Genetic Variants Of CYP2A13: Alteration On Coumarin 7-hydroxylation And Association With The Susceptibility To Lung Cancer

Cytochrome P450 (CYP450) is a superfamily of monooxygenases that play an important role in the metabolism of various endogenous and exogenous compounds and the activation of environmental carcinogens. Human CYP450 are highly polymorphism, which mostly caused by single nucleotide polymorphisms (SNPs). SNP of human CYP450 can change the synthesis and activity of human CYP450 protein, and lead to the variation of human susceptibility to the xenobiotics toxicityAFBi is a mycotoxin synthesized by Aspergillus flavus and Aspergillus parasiticus and is considered by the WHO and NIH as an unavoidable food contaminant and a liver carcinogen, besides its acute toxicity (aflatoxicosis). Coumarin is a natural product widely used in perfumes, toiletries, tobacco products and the treatments of some diseases such as lymphedema. It has been reported that coumarin may act as a co-mutagen with aflatoxin Bi when incubated with human liver S9.CYP2A13 is highly expressed in human respiratory tract and efficient in metabolizing AFBi and coumarin. CYP2A13 has been reported as a key enzyme in AFBi metabolism in human lung. And it is reasonable to further speculate that functional genetic polymorphisms of CYP2A13 may also have a significant impact on human susceptibility to lung cancers related to coumarin and AFB1 exposure. The study of effects of genetic polymorphisms of CYP2A13 on AFB1 and coumarin metabolism should be useful in designing and interpreting molecular epidemiologica studies related to CYP2A13 genetic polymorphisms.The recombinant human metabolism enzyme is a useful tool for the xenobiotics metabolism study in vitro, which can provide interpretation and evidence for metabolism study in vivo, and even predict the situation in vivo to some extent. Using heterologously expressed proteins of variants of a human metabolism enzyme, the variation of the xenobiotic metabolism activities caused by genetic polymorphisms could be valued, the results of which should aid in the designing and interpreting molecular epidemiological studies.In the present study, wild-type and variant human CYP2A13 were expressed in Sf9 cells to determine the functional consequences, if any, that CYP2A13 variants might have on CYP2A13-catalysed coumarin 7-hydroxylation. A compute model was built to elucidate the mechanism underlying the different catalytic activity of CYP2A13 for coumarin. Finally, the frequency distribution of CYP2A13 polymorphisms in a Chinese Han population was reported in this work, and an association study was conducted to investigate the correlation of CYP2A13 polymorphisms with human susceptibility to lung cancers.1. Construction and expression of Human CYP2A13 and its mutantsThere are several expression system used to heterologously express CYP450 protein, including E.coli, yeast, mammalian cell and baculovirus infected insect cells. Bac-to-Bac(?) baculovirus infected Sf9 insect cell expression system were effective in eukaryotic protein expression and CYP2A13s were successfully expressed by this system in this experiment.The full-length CYP2A13 gene was amplified from total RNA of a Chinese human lung by using reverse transcription-polymerase chain reaction (RT-PCR). Xba I site and Xho I sites were introduced at the 5’terminus and 3’terminus respectively, and a his-tag sequence was added at the 3’-side of CYP2A13 sequence. After sequencing confirmation, the correct human CYP2A13 gene was used as template to obtain CYP2A13 alleles by site-directed mutation PCR. The correct CYP2A13 alleles were verified by sequencing, and inserted into the pFastBacTM1 vector to generate recombinant pFastBacTM1-CYP 2A13s. Recombinant pFastBacTM1-CYP2A13s was then transformed into MAX Efficiency DH10BacTM competent Escherichia coli to generate recombinant bacmid (Bacmid-CYP2A13s) through transposition. After identification of successful transposition by using PCR, the Sf9 cells were transfected with recombinant Bacmid-CYP2A13s and generated the recombinant baculovirus, which is the P1 Viral Stock. High-titer baculovirus were obtained by subsequently twice viral stock amplification. The expression conditions were optimized to obtain the highest production, including the multiplicity of infection (MOI). CYP2A13 proteins were expressed with optimized expression conditions and detected using anti-His antibody. Microsomal P450 content was determined by reduced CO-difference spectrum.2. Coumarin 7-hydroxylation by CYP2A13s and kinetic analysisCYP2A13 has been shown to be predominantly expressed in human respiratory tract, at the highest level in the nasal mucosa and a relatively lower levels in the lung and trachea. In 2005, CYP2A13 was first reported as an efficient enzyme in metabolizing AFB1 to its carcinogenic and toxic epoxides, including AFM1-8,9-epoxide and AFB1-8,9-epoxide. Compared with CYP1A2, the level of AFM1-8,9-epoxide formation by CYP2A13 is the same as that by CYP1A2 and the level of AFB1-8,9-epoxide formation by CYP2A13 is about one third of that by CYP1A2. A striking difference can be observed in the sensitivity to AFB1-induced toxicity between the CHO cells expressing CYP2A13 and other AFBi-activiating CYP enzymes. The CHO-2A13 cells are at least 100 times more sensitive to AFB1 toxicity, compared to CHO-3A4 cells and CHO-1A2 cells. These results not only confirm CYP2A13 is able to efficiently metabolize AFB1, but also suggest that CYP2A13 is much more efficient than CYP1A2 and CYP3A4 in activating AFB1 to its cytotoxic metabolites. It has been reported that coumarin may act as a co-mutagen with aflatoxin B1 and could enhance the AFB1-induced toxicity when co-incubated with human liver S9. A published study suggests that missense genetic polymorphisms of CYP2A13 is very unlikely to be an important modifier in human lung cancer risk AFB1 exposure. It is meaningful to investigate the effects of genetic polymorphisms of CYP2A13 on coumarin metabolism and the results should be useful in designing and interpreting molecular epidemiologica studies related to CYP2A13 genetic polymorphisms.In this work, we obtained active wild-type and variant human CYP2A13 protein with a Bac-to-Bac(?) expression system, and coumarin was metabolized to 7-hydroxy coumarin. Metabolite were isolated on a HPLC system and identified by fluorescence detector. CYP2A13*2, CYP2A13*5, CYP2A13*6, and CYP2A13*9 were found to have a modest increased catalytic efficiency in vitro for coumarin 7-hydroxylation, which suggest that it is unlikely that these modest changes would cause a detectable functional significance in vivo. And 7-hydroxylation of coumarin by CYP2A13*4 was not detectable. A compute model was built to elucidate the mechanism underlying the different catalytic activity of CYP2A13 for coumarim, which is consistent with the kinetic analysis. The results suggest that the effects of genetic polymorphisms of CYP2A13 on AFB1-induced toxicity when co-incubated with coumarin are modest. This is the first report to characterize coumarin metabolism by variant CYP2A13 enzymes expressed in sf9 with full length and elucide the mechanisms underlying the different catalytic activity of CYP2A13 variants for coumarin by conducting computer modeling studies. In addition, this is also the first report of mechanisms of the in stability of CYP2A13*4 variant.Combined with pubished studies about the influence of CYP2A13 polymorphisms on metabolisms of other chemicals, we assume that CYP2A13*2, CYP2A13*5, CYP2A13*6, CYP2A13*8 and CYP2A13*9 might not be associated with human susceptibility to lung cancers. 3. Frequency distribution of genetic variants of human CYP2A13 in a Chinese Han Population, and relationship between lung cancer risk and CYP2A13 genetic polymorphismsLike other CYP450s, CYP2A13 are highly polymorphism. The genetic variation of CYP2A13 can lead to the change on xenobiotics toxicity and activity. Some polymorphisms in CYP2A13 associated with a substantial reduction in lung cancer risk were reported. Recently, A total of approximately 55 single nucleotide polymorphisms (SNPs), including 9 SNPs in the coding region, have been identified in CYP2A13 gene. Variants caused by these SNPs showed an activity change in environmental toxicant. This study is to find out the association between CYP2A13 polymorphism and human lung cancer risk.To investigate the distribution frequency of CYP2A13 alleles and SNPs in a Chinese Han population, a two-step ASA strategy was applied to genomic DNAs of 140 unrelated volunteers. Five DNA sample, analyzed by sequencing, were used as positive controls, and the results of ASA of 5 positive controls were consistent with the results of sequencing. The frequence of the variant CYP2A13*2 allele, variant CYP2A13*3 insertion, variant CYP2A13*4 allele, variant CYP2A13*5 allele, variant CYP2A13*6 allele, variant CYP2A13*7 allele, variant CYP2A13*8 allele and variant CYP2A13*9 allele was 6.8%,1.1%,4.0%,1.1%,1.5%,4.0%,1.1% and 0.4%, respectively. Japanese have much higher distribution frequency in CYP2A13*3 variant (4.9%) than the Chinese Han population has (1%), and the CYP2A13*4 allele frequency reported in Japanese (0.3%) was significantly lower than that in the Chinese Han population (3.75%). There were no significant differences of CYP2A13*7 allele, CYP2A13*8 allele and CYP2A13*9 allele between the French Caucasian reported previously and the Chinese Han population.An association study was performed to investigate the correlation of CYP2A13 polymorphisms with human susceptibility to lung cancers. We found that CYP2A13*2, CYP2A13*5, CYP2A13*6, CYP2A13*8 and CYP2A13*9 are not associated with the individual variation of susceptibility to lung cancers, which coincided with the results of kinetic analysis. However, CYP2A13*3, CYP2A13*4 and CYP2A13*7 are also found not associated with the individual variation of susceptibility to lung cancers, which might be caused by the following facts:1) CYP2A13*3, CYP2A13*4 and CYP2A13*7 exist in Chinese population mainly as heterozygote, and the active wild-type CYP2A13 could weaken the protection caused by CYP2A13*3, CYP2A13*4 and CYP2A13*7. therefore, it is hard to examine the contribution of the in the CYP2A13 null allele to the risk of lung cancer in a small sample size epidemiological study; 2) there are other enzymes, polymorphisms of which could significantly affect the activity of metabolism of CYP450, such as POR, of which some missense polymorphic variants could cause a ten-fold increase in the activity of wild-type CYP2A13; 3) there are other enzymes responsible for the activation of AFB1 or NNK in lung, and the genetic polymorphisms of the enzymes are determinants in the individual variation of lung carcinogenesis.Our results suggest that when designing NNK or AFB1 exposure-related molecular epidemiological studies, there might be other factors should be considered besides CYP2A13.