Medical Biochemistry

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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.
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.
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.
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.
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.
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
Fusaric acid dampens the Nrf2-mediated stress response in human embryonic kidney cells.
(2016) Govender, Melissa.; Chuturgoon, Anil Amichund.; Nagiah, Savania.
Abstract available in PDF file.
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.
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.
Fusaric acid induces mitochondrial stress in human hepatocellular carcinoma (HepG2) cells.
(2015) Abdul, Naeem Sheik.; Chuturgoon, Anil Amichund.; Nagiah, Savania.
Abstract available in PDF file.

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