Medical Biochemistry

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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.
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.
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.
An investigation into the molecular and Epigenetic alterations associated with Fumonisin B1-induced toxicity in human liver (HEPG2) cells.
(2020) Arumugam, Thilona.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
The contamination of agricultural commodities with Fusarium mycotoxins is a global issue in food safety, with fumonisin B1 (FB1) being the most prevalent contaminant. FB1 is not only phytotoxic, but it induces a wide range of toxic effects in animals and humans and is associated with carcinogenesis in animals and humans. Intense research has uncovered several mechanisms by which FB1 induces toxicity. Recent evidence suggests that epigenetic mechanisms may also contribute to the toxic effects of FB1. Epigenetic modifications including DNA methylation, histone methylation, N-6- methyladenosine (m6A) RNA methylation, and non-coding RNAs such as microRNAs (miRNA) and long non-coding RNA (lncRNA) are central mediators of cellular function and cellular stress responses and disruption may be pertinent in FB1-induced toxicities. This study aimed to determine the epigenetic mechanisms of FB1-induced hepatotoxicity by specifically investigating changes in DNA methylation, histone 3 lysine 4 trimethylation (H3K4me3), m6A RNA modification, and noncoding RNA in human hepatoma (HepG2) cells. The effect of these FB1-induced epigenetic modifications on stress responses was further investigated. FB1 impairs DNA repair processes via epigenetic mechanism. FB1 reduced the expression of histone demethylase, KDM5B, which subsequently increased the total H3K4me3 and the enrichment of H3K4me3 at the PTEN promoter region; this led to an increase in PTEN transcript levels. However, miR-30c inhibited PTEN translation. Thus, PI3K/AKT signaling was activated, inhibiting CHK1 activity via phosphorylation of its serine 280 residue. This hampered the repair of oxidative DNA damage that occurred as a result of FB1 exposure. Exposure to FB1 not only induced oxidative DNA damage but elevated levels of intracellular ROS triggering cell injury. In response to oxidative injury, cells induce Keap1/Nrf2 signaling which is regulated by epigenetic mechanisms. FB1 elevated global m6A RNA levels which were accompanied by an increase in m6A “writers”: METTL3 and METTL14, and “readers”: YTHDF1, YTHDF2, YTHDF3 and YTHDC2 and a decrease in m6A “erasers”: ALKBH5 and FTO. Hypermethylation occurred at the Keap1 promoter, resulting in a reduction of Keap1 transcripts. The hypomethylation of Nrf2 promoters and decrease in miR-27b expression led to an increase in Nrf2 mRNA expression. m6A-Keap1 and m6A-Nrf2 levels were both elevated; however, protein expression of Keap1 was reduced whereas Nrf2 was increased. Collectively, these epigenetic modifications (promoter methylation, miRNA-27b and m6A RNA) activated antioxidant signaling by reducing Keap1 expression and increasing Nrf2 expression. If cells are unable to cope with stress, p53-mediated apoptosis is activated. Crosstalk between the lncRNA, HOXA11-AS, miR-124 and DNA methylation can influence p53 expression and apoptosis. FB1 upregulated HOXA11-AS leading to the subsequent decrease in miR-124 and increase in SP1 and DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B). This promoted global DNA methylation and hypermethylation of p53 promoters, thereby reducing p53 expression and caspase activity. Taken together, the data suggests that FB1 inhibits p53-dependent apoptosis via HOXA11- AS/miR-124/DNMT axis. Collectively, this study provides novel insights into additional mechanisms of FB1-induced toxicities by epigenetically modulating stress response mechanisms.

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