Savania Nagiah

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). J. Cell. Biochem. 116: 1939–1946, 2015.

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 [IC50 = 1.5 μM (24 h) and 9.4 μM (48 h) determined using MTT assay] and 25 μM 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 OTA+resveratrol significantly decreased OGG1 expression (P < 0.05). OGG1 expression increased during 48‐h exposure to resveratrol and OTA+resveratrol (P < 0.05). Comet tail lengths doubled in 48‐h OTA‐treated cells, whereas at both time periods, OTA+resveratrol yielded shorter comet tails (P < 0.0001). During 24‐ and 48‐h exposure, OTA, resveratrol, and OTA+resveratrol significantly decreased mRNA expression of Nrf2 (P < 0.05). Luminometry analysis of GSH revealed an increase by OTA+resveratrol 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 OTA+resveratrol (P < 0.0011) and during 48‐h exposure to resveratrol (P < 0.0005). J. Cell. Biochem. 116: 2947–2955, 2015.

Oxidative stress and air pollution exposure during pregnancy A molecular assessment

Chronic air pollution exposure during pregnancy can cause oxidative stress leading to adverse birth outcomes. The aim of this study was to assess and compare oxidative stress response in peripheral lymphocytes isolated from pregnant women from a highly industrialized locale (south Durban (SD); n = 50) and a control with lower air pollutant levels (north Durban (ND); n = 50). Oxidative stress response was measured by quantifying malondialdehyde (MDA) levels and a SuperArray gene panel. Mitochondrial function (adenosine triphosphate (ATP) levels and mitochondrial depolarization), DNA integrity (comet assay and mitochondrial DNA (mtDNA) viability) and DNA repair (OGG1) were assessed. Antioxidant response was assessed by quantification of glutathione (GSH) and SOD2, nuclear factor erythroid 2-related factor 2 (Nrf2) and uncoupling protein 2 (UCP2) protein and messenger RNA (mRNA) expression. Levels of MDA (p = 0.9), mitochondrial depolarization (p = 0.88), ATP (1.89-fold), SOD2 (1.23-fold) and UCP2 (1.58-fold) gene expression were elevated in the SD group with significantly higher UCP2 protein levels (p = 0.05) and longer comet tail length (p = 0.0004). The expression of Nrf2 protein (p = 0.03) and mRNA levels (−1.37-fold), GSH concentration (p < 0.0001), mtDNA amplification (−2.04-fold) and OGG1 mRNA (−2.78-fold) activity were decreased in the SD group. Of the 84 oxidative stress-related genes evaluated, 26 were differentially regulated. Pregnant women exposed to higher air pollutant levels showed increased markers for oxidative stress and compromised DNA integrity and repair.

Fusaric acid induces oxidative stress and apoptosis in human cancerous oesophageal SNO cells

ABSTRACT Oesophageal cancer (OC) is a global problem incrementally incident among black South African males. The high incidence of OC may be due to the consumption of corn as a staple, often contaminated with mycotoxins. Fusaric acid (FA), a neglected mycotoxin, is known to disrupt mitochondrial energy metabolism, chelates divalent metal cations and induces cell death in plants. This study investigated FA‐induced cytotoxicity and apoptotic induction in the SNO OC cell line. Cells were treated with FA (IC50 = 78.81 &mgr;g/mL; 24 h; MTT assay) and assayed for oxidative stress and membrane damage (TBARS, LDH cytotoxicity and glutathione), apoptotic induction (ATP levels, caspase‐8, ‐9, ‐3/7 activities) (Luminometry), single strand DNA and nuclear fragmentation (Comet and Hoechst assay). Additionally, relative expression of pro‐ and anti‐apoptotic proteins were determined (Western Blotting). Significant antioxidant depletion was consistent with a concomitant increase in ROS‐induced lipid peroxidation and extracellular LDH levels. FA induced apoptosis by significantly increasing Bax expression and caspase‐8, ‐9 and ‐3/7 activities whilst decreasing ATP levels and Bcl‐2 expression. Further, FA significantly increased comet tail lengths, PARP‐1 expression and late stage apoptotic body formation in SNO cells. This study shows that FA is cytotoxic and induces increased apoptosis in SNO cells. HIGHLIGHTSFusaric acid (FA) reduced metabolic activity and cell viability in SNO cells.FA increased ROS‐induced lipid peroxidation with a concomitant decrease in glutathione.FA decreased ATP levels and increased LDH leakage from SNO cells.FA induced apoptosis by increasing caspase‐3/7, ‐8 and ‐9 activities and DNA damage.

Synthesis, in vitro evaluation, and 68Ga-radiolabeling of CDP1 toward PET/CT imaging of bacterial infection

Bacterial infections are a major concern in the human health sector due to poor diagnosis and development of multidrug‐resistant strains. PET/CT provides a means for the non‐invasive detection and localization of the infectious foci; however, the radiotracers available are either cumbersome to prepare or their exact contribution toward the imaging is not yet established. Human antimicrobial peptides are of interest for development as PET radiotracers as they are an integral component of the immune system, non‐immunogenic toward the recipient, and show selectivity toward pathogens such as bacteria. Herein we report on the potential of LL37, a human cathelicidin antimicrobial peptide, as a radiotracer for bacterial imaging. Bifunctional chelator 1,4,7‐triazacyclononane,1‐glutaric acid‐4,7‐acetic acid was utilized to functionalize the antimicrobial peptide, which in turn was capable of chelating gallium. The synthesized natGa‐CDP1 showed bacterial selectivity and low affinity toward hepatic cells, which are favorable characteristics for further preclinical application.

Trigonella foenum-graecum Seed Extract, 4-Hydroxyisoleucine, and Metformin Stimulate Proximal Insulin Signaling and Increase Expression of Glycogenic Enzymes and GLUT2 in HepG2 Cells

BACKGROUND Fenugreek (Trigonella foenum-graecum) is globally recognized for its medicinal properties and hypoglycemic effects. The seed extract as well as its active compound, 4-hydroxyisoleucine (4-OH-Ile), have been shown to reduce hyperglycemic insulin resistance. The mechanism by which this occurs has not been investigated in human liver cells (HepG2) in comparison to the antihyperglycemic drug, metformin. METHODS We investigated the effects of an aqueous fenugreek seed extract (FSE), 4-OH-Ile, and metformin in HepG2 cells relative to insulin as a positive control. Cells were treated with FSE and 4-OH-Ile at 100 ng/mL under normoglycemic (5 mM glucose) and hyperglycemic (30 mM glucose) conditions for 72 hr. Tyrosine phosphorylation of insulin receptor-β (IR-β), protein kinase B (Akt), glycogen synthase kinase-3α/β (GSK-3α/β), and glucose transporter 2 (GLUT2) was determined by western blotting. Gene expression of sterol regulatory element-binding protein 1c (SREBP1c), GLUT2, glycogen synthase (GS), and glucokinase (GK) was evaluated by quantitative polymerase chain reaction, and supernatant glucose levels were measured using the Piccolo biochemistry analyzer. RESULTS Under normo- and hyperglycemic conditions, FSE, 4-OH-Ile, insulin (100 ng/mL), and metformin (2 mM) caused a significant increase in phosphorylation of IR-β, Akt, GSK-3α/β, and GLUT2. Glucose uptake, however, was most significantly increased in FSE-treated cells during both conditions. FSE induced the most significant changes in downstream insulin signaling, GS, GK, SREBP1c, and GLUT2 expression compared to 4-OH-Ile, metformin, and insulin. In addition, FSE significantly increased glucose uptake. CONCLUSIONS Collectively, these findings provide a mechanism by which FSE exerts antihyperglycemic effects similar to metformin and insulin that occurs via enhanced insulin signaling, gene expression, and increasing glucose uptake.

Fusaric Acid immunotoxicity and MAPK activation in normal peripheral blood mononuclear cells and Thp-1 cells

Fusaric acid (FA), a food-borne mycotoxin, is a potent divalent metal chelator. The human immune system is complex and susceptible to environmental insult however, the immunotoxity of FA remains unknown. We investigated the immunotoxicity of FA on human peripheral blood mononuclear cells (PBMCs) and Thp-1 cells. FA was cytotoxic to PBMCs (IC50-240.8 μg/ml) and Thp-1 (IC50-107.7 μg/ml) cells at 24 h. FA induced early apoptosis but significantly decreased caspase activity in PBMCs, a characteristic of paraptosis. In Thp-1 cells, FA induced apoptosis and increased caspase −9 and −3/7 activities. In PBMCs, FA maintained mitochondrial membrane potential and decreased protein expression of Bax whilst increasing expression of p-Bcl-2; FA induced oxidative stress and depleted ATP levels in both cell types. In Thp-1 cells, FA increased mitochondrial membrane depolarization and decreased p-Bcl-2 expression. In PBMCs, FA significantly up-regulated the MAPK protein expression of p-ERK and p-JNK but down-regulated p-p38 expression. In Thp-1 cells, FA up-regulated MAPK protein expression of p-ERK whilst p-JNK and p-p38 expression were down-regulated. In conclusion FA induced programmed cell death and altered MAPK signaling in healthy PBMCs and Thp-1 cells strongly suggesting a possible mechanism of FA induced immunotoxicity in vitro.

Toxicity assessment of mycotoxins extracted from contaminated commercial dog pelleted feed on canine blood mononuclear cells

Raw ingredients of pet food are often contaminated with mycotoxins. This is a serious health problem to pets and causes emotional and economical stress to the pet owners. The aim of this study was to determine the immunotoxicity of the most common mycotoxins (aflatoxin, fumonisin, ochratoxin A and zearalenone) by examining 20 samples of extruded dry dog food found on the South African market [10 samples from standard grocery store lines (SB), 10 from premium veterinarian lines (PB)]. Pelleted dog food was subjected to extraction protocols optimized for the above mentioned mycotoxins. Dog lymphocytes were treated with the extracts (24 h incubation and final concentration 40 μg/ml) to determine cell viability, mitochondrial function, oxidative stress, and markers of cell death using spectrophotometry, luminometry and flow cytometry. Malondialdehyde, a marker of oxidative stress showed no significant difference between SB and PB, however, GSH was significantly depleted in SB extract treatments. Markers of apoptosis (phosphatidylserine externalization) and necrosis (propidium iodide incorporation) were elevated in both food lines when compared to untreated control cells, interestingly SB extracts were significantly higher than PB. We also observed decreased ATP levels and increased mitochondrial depolarization in cells treated with both lines of feed with SB showing the greatest differences when compared to the control. This study provides evidence that irrespective of price, quality or marketing channels, pet foods present a high risk of mycotoxin contamination. Though in this study PB fared better than SB in regards to cell toxicity, there is a multitude of other factors that need to be studied which may have an influence on other negative outcomes.

Inverse association between microRNA-124a and ABCC4 in HepG2 cells treated with antiretroviral drugs.

Abstract The ATP-binding cassette (ABC) super-family of drug transporters regulates efflux of xenobiotic compounds. The subfamily, multi-drug resistance proteins (MRPs) transports cyclic nucleotides and xenobiotics. Epigenetic modulation of drug transporters is scarcely described. The regulatory role of microRNA (miR)-124a on drug transporter gene ABCC4 was only recently reported. Our study investigated the differential regulation of miR-124a by nucleoside reverse transcriptase inhibitors (NRTIs): Zidovudine (AZT), Stavudine (d4T) and Tenofovir (TFV); at 24 h and 120 h treatments in HepG2 cells. ABCC4 mRNA (qPCR) and ABCC4 protein (western blot) were quantified. Cytotoxicity was evaluated by lactate dehydrogenase (LDH) levels. All NRTIs elevated miR-124a levels at 24 h, with a concomitant decline in ABCC4 mRNA levels (p<0.05). At 120 h, d4T and TFV elevated miR-124a and depleted ABCC4 mRNA levels (p<0.0001), while the inverse was observed with AZT (p<0.005). ABCC4 protein was increased by d4T and TFV at 24h. A significant reduction in protein levels was observed at 120 h in all three treatments (p<0.005). The disjoint in mRNA and protein levels is likely due to ABCC4 being a membrane bound protein. Following prolonged exposure, membrane integrity was compromised as evidenced by increased LDH leakage (p<0.005). We conclude antiretroviral drugs have varying effects on miR-124a and ABCC4.

Atorvastatin induces MicroRNA-145 expression in HEPG2 cells via regulation of the PI3K/AKT signalling pathway

The use of statins as a potential cancer drug has been investigated; however the molecular mechanisms involved in their anti-oxidant, anti-proliferative and anti-cancer effects remain elusive. In our study, we investigated the involvement of downstream mevalonate products that mediate the anti-oxidant and anti-proliferative effects of Atorvastatin (Ato), and its effect on microRNA-145 expression in HepG2 hepatocellular carcinoma cells. An amorphous soluble form of Ato was prepared and found to be cytotoxic in vitro [IC50 (1.2 mM); 48 h]. Atorvastatin induced a dose-dependent increase in cell mortality with a concomitant depletion of intracellular ATP levels (p = 0.005); significantly increased extracellular nitrite levels (p = 0.001) and decreased lipid peroxidation (p = 0.0097) despite a decrease in GSH. The intrinsic apoptotic pathway was activated via increased caspase -9 (p < 0.0001) and -3/7 (p = 0.0003) activities. Increased protein expression of pGSK3-(α/β) (p = 0.0338), p53 (p = 0.0032), Mdm2 (p < 0.0001), with significantly diminished levels of PI3K (p = 0.0013), pAKT (p = 0.0035), and Akt (p = 0.0077), indicated that Ato-mediated cell death occurred via inhibition of the PI3K/Akt pathway. Additionally, the expression of PI3K (p = 0.0001) and c-myc (p = 0.0127) were also downregulated, whilst and miRNA-145 (p = 0.0156) was upregulated. In conclusion our data strongly indicates a plausible mechanism involved in the cytotoxic effects of Ato and is the first study to show that Ato modulates miR-145 expression in hepatocytes. ≤ .