Study On The Detection Of BPDE Metabolized From B(a)P And BPDE-induced DNA Damage

B(a)P was also named 3,4- benzo(a)pyrene, composed of a benzene ring and a pyrene molecule, and was a pervasive chemical pollutant in our industrial and living environment. In 1933, the British scholar dissociated benzo(a)pyrene from coal tar, and succeeded in inducing the skin cancer of mice, making benzo(a)pyrene become the first discovered environmental chemical carcinogen. It was identified from generous animal experiments that B(a)P exhibits carcinogenic properties in the part or all of the body. Epidemiologic studys signed that there were many ways that B(a)P could be absorpted, such as passing skin, respiratory passage and so on. Besides carcinogenicity,B(a)P also could cause aberration and mutagenesis. Moreover, it was thought to interfere with endocrine system of human. In 1983, the International Agency for Research on Cancer confirmed B(a)P to be an environmental carcinogen.Although B(a)P was thought to be carcinogenic agent with high activity, it was not the direct acting carcinogen, and it must be activated by mixed function oxidase in the microsome to possess the ability of carcinogenesis. B(a)P was metabolically activated via a three-step process. First, cytochromes P450 catalyze the formation of (7R,8S)-epoxy-7,8-dihydrobenzo[a]pyrene (BaP-7,8-oxide). This is converted to (7R,8R)-dihydroxy-7,8-dihydrobenzo[a]pyrene (BaP-7,8-diol), catalyzed by epoxide hydrolase. BaP-7,8-diol then undergoes another oxidation step, catalyzed by cytochromes P450 and other enzymes, producing mainly (7R,8S)-dihydroxy-(9S,10R)-epoxy -7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). BPDE reacted with biomacromolecule in the body, such as DNA, protein, producing covalent compound, so BPDE was believed to be the ultimate genotoxic metabolite of B(a)P. With the development of studies in B(a)P, popeple payed close attention to the carcinogenicity of BPDE, but the specific mechanism of action was still indefinite.BPDE reacted with DNA producing a major adduct at the N2 position of deoxyguanosine (BPDE-N2-dG). BPDE adducts could prevent DNA from duplicating, result in blocking the duplication, decrease accuracy of duplication and induce the incorrect leak of bases in corresponding site which cotained adducts, at last there would be appeared G-T transformation and partial G-A mutation. So BPDE adducts were thought to play an important role in the early stages of carcinogenesis, and also in the process of growth of cancer.B(a)P was commonly found in coal tar, the smoke from burning carbon, coal and petroleum, vehicle exhaust fumes, industrial waste and tobacco smoke. In addition, broiled and smoked foods also contained considerable B(a)P. So except occupational exposure, in the living enviroment we usually got touch with B(a)P. In light of healthy perniciousness of human from BPDE, which was the ultimate genotoxic metabolite of B(a)P, a plethora of studies had addressed the detection of DNA and protein adducts in human, cell lines and animal exposed to BPDE, but many of them layed particular emphasis on qualitative and quantitative analysis of BPDE-DNA adducts, protein adducts or hydrolysis products of adducts, which the pretreatment were too many so that the recovery rates of BPDE adducts were prone to low. If we choosed directly detect BPDE, not only the methods were simple, easily to operate and could be served as molecular biomarkers suitable for use in risk assessment, but also provided the metabolism activated information of chemical poisons.In this study, we detected the purified BPDE in DMSO solution and BPDE in cell lysate using high performance liquid chromatography with ultraviolet detector (HPLC-UVD) and high performance liquid chromatography with fluorescence detector (HPLC-FD), settling a good foundation to investigate qualitation and quantitative analysis of BPDE further; then detected BPDE induced the DNA damage of HepG2 cell cultured in vitro by single cell gel electrophoresis, which were reflected the damage of genetic material from BPDE; at last explored metabolism of B(a)P prototype, HepG2 cells were treated with B(a)P alone, or pretreated with different concentrations of PCB153 then co-treated with B(a)P and PCB153, then using high performance liquid chromatography with ultraviolet detector and fluorescence detector, detected the density of B(a)P metabolismed in HepG2 cells, with the hope of illuminating the effect of PCB153 in the metabolic process of B(a)P.PartⅠTechniques of detecting BPDE metabolized from B(a)P1. Technique of detecting BPDE metabolized from B(a)P with HPLC-UV detectorWe detected purified BPDE in DMSO solution and BPDE in cell lysate using high performance liquid chromatography with ultraviolet detector (HPLC-UVD). The results were as follows: it was showed a good linear correlation between the concentrations of BPDE in DMSO solution and its peak areas, the linear response with the concentration of BPDE in DMSO solution and its peak area was obtained at the range of 1.00~20.00μg/ml, the equation of linear regression was Y=637138X-124587, the correlation coefficient was 0.9982, the lowest detection limit was 0.0625μg/ml. The equation of linear regression of BPDE in cell lysate was Y=424298X+24598, the correlation coefficient was 0.9921. The intra-assay and inter-assay relative standard deviations were 2.9～3.6% and 4.3～5.9%, respectively. BPDE chemical property was extremely instable and was sensitive to light, moisture and acidic PH. In the same density, the areas between purified BPDE and BPDE in cell lysate were different. According to the data, we discovered the content of BPDE in in cell lysate was lower than that of purified BPDE, but significantly higher than the natural change in the environment.2. Technique of detecting BPDE metabolized from B(a)P with HPLC-fluorescence detectorWe detected purified BPDE in DMSO solution and BPDE in cell lysate using high performance liquid chromatography with fluorescence detector (HPLC-FD). The results were as follows: it was showed a good linear correlation between the concentrations of BPDE in DMSO solution and its peak areas, the linear response with the concentration of BPDE in DMSO solution and its peak area was obtained at the range of 0.01～1.00μg/ml, the equation of linear regression was Y=2E+07X+178569, the correlation coefficient was 0.9988, the lowest detection limit was 0.005μg/ml. The equation of linear regression of BPDE in cell lysate was Y=424298X+24598, the correlation coefficient was 0.9921. The intra-assay and inter-assay relative standard deviations were 3.2～4.6% and 5.9～7.4%, respectively. In the same density, the areas between purified BPDE and BPDE in cell lysate were different. According to the data, we discovered the content of BPDE in in cell lysate was lower than that of purified BPDE, but significantly higher than the natural change in the environment.PartⅡToxicological study on BPDE induced DNA damage in HepG2 cell HepG2 cells were treated with different concentrations of BPDE (0.05μmol/L, 0.1μmol/L, 0.5μmol/L, 1μmol/L, and 2μmol/L) for 24 hours. 50μmol/L B(a)P was used as positive control. The damage of HepG2 cells were detected by single cell gel electrophoresis. The results showed that: in this part of study, the significant differences of Olive tail moment were found between the different concentrations groups of BPDE and normal group(P<0.01), and the significant differences of Olive tail moment were also among the groups of the different concentrations BPDE(P<0.01), in the same time, the significant difference was present between the highest BPDE and positive control. BPDE caused increase in Olive tail moment in a dose-dependent fashion. Statistical significant increases of Olive tail moment were observed in HepG2 cell treated with 0.05, 0.1, 0.5, 1 and 2μmol/L BPDE for 24 hours. These results demonstrated that BPDE could induce DNA damage which were in a dose-dependent fashion.PartⅢThe exploratory study on the metabolism of B(a)P prototypeHepG2 cells were treated with 50μmol/L B(a)P for 72 hours alone, or pretreated with different concentrations of PCB153(0.1μmol/L, 1μmol/L, 10μmol/L, 100μmol/L) for 48 hours then co-treated with 50μmol/L B(a)P and PCB153 for another 24 hours. The density of B(a)P metabolismed in the HepG2 cells were detected by high performance liquid chromatography with ultraviolet detector and fluorescence detector (HPLC-UVD﹑FD).The results were as follows: Compared with group treated with B(a)P alone, enhanced concentrations of B(a)P detected in the HepG2 cells were found in combined treatment groups which PCB153 were at low concentrations (0.1μmol/L, 1μmol/L), and with increasing the concentrations of PCB153, the concentrations of B(a)P detected in the HepG2 cells were also increasing. Although at high concentrations of PCB153 (10μmol/L, 100μmol/L), the concentrations of B(a)P detected in the HepG2 cells were lower than that in the group treated with B(a)P alone , which were decreased with increasing the concentrations of PCB153. So we supposed that when PCB153 induced the enzyme activity of P450, there would be some range of density. Between the range of 10μmol/L and 100μmol/L, the enzyme activity of P450 could be enhanced by PCB153, thus enhanced the metabolism of B(a)P in the HepG2 cells. Once out of this range, PCB153 could not enhance the enzyme activity of P450.