Doctoral Degrees (Medical Biochemistry)

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An assessment of gene polymorphisms in young South African Indians with coronary artery disease and the effect of atorvastan in vitro.
(2012) Phulukdaree, Alisa.; Chuturgoon, Anil Amichund.
The global burden of heart disease increases every year. It has been estimated that by the year 2020, coronary artery disease (CAD) will be the number one cause of death worldwide. Indian populations throughout the world have the highest prevalence of CAD and early onset of the disease compared to other ethnic groups. Glutathione S-transferases (GSTs) detoxify environmental agents which influence the onset and progression of disease. Dysfunctional detoxification enzymes are responsible for prolonged exposure to reactive molecules and can contribute to endothelial damage, an underlying factor in CAD. Uncoupling proteins (UCPs) 2 and 3 play an important role in the regulation of oxidative stress which contributes to chronic inflammation. Coronary artery disease is a chronic inflammatory disorder characterized by elevated levels of C-reactive protein (CRP) and pro-inflammatory cytokines such as interleukin 6 (IL-6). Polymorphisms of these genes have been linked to CAD and other chronic diseases. Statins, metabolised in the liver, are the most commonly used drug to control atherosclerosis progression in CAD patients. The pleiotropic effects of statins have been attributed to both favourable and adverse outcomes in CAD patients particularly related to myopathy and hepatotoxicity. All patients (n=102) recruited into this study were South African Indian males. A corresponding age-, gender- and ethnicity-matched control group (n=100) was also recruited. The frequency of the GSTM1 +/0, GSTP1 A105/G105, IL6 -174G/C and CRP -390C/A/T genotypes was assessed by polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP). For the in vitro study, the biological effect of atorvastatin on HepG2 cells was assessed. The metabolic activity, cytotoxicity, oxidative stress and nitric oxide production was assessed by the ATP, lactate dehydrogenase (LDH), thiobarbituric acid reactive substance (TBARS) and Griess assays, respectively. The profile of 84 microRNA (miRNA) species was evaluated using the miRNA Pathway Finder PCR SuperArray. The predicted targets of up-regulated miRNAs were determined using the online software, Targetscan. The mRNA levels of guanidinoacetoacetate (GAMT), arginine glycine aminotransferase (AGAT) and spermine oxidase (SMO) were determined using quantitative PCR. Western blotting was used to determine GAMT and phosphorylated p53 levels in treated cells. The GSTM1 0/0 and GSTP1 A105/A105 genotypes occurred at higher frequencies in CAD patients compared with the control group (36% vs. 18% and 65% vs. 48%, respectively). A significant association with CAD was observed in GSTM1 0/0 (odds ratio (OR)=2.593; 95% confidence interval (CI) 1.353 - 4.971; p=0.0043) and GSTP1 A105/A105 OR=0.6011; 95% CI 0.3803 - 0.9503; p=0.0377). We found a significant association between smoking and CAD; the presence of either of the respective genotypes together with smoking increased the CAD risk (GSTP1 A105 relative risk (RR)=1.382; 95% CI 0.958 - 1.994; p=0.0987 and GSTM1 null RR=1.725; 95% CI 1.044 - 2.851; p=0.0221). The UCP2 -866G/A and UCP3 -55C/C genotypes occurred at highest frequency in CAD patients (59% vs. 52% and 66% vs. controls: 63% respectively) and did not influence the risk of CAD. Homozygous UCP3 -55T/T genotype was associated with highest fasting glucose (11.87±3.7mmol/L vs. C/C:6.11±0.27mmol/L and C/T:6.48±0.57mmol/L, p=0.0025), HbA1c (10.05±2.57% vs. C/C:6.44±0.21% and C/T:6.76±0.35%, p=0.0006) and triglycerides (6.47±1.7mmol/Lvs. C/C:2.33±0.17mmol/L and C/T:2.06±0.25mmol/L, p<0.0001) in CAD patients. A significant association between the G allele of the IL6 -174 polymorphism and non-diabetic CAD patients was found (p=0.0431 odds ratio: 1.307, 95% CI: 1.047-1.632). A significant association with the C allele of the -390 CRP triallelic variants and CAD (p=0.021 odds ratio: 1.75, 95% CI: 1.109-2.778) was also found using a contingency of the C allele vs. the minor A and T allele frequencies. The strength of the association of the C allele with non- diabetic CAD subjects was much higher (p=0.0048 odds ratio: 2.634, 95% CI: 1.350-5.138). Circulating median levels of IL-6 (0.9 (0.90, 0.91) pg/ml and 0.9 (0.87, 0.92) pg/ml) and CRP (5.65 (1.9, 8.2) mg/l and 2.90 (1.93, 8.35) mg/l) were similar between CAD patients and controls, respectively. A similar finding was observed between controls and non-diabetic CAD subjects. Levels of IL-6 and CRP in CAD subjects were not significantly influenced by polymorphic variants of IL-6 and CRP. In the control group, the level of IL-6 was significantly influenced by the IL6 -174 G allele (p=0.0002) and the CRP -390 C allele (p=0.0416), where subjects with the homozygous GG (0.9 (0.9, 1,78) pg/ml) and CC (0.9 (0.9, 0.95) pg/ml) genotype had higher levels than the C allele carriers (0.9 (0.64, 0.91) pg/ml) or A and T carriers (0.9 (0.69, 0.91) pg/ml) combined. The lowest measure of proliferation/metabolism in HepG2 cells was observed at 20μM atorvastatin, with 82±9.8% viability. The level of cytotoxicity was increased in statin treated cells from 0.95±0.02 units to 1.11±0.03 units (p=0.001) and malondialdehyde levels was reduced from 0.133±0.003 units to 0.126±0.005 units (p=0.009) whilst nitrite levels were elevated (0.0312±0.003 units vs. control: 0.027±0.001 units, p=0.044). MicroRNAs most significantly upregulated by atorvastatin included miR-302a-3p (3.05-fold), miR-302c-3p (3.61-fold), miR-124-3p (3.90-fold) and miR-222-3p (4.4-fold); miR-19a-3p, miR-101-3p and let-7g were downregulated (3.63-fold, 2.92-fold, 2.81-fold, respectively). A list of miRNA targets identified included those with a role in metabolism and inflammation. The miR-124a specifically targets the mRNA of GAMT and SMO.
Antibiotic resistance in the food chain : a case study of Campylobacter spp. in poultry.
(2013) Bester, Linda Antionette.; Essack, Sabiha Yusuf.
The sub-therapeutic use of antibiotics for growth promotion in food animal production, has engendered substantial debate on the dissemination of antibiotic resistance via the food chain, specifically, the probability of antibiotic use in food production creating a reservoir of resistant bacteria and/or resistance genes that may spread to humans thereby limiting the therapeutic value of antimicrobial drugs. In the absence of any surveillance programme on food-borne bacteria in South Africa, this study focussed on Campylobacter spp. in poultry and encompassed a literature review on the prevailing debate on the dissemination of antibiotic resistance via the food chain, a phenotypic observational study on the prevalence and antibiotic resistance profiles of Campylobacter spp. isolated within and across different poultry farming systems and a genotypic component that covered identification methods, plasmid profile determination and strain typing. Identification methods for Campylobacter spp., viz, biochemical tests and matrix assisted laser desorption ionization- time of flight (MALDI-TOF) mass spectrometry was compared to the PCR which is considered the gold standard as a molecular method of identification. The MALDI-TOF was shown to be superior to the biochemical tests for the identification of C. coli but equivalent to the biochemical tests for C. jejuni. Of the 363 samples collected in total, the frequency of thermophilic Campylobacter was 68 % in rural farms (or informally reared poultry), 47 % in both commercial free-range and industrial broilers and the highest in industrial layers at 94 %. Antibiotic resistance analysis showed that isolates from the rural farming systems were significantly (P < 0.01) more susceptible to ciprofloxacin, tetracycline and erythromycin when compared to the other farming systems. Significant (P < 0.001) antibiotic resistance differences were detected between broilers (5 - 8 week lifespan), and layers (36 - 52 week lifespan) for gentamicin, ciprofioxacin and tetracycline. Plasmids were fonnd be harboured by isolates in all the farming systems; in 84 % of isolates from free-range broilers, 77 % of isolates from industrial broilers, 83 % of isolates from industrial layer hens and 75 % of isolates from the rural farming system. The PFGE genotyping of 42 Campylobacter isolates generated 39 SmaI types. Substantial and substantive genetic diversity was observed between and within farming systems. The lack of correlations amongst the parameters within and between farming systems attested to the diversity and complexity of phenotypes and genotypes and indicated de novo evolution in response to antibiotic selection pressure and animal husbandry practices.
An investigation into the biochemical, molecular and epigenetic effects of fumonisin B1 in liver (HEPG2) cells.
(2014) Chuturgoon, Anil Amichund.; Moodley, Devan.; Phulukdaree, Alisa.
Fumonisins are carcinogenic mycotoxins that occur world wide in maize and maizebased products intended for human consumption. Consumption of fumonisincontaminated maize as a staple diet has been associated with oesophageal and liver cancer in South Africa and China. Fumonisin B1 (FB1) inhibits sphingolipid biosynthesis and has been implicated in cancer promoting activity in animals and humans. FB1 disrupts DNA methylation and induces chromatin modifications in human hepatoma (HepG2) cells. In this study FB1 (IC50=200μM) altered liver enzyme expression of DNA methyltransferases and demethylases. DNA methyltransferase activities of DNMT1, 3a and 3b were significantly decreased, whilst both DNA methylase (MBD2) activity and expression was significantly up-regulated resulting in global DNA hypomethylation. In addition the histone demethylases, KDM5B and KDM5C, expression was increased. FACS data confirmed FB1 significantly increased global DNA hypomethylation – a process that causes chromatin instability. Next the effect of FB1 on miRNA expression was evaluated; FB1 significantly down-regulated (11 fold) expression of miR-27b. MiR-27b modulates expression of human cytochrome P450 (CYP1B1) that catalyzes the metabolic activation of many procarcinogens. In order to directly assess the effect of miR-27b on CYP1B1 mRNA levels, liver cells were transfected with the mimic to miR-27b. CYP1B1 mRNA and protein expression was significantly up-regulated by 1.8- fold and 2.6- fold respectively. CYP1B1 is post-transcriptionally regulated by miR-27b suggesting that FB1- induced modulation of miR-27b in hepatic cells may be an additional mode of hepatic neoplastic transformation. Finally, the effect of FB1 on the apoptotic pathway in HepG2 cells was investigated using an mRNA expression array panel of pro- and anti- apoptotic molecules. FB1 significantly increased an AIP family member - BIRC 8/ILP-2 (8-fold) in an apoptosis array. In addition, ILP2 protein expression was increased (2.3-fold) with a corresponding decrease in Smac/DIABLO protein levels (1.7-fold). Further analysis showed an FB1 (0μM, 50μM, 100μM, 200μM) dosedependent increase in BIRC-8/ILP-2 mRNA and protein expression in HepG2 cells. This data suggests that FB1 modulates apoptosis in a complex dose-dependent regulation of pro- and anti-apoptotic molecules – and it is not a matter of simply switching on or off. In conclusion, the data shows that FB1 possess epigenetic properties by inducing global DNA hypomethylation, modulating miRNA expression, and increasing expression of the AIP protein family (BIRC8/ILP-2) that may lead to liver tumourigenesis.
Biochemical, apoptotic and cellular stress studies in rheumatoid arthritis.
(2009) Moodley, Devapregasan.; Mody, Girish Mahasukhlal.; Chuturgoon, Anil Amichund.
Abstract available in PDF file.
An investigation into the clinical, biochemical, immunological and epigenetic factors in black South Africa women with preeclampsia and HIV.
(2015) Maharaj, Niren Ray.; Chuturgoon, Anil Amichund.; Moodley, Jagidesa.
Introduction: Preeclampsia (PE) and HIV/AIDS contribute significantly to adverse maternal and perinatal outcomes globally. The treatment of PE remains empiric, however in HIV infection, highly active antiretroviral therapy (HAART) is used routinely for maternal health and the prevention of vertical transmission in pregnancy. The relationship between PE and HIV /HAART remains controversial and despite extensive research, the pathophysiology of both conditions is not fully understood. Contemporary and new research strategies include immunological and genetic aspects of PE and HIV, and clinical and biochemical effects associated with HAART. Aims and Objectives: The aims of the study were to determine: (1) the association of HIV and HAART with clinical and biochemical indices in women with PE, (2) the effects of HAART in relation to inflammatory cytokines in PE and HIV, and (3) the role of gene polymorphisms in preeclamptic women with HIV on HAART. The specific objectives of the study were to identify significant differences (p>0.05) in the routine clinical and laboratory indices between the preeclamptic groups, (2) to identify significant changes in pro-inflammatory cytokines IL2, TNFα, IFNɣ and IL6 in relation to HIV / HAART and preeclampsia, and (3) to determine the association of single nucleotide polymorphisms (SNP) miRNA 27a (rs 895819T>C) and miRNA 146a ( rs 2910164G>C) with preeclampsia risk and susceptibility to associated factors. Materials and Methods: A prospective cohort study was conducted between July 2013 and September 2014 at Prince Mshiyeni Memorial Hospital in Durban, South Africa. To maintain ethnographic and anthropometric consistency, a standard cohort of one hundred and ninety three (193) Black women of Zulu ethnicity comprising 4 groups: i.e. uninfected normotensive (50; 26%), infected normotensive (45; 23%), uninfected preeclamptic (53; 28%) and infected preeclamptic women (45; 23%) was recruited during pregnancy after ethical approval and informed consent and followed until delivery. Women with gestational hypertension, renal disease, diabetes mellitus, chronic hypertension and collagen vascular disease were excluded. Specific patient characteristics, clinical features, laboratory indices, and complications were analysed descriptively. Serum levels of pro-inflammatory cytokines TNF-α, IFN- γ, IL2 and IL6 were determined, using commercially available kits and Cytometric Bead Array (CBA). Genotyping using PCR and the TaqMan® SNP genotyping assay for single nucleotide polymorphisms miRNA 27a rs895819T>C and miRNA 146a rs 2910164G>C was performed and analysed in relation to preeclampsia risk, susceptibility to related clinical features and pro-inflammatory cytokines. Comparative data was recorded and analysed descriptively. Results: Our results indicate that the clinical features, laboratory indices, and complications among HIV infected preeclamptic women on HAART is similar to uninfected preeclamptic women. However, gammaglutamyl transferase (GGT), a hepatic enzyme, was markedly elevated (p=0.001) in HIV infected preeclamptic women on HAART. There were significant decreases in pro-inflammatory cytokines IL2 (p=0.010), TNF-α (p=0.045), and IL6 (p=0.005), in normotensive women on HAART compared with uninfected women and significant decreases in IL2 (p=0.000) and TNFα (p=0.000) in preeclamptic women on HAART compared with uninfected preeclamptic women. In the genotype analyses, we found that the variant genotypes (GC/CC) in miRNA 146a were significantly associated with lower severe preeclampsia risk (OR: 0.34, 0.12-0.99; p=0.048), especially in the presence of HIV/HAART (OR: 0.11; 0.02-0.68, p= 0.018), and the variant genotype (TC/CC) in miRNA 27a was associated with an elevated BMI in preeclamptic women (32.57 vs 29.25; p= 0.06), especially in the presence of HIV/HAART (33.47 vs 27.87; p=0.005). Conclusion: The effects of HIV and HAART influence biochemical, immunological and genetic aspects in women with preeclampsia. Gammaglutamyl transferase, a hepatic enzyme, was markedly elevated and suggests a possible compounding effect on the liver, which is a target organ for preeclampsia and the side effects of HAART. Current management guidelines remain appropriate, however serial hepatic function tests are necessary in clinical practice. The prevention of obesity is also necessary to reduce long term cardiovascular complications associated with these conditions. The immune reconstitutive effects of HAART include a reduction in pro-inflammatory cytokines in normotensive women as well as in preeclamptic women, which may have a bearing on the clinical course of preeclampsia in women on HAART. MiRNA 27a and miRNA 146a are endogenous small RNAs that post transcriptionally regulate gene expression and are implicated in a plethora of pathophysiological processes related to preeclampsia and its related features. TC/CC and GC/CC genotype variants in the functional single nucleotide polymorphisms (SNP) microRNA27a (rs 895819 T>C) and microRNA 146a (rs 2910164G>C were associated with obesity and severe preeclampsia respectively. Obesity is a wellrecognised risk factor for preeclampsia, and severe preeclampsia is associated with increased morbidity and mortality. These findings demonstrate the importance of molecular genetics and inflammatory mediators in preeclampsia, the pathogenesis of which remains multifactorial in origin.
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.
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.
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.
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.
Quantification of circulating cell free fetal DNA and cell free total DNA in normal pregnancy and in pregnancy-related hypertension in Black South African Women.
(2016) Eche, Simeon.; Mackraj, Irene.; Moodley, Jagidesa.
Abstract available in PDF file.
An in vitro investigation into the anti-proliferative and anti-inflammatory properties of centella asiatica (linnaeus) urban (leaf) and withania somnifera (linnaeus) dunal (root) extracts.
(2017) Naidoo, Dhaneshree Bestinee.; Chuturgoon, Anil Amichund.
Abstract available in PDF file.
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.
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.
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.
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.
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.
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.
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.
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.
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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