Chem Res Toxicol. 2022 Sep 19;35(9):1519-1532. doi: 10.1021/acs.chemrestox.2c00177. Epub 2022 Sep 6.
Epidemiological and mechanistic studies suggest that processed and red meat consumption and tobacco smoking are associated with colorectal cancer (CRC) risk. Several classes of carcinogens, including -nitroso compounds (NOCs) in processed meats and heterocyclic aromatic amines (HAAs) and polycyclic aromatic hydrocarbons (PAHs) in grilled meats and tobacco smoke, undergo metabolism to reactive intermediates that may form mutation-inducing DNA adducts in the colorectum. Heme iron in red meat may contribute to oxidative DNA damage and endogenous NOC formation. However, the chemicals involved in colorectal DNA damage and the paradigms of CRC etiology remain unproven. There is a critical need to establish physicochemical methods for identifying and quantitating DNA damage induced by genotoxicants in the human colorectum. We established robust nano-liquid chromatography/high-resolution accurate mass Orbitrap tandem mass spectrometry (LC/HRAMS) methods to measure DNA adducts of nine meat and tobacco-associated carcinogens and lipid peroxidation products in the liver, colon, and rectum of carcinogen-treated rats employing fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues. Some NOCs form -carboxymethyl-2'-deoxyguanosine, -methyl-2'-deoxyguanosine, and unstable quaternary -linked purine/pyrimidine adducts, which generate apurinic/apyrimidinic (AP) sites. AP sites were quantitated following derivatization with -(pyridin-3-yl-methyl)hydroxylamine. DNA adduct quantitation was conducted with stable isotope-labeled internal standards, and method performance was validated for accuracy and reproducibility. Limits of quantitation ranged from 0.1 to 1.1 adducts per 10 bases using 3 μg of DNA. Adduct formation in animals ranged from ∼1 in 10 to ∼1 in 10 bases, occurring at comparable levels in fresh-frozen and FFPE specimens for most adducts. AP sites increased by 25- to 75-fold in the colorectum and liver, respectively. Endogenous lipid peroxide-derived 3-(2-deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3)-one (MdG) and 6-oxo-MdG adduct levels were not increased by carcinogen dosing but increased in FFPE tissues. Human biomonitoring studies can implement LC/HRAMS assays for DNA adducts and AP sites outlined in this work to advance our understanding of CRC etiology.
流行病学和机制研究表明，加工肉类和红色肉类的消费以及吸烟与结直肠癌（CRC）风险有关。几类致癌物质，包括加工肉类中的亚硝胺（NOC）和烤肉中的杂环芳香胺（HAA）和多环芳烃（PAH）以及烟草烟雾，经过代谢转化为可能在结直肠中形成致突变 DNA 加合物的反应性中间体。红色肉类中的血红素铁可能导致氧化 DNA 损伤和内源性 NOC 的形成。然而，涉及结直肠癌 DNA 损伤的化学物质和 CRC 病因学的范例仍然未经证实。迫切需要建立用于鉴定和定量分析遗传毒性物质在人结直肠中诱导的 DNA 损伤的物理化学方法。我们建立了强大的纳升液相色谱/高分辨率精确质量轨道阱串联质谱（LC/HRAMS）方法，用于测量用致癌剂处理的大鼠的肝脏、结肠和直肠中 9 种与肉类和烟草相关的致癌物质和脂质过氧化产物的 DNA 加合物，使用新鲜冷冻和福尔马林固定石蜡包埋（FFPE）组织。一些 NOC 形成β-羧甲基-2'-脱氧鸟苷、β-甲基-2'-脱氧鸟苷和不稳定的季铵嘌呤/嘧啶加合物，产生无嘌呤/无嘧啶（AP）位点。AP 位点通过与（吡啶-3-基-甲基）羟胺衍生化进行定量。使用稳定同位素标记的内标进行 DNA 加合物定量，并且针对准确性和重现性验证了方法性能。使用 3μg 的 DNA，定量下限范围为每个 10 个碱基 0.1 至 1.1 个加合物。动物中的加合物形成范围为每个 10 个碱基约 1 至约 1，对于大多数加合物，在新鲜冷冻和 FFPE 标本中以可比水平发生。AP 位点分别在结直肠和肝脏中增加了 25 至 75 倍。内源性脂质过氧化物衍生的 3-(2-脱氧-β-d-赤-戊呋喃糖基)嘧啶并[1,2-α]嘌呤-10(3)-酮（MdG）和 6-氧代-MdG 加合物水平不因致癌剂给药而增加，但在 FFPE 组织中增加。人类生物监测研究可以实施本工作中概述的用于 DNA 加合物和 AP 位点的 LC/HRAMS 测定，以推进我们对 CRC 病因的理解。
