Targeting PAX8 sensitizes ovarian cancer cells to ferroptosis by inhibiting glutathione synthesis.

Ovarian cancer is a malignant tumor originating from the ovary, characterized by its high mortality rate and propensity for recurrence. In some patients, especially those with recurrent cancer, conventional treatments such as surgical resection or standard chemotherapy yield suboptimal results. Consequently, there is an urgent need for novel anti-cancer therapeutic strategies. Ferroptosis is a distinct form of cell death separate from apoptosis. Ferroptosis inducers have demonstrated promising potential in the treatment of ovarian cancer, with evidence indicating their ability to enhance ovarian cancer cell sensitivity to cisplatin. However, resistance of cancer cells to ferroptosis still remains an inevitable challenge. Here, we analyzed genome-scale CRISPR-Cas9 loss-of function screens and identified PAX8 as a ferroptosis resistance protein in ovarian cancer. We identified PAX8 as a susceptibility gene in GPX4-dependent ovarian cancer. Depletion of PAX8 rendered GPX4-dependent ovarian cancer cells significantly more sensitive to GPX4 inhibitors. Additionally, we found that PAX8 inhibited ferroptosis in ovarian cancer cells. Combined treatment with a PAX8 inhibitor and RSL3 suppressed ovarian cancer cell growth, induced ferroptosis, and was validated in a xenograft mouse model. Further exploration of the molecular mechanisms underlying PAX8 inhibition of ferroptosis mutations revealed upregulation of glutamate-cysteine ligase catalytic subunit (GCLC) expression. GCLC mediated the ferroptosis resistance induced by PAX8 in ovarian cancer. In conclusion, our study underscores the pivotal role of PAX8 as a therapeutic target in GPX4-dependent ovarian cancer. The combination of PAX8 inhibitors such as losartan and captopril with ferroptosis inducers represents a promising new approach for ovarian cancer therapy.

Effect of ADHI on hepatic stellate cell activation and liver fibrosis in mice.

The relationship between alcohol dehydrogenase (ADH) and liver fibrosis has been studied, but the mechanism of ADH involvement in liver fibrosis remains unclear. The aim of the present study was to explore the role of ADHI, the classical liver ADH, in hepatic stellate cell (HSC) activation and the effect of 4-methylpyrazole (4-MP), an ADH inhibitor, on liver fibrosis induced by carbon tetrachloride (CCl4) in mice. The results showed that overexpression of ADHI significantly increases proliferation, migration, adhesion and invasion rates of HSC-T6 cells as compared with controls. When HSC-T6 cells were activated by ethanol, TGF-ß1 or LPS, the expression of ADHI was elevated significantly (P < 0.05). Overexpression of ADHI significantly increased the levels of COL1A1 and α-SMA, markers of HSC activation. Moreover, the expression of COL1A1 and α-SMA was decreased significantly by transfection of ADHI siRNA (P < 0.01). In a liver fibrosis mouse model ADH activity increased significantly and was highest in the 3rd week. The activity of ADH in the liver was correlated with its activity in the serum (P < 0.05). 4-MP significantly decreased ADH activity and ameliorated liver injury, and ADH activity was positively correlated with the Ishak score of liver fibrosis. In conclusion, ADHI plays an important role in the activation of HSC, and inhibition of ADH ameliorates liver fibrosis in mice.

The POR rs10954732 polymorphism decreases susceptibility to hepatocellular carcinoma and hepsin as a prognostic biomarker correlated with immune infiltration based on proteomics.

The effect of the cytochrome P450 oxidoreductase (POR) rs10954732 (G > A) polymorphism on hepatocellular carcinoma (HCC) susceptibility is unknown. Here we found that A allele carriers showed a 69% decrease in susceptibility to HCC with overall survival (OS) prolonged to 199%, accompanied by lower activity for cytochrome P450 2E1. A total of 222 differentially expressed proteins were mainly enriched in neutrophil and T cell activation and involved in the immune and inflammatory responses, constituting the altered immune tumor microenvironment related with A allele by proteomics analysis. Hepsin (HPN) showed significant down-regulation in HCC and up-regulation in A allele carriers. A lower HPN level was associated with increased susceptibility to HCC and a worse prognosis. Moreover, HPN is a potential independent prognostic biomarker for HCC and is strongly associated with clinicopathological features, tumor-infiltrating status of immune cells both in our discovery cohort and database surveys. Our findings provide a new potential mechanism by which HPN may play an important role in the susceptibility of rs10954732 A allele carriers to HCC and their prognosis through tumor immune infiltration, thus offering potential insights for future studies on tumor immunotherapy.

Use of proteomics to identify mechanisms of hepatocellular carcinoma with the CYP2D6*10 polymorphism and identification of ANGPTL6 as a new diagnostic and prognostic biomarker.

BACKGROUND: Although an association between the cytochrome P4502D6 (CYP2D6) *10 (100C>T) polymorphism and hepatocellular carcinoma (HCC) is known, the mechanism remains unclear. Here we aimed to explore mechanisms of CYP2D6*10 (100C>T) polymorphism conferring to HCC, and screen markers for HCC. METHODS: Label-free global proteome profiling with 34 normal livers and peritumor tissue from 61 HCC patients was performed, and angiopoietin-like protein-6 (ANGPTL6) was evaluated in 2 liver samples validation cohorts and 2 blood specimens validation cohorts. RESULTS: We found a significantly decreased frequency of TT in HCC patients which reduced HCC susceptibility by 69.2% and was accompanied by lowered enzymatic activity for CYP2D6. Proteomic analysis revealed 1342 differentially expressed proteins (DEPs) that were associated with HCC and 88 DEPs were identified as 100 TT-related proteins, likely underlying the susceptibility to HCC. Twenty-two upregulated DEPs and 66 downregulated DEPs were mainly related to lipid metabolism and the extracellular matrix, respectively. High ANGPTL6 was associated with a higher risk to HCC and worse prognosis. ANGPTL6 was both an independent risk factor and an independent prognostic factor for HCC and exhibited strong potential for predicting HCC occurrence, with comparable AUC values and higher sensitivity compared with alpha-fetoprotein. CONCLUSIONS: The TT genotype-associated decreased risk of HCC appears to be related to lowered CYP2D6 activity and altered protein expression in the tumor microenvironment, and ANGPTL6 is a promising new diagnostic and prognostic biomarker for HCC. Our findings reveal new mechanistic insights for polymorphisms related to HCC risk and provide avenues for screening for HCC.

Variation in the expression of cytochrome P450-related miRNAs and transcriptional factors in human livers: Correlation with cytochrome P450 gene phenotypes.

Cytochrome P450 (CYP) gene expression exhibits large interindividual variation attributable to diverse regulatory factors including microRNAs (miRNAs) and hepatic transcription factors (TFs). We used real-time qPCR with 106 human liver samples to measure the expression and interindividual variation of seven miRNAs and four TFs that have been reported to regulate the expression of CYPs; we also identified factors that influence their expression. The results show that expression of the seven miRNAs and the four TFs exhibits a non-normal distribution and the expression variability is high (89- to 618-fold for miRNA and 12- to 85-fold for TFs). Age contributed to the interindividual variation for miR-148a, miR-27b and miR-34a, whereas cigarette smoking and alcohol consumption significantly reduced HNF4α mRNA levels. Association analysis showed significant correlations among the seven miRNAs as well as the four TFs. Furthermore, we systematically evaluated the impact of the seven miRNAs and four TFs on protein content, mRNA levels, translation efficiency and activity of 10 CYPs. The results show that numerous associations (positive and negative) are present between the seven miRNAs or the four TFs and the 10 CYP phenotypes (as indicated by mRNA, protein and activity); specifically, miR-27b, miR-34a and all four TFs played key roles in the interindividual variation of CYPs. Our results extend previous findings and suggest that miR-27b and miR-34a may be potential direct or indirect master regulators of CYP expression and thereby contribute to the interindividual variations in CYP-mediated drug metabolism.

From Genotype to Phenotype: Content and Activities of Cytochromes P450 2A6 in Human Liver In Vitro and Predicted In Vivo.

Unraveling the molecular mechanisms by which genetic variants of cytochrome P450 2A6 lead to different metabolic phenotypes remains a long-standing but important challenge. CYP2A6 is an enzyme involved in the metabolism of several clinical drugs as well as the metabolic activation of carcinogenic nitrosamines. Herein, CYP2A6 genotypes and phenotypes, as indicated by protein content [by liquid chromatography-mass spectrometry (MS)/MS] and metabolic activities [Vmax, clearance (CL)], were determined for 90 human liver samples. We determined the median, range, and interindividual and intraindividual variation of CYP2A6 content and activity at the microsomal, liver tissue, and whole liver level and predicted hepatic in vivo clearance by in vitro-in vivo extrapolation based on CYP2A6-mediated coumarin metabolism by each CYP2A6 genotype. These results reveal how different CYP2A6 genotypes yield different phenotypic traits in protein content and enzyme activity. For relative Vmax, CL, and protein content, the intraindividual percentage coefficients of variation (ICVs) were 41.0% (18.8%-125.1%), 28.5% (2.39%-133.5%), and 27.8% (2.68%-88.0%), respectively. The high ICVs implied large intraindividual variation at different levels, sometimes in a genotype-dependent manner. Intergenotype analysis revealed that the CYP2A6*4 allele demonstrated the most obvious effect on phenotypic outcomes, both in protein content and in metabolic activity. Indeed, decreased CYP2A6 protein content with the CYP2A6*4 genotype might explain the decreased metabolic activity from the molecular to the organismal level. These findings may allow useful predictions for CYP2A6-mediated drug metabolism on an individual patient basis in accord with the goal of achieving personalized medicine. SIGNIFICANCE STATEMENT: We provide the median, range, and interindividual and intraindividual variation in CYP2A6 content at the microsomal, liver tissue, and whole liver level by liquid chromatography-mass spectrometry (MS)/MS as well as activities at the protein, microsomal, liver tissue, and whole liver level both in vitro and at the organismal level based on CYP2A6-mediated coumarin metabolism with each CYP2A6 genotype, thereby allowing us to elucidate how different CYP2A6 genotypes yield differing phenotypic traits (protein content and enzyme activity), facilitating the development of personalized medicine.

A novel mass spectrometry method for the absolute quantification of several cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase enzymes in the human liver.

Cytochrome P450 (CYP450) and 5'-diphosphate glucuronosyltransferases (UGT) are the two major families of drug-metabolizing enzymes in the human liver microsome (HLM). As a result of their frequent abundance fluctuation among populations, the accurate quantification of these enzymes in different individuals is important for designing patient-specific dosage regimens in the framework of precision medicine. The preparation and quantification of internal standards is an essential step for the quantitative analysis of enzymes. However, the commonly employed stable isotope labeling-based strategy (QconCAT) suffers from requiring very expensive isotopic reagents, tedious experimental procedures, and long labeling times. Furthermore, arginine-to-proline conversion during metabolic isotopic labeling compromises the quantification accuracy. Therefore, we present a new strategy that replaces stable isotope-labeled amino acids with lanthanide labeling for the preparation and quantification of QconCAT internal standard peptides, which leads to a threefold reduction in the reagent costs and a fivefold reduction in the time consumed. The absolute amount of trypsin-digested QconCAT peptides can be obtained by lanthanide labeling and inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis with a high quantification accuracy (%RE < 20%). By taking advantage of the highly selective and facile ICP-OES procedure and multiplexed large-scale absolute target protein quantification using biological mass spectrometry, this strategy was successfully used for the absolute quantification of drug-metabolizing enzymes. We obtained good linearity (correlation coefficient > 0.95) over concentrations spanning 2.5 orders of magnitude with improved sensitivity (limit of quantification = 2 fmol) in nine HLM samples, indicating the potential of this method for large-scale absolute target protein quantification in clinical samples. Graphical abstract.

High CYP2E1 activity aggravates hepatofibrosis by limiting macrophage polarization towards the M2 phenotype.

Cytochrome P450 2E1 (CYP2E1) is an important drug-metabolizing enzyme that has been recognized as one of the risk factors for hepatofibrosis. Macrophages play key roles in regulating hepatofibrosis progression and resolution. However, whether CYP2E1 is involved in the regulation of macrophage polarization during hepatofibrosis is still unclear. Herein, we measured CYP2E1 activity and the expression of CD163 (an M2 marker) and CD68 (a pan-macrophage marker) in hepatofibrotic tissue from HCC patients (n = 26) with comparison to normal liver tissue (n = 26). The relationship of CYP2E1 activity in vivo and the CD163/CD68 ratio (an indicator of M2 polarization), as well as the extent of hepatofibrosis, were evaluated in diethylnitrosamine (DEN)-treated Sprague-Dawley rats. Strikingly, CYP2E1 activity and expression of CD68 increased and the CD163/CD68 ratio decreased, especially in hepatofibrotic tissue with higher CYP2E1 activity. Expression of α-SMA, Ki67, and PCNA were positively correlated with CYP2E1 activity and inversely correlated with the CD163/CD68 ratio. Furthermore, CYP2E1 activity showed an inverse correlation with the CD163/CD68 ratio. Overall, high CYP2E1 activity aggravates hepatofibrosis by restraining M2 macrophage polarization, providing a novel insight for understanding the profibrotic activity of CYP2E1 and a promising avenue for hepatofibrosis therapy.

CYP3A4/5 Activity Probed with Testosterone and Midazolam: Correlation between Two Substrates at the Microsomal and Enzyme Levels.

Testosterone (TST) and midazolam (MDZ) are widely used as probes to detect CYP3A4/5 activity, but the data acquired with these two substrates do not correlate well at the microsomal level (per milligram of microsomal protein), and the reason is unclear. In this study, CYP3A4/5 activity was probed with TST and MDZ at the microsomal and enzyme levels (per picomole of CYP3A4/5) in 72 human liver samples. Correlation coefficients were lower in Vmax and CLint at the microsomal level, as compared with those at the enzyme level ( Vmax 0.658 vs 0.883; CLint no correlation vs 0.796). Compared with TST, MDZ was found to correlate better with the content of CYP3A4/5 (no correlation vs 0.431) and CYP3A5 (no correlation vs 0.447), and huge variations in enzyme content existed among different genotypes, which explained the lower degree of correlation at the microsomal level. In addition, different genotypes had varying effects on activity at the enzyme level, whereas the difference between activity at the enzyme level probed with TST and that probed with MDZ was not obvious ( P > 0.05), indicating that the effect of gene polymorphisms on correlation between activity probed with these two substrates was limited at the enzyme level. In conclusion, our study demonstrates a high degree of correlation between CYP3A4/5 activity probed with TST and MDZ at the enzyme level but not at the microsomal level and allows us to correctly understand the influence of gene polymorphisms on the correlations.

Inhibitory effect of chlormethiazole on the toxicokinetics of diethylnitrosamine in normal and hepatofibrotic rats.

The effect of chlormethiazole (CMZ) at single and multiple doses on the toxicokinetics of diethylnitrosamine (DEN) was investigated in normal rats and those with DEN-induced liver fibrosis. Twelve rats were treated with DEN (50 mg/kg) alone and in combination with a single dose of CMZ (10, 50, or 100 mg/kg) by intraperitoneal (i.p.) injection. In a multiple dose test, six rats were treated with CMZ (50 mg/kg) for 7 d with addition of DEN (50 mg/kg) on days 1 and 7. Lastly, 12 rats were treated with DEN (50 mg/kg) by i.p. injection twice a week for 4 consecutive weeks, followed by weekly injections for another 8 weeks to build the model of liver fibrosis. Following this induction, the 12 rats were given CMZ (50 mg/kg) combined with DEN (50 mg/kg) to study the inhibitory effect of CMZ on DEN metabolism in hepatofibrotic rats. Serial blood samples were also collected and analyzed by a validated high-performance liquid chromatography (HPLC) method. A single-dose CMZ treatment decreased DEN clearance (CL), prolonged the t1/2, and increased the 'area under the curve' (AUC) for DEN in normal and hepatofibrotic rats relative to rats that did not receive CMZ. Treatment with CMZ for 7 d further prolonged the t1/2 for DEN but did not alter the CL and AUC relative to a single CMZ treatment. These results suggest that CMZ significantly inhibits the metabolism of DEN in normal and hepatofibrotic rats.

Effect of P450 Oxidoreductase Polymorphisms on the Metabolic Activities of Ten Cytochrome P450s Varied by Polymorphic CYP Genotypes in Human Liver Microsomes.

Background/ Aims: Little is known about the effect of P450 oxidoreductase (POR) gene polymorphisms on the activities of CYPs with multiple genotypes. METHODS: We genotyped 102 human livers for 18 known POR single nucleotide polymorphisms (SNPs) with allelic frequencies greater than 1% as well as for 27 known SNPs in 10 CYPs. CYP enzyme activities in microsomes prepared from these livers were determined by measuring probe substrate metabolism by high performance liquid chromatograph. RESULTS: We found that the effects of the 18 POR SNPs on 10 CYP activities were CYP genotype-dependent. The POR mutations were significantly associated with decreased overall Km for CYP2B6 and 2E1, and specific genotypes within CYP1A2, 2A6, 2B6, 2C8, 2D6 and 2E1 were identified as being affected by these POR SNPs. Notably, the effect of a specific POR mutation on the activity of a CYP genotype could not be predicted from other CYP genotypes of even the same CYP. When combining one POR SNP with other POR SNPs, a hitherto unrecognized effect of multiple-site POR gene polymorphisms (MSGP) on CYP activity was uncovered, which was not necessarily consistent with the effect of either single POR SNP. CONCLUSIONS: The effects of POR SNPs on CYP activities were not only CYP-dependent, but more importantly, CYP genotype-dependent. Moreover, the effect of a POR SNP alone and in combination with other POR SNPs (MSGP) was not always consistent, nor predictable. Understanding the impact of POR gene polymorphisms on drug metabolism necessitates knowing the complete SNP complement of POR and the genotype of the relevant CYPs.

Higher Activity of Alcohol Dehydrogenase Is Correlated with Hepatic Fibrogenesis.

Hepatofibrosis can progress to cirrhosis and hepatocellular carcinoma (HCC). Prevention, stabilization, and reversal of disease progression are vital for patients with hepatofibrosis, and identifying the risk factors for hepatofibrosis is urgently needed. In this study, we examined the activities of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in the fibrotic livers of HCC patients (n = 88) and comparied these results with activities in patients with normal livers (n = 74). A fibrosis-carcinoma rat model was used to study the activity of ADH in fibrosis and HCC and the relationship between innate ADH activity and the extent of hepatofibrosis or HCC. Substantial interindividual variations were found in the activities of ADH and ALDH in normal livers. The activity levels of total ADH, ADHI, and ADHII in fibrotic livers were significantly higher than those in normal livers (P < 0.001), whereas the activity of ALDH was slightly greater. The positive rates of ADHI and ADHII were 84.1% and 77.3%, respectively; the areas under the receiver operator characteristics (ROC) curve were 0.943 and 0.912, respectively. For the rat model compared with controls, ADH activity in liver was significantly increased at the fibrotic and HCC stages, and no significant difference was noted between ADH activity in the liver at these two stages. The innate activity of ADH in serum was well correlated with the extent of hepatofibrosis as indicated by Masson area%, Ki67+%, proliferating cell nuclear antigen +%, and GST-p average density at fibrotic stage but not at HCC stage. A higher level of activity of ADH is a risk factor for hepatofibrogenesis and might be a prevention target for hepatofibrosis.

Higher CYP2E1 Activity Correlates with Hepatocarcinogenesis Induced by Diethylnitrosamine.

Hepatocellular carcinoma (HCC) is one of the most common types of primary liver cancer and the third most frequent cause of cancer death worldwide. Diethylnitrosamine (DEN) is one of the recognized risk factors for hepatocarcinogenesis likely due to CYP2E1-mediated metabolic activation. However, CYP2E1-mediated DEN metabolic activity in non-neoplastic liver tissue from HCC patients has not been determined; the role of CYP2E1 activity, in particular CYP2E1 constitutive activity and CYP2E1 inhibited activity, with respect to the hepatocarcinogenesis induced by DEN is not yet clear. Herein, we determined CYP2E1-mediated DEN metabolic activity in non-neoplastic liver tissue from HCC patients and found that CYP2E1-mediated DEN metabolic activity was significantly elevated with a 43.3% positive rate, and clinicopathologic parameters did not affect the activity. Then, using a Sprague-Dawley rat liver tumor model induced by DEN, the relationship between CYP2E1 constitutive/inhibited activity and hepatocarcinogenesis was explored. The results showed that the CYP2E1 constitutive activity was strongly correlated with tumor incidence and severity of liver tumorigenesis (nodule numbers and size), whereas inhibition of CYP2E1 activity decreased hepatocyte proliferation, liver injury, and liver carcinogenesis in the presence of DEN. In conclusion, the higher CYP2E1 activity would lead to an increased incidence of HCC as a result of CYP2E1-mediated DEN activation. Therefore, higher CYP2E1 activity might be a risk factor for HCC induced by DEN.

From hepatofibrosis to hepatocarcinogenesis: Higher cytochrome P450 2E1 activity is a potential risk factor.

Hepatofibrosis is an important susceptibility factor for hepatocarcinogenesis. However, only a handful of cases of hepatofibrosis will develop into hepatocellular carcinoma (HCC). As cytochrome P450 2E1 (CYP2E1) is involved in the metabolism and activation of many known environmental toxicants and procarcinogens, this enzyme may play a role in the development of hepatocarcinogenesis subsequent to hepatofibrosis. Herein, we evaluated whether higher CYP2E1 activity is a risk factor for the development of hepatocarcinogenesis from hepatofibrosis. CYP2E1 activity in fibrotic tissues from 72 HCC patients and in normal liver tissues from 59 control subjects was determined along with the severity of hepatofibrosis in hepatocarcinogenesis patients. Similarly, using a rat diethylnitrosamine-induced hepatocarcinogenesis model, CYP2E1 activity at the hepatofibrosis and hepatocarcinogenesis stages was determined, the correlation between CYP2E1 activity at the hepatofibrosis stage and hepatocarcinogenesis was explored, and the impact of inhibition of CYP2E1 activity on hepatocarcinogenesis was studied. The results showed that while CYP2E1 activity in HCC patients with underlying hepatofibrosis was increased, the severity of hepatofibrosis did not correlate with CYP2E1 activity. In the rat hepatocarcinogenesis model, unexpectedly, CYP2E1 activity was found to decrease from hepatofibrosis to hepatocarcinogenesis. Importantly, however, hepatofibrotic rats with higher CYP2E1 activity developed a more severe form of HCC. Moreover, inhibition of CYP2E1 activity could decrease the occurrence and development of HCC in rats. In conclusion, higher CYP2E1 activity may be a risk factor for hepatocarcinogenesis from hepatofibrosis, which raises the possibility of screening patients with hepatofibrosis for CYP2E1 activity to better estimate their risk for hepatocarcinogenesis.

Content and Activities of UGT2B7 in Human Liver In Vitro and Predicted In Vivo: A Bottom-Up Approach.

UDP-glucuronosyltransferase 2B7 (UGT2B7) is one of the most significant isoforms of UGTs in human liver. This research measured UGT2B7 protein content and activities, including maximum velocity (Vmax) and intrinsic clearance (CLint), in human liver at isoform, microsomal, liver tissue, and liver levels and identified the factors that influence expression. We determined absolute protein content by liquid chromatography-tandem mass spectroscopy and activities using the probe drug zidovudine in 82 normal human liver microsomes. Using a bottom-up method for derivation, we showed UGT2B7 content at the microsomal, liver tissue, and liver levels, as well as activities at the isoform, microsomal, liver tissue, and liver levels in vitro, and predicted hepatic clearance in vivo, with median, range, variation, and 95% and 50% prediction intervals. With regard to the intrinsic activities, the maximum velocity (Vmax) had a median (range) of 7.5 (2-24) pmol/min per picomole of 2B7, and the CLint was 0.08 (0.02-0.31) µl/min per picomole of 2B7. Determinations at liver level showed larger variations than at microsomal level, so it was more suitable for evaluating individual differences. By analyzing factors that affect UGT2B7, we found that: 1) The content at the liver tissue and liver levels correlated positively with activities; 2) the mutant heterozygotes of -327G>A, -900A>G, -161C>T may lead to decreased protein content and increased intrinsic CLint; and 3) the transcription factor pregnane X receptor mRNA expression level was positively associated with the measured protein content. In all, we showed that protein content and activities at different levels and the factors that influence content provide valuable information for UGT2B7 research and clinically individualized medication.

Intraindividual Variation and Correlation of Cytochrome P450 Activities in Human Liver Microsomes.

We systematically studied and explored intraindividual variation and correlation in the activities of cytochrome P450 (CYP) using 105 normal liver samples. The intraindividual percentage coefficients of variation of relative Km, Vmax, and CLint were 45.9% (18.3-140%), 44.0% (16.8-127%), and 52.0% (24.2-134%). Overall values of relative Km, Vmax, and CLint for 10 CYPs were sorted. The order, from highest to lowest, was CYP2D6, 3A4/5, 2C19, 2C9, 2B6, 1A2, 2A6, 2C8, and 2E1 for Km; CYP3A4/5, 2C19, 2D6, 2C8, 2E1, 2B6, 1A2, 2A6, and 2C9 for Vmax; and CYP2D6, 2B6, 3A4/5, 2C19, 2C9, 1A2, 2E1, 2C8, and 2A6 for CLint. CYP2A6*4, 2B6 785A>G, and 2D6 100C>T contributed to intraindividual variation of CYP activities. The correlations and similarities among 10 CYP activities were analyzed, and it was found that the highest correlation and similarity for Vmax, Km, and CLint occurred between CYP2C9 and 2C8, CYP2C9 and 1A2, and CYP2C9 and 2C8, respectively. In conclusion, CYP activities exhibit considerable intraindividual variation, with some notable correlations, which might be helpful to comprehensively understand the internal connection among CYP activities and to guide the rational use of drugs.

From Genotype to Phenotype: Cytochrome P450 2D6-Mediated Drug Clearance in Humans.

How genotypic variation results in phenotypic differences is still a challenge for biology. In the field of drug metabolism, the means by which specific cytochrome P4502D6 (CYP2D6) genotypes yield different phenotypes at various levels (molecular, cellular, and organismal) is an important question, as differences in CYP2D6 activity can contribute to adverse drug reactions. Herein, the genotype of CYP2D6 was determined along with the absolute content of CYP2D6 and microsomal protein per gram of liver in human liver microsomes, the molecular, cellular (microsomal, tissue, organ), and organismal phenotype of CYP2D6 determined; the effect of genotype on each phenotype of CYP2D6-mediated dextromethorphan clearance (CL) was delineated, and the overall genotype-phenotype relationship for CYP2D6 was charted. We demonstrate that changes in the cellular and organismal CL phenotypes are markedly greater than changes seen at the molecular level. With individuals carrying the 1661CC polymorphism, for example, the most noticeable change took place in organ CL phenotype (4.17-fold), followed by tissue (3.75-fold), organism (3.69-fold), microsomal (3.09-fold), and molecular (1.66-fold) phenotypes. In addition, the biggest intragenotype individual coefficient of variation in organismal phenotype was observed in the 1661GG individuals, which reached 104.5%, followed by that of 100TT, 100CT, 1661GC, 100CC, and 1661CC polymorphisms (102.7%, 62.4%, 53.5%, 49.7%, and 44.8%, respectively). Our study has allowed us to chart the genotype-phenotype relationship for CYP2D6 from the molecular to the organismal level as well as allowed us to determine intragenotype individual variation in phenotype with each genotype.

Physiological Content and Intrinsic Activities of 10 Cytochrome P450 Isoforms in Human Normal Liver Microsomes.

Due to a lack of physiologic cytochrome P450 (P450) isoform content, P450 activity is typically only determined at the microsomal level (per milligram of microsomal protein) and not at the isoform level (per picomole of P450 isoform), which could result in the misunderstanding of variations in P450 activity between individuals and further hinder development of personalized medicine. We found that there were large variations in protein content, mRNA levels, and intrinsic activities of the 10 P450s in 100 human liver samples, in which CYP2E1 and CYP2C9 showed the highest expression levels. P450 gene polymorphisms had different effects on activity at two levels: CYP3A5*3 and CYP2A6*9 alleles conferred increased activity at the isoform level but decreased activity at the microsomal level; CYP2C9*3 had no effect at the isoform level but decreased activity at the microsomal level. The different effects at each level stem from the different effects of each polymorphism on the resulting P450 protein. Individuals with CYP2A6*1/*4, CYP2A6*1/*9, CYP2C9*1/*3, CYP2D6 100C>T TT, CYP2E1 7632T>A AA, CYP3A5*1*3, and CYP3A5*3*3 genotypes had significantly lower protein content, whereas CYP2D6 1661G>C mutants had a higher protein content. In conclusion, we first offered the physiologic data of 10 P450 isoform contents and found that some single nucleotide polymorphisms had obvious effects on P450 expression in human normal livers. The effects of gene polymorphisms on intrinsic P450 activity at the isoform level were quite different from those at the microsomal level, which might be due to changes in P450 protein content.

Correlation of Cytochrome P450 Oxidoreductase Expression with the Expression of 10 Isoforms of Cytochrome P450 in Human Liver.

Human cytochrome P450 oxidoreductase (POR) provides electrons for all microsomal cytochromes P450 (P450s) and plays an indispensable role in drug metabolism catalyzed by this family of enzymes. We evaluated 100 human liver samples and found that POR protein content varied 12.8-fold, from 12.59 to 160.97 pmol/mg, with a median value of 67.99 pmol/mg; POR mRNA expression varied by 26.4-fold. POR activity was less variable with a median value of 56.05 nmol/min per milligram. Cigarette smoking and alcohol consumption clearly influenced POR activity. Liver samples with a 2286822 TT genotype had significantly higher POR mRNA expression than samples with CT genotype. Homozygous carriers of POR2286822C>T, 2286823G>A, and 3823884A>C had significantly lower POR protein levels compared with the corresponding heterozygous carriers. Liver samples from individuals homozygous at 286823G>A, 1135612A>G, and 10954732G>A generally had lower POR activity levels than those from heterozygous or wild-type samples, whereas the common variant POR*28 significantly increased POR activity. There was a strong association between POR and the expression of P450 isoforms at the mRNA and protein level, whereas the relationship at the activity level, as well as the effect of POR protein content on P450 activity, was less pronounced. POR transcription was strongly correlated with both hepatocyte nuclear factor 4 alpha and pregnane X receptor mRNA levels. In conclusion, we have elucidated some potentially important correlations between POR single-nucleotide polymorphisms and POR expression in the Chinese population and have developed a database that correlates POR expression with the expression and activity of 10 P450s important in drug metabolism.

LC-MS/MS Analysis and Pharmacokinetics of Sodium (±)-5-Bromo-2-(α-hydroxypentyl) Benzoate (BZP), an Innovative Potent Anti-Ischemic Stroke Agent in Rats.

A rapid, sensitive and selective liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of sodium (±)-5-Bromo-2-(α-hydroxypentyl) benzoate (BZP) and its active metabolite 3-butyl-6-bromo-1(3H)-isobenzofuranone (Br-NBP) in rat plasma using potassium 2-(1-hydroxypentyl)-benzoate (PHPB) and l-3-n-butylphthalide (NBP) as internal standards (IS). Chromatographic separation was achieved on a Hypersil GOLD C18 column using a gradient elution of ammonium acetate and methanol at a flow rate of 0.2 mL/min. Good linearity was achieved within the wide concentration range of 5-10,000 ng/mL. The intra-day and inter-day precision was less than 8.71% and the accuracy was within -8.53% and 6.38% in quality control and the lower limit of quantitation samples. BZP and Br-NBP were stable during the analysis and the storage period. The method was successfully applied to pharmacokinetic studies of BZP in Sprague-Dawley rats for the first time. After a single intravenous administration of BZP at the dose of 0.75 mg/kg, the plasma concentration of BZP and Br-NBP declined rapidly and the AUC0-t of BZP was significantly greater in female rats compared to male rats (p < 0.05). The data presented in this study serve as a firm basis for further investigation of BZP in both preclinical and clinical phases.