Initiation but no execution - modulation of peripheral blood lymphocyte apoptosis in rheumatoid arthritis - a potential role for heat shock protein 70

<p>Abstract</p> <p>Background</p> <p>Rheumatoid arthritis (RA) is a chronic autoimmune disease, which causes synovial damage. Persistence of lymphocyte infiltrates in the rheumatoid synovium has been attributed to abnormal apoptosis. While not comprehensively investigated, perturbations in peripheral blood lymphocyte (PBL) apoptosis may also be involved in perpetuation of autoimmune processes in RA.</p> <p>Methods</p> <p>We investigated total, CD4+ and CD19+ PBL apoptosis in our study cohort by monitoring the translocation of phosphatidylserine using the Annexin-V assay. To examine the role of death receptor mediated apoptosis as well as activation-induced-cell-death (AICD), PBLs were labeled with CD95/Fas and CD69 markers and enumerated by flow cytometry. Proteolytic activity of initiator and executioner caspases was determined by luminometry. DNA fragmentation assays were used to examine whether apoptotic signals were transduced to the nucleus. Quantitative PCR arrays were used to investigate apoptotic pathways associated with RA-PBLs. Since heat-shock-protein-70 (HSP70) is an inducible protein which modulates apoptotic signals, we determined HSP70 levels by intra-cellular flow cytometry and western blots.</p> <p>Results</p> <p>The RA-PBLs showed signs of elevated apoptosis whilst in circulation. These include increases in the loss of plasma membrane asymmetry, indicated by increased externalization of phosphatidylserine (especially in B-lymphocytes). RA-PBLs showed a bias to CD95/Fas mediated apoptotic pathways, but low levels of the CD69 marker suggested that this was not associated with immune activation. Although downstream markers of apoptosis such as caspase-3/7 activity, were increased, no DNA fragmentation was observed in RA-PBLs. Interestingly, elevated levels of apoptosis did not correlate with absolute lymphocyte counts in RA patients. Levels of HSP70 were highly elevated in RA-PBLs compared to controls.</p> <p>Conclusion</p> <p>The results suggest that while apoptosis may be initiated in RA-PBLs, they may lack commitment to fully executing the apoptotic program. This may be related to inhibition on apoptotic transduction by HSP70. This study provides evidence that abnormalities in RA-PBLs apoptosis may occur whilst still in circulation and may contribute to pathogenesis of the disease.</p

An investigation into the biochemical, molecular and epigenetic effects of fumonisin B1 in liver (HEPG2) cells.

Ph. D. University of KwaZulu-Natal, Durban 2014.Fumonisins are carcinogenic mycotoxins that occur world wide in maize and maizebased products intended for human consumption. Consumption of fumonisincontaminated maize as a staple diet has been associated with oesophageal and liver cancer in South Africa and China. Fumonisin B1 (FB1) inhibits sphingolipid biosynthesis and has been implicated in cancer promoting activity in animals and humans. FB1 disrupts DNA methylation and induces chromatin modifications in human hepatoma (HepG2) cells. In this study FB1 (IC50=200μM) altered liver enzyme expression of DNA methyltransferases and demethylases. DNA methyltransferase activities of DNMT1, 3a and 3b were significantly decreased, whilst both DNA methylase (MBD2) activity and expression was significantly up-regulated resulting in global DNA hypomethylation. In addition the histone demethylases, KDM5B and KDM5C, expression was increased. FACS data confirmed FB1 significantly increased global DNA hypomethylation – a process that causes chromatin instability. Next the effect of FB1 on miRNA expression was evaluated; FB1 significantly down-regulated (11 fold) expression of miR-27b. MiR-27b modulates expression of human cytochrome P450 (CYP1B1) that catalyzes the metabolic activation of many procarcinogens. In order to directly assess the effect of miR-27b on CYP1B1 mRNA levels, liver cells were transfected with the mimic to miR-27b. CYP1B1 mRNA and protein expression was significantly up-regulated by 1.8- fold and 2.6- fold respectively. CYP1B1 is post-transcriptionally regulated by miR-27b suggesting that FB1- induced modulation of miR-27b in hepatic cells may be an additional mode of hepatic neoplastic transformation. Finally, the effect of FB1 on the apoptotic pathway in HepG2 cells was investigated using an mRNA expression array panel of pro- and anti- apoptotic molecules. FB1 significantly increased an AIP family member - BIRC 8/ILP-2 (8-fold) in an apoptosis array. In addition, ILP2 protein expression was increased (2.3-fold) with a corresponding decrease in Smac/DIABLO protein levels (1.7-fold). Further analysis showed an FB1 (0μM, 50μM, 100μM, 200μM) dosedependent increase in BIRC-8/ILP-2 mRNA and protein expression in HepG2 cells. This data suggests that FB1 modulates apoptosis in a complex dose-dependent regulation of pro- and anti-apoptotic molecules – and it is not a matter of simply switching on or off. In conclusion, the data shows that FB1 possess epigenetic properties by inducing global DNA hypomethylation, modulating miRNA expression, and increasing expression of the AIP protein family (BIRC8/ILP-2) that may lead to liver tumourigenesis

GST polymorphisms and early-onset coronary artery disease in young South African Indians

Background. Glutathione S-transferases (GSTs) detoxify environmental agents which influence the onset and progression of disease. Dysfunctional detoxification enzymes are responsible for prolonged exposure to reactive molecules and can contribute to endothelial damage, an underlying factor in coronary artery disease (CAD).Objectives. We aimed to assess 2 common polymorphic variant isoforms in GSTM1 and GSTP1 of GST in young CAD patients.Methods. All patients (N=102) were South Africans of Indian ancestry, a population associated with high CAD risk. A corresponding age-, sex- and race-matched control group (N=100) was also recruited. Frequency of the GSTM1 +/0 (v. +/0 and 0/0) and GSTP1 A105/G105 (v. wild-type A105/A105) genotypes was assessed by differential polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP), respectively.Results. The GSTM1 0/0 and GSTP1 A105/A105 genotypes occurred at higher frequencies in CAD patients compared with the control group (36% v. 18% and 65% v. 48%, respectively). A significant association with CAD was observed in GSTM1 0/0 (odds ratio (OR)=2.593; 95% confidence interval (CI) 1.353 - 4.971; p=0.0043) and GSTP1 A105/A105 OR=0.6011; 95% CI 0.3803 - 0.9503; p=0.0377). We found a significant association between smoking and CAD; the presence of either of the respective genotypes together with smoking increased the CAD risk (GSTP1 A105 relative risk (RR)=1.382; 95% CI 0.958 - 1.994; p=0.0987 and GSTM1 null RR=1.725; 95% CI 1.044 - 2.851; p=0.0221).Conclusion. Our findings support the association of genotypes GSTM1 0/0 and GSTP1 A105/A105 and smoking with CAD.S Afr Med J 2012;102(7):627-630

Mitochondrial and oxidative stress response in HepG2 cells following acute and prolonged exposure to antiretroviral drugs

Chronic HIV treatment with antiretroviral drugs has been associated with adverse health outcomes. Mitochondrial toxicity exhibited by nucleoside reverse transcriptase inhibitors (NRTIs) is pinpointed as a molecular mechanism of toxicity. This study evaluated the effect of NRTIs: Zidovudine (AZT, 7.1 μM), Stavudine (d4T, 4 μM) and Tenofovir (TFV, 1.2 μM), on mitochondrial (mt) stress response, mtDNA integrity and oxidative stress response in human hepatoma cells at 24 and 120 h. Markers for mt function, mt biogenesis, oxidative stress parameters, and antioxidant response were evaluated by spectrophotometry, luminometry, flow cytometry, qPCR and western blots. We found that AZT and d4T reduced mtDNA integrity (120 h, AZT: 76.1%; d4T:36.1%, P < 0.05) and remained unchanged with TFV. All three NRTIs, however, reduced ATP levels (AZT: 38%; d4T: 56.4%; TFV: 27.4%, P = 0.01) and mt membrane potential at 120 h (P < 0.005). Oxidative damage and reactive oxygen species (ROS) were increased by TFV and AZT at 24 h, and by d4T at 120 h (P < 0.05). Antioxidant response molecules and mt biogenesis markers were elevated by all NRTIs, with TFV causing the most significant increase (P < 0.05). Data from this study suggest that AZT, d4T and TFV alter mt function. TFV, however, achieves this independently of mtDNA depletion. Furthermore, AZT exerts toxicity soon after exposure as noted from changes at 24 h and d4T exerts greater toxicity over prolonged exposure (120 h).National Research Foundation Innovation Doctoral Scholarship ; Grant number : 84538 ; Grant sponsor : University of KwaZulu Natal, College of Health Sciences Masters and Doctoral Research Scholarship.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1097-46442016-09-30hb2016Physiolog

Patulin triggers NRF2-mediated survival mechanisms in kidney cells

Patulin (PAT), a mycotoxin contaminant of apples and apple products, has been implicated in nephrotoxicity. PAT depletes glutathione (GSH) and elevates reactive oxygen species (ROS). The antioxidant (AO) response is activated by Nuclear erythroid 2-related factor (NRF2) and enhanced by Silent information regulator 3 (SIRT3). The effects of PAT on these molecules have yet to be examined. We investigated the effects of PAT on AO response survival pathways in human embryonic kidney cells (HEK293). PAT cytotoxicity on HEK293 cells was evaluated (MTT assay; 24 h; [0–100 μM]) to determine an IC50. GSH levels were measured using luminometry. Intracellular ROS was evaluated by flow cytometry. Protein expression of Keap1, NRF2, SIRT3 and PGC-1α was quantified by western blotting and gene expression of SOD2, CAT and GPx was evaluated by qPCR. PAT caused a dose dependent decrease in HEK293 cell viability and a significant increase in levels of intracellular ROS (p = 0.0006). A significant increase in protein expression (p = 0.029) was observed. PAT increased gene expression of SOD2 and CAT (p = 0.0043), however, gene expression of GPx was significantly reduced (p = 0.0043). These results show the up-regulation of NRF2 mediated AO mechanisms in response to PAT toxicity.National Research Foundation (Grant UID: 90102), and the UKZN College of Health Sciencehttp://www.elsevier.com/locate/toxicon2016-06-01hb201

The phytoalexin resveratrol ameliorates ochratoxin a toxicity in human embryonic kidney (HEK293) cells

Ochratoxin A (OTA) is a nephrotoxic mycotoxin produced by Aspergillus and Penicillium fungi. It contaminates human and animal food products, and chronic exposure is associated with renal fibrosis in humans (Balkan endemic nephropathy). Resveratrol, a phytoalexin, possesses anti-cancer and antioxidant properties. We investigated the mechanism of cellular oxidative stress induced by OTA, and the effect of resveratrol in human embryonic kidney (HEK293) cells over 24 and 48 h. Cells were exposed to OTA [IC50¼1.5 mM (24 h) and 9.4 mM (48 h) determined using MTT assay] and 25mM resveratrol. Glutathione was quantified by luminometry and gene expression of Nrf2 and OGG1 was determined by qPCR. Protein expression of Nrf2, LonP1, SIRT3, and pSIRT1 was assessed by Western blot, DNA damage (comet assay), and intracellular reactive oxygen species (flow cytometry). At 24 h, resveratrol increased mRNA expression of the DNA repair enzyme, OGG1 (P<0.05), whereas OTA and OTAþresveratrol significantly decreased OGG1 expression (P<0.05). OGG1 expression increased during 48-h exposure to resveratrol and OTAþresveratrol (P<0.05). Comet tail lengths doubled in 48-h OTA-treated cells, whereas at both time periods, OTAþresveratrol yielded shorter comet tails (P<0.0001). During 24- and 48-h exposure, OTA, resveratrol, and OTAþresveratrol significantly decreased mRNA expression of Nrf2 (P<0.05). Luminometry analysis of GSH revealed an increase by OTAþresveratrol for 24 and 48 h (P<0.05 and P<0.001, respectively). Western blot analysis showed decreased Nrf2 protein expression during 24-h exposure, but increased Nrf2 expression during 48 h. LonP1 protein expression increased during 24-h exposure to OTA (P<0.05) and OTAþresveratrol (P<0.0011) and during 48-h exposure to resveratrol (P<0.0005).http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1097-46442016-12-31hb201

Association of -308 TNF-alpha promoter polymorphism with viral load and CD4 T-helper cell apoptosis in HIV-1 infected black South Africans

Objective. To determine whether the -308 TNF-α promoter polymorphism is associated with markers of HIV progression in the South African population. Methods. Polymerase chain reaction-restriction fragment length polymorphism was used to detect the -308 TNF-α polymorphism in 75 patients and 76 healthy controls. Serum TNF-α concentrations were measured using ELISA in each cohort. CD4+ T cell apoptosis and HIV-1 RNA viral load were determined using Annexin-V-FITC assay and Nuclisens Easy Q HIV-1 assay respectively. CD4 + T cell counts were measured flow cytometrically. Results. The frequency of -308 G allele was similar in the HIV-1 and control cohorts. The -308GG genotype was associated with lower TNF-α concentrations and markers of increased HIV progression indicated by higher TH lymphocyte apoptosis, lower TH lymphocyte count and higher plasma viral load, irrespective of treatment. Conclusion. The presence of the TNF-α -308 G allele in HIV-1 patients may be associated with increased risk of HIV-1 progression. Further research is required to investigate the nature of this association. S Afr J HIV Med 2012;13(2):72-77