| 1BackgroundHuman diseases, especially human cancers have been associated with environmental causes. The environment is swarmped with food additives, insecticides, pesticides and industrial chemicals. These chemicals are endangering people's health through various all sorts of ways. For example, they can modify endogenous pathways and induce malignant transformation of human cells. Resulting from environmental contamination, the worldwide incidence of many forms of cancer have increased. Insight into the intricate causes of different types of cancer requires detailed analysis of mechanism inherent in each specific type of carcinoma. Challenges involve toxicological approaches to integrate recent advances to elucidate the causation and ultimate prevention of human cancer. Significant and promising fruits of exploratory studies on exposure levels to chemical carcinogens that produce tumor development provide useful clues and contribute to the evaluation of human carcinogens. However, The molecular mechanisms underlying carcinogenicity are still not very clear. In addtion, more important thing is how to prevent cancers using these kownl edges.In the post-genome era, epigenetics becomes a hot spot. The term "epigenetics" is defined as the study of regulation of gene activity that is not dependent on gene sequence and involves heritable and non-heritable alterations in gene activity and transcriptional potential of cells. That is to say, epigenetics refers to changes in phenotype or gene expression caused by mechanisms other than changes of DNA sequence. These changes may remain through cell divisions and last for multiple generations without changes in the underlying DNA sequence. Instead, non-genetic factors, DNA methylation and histone modification, for instance, cause different gene expression. The combinatorial pattern of these factors, which is interpreted by the cell as an epigenetic code, accounts for why the differentiated cells in a multi-cellular organism express only the genes which are required for their activity. The past several years have witnessed an explosive increase in our knowledge about epigenetics. The introduction of new biologic technologies, such as chromatin immunoprecipitation (ChIP) analysis which has accelerated the discovery of a growing list of epigenetic events associated with carcinogenicity.Cancer is related to both genetic and epigenetic mutations. Most people thought genetic mutations include base substitution, insertion as well as DNA strand breaks cause cells loss of regulation, thus induce cancer. At present, many researchs found that epigenetic modifications, such as cytosine methylation, are also factors of cancer. Changes of epigenetic modificantions are found in lung cancer, prostate cancer and breast cancer. Although epigenetic modifications are determined in early life, they can be modified with environmental stimuli. In such way, external environmental factors affect gene expressions and chromatin structures. Therefore, epigenetic alterations might be a key factor for pollutants to cause cancers.Chromium and its compounds have been widely used in the manufacture of a large number of industrial products, such as stainless steel. However, high volume utilization and inappropriate disposal of chromium waste products have created abundant sources of heavy environmental pollution. Exposure to chromium impacts millions of people residing in the vicinity of many toxic spots and users of chromium products. Chromium is a potent human mutagen and carcinogen. The capability of chromium to cause cancers has been known for more than a century, and numerous epidemiological studies have been performed to determine its carcinogenicity. Benzo[a]pyrene(B[a]P), which belongs to polycyclic aromatic hydrocarbons(PAHs), is also a procarcinogen and an environment toxicant present in car exhaust, incomplete fossil fuel combustion, municipal waste incineration, tobacco smoke, certain foods and occupational exposures. The carcinogenicity of PAHs has been well established. International Agency for Research of Cancer sets B[a]P as Class2group A human carcinogen. Joint toxic action of two or more chemicals is an important research field of environmental toxicology. Chemicals are always coexistence in the environment and interact with each other to cause antagonistic or adduct effect when target an object. The study of a single chemical is not enough to show the situation of the real world, thus isn't proper as reference of environmental standards and capacity. It is necessary to pay more attention to joint toxic action of chemicals. Cr(VI) and B[a]P are widely pollutants exist in the environment and usually co-pollute our sociaty. So it is meaningful to find the mechanisms underlying joint action of Cr(VI) and B[a]P.This article armed at studying the mechanisms of joint toxic action of Cr(VI) and B[a]P, including genotoxic and epigenetic effects, and analyzing their roles in it. Our studies have an important theoretic significance in elucidating joint action of Cr(VI) and B[a]P.For the comprehensive evaluation of the safty of both chemicals provide the experimental basis and new prospect.2Methods2.1The biological characteristics of16HBE cells and the detection of cytotoxicity and oxidative damage16HBE cells were treated with Cr(Ⅵ),B[a]P respectively or jointly for6h,12h,24h, and detected with cell count kit8(CCK-8) at the end point. Acording to the result of CCK-8, we set3group and15doses:group1(Cr(VI):0.3μM,0.6μM,1.2μM,2.5μM and5μM), group2(B[a]P:2.5μM,5μM,10μM,20μM and40μM), and group3(B[a]P-Cr(VI):1.2-0.1μM,2.5-0.3μM,5-0.6μM,10-1.2μM和20-2.5μM). The biological characteristics of normal cells and treated cells were compared. DNA damage of cells of different groups were detected with single cell gel electrophoresis. Real-time Q-PCR was used to quantitate the expression of Oxidative damage repair gene(OGG1,MGMT,MYH,MTH1) and base repair gene(XRCCl,MLH1,MSH6, PARP-1)2.2The detection of apoptosis and cell cycle of16HBE cells16HBE cells were treated with Cr(Ⅵ),B[a]P respectively or jointly for24h, and cell cycle and apotosis were assessed by both flow cytometer and single cell gel electrophoresis. Real-time Q-PCR was used to quantitate the expression of apoptosis related gene(Caspase3,bax,Bcl-2).2.3The detection of overall genomic DNA methylation of16HBE cells16HBE cells were treated with Cr(Ⅵ),B[a]P respectively or jointly for24h, and overall genomic DNA methylation of16HBE cells were detected both by5-methyl cytosine immunofluorescence and bisulfite modification. The content of DNA methyltransferases and methyl-binding proteins were detected by western blot.2.4The detection of histone acetylation levels of16HBE cells16HBE cells were treated with Cr(Ⅵ), B[a]P respectively or jointly for24h, and histone acetylation levels of H3and H4were detected by immunofluorescence and western blot. The content of histone deacetylases were also detected by western blot.2.5The detection of histone biotinylation levels of16HBE cells16HBE cells were treated with Cr(Ⅵ),B[a]P respectively or jointly for24h, and real-time Q-PCR was used to quantitate the expression of histone biotinylation related gene(BTD,HCS). The content of histone biotinylation related proteins were detected by western blot while immunofluorescence were used to determine location of these proteins in cells. Histone biotinylation levels were detected by western blot.3Results3.1The biological characteristics of16HBE cells and the detection of cytotoxicity and oxidative damageAs observed in the cell counting kit-8, exposure to Cr(Ⅵ),B[a]P respectively or jointly decreased cell viability in a dose and size dependent manner. Morphological observation found that, normal cells were elongated and loose, while treated cells were square and closely.The damage indexs(tail DNA%,tail length,tail moment,OTM) of treated cells were increased in a dose and size dependent manner. Specifically, B[a]P caused highest DNA damage, while Cr(Ⅵ) induced more high grade injured cells. The degree of DNA damage of Cr(Ⅵ) and B[a]P jointly was similar to Cr(VI) treatment.Cr(Ⅵ) increased expression of MUTYH and MTH1, inhibited expression of MGMT; B[a]P increased expression of all oxidative damage repair genes except OGG1.The joint action of Cr(VI) and B[a]P was similar to Cr(Ⅵ) treatment.Base repair genes were not sensitive to Cr(Ⅵ) treatment while B[a]P increased expression of all4genes in a dose and size dependent manner. The joint action of Cr(VI) and B[a]P was similar to B[a]P treatment.3.2The detection of apoptosis and cell cycle of16HBE cellsThe detection of apoptosis showed that the results of3groups of chemicals treatment were similar. Low dose of chemicals increased proportion of apoptotic cells compared with normal cells, while high dose of chemicals decreased it. The detection of cell cycle showed that Cr(Ⅵ) decreased G1and G2phases but increased S phase gradually, while B[a]P changed cell cycle sharply. The joint action of Cr(Ⅵ) and B[a]P was similar to Cr(Ⅵ) treatment.Cr(Ⅵ) increased expression of Caspase3and bax but inhibited Bcl-2, while B[a]P increased expression of Caspase3and bax but had no influence to Bcl-2. The joint action of Cr(Ⅵ) and B[a]P was similar to B[a]P treatment.3.3The detection of overall genomic DNA methylation of16HBE cellsLow dose of Cr(Ⅵ) can significantly reduce the overall cellular methylation degree, while high dose of Cr(Ⅵ) increased it. B[a]P decreased the overall cellular methylation in a dose and size dependent manner. Low dose of combination of Cr(Ⅵ) and B[a]P reduced the overall cellular methylation of16HBE cells, and with the dose increased, it changed irregularily.Cr(Ⅵ) increased the content of Dnmt3b in cells in a dose and size dependent manner, while high dose of Cr(Ⅵ) increased the expression of MBD2. B[a]P increased significantly the content of Dnmtl and MBD2while reduced Dnmt3b. The combination of Cr(Ⅵ) and B[a]P reduced Dnmtl and MBD2but increased Dnmt3b.3.4The detection of histone acetylation levels of16HBE cellsCr(Ⅵ) reduced H3and H4acylation levels gradually but had no effect to histone acetylation enzymes, such as HDAC2and HDAC3. The effect of B[a]P to H3and H4acylation levels was irregular, while the content of HDAC2and HDAC3were increased in a dose and size dependent manner. The combination of Cr(Ⅵ) and B[a]P increased H3and H4acylation levels, while the content of HDAC2and HDAC3were increased at low dose, but decreased at high dose.3.5The detection of histone biotinylation levels of16HBE cellsCr(Ⅵ) reduced the expression fo both BTD and HCS. The content of BTD in cells was reduced but HCS remained its level. In normal cells, BTD was distributed in both nucleus and cytoplasm, but it focused to nucleus when treated with chemicals. HCS was always located in nucleus whether treated with chemicals or not. The level of histone biotinylation was increased in cells treated with low dose of Cr(VI) but decreased at high dose.B[a]P increased the expression of both BTD and HCS, but the content of HCS in cells had no change while BTD increased. The level of histone biotinylation was increased in cells treated with low dose of B[a]P but decreased at high dose.The combination of Cr(Ⅵ) and B[a]P had no effect to the expression of both BTD and HCS. But the content fo HCS was increased in a dose and size dependent manner, while BTD was reduced. The level of histone biotinylation was changed the same as cells treated with Cr(VI).4Conclusions4.1The main factor of damage in cells treated with Cr(Ⅵ) is ROS which were created during a series of reduction reactinos. The damage caused by Cr(Ⅵ) to16HBE cell is weaker than B[a]P. However, Cr(Ⅵ) competes with B[a]P and dominates the damage effect when cells treat with both Cr(Ⅵ) and B[a]P.4.2Changes of cell apoptosis were the same no matter cells were treated with Cr(VI) or B[a]P respectively or jointly. When cells were injured, cell cycle arrest period allways occurs at S phase. The effect of joint action of Cr(Ⅵ) and B[a]P is similar to Cr(VI) treatment.4.3Both low dose of Cr(Ⅵ) and high dose of B[a]P reduced the overall level of methylation of16HBE cells, which causes loss of chormatin stability and gene imprinting. The effect of joint action of Cr(Ⅵ) and B[a]P to DNA methylation is between the effect of both chemical treatment respectively. 4.4Cr(Ⅵ) reduced histone acetylation levels while B[a]P impacts it irregularly. But the detection of joint action of Cr(Ⅵ) and B[a]P showed that histone acetylation levels were increased.4.5Both Cr(Ⅵ) and B[a]P increased histone biotinylation levels. In normal cells, HCS is mainly responsilbe for biotin activity in nucleus, while BTD is responsible for biotin activity both in nuleus and cytoplasm. Cr(Ⅵ) influences changes of histone biotinylation when cells treated with both Cr(Ⅵ) and B[a]P.
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