Genetic diversity of cytochrome P450 1A2 with different metabolic activities in domestic cats.
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ABSTRACT:
SUBMITTER: Sugiyama S
PROVIDER: S-EPMC6656809 | biostudies-literature | 2019 Jul
REPOSITORIES: biostudies-literature
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Project description:Knowledge of genetic polymorphisms of cytochrome P450 (CYP), the most important xenobiotic metabolizing enzyme, is very limited in cats. Preliminarily, we investigated genetic polymorphisms in CYP2A13, one of the major CYP isoforms in the liver and lung. Four synonymous and three non-synonymous polymorphic variants were identified in feline CYP2A13 in domestic cats in Japan, without an obvious major type. Metabolic parameters, Km and Vmax, of coumarin hydroxylation of CYP2A13 were shown to range within two times for the identified non-synonymous polymorphic variants by using heterologous coexpression system in Escherichia coli. The results confirmed the polymorphic nature of CYP2A13 as a basis for effective application of medicines and prevention of adverse reactions in treatment of domestic cats.
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Project description:Knowledge on genetic polymorphisms of metabolising enzymes including cytochrome P450 (CYP) is very limited in cats. We investigated polymorphisms in CYP3A131, one of the major CYP isoforms in the feline liver and small intestine. Eight non-synonymous variants and one synonymous variant of feline CYP3A131 were identified in 29 cats. A major non-synonymous type was not observed. Metabolic parameters (Km and Vmax) of dibenzylfluorescein hydroxylation were ranged within about 2 times for the identified non-synonymous variants by using a heterologous coexpression system of CYP3A131 and feline cytochrome P450 reductase in Escherichia coli. The results confirmed the polymorphic nature of CYP3A131 as a basis for effective application of medicines and prevention of adverse reactions in the treatment of domestic cats.
| S-EPMC6483910 | biostudies-literature
Project description:Cytochrome P450 1A2 (CYP1A2) is one of the CYP450 mixed-function oxidase system that is of clinical importance due to the large number of drug interactions associated with its induction and inhibition. In addition, significant inter-individual differences in the elimination of drugs metabolized by CYP1A2 enzyme have been observed which are largely due to the highly polymorphic nature of CYP1A2 gene. However, there are limited studies on CYP1A2 phenotypes and CYP1A2 genotypes among Emiratis and thus this study was carried out to fill this gap. Five hundred and seventy six non-smoker Emirati subjects were asked to consume a soft drink containing caffeine (a non-toxic and reliable probe for predicting CYP1A2 phenotype) and then provide a buccal swab along with a spot urine sample. Taq-Man Real Time PCR was used to determine the CYP1A2 genotype of each individual. Phenotyping was carried out by analyzing the caffeine metabolites using High Performance Liquid Chromatography (HPLC) analysis. We found that 1.4%, 16.3% and 82.3% of the Emirati subjects were slow, intermediate and rapid CYP1A2 metabolizers, respectively. In addition, we found that 1.4% of the subjects were homozygote for derived alleles while 16.1% were heterozygote and 82.5% were homozygote for the ancestral allele. The genotype frequency of the ancestral allele, CYP1A2*1A/*1A, is the highest in this population, followed by CYP1A2 *1A/*1C and CYP1A2 *1A/*1K genotypes, with frequencies of 0.825, 0.102 and 0.058, respectively. The degree of phenotype/genotype concordance was equal to 81.6%. The CYP1A2*1C/*1C and CYP1A2*3/*3 genotypes showed significantly the lowest enzyme phenotypic activity. The frequency of slow activity CYP1A2 enzyme alleles is very low among Emiratis which correlates with the presence of low frequencies of derived alleles in CYP1A2 gene.
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Project description:P450 1A2 is responsible for the metabolism of clinically important drugs and the metabolic activation of environmental chemicals. Genetic variations of P450 1A2 can influence its ability to perform these functions, and thus, this study aimed to characterize the functional significance of three P450 1A2 allelic variants containing nonsynonymous single nucleotide polymorphisms (P450 1A2*8, R456H; *15, P42R; *16, R377Q). Variants containing these SNPs were constructed and the recombinant enzymes were expressed and purified in Escherichia coli. Only the P42R variant displayed the typical CO-binding spectrum indicating a P450 holoenzyme with an expression level of ? 170 nmol per liter culture, but no P450 spectra were observed for the two other variants. Western blot analysis revealed that the level of expression for the P42R variant was lower than that of the wild type, however the expression of variants R456H and R377Q was not detected. Enzyme kinetic analyses indicated that the P42R mutation in P450 1A2 resulted in significant changes in catalytic activities. The P42R variant displayed an increased catalytic turnover numbers (k cat) in both of methoxyresorufin O-demethylation and phenacetin O-deethylation. In the case of phenacetin O-deethylation analysis, the overall catalytic efficiency (k cat/K m) increased up to 2.5 fold with a slight increase of its K m value. This study indicated that the substitution P42R in the N-terminal proline-rich region of P450 contributed to the improvement of catalytic activity albeit the reduction of P450 structural stability or the decrease of substrate affinity. Characterization of these polymorphisms should be carefully examined in terms of the metabolism of many clinical drugs and environmental chemicals.
| S-EPMC4354321 | biostudies-literature
Project description:Hepatocellular carcinoma (HCC) occurs more frequently in men than in women. It is commonly agreed that estrogen plays important roles in suppressing HCC development, however, the underlying mechanism remains largely unknown. Since estrogen is mainly metabolized in liver and its metabolites affect cell proliferation, we sought to investigate if the liver-specific cytochrome P450 1A2 (CYP1A2) mediated the inhibitory effect of estrogen on HCC. In this study, the expression of estrogen-metabolizing enzyme CYP1A2 was determined in HCC tissues and cell lines. Cell proliferation and apoptosis were assessed in cells with or without CYP1A2 overexpression. The levels of 17β-estradiol (E2) and its metabolite 2-methoxyestradiol (2-ME) were determined. A xenograft tumor model in mice was established to confirm the findings. It was found that CYP1A2 expression was greatly repressed in HCC. E2 suppressed HCC cell proliferation and xenograft tumor development by inducing apoptosis. The inhibitory effect was significantly enhanced in cells with CYP1A2 overexpression, which effectively conversed E2 to the cytotoxic 2-ME. E2 in combination with sorafenib showed an additive effect on HCC. The anti-HCC effect of E2 was not associated with estrogen receptors ERα and ERβ as well as tumor suppressor P53 but enhanced by the approved anti-HCC drug sorafenib. In addition, HDAC inhibitors greatly induced CYP1A2 promoter activities in cancer cells, especially liver cancer cells, but not in non-tumorigenic cells. Collectively, CYP1A2 metabolizes E2 to generate the potent anti-tumor agent 2-ME in HCC. The reduction of CYP1A2 significantly disrupts this metabolic pathway, contributing the progression and growth of HCC and the gender disparity of this malignancy.
| S-EPMC4836701 | biostudies-literature
Project description:A unique cytochrome P450 (CYP) oxidoreductase (CPR) sustains activities of human microsomal CYPs. Its function requires toggling between a closed conformation enabling electron transfers from NADPH to FAD and then FMN cofactors and open conformations forming complexes and transferring electrons to CYPs. We previously demonstrated that distinct features of the hinge region linking the FAD and FMN domain (FD) modulate conformer poses and their interactions with CYPs. Specific FD residues contribute in a CYP isoform-dependent manner to the recognition and electron transfer mechanisms that are additionally modulated by the structure of CYP-bound substrate. To obtain insights into the underlying mechanisms, we analyzed how hinge region and FD mutations influence CYP1A2-mediated caffeine metabolism. Activities, metabolite profiles, regiospecificity and coupling efficiencies were evaluated in regard to the structural features and molecular dynamics of complexes bearing alternate substrate poses at the CYP active site. Studies reveal that FD variants not only modulate CYP activities but surprisingly the regiospecificity of reactions. Computational approaches evidenced that the considered mutations are generally in close contact with residues at the FD-CYP interface, exhibiting induced fits during complexation and modified dynamics depending on caffeine presence and orientation. It was concluded that dynamic coupling between FD mutations, the complex interface and CYP active site exist consistently with the observed regiospecific alterations.
| S-EPMC10377444 | biostudies-literature
Project description:The cytochrome P450 (CYP) 1A2 gene is involved in the metabolism of several carcinogens and clinically important drugs, generating a high potential for pharmacokinetic interactions. Since no data are available for Tibetan aborigines, the present study aimed to investigate the distribution of variant CYP1A2 alleles in a population living in Tibetan region of China. Genotyping analyses of CYP1A2 were conducted in 96 unrelated, healthy volunteers of Tibetan ancestry using direct sequencing assays. A total of 14 different CYP1A2 polymorphisms, including two novel variants (1690G>A and 2896C>T) in the intron region and a novel non‑synonymous one (795G>C, Gln265His) were detected. CYP1A2*1A (6.77%), CYP1A2*1B (58.33%) and CYP1A2*1F (14.58%) were the most frequent defective alleles identified in the sample. The frequencies of the prevalent genotypes CYP1A2*1A/*1B, *1B/*1B, *1B/*1F were 13.54%, 16.67% and 29.17%, respectively. In addition, the novel non‑synonymous variant 795G>C (Gln265His) was predicted to be benign by PolyPhen‑2 and SIFT tools. The present study provides useful information on the pattern of CYP1A2 polymorphisms in Chinese Tibetan population. The current results may have potential benefits for the development of personalized medicine in the Tibetan population.
| S-EPMC5482113 | biostudies-literature
Project description:Background: Bilirubin (BR) is metabolized mainly by uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) through glucuronidation in the liver. Some studies have shown that several subtypes of cytochrome P450 (CYP) enzymes, including CYP1A2, are upregulated by inducers and proposed to be alternative BR degradation enzymes. However, no information is available on the BR degradation ability of CYP in normal rats without manipulation by CYP inducers. Methods: Quantitative real-time polymerase chain reaction (QRT-PCR), western blot, immunofluorescence, and confocal microscopy were used to find expression of CYP1A2 in the brain and the liver. BR metabolites in microsomal fractions during development were examined by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (LC-MS/MS). Results: In the present study, we observed that CYP1A2 mRNA levels increased at postnatal days (P)14 and P30 with respect to the level at P7 both in liver and brain, this increment was especially pronounced in the brain at P14. The expression of CYP1A2 in the brainstem (BS) was higher than that in the cerebellum (CLL) and cortex (COR). Meanwhile, the CYP1A2 protein level was significantly higher in the COR than in the brainstem and CLL at P14. The levels of BR and its metabolites (m/z values 301, 315, 333 and biliverdin) were statistically unaltered by incubation with liver and brain microsomal fractions. Conclusion: Our results indicated that the region-specific expression of CYP1A2 increased during development, but CYP family enzymes were physiologically incapable of metabolizing BR. The ability of CYPs to oxidize BR may be triggered by CYP inducers.
| S-EPMC6813656 | biostudies-literature
Project description:BackgroundHuman cytochrome P450 (CYP) enzymes mediate the first step in the breakdown of most drugs and are strongly involved in drug-drug interactions, drug clearance and activation of prodrugs. Their biocatalytic behavior is a key parameter during drug development which requires preparative synthesis of CYP related drug metabolites. However, recombinant expression of CYP enzymes is a challenging bottleneck for drug metabolite biosynthesis. Therefore, we developed a novel approach by displaying human cytochrome P450 1A2 (CYP1A2) and cytochrome P450 reductase (CPR) on the surface of Escherichia coli.ResultsTo present human CYP1A2 and CPR on the surface, we employed autodisplay. Both enzymes were displayed on the surface which was demonstrated by protease and antibody accessibility tests. CPR activity was first confirmed with the protein substrate cytochrome c. Cells co-expressing CYP1A2 and CPR were capable of catalyzing the conversion of the known CYP1A2 substrates 7-ethoxyresorufin, phenacetin and the artificial substrate luciferin-MultiCYP, which would not have been possible without interaction of both enzymes. Biocatalytic activity was strongly influenced by the composition of the growth medium. Addition of 5-aminolevulinic acid was necessary to obtain a fully active whole cell biocatalyst and was superior to the addition of heme.ConclusionWe demonstrated that CYP1A2 and CPR can be co-expressed catalytically active on the cell surface of E. coli. It is a promising step towards pharmaceutical applications such as the synthesis of drug metabolites.
| S-EPMC4736170 | biostudies-literature
Project description:Human CYP1A2 is an important drug-metabolizing enzyme, similar in sequence to CYP1A1 but with distinct substrate specificity. We have previously shown that residue 382 affected CYP1A1 and CYP1A2 specificities with alkoxyresorufins. To determine whether this residue is also important for the metabolism of other substrates, we have investigated phenacetin oxidation by single (T124S, T223N, V227G, N312L, and L382V) and multiple (L382V/T223N, L382V/N312L, L382V/T223N/N312L, and L382V/T124S/N312L) mutants of CYP1A2. The enzymes were expressed in Escherichia coli and purified. All the CYP1A2 mutants that contained the L382V substitution displayed much higher activities than the wild-type enzyme, with k(cat) values 3-fold higher, in contrast to other mutants, for which k(cat) decreased. Likewise, a significant increase in specificity, expressed as the k(cat)/K(m) ratio, was observed for the mutants containing the L382V substitution. The efficiency of coupling of reducing equivalents to acetaminophen formation was decreased for all the single mutants except L382V, for which the coupling increased. This effect was also observed with multiple CYP1A2 mutants containing the L382V substitution. Low activities of the four other single mutants were likely caused by dramatically increased uncoupling to water. In contrast, the increase in activity of the L382V-containing mutants resulted from decreased water formation. This finding is consistent with molecular dynamics results, which showed decreased phenacetin mobility leading to increased product formation. The results of these studies confirm the importance of residue 382 in CYP1A2-catalyzed oxidations and show that a single residue substitution can dramatically affect enzymatic activity.
| S-EPMC2908983 | biostudies-literature