Cr（Ⅵ）和B[a]P对16HBE细胞的联合毒性效应及表观遗传改变的研究
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摘要
1研究背景
人类的疾病,尤其是各种癌症,总是与环境因素有着不可分割的联系。我们周围的环境日益复杂,伴随着的是大量的食品添加剂、农药、家用化学品等等。这些物质通过各种途径危害着人们的健康,例如通过改变人类细胞的生物途径而导致细胞的恶性转化,最终导致癌细胞的形成。环境污染物增多的结果就是每年各种不同癌症病例的不断增加。因此,对于这些化学物质是通过怎样的机理以及复杂的途径导致各种癌症的形成,引起了世界各地许多科研工作者的高度关注。虽然目前有许多关于化学物质暴露水平与致癌相关的探索性研究获得了重要成果,但是人们对于致癌的分子机制以及成因仍然不是很清楚。更关键的是,如何将这些环境污染物致癌的毒理学机制和成因与最终预防癌症的发生联系起来。
在后基因时代,表观遗传学在生命科学领域中普遍受到关注。表观遗传是指没有DNA序列变化的、可通过有丝分裂和减数分裂在细胞和世代间传递的基因表达改变。表观遗传学研究主要涉及DNA甲基化、组蛋白翻译后修饰、染色质重塑以及MicroRNA、SiRNA等。也就是说,与遗传学不同,各种表观遗传现象不会涉及基因杂合、基因突变或者微卫星不稳等等,但是也能在细胞分裂生长时与基因序列一样传给分裂细胞。虽然没有导致基因序列的变化,但是这些表观遗传现象,如DNA甲基化的变化却会引起基因的表达发生变化。这也就可以解释,在一个多细胞的组织或器官中,为什么拥有相同基因序列的细胞会根据需要发展成为具有完全不同功能的细胞。表观遗传修饰,例如DNA甲基化以及组蛋白修饰,与染色质重组复合蛋白一起决定了染色质结构的凝聚状态和基因的转录活性。
癌症是一种与基因变异和表观遗传变异都相关的疾病。多数人对于癌症的理解为,这是一种由于基因变异导致体细胞失去正常调控而引起的疾病。这种变异包括碱基替代、插入和切除,以及由于DNA链的断裂造成的序列重组等等。而目前越来越多的研究发现,在有丝分裂过程中,伴随着DNA复制一同稳定继承下来的表观遗传修饰也是引起癌症的一个因素,例如胞嘧啶甲基化的改变。目前被证实的与表观遗传调控基因而导致肿瘤生成的包括了肺癌、前列腺癌以及乳腺癌等等。尽管表观遗传修饰在生命早期就已经确定下来了,但是随着内源的或周围环境的刺激,其发生的改变可以是伴随一生的。通过表观遗传这样一种途径,外在环境刺激就影响到了基因的表达和染色质结构。因此,表观遗传修饰的改变有可能是环境污染物质诱导肿瘤生成的一个关键因素。
六价铬(Cr(VⅥ))是工业上常用的一种材料,在电子产品中就有广泛的用途,如用于色素中的着色剂及冷却水循环系统中,如吸热帮浦、工业用冷冻库及冰箱热交换器中的防腐蚀剂。但是其毒性很强,可能导致敏感以及造成遗传性基因缺陷。长期或短期接触以及吸入时将会有致癌危险,对环境也有持久性危险。对铬类产品的研究也发现铬暴露容易引起与呼吸相关的癌症,如肺癌。苯并芘(benzo(a)pyrene,B [a]P)也是一种强致癌物,可引起肺癌、胃癌、皮肤癌等,属于多环芳烃类化合物(PAH),广泛存在于煤焦油、烟草燃烧生成的烟雾、油炸食物以及汽车尾气中。国际癌症研究机构将其列为第二类A组人类致癌物质。
联合毒性是环境毒理学的一个重要研究方向,即指两种或两种以上毒物共同作用于目标时其保留或改变各自毒性作用的现象。由于自然环境的复杂性,有毒化合物不可能单独存在,更多的是两种或多种化合物共存,它们作用于生物体时往往会因为污染物之间发生交互作用,产生协同或者拮抗以及加合的效应而表现出与单一作用完全不同的联合毒性来。单个污染物的研究虽然具有一定的参考价值,但作为制定环境标准以及环境容量的依据,就显得证据不足,因此混合化合物对机体的联合作用越来越受到人们的重视。而且,由于污染多有伴生性和综合性的特点,研究几种环境污染物的联合损伤效应更加接近环境真实性,国际上对于这种联合毒性的研究也多有报道。Cr(VⅥ)和B[a]P都是广泛存在于环境中的污染物。目前国际上已经开始关注两者的联合毒性效应,因为现代社会同时受到多环芳烃化合物和重金属污染的情况已经非常普遍了,而Cr(VⅥ)和B[a]P分别是这两类物质的代表。
本研究为了解Cr(VⅥ)和B[a]P对于支气管上皮细胞的联合毒性效应,包括遗传毒性和表观遗传修饰变化,分析这两种环境污染物的联合毒性效应,并探讨表观遗传效应在联合毒性中所起的作用,为Cr(VⅥ)和B[a]P的环境卫生标准以及容许浓度提供实验基础,也为化学致癌的防治提供新的思路。
2研究方法
2.116HBE细胞的生物学特征研究、细胞毒性及氧化损伤的检测
分别用Cr(VⅥ)、B[a]P以及两者联合处理细胞6h、12h以及24h,采用CCK-8测定终点,根据测定结果确定染毒剂量和染毒时间,最终设置溶剂对照组(Ctrl)、Cr(VⅥ)处理组(0.3gM、0.6μM、1.2μM、2.5μM和5μM)、B[a]P处理组(2.5μM、5μM、10μM、20μM和40μM)和联合毒物处理组(B[a]P-Cr(VⅥ)：1.2-0.1μM、2.5-0.3μM、5-0.6μ,M、10-1.2μM和20-2.5μM)共3组15个剂量。比较正常细胞和染毒细胞的形态学差异；用单细胞凝胶电泳实验检测各组细胞的DNA损伤程度；用荧光定量PCR(Q-PCR)对氧化损伤修复基因(OGG1、MGMT、MYH、MTH1)(?)(?)碱基修复基因(XRCC1、MLH1、MSH6、PARP-1)进行检测。
2.216HBE细胞的凋亡和周期变化的检测
分别用Cr(VⅥ)、B[a]P以及两者联合处理细胞24h,采用流式细胞仪或单细胞凝胶电泳实验检测细胞的凋亡情况和周期变化情况,用荧光定量PCR (Q-PCR)对凋亡相关基因(Caspase3、bax、Bcl-2)进行检测。
2.3整体基因组DNA甲基化的检测
分别用Cr(VⅥ)、B[a]P以及两者联合处理细胞24h,采用5-甲基胞嘧啶抗体免疫荧光法和亚硫酸氢盐修饰法检测16HBE细胞整体基因组DNA甲基化的变化趋势,并用western blot法检测DNA甲基转移酶和甲基化结合蛋白在细胞中的含量变化。
2.4组蛋白乙酰化水平的检测
分别用Cr(VI)、B[a]P以及两者联合处理细胞24h,采用细胞免疫荧光法以及western blot法检测各处理组细胞的组蛋白H3和H4整体乙酰化水平,并用western blot法检测组蛋白去乙酰化酶在细胞中的含量变化。
2.5组蛋白生物素化水平的检测
分别用Cr(VⅥ)、B[a]P以及两者联合处理细胞24h,采用Western blot法检测各处理组细胞的组蛋白生物素化水平,以Q-PCR法和Western blot法检测组蛋白生物素化相关蛋白在不同处理组中的表达变化和在细胞中的含量变化,并用细胞免疫荧光法对组蛋白生物素化相关蛋白在细胞中的位置进行观察。
3研究结果
3.116HBE细胞的生物学特征研究、细胞毒性及氧化损伤的检测
用不同浓度的Cr(VⅥ)、B[a]P以及两者联合处理细胞后发现细胞的存活率呈剂量依赖性降低,且时间越长,细胞存活率越低。通过形态学观察,发现正常细胞形态完整,呈长形,胞质较多,排列较为疏松；而染毒后细胞呈方形,胞质减少,排列非常紧密。
无论是Cr(VⅥ)、B[a]P还是两者联合处理细胞,各项损伤指标(尾部DNA%、尾长、尾矩、OTM值)均呈剂量依赖性增高,且随着染毒浓度的提高,根据尾矩对细胞损伤程度分级,高等级损伤的细胞所占比例越来越大。具体而言,B[a]P导致的细胞综合损伤程度最高,Cr(VⅥ)染毒对细胞损伤程度最小,但是最高剂量组中重度损伤细胞所占比例大于其他两类染毒方式；联合染毒对细胞的综合损伤程度类似于Cr(VⅥ),但是在低剂量时就出现重度损伤细胞,早于其他两类染毒方式。
Cr(VⅥ)促进MUTYH和MTH1的表达,抑制MGMT的表达,对OGG1的影响不明显；B[a]P对OGG1、MUTYH和MTH1都有促进表达的作用,同样抑制MGMT的表达；联合染毒的效果偏向于Cr(VⅥ)染毒,促进MUTYH和MTH1的表达,抑制MGMT的表达,对OGG1的影响不明显。
Cr(VⅥ)对碱基修复基因的表达影响不大,高剂量染毒才能导致PARP-1、MLH1和MSH6的表达显著提高,而对PARG和XRCC1的表达几乎无影响；B[a]P对该类基因的表达影响非常明显,最低剂量就可以导致4个基因表达的升高；联合染毒的效果类似于B[a]P,促进4个基因的表达,对XRCC1、MLH1和MSH6的影响都具有统计学意义(p<0.05)。
3.216HBE(?)田胞的凋亡和周期变化的检测
凋亡检测结果显示,3类染毒方式对细胞凋亡的影响都相似,与溶剂对照组相比较,低剂量的毒物都能够引起较高比例的凋亡细胞产生；随着染毒剂量的增加,凋亡细胞的比例逐渐降低。周期检测结果显示,随着Cr(VⅥ)浓度的增加,细胞的G1期和G2期逐渐缩短,而S期逐渐加长；B[a]P染毒对细胞周期影响非常明显,最低剂量组(2.5}μM)就能够明显缩短G1期和(G2期,延长s期,随着B[a]P剂量的增加G1期有所延长,而S期有所降低。
Cr(Ⅵ)可以促进Caspase3和bax的表达,抑制Bcl-2的表达；而B[a]P同样促进Caspase3(?)口bax的表达,但是对Bcl-2的影响不大；联合染毒的效果类似于B[a]P,对Caspase3和bax有促进作用,而对Bcl-2无明显影响。
3.3整体基因组DNA甲基化的检测
低剂量Cr(VⅥ)染毒可以明显降低细胞的整体甲基化程度,随着Cr(VⅥ)浓度的增加,细胞整体甲基化程度逐渐回升,而最高剂量组(5μM)的整体甲基化程度较溶剂对照组有显著性提高；B[a]P可以导致细胞整体甲基化程度的降低,随着B[a]P浓度的增加,整体甲基化程度逐渐降低,具有剂量效应关系；低剂量的联合毒物降低了细胞整体甲基化水平,随着染毒剂量增加,整体甲基化程度逐步回升,在染毒剂量为5-1.2μM时达到最高,然后再次降低。
Cr(VⅥ)染毒对Dnmtl的影响不明显,增加了Dnmt3b在细胞中的含量,且具有剂量效应关系,而在最高剂量时才使MBD2的含量有明显增加；B[a]P染毒明显增加了Dnmtl和MBD2在细胞中的含量,而随着B[a]P浓度的增加,Dnmt3b的含量逐渐减少,具有剂量效应关系；联合染毒降低了Dnmtl和MBD2在细胞中的含量,而增加了Dnmt3b的含量。
3.4组蛋白乙酰化水平的检测
随着Cr(VⅥ)染毒剂量的提高,H3和H4乙酰化水平逐渐降低。用Western blot法对组蛋白乙酰化相关蛋白进行检测后发现,Cr(VⅥ)对HDAC2和HDAC3的影响都不大。
但是对于B[a]P处理过的细胞进行检测后发现,虽然HDAC2和HDAC3的含量有剂量依赖性的提高,但是H3和H4的整体乙酰化水平变化并无明显规律,并且H4组蛋白乙酰化程度还略有提高。
Cr(VⅥ)和B[a]P联合处理细胞后发现,组蛋白乙酰化的水平整体趋势是增加的。对相关蛋白的检测发现,与溶剂对照组相比较,HDAC2和HDAC3的含量在低剂量时有所提高,然后随着染毒浓度的继续提高而逐渐减少,最高剂量组(20,2.5μM)的含量变化较对照组明显。
3.5组蛋白生物素化水平的检测
BTD在细胞中的含量变化随着Cr(VⅥ)浓度的提高而显著性降低,但是HCS的含量却几乎没有变化。在正常细胞中BTD蛋白均匀分布在细胞核和细胞质中,随着BTD含量的降低,细胞质中的BTD越来越少,大多集中到了细胞核中,最后基本上只有在细胞核中才能发现BTD。而HCS在正常细胞中也几乎绝大部分存在于细胞核之中,由于在Cr(VⅥ)处理过程中HCS的含量没有变化,所以在其他Cr(VⅥ)处理组中HCS的位置也没有什么变化,都集中在细胞核中。
B[a]P染毒后,细胞中HCS蛋白含量几乎没有变化,而BTD的含量随着B[a]P浓度的提高而降低。经B[a]P染毒的细胞其组蛋白生物素化水平是随着B[a]P浓度的提高而逐渐升高的,到B[a]P浓度为10μM时达到最高,然后开始降低。
Cr(VⅥ)和B[a]P联合处理细胞后发现,HCS在细胞中的含量随联合毒物浓度的提高而显著提高,而BTD的含量在降低到一定程度后又开始回升。虽然BTD和HCS在细胞中的含量变化与Cr(VⅥ)单独处理时不同,但是组蛋白生物素化的水平变化趋势却是类似的。
4结论
4.1Cr(VⅥ)暴露对细胞的损伤主要是因为其在细胞内发生一系列还原反应导致过量自由基产生,而其最终产物Cr(Ⅲ)与DNA形成加合物导致的损伤所占比例不大。Cr(VⅥ)单独暴露所导致的细胞损伤要弱于B[a]P,但是两者联合处理时Cr(VⅥ)占据主导地位。
4.2联合染毒对细胞凋亡的影响与两者单独处理时没有差别,低剂量时细胞凋亡程度显著增加,高剂量时凋亡程度降低。经Cr(VⅥ)和B[a]P染毒后,周期停滞时期主要发生在S期。联合染毒对细胞周期的影响结果也与Cr(Ⅵ)单独处理时情况一致。
4.3低剂量Cr(VⅥ)降低了细胞的整体甲基化水平,导致染色质稳定性失衡以及基因印迹丢失。高剂量B[a]P可以降低细胞的整体甲基化水平。Cr(VⅥ)和B[a]P联合处理细胞时对DNA甲基化水平的影响没有显著性变化。
4.4Cr(VⅥ)可以降低细胞组蛋白乙酰化水平,但是B[a]P对其的影响却没有规律。而两者联合处理导致了组蛋白乙酰化水平的提高,并且对组蛋白乙酰化相关蛋白的影响趋势与两者单独处理时相反。
4.5无论是Cr(VⅥ)还是B[a]P都可以提高组蛋白生物素化的水平。在正常细胞中,HCS主要对细胞核内的生物素活动负责,BTD是细胞内生物素活动的全权管理者,但是在组蛋白生物素化修饰方面却是辅助者。联合染毒时,Cr(VⅥ)对组蛋白生物素化的影响占据了主导地位。
1Background
Human 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.
2Methods
2.1The biological characteristics of16HBE cells and the detection of cytotoxicity and oxidative damage
16HBE 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 cells
16HBE 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 cells
16HBE 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 cells
16HBE 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 cells
16HBE 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.
3Results
3.1The biological characteristics of16HBE cells and the detection of cytotoxicity and oxidative damage
As 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 cells
The 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 cells
Low 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 cells
Cr(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 cells
Cr(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).
4Conclusions
4.1The 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.
人类的疾病,尤其是各种癌症,总是与环境因素有着不可分割的联系。我们周围的环境日益复杂,伴随着的是大量的食品添加剂、农药、家用化学品等等。这些物质通过各种途径危害着人们的健康,例如通过改变人类细胞的生物途径而导致细胞的恶性转化,最终导致癌细胞的形成。环境污染物增多的结果就是每年各种不同癌症病例的不断增加。因此,对于这些化学物质是通过怎样的机理以及复杂的途径导致各种癌症的形成,引起了世界各地许多科研工作者的高度关注。虽然目前有许多关于化学物质暴露水平与致癌相关的探索性研究获得了重要成果,但是人们对于致癌的分子机制以及成因仍然不是很清楚。更关键的是,如何将这些环境污染物致癌的毒理学机制和成因与最终预防癌症的发生联系起来。
在后基因时代,表观遗传学在生命科学领域中普遍受到关注。表观遗传是指没有DNA序列变化的、可通过有丝分裂和减数分裂在细胞和世代间传递的基因表达改变。表观遗传学研究主要涉及DNA甲基化、组蛋白翻译后修饰、染色质重塑以及MicroRNA、SiRNA等。也就是说,与遗传学不同,各种表观遗传现象不会涉及基因杂合、基因突变或者微卫星不稳等等,但是也能在细胞分裂生长时与基因序列一样传给分裂细胞。虽然没有导致基因序列的变化,但是这些表观遗传现象,如DNA甲基化的变化却会引起基因的表达发生变化。这也就可以解释,在一个多细胞的组织或器官中,为什么拥有相同基因序列的细胞会根据需要发展成为具有完全不同功能的细胞。表观遗传修饰,例如DNA甲基化以及组蛋白修饰,与染色质重组复合蛋白一起决定了染色质结构的凝聚状态和基因的转录活性。
癌症是一种与基因变异和表观遗传变异都相关的疾病。多数人对于癌症的理解为,这是一种由于基因变异导致体细胞失去正常调控而引起的疾病。这种变异包括碱基替代、插入和切除,以及由于DNA链的断裂造成的序列重组等等。而目前越来越多的研究发现,在有丝分裂过程中,伴随着DNA复制一同稳定继承下来的表观遗传修饰也是引起癌症的一个因素,例如胞嘧啶甲基化的改变。目前被证实的与表观遗传调控基因而导致肿瘤生成的包括了肺癌、前列腺癌以及乳腺癌等等。尽管表观遗传修饰在生命早期就已经确定下来了,但是随着内源的或周围环境的刺激,其发生的改变可以是伴随一生的。通过表观遗传这样一种途径,外在环境刺激就影响到了基因的表达和染色质结构。因此,表观遗传修饰的改变有可能是环境污染物质诱导肿瘤生成的一个关键因素。
六价铬(Cr(VⅥ))是工业上常用的一种材料,在电子产品中就有广泛的用途,如用于色素中的着色剂及冷却水循环系统中,如吸热帮浦、工业用冷冻库及冰箱热交换器中的防腐蚀剂。但是其毒性很强,可能导致敏感以及造成遗传性基因缺陷。长期或短期接触以及吸入时将会有致癌危险,对环境也有持久性危险。对铬类产品的研究也发现铬暴露容易引起与呼吸相关的癌症,如肺癌。苯并芘(benzo(a)pyrene,B [a]P)也是一种强致癌物,可引起肺癌、胃癌、皮肤癌等,属于多环芳烃类化合物(PAH),广泛存在于煤焦油、烟草燃烧生成的烟雾、油炸食物以及汽车尾气中。国际癌症研究机构将其列为第二类A组人类致癌物质。
联合毒性是环境毒理学的一个重要研究方向,即指两种或两种以上毒物共同作用于目标时其保留或改变各自毒性作用的现象。由于自然环境的复杂性,有毒化合物不可能单独存在,更多的是两种或多种化合物共存,它们作用于生物体时往往会因为污染物之间发生交互作用,产生协同或者拮抗以及加合的效应而表现出与单一作用完全不同的联合毒性来。单个污染物的研究虽然具有一定的参考价值,但作为制定环境标准以及环境容量的依据,就显得证据不足,因此混合化合物对机体的联合作用越来越受到人们的重视。而且,由于污染多有伴生性和综合性的特点,研究几种环境污染物的联合损伤效应更加接近环境真实性,国际上对于这种联合毒性的研究也多有报道。Cr(VⅥ)和B[a]P都是广泛存在于环境中的污染物。目前国际上已经开始关注两者的联合毒性效应,因为现代社会同时受到多环芳烃化合物和重金属污染的情况已经非常普遍了,而Cr(VⅥ)和B[a]P分别是这两类物质的代表。
本研究为了解Cr(VⅥ)和B[a]P对于支气管上皮细胞的联合毒性效应,包括遗传毒性和表观遗传修饰变化,分析这两种环境污染物的联合毒性效应,并探讨表观遗传效应在联合毒性中所起的作用,为Cr(VⅥ)和B[a]P的环境卫生标准以及容许浓度提供实验基础,也为化学致癌的防治提供新的思路。
2研究方法
2.116HBE细胞的生物学特征研究、细胞毒性及氧化损伤的检测
分别用Cr(VⅥ)、B[a]P以及两者联合处理细胞6h、12h以及24h,采用CCK-8测定终点,根据测定结果确定染毒剂量和染毒时间,最终设置溶剂对照组(Ctrl)、Cr(VⅥ)处理组(0.3gM、0.6μM、1.2μM、2.5μM和5μM)、B[a]P处理组(2.5μM、5μM、10μM、20μM和40μM)和联合毒物处理组(B[a]P-Cr(VⅥ)：1.2-0.1μM、2.5-0.3μM、5-0.6μ,M、10-1.2μM和20-2.5μM)共3组15个剂量。比较正常细胞和染毒细胞的形态学差异；用单细胞凝胶电泳实验检测各组细胞的DNA损伤程度；用荧光定量PCR(Q-PCR)对氧化损伤修复基因(OGG1、MGMT、MYH、MTH1)(?)(?)碱基修复基因(XRCC1、MLH1、MSH6、PARP-1)进行检测。
2.216HBE细胞的凋亡和周期变化的检测
分别用Cr(VⅥ)、B[a]P以及两者联合处理细胞24h,采用流式细胞仪或单细胞凝胶电泳实验检测细胞的凋亡情况和周期变化情况,用荧光定量PCR (Q-PCR)对凋亡相关基因(Caspase3、bax、Bcl-2)进行检测。
2.3整体基因组DNA甲基化的检测
分别用Cr(VⅥ)、B[a]P以及两者联合处理细胞24h,采用5-甲基胞嘧啶抗体免疫荧光法和亚硫酸氢盐修饰法检测16HBE细胞整体基因组DNA甲基化的变化趋势,并用western blot法检测DNA甲基转移酶和甲基化结合蛋白在细胞中的含量变化。
2.4组蛋白乙酰化水平的检测
分别用Cr(VI)、B[a]P以及两者联合处理细胞24h,采用细胞免疫荧光法以及western blot法检测各处理组细胞的组蛋白H3和H4整体乙酰化水平,并用western blot法检测组蛋白去乙酰化酶在细胞中的含量变化。
2.5组蛋白生物素化水平的检测
分别用Cr(VⅥ)、B[a]P以及两者联合处理细胞24h,采用Western blot法检测各处理组细胞的组蛋白生物素化水平,以Q-PCR法和Western blot法检测组蛋白生物素化相关蛋白在不同处理组中的表达变化和在细胞中的含量变化,并用细胞免疫荧光法对组蛋白生物素化相关蛋白在细胞中的位置进行观察。
3研究结果
3.116HBE细胞的生物学特征研究、细胞毒性及氧化损伤的检测
用不同浓度的Cr(VⅥ)、B[a]P以及两者联合处理细胞后发现细胞的存活率呈剂量依赖性降低,且时间越长,细胞存活率越低。通过形态学观察,发现正常细胞形态完整,呈长形,胞质较多,排列较为疏松；而染毒后细胞呈方形,胞质减少,排列非常紧密。
无论是Cr(VⅥ)、B[a]P还是两者联合处理细胞,各项损伤指标(尾部DNA%、尾长、尾矩、OTM值)均呈剂量依赖性增高,且随着染毒浓度的提高,根据尾矩对细胞损伤程度分级,高等级损伤的细胞所占比例越来越大。具体而言,B[a]P导致的细胞综合损伤程度最高,Cr(VⅥ)染毒对细胞损伤程度最小,但是最高剂量组中重度损伤细胞所占比例大于其他两类染毒方式；联合染毒对细胞的综合损伤程度类似于Cr(VⅥ),但是在低剂量时就出现重度损伤细胞,早于其他两类染毒方式。
Cr(VⅥ)促进MUTYH和MTH1的表达,抑制MGMT的表达,对OGG1的影响不明显；B[a]P对OGG1、MUTYH和MTH1都有促进表达的作用,同样抑制MGMT的表达；联合染毒的效果偏向于Cr(VⅥ)染毒,促进MUTYH和MTH1的表达,抑制MGMT的表达,对OGG1的影响不明显。
Cr(VⅥ)对碱基修复基因的表达影响不大,高剂量染毒才能导致PARP-1、MLH1和MSH6的表达显著提高,而对PARG和XRCC1的表达几乎无影响；B[a]P对该类基因的表达影响非常明显,最低剂量就可以导致4个基因表达的升高；联合染毒的效果类似于B[a]P,促进4个基因的表达,对XRCC1、MLH1和MSH6的影响都具有统计学意义(p<0.05)。
3.216HBE(?)田胞的凋亡和周期变化的检测
凋亡检测结果显示,3类染毒方式对细胞凋亡的影响都相似,与溶剂对照组相比较,低剂量的毒物都能够引起较高比例的凋亡细胞产生；随着染毒剂量的增加,凋亡细胞的比例逐渐降低。周期检测结果显示,随着Cr(VⅥ)浓度的增加,细胞的G1期和G2期逐渐缩短,而S期逐渐加长；B[a]P染毒对细胞周期影响非常明显,最低剂量组(2.5}μM)就能够明显缩短G1期和(G2期,延长s期,随着B[a]P剂量的增加G1期有所延长,而S期有所降低。
Cr(Ⅵ)可以促进Caspase3和bax的表达,抑制Bcl-2的表达；而B[a]P同样促进Caspase3(?)口bax的表达,但是对Bcl-2的影响不大；联合染毒的效果类似于B[a]P,对Caspase3和bax有促进作用,而对Bcl-2无明显影响。
3.3整体基因组DNA甲基化的检测
低剂量Cr(VⅥ)染毒可以明显降低细胞的整体甲基化程度,随着Cr(VⅥ)浓度的增加,细胞整体甲基化程度逐渐回升,而最高剂量组(5μM)的整体甲基化程度较溶剂对照组有显著性提高；B[a]P可以导致细胞整体甲基化程度的降低,随着B[a]P浓度的增加,整体甲基化程度逐渐降低,具有剂量效应关系；低剂量的联合毒物降低了细胞整体甲基化水平,随着染毒剂量增加,整体甲基化程度逐步回升,在染毒剂量为5-1.2μM时达到最高,然后再次降低。
Cr(VⅥ)染毒对Dnmtl的影响不明显,增加了Dnmt3b在细胞中的含量,且具有剂量效应关系,而在最高剂量时才使MBD2的含量有明显增加；B[a]P染毒明显增加了Dnmtl和MBD2在细胞中的含量,而随着B[a]P浓度的增加,Dnmt3b的含量逐渐减少,具有剂量效应关系；联合染毒降低了Dnmtl和MBD2在细胞中的含量,而增加了Dnmt3b的含量。
3.4组蛋白乙酰化水平的检测
随着Cr(VⅥ)染毒剂量的提高,H3和H4乙酰化水平逐渐降低。用Western blot法对组蛋白乙酰化相关蛋白进行检测后发现,Cr(VⅥ)对HDAC2和HDAC3的影响都不大。
但是对于B[a]P处理过的细胞进行检测后发现,虽然HDAC2和HDAC3的含量有剂量依赖性的提高,但是H3和H4的整体乙酰化水平变化并无明显规律,并且H4组蛋白乙酰化程度还略有提高。
Cr(VⅥ)和B[a]P联合处理细胞后发现,组蛋白乙酰化的水平整体趋势是增加的。对相关蛋白的检测发现,与溶剂对照组相比较,HDAC2和HDAC3的含量在低剂量时有所提高,然后随着染毒浓度的继续提高而逐渐减少,最高剂量组(20,2.5μM)的含量变化较对照组明显。
3.5组蛋白生物素化水平的检测
BTD在细胞中的含量变化随着Cr(VⅥ)浓度的提高而显著性降低,但是HCS的含量却几乎没有变化。在正常细胞中BTD蛋白均匀分布在细胞核和细胞质中,随着BTD含量的降低,细胞质中的BTD越来越少,大多集中到了细胞核中,最后基本上只有在细胞核中才能发现BTD。而HCS在正常细胞中也几乎绝大部分存在于细胞核之中,由于在Cr(VⅥ)处理过程中HCS的含量没有变化,所以在其他Cr(VⅥ)处理组中HCS的位置也没有什么变化,都集中在细胞核中。
B[a]P染毒后,细胞中HCS蛋白含量几乎没有变化,而BTD的含量随着B[a]P浓度的提高而降低。经B[a]P染毒的细胞其组蛋白生物素化水平是随着B[a]P浓度的提高而逐渐升高的,到B[a]P浓度为10μM时达到最高,然后开始降低。
Cr(VⅥ)和B[a]P联合处理细胞后发现,HCS在细胞中的含量随联合毒物浓度的提高而显著提高,而BTD的含量在降低到一定程度后又开始回升。虽然BTD和HCS在细胞中的含量变化与Cr(VⅥ)单独处理时不同,但是组蛋白生物素化的水平变化趋势却是类似的。
4结论
4.1Cr(VⅥ)暴露对细胞的损伤主要是因为其在细胞内发生一系列还原反应导致过量自由基产生,而其最终产物Cr(Ⅲ)与DNA形成加合物导致的损伤所占比例不大。Cr(VⅥ)单独暴露所导致的细胞损伤要弱于B[a]P,但是两者联合处理时Cr(VⅥ)占据主导地位。
4.2联合染毒对细胞凋亡的影响与两者单独处理时没有差别,低剂量时细胞凋亡程度显著增加,高剂量时凋亡程度降低。经Cr(VⅥ)和B[a]P染毒后,周期停滞时期主要发生在S期。联合染毒对细胞周期的影响结果也与Cr(Ⅵ)单独处理时情况一致。
4.3低剂量Cr(VⅥ)降低了细胞的整体甲基化水平,导致染色质稳定性失衡以及基因印迹丢失。高剂量B[a]P可以降低细胞的整体甲基化水平。Cr(VⅥ)和B[a]P联合处理细胞时对DNA甲基化水平的影响没有显著性变化。
4.4Cr(VⅥ)可以降低细胞组蛋白乙酰化水平,但是B[a]P对其的影响却没有规律。而两者联合处理导致了组蛋白乙酰化水平的提高,并且对组蛋白乙酰化相关蛋白的影响趋势与两者单独处理时相反。
4.5无论是Cr(VⅥ)还是B[a]P都可以提高组蛋白生物素化的水平。在正常细胞中,HCS主要对细胞核内的生物素活动负责,BTD是细胞内生物素活动的全权管理者,但是在组蛋白生物素化修饰方面却是辅助者。联合染毒时,Cr(VⅥ)对组蛋白生物素化的影响占据了主导地位。
1Background
Human 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.
2Methods
2.1The biological characteristics of16HBE cells and the detection of cytotoxicity and oxidative damage
16HBE 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 cells
16HBE 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 cells
16HBE 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 cells
16HBE 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 cells
16HBE 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.
3Results
3.1The biological characteristics of16HBE cells and the detection of cytotoxicity and oxidative damage
As 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 cells
The 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 cells
Low 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 cells
Cr(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 cells
Cr(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).
4Conclusions
4.1The 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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