Medical Biochemistry

Permanent URI for this communityhttps://hdl.handle.net/10413/7035

Browse

Now showing 1 - 20 of 68

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.
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
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.
Analysis of a multidrug resistant acinetobacter SPP. outbreak in the intensive care unit of King Edward VIII Hospital.
(2000) Deedat, Fathima.; Sturm, Adriaan Willem.
The study arose out of a need to investigate and control a nosocomial outbreak caused by multidrug resistant Acinetobacter spp in the fifteen-bed intensive care unit of King Edward VIII Hospital. Following the discovery of the index case, four other patients were found to have a similar strain of Acinetobacter spp. All fifteen patients in the ward were subsequently screened for the organism. Forty-seven isolates were obtained from 12 patients. Eight of the patients were infected with the organism and six of these eight patients subsequently died. Swabs from the ward environment were also screened for the organism, which was found in patients' baths, suction water and urine collection jars. The outbreak was aborted by the use of strict infection control techniques. Minimum inhibitory concentrations (MICs) of 20 of the 47 isolates were determined for the following antimicrobials: imipenem, ciprofloxacin, gentamicin, amikacin, netilmycin,cefotaxime, ceftazidime and tetracycline. The same 20 isolates were further typed using ribotyping. Seven different antibiogram patterns were obtained using the MIC data. The majority of isolates (11) fit into a Single type, and showed resistance to all drugs tested, except for susceptibility to tetracycline and netilmycin only. Ribotyping revealed 5 different types. There were 9 isolates of ribotype a, 2 of ribotype b, 3 of ribotype c, 5 of ribotype d and 1 of ribotype e. In conclusion, this study describes a nosocomial outbreak with a multidrug resistant Acinetobacter spp. in an intensive care unit. The results showed that there was no correlation between the two typing methods used, ribotyping was more discriminatory than antibiogram types, with the majority of strains belonging to two different ribotypes.
Antibiotic resistance in the food chain : a case study of Campylobacter spp. in poultry.
(2013) Bester, Linda Antionette.; Essack, Sabiha Yusuf.
The sub-therapeutic use of antibiotics for growth promotion in food animal production, has engendered substantial debate on the dissemination of antibiotic resistance via the food chain, specifically, the probability of antibiotic use in food production creating a reservoir of resistant bacteria and/or resistance genes that may spread to humans thereby limiting the therapeutic value of antimicrobial drugs. In the absence of any surveillance programme on food-borne bacteria in South Africa, this study focussed on Campylobacter spp. in poultry and encompassed a literature review on the prevailing debate on the dissemination of antibiotic resistance via the food chain, a phenotypic observational study on the prevalence and antibiotic resistance profiles of Campylobacter spp. isolated within and across different poultry farming systems and a genotypic component that covered identification methods, plasmid profile determination and strain typing. Identification methods for Campylobacter spp., viz, biochemical tests and matrix assisted laser desorption ionization- time of flight (MALDI-TOF) mass spectrometry was compared to the PCR which is considered the gold standard as a molecular method of identification. The MALDI-TOF was shown to be superior to the biochemical tests for the identification of C. coli but equivalent to the biochemical tests for C. jejuni. Of the 363 samples collected in total, the frequency of thermophilic Campylobacter was 68 % in rural farms (or informally reared poultry), 47 % in both commercial free-range and industrial broilers and the highest in industrial layers at 94 %. Antibiotic resistance analysis showed that isolates from the rural farming systems were significantly (P < 0.01) more susceptible to ciprofloxacin, tetracycline and erythromycin when compared to the other farming systems. Significant (P < 0.001) antibiotic resistance differences were detected between broilers (5 - 8 week lifespan), and layers (36 - 52 week lifespan) for gentamicin, ciprofioxacin and tetracycline. Plasmids were fonnd be harboured by isolates in all the farming systems; in 84 % of isolates from free-range broilers, 77 % of isolates from industrial broilers, 83 % of isolates from industrial layer hens and 75 % of isolates from the rural farming system. The PFGE genotyping of 42 Campylobacter isolates generated 39 SmaI types. Substantial and substantive genetic diversity was observed between and within farming systems. The lack of correlations amongst the parameters within and between farming systems attested to the diversity and complexity of phenotypes and genotypes and indicated de novo evolution in response to antibiotic selection pressure and animal husbandry practices.
The antiproliferative and apoptosis inducing effects of Moringa oleifera aqueous leaf extract and its synthesised gold nanoparticles - modulation of oncogenes and tumour suppressor genes in human cancer cell lines.
(2015) Tiloke, Charlette.; Chuturgoon, Anil Amichund.; Phulukdaree, Alisa.
Cancer is one of the leading causes of global mortality. In South Africa (SA), the burden of cancer (lung and oesophageal) continues to increase. Moringa oleifera (MO), indigenous to India, is found widely in SA and used in traditional treatments of cancer. Gold nanoparticles (AuNP’s) are showing potential in cancer therapies and can be synthesised using plants extracts such as MO leaf extract (MOE). This study investigated the antiproliferative effect of MOE and AuNP’s synthesised from MOE (MLAuNP) in A549 lung and SNO oesophageal cancer cells. MO crude aqueous leaf extract was prepared and cytotoxicity (MTT assay) was assessed in A549, SNO cells and normal peripheral blood mononuclear cells (PBMCs) (24h). Oxidative stress, DNA fragmentation and apoptotic markers were determined. A one-pot green synthesis technique using MOE to synthesise MLAuNP was then conducted. A549, SNO cells and PBMCs were also exposed to MLAuNP and CAuNP to evaluate cytotoxicity and apoptotic markers. MOE was cytotoxic to A549 cells. MOE (IC50: 166.7μg/ml, 24h) significantly increased lipid peroxidation, decreased glutathione (GSH) and Nrf2 levels leading to DNA fragmentation. MOE induced apoptosis by significantly increasing p53, caspase-9, enhancing caspase-3/7 activities and Smac/DIABLO expression. MOE significantly cleaved PARP-1 into 89kDa and 24kDa fragments. MOE was not cytotoxic to PBMCs but in SNO cells (IC50: 389.2μg/ml, 24h), it significantly increased lipid peroxidation, DNA fragmentation, decreased GSH, catalase and Nrf2 levels. Apoptosis was confirmed by the significant increase in phosphatidylserine (PS) externalisation, caspase-9, enhanced caspase-3/7 activities and significant decrease in ATP levels. MOE significantly increased p53, Smac/DIABLO and cleavage of PARP-1, resulting in an increase in the 24kDa fragment. MLAuNP was successfully synthesised. MLAuNP and CAuNP were not cytotoxic to PBMCs, whilst its pro-apoptotic properties were confirmed in A549 (IC50: MLAuNP - 98.46μg/ml; CAuNP - 121.4μg/ml) and SNO (IC50: MLAuNP - 92.01μg/ml; CAuNP - 410.4μg/ml) cells. MLAuNP significantly increased caspase activity in SNO cells while MLAuNP significantly increased PS externalisation, mitochondrial depolarisation, caspase-9, caspase-3/7 activities and decreased ATP levels. Also, MLAuNP significantly increased p53, Bax, Smac/DIABLO, PARP-1 24kDa fragment and enhanced SRp30a levels. Conversely, MLAuNP significantly decreased Bcl-2, Hsp70, Skp2, Fbw7α, c-myc levels and activated alternate splicing with caspase-9a splice variant being increased. These findings indicate that MOE exerts antiproliferative effects in cancerous A549 and SNO cells by increasing oxidative stress, DNA fragmentation and inducing apoptosis. MLAuNP also possessed antiproliferative properties in SNO cells and induced apoptosis in A549 cells by modulating oncogenes, tumour suppressor genes and activating alternate splicing of caspase-9. MOE and MLAuNP showed potential use as a complementary and alternative treatment for lung and oesophageal cancer. MOE fractionation studies are further recommended to identify the bioactive compounds responsible for the antiproliferative effect seen in A549 and SNO cells. In addition, membrane transport proteins as well as cell cycle analysis will provide further insight into MOE and MLAuNP antiproliferative effect.
Antiproliferative effect of a novel synthesized carbazole compound on A549 lung cancer cell line.
(2014) Molatlhegi, Refilwe P.; Chuturgoon, Anil Amichund.
Increased death rates due to lung cancer have necessitated the search for potential novel anticancer compounds such as carbazole derivatives. Carbazoles are aromatic heterocyclic compounds with anticancer, antibacterial and anti-inflammatory activity. The study investigated the ability of the novel carbazole compound (Z)-4-[9-ethyl-9aH-carbazol-3-yl) amino] pent-3-en-2-one (ECAP) to inhibit the proliferation of lung cancer cells and its mechanism of action. ECAP was synthesized as a yellow powder with melting point of 240-247 °C. The 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lipid peroxidation and comet assays were used to assess the anti-proliferative effects of the compound on A549 lung cancer cell line. Protein expression was determined using western blots, apoptosis was measured by luminometry for caspase-3/7, -8 and -9 and flow cytometry was used to measure phosphatidylserine externalisation. ECAP induced a p53 mediated apoptosis of lung cancer cells by significantly down-regulating the expression of antioxidant defense proteins, Hsp70 (p < 0.02) and Bcl-2 (p < 0.0006), thereby up-regulating reactive oxygen species (ROS) production. This resulted in DNA damage (p < 0.0001) and subsequent up-regulation of Bax and caspase activity consequently inducing apoptosis of lung cancer cells. These results demonstrate the potential anticancer effects of ECAP on cultured lung cancer cells. However, further investigation and characterization is required to fully understand the possible use of carbazole compound (Z)-4-[9-ethyl-9aH-carbazol-3-yl) amino] pent-3- en-2-one as potential lung cancer treatment.
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.
An assessment of gene polymorphisms in young South African Indians with coronary artery disease and the effect of atorvastan in vitro.
(2012) Phulukdaree, Alisa.; Chuturgoon, Anil Amichund.
The global burden of heart disease increases every year. It has been estimated that by the year 2020, coronary artery disease (CAD) will be the number one cause of death worldwide. Indian populations throughout the world have the highest prevalence of CAD and early onset of the disease compared to other ethnic groups. 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 CAD. Uncoupling proteins (UCPs) 2 and 3 play an important role in the regulation of oxidative stress which contributes to chronic inflammation. Coronary artery disease is a chronic inflammatory disorder characterized by elevated levels of C-reactive protein (CRP) and pro-inflammatory cytokines such as interleukin 6 (IL-6). Polymorphisms of these genes have been linked to CAD and other chronic diseases. Statins, metabolised in the liver, are the most commonly used drug to control atherosclerosis progression in CAD patients. The pleiotropic effects of statins have been attributed to both favourable and adverse outcomes in CAD patients particularly related to myopathy and hepatotoxicity. All patients (n=102) recruited into this study were South African Indian males. A corresponding age-, gender- and ethnicity-matched control group (n=100) was also recruited. The frequency of the GSTM1 +/0, GSTP1 A105/G105, IL6 -174G/C and CRP -390C/A/T genotypes was assessed by polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP). For the in vitro study, the biological effect of atorvastatin on HepG2 cells was assessed. The metabolic activity, cytotoxicity, oxidative stress and nitric oxide production was assessed by the ATP, lactate dehydrogenase (LDH), thiobarbituric acid reactive substance (TBARS) and Griess assays, respectively. The profile of 84 microRNA (miRNA) species was evaluated using the miRNA Pathway Finder PCR SuperArray. The predicted targets of up-regulated miRNAs were determined using the online software, Targetscan. The mRNA levels of guanidinoacetoacetate (GAMT), arginine glycine aminotransferase (AGAT) and spermine oxidase (SMO) were determined using quantitative PCR. Western blotting was used to determine GAMT and phosphorylated p53 levels in treated cells. The GSTM1 0/0 and GSTP1 A105/A105 genotypes occurred at higher frequencies in CAD patients compared with the control group (36% vs. 18% and 65% vs. 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). The UCP2 -866G/A and UCP3 -55C/C genotypes occurred at highest frequency in CAD patients (59% vs. 52% and 66% vs. controls: 63% respectively) and did not influence the risk of CAD. Homozygous UCP3 -55T/T genotype was associated with highest fasting glucose (11.87±3.7mmol/L vs. C/C:6.11±0.27mmol/L and C/T:6.48±0.57mmol/L, p=0.0025), HbA1c (10.05±2.57% vs. C/C:6.44±0.21% and C/T:6.76±0.35%, p=0.0006) and triglycerides (6.47±1.7mmol/Lvs. C/C:2.33±0.17mmol/L and C/T:2.06±0.25mmol/L, p<0.0001) in CAD patients. A significant association between the G allele of the IL6 -174 polymorphism and non-diabetic CAD patients was found (p=0.0431 odds ratio: 1.307, 95% CI: 1.047-1.632). A significant association with the C allele of the -390 CRP triallelic variants and CAD (p=0.021 odds ratio: 1.75, 95% CI: 1.109-2.778) was also found using a contingency of the C allele vs. the minor A and T allele frequencies. The strength of the association of the C allele with non- diabetic CAD subjects was much higher (p=0.0048 odds ratio: 2.634, 95% CI: 1.350-5.138). Circulating median levels of IL-6 (0.9 (0.90, 0.91) pg/ml and 0.9 (0.87, 0.92) pg/ml) and CRP (5.65 (1.9, 8.2) mg/l and 2.90 (1.93, 8.35) mg/l) were similar between CAD patients and controls, respectively. A similar finding was observed between controls and non-diabetic CAD subjects. Levels of IL-6 and CRP in CAD subjects were not significantly influenced by polymorphic variants of IL-6 and CRP. In the control group, the level of IL-6 was significantly influenced by the IL6 -174 G allele (p=0.0002) and the CRP -390 C allele (p=0.0416), where subjects with the homozygous GG (0.9 (0.9, 1,78) pg/ml) and CC (0.9 (0.9, 0.95) pg/ml) genotype had higher levels than the C allele carriers (0.9 (0.64, 0.91) pg/ml) or A and T carriers (0.9 (0.69, 0.91) pg/ml) combined. The lowest measure of proliferation/metabolism in HepG2 cells was observed at 20μM atorvastatin, with 82±9.8% viability. The level of cytotoxicity was increased in statin treated cells from 0.95±0.02 units to 1.11±0.03 units (p=0.001) and malondialdehyde levels was reduced from 0.133±0.003 units to 0.126±0.005 units (p=0.009) whilst nitrite levels were elevated (0.0312±0.003 units vs. control: 0.027±0.001 units, p=0.044). MicroRNAs most significantly upregulated by atorvastatin included miR-302a-3p (3.05-fold), miR-302c-3p (3.61-fold), miR-124-3p (3.90-fold) and miR-222-3p (4.4-fold); miR-19a-3p, miR-101-3p and let-7g were downregulated (3.63-fold, 2.92-fold, 2.81-fold, respectively). A list of miRNA targets identified included those with a role in metabolism and inflammation. The miR-124a specifically targets the mRNA of GAMT and SMO.
Atorvastatin induces apoptosis via the P13K/AKT signalling pathway in human hepatocellular carcinoma (HEPG2) cells.
(2016) Docrat, Taskeen Fathima.; Chuturgoon, Anil Amichund.
Abstract available in PDF file.
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.
A biochemical assessment of Ochratoxin A stress responses in vitro, with resveratrol as a possible therapeutic intervention.
(2017) Raghubeer, Shanel.; Chuturgoon, Anil Amichund.
Abstract available in PDF file.
A biochemical assessment of stress response following acute and prolonged exposure to antiretroviral drugs (nucleoside reverse transcriptase inhibitors) in vitro.
(2015) Nagiah, Savania.; Chuturgoon, Anil Amichund.; Phulukdaree, Alisa.
Nucleoside reverse transcriptase inhibitors (NRTIs) are the most extensively used antiretroviral (ARV) drugs in highly active antiretroviral therapy (HAART). The long term use of HAART is associated with changes to metabolic parameters leading to lipodystrophy and metabolic syndrome, as well as toxicity to high energy demand organs e.g. liver, kidney, heart, and nervous system. Underlying the myriad of NRTI-associated adverse health outcomes is mitochondrial (mt) toxicity. Although inhibition of mtDNA synthesis was one of the first identified mechanisms of toxicity, it did not provide a holistic explanation for all NRTIs. Furthermore, variations in adaptive stress responses were observed following acute and chronic exposure to NRTIs. Insight gained from the molecular changes induced by NRTIs will enable effective management and limit adverse health outcomes. The human hepatoma (HepG2) cell line was used as an in vitro model to investigate changes to mt function, cellular redox status, and antioxidant response following acute [24 hour (h)] and prolonged (120 h) exposure to NRTIs – Zidovudine (AZT, 7.1μM); Stavudine (d4T, 4μM); Tenofovir (TFV, 1.2μM). Long term exposure to AZT and d4T reduced mtDNA levels (120h, AZT: 76.1%; d4T:36.1%, p<0.05) and mt function was compromised as evidenced by reduced ATP levels (AZT: 38%; d4T: 56.4%) and increased mt membrane depolarisation (p<0.02). Tenofovir compromised mt function at 120 h independently of depleting mtDNA levels. Oxidative stress parameters were significantly elevated by AZT and TFV at 24h; and all NRTIs at 120 h (p<0.05). Endogenous antioxidant response was highest in TFV in both time periods (120h; p<0.05). Once NRTI induced oxidative stress in HepG2 cells was established, protein homeostatic response to oxidative stress was investigated. Lon protease expression and related endoplasmic reticulum (ER) stress was evaluated. The data showed that ATP-dependent protein homeostasis responses Lon, heat shock protein 60 (HSP60) and ER stress were significantly increased at 24 h (>2 fold); but significantly decreased at 120 h for all NRTIs (p<0.005). The compromised ATP-dependent stress response then led to the assessment of an ATP- dependent drug transporter responsible for efflux of xenobiotics in hepatocytes. The transporter, ATP-binding cassette C4 (ABCC4), is regulated by microRNA (miR-) 124a. Regulation of ABCC4 by miR-124a has implications for bio-accumulation and resultant toxicity. An inverse relationship between miR-124a and ABCC4 mRNA levels in all treatments at both time periods was observed. All NRTIs elevated miR-124a levels at 24 h (p=0.0009) and this observation was consistent in d4T and TFV treated HepG2 cells at 120 h (p<0.0001). This was accompanied with a concomitant decline in ABCC4 mRNA levels (p<0.0001) relative to the control. Prolonged exposure to AZT caused a decrease in miR-124a and elevated ABCC4 mRNA levels. Protein expression of multi-drug resistance protein 4 (MRP4), coded for by ABCC4, did not correlate to mRNA expression. At 120 h, all NRTIs caused significant depletion of MRP4 (possibly due to oxidative cell membrane damage or ATP depletion). In conclusion, all three NRTIs compromised mt function and induced oxidative damage in HepG2 cells, with greater toxicity over 120 h. Reduced ATP levels compromised the ATP-dependent stress response proteins and xenobiotic detoxification. Tenofovir could be considered a safer alternative as it elicited the highest antioxidant response in spite of reduced mt function.
A Biochemical assessment of the potential of Spirulina Platensis to Ameliorate the adverse effects of highly active Antiretroviral therapy In Vitro.
(2022) Sibiya, Thabani.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
The human immunodeficiency virus (HIV) has been one of the prevalent causes of diseases on a global scale over four decades of its emergence. It is estimated that about 37.7 million people are infected with HIV globally, and 8.2 million persons are in South Africa. The highly active antiretroviral therapy (HAART) involves combining various types of therapies that are dependent on the infected person’s viral load. HAART helps to regulate the viral load and prevents its associated symptoms from progressing into acquired immune deficiency syndrome (AIDS). Despite its success in prolonging HIV-infected patients' lifespan, the long-term use of HAART promotes metabolic syndrome (MetS) through an inflammatory pathway, excess production of reactive oxygen species (ROS), and mitochondrial dysfunction. Interestingly, Spirulina platensis (SP), a blue-green microalga commonly used as a traditional food by Mexican and African people, has been demonstrated to mitigate MetS by regulating oxidative stress and inflammation. This study examined the protective role of SP against HAART-induced oxidative stress and inflammation in human hepatoma (HepG2) liver cells. The first published manuscript (appendix A) is a literature review on the potential of SP to ameliorate adverse effects of HAART: An update focusing on highlighting the potential positive synergistic effects of SP and HAART. This review provides introductory background of spirulina and its protective attributes. Thereafter, a study in an in vitro model was carried out by measuring oxidative stress, antioxidant, and inflammation markers. The HepG2 cell line was used as an in vitro model. Changes were investigated in cellular redox status, inflammation, and antioxidant response. The data analysis followed prolonged [96 hours (hrs)] exposure to HAART and acute (24 hrs) exposure to SP. HAART (Lamivudine (3TC): 1.51 μg/ml, tenofovir disoproxil fumarate (TDF): 0.3 μg/ml and Emtricitabine (FTC): 1.8 μg/ml) in HepG2 cells was investigated for 96 hrs and thereafter, treated with 1.5 μg/ml SP for 24 hrs. The HepG2 cells that served as control contained complete culture medium (CCM) only. 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) assay was used to determine cell viability following SP treatment. Cellular redox status was assessed using the quantification of intracellular reactive oxygen species (ROS), lipid peroxidation, and lactate dehydrogenase (LDH) assay. The fluorometric, JC-1 assay was used to determine mitochondrial polarisation. Protein expression was determined using western blots. Quantitative Polymerase Chain Reaction (qPCR) was also employed for micro-RNA and gene expressions. The findings from these investigations led to further analyses as depicted and described in our second, third, and fourth manuscripts. In the second published manuscript (chapter three), antioxidant markers and Nuclear erythroid 2 related factor 2 (NRF-2), a key regulator of antioxidants, was investigated. The results show that SP exposure induces an antioxidant response. The results further reveal that prolonged exposure with HAART followed by SP treatment induced an antioxidant response through upregulating NRF-2 (p < 0.0001), CAT (p < 0.0001), and NQO-1 (p < 0.0001) mRNA expression. Furthermore, NRF-2 (p = 0.0085) and pNRF-2 (p < 0.0001) protein expression was upregulated in the HepG2 cells postexposure to HAART-SP. In the third manuscript (chapter four), microRNAs and genes involved in inflammatory response were analysed. SP prevented the inhibition of microRNAs involved in the regulation of inflammation. MiR- 146a (p < 0.0001) and miR-155 (p < 0.0001) levels increased in SP treated cells. However, only miR- 146a (p < 0.0001) in HAART-SP indicated an increase, while miR-155 (p < 0.0001) in HAART-SP treatment indicated a significant decrease expression. SP may mitigate the inhibition of selected miRNAs that regulate inflammation in HAART treated HepG2 cells. Further, analysis revealed that Cox-1 mRNA expression was significantly increased in HAART-SP treated cells (p < 0.0001). Moreover, HepG2 cells exposed to HAART-SP treatment showed a significant decreased Cox-2 (p < 0.0001) expression, therefore, SP potentially controls inflammation by regulating microRNA and gene expressions. Moreover, the positive synergistic effect is indicated by normalised intracellular ROS levels (p < 0.0001) in HAART-SP treated cells. In the fourth manuscript (chapter five), it was shown how SP mitigates inflammation induced by HAART in HepG2 liver cells. SP inhibits the inflammatory pathway, by significantly decreasing iNOS (p < 0.0001), IκB-α (p < 0.0001), NF-κB (p < 0.0001), IL-1β (p = 0.0002) and TNF-α (p = 0.0074) mRNA levels. The HAART-SP post treatments reduced inflammation as evidenced by decreased mRNA levels of NF-κB (p < 0.0001), IL-1β (p < 0.0001), IL-12 (p < 0.0001), TNF-α (p < 0.0001). Furthermore, NF-κB (p < 0.0001) protein expression was downregulated. Thus, SP has the potential to inhibit inflammation induced by HAART (3TC, TDF and FTC) in HepG2 cells. Finally, the overall results show that SP mitigates HAART-adverse drug toxicity in HepG2 cells, by activating the antioxidant response in HepG2 cells.
Biochemical, apoptotic and cellular stress studies in rheumatoid arthritis.
(2009) Moodley, Devapregasan.; Mody, Girish Mahasukhlal.; Chuturgoon, Anil Amichund.
Abstract available in PDF file.
Biotyping Saccharomyces cerevisiae strains using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS)
(2011) Moothoo-Padayachie, Anushka.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.; Govender, Thavendran.; Govender, Patrick.
In clinical diagnosis and fermentation industries there is a need for a method that allows for the differentiation of yeast to the strain level (biotyping). The ideal biotyping method should be accurate, simple, rapid and cost-effective, and capable of testing a large number of yeast isolates. Matrix assisted laser desorption/ionization time of flight mass spectrometry has emerged as a powerful biotyping tool for the identification of bacteria and clinical yeast isolates, mainly Candida. It has been found that the MALDI-TOF MS signals from yeast are harder to obtain than from bacteria. It has been reported by several research studies that a cell lysis step is required to obtain a mass spectral signal for clinical Candida strains. To date an optimized sample preparation protocol has not been devised for the biotyping of S. cerevisiae strains. Studies on the identification of yeast using MALDI-TOF MS have focused primarily on clinical Candida yeast isolates but have included very few S. cerevisiae strains. Furthermore these yeast identification studies using MALDI-TOF MS have only achieved identification to the species and not strain level. A major limiting attribute of MALDI-TOF MS for the accurate identification of microbes, is its dependency on a comprehensive mass spectral database. Bruker Daltonics is a pioneer and leader in providing innovative life science tools based on mass spectrometry thus the Bruker Daltonics mass spectral database and state-of-the-art instruments and accompanying software were selected for this study. The Bruker Daltonics mass spectral database currently holds three thousand seven hundred and forty microorganisms of which only a mere seven are S. cerevisiae strains. Initially in this study, a number of parameters of a generic ethanol/formic acid protein extraction procedure as originally described by Bruker Daltoincs were considered in the development of a sample preparation protocol that yielded characteristic and highly reproducible MALDI-TOF mass spectra. The parameters considered included cell number, alcohol fixation, matrix solution and media. It was found that using the optimized sample preparation protocol unique and highly reproducible mass spectral profiles were obtained for all three S. cerevisiae strains. Multivariate analysis confirmed that the differences between all three S. cerevisiae strains were statistically significant. For quality assurance, the spectra of the three strains were sent for evaluation by Bruker Daltonics and were deemed suitable for the purpose of biotyping. The newly created ethanol/formic acid extraction procedure was used to generate an S. cerevisiae mass spectral database comprising of forty-five S. cerevisiae strains within a local context but also of global significance. The accuracy of the mass spectral database was assessed using blind coded S. cerevisiae strains obtained from the Agricultural Research Council Infruitec-Nietvoorbij (Institute for Deciduous Fruit, Vines and Wine), Stellenbosch, South Africa. It was found that S. cerevisiae identification to the species and more importantly strain level was achievable with relatively good accuracy. To determine the potential application of MALDI-TOF MS as an accurate method for S. cerevisiae strain identification in industry, blind coded S. cerevisiae strains were obtained from Natal Cane Products and subjected to MALDITOF MS analysis. It was found that four of the pure cultures submitted were correctly identified to the strain level and the three S. cerevisiae strains incorrectly identified may have been contaminants or the result of incorrect optimization conditions for the fermentation. Thus MALDITOF MS was shown to be an accurate identification tool, that may also be used to detect contaminants or incorrect environmental conditions which can result in substantial losses.
Characterisation of Fumonisin B1 toxicity in a cancerous liver cell line- induction of tissue transglutaminase and the endoplasmic reticulum stress pathway.
(2013) Anderson, Samantha Mary.; Chuturgoon, Anil Amichund.; Phulukdaree, Alisa.
Fumonisin B1 (FB1) is a mycotoxin which is well characterised as a contaminant of maize and maize-based products worldwide, especially in South Africa. Its toxic effects have been associated with hepatotoxicity, nephrotoxicity and carcinogenicity. Tissue transglutaminase (TG2) is a unique and ubiquitous enzyme that catalyses the post-translational modification of proteins and has GTPase activity. Tissue transglutaminase is an important enzyme in a number of biological processes such as cell differentiation and proliferation, extracellular matrix organisation, cell signalling and apoptosis. This study investigated the possible role of TG2 induction by FB1 and the effect FB1 toxicity has on the endoplasmic reticulum (ER) stress pathway in HepG2 cells. A SDS-PAGE adaption of the TG2 activity assay confirmed TG2 crosslinking activity by FB1 incorporation into fibronectin in the presence of calcium and TG2. This interaction was validated using fluorescent microscopy where FB1 incorporated into the HepG2 cell’s cytoplasmic vesicles and plasma membrane. The up-regulation of TG2 in HepG2 cells treated with FB1 was further investigated using western blotting and showed increased TG2 up-regulation. Fumonisin B1 disrupts membrane-bound sphingolipids as a mechanism of toxicity; FB1 was shown to cause cytoskeletal damage and disrupted the cell’s membranes leading to cell stress. The protein kinase RNA-like endoplasmic reticulum kinase (PERK) ER stress pathway was induced as a result of FB1 exposure and investigated using western blotting and quantitative polymerase chain reaction. After 72hours with 50μM and 100μM FB1 total PERK decreased, phosphorylated eukaryotic initiating factor α remained activated with a significant increase in messenger RNA (mRNA) expression (p<0.05) and transcription factor CCAAT-enhancer-binding protein homologous protein mRNA was significantly induced (p<0.05). The involvement of nuclear factor kappa B (NFkB) and TG2 in ER stress induced apoptosis was investigated through western blotting and quantitative polymerase chain reaction. After 72hours, an up-regulation of both nuclear NFkB and nuclear TG2 was observed; with a corresponding significant increase in nuclear TG2 mRNA expression (p<0.05). A significant increase in transcription factor, Sp1 mRNA expression (p<0.05) was observed after 72hours. Data suggests PERK activation leads to NFkB induction and nuclear translocation; which promoted nuclear TG2 transcription. The activation of TG2 resulted in Sp1 crosslinks that could act as potential inducers of FB1 induced apoptosis. Flow cytometry was used to measure apoptosis and mitochondrial depolarisation. Caspase activity was measured using the Caspase-Glo® assays and ATP concentration was measured using CellTitre-GloTM assay. After 72hours caspases 3/7 and 8 showed a significant decrease in activity at 100μM FB1 (p<0.05) and a decrease in caspase 9. After 72hours with 10μM FB1 treatment a significant increase in phosphatidylserine externalisation (p<0.05), a significant decrease in healthy/live cells (p<0.05) and a significant increase in depolarised mitochondria (p<0.05) were observed. There was also a significant increase in Sp1 mRNA expression (p<0.05). However, at 50μM FB1 treatment there was a decrease in phosphatidylserine externalisation, a significant increase in live cells (p<0.05) and a significant decrease in depolarised mitochondria (p<0.05). Data suggests that ER stress persisted in HepG2 cells with no apoptosis or cell recovery occurring at high chronic doses of FB1 whilst ER stress induced apoptosis at low chronic doses of FB1 in HepG2 cells. Fumonisin B1 may be a possible substrate for TG2 crosslinking activity due to its primary amine group, since this mycotoxin has the potential to induce TG2 expression and activation. Further studies are required to determine the role of FB1 in the inositol-requiring protein 1α and activating transcription factor 6 arms of the ER stress pathway.
Current Antiretroviral Drugs- An investigation of metabolic syndrome promotion in HepG2 cells.
(2022) Mohan, Jivanka.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
Metabolic Syndrome (MetS) affects more than 20% of adults globally. Furthermore, the prevalence of MetS in HIV-infected patients on chronic antiretroviral (ARV) therapy continues to rise rapidly. This is alarming as a significant portion of people are HIV-infected worldwide, with the highest incidence experienced in Sub-Saharan Africa. An estimated 21% of people receiving ARV treatment display insulin resistance associated with mitochondrial dysfunction and inflammation. The current study aimed to determine the disruptions of metabolic processes associated with ARV use (Tenofovir disoproxil fumarate (TDF), Lamivudine (3TC) and Dolutegravir (DTG)) following a 120-h exposure period in HepG2 liver cells. Thereafter mitochondrial stress, inflammasome activation and insulin resistance promotion were assessed. Following HepG2 cellular ARV exposure, it was found that mitochondrial stress proteins SIRT3 and UCP2 expressions were significantly suppressed. Due to these aberrations, endogenous cellular attempts to activate the antioxidant responses (pNrf2, SOD2, CAT) and mitochondrial maintenance systems (PINK1 and p62) in selected singular and combinational ARV treatments seemed insufficient. This resulted in lipid oxidative damage and reduced ATP production. These results indicate that ARVs induce mitochondrial dysfunction in liver cells. Furthermore, it was deduced that combinational ARV exposure promoted inflammasome activation at a genomic level. This was seen in increased expression of NLRP3 mRNA expression and caspase-1 activity with coinciding elevation in IL-1β in mRNA expression. Additionally, JNK expression was upregulated, with correlating increases in p-IRS1 protein expression and decreased IRS1 mRNA expression being observed. Consequently, both PI3K and AKT mRNA expression was suppressed, whilst miR-128a expression was significantly upregulated. It can be deduced that the combinational use of ARVs induced mitochondrial dysfunction and subsequently prompted inflammasome activation. This led to dysregulation of the IRS1/PI3K/AKT insulin signalling pathway and the initiation/promotion of insulin resistance. This is further supported through miRNA activation, suggesting possibilities for future studies on in vivo ARV use and related epigenetic changes.
The effects of fumonisin B¹ in preeclampsia.
(2012) Serumula, Metse Regina.; Chuturgoon, Anil Amichund.; Moodley, Jagidesa.
Preeclampsia is the leading cause of foetal and maternal mortality and morbidity in developing countries. In South Africa, maize is a dietary staple for most black African populations and is susceptible to contamination by mycotoxins such as fumonisin B1 (FB1).Fumonisin B1 is a ubiquitous secondary metabolite of Fusarium fungi produced predominantly by Fusarium verticillioides. This mycotoxin shares structural similarities with the backbone of sphingoid bases (sphinganine and sphingosine) which are substrates for the biosynthesis of complex sphingolipids. The mechanism of FB1 toxicity therefore is centred on the disruption of this process. The aim of the present study was to elucidate the possible causal link between FB1 and preeclampsia. Following ethical approval, 20 normotensive and 20 preeclamptic patients were recruited into the study. Blood and placental tissue were collected and processed for further analysis. The presence of FB1 was verified using standard immunohistochemical and electrophoretic techniques. The levels of FB1 and sphingolipids were quantified using high performance liquid chromatography (HPLC). Western blotting was conducted to confirm the presence of FB1 in the serum. Placental tissue apoptosis was evaluated using Hoechst staining and other markers. Lipid peroxidation was measured in serum and placental tissue of both groups. Fumonisin B1 was immunolocalised within the endothelial cells and mesenchymal cells of placentas from both groups, while FB1 was present in cytotrophoblastic cells of preeclamptic patients only. In addition, FB1 concentrations were significantly higher in preeclamptic compared to normotensive serum samples. Sphinganine was significantly elevated in preeclamptic serum samples whilst there was no statistical difference in the sphingosine levels between the groups. Chromatin condensation was higher in the preeclamptic patients. Caspase 3 and Fas were present with greater intensity in preeclamptic samples. The levels of lipid peroxidation were significantly higher in both serum and placental tissue of preeclamptic patients. This study has demonstrated not only the presence of FB1 in the serum and placental tissues of pregnant women but also the potential effects of this mycotoxin in the humans.
The effects of Sutherlandia frutescens in cultured renal proximal and distal tubule epithelial cells.
(2009) Phulukdaree, Alisa.; Chuturgoon, Anil Amichund.
Sutherlandia frutescens (SF), an indigenous medicinal plant to South Africa (SA), is traditionally used to treat a diverse range of illnesses including cancer and viral infections. The biologically active compounds of SF are polar, thus renal elimination increases susceptibility to toxicity. This study investigated the antioxidant potential, lipid peroxidation, mitochondrial membrane potential and apoptotic induction by SF on proximal and distal tubule epithelial cells. Cell viability was determined using the MTT assay. Mitochondrial membrane potential was determined using a flow cytometric JC-1 Mitoscreen assay. Cellular glutathione and apoptosis were measured using the GSH-GloTM Glutathione assay and Caspase-Glo® 3/7 assay, respectively. The IC50 values from the cell viability results for LLC-PK1 and MDBK was 15 mg/ml and 7 mg/ml, respectively. SF significantly decreased intracellular GSH in LLC-PK1 (p < 0.0001) and MDBK (p < 0.0001) cells. Lipid peroxidation increased in LLC-PK1 (p < 0.0001) and MDBK (p < 0.0001) cells. JC-1 analysis showed that SF promoted mitochondrial membrane depolarization in both LLC-PK1 and MDBK cells up to 80% (p < 0.0001). The activity of caspase 3/7 increased both LLC-PK1 (11.9-fold; p < 0.0001) and MDBK (2.2-fold; p < 0.0001) cells. SF at high concentrations plays a role in increased oxidative stress, altered mitochondrial membrane integrity and promoting apoptosis in renal tubule epithelia.

Browse

Browsing Medical Biochemistry by Title