pi6 and Rb protein levels but not mRNA levels in lungs. Accordingly, the SO2-induced decreases of CYP1A1/2 might not influence the metabolic activation of B(a)P. However, when B(a)P and SO2 were given in the combinations, one might postulate that a synergistic effect on the expressions of proto-oncogenes (c-fos, c-jun, and c-myc) and tumor suppressor genes (p53, pi6, and Rb) between SO2 and B(a)P, which might be one of the possible mechanisms of combination effects between B(a)P and the air pollutants.In order to study the possible carcinogenic molecular mechanism of SO2 and its derivatives, the dose-response and time-response relationships of SO2 derivatives on transcriptional and translational regulations of some proto-oncogenes and tumor suppressor genes in human bronchial epithelial cells BEP2D were investigated. The mRNA and protein levels of those proto-oncogenes (c-fos, c-jun, c-myc, and K-ras) and tumor suppressor genes (p53, pi6, and Rb) were analyzed in BEP2D cells using real-time RT-PCRassay and Western blot analysis, respectively. It showed that dose-dependentinductive expressions of c-fos, c-jun, c-myc, K-ras, and p53 mRNA andprotein and dose-dependent repressive expressions of pl6 and Rb mRNA andprotein on BEP2D cells after SO2 derivatives exposure. The expressions ofthose genes differed at different post-exposure time. BEP2D cells wereexposed for 4 h (0.1 mM SO2 derivatives), rinsed, and maintained in normalgrowth media for 0, 0.5, 1, 4, or 24h post-treatment. The highest inductionsof c-fos and c-jun were observed at 0.5h or Oh, respectively. It showed highexpressions of c-fos and c-jun at different post-exposure time, and significantinduction could be observed at 24h except that the mRNA levels of c-jun wasdecreased to the base line. The highest inductions of p53 and K-ras wereobserved at Oh or 0.5h, respectively. SO2 derivatives elicited similar increasein c-myc expressions in BEP2D cells at 0 and 0.5 h following exposure. Itshowed time-dependent decreases of c-myc, K-ras, and p53 expressions afterpost-exposure. The expressions of c-myc, K-ras, and p53 were decreased tothe base line at 24h, 4h, and 0.5h, respectively. The post-exposure time didnot affect the expressions of pl6 and Rb. The expressions of pi6 and Rbwere decreased at Oh, and the expressions were similar to the initial levels atother post-exposure time. It indicated that the change of c-fos, c-jun, pl6, andRb expressions might involve in the permanent damage of SO2 and itsderivatives. However, the transient inductions of c-myc, K-ras, and p53 mighthave relation with the acute damage of SO2 and its derivatives. Thealterations of proto-oncogenes and tumor suppressor genes expressions bySO2 derivatives would disorder the regulations of cellular proliferation,differentiation, and apoptosis in BEP2D. It offer a possible molecularmechanism of the increase of tumor incidence in mice long-term exposed toSO2. It implied that the possibility of carcinogenesis increased after long-term SO2 exposure in human bronchial epithelial cell.In conclusion, the toxicological mechanism of SO2 on human being or animal involved not only in its genetic toxicity, oxidative damage, and DNA damage, but also in the alterations of a serious of genes transcriptional and translational expressions. For example, it alters the biotransformation of xenobiotics by the CYP450s suppression and disorders the regulations of cellular proliferation, differentiation, and apoptosis by alterations of proto-oncogenes and tumor suppressor genes expressions. Meanwhile, the mechanisms of combination effects between B(a)P and SO2, which usually present in the air at the same time, were studied in the present study. It suggests that the alterations of proto-oncogenes and tumor suppressor genes might share the mechanism of the co-carcinogenesis of SO2 with B(a)P.