Browsing by Author "Chuturgoon, Anil Amichund."

Now showing 1 - 20 of 75

An investigation into kojic acid-associated mitochondrial toxicity and inflammation in melanoma cells (SK-MEL-1).
(2023) Suritham, Tamzin Kimera.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
ojic acid (KA), 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one, is used in agriculture, food, and cosmetics. KA is known to have antimicrobial, antifungal, antioxidant, and anti-inflammatory properties. The cosmetic industry's increasing interest in KA is due to its ability to inhibit tyrosinase activity resulting in skin lightening. The mitochondria play a key role in maintaining homeostasis and ensuring efficient melanin production. Therefore, mitochondrial dysfunction has severe effects on the skin. This study investigates mitochondrial stress, antioxidant responses, protein kinase signalling and inflammation in human melanoma (SK-MEL-1) cells. The mitochondria are important in processing metabolites and supplying the cell with energy in the form of ATP. KA interacts with key mitochondrial homeostasis proteins. Our results found an increase in macromolecule damage specifically lipid peroxidation and protein oxidation. Due to oxidative conditions, increased Nrf2 expression was observed. LON protease is ATP-dependent and regulated by Sirtuin 3 expression. Mitochondrial function was affected illustrated by decreased ATP production leading to decreased LON protease and Sirtuin 3 protein expression. Following increased oxidative stress, KA suppressed the expression of protein kinases but increased inflammatory mediators. There was decreased expression of phospho-Akt, Akt, phospho-GSK3β, p38 and ERK1/2. The mediation of the NLRP3 inflammasome involves priming and activation. At concentrations with high proliferation, NFκB gene and protein expression was activated. The protein kinase signalling pathways are known as mediators of inflammation; however, protein and gene expression of inflammatory mediators was increased following KA treatment. The inflammasome was subsequently activated as shown by an increase in intracellular caspase 1 levels as well as NLRP3, ILβ and IL-6 expression. KA induced mitochondrial stress and suppressed mitochondrial homeostasis proteins. The increased Nrf2 expression could have further downregulated LON protease expression and increased macromolecule damage. Oxidative conditions could have activated the inflammasome pathway independent of protein kinase signalling. In conclusion, KA displayed mitochondrial toxicity following acute exposure by suppressing mitochondrial homeostasis, protein kinase pathways and initiating inflammation. 1
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.
Apoptosis in peripheral blood mononuclear cells of human immunodeficiency virus (HIV) infected patients undergoing highly active antiretroviral therapy.
(2008) Karamchand, Leshern.; Chuturgoon, Anil Amichund.; Dawood, Halima.
Highly active antiretroviral therapy (HAART) is currently the only treatment that effectively reduces the morbidity and mortality of individuals infected with Human Immunodeficiency Virus-1 (HIV-1). Standard HAART regimens typically comprise 2 nucleoside reverse transcriptase inhibitors and either one non-nucleoside reverse transcriptase inhibitor or a protease inhibitor. These drugs bind to and inhibit the HIV-1 Reverse Transcriptase and Protease enzymes respectively, thereby suppressing viral replication. The nucleoside reverse transcriptase inhibitors promote mitochondrial (mt) dysfunction by strongly inhibiting mt polymerase gamma (Pol-y) and subsequently, mtDNA replication. In contrast, the non-nucleoside reverse transcriptase inhibitors, efavirenz (EFV) and nevirapine (NVP) do not inhibit Pol-y although EFV has been shown to induce mt depolarisation ( mlow) in vitro at supra-therapeutic concentrations. However, the capacity of non-nucleoside reverse transcriptase inhibitor drugs to induce mt toxicity in vivo previously remained undetermined. The objective of this study was to determine the influence of EFV and NVP on peripheral lymphocyte mt transmembrane potential (Avj/m) and apoptosis in HIV-1-infected patients treated with these non-nucleoside reverse transcriptase inhibitors. Thirty-two HIV-1-infected patients on HAART between 4 and 24 months (12 on EFV, 20 on NVP) and 16 HAART-naive HIV-1-infected patients were enrolled into this study. All participants were black South African patients. Spontaneous peripheral lymphocyte apoptosis and mlow were measured ex vivo by flow cytometry for all patients. CD4 T-helper apoptosis for the EFV and NVP cohorts was 19.38% ± 2.62% and 23.35% ± 1.51% (mean ± SEM), respectively, whereas total lymphocyte mlow was 27.25% ± 5.05% and 17.04% ± 2.98%, respectively. Both parameters for each cohort were significantly lower (P < 0.05) than that of the HAART-naive patients. The NVP cohort exhibited both a significant time dependent increase in peripheral lymphocyte ö¿mlow (P = 0.038) and correlation between Thelper apoptosis and low (P = 0.0005). These trends were not observed in the EFV cohort. This study provides evidence that both EFV and NVP induce peripheral lymphocyte ö¿ m low in HIV-1-infected patients on non-nucleoside reverse transcriptase inhibitor-based HAART, which in the case of NVP is sufficient to induce the apoptosis cascade.
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.
The biochemical effects of Sutherlandia Frutescens in cultured H9 cancerous T cells and normal human T lymphocytes.
(2008) Ngcobo, Mlungisi.; Chuturgoon, Anil Amichund.
Indigenous plants have long been used by African populations in their cultural lives and health care. Sutherlandia frutescens (SF) is a popular traditional medicinal plant found in various parts of southern Africa and used for treatment or management of different diseases, including cancer and HIV/AIDS. In this study, the biochemical effects of various dilutions (1/50, 1/150, 1/200, and 1/300) of SF 70% ethanol (SFE) and deionised water (SFW) extracts in cancerous H9 and normal T cells were examined. Untreated, 70% ethanol-treated and camptothecin (CPT, 20jiiM) treated cells were used as reference samples for comparison. Cytotoxicity, apoptotic enzymes activity, oxidant scavenging and antioxidant promoting abilities, cellular morphology and cytokine signalling effects were assessed using the methylthiazol tetrazolium (MTT) assay, adenosine triphosphate (ATP) assay, caspase-3/-7 activity assay, thiobarbituric acid reactant substance (TBARS) and glutathione (GSH) assays, fluorescence microscopy and an ELISAbased cytokine analyses assay respectively. Sutherlandia frutescens ethanol and water extract dilutions (1/50 and 1/200) were shown to be cytotoxic to H9 T cells in a dose- and time-dependent manner with the SFE extract having an average IC50 of 1/40 after 24 hours while SFW extract reached a similar IC50 only after 48 hours. In normal T cells, the SFE extract induced proliferation after 24 hours but this was reverse after 48 hours. The SFW extract dilutions did not significantly change cell viability after 24 hours but significantly increased cell viability after 48 hours. Both SFE and SFW extracts dilutions induced a dose- and time-dependent inhibition of caspase-3/-7 activity in both H9 and normal T cells. Both types of extracts were also shown to efficiently remove lipid peroxides from supernatants of treated cell lines, with SFW extract having a more lasting effect. In the GSH assay, the SFE and SFW extract dilutions reduced GSH levels in H9 T cells, with the SFW extract dilutions being more effective. In normal T cells, the higher dilutions (1/150 and 1/300) of SFW extract increased GSH levels significantly while lower dilutions (1/50) of both SFE and SFW extracts significantly inhibited GSH levels. Lower dilutions (1/50) of SFE and SFW extracts induced chromatin condensation in both H9 and normal T cells after 48 hours incubation. Using treated peripheral blood mononuclear cells (PBMCs) supernatants, SFE and SFW extract dilutions were shown to reduce the levels of pro-inflammatory cytokines IL 1 p and TNF-a in a dose-dependent manner. These results further confirmed the anticancer abilities of SF and showed that higher concentrations of this medicinal plant can be toxic to normal T cells in vitro while lower concentrations can stimulate the immune cells. Therefore further studies should be conducted with regards to the effects of SF on the immune system in both in vitro and in vivo systems.
Biochemical, apoptotic and cellular stress studies in rheumatoid arthritis.
(2009) Moodley, Devapregasan.; Mody, Girish Mahasukhlal.; Chuturgoon, Anil Amichund.
Abstract available in PDF file.
Cellular and biochemical effects induced by antiretroviral drugs.
(2013) Anwar, Khawar.; Pillay, Tahir S.; Chuturgoon, Anil Amichund.
In the treatment of HIV/AIDS, protease inhibitors (PIs) and nucleoside/nucleotide analogue reverse transcriptase inhibitors (NRTIs) are the major components of highly active antiretroviral therapy (HAART). The side effects of these drugs include various metabolic disorders including insulin resistance, dyslipidaemia and lipodystrophy. The precise mechanistic basis of these remains largely unknown. In this study we aimed to understand the molecular basis of these metabolic effects by analysing the effects on lipoprotein lipase (LPL) activity, insulin signaling and the cellular metabolic profile. It was previously shown by this group that indinavir inhibits insulin signaling at a proximal level. The study was extended to a wider range of ARVs and in particular, the effects of sodium salicylate (NaSal) and berberine chloride (BBR) were analysed to determine if they could reverse the effects of the drugs on insulin signalling. In addition, Chinese hamster ovary cells transfected with the human insulin receptor (CHO-IR) were used for the first time to study the effects of NRTIs on the insulin signaling pathways. The high level of expression of insulin receptor facilitated sensitive detection of any alteration in the phosphorylation of signaling proteins as compared to 3T3-L1 adipocytes. Three PIs, indinavir, nelfinavir and ritonavir were used in this study. Indinavir and nelfinavir treatment significantly reduced the insulin-stimulated phosphorylation of the IRβ, IRS-1, Akt and MAPK in CHO-IR cells. However phosphorylation of GSK-3α\β was not affected by the PIs. Ritonavir also decreased (not statistically significant) the phosphorylation of IR-β and IRS-1 but its inhibitory effect on MAPK was the same as by the other PIs. NRTI’s did not inhibit insulin-stimulated tyrosine phosphorylation of IRβ and IRS-1 but reduced phosphorylation at MAPK and Akt. In order to understand the role of NFκ-B pathway in blocking insulin-stimulated tyrosine phosphorylation, IKK-16, a selective inhibitor of IkB kinase (IKK) was used but no significant involvement of this pathway was found in blocking tyrosine phosphorylation at IRS-1. Similarly, NaSal and BBR were also used to reverse the effects induced by PIs and NRTIs in CHO-IR cells but no significant change was observed on Akt and MAPK. NaSal and BBR reduced (but not significantly) the effects of PIs (indinavir and nelfinavir) on IR-β and IRS-1. These findings suggest that PIs induce insulin resistance by affecting multiple steps in the signaling pathway. At the proximal end of the insulin signalling pathway, protease inhibitors affect IR-β and IRS-1 while at the distal end they affect phosphorylation of Akt and MAPK. CHO-IR cells were also used to measure LPL activity using a colorimetric method employing pNPB as substrate. The most commonly used ARVs were tested. These included four PIs and six NRTIs .The results showed that NRTIs stavudine and emtricitabine significantly inhibited the LPL activity from the CHO-IR cells. PIs indinavir and nelfinavir were also found to decrease LPL activity extracellularly when added to the assay reaction in vitro. Similarly nelfinavir and atazanavir sulfate inhibited the activity of the LPL from the CHO-IR cells after 16 hour treatment. This suggested that these drugs may interfere with the enzyme activity intracellularly either at the level of its synthesis or its transportation from cytoplasm to the cell surface. These finding suggests that protease inhibitors may play a role in inhibiting lipoprotein lipase activity in vivo, and may thereby induce metabolic disorders in HIV-positive patients being treated with protease inhibitors. Metabolomic analysis was performed on the supernatant of cells treated with PIs and NRTIs, with and without insulin stimulation. Many significant alterations and trends in amino acids and organic acids levels in CHO-IR cells supernatants (treated with PIs) were recorded using 1H-NMR. For example, PIs decreased the synthesis of threonine, phenylalanine, lysine, arginine, isoleucine butyrate, glutamate, histidine and 2-oxo-isovalerate. Furthermore overproduction of lactate and ketones were observed in the nelfinavir treated cells. This may be a consequence of the secondary effects of insulin resistance induced by the PIs. Similarly NRTIs (stavudine and tenofovir) treatment also induced changes in the levels of many amino acids and organic acids .NRTIs decreased the synthesis of acetate, acetoacetate, histidine, methionine, phenylalanine and tryptophan while significant increase in the acetone was observed in stavudine-treated cells. Taken together, the results of this study suggest that PIs and NRTIs, inhibits LPL activity and synthesis, affects insulin signalling pathways at different levels and alters the synthesis of different cellular metabolites which may affect the signalling pathways of insulin.
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 cytotoxic effects of deoxynivalenol and fumonisin B1 on the HT-29 human colonic adenocarcinoma cell line.
(2005) Reddy, Krishnaveni.; Chuturgoon, Anil Amichund.
The human population can be considered as a subject of combined exposure to chemicals against which the gastrointestinal tract represents the first barrier. The most relevant are those compounds that occur in plants which are used as foods, medicines and beverages. Of special interest are the mycotoxins deoxynivalenol (DON) and fumonisin B1 (FB1), two of the most commonly encountered food-borne mycotoxins and curcumin, a popular spice and pigment reported to have antineoplastic properties. In this study, the HT-29 cell line was used to assess the toxicity of the mycotoxins DON and FB1 (5uM and 50uM) as well as the possible cytoprotective effects of curcumin (50uM) on colonic cells. Mixtures of both mycotoxins were also assessed to determine any possible interaction. Cytotoxicity, DNA fragmentation, cellular morphology and cell surface alterations were evaluated using the methylthiazol tetrazolium (MTT) bioassay, the single cell gel electrophoresis (SCGE) assay, fluorescence microscopy and scanning electron microscopy respectively. Deoxynivalenol displayed cytotoxic and genotoxic effects as well as induced morphological features of apoptosis and cell surface alterations that worsened with increasing concentration. Fumonisin B1 exhibited a proliferative effect at the high concentration however DNA damage and cell surface alterations worsened with decreasing concentration. Mixtures of DON and FB1 displayed similar effects to those exhibited by DON in terms of cytotoxicity, DNA fragmentation, morphology and cell surface alterations indicating that DON is able to antagonise the effects of FB1 at the concentrations tested. Curcumin appeared to exhibit a protective effect that was prominent when co-administered with the 50uM toxin concentration. Low concentrations of DON and FB1 (5uM) were sufficient to induce apoptosis in this cell line and suggest a danger from natural contamination by these toxins. Curcumin, however, warrants further investigation with regards to its cytoprotective activities in the presence of these mycotoxins as it could present a promising candidate for a natural chemoprotective agent in the armamentarium against mycotoxin induced cancers.
The cytotoxic effects of T-2 toxin on normal human lymphocytes.
(1998) Moodley, Therishnee.; Chuturgoon, Anil Amichund.; Dutton, Michael Francis.
T -2 toxin is an immunosuppressive mycotoxin that has been conjoined with several symptoms and diseases as early as the turn of the century, but whose mechanisms of action are still being investigated. Accordingly, this study was an attempt to determine the cytotoxic effects of T -2 toxin on normal human lymphocytes in vitro, with particular emphasis on mitochondrial viability, cellular and nuclear morphology as well as the localisation of the subcellular sites of toxin interaction. The cytotoxicity of T -2 toxin was assessed with the use of a methylthiazol tetrazolium (MTT) assay. This assay targeted the succinate dehydrogenase activity of the lymphocytic mitochondria, over a range of concentrations of T-2 toxin at various incubation times. The morphology of treated lymphocytes was analysed with the use of transmission electron microscopy and the localisation of the toxin was accomplished via immunocytochemistry. DNA fragmentation studies formed an integral part of the analyses. The cytotoxicity assay indicated that not only was cell viability inversely proportional to both the dose and exposure time, but that the eftects of the different doses were only evident at prolonged incubation times (12-24 hours). The electron microscopy studies showed that T-2 toxin (1,56 ug/ml) induced apoptosis (cell suicide) in normal human lymphocytes. This was determined by the observation of chromatin condensation and nuclear disintegration within the toxin treated lymphocytes. Apoptosis seemed to occur independently of mitochondrial damage at 6 hours of exposure to T-2 toxin. The presence of polyribosomes within the treated lymphocytes indicated that protein synthesis was not inhibited. Anti-T-2 toxin conjugated gold label was present in all areas of damage, particularly within the nuclei of the T-2 toxin treated lymphocytes. The DNA fragmentation results showed that T-2 toxin induced fragmentation in lymphocytes, the extent of which was directly proportional to the exposure time. It appears that the early signs of T-2 toxin induced apoptosis in normal human lymphocytes can be determined by damage to the nucleus.
A cytotoxic evaluation of aflatoxin B1, zearalenone and their epoxide derivatives using human cell lines.
(1996) Pillay, Dharmarai.; Chuturgoon, Anil Amichund.; Dutton, Michael Francis.
Since the discovery of mycotoxins in food, the thrust of biochemical and toxicological research has been carried out on animals which has proven to be uncoordinated and not easily extrapolated to humans. Over the last decade, there have been increasing pressures to review and reduce the use of animals in experimental toxicological studies. Consequently in this study aflatoxin B1 (AFB1), zearalenone (Zea) and their epoxide derivatives have been evaluated using in vitro assays. The HepG2, A549 and Hela cell lines were used for assessing the cytotoxicity, effects on cellular metabolism and sites of action of AFB1, Zea and their derivatives. The cytotoxicity of these mycotoxins was evaluated using the methylthiazol tetrazolium (MTT) reduction assay. Cells, treated with mycotoxins were prepared for transmission electron mlcroscopy (TEM), immunocytochemistry (ICC), scanning electron microscopy (SEM), confocal and light microscopy. From the cytotoxicity assay it was found that the epoxide derivatives were more toxic than the parent toxin when exposed to HepG2 cells with no significant differences in toxicity levels in A549 and Hela treated cells. Both epoxide derivatives displayed a regression of hepatoma cell proliferation at high doses (25ug/ml) while lower concentrations (<12.5ug/ml) enhanced cell growth. Microscopy analyses showed distinct cellular alterations. When exposed to AFB1 (12.5ug/ml) hepatoma cells showed prominent ultrastructural alterations such as areas of cytoplasmic lysis and increased numbers of secondary lysosomes while cells exposed to Zea (l2.5ug/ml) displayed numerous ovoid mitochondria and proliferation of rough endoplasmic reticulum which is indicative of enhanced protein synthesis. The presence of label in toxin treated cells is suggestive of the effects of these mycotoxins. Such cellular changes may lead to altered metabolism and cell function.
Deoxynivalenol downregulates NRF2-induced cytoprotective response in human hepatocellular carcinoma (HepG2) cells.
(2017) Ndlovu, Siqiniseko Sinikiwe.; Chuturgoon, Anil Amichund.; Nagiah, Savania.
Deoxynivalenol (DON) is a mycotoxin produced by Fusarium species that commonly infect agricultural foods. DON exhibits multiple toxic effects in both animals and humans, binding to the A site of the 28S ribosome and inhibits peptidyl transferase and protein elongation. It induces cytotoxicity through oxidative stress and inhibition of protein synthesis. Liver cells possess the antioxidant signalling mediator - Nuclear erythroid-2-Related factor (NRF2) that is activated in response to oxidative stress. There is no sufficient work done to show if the HepG2cells have an ability to withstand the molecular modifications induced by DON. The aim of the study was to investigate the cytotoxicity of DON and its effect on the NRF2 antioxidant response in HepG2 cells. The MTT assay was used to determine a dose response of DON (72 hr) on cell viability and to generate an IC50 value to use in subsequent assays. The intracellular concentration of GSH and ATP was determined using Luminometry. Lipid peroxidation and membrane damage were assessed by TBARS and LDH cytotoxicity assays respectively. Protein expression of NRF2, phosphorylated (p-)NRF2, catalase (CAT), superoxide dismutase (SOD)2, and Sirtuin (Sirt)3 was quantified by Western Blotting. The mRNA expressions of GPx, CAT and SOD2 were quantified using qPCR. DON decreased cell viability in a dose-dependent manner with an IC50 value of 26.17 μM. DON caused a significant decrease in the intracellular GSH concentration (1.77-fold, p= 0.0005). There was a significant decrease in the intracellular ATP content (1.92-fold, p= 0.0002).The study shows an induced lipid peroxidation and membrane damage in HepG2 cells by DON, as there was a significant increase in extracellular levels of both MDA (1.89-fold, p=0.0020) and LDH (1.35-fold, p=0.0207). DON reduced total NRF2 expression (0.30-fold, p= 0.0017), however activated p-NRF2 was significantly up-regulated (3.54-fold, p= 0.0085). There was a downregulation in the NRF2 target antioxidant proteins: CAT (0.33-fold, p= 0.005) with a concomitant decrease in CAT mRNA levels (0.02-fold, p= 0.0003), SOD2 (0.02-fold, p= 0.0137), with a parallel trend in the levels of SOD2 mRNA (0.06-fold, p= 0.0020) by DON. This toxin also significantly decreased the mRNA expression of GPx levels (0.03-fold, p= 0.0006). The expression of a mitochondrial stress response Sirt3 was significantly decreased (0.14-fold, p= 0.0058). Taken together, the data shows that DON causes oxidative stress and downregulates the NRF2-induced cytoprotection in HepG2 cells. Keywords: Deoxynivalenol Antioxidant response NRF-2
Determination of exposure of humans to selected mycotoxins with particular reference to aflatoxins.
(1995) Early, Deborah Angeline.; Dutton, Michael Francis.; Chuturgoon, Anil Amichund.
Mycotoxins are poisonous secondary metabolites commonly produced by fungi and are involved in human disease conditions known as mycotoxicoses. There is evidence to show that food eaten by the rural Black population of Southern Africa is contaminated with mycotoxins. A tenuous relationship exists between the occurrence of mycotoxins in foods and certain disease conditions in humans. In order to verify this relationship, efforts have, in the past, been made to detect mycotoxins and their metabolites in physiological fluids and tissues. The difficulty with this approach is that mycotoxins in the body have short half lives, being rapidly excreted or metabolised to other forms. More recently it has been shown that aflatoxin B1, as its activated epoxide, can conjugate with macromolecules such as nucleic acids and proteins. These survive for much longer than the free toxins and by suitable methods can be isolated and measured. This allows for a much better estimate of exposure of the individual to aflatoxin. This study reviews and evaluates screening methods for the detection and analysis of mycotoxin contamination in rural foodstuffs such as maize and groundnuts. Methods for the production of aflatoxin-lysine and protein adducts are motivated and developed then used in the identification of naturally occurring adducts in humans. Isolation and quantitative analysis techniques are proposed to routinely screen patients for evidence of aflatoxin exposure.