Masters Degrees (Medical Biochemistry)

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

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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.
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-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.
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.

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Browsing Masters Degrees (Medical Biochemistry) by Author "Ghazi, Terisha."