Expression Of The Cyp450 Enzymes Related To Aflatoxin B1 Metabolism And Their Influencing Factors Analysis

Aflatoxin B₁ (AFB₁), classified as a Group I carcinogen, is the most prevalent and carcinogenic product of Aspergillus flavus and Aspergillus parasiticus, and can contaminate a number of agricultural products and foods. Although the liver is clearly the principal target organ of AFB₁, there are evidences strongly support a role for AFB₁ in the induction of human respiratory tumorigenesis. Cytochrome P450 (CYP450) enzymes are the major enzymes for metabolic activation of AFB₁ to epoxide, a reactive electrophilic intermediate associated with AFB₁'s carcinogenicity. Many P450 isforms are capable of bioactivating AFB₁ and CYP1A2 is regarded as the major AFB₁-metabolizing enzymes in human liver. However, some recent studies demostrated that CYP2A13 had a significant activity in metabolizing AFB₁ to varieties reactive intermediates including epoxides. Considering CYP2A13 is predominantly expressed in the human respiratory tract, while CYP1A2 is almost exclusively expressed in human liver, suggesting CYP2A13 may related to AFB₁ metabolizing in situ and inducing tumorigenesis, evidences for AFB₁ bioactivating by CYP2A13 is not sufficient yet. Thus, it's necessary to identify the association ]between AFB₁ and CYP2A13, and further to obtain their key metabolites via metabolism in vitro, which will help to illustrates the mechanism of AFB₁-induced tumorigenesis in respiratory system.A bulk of single ingredient and functional CYP450 protein is needed to reconstitute a metabolic system in vitro for AFB₁ metabolism. Expression of CYP450 in heterologous systems is the chief method to obtain single pure enzymes. Insect cells has the similar mode to mammalian cells for post-transcription and post-translation products modification, it can yield high level of unmodified, native CYP450 proteins. NADPH-Cytochrome P450 reductase (POR) , a protein which can transfer electrons form NADPH to hemeproteins, should take into considerations for the reaction activity of eukaryotic P450s, the activity maybe hampered if CYP450s expression alone. Previous studies showed that functional CYP450s can be gotten through addition of cofactors (i.e.,δ-aminolevulinic acid (δ-ALA), ferric citrate, Hemin), and the approach of co-expression-by-co-infection enables the production of enzymatically active CYP450 in the natural microsomal membrane, which can subsequently provide a available way to reconstitute catalytically active systems in vitro. Considerating the complicate of CYP450s expression in vitro, systematical studies should be taken to find the favourable conditions for stable and efficient expression of CYP450s.ObjectivesBaculovirus/Sf9 system was utilized for expression of CYP2A13 and other major AFB₁ activation-related enzymes, in which different cofactors including Hemin,δ-ALA and Fe³⁺ were studied whether they could affect the efficiency and activity of CYP450s and POR expression. Then we tried the co-expression of CYP450s and POR to get a more stable and efficient expression system, which can offer us a reliable research platform for reconstructing metabolism system in vitro, explaining the bioactivities of AFB₁ by CYP2A13 and identifying key metabolites. MethodsCYPs cDNA were transferred into pFastBac1 vector via restriction enzymes and Quik T4 DNA ligase. After verified by PCR and double enzymes digestion, CYP450s cDNA were inserted into a baculovirus shuttle vector, and generated recombinant bacmids following transposition, then the recombinant bacmids were verified by PCR. Recombinant baculovirus were generated by transfecting the recombinant bacmid DNA into the insect cell line Sf9 cells; CYP1A2, CYP3A4, CYP2A6, CYP2A13 and POR were expressed in Sf9 cells infected with the virus particles by addingδ-ALA and Fe³⁺, then the target proteins were detected by Western blotting analysis; CYP contents were measured by standard reduced CO-difference spectroscopy , POR activity was analysised by Cytochrome C Reductase Assay.Different concentrations and combinations ofδ-ALA, Fe³⁺ and Hemin were added into the Baculovirus/Sf9 system to expression CYP450s, then proteins were detected and evaluated by Western blotting, CO-difference spectroscopy and Cytochrome C assay. In addition, different CYP:POR ratios were tried in co-expression-by-co-infection to find the optimization conditions for heterologous expression.Results1. Recombinant plasmids of pFastBac 1-CYPs(POR) were get via restriction enzymes and Quik T4 DNA ligase by choosing suitable and correct restriction enzyme sites , then they were verified by PCR and double enzymes digestion; Reconstructed Ac-Bacmid-CYPs(POR)were get subsequently, then transfected into Sf9 cells to obtain the recombinant baculovirus particles.2. Tradition conditions were used to express CYP1A2 , CYP3A4, CYP2A6, CYP2A13 and POR in vitro. A single protein band was detected by immunoblot analysis with the expected molecular weight in the microsomes prepared from the Sf9 cells that were infected with the expression vectors containing CYP450 cDNAs; All the microsome samples expressing the recombinant CYP450s proteins displayed the characteristic absorption peak at 450 nm. As determined by CO-difference spectroscopy analysis. CYP450 contents for CYP1A2 , CYP3A4, CYP2A6 and CYP2A13 were 0.21nmol, 0.057 nmol, 0.027 nmol and 0.023 nmol per mg microsome proteins. While Cytochrome C assay revealed that POR activity was 1519.47 Unit per mg microsome proteins, and it can still keep the similar acitivity (1505.01 Unit per 1mg microsome proteins) even though they were stored at -70℃for more than three months.3. Compared with addingδ-ALA and Fe³⁺ alone, adding 0.1mMδ-ALA and 0.1mM Fe³⁺ simultaneously can significantly improve the expression of CYP1A2 (40% and 65% higher, respectively), they also increased 32% higher than adding Hemin alone. The expression level of POR was much higher when addingδ-ALA or (and) Fe³⁺, although it may not necessary to adding co-factors, it was 2.13, 1.71, 1.86 times higher than without co-factors adding. The activity of POR withδ-ALA or (and) Fe³⁺ addition was higher too, and the value were 7.1, 4.5, 5.4 times higher than without co-factors. Different concentrations and combinations ofδ-ALA and Fe³⁺ could also affect the activity of CYP450, one-way ANOVA analysis showed that the activity of CYP2A6 adding 0.35mMδ-ALA and 0.1mM Fe³⁺ was significantly higher than other groups.4. Different ratio of CYP:POR can also affect the expression when co-expression in the Baculovirus/Sf9 system, the current study revealed that the protein expression level of CYP1A2 and POR were much higher when the CYP1A2:POR ratio was 2.5:1 or 3:1, and 50% higher than the ratio of 2:1 and 4:1. In the system of 2.5:1 ( CYP1A2:POR) , expression of CYP1A2 and POR in adding 0.1 and 0.35mMδ-ALA were much higher than 0.6mMδ-ALA, and 0.1mM Fe³⁺ as well. However, the expression decreased when co-addtion ofδ-ALA and Fe³⁺ were added in the system, and the detail mechanism should be further discussed..Conclusions1. A certain amount of active CYP450s can be obtained under traditional conditions but huge differences of the activities existed among the CYP450s; POR was stable for a considerable time when it stored at -70℃.2. Co-factors can affect the expressions and activities of CYP450s and POR obviously. Co-addition ofδ-ALA and Fe³⁺ can significantly increase the expression and activity of CYP450s. But there is little difference for POR except the addition ofδ-ALA.3. The ratio of 2.5:1 and 3:1 for CYP1A2:POR were benefit for their mutual expressions, and the low concentrations ofδ-ALA or Fe³⁺ were suitable for their co-expression, however, the expression decreased instead when they were co-addition in ths system.In summary, different factors could affect the expression and activity of CYP450s and/or POR proteins in Baculovirus/Sf9 system. It is necessary to optimize the conditions in practise in order to ensure the stabilization and high-performance of the expression.