Doctoral Degrees (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.
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    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.
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    Predictive anthropometric measurements, associated factors, outcomes, and genetic factors involved in maternal overweight and obesity in HIV-infected and HIV-uninfected black South African pregnant women.
    (2022) Erasmus, Christen Renee.; Chuturgoon, Anil Amichund.
    Background: The proportion of overweight and obese people living with human immunodeficiency virus (HIV) infection have increased globally and are both epidemics that are endemic to countries like South Africa. Targeting these two epidemics in pregnant women, need to be a priority in maternal health research, with the findings from these studies aimed to eventually translate into improving maternal health outcomes. Aims and objectives: This study aimed to evaluate the anthropometric differences, factors, outcomes, and epigenetic factors involved in pregnant black South African pregnant women with a body mass index (BMI) ≥25.0 kg/m2 in comparison to those with a BMI <25 kg/m2. The specific study objectives were to: (i) investigate the relationship between maternal BMI and maternal anthropometric measurements among black South African pregnant women; (ii) identify what measurement cut-offs accurately predict each nutritional status group; (iii) investigated the anthropometric differences between pregnant women living with and without HIV; (iv) investigate the differences between the pregnant women with a BMI of ≥ 25.0 kg/m2 compared to those with a BMI <25.0 kg/m2; (v) investigate the factors associated with overweight and obesity in both HIV-infected and HIV-uninfected pregnant South African women; (vi) investigate the maternal health outcomes associated with overweight and obesity in both HIV-infected and HIV-uninfected pregnant South African women; and (vii) investigate whether maternal BMI and HIV status had an effect on the mRNA expression of adiponectin (ADIPOQ), leptin (LEP), leptin receptor (LEPR), fat mass and obesity-associated (FTO), and ghrelin (GHRL) in visceral adipose tissue (VAT) and in whole blood (WB) obtained from pregnant black South African women. Method: A cross-sectional study design was employed. Sample selection was conducted at Prince Mshiyeni Memorial Regional Hospital, which is situated in Umlazi within the eThekweni municipality, KwaZulu-Natal, South Africa. The catchment area for the hospital includes both rural and urban geographical areas. Pregnant women admitted to the labour ward were approached to participate in this study. The inclusion criteria for this study were as follows: (1) ≥ 18 years of age; (2) pregnant females; (3) black South African citizen; (4) clinically stable; (5) able to stand without assistance; (6) given verbal and written consent to participate in the study; and (7) gave consent to obtain a VAT sample during their c-section operation. The participants were categorized x according to BMI (kg/m2) into two groups: (1) overweight/obese pregnant women (≥25kg/m2); and (2) non-overweight/non-obese pregnant women (<25kg/m2). A total of 458 pregnant women were approached to participate in the study, but 245 subjects met the inclusion criteria and of these 45 declined to participate. Hence, a total of 200 subjects met all the inclusion criteria, but of these participants only 79 subjects were able to provide a VAT sample. The statistical tests that were applied included: (i) Fisher’s exact test and the χ 2 test; (ii) Pearson correlation coefficient; (iii) the Spearman’s rank-order correlation coefficient; (iv) the Mann Whitney t-test; (v) one-way ANOVA; (vi) area under the curve of the receiver operator characteristic curves to determine the cut-off values; and (v) simple logistic regression was performed to select the variables for multiple logistic regression analysis, and only variables with a p-value <0.05. A p-value of <0.05 was considered statistically significant. Results: Maternal age was significantly positively associated with changes in maternal anthropometric measurements. Maternal BMI was significantly positively correlated with other maternal anthropometric measurements including mid upper arm circumference (MUAC) (left and right), tricep skinfold (TSF) (right), subscapular skinfold (SSF) (right), mid arm muscle circumference (MAMC) (right), wrist circumference (WC) (right), but significantly negatively correlated with frame size. The anthropometric methods that were accurate for assessing obesity in pregnancy included TSF (right) (cut-off of ≥20.75 mm), SSF (right) (cut-off of ≥21.75 mm), MAMC (right) (cut-off of ≥25.23 cm), and WC (right) (cut-off of ≥16.25cm). Also, SSF (right) (cut-off of ≥15.75mm) and MAMC (right) (cut-off of ≥23.35cm) could be used to assess for overweight nutritional status. Lastly, frame size could be used to assess for underweight (cut-off of ≥10.05) and normal (cut-off of ≥9.95) nutritional status. The HIV-infected pregnant women did not differ anthropometrically to the HIV-uninfected pregnant women. The demographic characteristics, food frequency intake, physical activity and lifestyle characteristics were not significantly different between the participants with a BMI of ≥ 25.0 kg/m2 compared to those with a BMI of <25 kg/m2. The dietary pattern of the overweight/obese participants showed that there was a higher intake of saturated fat, higher in salt, higher in sugar, higher in animal protein, lower in dairy, higher in legumes, higher in starch, higher in vegetables, and had a similar intake of fruit in comparison to the non-overweight/non-obese participants. Also, maternal age was significantly different between those with a BMI ≥25 kg/m2 compared to those with a BMI <25 kg/m2, where the overweight and obese participants were significantly older (p=0.0173). Multiple logistic regression analysis showed that maternal age (OR:1.061; 95%CI 1.008-1.117; p=0.023) and gestational age (OR:1.121; 95%CI 1.005-1.251; p=0.041) were significantly associated with maternal overweight and obesity in both HIV-infected and HIV-uninfected pregnant women. For maternal health outcomes, multiple logistic regression analysis showed that HPT disorders (OR:0.273; 95%CI 0.124-0.601; p=0.001) and anaemia (OR:2.420; 95%CI 1.283-4.563; p=0.006) were significantly associated with maternal overweight and obesity in both HIV-infected and HIVuninfected pregnant women. The overweight and obese HIV-infected pregnant women (OR:0.233; 95% CI 0.075-0.717; p=0.011) had increased odds for developing HPT disorders compared to HIV-uninfected overweight and obese pregnant women (OR:0.471; 95% CI 0.172-1.291; p=0.143). It was identified that there were statistically significant differences for ADIPOQ (p <0.001), LEP (p=0.0105) and LEPR (p=0.0220) where mRNA expression was greater in the VAT compared to WB. The mRNA expression of FTO was similar in VAT and WB (p=0.4039). There were no significant differences in mRNA expression for ADIPOQ, LEP, LEPR and FTO between all the BMI and HIV status groups. However, there were patterns identified that allude to BMI, HIV, and the combination of BMI and HIV which showed that there was an effect on the mRNA expression of ADIPOQ, LEP, LEPR and FTO in the pregnant women. The pregnant women with a BMI ≥25.0 kg/m2 showed a downregulatory pattern for ADIPOQ, LEP, LEPR and FTO in VAT and WB. The HIV-infected pregnant women showed a downregulatory pattern for ADIPOQ, LEP, LEPR and FTO in VAT and WB. The HIV-infected pregnant women with a BMI ≥25.0kg/m2 had the lowest mRNA expression for ADIPOQ, LEP, LEPR and FTO in VAT and WB. The mRNA expression of GHRL in the VAT and WB samples from the pregnant women was undetectable. Conclusion: Maternal nutritional status can be accurately predicted by using surrogate maternal anthropometric measurements such as MUAC, TSF, SSF, MAMC, WC, and frame size. Pregnant women living with HIV do not differ anthropometrically to pregnant women living without HIV. Maternal overweight and obesity in both HIV-infected and HIV-uninfected pregnant black South African women was significantly associated with maternal age, gestational age, HPT disorders and anaemia. Maternal overweight/obesity decreased the odds for anaemia during pregnancy but increased the odds for the development of HPT disorders during pregnancy, especially in the HIVinfected pregnant women. Pregnant black South African women presenting with overweight/obesity and being HIV-infected showed to have the worst downregulatory effect on mRNA expression of ADIPOQ, LEP, LEPR, and FTO. The downregulation of these genes may result in the dysregulation of metabolic pathways that usually control weight gain during pregnancy.
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    Structural and functional characterization of the egress and invasion machinery of the Malaria parasite: proposing a new way forward in Malaria therapeutics from an atomistic perspective.
    (2018) Munsamy, Geraldene.; Soliman, Mahmoud Elsayed Soliman.
    ABSTRACT The past decade has witnessed numerous efforts to control the invasive tactics of the malarial parasite, including focused research towards selective malarial inhibitors of Plasmodium falciparum, the most lethal strain of the Plasmodium species. The recent discovery of the key mediators of egress and erythrocyte invasion of the malaria parasite has opened a new avenue that may be harnessed for the development of effective therapeutics that may permanently eradicate the malaria virus. These new parasitic targets of P. falciparum are PIX and PX and have gained considerable attention in drug discovery pipelines however, the absence of crystal structures of these enzymes evidenced a lack in structural information, as there is currently little known regarding the structural dynamics, active site domains and the mechanism of inhibition of these enzymes. This has therefore led to the modeling of the 3D protein structure of each enzyme to gain a fundamental understanding regarding the structural and functional characteristics that may be visualized from an atomistic perspective. The emergence of new drug targets has led to the integral use of computational techniques including molecular modeling, molecular docking, virtual screening protocols and molecular dynamic simulations which allow chemists to evaluate and assess millions of compounds and thus funnel out potential lead drugs. These in silico techniques further justify the current use of Computer-Aided Drug Design as a cost-effective approach to fast track the drug discovery process. The above-mentioned techniques, amongst a vast range of other computational tools were integrated in this study to provide insight into conformational changes that elucidate potential inhibitory mechanisms, identification of the active site cleft, characterization and pharmacophoric features leading to novel small molecule inhibitors. This study focused on analysing the flap dynamics specific to the aspartic protease family of enzymes using a defined set of parameters to map out the binding domain for the design of potential antimalarial drugs. To gain a molecular perspective of the conformational binding of two proposed experimental drugs which showed substantial inhibitory activity against PIX and PX molecular dynamic simulations were performed and further evaluated employing in silico thermodynamic analysis to provide insight into the proposed binding of mode of each inhibitor, highlighting the key moieties required for binding. A pharmacophoric model was also generated using in silico tools to screen for tailored inhibitors specific to PIX. The aim of this study was to generate fundamental insight into the structural and functional characterization of two prominent targets that play an indispensable role in survival of the malaria virus. The implementation of the information extracted from this study, may provide a structural outline for molecular biologists, and pharmaceutical scientists to aid in the design of novel antimalarial therapeutics.
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    Multidimensional computational modeling of Potent BACE1 (β-Secretase) inhibitors towards Alzheimer’s disease treatment.
    (2021) Ugbaja, Samuel Chima.; Kumalo, Hezekiel M.; Lawal, Monsurat Motunrayo.
    Alzheimer’s disease (AD), as a progressive multifactorial neurodegenerative abnormality of the brain, is often connected with loss or death of neurons as its primary pathogenesis. Another kind of dementia is associated with memory loss and unstable and irrational behaviors, especially among the elderly above 60 years. In South Africa, there are over four million people above the age of 60 years, with an approximation of one hundred and eighty-seven thousand living with dementia. The two distinguishing features (hallmarks) of AD are neurofibrillary tangles and β-amyloid plaques. The β-amyloid plaques result when amyloid precursor protein (APP) is cleaved by β-amyloid precursor protein cleaving enzyme1 (BACE1), otherwise known as β-secretase. Since 1999 the first BACE1 was discovered, it has become a major interest in attempting to develop drugs for the inhibition or reduction of the β-amyloid aggregates in the brain. Reducing or inhibiting the accumulation of β-amyloid has long been the target in the design of drugs for AD treatment. Having a good knowledge of the characteristic properties (BACE1) would assist in the design of potent selective BACE1 inhibitors with fewer or no side effects. Hitherto, only five drugs have been approved by the Food and Drug Administration (FDA) for the remediation of Alzheimer’s disease, and none of the approved drugs targets BACE1. In about twenty years of its discovery, several past and ongoing studies have focused on BACE1 therapeutic roles as a target in managing AD. Several attempts have previously beenmade in designing some small drugmolecules capable of good BACE1 inhibition. Some of the initially discovered BACE1 inhibitors include verubecestat, lanabecestat, atabecestat, and umibecestat (CNP-520). Although these inhibitors significantly lowered β-amyloid plaques in persons having neurological Alzheimer’s at its clinical trials (phase 3), they were suddenly terminated for some health concerns. The termination contributed to the reasons why there are insufficient BACE-targeted drugs for AD treatment. Lately, a novel potent, orally effective, and highly selective AM-6494 BACE1 inhibitor was discovered. This novel BACE1 inhibitor exhibited no fur coloration and common skin alteration, as observed with some initial BACE1 inhibitors. AM-6494 with an IC50 value of 0.4 nM in vivo is presently selected and at the preclinical phase trials. Before this study, the inhibition properties of this novel BACE1 inhibitor at the atomistic and molecular level of BACE1 inhibition remained very unclear. The first manuscript (chapter two) is a literature review on Alzheimer's disease and β-secretase inhibition: An update focusing on computer-aided inhibitor design. We provide an introductory background of the subject with a brief discussion on Alzheimer’s pathology. The review features computational methods involved in designing BACE1 inhibitors including the discontinued drugs. Using the topical keywords BACE1, inhibitor design, and computational/theoretical study in theWeb of Science and Scopus database, we retrieved over 49 relevant articles. The search years are from 2010 and 2020, with analysis conducted from May 2020 to March 2021. Our second manuscript (chapter three) reviewed BACE1 exosite-binding antibody and allosteric inhibition as an alternative therapeutic development. We studied BACE1 biological functions, the pathogenesis of the associated diseases, and the enzymatic properties of the APP site cleavage. We suggested an extensive application of advanced computational simulations in the investigation of anti-BACE1 body and allosteric exosites. It is believed that this investigation will further help in reducing the associated challenges with designing BACE1 inhibitors while exploring the opportunities in the design of allosteric antibodies. The review also revealed that some molecules exhibited dual binding sites at the active site and allosteric site. As a result, we recommend an extensive investigation of the binding free energy beyond molecular docking (such as advanced molecular dynamic simulations) as this promises to reveal the actual binding site for the compounds under investigation. Chapter four contains the detailed computational science techniques which cover the application of the vitally essential methods of molecular mechanics (MM), quantum mechanics (QM), hybrid of QM/MM, basis sets, and other computational instruments employed in this study. In the third manuscript (chapter five), we carried out computational simulations of AM-6494 and CNP- 520.CNP520 was one of the earliest BACE1 drugs that were terminated, chosen in this study forcomparative reasons. This simulation was to elucidate and understand the binding affinities of these two inhibitors at the atomistic level. We explored the quantum mechanics (QM) density functional theory (DFT) and hybrid QM/MM of Our Own N-layered Integrated molecular Orbital and Molecular Mechanics (ONIOM) in these simulations. These computational approaches helped in predicting the electronic properties of AM-6494 and CNP-520, including their binding energies when in complex with BACE1. Considering the debates on which protonated forms of Asp 32 and Asp 288 gives a more favorable binding energy, we analysed the two forms which involved the protonation and un-protonation of Asp 32 and Asp 228.The ONIOM protonated model calculation gave binding free energy of -33.463 kcal/mol (CNP-520)and 62.849 kcal/mol (AM-6494) while the binding free energy of -59.758 kcal/mol was observed for the unprotonated AM-6494 model. These results show the protonated model as a more favourable binding free energy when compared with the un-protonation AM-6494 model. Further thermochemistry processes coupled with molecular interaction plots indicate that AM-6494 has better inhibition properties thanCNP-520.However, it was observed that the protonation and the un-protonation of Asp 32 and Asp 228 modelscould adequately illustrate the interatomic binding of the ligands-BACE1 complex. To further explicate the binding mechanism, conformational and structural dynamism of AM-6494 relative to CNP-520 in complex with BACE1, we carried out advanced computational simulations in the fourth manuscript (chapter six). The extensive application of accelerated molecular dynamics simulations, as well as principal component analysis, were involved. From the results, AM-6494 further exhibited higher binding affinity with van der Waals as the predominant contributing energy relative to CNP-520. Furthermore, conformational analysis of the β-hairpin (flap) within the BACE1 active site exhibited efficient closed flap conformations in complex withAM-6494 relative to CNP-520, whichmostly alternated between closed and semi-open conformational dynamics. These observations further elucidate that AM- 6494 shows higher inhibitory potential towards BACE1. The catalytic dyad (Asp32/228), Tyr14, Leu30, Tyr71, and Gly230 constitute essential residues in both AM-6494 potencies CNP-520 at the BACE1 binding interface. The results from these extensive computational simulations and analysis undoubtedly elucidate AM-6494 higher inhibition potentials that will further help develop new molecules with improved potency and selectivity for BACE1. Besides, grasping the comprehensive molecular mechanisms of the selected inhibitors would also help in fundamental pharmacophore investigation when designing BACE1 inhibitors. Finally, the implementation of computational techniques in the designing of BACE1 inhibitors has been quite interesting. Nevertheless, the designing of potent BACE1 inhibitors through the computational application of the QM method such as the density functional theory (DFT), MM, and a hybrid QM/MM method should be extensively explored. We highly recommend that experimentalists should always collaborate with computational chemists to save time and other resources. ISIZULU ABSTRACT Iqoqa Isifo se-Alzheimer (AD), njengoba siqhubeka siyinhlanganisela yezimbangela ze- neurodegenerative engajwayelekile ebuchosheni, isikhathi esiningi kuxhumana nokulahleka noma ukufa kwama-neurons njengongqaphambili we-pathogenesis. Kungolunye uhlobo lwedementia oluhambisana nokulahlekelwa ukukhumbula kanyenokuxenga kanye nokuphanjanelwa ingqondo, ikakhulukazi kubantu abadala esebeneminyaka engaphezulu kuka-60. ENingizimu Afrikha, kunabantu abangaphezulu kwezigidi ezine abangephezulu kweminyaka ewu-60, ngokuhlawumbisela nje abayinkulungwane namashumi ayisishayangolombili nesikhombisa baphila nedemetia. Zimbili izimpawu ezihlukanisekayo ze-AD ziba-ama-neurofibrillary tangles kanye ne-B-amyloid plaques. I-B-amyloid plaques ingumphumela ngesikhathi i-amyloid eyiprotheni egijimayo iqhwakele oketshezini i-enzyme1 (BACEI), ngale kwalokho yaziwanjenge B-secretase. Kusukela ngo 1999 i-BAC1 yatholakala, isiphenduke ungqaphambili emizamweni yokwakha isidakamizwa sokwehlisa i-B-amyloid ngokwezinga lengqondo. Ngokunciphisa ukwanda kwe-B-amyloid isiphenduke okuqondiwe mayelana nokuqopha isidakamizwa ukuze kwelashwe i-AD. Ukuba nolwazi oluhle oluthinta isici sezakhi ze-BACE1 kuzosiza ekubazeni amandla akhethiwe i-BACE1 ukuvimbela imiphumela engaqondiwe. Kuze kube manje mihlanu imithi esiphasisiwe ngabezokuphatha ukudla kanye nezidakamizwa (FDA) ukwelapha isifo se-Alzheimer kanye nokuthi azikho kulezi eziphasisiwe izidakamizwa ebhekana ngqo ne-BACE1. Emva kokuba selitholakele lapho nje eminyakeni engu 20, sekunezinye esikhathini esedlule kanye nezifundo ezisaqhubeka zigxile ngokubheka kakhulu iqhaza lokwelapha i-BAC1 njengokuqondiswe ekungameleni u-AD. Imizamo eminingana yenziwa esikhathini esedlule ukuqopha uketshezi lwezidakamizwa olukwazi ukuvimba kahle i-BACE1. i-B-amyloid plaques kumuntu one-neurological ye-Alzheimer’s kumzamo (isigaba 3), kwabuye kwanqanyulwa ngenxa yokukhathazeka ngokwezempilo. Ukunqanyulwa kwanikela kuzizathu zokusilele kwezidakamizwa okuqondene nokulashwa kwe-AD. Kamuva, i-novel enamandla, ngisho ngawo umlomo kanye neyakhethwa ngezinga eliphezulu i-AM-6494 BACE1 evikelayo yatholakala. Le noveli i-BACE1 evimbayo yabukisa hhayi ukushintsha kombala woboya kanye nokushintsha kwesikhumba okujwayelekile, njengoba kubukwa nezivimbo zokuqala ze-BACE1. I-AM-6494 ne-IC50 enobumqoka buka 0.4nM kuyo i-vivo ekhethwa ngokwamanje kanye nesigaba sembulambethe yemizamo. Ngaphambi kwalesi sifundo, izakhi zesivimbela zale noveli i-BACE1zivimba ngokwe-atomistic kanye neqophelo le-molecular ye-B ACE1evimbayo kusale nje kungacacile. Umqulu wokuqala (isahluko sesibili) ukubuyekezwa kwesifo se-Alzheimer’s kanye no-B-secretase ovimbayo: ezikhumbuzayo ezigxile ngokusizwa yikhompuyutha eyisivimbo ngokwakhiwa. Sethula isendlalelo sesifundo kanye nengxoxo kafushane nezimbangela nemiphumela ye-Alzheimer. Ukubukezwa kwezimpawu zendlela zobukhompuyutha kufaka ekuqopheni isivimbo se-BACE1 nokuqhutshekiswa kwesidakamizwa. Ngokusebenzisa ofeleba begama BACE1, kusho ukwakha isivimbo, kanye nesifundo senjulalwazi kulwembu lobuchwepheshe kanye ne-Scopus sesizindalwazi. Sathola amaphepha acwaningiwe anokuhlobana angaphezulu kuka 49. Unyaka wokuthungatha usukela ku2010 kuya ku2020, nohlaziyo lwenziwa kusukela kuNhlaba 2020 kuya kuNdasa 2021. Umqulu wethu wesibili (isahluko sesithathu) sabuyekeza i-BACE ehlanganisa i-exosite antibody kanye ne-allosteric yokuthuthukisa ukwelashwa. Sakufunda ukusebenza kwesayensi yokuphila ye-BACE1, i-pathogenesis ehambisana nezifo kanye nezakhi zama-enzymatic esizinda sokuhlukana se-APP. Saphakamisa ukufakwa okunzulu nokucokeme kokulinganisa ngobuchwepheshe bekhompuyutha ekuphenyeni ama-anti-BACE1 omzimba kanye ne-allosteric ye-exosites. Kuyakholeka ukuthi uphenyo luzoqhubeka nokusiza ekwehliseni izinselelo ezihambisana nokwakha isithiyo se-BACE1 ngesikhathi kuhlolwa amathuba okwakheka kwe-allosteric yama-antibodies. Ubuyekezo luphinde lwaveza uketshezi olubukisa isizinda sokuhlanganisa kabili kusizinda esikhuthele kanye nesizinda se-allosteric. Umphumela, kube ukwenza isincomo mayelana nocwaningo olunzulu oluzohlanganisa umfutho okhululekile odlulele ku-molecular docking (njengesicokeme se-molecular yokuhlukahlukana kokulinganisa) njengoba lokhu kuthembisa ukuveza isiza esibopha ngempela ama-compounds angaphansi Isahluko sesine siqukethe imininingwane ngamaqhinga e-computational sayensi efaka isicelo esibalulekile sezindlela ezibalulekile ze-molecular mechanics (MM), i-quantum mechanics (QM), i-hybrid ye-QM/MM, ngesisekelo samasethi kanye namanye amathuluzi ekhompuyutha akhethwa kulesi sifundo. Kumqulu wesithathu (isahluko sesihlanu), siqhube isilinganiso se-computational ye-AM-6494 kanye CNP-520.I-CNP-520 kwakungenye yezidakamizwa zokuqala zeBACE1 ezashatshalaliswa, zakhethwa kulesisifundo ngezizathu zokuqhathanisa. Ukulinganisa kwakuchaza kanye nokuqonda ukusondelana ngokuhlanganiswa kwezithiyo ezimbili kusigaba se-atomistic. Kwahlolwa i-quatum mechanics (QM) yesisindo yokusebenza kwenjulalwazi (DFT) kanye ne-hybrid QM/MM yokwethu okuno-N oluwugqinsi lwe-molecular Orbital kanye ne-Molecular Mechanics (ONIOM) kulolu linganiso. Lezi zindlelakwenza ze-computational zasiza ekuqageleni kwezakhiwo zama-electronic e-AM-6494 kanye CNP-520, kungena namandla okuhlanganisa ngesikhathi kuba lukhuni ne-BACE1. Ngokucabanga izinkulumo mpikiswano mayelana nokuma kwe-protonated ye-Asp32 kanye Asp288 kunika ukuvumelana namandla okuhlanganisa, nokuhlaziya izimo ezimbili ezifaka i-protonation kanye ne-unprotonation ye-Asp32 kanye Asp228. I-ONIOM ye-protonated yomfanekiso wokubala wanikeza amandla akhululekile okuhlanganisa -33,463kcal/mol (NP-520) kanye 62.849 kcal /mol kwavela i-unprotonate ye-AM6494. Imiphumela itshengisa ukuthi i-protonated iyisifanekiso njengoba kuyisona esivumela ukuhlanganiswa ngokukhululeka ngesikhathi lapho bekuqhathanisa ne-unprotonation yomfanekiso u-AM-649. Kuqhutshelwa phambili nemisebenzi ye-thermochemistry kuhlangana nokudlelana ne-molecular plots kutshengisa ukuthi i-AM-649 inezakhiwo ezinhle zokuvimba kune CNP-520. Yize kunjalo kwabonakala ukuthi i-protonation kanye ne-unprotonation ye-Asp32 kanye neyomfanekiso owu- Asp228 bekungatshengisa ngokwenele ukuhlanganisa ngokwe-interatomic yama-ligands EBACE1 ebilukhuni. be-BACE1 ngokwedlulele isilinganiso se-computational. Ukwenza ngokujulile kuphangiswa isilinganiso se-molecular ngokuhlukana, kwakakwa nohlaziyo olusemqoka lwezingxenyana. Imiphumela ye-AM-6494 yaqhubeka yatshengisa ukusondelana kokuhlanganiswayo no-van der Waals njengohamba phambili ekunikeleni amandla ahlobene ne-CNP-520. Ukuvuma kohlaziyo lwe-B-hairpin ngaphakathi ku-BACE1 kutshengiswa esizeni esiphilayo esivala ngendlela umnyakazo wokuvuma kobunkimbinkimbi be-AM-6494 ehlobene neCNP-520, ngokuvamile eshitshashintshayo phakathi kwevalekile kanye nezishaya sakuvuleka kokuvuma okunhlobonhlobo. Lokhu kuhlolwa kuqhubeke kwachazwa ngokuthi i-AM-6494 itshengisa ukuvimba okukhulu nokunethemba mayelana ne-BACE1. Isikhuthazizinguquko se-dyad (Asp32/228), Tyr14, Leu 30, Tyr 71, kanye ne-Gly230 kwakha izinsalela ezibalulekile nxazombili kuAM-6494ne-potencies yeCNP-520 kuBACE1 nesixhumanisi esihlanganisayo. Imiphumela ivela kulama-computational anzulu ayisilinganiso kanye nohlaziyo olucacisa ngokungangabazi i-AM-6494 enesivimbelo esiphakeme esingakwazi ukuqhubeka nokusiza intuthuko yama-molecules amasha anamandla athuthukile kanye nakhethelwe i-BACE1. Ngaphandle kwalokhu, ukucosha izinkambiso ezibanzi ze-moleculor mayelana nezivimbo ezikhethiwe kuzosiza mayelana nophenyo olubalulekile lwe- pharmacophore ngesikhathi kuqoshwa izivimbo se-BACE1. Ekugcineni, ukwenziwa kwe-computational ngokwamacebo ekubazeni izivimbo ze-BACE1 kube into ehlaba umxhwele. Nokho ukubaza izivimbo ezinamandla ze-BACE1 ngokusebenzisa i-computational yendlela ye-QM njengenjulalwazi yesisindo esisebenzayo (DFT), MM, kanye nendlela ye-hybrid QM/MM kufanele iphenywe kanzulu. Sincoma kakhulu ukuthi ongoti abenza izibonisi kufanele njalo bahlangane nama-computational chemists ukonga isikhathi kanye nezinye izinsiza.
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    A pilot study: bisphenol A-glucuronide and their association with sex steroid hormones and 25 Hydroxy vitamin D among mother and child pairs.
    (2021) Gounden, Verena.; Chuturgoon, Anil Amichund.; Naidoo, Rajen.
    Bisphenol A (BPA) is an endocrine disruptor that has become ubiquitous in our environment. It is utilised in numerous consumer products related to the manufacture of plastics. Exposure to BPA has been linked to a wide range of disease including disorders of immune, reproductive and neurological development as well as malignancy. The in-utero stage is particularly vulnerable to the effects of BPA exposure. Maternal exposure has been shown to be positively correlated to BPA levels in the foetus and in early infancy. There is a paucity of data on the extent of exposure to BPA in sub-Saharan populations. As an endocrine disruptor BPA has been shown to affect steroid hormone function and production. However, the mechanism of BPAs action on steroid hormones have not been fully elucidated. The objectives of this study were to describe the extent of BPA exposure in maternal-child pairs in a local cohort, to determine the effect BPA exposure on their steroid hormone concentrations and to elucidate further mechanisms of BPA action via methylation studies of promoter regions of enzymes involved in steroid metabolism. Method: Matched maternal and cord blood samples collected as part of the Maternal and Child Environment birth cohort study were utilised for the purpose of the study. BPA and its metabolite BPA glucuronide (BPA-g) were analysed in the serum maternal and cord blood samples using an in-house developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Samples were also analysed for nine sex steroid hormones namely-: oestradiol (E2), total testosterone (TT), 11- deoxycorticosterone (11DOC), Dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS) androstenedione (Andro), 17-OH progesterone (17OHP), dihydrosterone (DHT) and progesterone (Prog) using LC-MS/MS. 25 hydroxy-Vitamin D (D2 and D3) concentrations were determined in the study cohort using high performance liquid chromatography (HPLC). The degree of the methylation status of the promoter regions of the CYP1B1 and CYP3A4 was assessed using quantitative PCR. A p value of <0.05 was considered significant. Statistical analysis was performed on Medcalc statistical software program version 18.11 (Medcalc, Belgium). Results: Significant exposure to BPA was described in this cohort with more than 75 percent of maternal and cord blood samples exhibiting detectable BPA and/or BPA-g levels. This study demonstrated a statistically significant positive correlation of maternal BPA and BPA-g concentrations with cord blood samples as well as a significant association with cord blood oestradiol and testosterone. A significant negative relationship with cord (p=0.03) and maternal BPA-g levels (p=0.04) and cord total 25OHD levels was noted. No significant association with CYP1B1 and CYP3A4 promoter methylation status and BPA concentrations was identified. Conclusion. This study is the first in South Africa to describe the extent of BPA exposure in a human cohort and in maternal-child pairs. It is also the first in Africa and one of the few studies worldwide to describe the relationship between steroid hormones and BPA in maternal and cord blood samples. The significant BPA exposure noted in this study has important implications with regards to public health strategies to limit BPA exposure as well as to prevent, identify and manage associated disease conditions.
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    The pathophysiology of cholesterol gallstones amongst Black South African women living with HIV.
    (2021) Mewa Kinoo, Suman.; Singh, Bhugwan.; Chuturgoon, Anil Amichund.
    Thirteen percent of South Africans are living with HIV and of those infected, 52% are on antiretroviral therapy (ART). ART has changed the course of this terminal illness to one of a chronic illness. However, the longer life span of people living with HIV has brought about numerous metabolic disorders particularly with change in cholesterol metabolism and risk of cardiovascular disease. Gallstone disease (GD) is also known to be triggered by cholesterol metabolism changes; thus, it is postulated that people living with HIV and ART may be at risk for developing gallstones as well. In South Africa (SA), there is evidence of an increase incidence of GD in black South Africans, a disease once with a low incidence amongst this population group which makes up over 80% of the country’s population. GD also ranks as one of the world’s most expensive disorders to health care systems and thus investigating a causative relationship between HIV, ART and GD has relevance to reduce the burden on our already constrained health care system in SA. Aim The aim of this study was to determine differences in clinical profiles and regulators of hepatic cholesterol and bile acid metabolism in HIV+ve Black South African women on ART presenting with gallstones compared to HIV-ve Black South African women with gallstones. Methods A case series study was conducted amongst all Black South African women undergoing cholecystectomy for gallstone disease over a 1-year period at King Edward VIII Hospital, Durban, SA. A total of 52 patients (34 HIV-ve and 18 HIV+ve) were assessed. Classical risk profiles (age, BMI, children, family history) and lipogram levels. (LDL-c, HDL-c, triglycerides, total cholesterol) were compared between the HIV+ve and HIV-ve women. Categorical variables were tested using either the Fisher’s exact test or Pearson’s Chi-square test. Means were compared using independent t-tests. For non-normally distributed data, the Mann-Whitney test was used. Statistical tests were two-sided, and p values of less than 0.05 were considered as statistically significant. Liver biopsies from five HIV+ve women and five HIV-ve women were analyzed for hepatic expression of key genes in cholesterol metabolism (LDLr, HMGCR, ABCA1) and transcriptional regulators of these genes (microRNA-148a, SREBP2) using quantitative PCR. The same five HIV+ve and five HIV-ve women were evaluated for gene expression of CYP7A1, HNF1α, HNF4α, LXRb, miR-194-5p and miR-122*_1 using RT-qPCR. Messenger RNA and miRNA levels were reported as fold change expressed as 2-ΔΔCt (RQ min; RQ max). Fold changes >2 and <0.5 were considered significant. Results The median age of HIV+ve vs HIV-ve women was 35 years and 50 years respectively (p=0.015). The HIV-ve group had a statistically significant number of patients in the overweight/obese category (BMI > 25kg/m2) compared to the normal weight category (BMI <25kg/m2) (p<0.001). The number of obese women in the HIV+ve group however did not reach statistical significance. Circulating total cholesterol was elevated in the HIV+ve group with significantly elevated LDL-c levels (3.16±0.64mmol/L) relative to uninfected women (2.10±0.74mmol/L; p=0.04). A scavenging receptor for LDL-c, LDLr was significantly decreased (0.18-fold) in this group, possibly contributing to higher LDL-c levels. Transcriptional regulator of LDLr, SREBP2 was also significantly lower (0.13-fold) in HIV+ve women. Regulatory microRNA, miR-148a-3p, was reduced in HIV+ve women (0.39-fold) with a concomitant increase in target ABCA1 (1.5-fold), which regulates cholesterol efflux. HIV+ve women displayed higher CYP7A1 [2.078-fold (RQ min: 1.278; RQ max: 3.381)], LXRb [2.595-fold (RQ min: 2.001; RQ max: 3.000)] and HNF1α [3.428 (RQ min: 1.806; RQ max: 6.507] levels. HNF4α [0.642-fold (RQ min: 0.266; RQ max: 1.55)], miR-194-5p [0.527-fold (RQ min: 0.37; RQ max: 0.752)] and miR-122*_1 [0.595-fold (RQ min: 0.332; RQ max: 1.066)] levels were lower in HIV-ve women. Conclusion HIV+ve women do not conform entirely to the normal known risk profile for GD. Black South African HIV+ve women with GD were significantly younger. Black South African HIV-ve women conform to the known risk factor of obesity with a statistically higher BMI whilst HIV+ve women do not. HIV+ve women also had fewer 1st degree relatives with GD compared to HIV-ve women, and less oestrogen exposure. HIV+ve women have a significant increase in circulating LDL-c coupled with reduced mRNA expression of hepatic LDLr. However, the suppression of miR-148, an epigenetic regulator of LDLr, was downregulated in the HIV+ve group. This would indicate a possible alternate pathway in the downregulation of LDLr in HIV+ve women linked with raised LDL-c and gallstone formation and will require further investigation. MiR-148a however did appear to regulate ABCA1 with an inverse relationship being observed in the HIV+ve woman. HIV+ve women displayed elevated expression of CYP7A1, HNF1α and LXRb. This could have been further influenced by ART and aging. HNF4α, which is known to cause upregulation of CYP7A1, was suppressed with upregulation of CYP7A1 and LXR, known to cause downregulation of CYP7A1 in humans as opposed to mice, also had the opposite effect in HIV+ve women. The best theoretical explanation for this will be an interruption in the enterohepatic circulation, as evident by HIV+ve patients known to have chronic inflammatory and relative malabsorptive disorders of the ileum, which may result in upregulation of CYP7A1 to produce more bile salts. However, these conclusions are drawn from a case series. Larger cohort studies are required into the effects of HIV on GD and the impact of ART on GD in order to put strategies in place to curb this disease process and reduce the morbidity from it and reduce the cost to the overburdened health system.
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    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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    An in vitro and in vivo evaluation of the immuno and neurotoxicological effects of fusaric acid on altered protein kinase signalling cascades.
    (2019) Dhani, Shanel.; Chuturgoon, Anil Amichund.
    Mycotoxins are naturally occurring toxins produced by moulds which contaminate numerous crops and foodstuffs including cereals, nuts and fruits. When consumed, mycotoxins pose a serious threat to both human and animal well-being, causing acute poisoning or chronic effects such as immune deficiency and cancer. Fusaric acid (FA), a ubiquitous mycotoxin produced by Fusarium species, is a frequent contaminate of staple grain-based foods, and is a specific inhibitor of dopamine-β-hydroxylase in human and animal hosts, thereby affecting the autonomic nervous system by causing significant alterations in catecholamine metabolism. The latter effect is believed to occur through the competitive binding of FA with tryptophan to albumin in circulation. Although several studies have reported the oral activity of FA in circulation and the nervous system, the immuno- and neurotoxic effects of FA are unknown. Therefore, in this study we aimed to investigate the immunotoxic and neurotoxic potential of FA, using in vitro human and in vivo murine models. On a daily basis, protein kinases are required to integrate developmental cues and environmental stimuli to decide cell fate (cell death or survival). Thus, alterations to mitogen-activated protein kinases (MAPKs) in the immunotoxicity of FA was assessed in vitro on healthy human peripheral blood mononuclear cells (PBMCs) and on the human acute monocytic leukemic (Thp-1) cell line at an acute exposure (1 day) (Chapter 2); with Thp-1 cells (IC50-107.7 μg/ml) showing a greater susceptibility to FA exposure than PBMCs (IC50-240.8 μg/ml). Notably, elevated stress-induced stimuli (increased oxidative stress and ATP depletion) activated the pro-apoptotic signalling of phosphorylatedextracellular signal-regulated kinase (p-ERK), resulting in initiation of intrinsic apoptosis evidenced by the decreased phosphorylation of B-cell lymphoma 2 (p-Bcl-2; an anti-apoptotic protein) and subsequent activation of caspase-9 and caspase-3/7 activities in Thp-1 cells. In contrast, a caspaseindependent (reduced caspase -8, -9 and -3/7 activities) form of cell death (paraptosis) was induced in PBMCs that was possibly mediated by ERK and c-Jun N-terminal kinase (JNK) in response to metabolic stress (decreased cellular ATP availability). The significant ATP depletion in immune cells then led to the assessment of the metabolic effects of FA in the brain; since the brain is exposed to the peripheral effects of FA and is a highly metabolic organ. Therefore, the next chapter (Chapter 3) investigated the neurometabolic effects of FA via the protein kinase B (Akt) and AMP-activated protein kinase (AMPK) signalling pathways (which are central regulators of cellular energy and metabolism) in C57BL/6 mice at acute (1 day) and prolonged exposure (10 days). Acute exposure to FA, augmented Akt signalling following the increased expressions of upstream regulators phosphatidylinositol 3-kinase (PI3K), mammalian target of rapamycin (mTOR) and p70 ribosomal S6 protein kinase (p70S6K). Activated Akt inhibited glycogen synthase kinase 3 (GSK3) activity with the simultaneous activation of AMPK, p53 phosphorylation and reduced glucose transporter (GLUT)-1 and -4 expression, potentially suppressing neuronal glucose entry. However, following prolonged exposure, FA dampened PI3K/Akt and AMPK signalling, but increased the expression of GLUT transporters (1 and 4) in mice brain. Despite the differential regulation of glucose receptors by the PI3K/Akt and AMPK pathways (at acute and prolonged exposures), neuronal ATP failed to rise despite the increased pyruvate dehydrogenase E1ß (PDHE1β) activity [a regulatory subunit of glycolysis and the tricarboxylic acid cycle (TCA) cycle] at both 1 and 10 days; suggesting that FA mediates ATP depletion independent of metabolic signalling. Given the evident neurometabolic disturbances mediated by FA and its importance as a risk factor for neurometabolic-related diseases, the next chapter (Chapter 4) investigated the neurotoxic potential of FA in C57/BL6 mice following acute (1 day) and prolonged exposures (10 days) and its influence on cyclic AMP (cAMP) response element binding (CREB) signalling, an essential transcription factor, commonly activated by MAPKs, that is responsible for the brain’s neuroprotective responses through the regulation of neurotrophic and metabolic signals in the brain. After an acute administration of FA, CREB signalling was enhanced with a simultaneous increase in brain derived neurotrophic factor (BDNF) expression; whilst FA suppressed CREB/BDNF signalling following a prolonged exposure. In contrast, protein expressions of MAPKs (ERK, JNK and p38) negatively correlated with CREB activity; inferring that FA induced MAPK-independent activation of CREB responses. Consistent with caspase activation in Thp-1 cells, FA increased caspase activities (8, -9 and -3/7) at 1- and 10 days postexposure, although to a lesser extent at a prolonged treatment. However, despite enhanced caspase activity, microanalysis of brain tissue showed no prominent histological markers of damage to extracellular tissue or neuronal cells. Although FA showed no significant neurotoxicity, alterations in glial cell density patterns at both acute and prolonged exposures were observed. Besides the neuroprotective roles of CREB/BDNF signalling in the brain, CREB and BDNF are also involved in memory development and psychiatric disorders. Therefore, although FA may have not been neurotoxic, dysregulation of CREB/BDNF signalling impacts normal brain functions which potentially plays a role in the development of neurological disorders with longer periods of exposure.Collectively, although FA demonstrated significant toxicity towards Thp-1 cells, FA did not display cytotoxicity to healthy immune and neuronal cells, suggesting that compromised cellular systems may be more vulnerable to the effects of FA. In addition, while FA did alter MAPK, PI3K/Akt, AMPK and CREB pathways, which are important regulators of cell survival/death and energy homeostasis, these pathways are also involved in several other fundamental cellular processes, including gene expression, cell differentiation, inflammation, and synaptic plasticity; thus, modifications to their activities could have severe outcomes in health and disease.
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    An investigation of the anti-hyperglycaemic, biochemical and molecular effects of 4-hydroxyisoleucine and fenugreek seed extract in comparison to metformin in vitro and in vivo.
    (2017) Naicker, Nikita.; Chuturgoon, Anil Amichund.; Nagiah, Savania.
    Type two diabetes mellitus (T2D) is a significant cause of premature death and disability, accompanied with negative socio-economic impacts. This metabolic disorder is characterized by hyperglycaemia and defective insulin signalling. Long-term exposure to hyperglycaemia gives rise to altered fat metabolism and reactive oxygen species (ROS) generation. These precursors are central to the progression of dyslipidaemia and attenuated antioxidant (AO) response and detoxification system, respectively. Diabetic dyslipidaemia and oxidative stress (OS) are risk factors for the onset and progression of cardiovascular disease (CVD) and other diabetic complications. The treatment regimen for T2D comprises self-care and anti-diabetic drugs such as metformin. However, due to the lack of compliance to self-care recommendations and some undesirable side effects of metformin, there is the necessity for alternate therapy. Natural products have been used for the treatment of many disorders, including T2D. Trigonella foenum-graecum commonly known as fenugreek is a plant that possesses anti-diabetic effects. These effects are attributed to its bioactive compound – 4-hydroxyisoleucine (4-OH-lle), which constitutes approximately 80% of the bio-composition of the fenugreek seed. Despite these effects, biochemical and molecular effects of 4-OH-lle on insulin signalling, lipid metabolism, and ROS production is not well-documented. This study investigated the effects of 4-OH-lle in comparison to metformin and fenugreek seed extract (FSE) on hyperglycaemic human hepatoma (HepG2) cells and C57BL/6 male mice. Treatments were conducted under normoglycaemic and hyperglycaemic conditions as follows; control, 4-OH-lle (in vitro: 100ng/ml; in vivo: 100mg/kg Body weight) metformin (in vitro: 20mM; in vivo: 20mg/kg Body weight) and FSE (in vitro: 100ng/ml; in vivo: 100mg/kg Body weight) treatment groups. The experiments included; blood glucose measurements, lipid profile analysis, spectrophotometric assays (in vitro), western blotting for protein expression and qPCR for mRNA expression. First, to validate the effects on insulin signalling and glucose sensing, glucose levels were measured with completion of an oral glucose tolerance test. 4-OH-lle treatment attenuated glucose levels, and elevated the mRNA levels of glycogen synthase (GS) and glucokinase (Gck). This was followed by the investigation of the protein and gene expression of insulin signalling regulators: insulin receptor β (IRβ), insulin receptor substrate 1 (IRS1), phosphorylated protein kinase B (pAkt), phosphorylated glycogen synthase kinase 3α/β (pGSK3α/β) and glucose transport 2 (GLUT2). In in vivo hyperglycaemia, 4-OH-lle increased the expression of the investigated proteins and genes. The results showed that 4-OH-lle was just as potent as MF, and FSE in stimulating the insulin signalling cascade. Second, the effect of 4-OH-lle on dyslipidaemia was investigated by measuring mRNA levels of sterol regulatory binding element 1c (SREBP1c) and fatty acid synthase (FAS) – key factors in fatty acid metabolism. Both genes were up-regulated and correlated with the changes in triglyceride and cholesterol levels. Next the protein expression of proprotein convertase subtilisin-like/kexin type (PCSK9) - a regulator of low density lipoprotein cholesterol (LDLc) and peroxisome proliferator-activated receptor gamma (PPARG) – a regulator of high density lipoprotein (HDLc) was evaluated. The data showed that 4-OH-lle down-regulated protein and mRNA expression of PCSK9 and up-regulated protein expression of PPARG. The reduction in PCSK9 levels correlated with the changes observed in low density lipoprotein receptor (LDLr) and LDLc, whereas the increase in PPARG correlated with the elevated mRNA expression of apolipoprotein A1 (Apo A1) and HDLc. Together these results provide substantial evidence for the regulatory effect of 4-OH-lle, in comparison to metformin, and FSE on PCSK9, PPARG and related lipid factors. Finally, the effect of 4-OH-lle on redox status and AO response was assessed by measuring nuclear factor E2-related factor 2 (Nrf2). In both models, there was an increase in the protein expression of phosphorylated Nrf2 accompanied by an increase in mRNA levels of superoxide dismutase 2 (SOD2) and glutathione peroxidase (GPx), and GSH levels. Mitochondria play a central role in contributing to elevated ROS levels. While nuclear responses like Nrf2 regulate ROS, mitochondria possess their own maintenance proteins. These include mitochondrial Lon protease 1 (LonP1), Sirtuin 3 (SIRT3) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) which play an integral role in combatting OS and mitochondrial dysfunction. The results showed that 4-OH-lle displayed a potent effect in inducing the AO response and increasing mitochondrial regulatory proteins. In conclusion, 4-OH-lle improved the compromised insulin signalling and the altered lipid profile as well as induced the AO response and mitochondrial maintenance proteins, in the presence of elevated glucose. Furthermore, the effect of 4-OH-lle was greater than the first-line drug; metformin and FSE, albeit in cultured human liver cells and a mouse model. Also, the crude seed extract displayed promising effects on all investigated parameters. Considering the active role of chronic hyperglycaemia in the onset and progression of CVD and diabetic complications, 4-OH-lle poses as a highly favourable alternate therapy in the treatment of T2D. Moreover, this has great importance in socio-economically challenged communities where T2D is a common disorder, access to healthcare facilities is limited, and plants serve as sources of easily accessible treatments.
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    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.
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    An investigation of stress-responses in pregnant women exposed to ambient air pollution in Durban, South Africa.
    (2017) Anderson, Samantha Mary.; Chuturgoon, Anil Amichund.
    Living or working within an unhealthy environment is attributed to 12.6 million deaths worldwide and 2.2 million deaths in Africa. Ambient air pollution (AAP) exposure is amongst the major contributors of environmental and air quality decay. Durban South Africa (SA) is a rapidly developing city that requires increased infrastructure, transportation, and energy production to support the growing urban population. This leads to air quality degradation, in addition to the heavy burden of human immunodeficiency virus (HIV) and obesity SA faces increase the susceptibility of pathological conditions including respiratory diseases and adverse birth outcomes. Infants in utero are particularly vulnerable to adverse AAP effects, attributed to oxidative stress (OS), inflammation and genetic susceptibility, due to their biological vulnerability, sensitivity to their environment and rapid differentiation and growth. South Durban (SD) comprises a complex mix of dense residential settlements and heavily industrialised areas with high levels of air pollution (AP). This makes SD an ideal location to investigate the effects of AAP, in particular, traffic-related AP (atmospheric oxides of nitrogen (NOx)), on OS and endoplasmic reticulum (ER) stress responses within third trimester pregnant women. A comparison sample of pregnant women, located within north Durban (ND) of similar socio-economic status were used for this study. The susceptibility of OS markers, including 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-OHdG) DNA adducts, lipid peroxidation (LP) and nitric oxide (NO) levels, on adverse birth outcomes, including low birthweight (LBW) and pre-term birth (PTB), were also determined. Additional risk factors such as HIV, obesity and single nucleotide polymorphisms (SNP) within genes of the antioxidant response pathway were investigated for OS and adverse birth outcome susceptibility. Atmospheric NOx pollution data were obtained from land use regression modelling that was previously reported. Atmospheric NOx and maternal serum 8-OHdG adducts were significantly elevated within SD living pregnant women. This induction of DNA damage was found to be the direct consequence of NOx exposure. Pregnant women carrying the variant and wild-type (wt) genotypes of glutathione S transferase (GST) P1 and M1 SNPs, respectively, increased the susceptibility of NOx induced OS. Exposure to increased NOx levels significantly reduced the gestational age (GA) of these pregnant women, with increased susceptibility for mothers carrying male neonates. The wt 8-oxoguanine glycosylase 1 (OGG1) Ser326Cys genotype was found to be associated with both HIV and obesity. Therefore pregnant women infected with HIV (HIV+) and carrying the wt genotype significantly increased the risk for HIV associated LBW and PTB. In addition, living within SD and being exposed to higher levels of AAP significantly increased the susceptibility for PTB. Comorbid HIV and obesity were identified as additional risk factors for birthweight (BW) reduction. Increased maternal serum NO levels were observed within HIV+ women, with reciprocal activity on malondialdehyde (MDA) levels. Increased levels of NO directly reduced BW, especially for HIV+ and SD living women. This suggests NO may play a key role in LBW aetiology as a consequence of HIV infection and traffic-related AP. HIV was shown to differentially modulate MDA’s effect on neonatal BW. Exposure to increased levels of NOx and HIV infection induced the expression of microRNA (miR)-144, which was shown to negatively regulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This transcription factor, Nrf2, was shown to significantly increase antioxidant gene expressions. Therefore the induction of miR-144 was implicated as a mechanism for increased OS due to HIV and NOx exposure. In addition, elevated ER stress genes were observed within HIV negative SD living patients. Hence, exposure to higher levels of AAP within SD led to increased ER stress, which may act reciprocally on the induction of ROS leading to increased OS. These findings indicate that exposure to atmospheric NOx, elevated AAP levels within SD and exposure to HIV infection resulted in increased OS with increased susceptibility towards adverse birth outcomes within pregnant women. Further studies into the mechanisms proposed within a larger population including multiple pollutants and gene interactions may give additional insight into the aetiology of adverse birth outcomes as a consequence of AAP exposure.
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    Temporal gene expression of Chlamydia trachomatis in keratinocytes at 37° versus 33°C.
    (2017) Mzobe, Gugulethu Favourate.; Joubert, Bronwyn C.; Sturm, Adriaan Willem.
    Abstract available in PDF file.
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    Molecular and biochemical aspects of the kallikrein-kinin system in oesophageal carcinoma.
    (1999) Dlamini, Zodwa Lawrentia.; Bhoola, Keshavlal Daya Narotam.
    Abstract available in PDF file.
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    The antiproliferative and apoptosis inducing effects of Moringa oleifera aqueous leaf extract and its synthesised gold nanoparticles - modulation of oncogenes and tumour suppressor genes in human cancer cell lines.
    (2015) Tiloke, Charlette.; Chuturgoon, Anil Amichund.; Phulukdaree, Alisa.
    Cancer is one of the leading causes of global mortality. In South Africa (SA), the burden of cancer (lung and oesophageal) continues to increase. Moringa oleifera (MO), indigenous to India, is found widely in SA and used in traditional treatments of cancer. Gold nanoparticles (AuNP’s) are showing potential in cancer therapies and can be synthesised using plants extracts such as MO leaf extract (MOE). This study investigated the antiproliferative effect of MOE and AuNP’s synthesised from MOE (MLAuNP) in A549 lung and SNO oesophageal cancer cells. MO crude aqueous leaf extract was prepared and cytotoxicity (MTT assay) was assessed in A549, SNO cells and normal peripheral blood mononuclear cells (PBMCs) (24h). Oxidative stress, DNA fragmentation and apoptotic markers were determined. A one-pot green synthesis technique using MOE to synthesise MLAuNP was then conducted. A549, SNO cells and PBMCs were also exposed to MLAuNP and CAuNP to evaluate cytotoxicity and apoptotic markers. MOE was cytotoxic to A549 cells. MOE (IC50: 166.7μg/ml, 24h) significantly increased lipid peroxidation, decreased glutathione (GSH) and Nrf2 levels leading to DNA fragmentation. MOE induced apoptosis by significantly increasing p53, caspase-9, enhancing caspase-3/7 activities and Smac/DIABLO expression. MOE significantly cleaved PARP-1 into 89kDa and 24kDa fragments. MOE was not cytotoxic to PBMCs but in SNO cells (IC50: 389.2μg/ml, 24h), it significantly increased lipid peroxidation, DNA fragmentation, decreased GSH, catalase and Nrf2 levels. Apoptosis was confirmed by the significant increase in phosphatidylserine (PS) externalisation, caspase-9, enhanced caspase-3/7 activities and significant decrease in ATP levels. MOE significantly increased p53, Smac/DIABLO and cleavage of PARP-1, resulting in an increase in the 24kDa fragment. MLAuNP was successfully synthesised. MLAuNP and CAuNP were not cytotoxic to PBMCs, whilst its pro-apoptotic properties were confirmed in A549 (IC50: MLAuNP - 98.46μg/ml; CAuNP - 121.4μg/ml) and SNO (IC50: MLAuNP - 92.01μg/ml; CAuNP - 410.4μg/ml) cells. MLAuNP significantly increased caspase activity in SNO cells while MLAuNP significantly increased PS externalisation, mitochondrial depolarisation, caspase-9, caspase-3/7 activities and decreased ATP levels. Also, MLAuNP significantly increased p53, Bax, Smac/DIABLO, PARP-1 24kDa fragment and enhanced SRp30a levels. Conversely, MLAuNP significantly decreased Bcl-2, Hsp70, Skp2, Fbw7α, c-myc levels and activated alternate splicing with caspase-9a splice variant being increased. These findings indicate that MOE exerts antiproliferative effects in cancerous A549 and SNO cells by increasing oxidative stress, DNA fragmentation and inducing apoptosis. MLAuNP also possessed antiproliferative properties in SNO cells and induced apoptosis in A549 cells by modulating oncogenes, tumour suppressor genes and activating alternate splicing of caspase-9. MOE and MLAuNP showed potential use as a complementary and alternative treatment for lung and oesophageal cancer. MOE fractionation studies are further recommended to identify the bioactive compounds responsible for the antiproliferative effect seen in A549 and SNO cells. In addition, membrane transport proteins as well as cell cycle analysis will provide further insight into MOE and MLAuNP antiproliferative effect.
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    An investigation into the TB/HIV manipulation of the T-cell immune response.
    (2015) Korb, Vanessa Claire.; Chuturgoon, Anil Amichund.; Moodley, Devapregasan.
    Abstract available in PDF file.
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    Hyperglycaemic-induced regulation of SIRT3 and downstream antioxidant profile.
    (2015) Gounden, Shivona.; Chuturgoon, Anil Amichund.; Moodley, D.
    Hyperglycaemia increases reactive oxygen species (ROS) production and mitochondrial dysfunction which are involved in metabolic disorders. Sirtuin 3 (SIRT3) is a primary mitochondrial deacetylase that regulates mitochondrial function and antioxidant (AO) defence. We investigated the role of SIRT3 in AO defence under hyperglycaemic conditions in HepG2 cells in the presence and absence of metformin and curcumin. We also examined cell protective mechanisms that counterbalance apoptotic stress under these oxidative conditions. HepG2 cells were cultured with 5mM (control), 19.9mM mannitol (OC), 10mM glucose, 30mM glucose (hyperglycaemic), 10mM nicotinamide (NAM) at 24hr and 72hr time points in the absence or presence of curcumin (5μM and 10μM) or metformin (3mM). Increased expressions of SIRT3, peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), mitochondrial AO enzymes glutathione peroxidase 1 (GPx1), superoxide dismutase 2 (SOD2), uncoupling protein 2 (UCP2) and mtDNA repair enzyme 7, 8-dihydro-8-oxoguanine (OGG1) were observed under hyperglycaemic conditions. The same trend was observed for all parameters following metformin and curcumin treatment. In addition, curcumin also increased expressions of nuclear factor-kappa B (NF-κB), lon protease (Lon) and heat shock protein 70 (Hsp70). These were optimally expressed in the 10μM curcumin-treated groups. We also showed that under hyperglycaemic conditions, apoptosis was initiated but may not have been fully executed due to the induction of stress proteins (heat shock protein 27, nuclear factor erythroid-derived 2-like 2) and AO defence that counterbalance apoptotic stress. The results suggest that SIRT3 modulates AO defence and confers resistance to oxidative stress (OS)- induced damage under hyperglycaemic conditions in HepG2 cells. Our data also suggests that metformin and curcumin may work synergistically with SIRT3, or through SIRT3-mediated mechanisms, to improve AO defence. Our model shows that hyperglycaemia may induce apoptosis; however, apoptotic stress may be counterbalanced by cell survival mechanisms that include stress response proteins and the downstream activation of AO defence. Mitochondria are susceptible to OS, which is involved in metabolic disorders. SIRT3 may, therefore, be therapeutically targeted as a potential cyto-protective factor. Modulation of SIRT3 function, by chemical or natural therapeutics, may also improve disease outcomes.
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    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.