Metabolic Mechanism Of Alkyl N-nitrosamines Catalyzed By Cytochrome P450

N-Nitrosamines(NAs) were an important environmental carcinogens which were widely existing in water, meat, tobacco, medicine, liquor and vegetables and it can induced a variety of tumors in animals and humans. NAs were metabolic activated by cytochrome P450(CYP450) in vivo and generate hydroxylated products, which further decomposed to active electrophiles to alkylated DNA bases, leading to the occurrence of cancer. Therefore, P450-catalyzed metabolic activation was a pivotal step in the carcinogenesis of NAs. In this study, the metabolic mechanism of NAs were investigated using quantum chemistry computations to provide a theoretical basis for further illuminating the carcinogenic mechanism of NAs.The mechanism of the metabolism of Nitrosoethylmethylamine(NMEA) and N-Nitrosoethylmethylamine(NMEA) occurring on the C?-H to form hydroxylation products by CYP450 activity centers of iron porphyrin and the C?-H hydroxylation process of Cpd 0 and Cpd I two kinds of iron porphyrin active centers were investigated using DFT(density functional theory) method. The geometric structure of each point in the reaction process was optimized by UB3 LYP functional combined with LANL2DZ(Fe)/6-31+G(d,p)(C,H,O,N,S) basis set, and the single point energy calculations were performed at the LANL2DZ(Fe)/6-311+G(d,p)(C,H,O,N,S) higher basis set. The results showed that the hydroxylation of NAs catalyzed by CYP450 included two steps, hydrogen abstraction and rebound reaction. Hydrogen abstraction was the rate-limiting step and was a typical hydrogen atom transfer(HAT) procedure, in which a hydrogen free radical transfered from NAs to the Fe O group of ferric porphyrin. Subsequently, the hydroxyl group on porphyrin rebounded to the NAs radical, resulting in the formation of hydroxylation product. Low-spin(LS) and high-spin(HS) states of ferric porphyrin equally participated the hydroxylation of NAs, there were no difference between the reaction enrgies, which exhibited obvious characters of two-state reactivity(TSR). In addition, when Cpd I catalyzed for NMEA, the relative reaction energy barrier was 13.27/13.92 kcal/mol(LS/HS). When Cpd 0 catalyzed for NMEA, the relative reaction energy barrier was 18.96/18.89 kcal/mol(LS/HS). So, Cpd I was the main catalyst compared with Cpd 0, and the reaction was more likely to occur.According to the “Di-region theory” of NAs carcinogenic mechanism, NAs molecular side chains excepted ?-position can be metabolized by hydroxylation, non-?-position can occur simultaneously and generated two metabolic electrophilic activity center, resulting in DNA interstrand crosslinking. And the mechanism of the three kinds of NAs(NMEA, NDEA and NMPA) metabolism occurring on the C?-H to form hydroxylation products by Cpd I were investigated at UB3 LYP functional combined with LANL2DZ(Fe)/6-31+G(d,p)(C,H,O,N,S) basis set. The results showed that the reaction process of C?-H hydroxylation was similar to that of C?-H, they were typical of the HAT(hydrogen atom transfer) process(that is, the hydrogen atom transfer from the NAs to the iron porphyrin Fe O), then the hydroxyl group rebounded to form the hydroxylation product. Both LS and HS were involved in the reaction, and the energy difference was not significant, the reaction conformed to the TSR. The C?-H hydroxylation energy barriers of the three kinds of NAs were 16~19 kcal/mol, which were 3~5 kcal/mol higher than the C?-H hydroxylation, this suggested that the hydroxylation of C?-H was more favorable than C?-H. In addition, extension of the analyses of HOMO-LUMO energy gaps(?EHOMO-LUMO) for the transition states and bond dissociation energies(BDE) of C?-H and C?-H hydroxylation showed that the ?EHOMO-LUMO of C?-H hydroxylation was about 14 kcal/mol lower than C?-H hydroxylation on LS, the BDE of C?-H hydroxylation was 6~11 kcal/mol lower than C?-H hydroxylation, it was further proved that the C?-H was more easily to be activated under the action of P450 than the C?-H.Interactions of different structure of NAs with CYP450 were investigated using molecular docking computations. Ten human CYP450 and five rat CYP450 with eight kinds of NAs were studied by molecular docking, by comparing the value of GOLD Score function combined docking conformation, it can get a reasonable structure of the docking system. Results showed that the higher scores P450 subtypes were 2A6 and 2E1, which was consistent with the higher efficiency of these two enzymes catalysing the metabolism of NAs observed in in vitro. As can be seen from the structure of the active center of the complex obtained from the docking, NAs can form hydrogen bond with CYP2A6 on 275 Threonine(Thr275) and with CYP2E1 on 270 Threonine(Thr270), it indicated that these residues played an important role in the metabolism of NAs; The distance between hydrogen atom of ?-carbon and the iron atom of the iron porphyrin was about 4 ?, and the distance between hydrogen atom of ?-carbon and iron atom of iron porphyrin was about 6 ?, C?-H was closer, further evidence that the ?-carbon atoms closed to the active center of iron porphyrin, and the reaction was more likely to occur.In this study, we not only clarified the metabolic mechanism of NAs catalyzed by CYP450, but also put forward that the non-alpha-metabolic activation may be an important factor that can not be ignored. It was of great significance to further reveal the carcinogenic mechanism of NAs, which provided a theoretical basis for the study of the relationship between the molecular structure and the carcinogenic activity.