Masters Degrees (Medical Biochemistry)

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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.
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.
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.
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.
Fusaric acid induces oxidative stress and apoptosis in human oesophageal cancer cells.
(2016) Devnarain, Nikita.; Chuturgoon, Anil Amichund.; Tiloke, Charlette.; Nagiah, Savania.
Abstract available in PDF file.
Fusaric acid-induced epigenetic modifications in vitro and in vivo: alternative mechanisms of hepatotoxicity.
(2019) Ghazi, Terisha.; Chuturgoon, Anil Amichund.; Nagiah, Savania.
The Fusarium-produced mycotoxin, Fusaric acid (FA), is a frequent contaminant of agricultural foods that exhibits toxicity in plants and animals with little information on its molecular and epigenetic mechanisms. Epigenetic modifications including DNA methylation, histone methylation, N-6-methyladenosine (m6A) RNA methylation, and microRNAs are central mediators of cellular function and may constitute novel mechanisms of FA toxicity. This study aimed to determine epigenetic mechanisms of FA-induced hepatotoxicity in vitro and in vivo by specifically investigating DNA methylation, histone 3 lysine (K) 9 trimethylation (H3K9me3), and m6A-mediated regulation of p53 expression in human liver (HepG2) cells and C57BL/6 mice livers. FA induced global DNA hypomethylation in HepG2 cells; decreased the expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) by inducing promoter hypermethylation and upregulated expression of miR-29b. Further, FA decreased the ubiquitination of DNMT1, DNMT3A, and DNMT3B by decreasing the expression of the ubiquitination regulators, UHRF1 and USP7. FA induced promoter hypomethylation of the demethylase, MBD2 and increased MBD2 expression contributing to global DNA hypomethylation in HepG2 cells. DNA methylation and H3K9me3 function in concert to regulate genome integrity and gene transcription. Sirtuin (Sirt) 1 is a histone deacetylase and direct target of miR-200a that regulates the repressive H3K9me3 mark by post-translationally modifying both H3K9Ac and the histone methyltransferase, SUV39H1. FA upregulated miR-200a and decreased Sirt1 expression in HepG2 cells and C57BL/6 mice livers. FA decreased the expression of SUV39H1 and histone demethylase, KDM4B which led to a decrease in H3K9me3 and an increase in H3K9me1. FA also decreased cell viability via apoptosis as evidenced by the significant increase in the activity of the executioner caspase-3/7. The tumor suppressor protein, p53 regulates cell cycle arrest and apoptosis in response to cellular stress. The expression of p53 is regulated at the transcriptional and post-transcriptional level by promoter methylation and m6A RNA methylation. In HepG2 cells, FA induced p53 promoter hypermethylation and decreased p53 expression. FA also decreased m6A-p53 levels by decreasing the expression of the methyltransferases, METTL3 and METTL14, and the m6A readers, YTHDF1, YTHDF3, and YTHDC2, thereby, decreasing p53 translation. In C57BL/6 mice livers FA, however, induced p53 promoter hypomethylation and increased p53 expression. FA increased m6A-p53 levels by increasing the expression of METTL3 and METTL14; and increased expression of YTHDF1, YTHDF3, and YTHDC2 increased p53 translation. In conclusion, this study provides evidence for alternative mechanisms of FA-induced hepatotoxicity (in vitro and in vivo) by modulating DNA methylation, H3K9me3, m6A RNA methylation, and epigenetically regulating p53 expression ultimately leading to genome instability and apoptotic cell death. These results provide insight into a better understanding of FA induced hepatic toxicity at the epigenetic and cellular level and may assist in the development of preventative and therapeutic measures against FA toxicity. It also suggests that exposure to FA may lead to the onset of human diseases via epigenetic changes/modifications. This is particularly relevant in under privileged communities where the food supply and storage conditions are inadequate.
HMG-CoA reductase inhibitor, atorvastatin induces apoptasis in human lung adenocarcinoma cell (A549).
(2015) Ramharack, Pritika.; Chuturgoon, Anil Amichund.; Nagiah, Savania.
Abstract available in PDF file.
An in vivo study to determine the effects of Ochratoxin A and Sutherlandia frutescens in male Wistar rats.
(2009) Durgiah, Raveshni.; Chuturgoon, Anil Amichund.
Ochratoxin A (OTA), a nephrotoxic mycotoxin, is a contaminant of several agricultural food products consumed by animals and humans. Apart from renal toxicity, in particular renal tumours, OTA may also result in teratogenicity, neurotoxicity and immunotoxicity. Sutherlandia frutescens, an indigenous medicinal plant, has shown significant potential in strengthening the immune system and in cancer treatment, with minimal side effects. The objective of this study was to determine the effects of OTA in male Wistar rats and ascertain if these effects may be reduced by S. frutescens. Rats were treated by intraperitoneal injection (i.p) with either a control (EtOH:dH20;30:70), S. frutescens (1.0mg/kg body weight), OTA (0.5mg/kg body weight) or a combination of OTA and S. frutescens for a period of 1 or 7 days (n=4). Genotoxicity and metabolic activity in peripheral blood mononuclear cells (PBMCs) were quantified using single cell gel electrophoresis (SCGE) and the methylthiazol tetrazolium (MTT) assay, respectively. Lymphocyte apoptosis and mitochondrial depolarisation were measured by flow cytometry. Fluorescence microscopy was utilised to determine renal tissue apoptosis (Hoechst staining) and OTA localisation using immunohistochemistry (IRC). SDS-PAGE and Western blot were utilised to determine protein expression in kidney tissue and serum. Ochratoxin A significantly reduced PBMC viability (14%) after 7 days, compared with Day 1 (p<0.001). Lymphocyte mitochondrial depolarisation was 56.5% and 66.2% in the OTA-only and combination groups, respectively after 7 days (p<0.001). Ochratoxin A produced an increase in DNA damage compared to the control (p<0.01). The renal tissue displayed typical signs of apoptosis such as chromatin condensation. Ochratoxin A was immunolocalised within the glomerulus. The protein analysis showed a decreased expression in the kidney mitochondrial protein fraction. Ochratoxin A preferentially bound to serum albumin and a 120kDa protein in the OTA-only and co-treatment groups after the 1-and 7-day regimes. Protein band intensities significantly decreased after the 7-day co-treatment (p<0.01). The data highlights that OTA toxicity is mediated by mitochondrial dysfunction. Furthermore, OTA disruptions in immune function may play a role in renal damage.
An investigation into marine bacterial species found in shark mouths in the Indian Ocean and their implications for human health.
(2015) Ramlakhan, Yathisha.; Chuturgoon, Anil Amichund.; Phulukdaree, Alisa.; Bester, Linda Antionette.; Singh, Sanil Duleep.
There is an ever increasing amount of pollution and waste being released into the environment. This is due to the increase in population, urbanisation and people migrating into cities. Approximately 2.4 billion people living in urban and rural areas have no access to basic sanitation. In the next 20 years, there will be a further increase of 2 billion people who will lack basic sanitation. In developing countries, 90% of untreated sewage is released into rivers, lakes and coastal waters. Apart from sewage, waste such as petroleum products, heavy metals and organochlorine also contribute to marine pollution. Companies that manufacture sugar/artificial sweeteners etc. and farming activities that utilize fertilizers for crops can cause eutrophication, as un-used fertilizers get washed into rivers. The marine water is a different environment to other aquatic and terrestrial environments. This then forces microbes to adapt, so they can be able to survive in the marine environment. The difference in the marine environment allows for the production of distinct bioactive metabolites such as secondary metabolites. These secondary metabolites come from algae and marine bacteria and these secondary metabolites are then exclusive to the marine waters. These secondary metabolites can be used for medical purposes, cosmetics, personal-care products etc. There is a huge problem with antibiotic resistance and research needs to be done to solve this resistance issue. Two common bacterial strains were isolated and identified from the mouth of sharks. The bacteria were identified as Bacillus cereus and Vibrio alginolyticus. They were isolated and cultured in broth for 3 days, till they reached the log phase of growth. The broth was then extracted for metabolites which the bacteria produced, using ethyl acetate. These metabolites were tested for cytotoxicity in the human liver hepatocellular carcinoma (Hep G2) cells. The concentrations that were determined to cause 50% cell death (IC50) in the cell viability assay on Hep G2 cells were 0.764 mg/ml and 0.918 mg/ml for B. cereus and V. alginolyticus, respectively. These values were then used for subsequent assays. Antibacterial testing was done for the bacterial extracts of Bacillus cereus and Vibrio alginolyticus. There was no antibacterial activity against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853. Assays that used flow cytometry was used to show if apoptosis/necrosis occurred. These were assays such as Annexin V and propidium staining. While assays that used luminometry showed the levels of ATP and determined whether apoptosis of the cells occurred. These were assays such as the ATP assay, mitochondrial depolarisation assay and determination of the caspase activities of caspase 3/7, 8 and 9. Additional assays, like the comet and TBARS assays, were done to show DNA fragmentation and oxidative stress of the cells, respectively. The results for the Annexin V/ propidium staining showed the control had a mean of 11.20 ± 1.0. Extract 1 (20.83 ± 0.8737) and extract 2 (25.37 ± 1.050) showed a higher percentage when compared to the control. Extract 2 was significant against the control (p<0.0273). For propidium staining, the control had a mean of 6.033 ± 0.4524. Extracts 1(11.57 ± 1.387) and 2 (11.43 ± 0.3215) showed a higher percentage when compared to the control. The Annexin V and propidium staining suggested that extract 1 and 2 had undergone both apoptosis and necrosis. For luminometry assays, the ATP assay showed that the control had a mean of 1.83x106 ± 5.82x104. Extracts 1 (1.5x106 ± 9.4x104) and extract 2 (1.4x106 ± 8.3x104) showed a decrease in ATP with reference to the control. In the mitochondrial depolarisation assay, the control had a mean of 14.83 ± 1.350. Extracts 1 (30.57 ± 0.75) and extract 2 (20.53 ± 8.56) showed a decrease in polarisation with reference to the control. For caspase 8 analysis, the control, extract 1 and extract 2 had means that were 4.23x104 ± 3.37x103, 52x103 ± 10.1x103 and 40x103±5.2x103, respectively. For caspase 9 analysis, the control, extract 1 and extract 2 had means that were 8.6x104 ± 4.6x103, 5.6x104 ± 4x103and 9.6x104 ± 5.6x104, respectively. The caspase 3/7 analysis showed that the control, extract 1 and extract 2 had means of 4.4x103 ± 0.57x103, 5.5x103 ± 0.19x103 and 5.8x103 ± 2 x103, respectively. Caspase 3/7 showed that apoptosis had occurred with the cells for all extracts used. Extract 1 showed a high caspase activity for caspase 8. This suggested that it followed the extrinsic pathway of apoptosis. Extracts 2 showed a high activity for caspase 9 which suggested that it followed the intrinsic pathway of apoptosis. The comet assay showed that the means of the control, extract 1 and extract 2 were 35.91 ± 21.93, 75.85 ± 11.43 and 60.48 ± 11.86, respectively. The extracts were significantly higher than the control (extract 1 and 2 p<0.0001). Extract 1 and 2 were compared to each other and had shown a significance between them (p<0.0001). The TBARS assay obtained the following MDA concentrations for the control, extract 1, extracts 2, negative and positive samples: 0,137, 0,132, 0,150, 0,088 and 20,502, respectively. The MDA concentration gives an indication of oxidative stress of the cells. From the cell viability assay, the secondary metabolites produced by B. cereus needed a lower concentration of extract to determine an IC50 value. This suggested that the secondary metabolites produced by B. cereus were more toxic than the secondary metabolites produced by V. alginolyticus. This was then further supported by assays such as mitochondrial depolarisation and the comet assay. The secondary metabolites that could be the reason why there were apoptosis and necrosis, are the toxins the bacteria produce. This is the enterotoxin or cereulide produced by B. cereus and TLH by V. alginolyticus. However, further studies need to be done to confirm if these toxins are the cause of cell death.
An investigation into the biochemical effects of Kojic acid (KA) on human hepatocellular carcinoma (HepG2) cells.
(2020) Suthiram, Kimera Tamzin.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
Kojic acid (KA) is a secondary metabolite and divalent metal chelator that is widely used in the beauty industry as a skin lightener. However, KA toxicity is not well-established in humans. This study aimed to determine the toxicity of KA by assessing oxidative stress, nuclear factor kappa B (NFκB) signalling, and mitogen-activated protein kinase (MAPK) signalling in human hepatoma (HepG2) cells following 24 h exposure. Cell viability was assessed using the methylthiazol tetrazolium (MTT) and crystal violet assays. To confirm cell death, apoptosis (caspase -8, -9, -3/7 luminometry), and Lactate dehydrogenase (LDH) leakage were assessed. Oxidative stress (TBARS), DNA damage (8-OHdG), and protein oxidation (protein carbonyls assay) to determine macromoleculedamage. An assessment of inflammatory and oxidative stress markers were carried out using mRNA expression GPx, NFκB, actor erythroid-2 factor-2 (Nrf2), phospho-Nrf2 (ser40), catalase (CAT), c-Jun-N-terminal kinase (JNK), p38, phospho-Sirtuin 1 (ser47) (phospho-sirt1), NFκB, phospho-NFκB (ser536), and activator protein 1 (AP-1) were assessed using Western Blot in HepG2 cells. KA decreased cell viability in HepG2 cells and elevated the activities of caspase -9 (p < 0.0001), caspase -8 (p = 0.0003) and caspase 3/7 (p < 0.0001) at lower concentrations [4.22 & 8.02 mM] which served as confirmation of apoptosis. Necrosis at the higher concentration [12.67 mM] was confirmed by the presence of LDH leakage indicating membrane damage. Increased cell death was further correlated with increased miRNA-29b expression (p = 0.009), a miRNA responsible for elevated apoptotic activity. Adenosine Triphosphate (ATP) production was increased significantly at 12.67 mM (p < 0.0001), while oxidative stress (Malondialdehyde (MDA) levels) was increased significantly at 4.22 mM (p < 0.0001). Macromolecules are susceptible to damage in the presence of oxidative stress. Due the elevation of MDA levels, DNA damage and protein oxidation assays were carried out. Protein carbonyls were significantly decreased (p < 0.0001), suggesting a potential cytoprotective effect. Due to the presence of oxidative stress, Nrf2, is activated and is responsible for the transcription of antioxidant genes. This was illustrated by an increase in activated Nrf2 at lower concentrations (4.22 & 8.02 mM), whilst at higher a concentration (12.67 mM) decreased phospho- (p > 0.0001). CAT was decreased significantly (p = 0.0002) and GPx significantly increased at lower concentration [4.22 mM] (1.51-fold). A key function of the MAPK pathway is the initiation of stress-activated protein kinases, p38 and JNK, in response to oxidative stress. KA significantly increased, p38 at lower concentration (p = 0.0011) and significantly decreased JNK1 (p = 0.0039) and JNK2 (p < 0.0001) activity. Regulation of reactive oxygen species (OS) production by Sirt-1 occur via the alteration of immune responses through NFκB signalling and AP-1. Inflammatory ediators, phospho-Sirt1 was significantly decreased (p < 0.0001), while AP-1 expression was elevated (p 15 which is in agreement with repressed inflammatory responses reflected by decreased NFκB expression. KA treatment resulted in increased MDA levels and antioxidant responses. MAPK signalling was elevated in response to oxidative stress suggesting the involvement in cell death, whilst inflammation was suppressed. In conclusion, KA displayed low toxicity in HepG2 cells.
An investigation into the effects of Sutherlandia Frutescens, L-Canavanine and aflatoxin B1 in the HepG2 human hepatocarcinoma cell line.
(2008) Pillay, Evashin.; Chuturgoon, Anil Amichund.
Aflatoxin B1 (AFB1), a potent hepatotoxic and hepatocarcinogenic mycotoxin synthesised by toxigenic fungi (Aspergillus flavus and Aspergillus parasiticus), is a common contaminant of many cereal commodities consequently posing a major threat to human and animal health. Sutherlandia frutescens (SF), a traditional medicinal plant endemic to Southern Africa, is commonly used by many cultures as a tonic for various health-related conditions. Incidentally, the present study aimed at investigating the potential hepatoprotective capacity of SF and L-canavanine (L-can, a major constituent of SF) against AFB1-induced cytotoxicity in human HepG2 cells and used a standard treatment procedure of 24 h. Cell viability was evaluated using the methyl thiazol tetrazolium (MIT) assay, which effectively demonstrated the ability of SF, when administered individually and in combination with AFB1, to be significantly cytotoxic to HepG2 cells in a dose-dependant manner. Reactive oxygen species (ROS) and consequent peroxidative damage caused by AFB1 are considered to be the main mechanisms leading to hepatotoxicity and was confirmed by the thiobarbituric acid reactive substances (TBARS) assay which revealed that AFB1 mediated a significant increase in lipid peroxidation. Additionally, comet assay analysis demonstrated the most pronounced effect to be observed following administration of AFB1. In contrast, AFB1-mediated genotoxicity was significantly reduced by SF and L-can. Such amelioration can be attributed to the marked increases in glutathione (OSH) levels observed after the co-administration of SF and L-can with AFB1. Cytoprotection by SF and L-can against AFB1-induced toxicity was further substantiated by the significant increases in heat shock protein 70 expression. Moreover, when SF and L-can were co-administered along with AFB1, analysis by flow cytometry revealed that AFB1 induced increases in apoptosis and necrosis were reduced. The findings of this study propose that SF and L-can may be selectively effective in alleviating AFB1-induced cytotoxicity and lends pharmacological credibility to the suggested ethnomedical uses of SF. However, the exact mechanism of action and the extracts efficacy in humans requires further authentication.

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