In the US alone, around 60,000 lives/year are lost to colon cancer. In order to study the mechanisms of colon carcinogenesis, in vitro model systems are required in addition to in vivo models. Towards this end, we have used the HT-29 colon cancer cells, cultured in Dulbecco’s Modified Eagle Medium (DMEM), which were exposed to benzo(a)pyrene (BaP), a ubiquitous and prototypical environmental and dietary toxicant at 1, 10, 100 nM and 1, 5, 10, and 25 μM concentrations for 96 h. Post-BaP exposure, growth, cytotoxicity, apoptosis, and cell cycle changes were determined. The BaP metabolite concentrations in colon cells were identified and measured. Furthermore, the BaP biotransformation enzymes were studied at the protein and mRNA levels. The BaP exposure–induced damage to DNA was assessed by measuring the oxidative damage to DNA and the concentrations of BaP-DNA adducts. To determine the whole repertoire of genes that are up- or downregulated by BaP exposure, mRNA transcriptome analysis was conducted. There was a BaP exposure concentration (dose)-dependent decrease in cell growth, cytotoxicity, and modulation of the cell cycle in the treatment groups compared to untreated or dimethylsulfoxide (DMSO: vehicle for BaP)-treated categories. The phase I biotransformation enzymes, CYP1A1 and 1B1, showed BaP concentration-dependent expression. On the other hand, phase II enzymes did not exhibit any marked variation. Consistent with the expression of phase I enzymes, elevated concentrations of BaP metabolites were generated, contributing to the formation of DNA lesions and stable DNA adducts, which were also BaP concentration-dependent. In summary, our studies established that biotransformation of BaP contributes to cytotoxicity, proliferation of tumor cells, and alteration of gene expression by BaP.

Graphical abstract

中文翻译：

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苯并 (a) 芘诱导的 HT-29 人结肠癌细胞的细胞毒性、细胞增殖、DNA 损伤和基因表达谱改变

仅在美国，每年就有大约 60,000 人死于结肠癌。为了研究结肠癌发生的机制，除了体内模型外，还需要体外模型系统。为此，我们使用了在 Dulbecco 改良鹰培养基 (DMEM) 中培养的 HT-29 结肠癌细胞，这些细胞暴露于苯并 (a) 芘 (BaP)，一种普遍存在的原型环境和饮食毒物，浓度为 1, 10 、100 nM 和 1、5、10 和 25 μM 浓度，持续 96 小时。确定了 BaP 后暴露、生长、细胞毒性、细胞凋亡和细胞周期变化。鉴定并测量了结肠细胞中的 BaP 代谢物浓度。此外，在蛋白质和 mRNA 水平上研究了 BaP 生物转化酶。通过测量 DNA 的氧化损伤和 BaP-DNA 加合物的浓度来评估 BaP 暴露引起的 DNA 损伤。为了确定由 BaP 暴露上调或下调的整个基因库，进行了 mRNA 转录组分析。与未处理或二甲基亚砜（DMSO：BaP 的载体）处理组相比，处理组中的细胞生长、细胞毒性和细胞周期调节存在 BaP 暴露浓度（剂量）依赖性降低。I 期生物转化酶 CYP1A1 和 1B1 显示 BaP 浓度依赖性表达。另一方面，II 期酶没有表现出任何显着变化。与 I 期酶的表达一致，产生了浓度升高的 BaP 代谢物，有助于形成 DNA 损伤和稳定的 DNA 加合物，这也是 BaP 浓度依赖性的。总之，我们的研究证实 BaP 的生物转化有助于细胞毒性、肿瘤细胞增殖和 BaP 基因表达的改变。

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