Maternal separation influences hepatic drug-metabolizing CYP450 gene expression without pathological changes in adult mice.

OBJECTIVES: The principal motive of this study is to explore the influence maternal separation (MS) exhibits on the mRNA expression of major drug metabolizing-cyp450s in parallel with the assessment of pathological changes that can be induced by MS in the livers of experimental mice. METHODS: Eighteen Balb/c mouse pups, comprising of both males and females, were separated from their mothers after birth. Following a six-week period during when the pups became adults, the mice were sacrificed and their livers were isolated for analysis of weight, pathohistological alterations, and the mRNA expression of drug metabolizing cyp450 genes: cyp1a1, cyp3a11, cyp2d9, and cyp2c29. RESULTS: The study demonstrated that MS markedly downregulated (p<0.05) the mRNA expression of all tested drug-metabolizing cyp450s in livers of female and male mice. Furthermore, the mRNA levels of major drug-metabolizing cyp450s were notably lower (p<0.05) in livers of female MS mice as compared with male MS mice. It was found that values of the total body weight and liver weight of MS mice did not vary significantly (p>0.05) from those of the control groups. Additionally, histological examination revealed that the hepatic tissue of MS mice was normal, similar to that of the control mice. CONCLUSIONS: In summary, MS downregulates the gene expression of major hepatic drug-metabolizing cyp450s without inducing pathological alterations in the livers of mice. These findings provide an explanation for the heterogeneity in pharmacokinetics and drug response of patients with early life stress.

Modulation of IRAK enzymes as a therapeutic strategy against SARS-CoV-2 induced cytokine storm.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the current pandemic coronavirus disease 2019 (COVID-19). Dysregulated and excessive production of cytokines and chemokines, known as cytokine storm, is frequently seen in patients with severe COVID-19 disease and it can provoke a severe systematic inflammation in the patients. The IL-1R/TLRs/IRAKs signaling network is a key pathway in immune cells that plays a central role in regulating innate immunity and inflammatory responses via stimulating the expression and production of various proinflammatory molecules including cytokines. Modulation of IRAKs activity has been proposed to be a promising strategy in the treatment of inflammatory disorders. In this review, we highlight the biochemical properties of IRAKs and their role in regulating inflammatory molecular signaling pathways and discuss the potential targeting of IRAKs to suppress the SARS-CoV-2-induced cytokine storm in COVID-19 patients.

Alteration of the Respiratory Microbiome in Hospitalized Patients with Asthma-COPD Overlap during and after an Exacerbation.

The immediate aim of this study was to comparatively examine the bacterial respiratory microbiome of patients in a stable state and during an exacerbation of asthma-COPD (chronic obstructive pulmonary disease) overlap (ACO). This prospective observational study took place in Jordan between 1 September 2021 and 30 April 2022. Sputum samples from patients with recognized ACO were acquired within 48 h of the exacerbation onset and again at 3 weeks following the exacerbation. The next-generation sequencing Illumina MiSeq was employed and uncovered significantly high bacterial diversity in the sputa. The results showed a significant decrease in the taxonomic richness in the sputum samples collected during the exacerbation episodes compared with those collected from patients in a stable state (p = 0.008), with an increase in the taxonomic evenness (p < 0.005). This change in the composition of the airway bacterial community suggests that the replacement of a significant portion of the airway microbiome with certain microorganisms may play a role in the decrease in microbial diversity observed during an ACO exacerbation. Greater knowledge of this link could allow for a more focused administration of antibiotics, especially during exacerbations, improving clinical efficacy and patient outcomes.

The Protective Effect of Metformin against Oxandrolone-Induced Infertility in Male Rats.

BACKGROUND: Oxandrolone is a synthetic testosterone analog that is widely used among bodybuilders and athletes. However, oxandrolone causes male infertility. Recently, it was found that metformin reduces the risk of infertility associated with diabetes mellitus. AIM: This study aimed to investigate the protective effects of metformin against oxandrolone-induced infertility in male rats. METHODS: Rats continuously received one of four treatments (n=7) over 14 days: control DMSO administration, oxandrolone administration, metformin administration, or co-administration of oxandrolone and metformin. Doses were equivalent to those used for human treatment. Subsequently, testicular and blood samples were collected for morphological, biochemical, and histological examination. In addition, gene expression of the testosterone synthesizing enzyme CYP11A1 was analyzed in the testes using RT-PCR. RESULTS: Oxandrolone administration induced male infertility by significantly reducing relative weights of testes by 48%, sperm count by 82%, and serum testosterone levels by 96% (ANOVA, P value < 0.05). In addition, histological examination determined that oxandrolone caused spermatogenic arrest, which was associated with 2-fold downregulation of testicular CYP11A1 gene expression. However, co-administration of metformin with oxandrolone significantly ameliorated toxicological alterations induced by oxandrolone exposure (ANOVA, P-value < 0.05). CONCLUSION: Metformin administration provided protection against oxandrolone-induced infertility in male rats. Further clinical studies are needed to confirm the protective effect of metformin against oxandrolone-induced infertility among athletes.

Molecular toxicological alterations in the mouse hearts induced by sub-chronic thiazolidinedione drugs administration.

Thiazolidinediones are well-known anti-diabetic drugs. However, they are not widely used due to their cardiotoxic effects. Therefore, in this study, we aimed to determine the molecular toxicological alterations induced in the mouse hearts after thiazolidinedione administration. Balb/c mice received doses clinically equivalent to those given to humans of the most commonly used thiazolidinediones, pioglitazone, and rosiglitazone for 30 days. After that, RNA samples were isolated from the hearts. The mRNA expression of cytochrome (cyp) p450 genes that synthesize the cardiotoxic 20-hydroxyeicosatetraenoic acid (20-HETE) in addition to 92 cardiotoxicity biomarker genes were analyzed using quantitative polymerase chain reaction array technique. The analysis demonstrated that thiazolidinediones caused a significant upregulation (p < 0.5) of the mRNA expression of cyp1a1, cyp4a12, itpr1, ccl7, ccr1, and b2 m genes. In addition, thiazolidinediones caused a significant (p < 0.05) downregulation of the mRNA expression of adra2a, bsn, col15a1, fosl1, Il6, bpifa1, plau, and reg3b genes. The most affected gene was itpr1 gene, which was upregulated by pioglitazone and rosiglitazone by sevenfold and 3.5-fold, respectively. In addition, pioglitazone caused significant upregulation of (p < 0.05) hamp, ppbp, psma2, sik1, timp1, and ucp1 genes, which were not affected significantly (p > 0.05) by rosiglitazone administration. In conclusion, this study showed that thiazolidinediones induce toxicological molecular alterations in the mouse hearts, such as the induction of cyp450s that synthesize 20-HETE, chemokine activation, inflammatory responses, blood clotting, and oxidative stress. These findings may help us understand the mechanism of cardiotoxicity involved in thiazolidinedione administration.

Targeting the intestinal TMPRSS2 protease to prevent SARS-CoV-2 entry into enterocytes-prospects and challenges.

The transmembrane protease serine 2 (TMPRSS2) is a membrane anchored protease that primarily expressed by epithelial cells of respiratory and gastrointestinal systems and has been linked to multiple pathological processes in humans including tumor growth, metastasis and viral infections. Recent studies have shown that TMPRSS2 expressed on cell surface of host cells could play a crucial role in activation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein which facilitates the rapid early entry of the virus into host cells. In addition, direct suppression of TMPRSS2 using small drug inhibitors has been demonstrated to be effective in decreasing SARS-CoV-2 infection in vitro, which presents TMPRSS2 protease as a potential therapeutic strategy for SARS-CoV-2 infection. Recently, SARS-CoV-2 has been shown to be capable of infecting gastrointestinal enterocytes and to provoke gastrointestinal disorders in patients with COVID-19 disease, which is considered as a new transmission route and target organ of SARS-CoV-2. In this review, we highlight the biochemical properties of TMPRSS2 protease and discuss the potential targeting of TMPRSS2 by inhibitors to prevent the SARS-CoV-2 spreading through gastro-intestinal tract system as well as the hurdles that need to be overcome.

SARS-CoV-2 entry in host cells-multiple targets for treatment and prevention.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new viral disease that has gained global attention owing to its ability to provoke community and health-care-associated outbreaks of severe infections in human populations. The virus poses serious challenges to clinical management because there are still no approved anti- SARS-CoV-2 drugs available. In this mini-review, we summarize the much updated published reports that demonstrate the mechanism of SARS-CoV-2 entry into host cells, and discuss the availability and development of attractive host-based therapeutic options for SARS-CoV-2 infections.

Identification and functional characterization of CYP4V2 genetic variants exhibiting decreased activity of lauric acid metabolism.

The objectives of the present study were to identify CYP4V2 genetic variants and characterize their functional consequences. A total of 26CYP4V2 genetic variants were identified, including seven novel variants in 60 randomly selected healthy subjects. Six protein-coding variants were studied, including three novel variants (L22V, R287T, and G410C) and three previously reported variants (R36S, Q259K, and H331P). The cDNA sequences encoding each amino acid variant and the wild-type CYP4V2 protein were cloned into the pcDNA/PDEST40 expression vector and transfected into eukaryotic 293T cells for overexpression of the CYP4V2 coding variants. CYP4V2 H331P and CYP4V2 G410C exhibited significant decreases in activity for lauric acid oxidation (20-30% of wild-type activity), when compared to the wildtype, which was correlated with low expression of CYP4V2 H331P and G410C substituted proteins. The other four CYP4V2 amino variants were comparable to wild-type CYP4V2 for lauric acid metabolism. The CYP4V2 H331P and G410C substitutions were predicted to cause a structural change through in silico analysis. In conclusion, the present study provides functional information about CYP4V2 genetic variants. These findings will be valuable for interpreting individual variations in phenotypes associated with CYP4V2 function in the clinical setting.

Inhibition of 20-hydroxyeicosatetraenoic acid (20-HETE) glucuronidation by non-steroidal anti-inflammatory drugs in human liver microsomes and recombinant UDP-glucuronosyltransferase enzymes.

20-hydroxyeicosatetraenoic acid (20-HETE) is an arachidonic acid metabolite which is known to increase platelet aggregation and cardiovascular risk. In this study, nine non-steroidal anti-inflammatory drugs (NSAIDs) selected by chemical structures were screened to determine their effects on the glucuronidation of 20-HETE using human liver microsomes (HLMs). Then, the combined effects of the selected NSAID and genetic polymorphisms in UDP-glucuronosyltransferase (UGT) were investigated. Among the tested NSAIDs, diclofenac was the strongest inhibitor of 20-HETE glucuronidation with an IC50 value of 3.5 µM. Celecoxib, naproxen, mefenamic acid, ibuprofen, and indomethacin showed modest inhibition with IC50 values of 77, 91, 190, 208, and 220 µM, respectively, while acetylsalicylic acid, rofecoxib, and meloxicam did not inhibit 20-HETE glucuronidation. Glucuronidation of 20-HETE by UGT2B7 and UGT1A9 recombinant enzymes was significantly inhibited by indomethacin, mefanemic acid, diclofenac, ibuprofen, naproxen, and celecoxib (P < 0.001). In addition, diclofenac exhibited a competitive inhibition mechanism with the Km value of 20-HETE glucuronidation increasing from 23.5 µM to 62 µM in the presence of 3.5 µM diclofenac. Diclofenac further decreased 20-HETE glucuronidation in HLMs carrying UGT2B7*2 alleles compared with the wild-type HLMs. The results from this study would be useful in understanding the alteration of 20-HETE levels in relation to NSAID and UGT genetic polymorphisms.

Association of HLA-DRB1 and -DQ Alleles and Haplotypes with Type 1 Diabetes in Jordanians.

BACKGROUND: The Human Leukocyte Antigen (HLA) class II genes, particularly the HLADR and -DQ loci, have been shown to play a crucial role in Type 1 Diabetes (T1D) development. OBJECTIVE: This study is the first to examine the contribution of the HLA-DR/DQ alleles and haplotypes to T1D susceptibility in Jordanians. METHODS: Polymerase chain reaction sequence-specific primers (PCR-SSP) were used to genotype 41 Jordanian healthy controls and 50 insulin-dependent diabetes mellitus (IDDM) patients. RESULTS: The following alleles were found to be significant high risk alleles in T1D Jordanian patients: DRB1*04 (OR=3.95, p<0.001), DRB1*0301(OR=5.27, p<0.001), DQA1*0301 (OR=5.67, p<0.001), DQA1*0501(OR=3.18, p=0.002), DQB1*0201(OR=2.18, p=0.03), DQB1*0302 (OR=5.67, p<0.001). However, Jordanians harboring the DRB1*0701 (OR=0.37, p=0.01), DRB1*1101 (OR=0.2, p=0.01), DQA1*0505 (OR=0.31, p=0.02), DQA1*0103 (OR=0.33, p=0.04), DQA1*0201 (OR=0.45, p=0.04), DQB1*0301 (OR=0.23, p=0.001), DQB1*0501 (OR=0.18, p=0.009) alleles had a significantly lower risk of developing T1D. CONCLUSION: A strong positive association of DRB1*04-DQA1*0301-DQBl*0302 (OR=5.67, p<0.001) and DRB1*0301-DQA1*0501-DQB1*0201 (OR=6.24, p<0.001) putative haplotypes with IDDM was evident in Jordanian IDDM patients whereas DRB1*1101-DQA1*0505- DQB1*0301 (OR=0.23, p=0.03) was shown to have a protective role against T1D in Jordanians. Our findings show that specific HLA class II alleles and haplotypes are significantly associated with susceptibility to T1D in Jordanians.

Molecular Functionality of Cytochrome P450 4 (CYP4) Genetic Polymorphisms and Their Clinical Implications.

Enzymes in the cytochrome P450 4 (CYP4) family are involved in the metabolism of fatty acids, xenobiotics, therapeutic drugs, and signaling molecules, including eicosanoids, leukotrienes, and prostanoids. As CYP4 enzymes play a role in the maintenance of fatty acids and fatty-acid-derived bioactive molecules within a normal range, they have been implicated in various biological functions, including inflammation, skin barrier, eye function, cardiovascular health, and cancer. Numerous studies have indicated that genetic variants of CYP4 genes cause inter-individual variations in metabolism and disease susceptibility. Genetic variants of CYP4A11, 4F2 genes are associated with cardiovascular diseases. Mutations of CYP4B1, CYP4Z1, and other CYP4 genes that generate 20-HETE are a potential risk for cancer. CYP4V2 gene variants are associated with ocular disease, while those of CYP4F22 are linked to skin disease and CYP4F3B is associated with the inflammatory response. The present study comprehensively collected research to provide an updated view of the molecular functionality of CYP4 genes and their associations with human diseases. Functional analysis of CYP4 genes with clinical implications is necessary to understand inter-individual variations in disease susceptibility and for the development of alternative treatment strategies.

The Nudix Hydrolase 15 (NUDT15) Gene Variants among Jordanian Arab Population

Background: Nudix Hydrolase 15 gene (NUDT15) encodes nucleotide triphosphate diphosphatase which metabolizes the purine analog drugs, such as anticancer thiopurine and anti-gout allopurinol. Genetic variants on Nudix Hydrolase 15 gene (NUDT15) gene effects the drug's hydrolyses and hence increases the susceptibility to drug-induced toxicity. The NUDT15 gene has been genotyped in various ethnic groups, however, it has not been genotyped among the Middle Eastern Arab Jordanian population. Aim: The current study aimed to identify NUDT15 genetic variants among Jordanian Arab population. Method: The DNA samples were isolated from leukocytes of 85 unrelated Jordanian Arab volunteers. The coding regions of NUDT15 gene; Exon 1,2 and 3, in addition to some regions of intron 1,2 and 3'UTR, were amplified using polymerase chain reaction (PCR). the PCR products were then subjected to purification and sequenced using Applied Biosystems Model (ABI3730x1). Results: Six NUDT15 genetic variants were found among the volunteers.The results were as followed: A novel synonymous variant 36A>G on exon 1 (6%, 95%CI= 3- 9%), the intronic IVS1 +116C>T variant on intron 1 (0.6%, 95%CI= 0-2%), the non-synonymous variant on exon 3; 415C>T (0.6%, 95%CI= 0-2%), A novel non-synonymous variant on exon 3; 404C>A (0.6%, 95%CI= 0-2%) , and two novel variants on 3'UTR ;502G>A (2%, 95%CI= 0.5-4%) and 588T>C (0.6%, 95%CI= 0-2%). NUDT15 36A>G wasfound to be the most common allele among Jordanians was. In silico softwares predicted that the novel NUDT15 404C>A was harmful and affected NUDT15 enzyme'sstability and function. Furthermore, the frequency of NUDT15 IVS1 +116C>T , among Jordanians, showed to be significantly lower from what was reported in other ethnicities with ap value > 0.05 on the other hand, the frequency of 415C>T variant showed to be similar to Europeans in contrast to Asians and Indians that showed to be significantly lower (p value > 0.05). Conclusions: The frequency of NUDT15 genetic variants is low among the Jordanian volunteers and significantly lower than other ethnic groups. The findings of this study may increase our understanding of the inter-individual variation in the response to purine analog drugs. Further clinical studies are needed to investigate the influence of novel NUDT15 404C>A on drug metabolism and response.

Effects of nonsteroidal anti-inflammatory drugs on the expression of arachidonic acid-metabolizing Cyp450 genes in mouse hearts, kidneys and livers.

Arachidonic acid (ARA) metabolites are involved in cardiovascular diseases and drug-induced cardiotoxicity. The present study aimed to investigate the effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on the gene expression of ARA-metabolizing cyp450 genes in the hearts, kidneys and livers of experimental mice. Thirty five Balb/c mice were divided into 5 groups, and each group contained 7 mice. Then, the groups were administered different NSAIDs, diclofenac mefenamic acid, ibuprofen, or meloxicam, for 14 days in doses equivalent to those used in human treatment. Subsequently, liver, kidney and heart samples were isolated for analysis of the expression of ARA-metabolizing cyp450 genes using real-time polymerase chain reaction. In addition, the histological alterations induced by mefenamic acid were examined. It was found that 20-HETE synthesizing gene cyp4a12 was upregulated (> 2.2 fold) in the hearts of NSAID-treated mice, which was associated with the 2-fold downregulation of the cardio-protective biomarker GATA4 gene and the induction of cox2 expression (p value < 0.05). In the kidneys, the expression of cyp4a12 was significantly reduced (p value <0.05) while cyp2c29 expression was upregulated by more than 2 fold. In the liver, all NSAIDs except diclofenac significantly decreased the expression of all genes tested (p value <0.05) and were associated with abnormal accumulation of fat in the liver. Furthermore, these molecular findings were in parallel to histological alterations induced in the liver, kidney, and heart after mefenamic acid administration. This study concluded that NSAIDs altered the expression of ARA-metabolizing cyp450 genes and induced histological alterations that may influence the function of the vital organs.

Alterations in the gene expression of drug and arachidonic acid-metabolizing Cyp450 in the livers of controlled and uncontrolled insulin-dependent diabetic mice.

BACKGROUND: Diabetic patients have lower capacity to metabolize drugs in comparison to normal people. Therefore, the present study aimed to investigate the alterations in gene expression of drug and arachidonic acid metabolizing cytochrome p450s (cyp450s) in the livers of controlled (CDM) and uncontrolled (UDM) insulin-dependent diabetic mice. METHODS: Balb/c mice were treated with single dose of streptozocin (240 mg/kg) to induce diabetes and compared with control group, which was treated with citric buffer (pH =4.5). After 3 days, the blood glucose level was measured to confirm the induction of diabetes. Normalization of blood glucose level in diabetic mice was achieved after 0.1 mL/kg Mixtard® insulin therapy for more 5 days. Then, the mice livers were isolated to extract RNA and convert it to cDNA. The gene expression of 14 genes, which play a major role in drug and arachidonic acid metabolism, were measured using quantitative real-time polymerase chain reaction technique. RESULTS: It was found that the gene expression was downregulated (ANOVA test, P-value <0.05) in the livers of UDM mice. The most downregulated genes were cyp4a12, cyp1a2, and slc22a1 with more than 10-fold reduction. The livers of CDM mice showed significantly (P-value <0.05) higher levels of mRNA than UDM mice, but still lower than the non-diabetic mice. CONCLUSION: This study concluded that hepatic gene expression of drug metabolizing and arachidonic acid- cyp450 enzymes is reduced in insulin-dependent diabetic mice, which can explain, at least in part, the variation in drug and fatty acid metabolism between normal and diabetic patients.

Sequence analysis of the N-acetyltransferase 2 gene (NAT2) among Jordanian volunteers.

The present study aimed to identify the NAT2 haplotypes, linkage disequilibrium, and novel NAT2 genetic variants among Jordanian population. We isolated the genomic DNA from 68 healthy, Arab, unrelated Jordanian volunteers to amplify the protein-coding region of NAT2 gene by polymerase chain reaction (PCR). Then, the amplified PCR products were sequenced using Applied Biosystems Model (ABI3730x1). It is found that the allele frequencies of known NAT2 genetic variants 191G>A, 282C>T, 341T>C, 481C>T, 590G>A, and 803A>G were 0.7, 26.5, 48.5, 35.3, 30.9, and 32.4%, respectively. The NAT2 allele frequencies were generally similar to those of white Europeans but different from those of Asian and African populations. The most common NAT2 haplotype was NAT2*5B with a frequency of 29.3%. According to the NAT2 haplotype frequencies, 72% (95% confidence interval 61.4-82.7%) of the volunteers were slow encoding NAT2 haplotype acetylators. The NAT2*5 represented variants 341T>C and 481C>T were in strong but not complete linkage disequilibrium (D' = 0.8, r2 = 0.63). In addition, this study found a novel nonsynonymous NAT2 436G>A genetic variant with low frequency (0.7%). However, this novel variant was predicted to be tolerated and not harmful to the NAT2 protein, using in silico prediction tools. It is concluded that the frequency of slow encoding NAT2 haplotype was high among Jordanian volunteers, which may have effects on drug responses and susceptibility to some diseases, such as cancers.

Effects of the genetic variants of organic cation transporters 1 and 3 on the pharmacokinetics of metformin in Jordanians.

BACKGROUND: Human response to the antidiabetic metformin is influenced by some factors, such as genetic variants in the SLC22A genes. This study aimed to determine the frequency of main SLC22A1 and SLC22A3 genetic variants and their influence on metformin pharmacokinetics among healthy unrelated Arab Jordanians. PATIENTS AND METHODS: The SLC22A1 and SLC22A3 genes were genotyped by DNA sequencing of exons 1, 3, 7, and 9 in the SLC22A1 gene and exons 6, 7, and 9 in the SLC22A3 gene. Then, a clinical pharmacokinetic study was conducted on 26 healthy volunteers. The pharmacokinetic parameters were calculated using non-compartmental model analysis. The study was an open-label, randomized study with single 1000 mg metformin administration. RESULTS: Results showed that volunteers with SLC22A3 rs8187722 variant had higher (χ2, p<0.05) metformin Cmax and AUC values than the wild SLC22A3 volunteers, whereas T½ and Kel were not affected. In addition, volunteers with the heterozygote SLC22A3 rs2292334 variant had significantly higher (χ2, p<0.05) metformin Cmax and AUC and lower Kel values than the wild-type SLC22A3 genotype. CONCLUSIONS: The SLC22A3 rs8187722 and rs2292334 genetic variants affected metformin pharmacokinetics among a clinical sample of Jordanians. The findings may increase our understanding of the inter-individual and inter-ethnic variations in metformin response.

Determination of major UDP-glucuronosyltransferase enzymes and their genotypes responsible for 20-HETE glucuronidation.

The compound 20-HETE is involved in numerous physiological functions, including blood pressure and platelet aggregation. Glucuronidation of 20-HETE by UDP-glucuronosyltransferases (UGTs) is thought to be a primary pathway of 20-HETE elimination in humans. The present study identified major UGT enzymes responsible for 20-HETE glucuronidation and investigated their genetic influence on the glucuronidation reaction using human livers (n = 44). Twelve recombinant UGTs were screened to identify major contributors to 20-HETE glucuronidation. Based on these results, UGT2B7, UGT1A9, and UGT1A3 exhibited as major contributors to 20-HETE glucuronidation. The Km values of 20-HETE glucuronidation by UGT1A3, UGT1A9, and UGT2B7 were 78.4, 22.2, and 14.8 µM, respectively, while Vmax values were 1.33, 1.78, and 1.62 nmol/min/mg protein, respectively. Protein expression levels and genetic variants of UGT1A3, UGT1A9, and UGT2B7 were analyzed in human livers using Western blotting and genotyping, respectively. Glucuronidation of 20-HETE was significantly correlated with the protein levels of UGT2B7 (r(2) = 0.33, P < 0.001) and UGT1A9 (r(2) = 0.31, P < 0.001), but not UGT1A3 (r(2) = 0.02, P > 0.05). A correlation between genotype and 20-HETE glucuronidation revealed that UGT2B7 802C>T, UGT1A9 -118T9>T10, and UGT1A9 1399T>C significantly altered 20-HETE glucuronide formation (P < 0.05-0.001). Increased levels of 20-HETE comprise a risk factor for cardiovascular diseases, and the present data may increase our understanding of 20-HETE metabolism and cardiovascular complications.

Molecular functionality of CYP2C9 polymorphisms and their influence on drug therapy.

CYP2C9 metabolizes approximately 20% of clinically used drugs, including the narrow therapeutic window drugs warfarin and phenytoin. More than 16,000 variants have been reported in the National Center for Biotechnology Information CYP2C9 database, as well as 58 alleles in the official P450 Nomenclature Committee website. Two single nucleotide polymorphisms represented by the CYP2C9*2 and CYP2C9*3 alleles have been studied extensively. However, in addition to these two alleles, other genetic factors and an individual's biological characteristics contribute to the overall drug phenotype. A major bottleneck for CYP2C9 pharmacogenomics in clinical field applications is the lack of knowledge regarding the numerous genetic polymorphisms and their molecular functionalities. An unmet gap exists between the ever-growing number of genetic variants and their molecular mechanisms. In the present review, functional changes of all known CYP2C9 protein coding alleles were predicted using in silico analyses and compared with the in vitro and in vivo data. We also summarize functional information from recently reported CYP2C9 variants. Regarding the previously known CYP2C9 variants, we provide an update on the functional information obtained from in vitro and in vitro data.

Expression of arachidonic acid-metabolizing cytochrome P450s in human megakaryocytic Dami cells.

Cytochrome P450s (P450s) are involved in the metabolism of arachidonic acid (ARA), and ARA metabolites are associated with various cellular signaling pathways, such as blood hemostasis and inflammation. The present study demonstrates the expression of ARA-metabolizing P450s in the human megakaryocytic Dami cells using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunublotting analysis followed by activity assays using ARA as a substrate. In addition to the previously identified CYP5A1, both protein and mRNAs of CYP1A1, 2U1, and 2J2 bands were detected. Ethoxyresorufin-O-deethylase (EROD) activity was observed in Dami cells, and its activity was significantly decreased after treatment with the P450 inhibitor SKF-525A when compared to the control groups (60% reduction, P < 0.001). CYP1A1 protein expression in Dami cells was induced by 3-methylenecholantheren. This increase in CYP1A1 protein level was correlated with enhanced EROD activity (fourfold increase vs. the control), as well as with increased metabolites, such as 20-hydroxyeicosatrienoic acid (20-HETE), 14, 15-EET (14-,15-epoxyeicosatrienoic acid), and 14, 15-dihydroxyeicosatrienoic acid (14, 15-DHET). The expression of soluble epoxide hydrolase, an enzyme responsible for the synthesis of DHETs from EETs, was confirmed by RT-PCR. Furthermore, 15 ARA metabolites, including 8,9-EET, 14,15-EET, and 20-HETE, were detected by LC-MS/MS in ARA-treated Dami cells, and their levels were decreased with the treatment of the SKF-525A. The present data suggest the possibility that the P450s play a role in the metabolism of ARA and other CYP-related substrates in human megakaryocytes and that P450 expression in megakaryocytic cell lines may predict their existences in platelets with functional activities.