Department of Toxicology, German Institute of Human Nutrition (DIfE) Potsdam-Rehbrücke, 14558 Nuthetal, Germany.
Chem Res Toxicol. 2012 Jul 16;25(7):1484-92. doi: 10.1021/tx300150n. Epub 2012 May 15.
5-Hydroxymethylfurfural (HMF), a heterocyclic product of the Maillard reaction, is a ubiquitous food contaminant. It has demonstrated hepatocarcinogenic activity in female mice. This effect may originate from sulfo conjugation of the benzylic alcohol yielding 5-sulfooxymethylfurfural (SMF), which is prone to react with DNA via nucleophilic substitution. Indeed, we showed that HMF induces gene mutations in Chinese hamster V79 cells engineered for the expression of human (h) sulfotransferase (SULT)1A1 but not in parental V79 cells. In order to identify potential DNA adducts, we incubated DNA samples with SMF or HMF in aqueous solution. Modified DNA was digested and surveyed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for adducts that may be formed by nucleosides either via nucleophilic substitution at the electrophilic carbon atom of SMF or via imine formation with the aldehyde group present in HMF and SMF. The most abundant adducts formed from SMF, N(6)-((2-formylfuran-5-yl)methyl)-2'-deoxyadenosine (N(6)-FFM-dAdo) and N(2)-((2-formylfuran-5-yl)methyl)-2'-deoxyguanosine (N(2)-FFM-dGuo), were synthesized, purified, and characterized by (1)H NMR. Imine adducts were only detected when DNA was incubated with very high levels of HMF following reduction of the imines to corresponding secondary amines by NaBH(3)CN. Sensitive techniques based on LC-MS/MS multiple reaction monitoring for the quantification of the adducts in DNA samples were devised using isotope-labeled [(15)N(5)]N(6)-FFM-dAdo and [(13)C(10),(15)N(5)]N(2)-FFM-dGuo as internal standards. Both 5-methylfurfuryl adducts were detected in DNA from V79-hSULT1A1 treated with HMF but not in DNA from V79 control cells. Considering the lack of other known mutagenic metabolites, we hypothesize that the hepatocarcinogenic potential of HMF originates from the formation of mutagenic SMF.
5-羟甲基糠醛（HMF）是美拉德反应的杂环产物，是一种普遍存在的食品污染物。它已被证明在雌性小鼠中具有肝癌致癌活性。这种效应可能源于苄醇的磺基结合，生成 5-磺基羟甲基糠醛（SMF），后者容易通过亲核取代与 DNA 反应。事实上，我们表明 HMF 会诱导表达人（h）磺基转移酶（SULT）1A1 的中国仓鼠 V79 细胞工程中的基因突变，但不会诱导亲本 V79 细胞中的基因突变。为了鉴定潜在的 DNA 加合物，我们将 SMF 或 HMF 与水溶液中的 DNA 孵育。用液相色谱-串联质谱法（LC-MS/MS）对修饰的 DNA 进行消化和检测，以寻找可能通过亲电碳原子的亲核取代形成的核苷加合物，或者通过存在于 HMF 和 SMF 中的醛基与亚胺形成的核苷加合物。从 SMF 形成的最丰富的加合物，N(6)-((2-糠醛-5-基)甲基)-2'-脱氧腺苷（N(6)-FFM-dAdo）和 N(2)-((2-糠醛-5-基)甲基)-2'-脱氧鸟苷（N(2)-FFM-dGuo）被合成、纯化，并通过（1）H NMR 进行了表征。只有当 DNA 与 HMF 孵育并通过 NaBH(3)CN 将亚胺还原为相应的仲胺时，才检测到亚胺加合物。设计了基于 LC-MS/MS 多重反应监测的灵敏技术，使用同位素标记的[(15)N(5)]N(6)-FFM-dAdo 和[(13)C(10),(15)N(5)]N(2)-FFM-dGuo 作为内标，对 DNA 样品中的加合物进行定量。HMF 处理的 V79-hSULT1A1 处理的 DNA 中均检测到 5-甲基糠醛加合物，但 V79 对照细胞的 DNA 中未检测到。考虑到缺乏其他已知的致突变代谢物，我们假设 HMF 的肝癌致癌潜力源于致突变 SMF 的形成。
