Masters Degrees (Medical Biochemistry)

Permanent URI for this collectionhttps://hdl.handle.net/10413/7039

Browse

Now showing 1 - 20 of 43

Ursolic acid as a potential inhibitor of mycobacterium tuberculosis cytochrome bc1 oxidase: a molecular modelling perspective.
(2021) Tembe, Ntombikayise.; Mhlongo, Ndumiso Nhlakanipho.; Kumalo, Hezekiel Mathambo.
Tuberculosis (TB) is a disease, caused by an infectious agent; Mycobacterium tuberculosis, which persists as a major problem globally, especially in developing countries such as Brazil, Indonesia, and South Africa. Individuals who are diabetic and human immunodeficiency virus (HIV) co-infected are at a higher risk of contracting TB. Hence, these risk factors are associated with a compromised immune system. Among these factors, various strains are involved in the pathogenesis of TB such as multidrug-resistant tuberculosis (MDR-TB) and extensively drugresistant tuberculosis (XDR-TB) strains. The emergence of these strains may result from failure to complete treatment within the stipulated period of six months. However, studies show that the protein QcrB; contributes more to TB pathogenesis. Therefore, there is an urgent need for the discovery of drugs that inhibit QcrB. The current FDA-approved anti-tubercular drugs such as, Lansoprazole sulfide (LSPZ) and Telacebec (Q203) which inhibit QcrB are bacteriostatic and have been linked to side effects including dementia, chronic kidney disease, and ischemic cardiac diseases [1], thus prompting a search for an alternative drug. Various natural compounds have been reported to possess several bioactivities that could be crucial in the management of tuberculosis (TB) disease. Warbugia salutaris, a medicinal plant has been found to exhibit inhibitory properties against M. tuberculosis. Numerous compounds are derived from W. salutaris. In this study, we focus solely on Ursolic acid (UA) and its derivative, Ursolic acid acetate (UAA). These two compounds possess antibacterial, anti-HIV, and antimycobacterial properties. This suggests that they could potentially possess inhibitory properties towards M.tuberculosis QcrB protein. In this study, computational methods are applied to investigate the inhibitory activity of UA and UAA on M. tuberculosis QcrB. Molecular Docking, Molecular Dynamics (MD) simulations, Radius of Gyration, Principal Component Analysis (PCA), and Molecular Mechanics-Generalized Born Surface Area (MM/GBSA) binding free energy calculations were performed in explicit solvent to accomplish our goal. The obtained results indicated that the (1) the binding of UA to QcrB induced a more stable and compacted conformation compared to LSPZ and Q203; (2) high total binding free energy estimated in the QcrB-UA system was due to numerous hydrophobic residues in the binding site of QcrB that interact with phenyl rings of UA resulting in hydrophobic packing. This implies that UA has a high binding affinity and, as a result, a strong inhibition of QcrB; (3) more H-bonds were observed in the QcrB-UA system than in the QcrBQ203 system; (4) rigidity was displayed mostly in Arg124 and Thr128; (5) Arg124 and Phe127 also contributed more to the total binding energy in QcrB-UA and QcrB-UAA. This implies that the ligands exert a high binding affinity in the porphyrin binding site than in the active site. The identification of a molecule that competes with the porphyrin ring for the binding site could be beneficial in QcrB pharmacological research; (6) UA could be a potential anti-tubercular agent through QcrB inhibition, although it is hepatotoxic within tolerable concentrations. However, observed potential hepatotoxicity was based on predictions. Although the preliminary findings of this report warrant further experimental validation, they lay a strong foundation for subsequent assessment and development of these natural compounds as antitubercular drugs.
1,4,7,10,13,16-Hexaazacyclooctadecane (Hexacyclen) Induced Nitrosative Stress and Downregulated NF-κB Cell Survival Pathway in Human Embryonic Kidney (Hek293) and Colorectal Adenocarcinoma (Caco2) Cells.
(2022) Nxumalo, Mthokozisi Bongani.; Khan, Rene Bernadette.; Khumalo, H.
Colorectal cancer (CRC) is the third most common malignancy detected and the second leading cause of cancer-related mortality. Mammalian cells require metals for the physiological process as they are part of the structure or co-factor of many proteins. However, excessive accumulation may manifest in toxicity. In addition, the promotion of oncogenesis and tumour growth has been associated with an increased presence of metals. Promising anticancer compounds that disrupt the onset and progression of carcinogenesis are currently being intensely investigated by the scientific community. Hexacyclen, a nitrogen electron donor and a potent metal ion chelator that binds various metal and transition metal cations, is one such anticancer drug. The cytotoxic effects of Hexacyclen on human colorectal adenocarcinoma cells (Caco2) and normal embryonic kidney cells (Hek293) were investigated in this work after acute exposure (48 hours). The toxicity of Hexacyclen was studied in Hek293 and Caco2 cells at different concentration ranges [(0-500 μM) and (0-50 μM), respectively]. The MTT (to determine IC20 and IC50), ATP and mitochondrial membrane potential (ΔΨM) assays were used to assess metabolic activity, while TBARS, NOS and GSH assays were used to assess oxidative activity. Caspase activity (-8, -9, -3/7), phosphatidylserine externalisation and LDH leakage were used to assess cell death by apoptosis. In addition, western blotting was used to examine the expression of antioxidant (SOD2, GPx, catalase), pro-and anti-apoptotic (p-p53, Bcl-2, HSP70, PARP, cPARP) and inflammatory (NF-κB, STAT3 and p-STAT3) proteins. From the dose-dependent MTT curve, an IC20 and IC50 of 6μM and 38μM (Hek293) and 1.2μM and 5μM (Caco2 cells) were determined. The decreased ATP concentration in Hek293 (p<0.05) and Caco2 (p>0.05) cells for both treatments was consistent with altered ΔΨM in both cell lines, indicating reduced metabolic activity. Elevated RNS was implied by increased iNOS particularly at the Caco2 IC50 (p<0.05) that promoted nitric oxide production at the IC20 (p>0.05) and IC50 (p<0.05) for Hek293 and Caco2 cells respectively. The decreased MDA in Hek293 cells (p>0.05) was associated with increased SOD2 (p<0.05) and GPx (p<0.05), while slightly increased MDA in Caco2 cells (p>0.05) accompanied increased SOD2 (p>0.05) and GPx (p<0.05 at the IC50 only). Furthermore, GSH levels were increased significantly in IC50-treated Hek293 and Caco2 cells (p<0.05), but downregulation of catalase in Hek293 and Caco2 cells was not significant. In this study, apoptosis was initiated by an increase in caspase-9 (IC50, p<0.05) but not caspase 8, which was decreased for both treatments in Hek293 cells (p<0.05). In Caco2 cells, caspase-8 (p<0.05) and caspase 9 (p>0.05) were increased. Anti-apoptotic Bcl-2 (p<0.05) and HSP70 (p<0.05 for Caco2 cells) were downregulated in both cell lines. The activity of p-p53 was not affected in IC20, whereas it was significantly reduced in IC50-treated (p<0.05) in Hek293 and Caco2 cells. Apoptosis was executed as caspase 3/7 was increased in all treatments (p<0.05), albeit non-significantly for IC20-treated Hek293 cells. Moreover, phosphatidylserine externalisation, an early apoptosis marker, was increased in both cell lines (p<0.05 for IC50-treated Hek293 cells), while LDH (a late marker) was increased for Hek293 cells (p<0.05) but not Caco2 cells (p>0.05). Interestingly, decreased cPARP/PARP activity was observed for IC50-treated cells (p<0.05) in both cell lines. Finally, the inflammatory markers NF-κB (p>0.05 for IC20-treated Hek293 cells) and p-STAT3/STAT3 (p>0.05 for IC20-treated Caco2 cells) were downregulated in this study. Hexacyclen induced apoptosis in Hek293 and Caco2 cells via an RNS-mediated mechanism. Intrinsic apoptosis was noted in Hek293 cells, while both pathways facilitated apoptosis in Caco2 cells. Interestingly, apoptosis proceeded concurrently with a reduction in the NF-κB cell survival pathway.
N, N Bis (2-Pyridylmethyl)-1, 2-Ethylenediamine Tetrahydrochloride Stimulates Intrinsic Apoptosis Mediated by Oxidative and Nitrosative Stress Induction of the NF-B/STAT3 Pathway in Human Hepatocellular Carcinoma (HepG2) Cells.
(2022) Ntanzi, Nosipho.; Khan, Rene Bernadette.
Introduction: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Its incidence is rising, and this trend is expected to continue for decades. Several cancer therapeutics have already been discovered and are being used to treat HCC, however, most of them cause severe side effects which decrease the treatment's effectiveness. Metal chelators such as ethylenediaminetetraacetic acid (EDTA) have previously demonstrated anti-cancer potential. N, Nbis (2-pyridylmethyl)-ethylenediamine tetrahydrochloride (H2pmen) is a tetradentate ligand that forms stable complexes with Fe, Cr, Cu(II), and Zn (II), and it has been shown to be a potentially effective reagent for metal chelation. This study investigated the antiproliferative and cytotoxic effects of H2pmen in the HepG2 cell line. Methods: The cell viability was determined by treating HepG2 cells with different concentrations (0–1000 μM) of H2pmen over 24h. MTT assay was used to obtain an IC50, which was then used in all subsequent assays. The cells were then assayed for oxidative stress and membrane damage (TBARS, NOS, GSH, and LDH cytotoxicity), apoptotic induction (ATP assay, JC-10 assay, Annexin v, Caspases), cytochrome P450 3A4 activity (Luminometry). Protein expression of iNOS, SOD2, Bax, Caspase-2, and STAT3 were identified using western blot analysis. The gene expression of GPx1, Nrf2, NF-κB, p53, and OGG1 was determined using qPCR. Results: H2pmen induced a dose-dependent decrease in cell viability (IC50 of 209 μg/ml), a significant increase in CYP34A activity (p0.05 at IC20 and IC50), a decrease in ATP production (at IC20 p0.05 and at IC50), a significant decrease in m (p0.05 at IC20 and at IC50). The ROSassociated membrane was induced, indicated by an increase in lipid peroxidation (p0.05 at IC20 and p≥0.05 at IC50), an increase in RNS production (p≥0.05 at IC20 and at IC50), an upregulation in iNOS protein expression (at IC20 where p0.05 and at IC50) and NF-κB gene expression (at IC20 where p0.05 and at IC50). Oxidative stress occurred due to a decrease in GSH levels (at IC20 and p≥0.05 at IC50), a significant downregulation in SOD2 protein expression, and upregulation in gene expression of GPx-1 (at IC20 where p≥0.05 and at IC50) and Nrf2 (at IC20 and at IC50 where p0.05). H2pmen initiated caspase-dependent apoptosis that was indicated by a decrease in Caspase-2 (p0.05at IC20 and at IC50), caspase-8 (at IC20 and p≥0.05 at IC50), a slight insignificant decrease at IC20 and an increase at the IC50 in caspase-9, a significant upregulation in Bax (p0.05 at IC20 and at IC50) protein expression and p53 (at IC20 where p0.05 and at IC50) gene expression. The significant increase in caspase-3/7 (p≥0.05 at IC20 and IC50), Annexin V levels (p≥0.05 at IC20 and at IC50), LDH (p≥0.05 at xviii IC20 and IC50), STAT3 (p0.05at IC20 at IC50), PARP1 (p0.05 at IC20 and at IC50), and OGG1 (p0.05 at IC20 and at IC50) shows that apoptosis was executed by H2pmen in HepG2 cells. Conclusion: H2pmen reduced cell viability of HepG2 cells, exerting a cytotoxic effect associated with decreased m and ATP, and increased LDH leakage. The chelating properties of H2pmen was linked to the induction of oxidative and nitrosative stress that affected lipids and DNA. The HepG2 cells mounted an antioxidant defense involving Nrf2 to counteract the depletion of SOD2 and GSH, with evidence of its effect associated with upregulation of GPx. The prevailing oxidative stress activated DNA repair enzymes (PARP1 and p53), while NF-κB and STAT3 pathways were also induced. Bax-induced MOMP and caspase-2 invoked VDAC triggered caspase-dependant apoptosis via the intrinsic pathway.
Momordica foetida facilitates glucose uptake independent of AMPK2 and PI3K to attenuate hyperglycemia-induced oxidative stress via a JNK-STAT3 mediated pathway in HepG2 cells.
(2022) Netshitangani, Tshamano Fulufhelo.; Khan, Rene Bernadette.
Introduction: The exponential rise in the global prevalence and incidence of type 2 diabetes is concerning. Hyperglycemia is a hallmark of type 2 diabetes that induces oxidative stress, leading to impairment of vital liver metabolic pathways. Metformin is the first-line treatment for type 2 diabetes mellitus. However, Momordica foetida has been used in folk medicine for the treatment and management of diabetes mellitus in various parts of the world including South Africa. Aim: In the present study, the cytoprotective effects of M. foetida on liver impaired glucose metabolism and oxidative stress damage were investigated on high glucose induced HepG2 cells, with Metformin as a positive drug control. Methods: The M. foetida leaves were used to prepare an aqueous lyophilized extract. The HepG2 cells were serum starved for 1 hour, then exposed to hyperglycemic conditions (30mM D-glucose) for 24 hours. Cells were treated with various concentrations (125 - 1000 μg/ml) of the lyophilized M. foetida aqueous extract for 24 hours, and the 3-(4,5- dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay evaluated the effects of high glucose and M. foetida on the metabolic activity of HepG2 cells. Antioxidants and prooxidants were assessed and quantified using luminometry, thiobarbituric acid reactive substances (TBARS) and nitric oxide synthase (NOS) assays. Western blot and quantitative real-time (qPCR) were used to observe the effects high glucose and M. foetida on signaling pathways and antioxidant response. Results: Glucose uptake in hyperglycemic conditions was mediated by increased gene expression of adenosine monophosphate-activated protein kinase alpha 2 (AMPKα2) (p˂0.05) and phosphatidylinositol 3‑kinase (PI3K) (p˂0.05), but glucose transporter 2 (GLUT2), glucokinase (GK) and glycogen synthase (GS) were downregulated (p˂0.05). Interestingly, an opposing response was noted for Metformin and M. foetida treatments, where AMPKα2 (p˂0.05) and PI3K (p˂0.05) were downregulated, whereas GLUT2, GK and GS were upregulated (p˂0.05) compared to the hyperglycemic control. When compared to the hyperglycemic conditions control, M. foetida treatments and Metformin showed an increase in glucose uptake. Hyperglycemic conditions induced toxicity indicated by increased extracellular lactate dehydrogenase (LDH) and decreased adenosine triphosphate (ATP), but Metformin and M. foetida decreased LDH activity back to xvi normoglycemic levels, indicating reduced cytotoxicity. Increased mitochondrial membrane potential (m) in hyperglycemic conditions was accompanied by increased lipid peroxidation (p˂0.05) and reactive nitrogen species (RNS) (p˂0.05). The m was increased further by M. foetida, with minimal effect on reactive oxygen species (ROS) production but effectively increasing RNS (p˂0.05). Oxidative damage was reduced in the hyperglycemic control but was increased by Metformin and M. foetida treatments prompting the activation of p53 in these cells (p˂0.05). Effective oxidative stress response was mounted by NRF2 (p˂0.05) and antioxidants SOD2 (p˂0.05) and GSH, but GPx1 and CAT (p˂0.05) were decreased. Interestingly, Metformin and M. foetida induced CAT (p˂0.05) and GPx1 (p˂0.05) in the antioxidant response, consequently decreasing GSH. Metformin decreased NRF2 (p˂0.05) and SOD2, while M. foetida increased NRF2 significantly and had no effect on SOD2 relative to the hyperglycemic control. Hyperglycemic conditions downregulated the oxidative stress response by MAPK (p-p38, pJNK and pERK1/2) (p˂0.05). However, Metformin upregulated pJNK (p˂0.05) and pERK1/2 (p˂0.05), but p-p38 (p˂0.05) was downregulated. Interestingly, M. foetida upregulated pJNK (p˂0.05), downregulated pERK1/2 (p˂0.05) and had no effect on p-p38. Hyperglycemic conditions also increased pSTAT3, which was downregulated by Metformin and M. foetida treatments (p˂0.05). Conclusion: Taken together, the results demonstrated that M. foetida enhanced the metabolic activity and reduced cell cytotoxicity in HepG2 cells. Furthermore, M. foetida facilitated glucose uptake independent of AMPK2 and PI3K. The main source of oxidative stress was increased RNS, which was alleviated by an effective MAPK/JNK and antioxidant response involving CAT.
Fusaric acid Fumonisin B1 CO -treatment regulates AMPK signalling and induces Apoptosis in HEPG2 cells.
(2019) Shilabye, Patane Sylvester.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
Background/Aim: Fusaric acid (FA) and Fumonisin B1 (FB1) are mycotoxins produced by Fusarium fungal species. These mycotoxins are major contaminants of maize and contribute to toxicity in animals and humans. The main mechanisms of FA and FB1 toxicity involve the induction of oxidative stress and apoptosis; however, FA was additionally found to chelate divalent cations, whereas FB1 inhibits sphingolipid synthesis. AMPK is an energy sensor involved in regulating cell proliferation. AMPK targets the transcription factors, p53 and FOXO3a that play a major role in apoptosis. To date numerous studies have investigated the individual effects of FA and FB1, however, their combined synergistic effects are unclear. This study investigated the effect of FA and FB1 co-treatment on AMPK-induced apoptosis in liver HepG2 cells. Methods: HepG2 cells were cultured and co-treated with various concentrations (5, 27, 100μM and combined 104μM FA and 200μM FB1 IC50s) of FA and FB1 for 24 hrs. Cytotoxic effects of FA and FB1 on HepG2 cells were determined using the MTT assay. The TBARS assay was used to determine oxidative stress. Western blot was used to determine protein expression of AMPK, p-AMPK and p53, whereas q-PCR was used to measure FOXO3a mRNA expression. LDH assay was used to measure membrane integrity. ATP levels and activity of caspases -3/7, -8 and -9 were measured using luminometry. Results: A combination of FA and FB1 decreased cell viability in a dose dependant manner. An IC50 of 27μM for FA and FB1 was obtained. ATP levels were significantly increased at 5μM and 27μM, whereas at 100μM and combined IC50s were significantly decreased (p<0.0001). Oxidative stress was significantly increased in FA and FB1 treated cells in a dose dependent manner (p<0.0001). The protein expression of total AMPK was decreased at 5μM, but increased at 27μM, 100μM and combined IC50s in relation to control (p<0.0001).p- AMPK showed a significant decrease (p<0.0001) in all FA and FB1 treated samples despite the increase in the expression of total AMPK. FOXO3a mRNA expression was decreased at 5μM and at combined IC50s, with the decrease being significant at 5μM. The results also indicated an increase at 27μM and 100μM (p<0.0001). p53 protein expressions were significantly decreased in all samples (p<0.0001). Caspase -3/7, -8 and -9 were significantly increased at 5-100μM and decreased at combined IC50s in HepG2 cells. In FA and FB1 samples, LDH levels were significantly decreased at 5μM and 27μM, and significantly increased at 100μM and combined IC50s (p<0.0001). Conclusion: FA and FB1 co-treatments suppressed AMPK signalling by downregulating p- AMPK and induced apoptosis and/necrosis in HepG2 cells.
Artemisia afra crude aqueous leaf extract indices oxidative stress and inflammation in human colon adenocarcinoma cells via the upregulation of the TNF-a,p38 and STAT3 pathway.
(2022) Mposula, Slindelo.; Khan, Rene Bernadette.
ABSTRACT Introduction: Artemisia afra (A. afra) is a widely used medicinal plant located in the southern African region. It is traditionally used to alleviate medical conditions such as coughs. Literature indicates a protective role by improving antioxidant capacity and reducing cell proliferation, which suggests anti-cancer potential. Colorectal carcinoma (CRC) is a global public health crisis and the second common cause of cancer-related fatalities. Current cancer treatment is deemed effective but not easily accessible and expensive in the southern African region. Therefore, the need for naturally derived anti-cancer agents remains to be investigated for accessible and affordable treatment. This study investigates the antiproliferative and antioxidant effects of A. afra crude aqueous leaf extract in the Caco-2 cell line. Materials and Methods: Caco-2 cells were treated with a range of A. afra concentrations (0-5000 μg/ml) for 48 hours. An IC50 was derived from the MTT assay and all subsequent assays compared the IC50 -treatement to an untreated control. Mitochondrial integrity was luminometrically assessed by measuring JC-10 fluorescence and ATP. Free radical production (TBARS, NOS) and membrane damage (LDH cytotoxicity), together with GSH quantitation were used to infer the presence of oxidative stress; antioxidant enzymes (SOD2, GPx-1, catalase, Nrf2) were also detected by western blotting. Apoptotic induction was verified by measuring phosphatidylserine externalisation, quantifying caspase activities and detecting pro- and anti-apoptotic proteins (Bax, Bcl2, cIAP, xIAP) by western blotting. Single strand DNA fragmentation was evaluated via the comet assay. Additionally, relative expression of DNA repair, inflammation and stress markers were determined using western blotting and qPCR. Results: Crude aqueous leaf extract of A. afra induced a dose-dependent reduction in cell viability, yielding an IC50 of 250 μg/ml. Decreased mitochondrial integrity (p = 0.697) was associated with significant depletion of intracellular ATP (p = 0.0043) and increased ROS production as validated by increased lipid peroxidation (p = 0.1638) and DNA oxidation (amplified OGG1). In addition, increased iNOS contributed to the production of RNS. Artemisia afra induced an antioxidant response that elevated Nrf2 at the mRNA and protein level, causing increased GSH (p = 0.0001), GPx-1 (p = 0.5067) and catalase, but SOD2 was decreased. Heightened levels of heatshock proteins (HSP27 and HSP70) correlate with increased ROS and upregulated phosphorylated p38 protein, but ERK and JNK protein expression was downregulated. Significant downregulation caspase-8 (p = 0.0252), caspase-9 (p = 0.0099) and caspases-3/7 (p = 0.0232) was associated with reduced Annexin-V) and extracellular LDH. In addition, the Bax/Bcl-2 ratio (p = 0.0033) and protein expression of inhibitors of apoptosis protein such as cIAP-1 and xIAP indicated reduced apoptotic activity in this study. Comet tail analysis indicated intact DNA, in congruence with decreased OGG1. Both TNF-α (p = 0.2323) and STAT-3 were upregulated, but NF-ĸB was decreased. In addition, cellular Myc and phosphorylated retinoblastoma were upregulated. Conclusion: The crude aqueous leaf extract of A. afra induced mitochondrial toxicity and ROS production. Despite a heightened antioxidant defense, ROS-mediated upregulation of TNF-, p38 and STAT3 promoted cell proliferation and inhibited apoptosis in Caco-2 cells. Taken together, A. afra is a cytotoxic and genotoxic agent that may induce cancer in human colorectal cells.
Terminalia phanerophlebia crude aqueous leaf extract activates the NRF2-mediated antioxidant defence to prevent oxidative stress in human hepatocellular carcinoma cells.
(2021) Nyahada, Marcilyn Rutendo.; Khan, Rene Bernadette.
Abstract available in PDF.
The prevalence of Methicillin-resistant Staphylococcus aureus in blood product hampers at the South African National Blood Service.
(2021) Seoraj, Varsha.; Chuturgoon, Anil Amichund.; Singh, Ravesh.; Van den Berg, Karin.
Background: Methicillin-resistant Staphylococcus aureus (MRSA), are strains of the Gram-positive cocci known to cause various health conditions. Patients suffer from abscesses, skin infections and more severe conditions such as osteomyelitis and septicaemia. These bacteria are highly resistant to antibiotics and bacteria such as these are a great risk to the public, especially since Staphylococcus aureus is an opportunistic bacterium. In 2016, a donor unit received for the production of eye serum at South African National Blood Service (SANBS), when quality controlled, tested positive for MRSA. The bacterial contamination was traced to a staff member at the clinic where the blood was donated. Little research has been conducted to determine if MRSA is a problem and if it could negatively affect the blood supply. Aim: The aim of this study was to determine whether blood services contribute to the spread of MRSA and other bacterial pathogens through the blood product hamper system. Materials and Methods: A cross-sectional study to determine the prevalence of MRSA on 850 blood product hampers moving between SANBS inventory laboratories and blood banks, was conducted at SANBS between August 2020 and May 2021. Hampers were swabbed with a Sigma-Transwab containing liquid Amies transport medium for the detection of MRSA. The swabs were cultured onto CHROMagar MRSA where a rose or mauve coloured colony confirmed the presence of MRSA. Bacterial contaminants which were detected during the testing procedure were isolated, and loaded onto the Vitek 2 Compact for bacterial identification. Results: A total of 696 hampers were processed as per the study protocol (81.9%). Out of the 850 hampers planned to be swabbed, 143 (16.8%) hampers were not swabbed as a result of staff not performing the procedure and swabs from 11 hampers were omitted (1.3%) as they did not comply too protocol requirements. Of the 696 hampers swabbed, MRSA was not detected (0%) however, bacterial growth other than MRSA was observed. The most common isolates detected were Aerococcus viridans, Rothia dentocariosa, followed by Bacillis spp as well as Stenotrophomonas maltophilia. Conclusion: The study findings have shown that an effective hamper cleaning system is needed to safeguard the integrity of our blood supply. The findings of this study should be taken into consideration throughout all provinces at SANBS, for the consistent and regular cleaning of hampers, which carry blood and blood products.
Exploring Iso-mukaadial acetates and other small compounds as inhibitors of recombinant Plasmodium falciparum lactate dehydrogenase.
(2021) Mabaso, Nonduduzo Hlengiwe.; Mhlongo, Ndumiso Nhlakanipho .; Pooe, Ofentse Jacob.
Malaria is a major killer disease in Sub-Saharan Africa, this disease is caused by a protozoan parasite of genus Plasmodium. It is a pressing health issue the public is facing, and the effectiveness of every treatment developed thus far is being jeopardized by the emergence of parasite drug resistance. This then creates a demand for new antiprotozoal medication, necessitating novel approaches that will assure the long- term discovery of the lead compounds. The investigation of compounds such as Iso-mukaadial acetate (IMA), Betulinic acid (BA), Ursolic acid (UA) and Oleanolic acid (OEA) which are isolated from plants shows to possess antimalarial activity. These compounds either originate from various plants or leaves, IMA which is isolated from a pepper bark tree, BA from bark of a plant species (white birch), UA from leaves of (lavender, rosemary), and OEA found in leaves and Olea europaea fruit. This study aims to investigate the inhibitory properties of these compounds against Plasmodium falciparum lactate dehydrogenase (PFLDH) an enzyme found in the parasite glycolytic pathway that converts pyruvate to lactate and in so doing, provides the energy needed for the survival of the malarial parasite. These methodologies were followed to conduct this study; Recombinant PfLDH was expressed and then purified for further analysis including colony PCR, expression, purification, interaction studies including Fourier transform infrared (FTIR) analysis and Ultraviolet-visible spectroscopy (UV-Vis), antimicrobial activity along with in silico analysis. The following results were obtained: Colony PCR confirmed the presence of a 951bp insert in the PKK223 plasmid. Metal affinity chromatography successfully purified PfLDH protein sized 34.9kDa which was confirmed by ExPasy ProtParam server. The following results were obtained from isolated compounds (BA and IMA) that were screened for IC50 to demonstrate overall activity against the asexual P. falciparum. BA and IMA had IC50 values of 1.27 and 1.03μg/ml against asexual P. falciparum, respectively. When compounds were incubated with protein, FTIR analysis showed a clear shift in the curve, which is indicative of an interaction between IMA and BA with PfLDH. UV-Vis showed that structural conformational change was induced, resulting in an interaction of the compounds with the aromatic side chains of PfLDH. The in silico analysis showed where these interactions occurred, highlighting the ligand atoms responsible for the interaction. Based on these findings, it is possible that these investigated compounds could be effective PfLDH inhibitors as they have binding affinities which are like the standard drug, chloroquine (QA).
Allicin ameliorates some deoxynivalenol-induced cytotoxic effects in human embryonic kidney (Hek293) cells, but also elicits synergistic and potentiating adverse effects.
(2020) Mamane, Yandisa Zintle.; Khan, Rene Bernadette.
Introduction: Deoxynivalenol (DON), a type B trichothecene produced by plant pathogenic fungi, especially Fusarium graminearum and F. culmorum, is a highly toxic mycotoxin found throughout South Africa. DON is consumed unintentionally through maize derived products and is rapidly becoming a potential health risk to humans and animals. It is a known immunosuppressant that induces apoptosis and oxidative stress and may cause liver lesions and kidney problems. Recently, dietary therapeutics have demonstrated a role against mycotoxin-induced cytotoxicity. Garlic (Allium sativum) is part of the Alliaceae family. The garlic bulb is used for medicine and as food consumption. The aqueous extract has recently demonstrated the potential to protect against mycotoxin-induced cell death and decrease reactive oxygen species (ROS). Aim: This study investigated the induction of apoptosis and oxidative stress by DON in Hek293 cells, and the ability of allicin to ameliorate these effects. Methods: Hek293 cells were treated with a range of allicin concentrations (0-150mM) over 24hrs. An EC50 of 1.7mM was obtained from the MTT assay and used in all subsequent assays. Hek293 cells were treated with 5μM DON, 1.7mM allicin (A), or a combination (DON+A) for 24hrs; untreated cells served as the control. Lipid peroxidation [malondialdehyde (MDA) and lactate dehydrogenase (LDH) assays] were used to indirectly quantify reactive oxygen species (ROS) and oxidative stress; reactive nitrogen species (RNS) were quantified using the nitrates assay. Apoptotic induction was determined by the detection of phosphatidylserine (annexin V) and DNA fragmentation. Necrotic cells were distinguished by propidium iodide uptake. Luminometric quantification of ATP, reduced glutathione (GSH), and caspase 9, 3/7, were used to verify these events. In addition, antioxidant enzymes protein expression of superoxide dismutase (SOD2), catalase and glutathione peroxidase (GPx1); as well as nuclear factor erythroid 2-related factor 2 (Nrf2) and heat shock protein (Hsp70), and apoptotic markers associated protein expression of p53, Bax, and poly (ADP-ribose) polymerase (PARP) were detected by western blotting. Results: DON-induced ROS production was suggested by the depletion of antioxidants including SOD2 (p < 0.0001), catalase (p < 0.0001) and GSH (p = 0.0886). Decreased lipid peroxidation indicated by the decreased MDA concentration (p < 0.0001) and reduced LDH (p = 0.0342) imply that the Hek293 cells were spared from the membrane-damaging effect of oxidative stress. A reduction in Hsp70 (p = 0.0056) and Nrf2 (p < 0.0001), and upregulation of GPx1 (p = 0.0362) protein expression was noted. In addition, increased nitrate concentration in all treatments compared to the control (p < 0.0001) suggested a shift to RNS production. Notably, allicin maintained Nrf2 protein expression similar to the control. The decrease in MDA concentration (p = 0.0109) by allicin was concurrent with depleted GSH (p = 0.0504)and increased SOD2, catalase and GPx1 (p < 0.0001), and suggests allicin induced an oxidative stress response. Allicin also protected DON-treated cells from oxidative stress by upregulating Hsp70 (p < 0.0001), catalase (p = 0.0006) and GPx1 (p = 0.0018), with concurrent decreased GSH (p = 0.0342) and ATP (p = 0.2028) concentration, which were also decreased by DON. In addition, allicin increased MDA (p < 0.0001) and LDH (p = 0.1267) towards control levels in the combined treatment. Apoptosis was reduced in the DON (p = 0.4631) and DON+A (p < 0.0488) treated cells in comparison to the control, necrosis was not evident in any treatment. The slight induction of p53 (p = 0.0008) and PARP-1 (p = 0.4036) by DON implies an attempt at DNA repair, but the Hek293 cells experienced reduced levels of apoptosis. Indeed, Bax expression was slightly reduced (p = 0.1071), caspases 9 (p = 0.0705) and 3/7 (p = 0.4431) activities were diminished, phosphatidylserine was not externalized, and PARP-1 was not cleaved. A non-fragmented DNA profile in allicin-treated and DON+A-treated Hek293 cells may be explained by increased expression of DNA repair proteins, PARP-1 (p = 0.0048 and p = 0.0004 respectively) and p53 (p < 0.0001). The upregulation of p53 is associated with an increase in Bax expression (p < 0.0001 and p = 0.0026 respectively). However, caspases 9 (p = 0.0596) and 3/7 (p = 0.0311) were not activated and apoptosis did not occur. Conclusion: DON treatment induced oxidative stress but not apoptosis in Hek293 cells at the concentration tested. In addition, its mechanism of toxicity in Hek293 cells appears to be more related to nitrosative stress and induction of DNA damage. Oxidative stress and not apoptosis is the possible mechanism of allicin-induced effects in Hek293 cells. Although allicin ameliorated some of the effects of DON in Hek293 cells, it also elicited synergistically or potentiating adverse effects that require further investigation.
An investigation into the biochemical effects of Kojic acid (KA) on human hepatocellular carcinoma (HepG2) cells.
(2020) Suthiram, Kimera Tamzin.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
Kojic acid (KA) is a secondary metabolite and divalent metal chelator that is widely used in the beauty industry as a skin lightener. However, KA toxicity is not well-established in humans. This study aimed to determine the toxicity of KA by assessing oxidative stress, nuclear factor kappa B (NFκB) signalling, and mitogen-activated protein kinase (MAPK) signalling in human hepatoma (HepG2) cells following 24 h exposure. Cell viability was assessed using the methylthiazol tetrazolium (MTT) and crystal violet assays. To confirm cell death, apoptosis (caspase -8, -9, -3/7 luminometry), and Lactate dehydrogenase (LDH) leakage were assessed. Oxidative stress (TBARS), DNA damage (8-OHdG), and protein oxidation (protein carbonyls assay) to determine macromoleculedamage. An assessment of inflammatory and oxidative stress markers were carried out using mRNA expression GPx, NFκB, actor erythroid-2 factor-2 (Nrf2), phospho-Nrf2 (ser40), catalase (CAT), c-Jun-N-terminal kinase (JNK), p38, phospho-Sirtuin 1 (ser47) (phospho-sirt1), NFκB, phospho-NFκB (ser536), and activator protein 1 (AP-1) were assessed using Western Blot in HepG2 cells. KA decreased cell viability in HepG2 cells and elevated the activities of caspase -9 (p < 0.0001), caspase -8 (p = 0.0003) and caspase 3/7 (p < 0.0001) at lower concentrations [4.22 & 8.02 mM] which served as confirmation of apoptosis. Necrosis at the higher concentration [12.67 mM] was confirmed by the presence of LDH leakage indicating membrane damage. Increased cell death was further correlated with increased miRNA-29b expression (p = 0.009), a miRNA responsible for elevated apoptotic activity. Adenosine Triphosphate (ATP) production was increased significantly at 12.67 mM (p < 0.0001), while oxidative stress (Malondialdehyde (MDA) levels) was increased significantly at 4.22 mM (p < 0.0001). Macromolecules are susceptible to damage in the presence of oxidative stress. Due the elevation of MDA levels, DNA damage and protein oxidation assays were carried out. Protein carbonyls were significantly decreased (p < 0.0001), suggesting a potential cytoprotective effect. Due to the presence of oxidative stress, Nrf2, is activated and is responsible for the transcription of antioxidant genes. This was illustrated by an increase in activated Nrf2 at lower concentrations (4.22 & 8.02 mM), whilst at higher a concentration (12.67 mM) decreased phospho- (p > 0.0001). CAT was decreased significantly (p = 0.0002) and GPx significantly increased at lower concentration [4.22 mM] (1.51-fold). A key function of the MAPK pathway is the initiation of stress-activated protein kinases, p38 and JNK, in response to oxidative stress. KA significantly increased, p38 at lower concentration (p = 0.0011) and significantly decreased JNK1 (p = 0.0039) and JNK2 (p < 0.0001) activity. Regulation of reactive oxygen species (OS) production by Sirt-1 occur via the alteration of immune responses through NFκB signalling and AP-1. Inflammatory ediators, phospho-Sirt1 was significantly decreased (p < 0.0001), while AP-1 expression was elevated (p 15 which is in agreement with repressed inflammatory responses reflected by decreased NFκB expression. KA treatment resulted in increased MDA levels and antioxidant responses. MAPK signalling was elevated in response to oxidative stress suggesting the involvement in cell death, whilst inflammation was suppressed. In conclusion, KA displayed low toxicity in HepG2 cells.
Fumonisin B1 induced antioxidant response in C57BL/6 male mice brain.
(2018) Sibiya, Thabani.; Chuturgoon, Anil Amichund.; Nagiah, Savania.; Ghazi, Terisha.
Background: Fumonisin B1 (FB1), a mycotoxin produced by the Fusarium species, contaminates maize. In South Africa maize is a dietary staple and FB1 endangers human and animal health. FB1 is known to have neurodegenerative effects; inhibits mitochondrial respiration, causes mitochondrial membrane depolarization and excessive ROS production. This study investigated the antioxidant response in mice brain after acute (24 hrs) and prolonged (10 days) exposure to FB1. Methods: Four groups (Control acute, FB1 acute, Control prolonged, FB1 prolonged) of C57BL/6 male mice (n=5 per group) were used. All controls were orally administered 0.1M PBS and FB1 groups were administered 5mg/kg of FB1. Following acute and prolonged exposure, the mice were euthanised by halothane anaesthesia. Brain tissues were harvested and stored in Qiazol and Cytobuster for RNA and protein isolation, respectively. Protein expression of CAT, pNrf2 and Nrf2 were determined using western blots. The mRNA expression of Nrf2, miR-141, SOD2, GPx, Tfam, LON, SIRT3 and Tau wwere determined using qPCR. Results: Protein expression of Nrf2 (Acute: *p=0,0144; prolonged: **p=0,0094) and pNrf2 (acute: *p=0,0132; prolonged: *p=0,0462) was significantly increased upon 24 hrs and significantly decreased upon 10 days in tissue exposed to FB1, while mRNA levels of Nrf2 were significantly reduced upon acute (***p=0,0001) and prolonged (**p=0,0013) exposure. FB1 induced a significant decrease in miR-141 levels in tissue following acute (**p=0,0019) and prolonged (***p=0,0004) exposure. FB1 increased the protein expression of CAT in tissue following acute (p=0,1206) and significantly increased expression upon prolonged (**p=0,0010) exposure. FB1 also significantly increased the mRNA expression of GPx in acute (***p=0,0001) and prolonged (**p=0,0024) exposure. FB1 significantly decreased the expression of SOD2 in mice brain following acute (**p=0,0070) and non-significantly decrease upon prolonged (p=0,2725) exposure. Tfam and LONP1 levels were significantly decreased upon acute (***p=0,0003, ***p=0,0005) and prolonged (*p=0,0196, *p=0,0117) exposure to FB1 respectively. However, SIRT3 expression was decreased upon acute (p=0,0594) and significantly increased upon prolonged (*p=0,0283) exposure to FB1.The mRNA expression of tau was significantly reduced upon acute (**p=0,0054) and prolonged (*p=0,0273) exposure to FB1. Conclusion: FB1 compromises antioxidant and mitochondrial survival responses in mice brain. This may have implications in FB1-induced neurodegeneration.
Betulinic acid enhances the antioxidant profile in a hyperglycaemic model.
(2019) Maharaj, Gopala.; Chuturgoon, Anil Amichund.; Sheik-Abdul, Naeem.
Type 2 diabetes mellitus (T2DM) is a global pandemic, with prevalence rapidly rising in South Africa. T2DM is characterized by insulin resistance, leading to hyperglycaemia which induces oxidative stress (OS) and inflammation with subsequent complications. Betulinic acid (BA), a ubiquitous plant triterpenoid, has many proven benefits including antioxidant (AO) properties. Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor which binds to triterpenes and promotes glucose uptake and stimulates cytoprotective and anti-inflammatory effects. This study investigated the potential of BA to modulate cytoprotective responses through PPARγ in response to hyperglycaemic (HG) induced OS in a human hepatoma (HepG2) liver cell model. HepG2 cells were cultured under normoglycaemic (NG) and HG conditions and subsequently treated with 5μM and 10μM BA. Spectrophotometric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays] and luminescent (ATP assay) principles were employed to assess viability of the chosen BA concentrations. Phosphorylation of the insulin receptor β-subunit (IRβ) was assessed via Western blot to confirm BA’s anti-HG effects. Intracellular reactive oxygen species (ROS) levels were assessed via fluorescence using the 2′,7′-dichlorodihydrofluorescein-diacetate (H2DCF-DA) assay, and oxidative stress biomarkers were quantified spectrophotometrically, via use of the thiobarbituric acid reactive substances (TBARS) assay for lipid peroxidation, and protein carbonyl assay (PCA). Intracellular AO potential was measured via luminometric quantification of reduced glutathione (GSH). Western blots quantifying protein expression of PPARγ, nuclear factor erythroid 2-related factor2 (NRF2), phosphorylated NRF2 (pNRF2), sirtuin3 (SIRT3), PPARγ coactivator 1α (PGC1α), superoxide dismutase 2 (SOD2), catalase (CAT), uncoupling protein 2 (UCP2), lon protease (LONP1) and nuclear factor κ-B (NFκB) as well as quantitative polymerase chain reaction (qPCRs) assessing gene expression of glutathione peroxidase (GPx1), NRF2, SIRT3, PGC1α and micro-RNA 124 (miR124) were run to elucidate the molecular mechanism behind the cytoprotective response of BA. The MTT, ATP and LDH assays confirmed cell viability, lack of toxicity and stable energy output, while TBARS, DCF and PCA confirmed a reduction of ROS and its biomarkers. A preliminary Western blot of IRβ confirmed BA’s anti-hyperglycaemic actions at a prime concentration of 5μM BA. Further, Western blots also confirmed an AO-induced protective mechanism at 5μM BA originating from the PPARγ/NRF2 positive feedback loop, further involving SIRT3 (p<0.0001), PGC1α (p=0.0025), LONP1 (p<0.0001), and AOs: SOD2 (p<0.0001), CAT (p=0.0003) and UCP2 (p<0.0001). The GSH assay and mRNA levels of PGC1α (p<0.0001), NRF2 (p<0.0001), SIRT3 (p<0.0001) and GPx1 (p<0.0001) further confirmed the mechanism, while miR124 levels (p=0.0093) hinted at epigenetic regulation between the transcription factors. Additionally, BA was found to downregulate NFκB (p<0.0001) in the HG state possibly combatting ROS-induced inflammation. In conclusion, BA illustrated cytoprotective effects on HG induced OS at an optimum concentration of 5μM, by upregulating the AO response and reducing ROS. Thus, BA may be considered an alternate and cheap adjunctive therapy to mitigate complications of T2DM.
Fusaric acid-induced epigenetic modifications in vitro and in vivo: alternative mechanisms of hepatotoxicity.
(2019) Ghazi, Terisha.; Chuturgoon, Anil Amichund.; Nagiah, Savania.
The Fusarium-produced mycotoxin, Fusaric acid (FA), is a frequent contaminant of agricultural foods that exhibits toxicity in plants and animals with little information on its molecular and epigenetic mechanisms. Epigenetic modifications including DNA methylation, histone methylation, N-6-methyladenosine (m6A) RNA methylation, and microRNAs are central mediators of cellular function and may constitute novel mechanisms of FA toxicity. This study aimed to determine epigenetic mechanisms of FA-induced hepatotoxicity in vitro and in vivo by specifically investigating DNA methylation, histone 3 lysine (K) 9 trimethylation (H3K9me3), and m6A-mediated regulation of p53 expression in human liver (HepG2) cells and C57BL/6 mice livers. FA induced global DNA hypomethylation in HepG2 cells; decreased the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) by inducing promoter hypermethylation and upregulated expression of miR-29b. Further, FA decreased the ubiquitination of DNMT1, DNMT3A, and DNMT3B by decreasing the expression of the ubiquitination regulators, UHRF1 and USP7. FA induced promoter hypomethylation of the demethylase, MBD2 and increased MBD2 expression contributing to global DNA hypomethylation in HepG2 cells. DNA methylation and H3K9me3 function in concert to regulate genome integrity and gene transcription. Sirtuin (Sirt) 1 is a histone deacetylase and direct target of miR-200a that regulates the repressive H3K9me3 mark by post-translationally modifying both H3K9Ac and the histone methyltransferase, SUV39H1. FA upregulated miR-200a and decreased Sirt1 expression in HepG2 cells and C57BL/6 mice livers. FA decreased the expression of SUV39H1 and histone demethylase, KDM4B which led to a decrease in H3K9me3 and an increase in H3K9me1. FA also decreased cell viability via apoptosis as evidenced by the significant increase in the activity of the executioner caspase-3/7. The tumor suppressor protein, p53 regulates cell cycle arrest and apoptosis in response to cellular stress. The expression of p53 is regulated at the transcriptional and post-transcriptional level by promoter methylation and m6A RNA methylation. In HepG2 cells, FA induced p53 promoter hypermethylation and decreased p53 expression. FA also decreased m6A-p53 levels by decreasing the expression of the methyltransferases, METTL3 and METTL14, and the m6A readers, YTHDF1, YTHDF3, and YTHDC2, thereby, decreasing p53 translation. In C57BL/6 mice livers FA, however, induced p53 promoter hypomethylation and increased p53 expression. FA increased m6A-p53 levels by increasing the expression of METTL3 and METTL14; and increased expression of YTHDF1, YTHDF3, and YTHDC2 increased p53 translation. In conclusion, this study provides evidence for alternative mechanisms of FA-induced hepatotoxicity (in vitro and in vivo) by modulating DNA methylation, H3K9me3, m6A RNA methylation, and epigenetically regulating p53 expression ultimately leading to genome instability and apoptotic cell death. These results provide insight into a better understanding of FA induced hepatic toxicity at the epigenetic and cellular level and may assist in the development of preventative and therapeutic measures against FA toxicity. It also suggests that exposure to FA may lead to the onset of human diseases via epigenetic changes/modifications. This is particularly relevant in under privileged communities where the food supply and storage conditions are inadequate.
Fumonisin B2 induces mitochondrial stress and mitophagy in Hek293 cells.
(2019) Mohan, Jivanka.; Chuturgoon, Anil Amichund.; Sheik-Abdul, Naeem.
Food insecurity poses a significant socio-economic problem in third world economies, particularly in countries that rely heavily on maize and maize products. Ubiquitous soil fungi parasitize agricultural commodities and produce mycotoxins. Fumonisin B2 (FB2), a neglected mycotoxin, is produced by several Fusarium species. The aim of this study was to investigate mitochondrial stress responses in human embryonic kidney (Hek293) cells exposed to FB2 for 24 hours (hr). Cell viability was assessed via the methylthiazol tetrazolium (MTT) assay and the half maximal inhibitory concentration (IC50) value (317.4 μM) was generated. Additional concentrations of 100 μM and 500 μM were selected to achieve a broader toxic profile of FB2. Reactive oxygen species (ROS) was quantified (fluorescence), mitochondrial membrane depolarisation (fluorescence) was assessed and adenosine triphosphate (ATP) concentration was evaluated (luminometry) to assess mitochondrial integrity. The relative expression of mitochondrial stress response proteins, Sirtuin 3 (SIRT3), Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), LON protease (LONP1), PTEN-induced putative kinase 1 (PINK1), ubiquitin-binding adaptor p62 (p62) and heat shock protein 60 (HSP60) was determined by western blots. Transcript levels of SIRT3, PINK1 and microRNA-27b (miR-27b) was assessed using quantitative PCR (qPCR). Results indicated that both low and high concentrations of FB2 that were within the naturally occurring concentration range of the compound were able to induce mitochondrial dysfunction. FB2 (IC50) downregulated mitochondrial stress proteins and upregulated mitophagy markers. Despite upregulation of mitochondrial stress maintenance proteins at the highest concentration (500 μM) of FB2, mitophagic markers increased with subsequent cell death; whilst at a lower concentration (100 μM) of FB2, mitochondrial stress protein expressions were upregulated resulting in decreased expression of mitophagic markers and cell proliferation. In conclusion, FB2 was cytotoxic to the kidney derived Hek293 cells via induction of mitochondrial stress and mitophagy. Keywords: Fumonisin B2, mitophagy, mitochondrial stress, PINK1, Nrf2, SIRT3, human kidney cells, microRNA
Aflatoxin B1 modulates oxidative stress and apoptosis in human embryonic kidney cells.
(2019) Dlamini, Nomali Zanele.; Khan, Rene Bernadette.
Introduction: Aflatoxin B1 (AFB1) is produced by filamentous fungal strains of Aspergillus flavus and Aspergillus parasiticus that infect field crops, therefore AFB1 is a frequent contaminant of dietary staples such as rice, maize and peanuts. Humans and animals are exposed to AFB1 through consumption of contaminated foods, predisposing them to various diseases. AFB1 is a potent hepatotoxin that has been classified by the International Agency of Research on cancer (IARC) as a group1 carcinogen. The carcinogenic effects of AFB1 have been attributed to the metabolism of this toxin to an epoxide that promotes the production of free radicals, mitochondrial toxicity and induction of cell death. With the increasing prevalence of kidney associated diseases in humans, and the AFB1-associated kidney toxicity observed in animals, this study investigated the cytotoxic effects/mechanism of AFB1 in human embryonic kidney (Hek293) cells. Methods: Hek293 cells were exposed to AFB1 (0-100μM) for 24hrs. The effect on cell viability was assessed using the methylthiazol tetrazolium (MTT) assay, which also produced the half maximal inhibitory concentration (IC50) used in subsequent assays. Free radical production was evaluated by quantifying malondialdehyde (MDA) and nitrate concentration, while DNA fragmentation was determined using the single cell gel electrophoresis (SCGE) assay and DNA gel electrophoresis. Damage to cell membranes was ascertained using the lactate dehydrogenase (LDH) assay. The concentration of ATP, reduced glutathione (GSH), necrosis, annexin V and caspase activity was measured by luminometry. Western blotting and quantitative PCR was used to assess the expression of proteins and genes associated with apoptosis and oxidative stress. Results and discussion: The MTT assay revealed a reduction in cell viability of Hek293 cells as the AFB1 concentration was increased, with a half maximum inhibitory concentration (IC50) of 32.60 μM. The decreased viability corresponded to decreased ATP concentration. The upregulation of Hsp70 indicated that oxidative stress was induced in the AFB1-treated cells. While this implies an increased production of free radicals, the accompanying upregulation of the antioxidant system indicates the activation of defense mechanisms to prevent cellular damage. Thus, membrane damage associated with increased radical formation was prevented as indicated by the reduced LDH release and necrosis. In addition, cytotoxic effects were evident as AFB1 activated the intrinsic pathway of apoptosis with corresponding increased DNA fragmentation, p53 and Bax upregulation and increased caspase activity, but externalisation of phosphatidylserine (PS), a major hallmark of apoptosis, did not occur in AFB1 treated Hek293 cells. Conclusion: The results suggest that AFB1 induced oxidative stress leading to cell death by the intrinsic pathway of apoptosis in Hek293 cells. Keywords : Aflatoxin B1 (AFB1), oxidative stress, apoptosis, Hek293 cells
Fumonisin B1-induced oxidative stress in human liver (HepG2) cells – an alternate mechanism of carcinogenesis.
(2017) Arumugam, Thilona.; Chuturgoon, Anil Amichund.; Nagiah, Savania.
Abstract available in pdf.
An investigation into the clinical outcomes of women with peripartum cardiomyopathy at Klerksdorp/Tshepong Hospital Complex.
(2015) Sigauke, Farai Russell.; Nyamande, Kennedy.
Introduction: Peripartum Cardiomyopathy (PPCM) is defined on the basis of 4 criteria:  The development of cardiac failure in the last month of pregnancy and up to 5 months after delivery;  Absence of an identifiable cause of cardiac failure,  Absence of a recognizable structural heart disease prior to the last month of pregnancy;  And left ventricular dysfunction demonstrated by classic echocardiographic criteria, such as depressed fractional shortening <30% or ejection fraction <45% and left ventricular end diastolic dimension >2.7 cm/m2. The incidence of PPCM is not always known because population-based estimates are not available as data is primarily based on case series from single centres. In the USA it ranges from 1 per 3000 to 1 per 4000. Some studies conducted in South Africa showed an incidence of 100-300 per 100 000 live births. Aim of the study: The aim of the study was to investigate the factors which contribute to the clinical outcomes of peripartum cardiomyopathy (PPCM) at Klerksdorp/Tshepong Hospital Complex in Klerksdorp South Africa, by means of quantitative research. Methods: A single centre, non randomised, retrospective cohort, chart review together with prospective assessment of patient outcomes was done on 34 patients attending Specialist Medical Outpatient Department Clinic at Klerksdorp/Tshepong Hospital Complex from January 2011 to September 2014. The patients would have delivered between January 2011 and March 2014. Recruitment followed the inclusion and exclusion criteria based on the definition of peripartum cardiomyopathy. The patient files were reviewed at the time of diagnosis and at six months looking at the background history, therapy, clinical progression and outcome. The last assessment was done with the patient. Demographic data, obstetric and medical history, clinical progression measured by serial signs and symptoms were collected. Chest radiograph, electrocardiograph and echocardiograms were also registered and correlated to the clinical outcome. Findings: A total of 38 patients were recruited with a diagnosis of peripartum cardiomyopathy. Four patients were excluded from the study. Of the 34 patients who completed the study 47% recovered, whilst 26.5% remained stable and 26.5% progressively declined. Three patients, 8.8% died. Thromboembolic phenomena were noted in 20.6% of patients whilst 33.3% were on anticoagulants. The period prevalence was 0.33% (3 per thousand live births). The mean proportion in different clinical outcome groups was compared using Tukey’s Studentised Range (HSD) test for result. There was a significant difference in the mean proportions between the three groups (p=0.0001). In the pairwise comparisons, the mean proportion of the recovered group was significantly higher than that of the stable (p<0.05). There was no difference in the mean proportion of the recovered and deteriorated groups (p>0.05). All the patients received standard cardiac failure treatment. Discussion: The prevalence of PPCM at Klerksdorp/Tshepong Hospital Complex was higher than other parts of South Africa. The clinical outcome distribution from the study faired with the reported 50% recovery, 25% stable and 25% progressive deterioration. The patients received standard medical therapy. Low usage of anticoagulants could have attributed to the high rate of thromboembolic events. Device therapy is indicated in refractory heart failure if resources permit. It was recommended that a high-quality, large, multicenter prospective study be conducted to better understand the clinical outcomes of PPCM and its influencing factors. Conclusion: PPCM is a rare condition, multifactorial in origin with a good clinical outcome in the majority of cases if treated appropriately. It should be considered in any patient who presents with acute dyspnea in the perinatal period. Patients with PPCM are being optimally treated at Klerksdorp/Tshepong Hospital Complex. Anticoagulation is recommended in patients with low ejection fractions. Further, large progressive studies are required to fully understand the aetiology and the effect of novel therapies.
Moringa oleifera crude aqueous leaf extract induces apoptosis in human hepatocellular carcinoma cells via the upregulation of NF-kB and IL-6/STAT3 pathway.
(2016) Shunmugam, Letitia.; Chuturgoon, Anil Amichund.; Tiloke, Charlette.
Abstract available in PDF file.
Fusaric acid induces DNA damage and post-translational modification Of p53 in hepatocellular carcinoma (HepG2) cells.
(2016) Ghazi, Terisha.; Chuturgoon, Anil Amichund.
Abstract available in PDF file.

Browse

Recent Submissions