Theoretical Study On Cytochrome P450- Mediated Metabolism And DNA Damage Of Typical Environmental Pollutants

The cytochrome P450 enzymes, play a vital role in the synthesis of endogeneous molecules and biotransformaiton of xenbiotics (metabolic detoxification/activation). The majority of chemical toxicants and carcinogens do not directly exert detrimental biological effects. In most cases, the activation of parent compounds to more electrophilic forms by P450s is necessary to produce molecules capable of reacting irreversibly with tissue nucleophiles, such as proteins and DNA. Since the versatility of P450s, and the importance of P450 enzymes and DNA in human health, it is quite essential to understand the underlying mechanisms. Although the experimental measurements always have some restricts, for example, hard to detect the short-lived transition states and intermediates, time and resource consuming, facing the 3R rules for the animal experiments, we can resort to theoretical computational approaches. In this work, we have applied density functional theory (DFT) methods to investigate some ambiguous reaction pathways, and also tried to establish reactivity prediction models based on mechanism, anticipating to provide useful information for chemical envrionmental and health risk assessments. The main conclusions are drawn.(1) To gain the insight into the metabolism of halogenated compounds by P450 enzymes, we have investigated the oxidative and reductive P450-mediated activation of tetra- and trichloromethane as halogenated models with DFT methods. We propose an oxidative halosylation (chlorine abstraction) mechanism for CCl4 under aerobic conditions by Compound Ⅰ (Cpd Ⅰ) of P450s, which follows the typical Groves-type hydrogen abstraction/radical rebound mechanism. By contract, the metabolism of CHCl3 occurs preferentially via an initial hydrogen-atom abstraction rather than halosylation. The reductive pathway based on the Mrcus theory shows that the electron transfer from the pentacoordinate ferrous-porphrin complex of P450 to the halogenated alkanes needs much lower barriers than the ones for chorine abstraction by Cpd Ⅰ in the oxidative pathway. Moreover, the studies highlight the substrate specific activation pathways by P450 enzymes leading to different products. These reactivity differences are rationalized, and reproduce experimental product distributions.(2) Many substances are bioactivated by P450s to form active compounds, an example is the conversion of olefinic substrates to epoxides, which are intermediates in the metabolic activation of many known or suspected carcinogens. We have calculated the activation energies for epoxidation by the active species Cpd Ⅰ of P450 enzymes for a diverse set of 36 olefinic substrates with state-of-the-art DFT methods. Activation energies can be estimated by the computationally less demanding method of calculating the ionization potentials of the substrates, which provides a useful and simple predictive model based on the reaction mechanism; however, the preclassification of these diverse substrates into weakly polar and strongly polar groups is a prerequisite for the construction of specific predictive models with good predictability for P450 epoxidation (weakly polar:R2=0.972, strongly polar:R2=0.936, for tranining set). This approach has been supported by both internal and external validations. Furthermore, the relation between the activation energies for the regioselective epoxidation and hydroxylation reactions of P450s and experimental data has been investigated. The results show that the computational and correction method used in this work, performs well in reproducing the experimental trends of the epoxidation and hydroxylation reactions.(3) Epoxide ring opening proceeds through a SN2-type mechanism with hard nucleophile DNA sites as the major facilitators of toxic effects. Thus, the quantitative prediction of chemical reactivity would enable a predictive assessment of the molecular potential to exert electrophile-mediated toxicity. We calculated the activation energies for reactions between epoxides and the guanine N7 site for a diverse set of 37 epoxides, including aliphatic epoxides, substituted styrene oxides, and PAH epoxides, using a state-of-the-art DFT method. It is worth noting that these activation energies for diverse epoxides can be further predicted by quantum chemically calculated nucleophilic indices from the theory of hard and soft acids and bases (HSAB), which is a less computationally demanding method than the exacting procedure for locating the transition state (simple epoxides:R2= 0.970, PAH epoxides:R2=0.976, for all data set). More importantly, the good qualitative/quantitative correlations between the chemical reactivity of epoxides and their bioactivity suggest that the developed model based on HSAB theory may aid in the predictive hazard evaluation of epoxides, enabling the early identification of mutagenicity/carcinogenicity relevant Sn2 reactivity.(4) We have chosen several representative furan-containing compounds, and calculated the key species of the metabolism reaction by the Cpd I of P450 enzymes. In general, O-addition to ortho-position (position 2, and 5) would lead to enediones, while O-addition to meta-position (position 3, and 4) would form epoxides. The position, size, and polarity of the substituents on the furan ring, may affect the reactivity of the ortho positions and itself. Specifically, when the compound has multiple substitutions, the intermediates possibly differ in quartet high-spin state and doublet low-spin state.