Tracing TET1 expression in prostate cancer: discovery of malignant cells with a distinct oncogenic signature.

BACKGROUND: Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) is involved in DNA demethylation and transcriptional regulation, plays a key role in the maintenance of stem cell pluripotency, and is dysregulated in malignant cells. The identification of cancer stem cells (CSCs) driving tumor growth and metastasis is the primary objective of biomarker discovery in aggressive prostate cancer (PCa). In this context, we analyzed TET1 expression in PCa. METHODS: A large-scale immunohistochemical analysis of TET1 was performed in normal prostate (NOR) and PCa using conventional slides (50 PCa specimens) and tissue microarrays (669 NOR and 1371 PCa tissue cores from 371 PCa specimens). Western blotting, RT-qPCR, and 450 K methylation array analyses were performed on PCa cell lines. Genome-wide correlation, gene regulatory network, and functional genomics studies were performed using publicly available data sources and bioinformatics tools. RESULTS: In NOR, TET1 was exclusively expressed in normal cytokeratin 903 (CK903)-positive basal cells. In PCa, TET1 was frequently detected in alpha-methylacyl-CoA racemase (AMACR)-positive tumor cell clusters and was detectable at all tumor stages and Gleason scores. Pearson's correlation analyses of PCa revealed 626 TET1-coactivated genes (r > 0.5) primarily encoding chromatin remodeling and mitotic factors. Moreover, signaling pathways regulating antiviral processes (62 zinc finger, ZNF, antiviral proteins) and the pluripotency of stem cells were activated. A significant proportion of detected genes exhibited TET1-correlated promoter hypomethylation. There were 161 genes encoding transcription factors (TFs), of which 133 were ZNF-TFs with promoter binding sites in TET1 and in the vast majority of TET1-coactivated genes. CONCLUSIONS: TET1-expressing cells are an integral part of PCa and may represent CSCs with oncogenic potential.

Novel insights into peptide amidation and amidating activity in the human circulation.

C-terminal α-amidation is the final and essential step in the biosynthesis of several peptide hormones. Peptidylglycine α-amidating monooxygenase (PAM) is the only known enzyme to catalyse this reaction. PAM amidating activity (AMA) is known to be present in human circulation, but its physiological role and significance as a clinical biomarker remains unclear. We developed a PAM-specific amidation assay that utilizes the naturally occurring substrate Adrenomedullin-Gly (ADM-Gly, 1-53). Using our amidation assay we quantified serum amidating activities in a large population-based cohort of more than 4900 individuals. A correlation of serum amidating activity with several clinical parameters including high blood pressure was observed. Increasing PAM-AMA was an independent predictor of hard outcomes related to hemodynamic stress such as cardiovascular mortality, atrial fibrillation and heart failure during long-term follow-up (8.8 ± 2.5 years). Moreover, results from an animal study in rats utilizing recombinant human PAM provide novel insights into the physiological role of circulating PAM and show its potential significance in circulating peptide amidation.

Prognostic role of high TET1 expression in patients with solid tumors: A meta-analysis.

BACKGROUND: Recently, increased expression of TET1 has been shown to inhibit tumor development in many studies. Therefore, a meta-analysis was conducted to assess the prognostic role of TET1 in solid tumors. METHODS: PubMed, Embase, and the Web of Science (last updated on June 13, 2019) were searched and 16 eligible studies involving 3100 patients were eventually taken forward into the meta-analysis. RESULTS: Pooled results indicated that higher TET1 expression in cancer tissues was associated with improved overall survival (OS) [hazard ratio (HR) = 0.736, 95% confidence interval (95% CI) = 0.542-0.998, P = .049]. In the subgroup analysis, higher TET1 expression in respiratory tumors (HR = 0.778, 95% CI = 0.639-0.946, P = .012) and breast cancer in Asian patients (HR = 0.326, 95% CI = 0.199-0.533, P < .001) were significantly associated with better OS. In addition, the association between high TET1 expression and prolonged OS was also statistically significant in the following subgroups; data source from samples (HR = 0.561, 95% CI = 0.384-0.819, P = .003), reported in text (HR = 0.539, 95% CI = 0.312-0.931, P = .027), TET1 protein (HR = 0.635, 95% CI = 0.409-0.984, P = .042), Asians (HR = 0.563, 95% CI = 0.376-0.844, P = .005). CONCLUSION: This meta-analysis displays that high expression levels of TET1 in tissues is significantly associated with better survival in patients with solid tumors. This finding can be used as evidence to the tone that TET1 may be a useful target for the treatment of patients with solid tumors in the future.

Variant analysis of HPD genes from two families showing elevated tyrosine upon newborn screening by tandem mass spectrometry (MS/MS).

Background Alterations in the structure and activity of 4-hydroxyphenylpyruvate dioxygenase (HPD) are causally related to two different metabolic disorders: recessively inherited tyrosinemia type III and dominantly inherited hawkinsinuria. The aim of this study was to provide a new perspective for the clinical understanding of the pathogenesis of tyrosinemia type III or hawkinsinuria. Case presentation A full-term newborn baby born after a safe pregnancy and childbirth with a birth weight of 3200 g and another full-term baby born after a safe pregnancy and childbirth with a birth weight of 2800 g are reported and analysed. DNA extraction, next-generation sequencing, bioinformatics analysis, Sanger sequencing and biochemical analysis were performed. One patient with a heterozygous HPD gene (NM_002150.2) c.460G > A mutation and one patient with a heterozygous HPD gene (NM_002150.2) c.248delG mutation showing elevated tyrosine levels upon newborn screening by tandem mass spectrometry (MS/MS) are reported. Conclusions The HPD gene may not be a strictly autosomal recessive pathogenic gene, which provides a new perspective for the clinical understanding of the pathogenesis of tyrosinemia type III or hawkinsinuria.

Elevated Expression of A-Raf and FA2H in Hepatocellular Carcinoma is Associated with Lipid Metabolism Dysregulation and Cancer Progression.

BACKGROUND: Identification of events leading to hepatocellular carcinoma (HCC) progression is essential for understanding its pathophysiology. The aims of this study are to identify and characterize differentially expressed proteins in serum of HCC-bearing rats and the corresponding controls during cancer initiation, progression and tumorigenesis. METHODS: Chemical carcinogens, N-Nitrosodiethylamine and 2-aminoacetylfluorine are administered to induce HCC to male Wistar rats. The 2D-Electrophoresis and PD-Quest analyses are performed to identify several differentially expressed proteins in serum of HCC-bearing animals. These proteins are further characterized by MALDI-TOF-MS/MS analyses. Using pathwaylinker a HCC-specific network is analyzed among the MALDITOF- MS/MS characterized proteins and their interactors. RESULTS: Carcinogen administration caused inflammation leading to liver injury and HCC development. Liver inflammation was confirmed by increase in the levels of TNF-α and IL-6 in carcinogen treated rats. We report significant increase in expression of two differentially expressed proteins, namely, A-Raf and Fatty Acid 2- Hydroxylase (FA2H), at early stage of HCC initiation, during its progression and at tumor stage. Real-time PCR analysis of mRNA for these proteins confirmed up-regulation of their transcripts. Further, we validated our experimental data with sera of clinically confirmed liver cancer patients. CONCLUSION: The study suggests that FA2H and A-Raf play a major role in the progression of HCC.

Reduced mRNA and Protein Expression Levels of Tet Methylcytosine Dioxygenase 3 in Endothelial Progenitor Cells of Patients of Type 2 Diabetes With Peripheral Artery Disease.

Endothelial progenitor cells (EPCs) with immunological properties repair microvasculature to prevent the complications in patients with diabetes. Epigenetic changes such as DNA methylation alter the functions of cells. Tet methylcytosine dioxygenases (TETs) are enzymes responsible for the demethylation of cytosine on genomic DNA in cells. We hypothesized that EPCs of diabetic patients with peripheral artery disease (D-PAD) might have altered expression levels of TETs. Subjects who were non-diabetic (ND, n = 22), with diabetes only (D, n = 29) and with D-PAD (n = 22) were recruited for the collection of EPCs, which were isolated and subjected to analysis. The mRNA and protein expression levels of TET1, TET2, and TET3 were determined using real-time PCR and immunoblot, respectively. The TET1 mRNA expression level in ND group was lower than that in the D and D-PAD groups. The TET3 mRNA level in the ND group was higher than that in the D group, which was higher than that in the D-PAD group. The TET1 protein level in the D-PAD group, but not the D group, was higher than that in the ND group. The TET2 protein level in the D-PAD group, but not the D group, was lower than that in the ND group. The TET3 protein level in the ND group was higher than that in the D group, which was higher than that in the D-PAD group, which is the lowest among the three groups. The changes of TETs protein levels were due to the alterations of their transcripts. These probably lead to epigenetic changes, which may be responsible for the reductions of EPCs numbers and functions in patients with the D-PAD. The expression pattern of TET3 mRNA and TET3 protein in EPCs may be a biomarker of angiopathy in diabetic patients.

Assessment of a combination of Serum Proteins as potential biomarkers to clinically predict Schizophrenia.

Schizophrenia (SZ) is a devastating psychiatric disorder. Validation of potential serum biomarkers during first-episode psychosis (FEP) is especially helpful to understand the onset and prognosis of this disorder. To address this question, we examined multiple blood biomarkers and assessed the efficacy to diagnose SZ. The expression levels of Neuregulin1 (NRG1), ErbB4, brain-derived neurotrophic factor (BDNF), DNA methyltransferases 1 (DNMT1) and ten-eleven translocation 1 (TET1) proteins in peripheral blood of 53 FEP patients and 57 healthy controls were determined by enzyme-linked immunosorbent assay (ELISA). Multivariable logistic regression including biomarker concentration as covariates was used to predict SZ. Differentiating performance of these five serum protein levels was analyzed by Receiver Operating Characteristic (ROC) curve analysis. We found that patients with SZ present a higher concentration of DNMT1, and TET1 in peripheral blood, but a lower concentration of NRG1, ErbB4 and BDNF than controls. Multivariable logistic regression showed that ErbB4, BDNF and TET1 were independent predictors of SZ, and when combined, provided high diagnostic accuracy for SZ. Together, our findings highlight that altered expression of NRG1, ErbB4, BDNF, DNMT1 and TET1 are involved in schizophrenia development and they may serve as potential biomarkers for the diagnosis of the schizophrenia. Therefore, our study provides evidence that combination of ErbB4, BDNF and TET1 biomarkers could greatly improve the diagnostic performance.

DNA Hydroxymethylation Levels Are Altered in Blood Cells From Down Syndrome Persons Enrolled in the MARK-AGE Project.

Down syndrome (DS) is caused by the presence of part or an entire extra copy of chromosome 21, a phenomenon that can cause a wide spectrum of clinically defined phenotypes of the disease. Most of the clinical signs of DS are typical of the aging process including dysregulation of immune system. Beyond the causative genetic defect, DS persons display epigenetic alterations, particularly aberrant DNA methylation patterns that can contribute to the heterogeneity of the disease. In the present work, we investigated the levels of 5-hydroxymethylcytosine and of the Ten-eleven translocation dioxygenase enzymes, which are involved in DNA demethylation processes and are often deregulated in pathological conditions as well as in aging. Analyses were carried out on peripheral blood mononuclear cells of DS volunteers enrolled in the context of the MARK-AGE study, a large-scale cross-sectional population study with subjects representing the general population in eight European countries. We observed a decrease in 5-hydroxymethylcytosine, TET1, and other components of the DNA methylation/demethylation machinery in DS subjects, indicating that aberrant DNA methylation patterns in DS, which may have consequences on the transcriptional status of immune cells, may be due to a global disturbance of methylation control in DS.

The Effect of Selenium Supplementation on Glucose Homeostasis and the Expression of Genes Related to Glucose Metabolism.

The aim of the study was to evaluate the effect of selenium supplementation on the expression of genes associated with glucose metabolism in humans, in order to explain the unclear relationship between selenium and the risk of diabetes. For gene expression analysis we used archival samples of cDNA from 76 non-diabetic subjects supplemented with selenium in the previous study. The supplementation period was six weeks and the daily dose of selenium was 200 µg (as selenium yeast). Blood for mRNA isolation was collected at four time points: before supplementation, after two and four weeks of supplementation, and after four weeks of washout. The analysis included 15 genes encoding selected proteins involved in insulin signaling and glucose metabolism. In addition, HbA1c and fasting plasma glucose were measured at three and four time points, respectively. Selenium supplementation was associated with a significantly decreased level of HbA1c but not fasting plasma glucose (FPG) and significant down-regulation of seven genes: INSR, ADIPOR1, LDHA, PDHA, PDHB, MYC, and HIF1AN. These results suggest that selenium may affect glycemic control at different levels of regulation, linked to insulin signaling, glycolysis, and pyruvate metabolism. Further research is needed to investigate mechanisms of such transcriptional regulation and its potential implication in direct metabolic effects.

Effects of electromagnetic fields exposure on plasma hormonal and inflammatory pathway biomarkers in male workers of a power plant.

PURPOSE: The potential health risks of electromagnetic fields (EMFs) have currently raised considerable public concerns. The aim of this study was to evaluate the effects of EMF exposure on levels of plasma hormonal and inflammatory pathway biomarkers in male workers of an electric power plant. METHODS: Seventy-seven male workers with high occupational EMF exposure and 77 male controls with low exposure, matched by age, were selected from a cross-sectional study. Moreover, high EMF exposure group was with walkie-talkies usage and exposed to power frequency EMF at the work places for a longer duration than control group. A questionnaire was applied to obtain relevant information, including sociodemographic characteristics, lifestyle factors, and EMF exposures. Plasma levels of testosterone, estradiol, melatonin, NF-κB, heat-shock protein (HSP) 70, HSP27, and TET1 were determined by an enzyme-linked immunosorbent assay. RESULTS: EMF exposure group had statistically significantly lower levels of testosterone (ß = -0.3 nmol/L, P = 0.015), testosterone/estradiol (T/E2) ratio (ß = -15.6, P = 0.037), and NF-κB (ß = -20.8 ng/L, P = 0.045) than control group. Moreover, joint effects between occupational EMF exposure and employment duration, mobile phone fees, years of mobile phone usage, and electric fees on levels of testosterone and T/E2 ratio were observed. Nevertheless, no statistically significant associations of EMF exposures with plasma estradiol, melatonin, HSP70, HSP27, and TET1 were found. CONCLUSIONS: The findings showed that chronic exposure to EMF could decrease male plasma testosterone and T/E2 ratio, and it might possibly affect reproductive functions in males. No significant associations of EMF exposure with inflammatory pathway biomarkers were found.

[Prognostic significance of indices of endogenous intoxication and of a monooxygenase system on the stages of surgical treatment in patients, suffering abdominal sepsis].

Some indices of endogenous intoxication and lymphocytic monooxygenase activity in the blood of patients, suffering abdominal sepsis (AS), were investigated, their prognostic significance was determined. In 28 patients the signs of AS were revealed, in 14 of a severe sepsis, in 11--of a septic shock. For peritoneal AS there were operated 37 patients, and for a pancreatogenic one--15. Relaparotomy "on demand" was performed in 12, and "the programmed" one--in 15 patients. A 30-days lethality in AS was 10.7%, in severe shock--28.6%, and in a septic one--63.6%. While AS occurrence a rising of metabolic activity of the monooxygenase system were registered in certain terms, and significant inhibition of this index--while severe state of the patients in a refractory shock occurrence. A safety correlational connections of indices in the blood and the lymphocytic monooxygenase system activity were determined in patients, who died.

Failure of pharmacogenetic-based dosing algorithms to identify older patients requiring low daily doses of warfarin.

INTRODUCTION: Warfarin doses vary greatly among patients and warfarin administration is accompanied by risk of bleeding. Genes responsible for its metabolism (CYP2C9) and effect on clotting (VKORC1) have been identified. It has been suggested that genotyping for variants in these genes can improve warfarin dosing and decrease bleeding complications. We evaluated performance of pharmacogenetic-based warfarin dosing estimation algorithms in old and very old patients. METHODS: Cross-sectional study of stable patients older than 65 years receiving warfarin with therapeutic International Normalized Ratio (INR) anticoagulation. Medical and laboratory data were reviewed and genotyping for CYP2C9 and VKORC1 performed. Warfarin dose estimates with and without genotype information were compared to clinically established therapeutic doses. RESULTS: Sixty-nine patients (32 men, 37 women; 41 nursing home residents; 28 senior care community residents) aged 81.4 ± 8.3 (mean ± S.D) years; ethnicity: Caucasian in 53, Asian in 10, Hispanic in 4, and African American in 2, received 3.3 ± 1.7 mg/d (range 0.7-9) warfarin achieving target INRs of 2.5 ± 0.2. Pharmacogenetic-based dose estimates (in combination with age, weight, height, smoking history, ethnicity/race, history of liver disease, selected co-medications such as amiodarone and enzyme inducers, baseline INR, clinical indication, and target INR), explained 50% of variability (P < .0001) compared with 12% without genetic data (P = .003). However, doses were overestimated in 15 of 16 patients requiring less than 2 mg/d (2.6 ± 0.9 mg/d compared with observed 1.5 ± 0.3 mg/d, P = .0001). Renal disease was a potential variable contributing to low warfarin requirements. DISCUSSION: The role of pharmacogenetic testing in the management of warfarin administration in patients is undergoing evaluation. Currently available pharmacogenetic- based warfarin dose estimation algorithms reduce variability in estimates for groups of older patients but consistently overestimate doses for older patients requiring the lowest doses of warfarin. CONCLUSIONS: Pharmacogenetic data add to our understanding of variability in warfarin dosing requirements but do not accurately identify older patients requiring the lowest warfarin doses. Therefore, the most prudent approach to warfarin therapy in older patients should include low initial doses in the absence of genotype variants associated with very low warfarin sensitivity and careful monitoring of INR responses.

Interactions of peptide amidation and copper: novel biomarkers and mechanisms of neural dysfunction.

Mammalian genomes encode only a small number of cuproenzymes. The many genes involved in coordinating copper uptake, distribution, storage and efflux make gene/nutrient interactions especially important for these cuproenzymes. Copper deficiency and copper excess both disrupt neural function. Using mice heterozygous for peptidylglycine alpha-amidating monooxygenase (PAM), a cuproenzyme essential for the synthesis of many neuropeptides, we identified alterations in anxiety-like behavior, thermoregulation and seizure sensitivity. Dietary copper supplementation reversed a subset of these deficits. Wildtype mice maintained on a marginally copper-deficient diet exhibited some of the same deficits observed in PAM(+/-) mice and displayed alterations in PAM metabolism. Altered copper homeostasis in PAM(+/-) mice suggested a role for PAM in the cell type specific regulation of copper metabolism. Physiological functions sensitive to genetic limitations of PAM that are reversed by supplemental copper and mimicked by copper deficiency may serve as indicators of marginal copper deficiency.

Fenugreek (Trigonella foenum graecum) seed polyphenols protect liver from alcohol toxicity: a role on hepatic detoxification system and apoptosis.

The present study investigates the hepatoprotective effect of fenugreek seed polyphenolic extract (FPEt) against ethanol-induced hepatic injury and apoptosis in rats. Chronic ethanol administration (6 g/kg/day x 60 days) caused liver damage that was manifested by the elevation of markers of liver dysfunction--aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), bilirubin and gamma-glutamyl transferase (GGT) in plasma and reduction in liver glycogen. The effects on alcohol metabolizing enzymes such as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) were studied and found to be altered in the alcohol-treated group. Ethanol administration resulted in adaptive induction of the activities of cytochrome p450 (cyt-p-450) and cytochrome-b5 (cyt-b5) and reduction in cytochrome-c-reductase (cyt-c-red) and glutathione-S-tranferase (GST), a phase II enzyme. Further, ethanol reduced the viability of isolated hepatocytes (ex vivo) as assessed by the trypan blue exclusion test and increased hepatocyte apoptosis as assessed by propidium iodide staining (PI). Treatment with FPEt restored the levels of markers of liver injury and mitigated the alterations in alcohol metabolizing and detoxification enzymes and the electron transport component cytochrome-c reductase. Increased hepatocyte viability and reduced apoptotic nuclei were observed in FPEt-treated rats. These findings demonstrate that FPEt acts as a protective agent against ethanol-induced abnormalities in the liver. The effects of FPEt are comparable with those of a known hepatoprotective agent, silymarin.

CYP2A6 genotype, phenotype, and the use of nicotine metabolites as biomarkers during ad libitum smoking.

CYP2A6 inactivates nicotine to cotinine and cotinine to 3-hydroxycotinine. We investigated which of plasma nicotine and metabolites were most related to CYP2A6 genotype and smoking levels. We assessed demographic and smoking histories in 152 Caucasian ad libitum smokers, measured breath carbon monoxide (CO) levels, and determined plasma nicotine, cotinine, and 3-hydroxycotinine by high-performance liquid chromatography and CYP2A6 genotypes by PCR. Cigarettes per day was most closely related to CO (r = 0.60, P < 0.001) followed by plasma cotinine (r = 0.53, P < 0.001), whereas plasma cotinine was most strongly correlated with CO levels (r = 0.74, P < 0.001), confirming that cotinine is a good indicator of smoking levels; this was not limited by CYP2A6 variants. 3-Hydroxycotinine/cotinine is reported to be a good marker of CYP2A6 activity, and we found that the 3-hydroxycotinine/(cotinine + nicotine) ratio was most correlated with CYP2A6 genotype (r = 0.38, P < 0.001). Inclusion of the CYP2A6*12A allele strengthened the correlation (r = 0.46, P < 0.001), suggesting that the identification of novel alleles will continue to improve this relationship. Nicotine metabolism is slower in smokers, and we have shown that CYP2A6 is reduced by nicotine treatment in monkeys. Here, we found that plasma nicotine levels were inversely correlated with CYP2A6 activity (3-hydroxycotinine/cotinine, r = -0.41, P < 0.001) among those without CYP2A6 variants, suggesting a reduction in metabolism with higher nicotine levels. Together, these findings (a) confirm the use of plasma cotinine and CO as indicators of Caucasians' smoking levels, and that this is not limited by CYP2A6 genetic variation; (b) indicate that 3-hydroxycotinine/cotinine and 3-hydroxycotinine/(cotinine + nicotine) are moderately good indicators of the CYP2A6 genotype; and (c) support that nicotine exposure may reduce its own metabolism.

Adrenocortical adenoma associated with inadequately treated congenital adrenal hyperplasia.

We report a 6 year-old boy with the simple virilizing form of 21-hydroxylase deficiency in whom an adrenal adenoma developed following 5 years of steroid treatment. Extremely high levels of basal serum 17alpha-hydroxyprogesterone as well as an exaggerated response of 17alpha-hydroxyprogesterone to adrenocorticotropic hormone confirmed congenital adrenal hyperplasia at 7 years of age. Initially elevated serum steroid levels were restrained by high dose hydrocortisone therapy, but he chronically tended to take inadequate doses of glucocorticoid. At 12 years of age an adenoma was found in the cortex of the hyperplastic right adrenal gland. The importance of early diagnosis and compliance with medication in the simple virilizing form of 21-hydroxylase deficiency is stressed.

Effects of whole deletion of CYP2A6 on nicotine metabolism in humans.

We investigated the effects of CYP2A6 genotypes on nicotine metabolism, focused from nicotine to cotinine and its additional 3'-hydroxylating resulted in trans-3'-hydroxycotinine formation. In the subjects genotyped by PCR-RFLP method, one cigarette smoking experiment was performed and urine samples were collected for 24 h. In all subjects who smoked, we detected nicotine, cotinine and trans-3'-hydroxycotinine in urine by GC-MS analysis. In whole deletion of CYP2A6, urinary excretion amounts of cotinine and trans-3'-hydroxycotinine were significantly smaller than those in the wild-type of CYP2A6*1. A lack of CYP2A6 reduces the formation of cotinine and trans-3'-hydroxycotinine, but not entirely reduces the trans-3'-hydroxycotinine formation. Unknown cotinine 3'-hydroxylating activity except CYP2A6 are suspected in humans.

Environmental and genetic determinants of aflatoxin-albumin adducts in the Gambia.

Aflatoxins together with chronic hepatitis B virus (HBV) infection contribute to the high incidence of hepatocellular carcinoma in developing countries. An understanding of the mechanism of interaction between these factors would provide a strong rationale for developing effective prevention strategies. In this study in The Gambia we examined the effect of environmental (place of residence and timing of sample collection) and host factors (age, sex, HBV status and interindividual variations in carcinogen metabolising enzymes) in determining blood aflatoxin-albumin adduct levels in 357 individuals of whom 181 were chronic HBV carriers. Samples were analysed for aflatoxin-albumin adducts, HBV status and genotypes of glutathione S-transferase (GST) M1, GSTT1, GSTP1 and epoxide hydrolase (EPXH). Urine samples were analysed for 6beta-hydroxycortisol:cortisol ratio as a marker of cytochrome P450 (CYP) 3A4 activity. Adduct levels were significantly higher in subjects resident in rural [geometric mean adduct level 34.9 pg aflatoxin B1-lysine equivalent (28.5-42.8; 95%CI)/mg albumin] than in periurban areas [22.2 pg (14.9-33.4)/mg] and were approximately twice as high in the dry season [mid-February to March; 83.2 pg (53.3-130.8)/mg] than the wet [July to August; 34.9 pg (28.5-42.8)/mg]. In contrast, HBV status, CYP3A4 phenotype, GSTT1, GSTP1 and EPXH genotypes were not associated with aflatoxin-albumin adduct level. However, mean adduct levels were significantly higher in non-HBV infected subjects with GSTM1 null genotype. The main factors which affect aflatoxin-albumin adduct levels in this population are environmental, notably place of residence and timing of sample collection. This study further emphasises the priority to reduce aflatoxin exposure in these communities by primary prevention measures.

Effects of chronic pulmonary overcirculation on pulmonary vasomotor tone.

BACKGROUND: A model of shunt-induced pulmonary hypertension was used to study the effects of pulmonary overcirculation on endothelial nitric oxide synthase (eNOS) and cytochrome P450-4A (cP450-4A) vasodilatory mechanisms and related hemodynamic responses. METHODS: An aortopulmonary shunt was constructed in 6-week-old piglets (n = 7, sham-operated controls n = 8). Hemodynamic measurements were made 4 weeks later under serial experimental conditions: baseline (fractional concentration of oxygen, 0.4); inhaled nitric oxide, 25 ppm (INO); hypoxia (fractional concentration of oxygen, 0.14); hypoxia + INO; N(omega)-nitro-L-arginine methylester (L-NAME 30 mg/kg intravenously, competitive NOS inhibitor); and L-NAME + INO. Lung protein levels of eNOS and cP450-4A and NOS activity were compared between groups. RESULTS: Shunted animals had a higher baseline pulmonary artery pressure (p < 0.05). L-NAME resulted in a greater increase in pulmonary vascular resistance in shunted animals (150% +/- 26% shunt versus 69% +/- 14% control; p = 0.01). The INO administered during baseline conditions decreased pulmonary vascular resistance only in control animals (p < 0.05). Protein levels of eNOS and NOS activity were similar in both groups; however, cP450-4A protein levels were decreased in the shunted group (p = 0.02). CONCLUSIONS: The NO production was preserved in shunted animals but they demonstrated greater vasodilatory dependence on NO, evidenced by an exaggerated increase in pulmonary vascular resistance after NOS inhibition. Loss of the cP450-4A vasodilatory system may be the driving force for NO dependency in the shunted pulmonary circulation.

Role of intestinal P-glycoprotein (mdr1) in interpatient variation in the oral bioavailability of cyclosporine.

Interpatient differences in the oral clearance of cyclosporine (INN, ciclosporin) have been partially attributed to variation in the activity of a single liver enzyme termed CYP3A4. Recently it has been shown that small bowel also contains CYP3A4, as well as P-glycoprotein, a protein able to transport cyclosporine. To assess the importance of these intestinal proteins, the oral pharmacokinetics of cyclosporine were measured in 25 kidney transplant recipients who each had their liver CYP3A4 activity quantitated by the intravenous [14C-N-methyl]-erythromycin breath test and who underwent small bowel biopsy for measurement of CYP3A4 and P-glycoprotein. Forward multiple regression revealed that 56% (i.e., r2 = 0.56) and 17% of the variability in apparent oral clearance [log (dose/area under the curve)] were accounted for by variation in liver CYP3A4 activity (p < 0.0001) and intestinal P-glycoprotein concentration (p = 0.0059), respectively. For peak blood concentration, liver CYP3A4 activity accounted for 32% (p = 0.0002) and P-glycoprotein accounted for an additional 30% (p = 0.0024) of the variability. Intestinal levels of CYP3A4, which varied tenfold, did not appear to influence any cyclosporine pharmacokinetic parameter examined. We conclude that intestinal P-glycoprotein plays a significant role in the first-pass elimination of cyclosporine, presumably by being a rate-limiting step in absorption. Drug interactions with cyclosporine previously ascribed to intestinal CYP3A4 may instead be mediated by interactions with intestinal P-glycoprotein.