Expression of genes involved in xenobiotic metabolism and transport in end-stage liver disease: up-regulation of ABCC4 and CYP1B1

BackgroundExpression of drug-metabolizing enzymes and drug transporters in liver is mainly regulated by a system of nuclear receptors. The aim of the current study was to investigate the expression of nuclear receptors, as well as these enzymes and transporters, in liver samples from patients suffering from end-stage liver disease of various etiologies (HCV infection, alcohol liver disease, and primary sclerosis cholangitis).MethodsGene expression was measured using quantitative real-time PCR with surgical specimens from livers of patients with end-stage liver disease, and non-tumoral liver tissue that served as control.ResultsOur study confirmed that the expression of most phase I enzymes is suppressed in end-stage liver disease, and is correlated with a decrease in NR1I2 and NR1I3, the main regulators of xenobiotic metabolism. While mRNA levels of phase II enzymes were generally unchanged, some ABC transporters were up-regulated. The most spectacular increases in expression were observed with ABCC4 (MRP4) – at the mRNA level, and CYP1B1 – at both the mRNA and protein levels. We also demonstrated that IL-6 can induce CYP1B1 expression independently of CYP1A1, in a human hepatocellular liver carcinoma cell line.ConclusionsAs CYP1B1 is an enzyme which converts various substrates into carcinogenous metabolites, its overexpression in liver may be one of the factors increasing the risk of hepatic cancers inpatients with liver disease. CYP1A1 and CYP1B1 are often referred to as model AHR target genes, but CYP1A1 was down-regulated in diseased liver samples. This points to the existence of differences in regulation of these two genes.     

Oxidative modulation of marcaine and lekoptin in H9C2 rat myoblasts

Aim： The cytotoxicity of marcaine was estimated in combination with a calcium channel blocker. In addition, the influence of marcaine and marcaine plus lekoptin on a model system using the H9C2 cardiac cell line was investigated. Methods： Cells were incubated for five hours with marcaine, lekoptin, or with both drugs simultaneously. Apoptotic cells were detected using the TUNEL assay and the alkaline comet assay. Mitochondrial cell function after drug uptake was examined using the MTT assay. The concentration of MDA （malondialdehyde） - the final product of fatty-acid peroxidation, was quantified spectrophotometrically. The expression of glutathione S-transferase n （GST-rI） was detected by immunofluorescence （IF） and Western blotting （WB） and inducible nitric oxide synthase （iNOS） was assessed by immuno-cytochemical staining （ABC）. Results： Incubation with marcaine resulted in the highest number of apoptotic cells. After incubation with both marcaine and lekoptin, moderate damage to cells （54.2%＋1.775% of DNA destruction） was observed. The highest levels of iNOS and GST-n expression were observed in cells treated with marcaine and marcaine plus lekoptin. The characteristic nuclear GST-n expression was observed in cells treated with both drugs. Conclusion： Lekoptin stimulated cells to proliferate. Marcaine caused membrane damage and ultimately cell death.     

Effects of direct stenting on epicardial and myocardial perfusion in patients with acute ST segment elevation myocardial infarction

BACKGROUND: Results of studies comparing direct stenting (DS) with conventional stenting (CS) after balloon predilatation in patients with acute myocardial infarction (MI) have been reported in the past, however they are conflicting. There are only few randomised studies that aim to answer whether DS improves epicardial and myocardial patency. AIM: To assess the effects of DS on epicardial and myocardial patency in patients with acute MI. METHODS: Consecutive patients with acute MI were randomised either to DS or CS strategy. Clinical exclusion criteria were as follows: clinical and electrocardiographic features of reperfusion, pulmonary oedema, cardiogenic shock, contradictions to coronarography, allergy to aspirin, ticlopidine, clopidogrel, heparin and stainless steel. Angiographic exclusion criteria were as follows: lesion <50% with correct patency in the infarct-related artery (IRA), lesion in the left main coronary artery, previously performed percutaneous coronary intervention in the target vessel, diameter of the IRA <2 mm or >4 mm. We assessed epicardial patency according to the TIMI (thrombolysis in myocardial infarction) scale and myocardial patency according to the TMPG (TIMI myocardial perfusion grade) scale. In addition, we analysed ST segment resolution in 12-lead electrocardiography (ECG). The ECG was performed before and 30 minutes after PCI. RESULTS: We analysed 300 consecutive patients with acute ST segment elevation MI. After exclusion of patients not suitable for the study design, the DS group comprised 110 patients and the CS group - 107 patients. Clinical and angiographic results were similar in both groups. Initial TIMI 0 (48.2% vs. 43.0%), initial TIMI 3 (31.8% vs. 28.0%), initial TMPG 0-1 (77.3% vs. 78.5%), final TIMI 3 (95.5% vs. 93.5%) and final TMPG 2-3 (68.2% vs. 60.8%) were similar in the DS and CS groups, respectively (p=NS). The incidence of no-reflow phenomenon was comparable in both groups (4.5% vs. 6.5%, NS). The inclusive rate of no-reflow phenomenon plus worsening patency in the IRA were 6.4% vs. 10.3% in the DS and CS groups respectively. The ST segment resolution > or = 50% was 58.1% in the DS group and 56.1% in the CS group (NS). CONCLUSIONS: Direct stenting does not significantly improve epicardial and myocardial patency in an unselected group of patients with acute ST segment elevation MI.     

Relationship between blood glucose on admission and prognosis in patients with acute myocardial infarction treated with percutaneous coronary intervention

BACKGROUND: Diabetes mellitus in patients with myocardial infarction affects in-hospital and late mortality. It has been shown that the glucose level on admission can also affect prognosis. This conclusion was based on an analysis performed on a heterogeneous group of patients, treated not only with percutaneous coronary intervention (PCI) but also with fibrinolysis. Moreover, the threshold values hyperglycaemia for the diagnosis of were also variable. AIM: To assess whether glucose level on admission affects in-hospital and one-year prognosis in patients with ST-segment elevation myocardial infarction (STEMI) treated with PCI. METHODS: Consecutive patients with STEMI treated with PCI were included in the analysis. Patients with STEMI complicated by cardiogenic shock were also included. Three groups according to the glucose level on admission were analysed: group I - <7.8 mmol/l (140 mg/dl), group II - 7.8-11.1 mmol/l (140-200 mg/dl), and group III - > or = 11.1 mmol/l (200 mg/dl). RESULTS: The incidence of diabetes mellitus in the total group (1027 patients) was 26.1%, and of cardiogenic shock - 9.2%. Group I consisted of 472 patients, group II - 307 patients, and group III - 248 patients. Compared with normoglycaemic patients, those with elevated glucose level were older, more often female, had more often hypertension, diabetes mellitus, cardiogenic shock, were more often treated with fibrinolysis before PCI but were less often smokers. Multivessel disease and initial patency of the infarct-related artery (TIMI 0-1) were more often observed in patients with higher glucose level. A trend towards a higher incidence of reocclusion was also more often present in patients with increased glucose level. Moreover, mean creatine kinase concentration was the highest and the left ventricular ejection fraction was the lowest in group III. During the in-hospital stay, the complication rate was as follows: stroke (1.1% vs. 1.3% vs. 4.4%), and mortality (2.8 vs. 4.9 vs. 13.3%) in groups I, II, and III, respectively. The same tendency was observed during the one-year follow-up period: stroke (1.3 vs. 2.9 vs. 6.9%), mortality (6.4 vs. 9.1 vs. 22.6%). The 1 mmol/l (18 mg/dl) increase of the baseline glucose level among various risk factors was an independent prognostic factor of higher -year mortality (HR=1.06; 95% CI 1.02-1.09). Diabetes mellitus did not affect prognosis among patients included in the analysis. CONCLUSION: Elevated glucose level on admission is associated with adverse prognosis in patients with STEMI treated with PCI.     

Persistent platelet activation is related to very early cardiovascular events in patients with acute coronary syndromes

INTRODUCTION: Persistent platelet function while on antiplatelet therapy affects outcomes in patients with acute coronary syndromes (ACS). AIM: To evaluate whether platelet reactivity measured by collagen-epinephrine (CEPI) or collagen-ADP (CADP) closure times (CT) with Platelet Function Analyzer 100 (PFA-100) is related to very early, in-hospital cardiovascular events in patients with ACS. METHODS: The study included 91 patients with ACS undergoing percutaneous coronary intervention (PCI) with stent implantation who were treated with aspirin and clopidogrel. Patients were stratified in accordance with both CEPI-CT (<190 s or >190 s), reflecting aspirin resistance, and our own cut-off point for CADP-CT measured at a mean of 6 days after admission. In-hospital events included re-infarction, cardiac arrest, recurrent angina, severe arrythmias, pulmonary oedema and cardiogenic shock. RESULTS: Patients were divided into 4 study groups: group 1 with CADP-CT <104 s (n=10, 11.0%), group 2 with CEPI-CT <190 s (n=10, 11.0%), group 3 with CADP-CT <104 s and CEPI-CT <190 s (n=9, 9.9%) and a control group with both CT values above the cut-off limits (n=62, 68.1%). The baseline clinical characteristics and received treatment of each subgroup were similar. A test for a trend between controls, group 1 or 2 and group 3 disclosed statistical significance (p <0.001). When analysed separately, only patients from group 3 had a higher incidence of negative outcomes compared to controls (p <0.005; relative risk RR - 9.0; 95% CI 2.4-33.9). CONCLUSIONS: Enhanced platelet function after PCI when measured under high shear rates by both PFA-100 cartridges is independently associated with the most unfavourable in-hospital clinical outcome.     

Identification of a novel circulating recombinant form (CRF) 36_cpx in Cameroon that combines two CRFs (01_AE and 02_AG) with ancestral lineages of subtypes A and G

An array of CRFs have been identified in Cameroon, the most notable being CRF02_AG. HIV-1 in the East Province of Cameroon is particularly diverse: in a recent study, we found a high proportion of unique recombinant forms (URFs). Herein we describe the analysis of the full-length sequences of two of these URFs, which, after preliminary analysis of gag, pol, and env fragments, appeared to be a novel CRF. This novel strain, CRF36_cpx, contains fragments that can be assigned to the CRF01_AE, CRF02_AG, and subtype A and G radiations. Forty percent of the genome can be classified as CRF02_AG, including regions in gag, pol, env, and the accessory genes. Twenty-seven percent is CRF01_AE, comprising the majority of gag, the beginning of env, and the end of env into the 3' LTR. Twenty percent of the genome can be assigned to subtype A, with segments in pol and env. The remaining 13% of the sequence is classifiable as subtype G, in pol and vpu. The subtype A and G lineages formed by the CRF36_cpx sequences are unique and appear ancestral in nature. CRF36_cpx is both the first to combine more than one CRF and the first to include fragments of CRF02_AG. The ancestral sequences present in CRF36_cpx represent a link to extinct strains, and, potentially, insight into the evolution of HIV-1.     

Photodynamic therapy with di-l-arginine protoporphyrinate on WiDr human colon adenocarcinoma xenografts in athymic nude mice

The fluorescence kinetics of a new photosensitizer for photodynamic therapy, di-l-arginine protoporphyrinate (PP(Arg)₂), was studied in the skin of healthy mice. Furthermore, induction of necrosis in WiDr human colon adenocarcinoma xenografts in athymic nude mice was studied after photodynamic therapy (PDT) with PP(Arg)₂.After intravenous administration of PP(Arg)₂ maximal fluorescence was reached after 72 h in normal mouse skin. Complete elimination of the drug from the mouse skin was not found even after 32 days.Exposure of WiDr tumours in mice to red light (λ = 632 nm, fluence 150 J/cm², fluence rate 250 mW/cm²) 24 and 72 h after intravenous administration of 10 mg/kg of PP(Arg)₂ caused extensive tumour necrosis. Epidermal damage and infiltration of inflammatory cells was seen 24 h after light exposure but not after 72 h.     

Methyl-CpG binding column-based identification of nine genes hypermethylated in colorectal cancer

DNA methylation is an epigenetic event that plays a role in gene expression regulation. Alterations in DNA methylation contribute to cancer development and progression. The aim of this study was to identify gene promoters aberrantly methylated in colorectal tumor tissue in comparison to normal colonic mucosa. Analyses were performed on two pooled DNA samples: from normal and cancerous tissue obtained from CRC patients. DNA was fractionated according to methylation degree with the use of affinity column containing methyl-CpG binding domain. To identify novel hypermethylated gene promoters, methylated DNA from normal and from cancerous tissues were analyzed with the use of promoter microarrays. We identified nine novel genes hypermethylated in colorectal cancer. The frequency of their promoter methylation was assessed in the larger group of patients (n = 77): KCNK12 (methylated in 41% of CRC patients), GPR101 (40%), CDH2 (45%), BARX1 (56%), CNTFR (22%), SYT6 (64%), SMO (21%), EPHA5 (43%), and GSPT2 (21%). The results of gene expression level analysis suggest the role of promoter methylation in downregulation of six out of nine genes examined. We did not find correlation between gene methylation and age, gender, tumor grade or stage. Importantly, in stage IV CRC methylation of GPR101 correlated with longer time to progression (P = 0.0042; HR = 2.5468; 95% CI 1.5391-10.0708).     

Association of the MDR1 (ABCB1) gene 3435C> T polymorphism with male infertility

Infertility is a common problem affecting one in six couples, and in 30% of infertile couples, the male factor is a major cause due to defective sperm quality. P-glycoprotein (P-gp), a product of the MDR1 (ABCB1) gene, may be a link between genetic and environmental factors contributing to the development of male infertility because pesticides (P-gp substrates) are well established factors of male infertility. The aim of the present study was to examine the effect of the MDR1 gene 3435C>T polymorphism on male infertility. In total, 162 male patients undergoing semen analysis due to initial infertility workup were included in the study. The control group consisted of 191 healthy males with proven fertility. MDR1 3435C>T genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Assessment of MDR1 genotypes among the infertile men showed that 17.9% of subjects were carriers of the CC genotype, 58.0% were CT and 24.1% were TT. Among fertile men, 30.4% of subjects were characterised by the CC genotype, 49.7% were CT and 19.9% were TT. In addition, the frequency of carriers of at least one T allele (i.e., CT and TT genotypes) among infertile and fertile subjects was 82.1% and 69.6%, respectively. The risk of infertility was significantly elevated by two-fold in individuals carrying at least one T allele (CT and TT genotypes: p = 0.009, OR = 2.00, 95% CI: 1.20–3.32). Furthermore, this elevated risk was still found when considering each of the CT and TT genotypes alone (TT genotype: p = 0.027, OR = 2.05, 95% CI: 1.09–3.86; CT genotype: p = 0.013, OR = 1.98, 95% CI: 1.16–3.36). This preliminary report suggests that P-gp may play some role in male infertility, mediating detrimental effects of environmental factors.