Medical Biochemistry

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Biotyping Saccharomyces cerevisiae strains using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS)
(2011) Moothoo-Padayachie, Anushka.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.; Govender, Thavendran.; Govender, Patrick.
In clinical diagnosis and fermentation industries there is a need for a method that allows for the differentiation of yeast to the strain level (biotyping). The ideal biotyping method should be accurate, simple, rapid and cost-effective, and capable of testing a large number of yeast isolates. Matrix assisted laser desorption/ionization time of flight mass spectrometry has emerged as a powerful biotyping tool for the identification of bacteria and clinical yeast isolates, mainly Candida. It has been found that the MALDI-TOF MS signals from yeast are harder to obtain than from bacteria. It has been reported by several research studies that a cell lysis step is required to obtain a mass spectral signal for clinical Candida strains. To date an optimized sample preparation protocol has not been devised for the biotyping of S. cerevisiae strains. Studies on the identification of yeast using MALDI-TOF MS have focused primarily on clinical Candida yeast isolates but have included very few S. cerevisiae strains. Furthermore these yeast identification studies using MALDI-TOF MS have only achieved identification to the species and not strain level. A major limiting attribute of MALDI-TOF MS for the accurate identification of microbes, is its dependency on a comprehensive mass spectral database. Bruker Daltonics is a pioneer and leader in providing innovative life science tools based on mass spectrometry thus the Bruker Daltonics mass spectral database and state-of-the-art instruments and accompanying software were selected for this study. The Bruker Daltonics mass spectral database currently holds three thousand seven hundred and forty microorganisms of which only a mere seven are S. cerevisiae strains. Initially in this study, a number of parameters of a generic ethanol/formic acid protein extraction procedure as originally described by Bruker Daltoincs were considered in the development of a sample preparation protocol that yielded characteristic and highly reproducible MALDI-TOF mass spectra. The parameters considered included cell number, alcohol fixation, matrix solution and media. It was found that using the optimized sample preparation protocol unique and highly reproducible mass spectral profiles were obtained for all three S. cerevisiae strains. Multivariate analysis confirmed that the differences between all three S. cerevisiae strains were statistically significant. For quality assurance, the spectra of the three strains were sent for evaluation by Bruker Daltonics and were deemed suitable for the purpose of biotyping. The newly created ethanol/formic acid extraction procedure was used to generate an S. cerevisiae mass spectral database comprising of forty-five S. cerevisiae strains within a local context but also of global significance. The accuracy of the mass spectral database was assessed using blind coded S. cerevisiae strains obtained from the Agricultural Research Council Infruitec-Nietvoorbij (Institute for Deciduous Fruit, Vines and Wine), Stellenbosch, South Africa. It was found that S. cerevisiae identification to the species and more importantly strain level was achievable with relatively good accuracy. To determine the potential application of MALDI-TOF MS as an accurate method for S. cerevisiae strain identification in industry, blind coded S. cerevisiae strains were obtained from Natal Cane Products and subjected to MALDITOF MS analysis. It was found that four of the pure cultures submitted were correctly identified to the strain level and the three S. cerevisiae strains incorrectly identified may have been contaminants or the result of incorrect optimization conditions for the fermentation. Thus MALDITOF MS was shown to be an accurate identification tool, that may also be used to detect contaminants or incorrect environmental conditions which can result in substantial losses.
Analysis of a multidrug resistant acinetobacter SPP. outbreak in the intensive care unit of King Edward VIII Hospital.
(2000) Deedat, Fathima.; Sturm, Adriaan Willem.
The study arose out of a need to investigate and control a nosocomial outbreak caused by multidrug resistant Acinetobacter spp in the fifteen-bed intensive care unit of King Edward VIII Hospital. Following the discovery of the index case, four other patients were found to have a similar strain of Acinetobacter spp. All fifteen patients in the ward were subsequently screened for the organism. Forty-seven isolates were obtained from 12 patients. Eight of the patients were infected with the organism and six of these eight patients subsequently died. Swabs from the ward environment were also screened for the organism, which was found in patients' baths, suction water and urine collection jars. The outbreak was aborted by the use of strict infection control techniques. Minimum inhibitory concentrations (MICs) of 20 of the 47 isolates were determined for the following antimicrobials: imipenem, ciprofloxacin, gentamicin, amikacin, netilmycin,cefotaxime, ceftazidime and tetracycline. The same 20 isolates were further typed using ribotyping. Seven different antibiogram patterns were obtained using the MIC data. The majority of isolates (11) fit into a Single type, and showed resistance to all drugs tested, except for susceptibility to tetracycline and netilmycin only. Ribotyping revealed 5 different types. There were 9 isolates of ribotype a, 2 of ribotype b, 3 of ribotype c, 5 of ribotype d and 1 of ribotype e. In conclusion, this study describes a nosocomial outbreak with a multidrug resistant Acinetobacter spp. in an intensive care unit. The results showed that there was no correlation between the two typing methods used, ribotyping was more discriminatory than antibiogram types, with the majority of strains belonging to two different ribotypes.
The effects of Sutherlandia frutescens in cultured renal proximal and distal tubule epithelial cells.
(2009) Phulukdaree, Alisa.; Chuturgoon, Anil Amichund.
Sutherlandia frutescens (SF), an indigenous medicinal plant to South Africa (SA), is traditionally used to treat a diverse range of illnesses including cancer and viral infections. The biologically active compounds of SF are polar, thus renal elimination increases susceptibility to toxicity. This study investigated the antioxidant potential, lipid peroxidation, mitochondrial membrane potential and apoptotic induction by SF on proximal and distal tubule epithelial cells. Cell viability was determined using the MTT assay. Mitochondrial membrane potential was determined using a flow cytometric JC-1 Mitoscreen assay. Cellular glutathione and apoptosis were measured using the GSH-GloTM Glutathione assay and Caspase-Glo® 3/7 assay, respectively. The IC50 values from the cell viability results for LLC-PK1 and MDBK was 15 mg/ml and 7 mg/ml, respectively. SF significantly decreased intracellular GSH in LLC-PK1 (p < 0.0001) and MDBK (p < 0.0001) cells. Lipid peroxidation increased in LLC-PK1 (p < 0.0001) and MDBK (p < 0.0001) cells. JC-1 analysis showed that SF promoted mitochondrial membrane depolarization in both LLC-PK1 and MDBK cells up to 80% (p < 0.0001). The activity of caspase 3/7 increased both LLC-PK1 (11.9-fold; p < 0.0001) and MDBK (2.2-fold; p < 0.0001) cells. SF at high concentrations plays a role in increased oxidative stress, altered mitochondrial membrane integrity and promoting apoptosis in renal tubule epithelia.
An in vivo study to determine the effects of Ochratoxin A and Sutherlandia frutescens in male Wistar rats.
(2009) Durgiah, Raveshni.; Chuturgoon, Anil Amichund.
Ochratoxin A (OTA), a nephrotoxic mycotoxin, is a contaminant of several agricultural food products consumed by animals and humans. Apart from renal toxicity, in particular renal tumours, OTA may also result in teratogenicity, neurotoxicity and immunotoxicity. Sutherlandia frutescens, an indigenous medicinal plant, has shown significant potential in strengthening the immune system and in cancer treatment, with minimal side effects. The objective of this study was to determine the effects of OTA in male Wistar rats and ascertain if these effects may be reduced by S. frutescens. Rats were treated by intraperitoneal injection (i.p) with either a control (EtOH:dH20;30:70), S. frutescens (1.0mg/kg body weight), OTA (0.5mg/kg body weight) or a combination of OTA and S. frutescens for a period of 1 or 7 days (n=4). Genotoxicity and metabolic activity in peripheral blood mononuclear cells (PBMCs) were quantified using single cell gel electrophoresis (SCGE) and the methylthiazol tetrazolium (MTT) assay, respectively. Lymphocyte apoptosis and mitochondrial depolarisation were measured by flow cytometry. Fluorescence microscopy was utilised to determine renal tissue apoptosis (Hoechst staining) and OTA localisation using immunohistochemistry (IRC). SDS-PAGE and Western blot were utilised to determine protein expression in kidney tissue and serum. Ochratoxin A significantly reduced PBMC viability (14%) after 7 days, compared with Day 1 (p<0.001). Lymphocyte mitochondrial depolarisation was 56.5% and 66.2% in the OTA-only and combination groups, respectively after 7 days (p<0.001). Ochratoxin A produced an increase in DNA damage compared to the control (p<0.01). The renal tissue displayed typical signs of apoptosis such as chromatin condensation. Ochratoxin A was immunolocalised within the glomerulus. The protein analysis showed a decreased expression in the kidney mitochondrial protein fraction. Ochratoxin A preferentially bound to serum albumin and a 120kDa protein in the OTA-only and co-treatment groups after the 1-and 7-day regimes. Protein band intensities significantly decreased after the 7-day co-treatment (p<0.01). The data highlights that OTA toxicity is mediated by mitochondrial dysfunction. Furthermore, OTA disruptions in immune function may play a role in renal damage.
An investigation into the effects of Sutherlandia Frutescens, L-Canavanine and aflatoxin B1 in the HepG2 human hepatocarcinoma cell line.
(2008) Pillay, Evashin.; Chuturgoon, Anil Amichund.
Aflatoxin B1 (AFB1), a potent hepatotoxic and hepatocarcinogenic mycotoxin synthesised by toxigenic fungi (Aspergillus flavus and Aspergillus parasiticus), is a common contaminant of many cereal commodities consequently posing a major threat to human and animal health. Sutherlandia frutescens (SF), a traditional medicinal plant endemic to Southern Africa, is commonly used by many cultures as a tonic for various health-related conditions. Incidentally, the present study aimed at investigating the potential hepatoprotective capacity of SF and L-canavanine (L-can, a major constituent of SF) against AFB1-induced cytotoxicity in human HepG2 cells and used a standard treatment procedure of 24 h. Cell viability was evaluated using the methyl thiazol tetrazolium (MIT) assay, which effectively demonstrated the ability of SF, when administered individually and in combination with AFB1, to be significantly cytotoxic to HepG2 cells in a dose-dependant manner. Reactive oxygen species (ROS) and consequent peroxidative damage caused by AFB1 are considered to be the main mechanisms leading to hepatotoxicity and was confirmed by the thiobarbituric acid reactive substances (TBARS) assay which revealed that AFB1 mediated a significant increase in lipid peroxidation. Additionally, comet assay analysis demonstrated the most pronounced effect to be observed following administration of AFB1. In contrast, AFB1-mediated genotoxicity was significantly reduced by SF and L-can. Such amelioration can be attributed to the marked increases in glutathione (OSH) levels observed after the co-administration of SF and L-can with AFB1. Cytoprotection by SF and L-can against AFB1-induced toxicity was further substantiated by the significant increases in heat shock protein 70 expression. Moreover, when SF and L-can were co-administered along with AFB1, analysis by flow cytometry revealed that AFB1 induced increases in apoptosis and necrosis were reduced. The findings of this study propose that SF and L-can may be selectively effective in alleviating AFB1-induced cytotoxicity and lends pharmacological credibility to the suggested ethnomedical uses of SF. However, the exact mechanism of action and the extracts efficacy in humans requires further authentication.
The effects of Tulbaghia violacea leaf, bulb and stalk extracts on Jurkat cells.
(2012) Mackenzie, Jared Stuart.; Myburg, Rene Bernadette.; Serumula, Metse Regina.
Studies have shown that the traditional healers have used Tulbaghia violacea (TV) (also known as ‘wild garlic’) for the treatment of a number of ailments including fever, tuberculosis, stomach problems, and oesophageal cancer. However, little is known with regards to the anticancer and antiproliferative properties of this plant. Therefore, this study investigated the effects of TV and domesticated garlic extracts on Jurkat cells, in order to determine whether or not these extracts possess anti-proliferative properties. Cultured Jurkat cells were treated with IC50 concentrations of garlic (14μg/ml), TV leaf (256μg/ml), TV bulb (225μg/ml) and TV stalk (216μg/ml) extracts as determined by the methylthiazol tetrazolium assay. Free radical production was measured using the thiobarbituric acid reactive substance (TBARS) and nitric oxide (NO) assays, while glutathione (GSH) concentration was measured using the GSH-Glo™ assay. The apoptosis inducing properties of each extract were measured using flow cytometry (Annexin V- Fluos and JC-1 assays) and luminometry (caspases 3/7, 8, 9 and ATP). Western blots were run to determine protein expression, while comet and DNA fragmentation assays were used to determine the level of DNA damage induced. Wild and domesticated garlic extracts induced a significant increase in malondialdehyde concentration ([MDA]), with TV bulb extract inducing the highest concentration (p<0.0001). A significant increase in NO concentration was observed in the bulb (p<0.0001) and stalk (p<0.001) extracts, and leaf (p<0.05) and stalk (p<0.05) TV extracts significantly increasing GSH concentration. The longest comet tails were observed in TV bulb extracts (p<0.0001) and comprised mainly of single strand breaks, while the comets induced following garlic exposure contained double strand breaks. All extracts, except TV leaf, increased the percentage of cells undergoing apoptosis. Tulbaghia violacea leaf induced a significant (p<0.0001) increase in percentage of cells undergoing necrosis, whereas TV bulb resulted in a significant (p<0.0001) decrease. All TV extracts induced caspase 3/7 and 9 activity, with the most significant increase in caspase 9 activity observed for TV leaf and bulb. No significant change in caspase 3/7 activity was evident for domesticated garlic. Cleavage of PARP and expression of NF B and HSP 70 occured for all extracts. However, HSP 70 was not differentially expressed. Exposure to wild and domesticated garlic extracts induced peroxidative lipid and DNA damage within the cells, indicating oxidative stress. This damage occurred in conjunction with increased percentage of cells undergoing apoptosis and expression of caspase 3/7. Therefore, these findings suggest that TV is inducing cell death through apoptosis in Jurkat cells using a number of mechanisms, including the induction of oxidative stress. This is of clinical significance, as cell death through apoptosis is the preferred method of action for anti-cancer drugs.
Silver nanoparticles of Albizia adianthifolia : the induction of apoptosis in a human lung carcinoma cell line.
(2012) Govender, Rishalan.; Chuturgoon, Anil Amichund.; Phulukdaree, Alisa.
Silver nanoparticles (AgNP), the most popular nano-compounds, possess unique chemical, physical and biological properties. Albizia adianthifolia (AA) – rich in saponins – is a plant of the Fabaceae family, found abundantly on the East coast of Africa. This plant is well known for its medicinal properties, and although the exact phytochemistry of AA is unknown, recent research suggests that AA can be used for the treatment of certain pathologies. The biological properties of a novel silver nanoparticle (AAAgNP) synthesised from an aqueous leaf extract of AA, were investigated on A549 lung carcinoma cells. Cell viability was determined by the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Cellular oxidative status (lipid peroxidation and glutathione (GSH) levels) were determined by the TBARS and GSH-Glo™ Glutatione assays respectively. ATP concentration was measured using the CellTitre-Glo™ assay. Caspase-3/-7, -8 and -9 activities were determined by Caspase-Glo® assays. Flow cytometry was used to measure apoptosis, mitochondrial (mt) membrane depolarisation, expression of CD95 receptors and intracellular smac/DIABLO levels. DNA fragmentation was assessed with the comet assay. The expression of p53, bax, PARP-1 and smac/DIABLO was evaluated by western blotting. Quantitative polymerase chain reaction was used to determine mRNA levels of bax and p53. AAAgNP caused a dose-dependent decrease in cell viability with a significant increase in lipid peroxidation (5-fold vs. control; p=0.0098) and decreased intracellular GSH (p=0.1184). A significant 2.5-fold decrease in cellular ATP was observed upon AAAgNP exposure (p=0.0040) with a highly significant elevation in mt membrane depolarisation (3.3-fold vs. control; p<0.0001). Apoptosis was also significantly higher (1.5-fold) in AAAgNP treated cells (p<0.0001) with a significant decline in expression of CD95 receptors (p=0.0416). AAAgNP caused a significant 2.5-fold reduction in caspase-8 activity (p=0.0024) with contrasting increases in caspase-3/-7 (1.7-fold vs. control; p=0.0180) and -9 activity (1.4-fold vs. control; p=0.0117). Western blots showed increased expression of smac/DIABLO (4.1-fold) in treated cells (p=0.0033). Furthermore, AAAgNP significantly increased the expression of p53, bax cleaved PARP-1 (1.2-fold; p=0.0498, 1.6-fold; p=0.0083 and 1.1-fold; p=0.0359 respectively). The expression of mRNA for both p53 and bax was also elevated post AAAgNP treatment, with 6-fold (p=0.0036) and 5-fold (p=0.0080) changes respectively compared to untreated cells. Data suggests that AAAgNP induces cell death in the A549 lung cells via the mt-mediated intrinsic apoptotic program. Further investigations are required to assess the potential use of AAAgNP in cancer treatment.
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.
The effects of Tulbaghia violacea (wild garlic) leaf and bulb extracts on an oesophageal cancer cell line (SNO)
(2012) Moonsamy, Suri.; Myburg, Rene Bernadette.; Serumula, Metse Regina.
Ethnopharmacological relevance: Indigenous plants such as Tulbaghia violacea(TV) and Allium sativum (garlic) are traditionally used as natural remedies to treat a variety of ailments, including cancer. This study investigated the effects of TV leaf and bulb extracts and garlic extract on a cancerous oesophageal cell line (SNO). Materials and methods: The methylthiazoltetrazolium (MTT) assay was used to determine the IC50 of TV leaf (TVL) (250μg/ml) and TV bulb extracts (TVB) (25μg/ml) and garlic (500μg/ml). Extracts were treated individually and in combination for a period of 24 hours. Oxidative damage and intracellular glutathione levels were assessed using the Thiobarbituric Acid Reactive Substances (TBARS) Assay and GSH-Glo™ Luminometry Assay, respectively. The CellTiter-Glo® Luminescent Cell Viability Assay was used to assess ATP activity. Induction of apoptosis and mitochondrial membrane potential were determined via the Caspase-Glo® 3/7 Assay, Caspase-Glo® 8 Assay, Caspase-Glo® 9 Assay and JC-1 Mitoscreen Assay, respectively. Morphological apoptotic changes were determined using the Hoechst 33342 stain. Expressions of p53, PARP and NFKB activities were determined by western blotting. Results: Bulb and leaf extracts of TV increased lipid peroxidation compared to the control (p>0.05), whilst garlic and combination of TV leaf and bulb (TVB + TVL) extracts significantly decreased lipid peroxidation relative to the control (p< 0.05). Endogenous glutathione levels significantly decreased in all TV treatments compared to the control (p<0.05).However, garlic was accompanied by insignificantly increased intracellular glutathione levels compared to the control (p> 0.05). The percentages of depolarised mitochondria in all treated cells were significantly decreased compared to untreated cells (p< 0.05). ATP levels increased significantly in garlic and combination (TVB + TVL) treated cells as compared to the control (p< 0.05), yet no significant differences were noted in TVL and TVB treatments (p> 0.05). Caspase8 and caspase 9 activities significantly increased in garlic and combination treated cells relative to the control (p<0.05). A similar trend was noted for caspase 3/7 activity in garlic and combination treatments (p< 0.05). However, initiator and executioner activities in TVL (p> 0.05) and TVB (p> 0.05) treatments did not significantly differ from the control (p> 0.05). All treatments (including garlic) resulted in increased DNA fragmentation and condensation. All treatments decreased p53 expression (p< 0.05), PARP expression (p< 0.05) and NFK B expression (p>0.05) compared to the control. Conclusions: All TV extracts and garlic induces apoptosis in the oesophageal cancerous SNO cell line through changes in oxidative stress, antioxidant systems, and nuclear chromatin condensation, as well as through induction of nuclear genes and signalling pathways. Since inhibition of apoptosis is a principal alteration in cancer, induction of apoptosis would result in a decrease in cancer cell growth. Thus, TV could be exploited as a potential anti-cancer agent.
The role of the uncoupling protein2 -866G/A polymorphism in oxidative stress markers associated with air pollution exposure during pregnancy.
(2012) Nagiah, Savania.; Phulukdaree, Alisa.; Chuturgoon, Anil Amichund.
Consistently high levels of air pollutants such as sulphur dioxide, particle matter and nitric oxides have been observed in the Durban South (DS) industrial basin. The adverse health outcomes associated with ambient air pollution (AAP) exposure have underlying molecular mechanisms. Oxidative stress is a known outcome of AAP exposure and contributes to the exacerbation of adverse AAP related outcomes such as chronic obstructive pulmonary disorder (COPD) and asthma. Pregnant women are at increased risk of developing oxidative stress due to increased energy expenditure. Oxidative stress during pregnancy is linked to adverse birth outcomes such as intrauterine growth retardation and low birth weight. The mitochondria are the most abundant source of endogenous reactive oxygen species (ROS), making these organelles extremely susceptible to oxidative damage. Alterations in mitochondrial function by air pollutants can contribute to oxidative stress. Uncoupling protein2 (UCP2) is an anion carrier located on the inner mitochondrial membrane that regulates mitochondrial ROS production by reducing mitochondrial membrane potential (Δψm) through mild uncoupling. Genetic variation in genes that play a role in oxidative stress response is likely to influence susceptibility to oxidative stress related health outcomes. The aim of this study was to evaluate air pollution associated oxidative stress response in women from the DS industrial basin and determine the functional relevance of a common -866G/A promoter polymorphism in the UCP2 gene. Fifty pregnant women from DS and 50 from north Durban (DN; control) were recruited. The thiobarbituric acid assay (TBARS) and comet assay were performed to measure oxidative stress and DNA fragmentation. Mitochondrial function was evaluated by JC-1 Mitoscreen and ATP luminometry. Quantitative PCR (qPCR) was performed to measure mitochondrial DNA (mtDNA) damage. Antioxidant response was determined by qPCR to measure mRNA expression of superoxide dismutase 2 (SOD2), nuclear factor erythroid 2-related factor 2 (Nrf2) and UCP2 mRNA expression. Western blots were performed to quantify UCP2 and Nrf2 protein expression. The samples were genotyped using PCR - restriction fragment length polymorphism. Results from the TBARS assay showed women from DS displayed elevated levels of MDA, a marker for oxidative stress (0.07±0.06μM; p = 0.56). ATP (1.89 fold) and Δψm (45.3±17.2%; p = 0.8) were also elevated in women from DS, favouring free radical production. DNA fragmentation, as indicated by comet tail length was also higher in DS when compared to the control group (0.57±0.16μm; p = 0.037). Analysis of mtDNA viability showed a 0.49 fold change in mtDNA amplification in women from the industrialized DS. All antioxidant genes, i.e. Nrf2 (0.73 fold), UCP2 (1.58 fold), SOD2 (1.23 fold), were up regulated in women from DS. Analysis of protein expression showed a significant increase in UCP2 expression (0.08±0.03RBI; p = 0.049) and a significant decline in Nrf2 levels (1.68±0.84RBI; p = 0.03). The homozygous G genotype was significantly more frequent in DS (37.5%) than in DN (18.6%; p = 0.047; OR: 2.57; 95% CI: 1.353 to 4.885). This genotype exhibited higher MDA levels, comet tail length, Δψm, SOD2, Nrf2, and UCP2 expression than the AA/GA in genotype in women from DS (p > 0.05). This study found that pregnant women from a more industrialized area exhibit higher markers for oxidative stress and conditions that favour mitochondrial free radical production.
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.
Isolation and characterisation of extended spectrum B-lactamases in South African Klebsiella pneumonia isolates.
(2012) Naidoo, Yashini.; Essack, Sabiha Yusuf.
The use of antibiotics and antimicrobial drugs has played a large role in the elimination of many infectious diseases, however the wide spread use of such drugs has given rise to the phenomenon of antimicrobial resistance and has rendered antibiotics ineffective to a broad range of bacteria. The aim of the study was to ascertain the differences if any in the phenotypic and genotypic resistance profiles of K. pneumoniae isolated from a single tertiary hospital in two surveillance studies undertaken at different times, viz., 2001 and 2007 with special emphasis on ESBLs. A correlation with antibiotic use was also undertaken. ESBL positives were identified and phenotypic resistance profiles were generated based on the resistance profiles of individual isolates by means of their MIC data. The molecular detection of ESBLs was carried out using representative isolates and sequencing was based on the phenotypic expression of the most common ESBL genes. The data was summarized using median values and interquartile ranges. Antibiotic use and susceptibility in 2000 was compared to that in 2007 using a Wilcoxon signed rank test for paired data since the same drugs were tested in both years. Of the isolates that were tested, sequencing revealed that TEM – 1 was identified in all isolates and SHV-1 and SHV-2 were identified in 60 % in the isolates collected in 2000 and 77 % and 11 % respectively in the isolates collected in 2007. SHV – 11 was present in 67% of isolates from 2007 and 55% of those were in combination with SHV – 1. Sequencing also revealed CTXM-15 present in one of the isolates collected in 2007. There was 100% susceptibility to cefoxitin and only one isolate in 2007 showing an intermediate result to imipenem. No novel β-lactamases were identified in this study; however the decrease in susceptibility over time is proof of bacterial evolution. The variety of β-lactamases and diversity of plasmid profiles in these two small populations provides proof to the claim that dissemination of resistance in Klebsiella pneumonia is effortless. Statistical analysis showed an increase in resistance from the year 2000 to 2007 however the correlation between overall antibiotic use and the increase in resistance did not reach statistical significance. It was observed that resistance increased despite only a slight increase in the use of a few antibiotics to which we attributed co-carriage of resistance genes.
The effects of fumonisin B¹ in preeclampsia.
(2012) Serumula, Metse Regina.; Chuturgoon, Anil Amichund.; Moodley, Jagidesa.
Preeclampsia is the leading cause of foetal and maternal mortality and morbidity in developing countries. In South Africa, maize is a dietary staple for most black African populations and is susceptible to contamination by mycotoxins such as fumonisin B1 (FB1).Fumonisin B1 is a ubiquitous secondary metabolite of Fusarium fungi produced predominantly by Fusarium verticillioides. This mycotoxin shares structural similarities with the backbone of sphingoid bases (sphinganine and sphingosine) which are substrates for the biosynthesis of complex sphingolipids. The mechanism of FB1 toxicity therefore is centred on the disruption of this process. The aim of the present study was to elucidate the possible causal link between FB1 and preeclampsia. Following ethical approval, 20 normotensive and 20 preeclamptic patients were recruited into the study. Blood and placental tissue were collected and processed for further analysis. The presence of FB1 was verified using standard immunohistochemical and electrophoretic techniques. The levels of FB1 and sphingolipids were quantified using high performance liquid chromatography (HPLC). Western blotting was conducted to confirm the presence of FB1 in the serum. Placental tissue apoptosis was evaluated using Hoechst staining and other markers. Lipid peroxidation was measured in serum and placental tissue of both groups. Fumonisin B1 was immunolocalised within the endothelial cells and mesenchymal cells of placentas from both groups, while FB1 was present in cytotrophoblastic cells of preeclamptic patients only. In addition, FB1 concentrations were significantly higher in preeclamptic compared to normotensive serum samples. Sphinganine was significantly elevated in preeclamptic serum samples whilst there was no statistical difference in the sphingosine levels between the groups. Chromatin condensation was higher in the preeclamptic patients. Caspase 3 and Fas were present with greater intensity in preeclamptic samples. The levels of lipid peroxidation were significantly higher in both serum and placental tissue of preeclamptic patients. This study has demonstrated not only the presence of FB1 in the serum and placental tissues of pregnant women but also the potential effects of this mycotoxin in the humans.
Characterisation of Fumonisin B1 toxicity in a cancerous liver cell line- induction of tissue transglutaminase and the endoplasmic reticulum stress pathway.
(2013) Anderson, Samantha Mary.; Chuturgoon, Anil Amichund.; Phulukdaree, Alisa.
Fumonisin B1 (FB1) is a mycotoxin which is well characterised as a contaminant of maize and maize-based products worldwide, especially in South Africa. Its toxic effects have been associated with hepatotoxicity, nephrotoxicity and carcinogenicity. Tissue transglutaminase (TG2) is a unique and ubiquitous enzyme that catalyses the post-translational modification of proteins and has GTPase activity. Tissue transglutaminase is an important enzyme in a number of biological processes such as cell differentiation and proliferation, extracellular matrix organisation, cell signalling and apoptosis. This study investigated the possible role of TG2 induction by FB1 and the effect FB1 toxicity has on the endoplasmic reticulum (ER) stress pathway in HepG2 cells. A SDS-PAGE adaption of the TG2 activity assay confirmed TG2 crosslinking activity by FB1 incorporation into fibronectin in the presence of calcium and TG2. This interaction was validated using fluorescent microscopy where FB1 incorporated into the HepG2 cell’s cytoplasmic vesicles and plasma membrane. The up-regulation of TG2 in HepG2 cells treated with FB1 was further investigated using western blotting and showed increased TG2 up-regulation. Fumonisin B1 disrupts membrane-bound sphingolipids as a mechanism of toxicity; FB1 was shown to cause cytoskeletal damage and disrupted the cell’s membranes leading to cell stress. The protein kinase RNA-like endoplasmic reticulum kinase (PERK) ER stress pathway was induced as a result of FB1 exposure and investigated using western blotting and quantitative polymerase chain reaction. After 72hours with 50μM and 100μM FB1 total PERK decreased, phosphorylated eukaryotic initiating factor α remained activated with a significant increase in messenger RNA (mRNA) expression (p<0.05) and transcription factor CCAAT-enhancer-binding protein homologous protein mRNA was significantly induced (p<0.05). The involvement of nuclear factor kappa B (NFkB) and TG2 in ER stress induced apoptosis was investigated through western blotting and quantitative polymerase chain reaction. After 72hours, an up-regulation of both nuclear NFkB and nuclear TG2 was observed; with a corresponding significant increase in nuclear TG2 mRNA expression (p<0.05). A significant increase in transcription factor, Sp1 mRNA expression (p<0.05) was observed after 72hours. Data suggests PERK activation leads to NFkB induction and nuclear translocation; which promoted nuclear TG2 transcription. The activation of TG2 resulted in Sp1 crosslinks that could act as potential inducers of FB1 induced apoptosis. Flow cytometry was used to measure apoptosis and mitochondrial depolarisation. Caspase activity was measured using the Caspase-Glo® assays and ATP concentration was measured using CellTitre-GloTM assay. After 72hours caspases 3/7 and 8 showed a significant decrease in activity at 100μM FB1 (p<0.05) and a decrease in caspase 9. After 72hours with 10μM FB1 treatment a significant increase in phosphatidylserine externalisation (p<0.05), a significant decrease in healthy/live cells (p<0.05) and a significant increase in depolarised mitochondria (p<0.05) were observed. There was also a significant increase in Sp1 mRNA expression (p<0.05). However, at 50μM FB1 treatment there was a decrease in phosphatidylserine externalisation, a significant increase in live cells (p<0.05) and a significant decrease in depolarised mitochondria (p<0.05). Data suggests that ER stress persisted in HepG2 cells with no apoptosis or cell recovery occurring at high chronic doses of FB1 whilst ER stress induced apoptosis at low chronic doses of FB1 in HepG2 cells. Fumonisin B1 may be a possible substrate for TG2 crosslinking activity due to its primary amine group, since this mycotoxin has the potential to induce TG2 expression and activation. Further studies are required to determine the role of FB1 in the inositol-requiring protein 1α and activating transcription factor 6 arms of the ER stress pathway.
An investigation into marine bacterial species found in shark mouths in the Indian Ocean and their implications for human health.
(2015) Ramlakhan, Yathisha.; Chuturgoon, Anil Amichund.; Phulukdaree, Alisa.; Bester, Linda Antionette.; Singh, Sanil Duleep.
There is an ever increasing amount of pollution and waste being released into the environment. This is due to the increase in population, urbanisation and people migrating into cities. Approximately 2.4 billion people living in urban and rural areas have no access to basic sanitation. In the next 20 years, there will be a further increase of 2 billion people who will lack basic sanitation. In developing countries, 90% of untreated sewage is released into rivers, lakes and coastal waters. Apart from sewage, waste such as petroleum products, heavy metals and organochlorine also contribute to marine pollution. Companies that manufacture sugar/artificial sweeteners etc. and farming activities that utilize fertilizers for crops can cause eutrophication, as un-used fertilizers get washed into rivers. The marine water is a different environment to other aquatic and terrestrial environments. This then forces microbes to adapt, so they can be able to survive in the marine environment. The difference in the marine environment allows for the production of distinct bioactive metabolites such as secondary metabolites. These secondary metabolites come from algae and marine bacteria and these secondary metabolites are then exclusive to the marine waters. These secondary metabolites can be used for medical purposes, cosmetics, personal-care products etc. There is a huge problem with antibiotic resistance and research needs to be done to solve this resistance issue. Two common bacterial strains were isolated and identified from the mouth of sharks. The bacteria were identified as Bacillus cereus and Vibrio alginolyticus. They were isolated and cultured in broth for 3 days, till they reached the log phase of growth. The broth was then extracted for metabolites which the bacteria produced, using ethyl acetate. These metabolites were tested for cytotoxicity in the human liver hepatocellular carcinoma (Hep G2) cells. The concentrations that were determined to cause 50% cell death (IC50) in the cell viability assay on Hep G2 cells were 0.764 mg/ml and 0.918 mg/ml for B. cereus and V. alginolyticus, respectively. These values were then used for subsequent assays. Antibacterial testing was done for the bacterial extracts of Bacillus cereus and Vibrio alginolyticus. There was no antibacterial activity against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853. Assays that used flow cytometry was used to show if apoptosis/necrosis occurred. These were assays such as Annexin V and propidium staining. While assays that used luminometry showed the levels of ATP and determined whether apoptosis of the cells occurred. These were assays such as the ATP assay, mitochondrial depolarisation assay and determination of the caspase activities of caspase 3/7, 8 and 9. Additional assays, like the comet and TBARS assays, were done to show DNA fragmentation and oxidative stress of the cells, respectively. The results for the Annexin V/ propidium staining showed the control had a mean of 11.20 ± 1.0. Extract 1 (20.83 ± 0.8737) and extract 2 (25.37 ± 1.050) showed a higher percentage when compared to the control. Extract 2 was significant against the control (p<0.0273). For propidium staining, the control had a mean of 6.033 ± 0.4524. Extracts 1(11.57 ± 1.387) and 2 (11.43 ± 0.3215) showed a higher percentage when compared to the control. The Annexin V and propidium staining suggested that extract 1 and 2 had undergone both apoptosis and necrosis. For luminometry assays, the ATP assay showed that the control had a mean of 1.83x106 ± 5.82x104. Extracts 1 (1.5x106 ± 9.4x104) and extract 2 (1.4x106 ± 8.3x104) showed a decrease in ATP with reference to the control. In the mitochondrial depolarisation assay, the control had a mean of 14.83 ± 1.350. Extracts 1 (30.57 ± 0.75) and extract 2 (20.53 ± 8.56) showed a decrease in polarisation with reference to the control. For caspase 8 analysis, the control, extract 1 and extract 2 had means that were 4.23x104 ± 3.37x103, 52x103 ± 10.1x103 and 40x103±5.2x103, respectively. For caspase 9 analysis, the control, extract 1 and extract 2 had means that were 8.6x104 ± 4.6x103, 5.6x104 ± 4x103and 9.6x104 ± 5.6x104, respectively. The caspase 3/7 analysis showed that the control, extract 1 and extract 2 had means of 4.4x103 ± 0.57x103, 5.5x103 ± 0.19x103 and 5.8x103 ± 2 x103, respectively. Caspase 3/7 showed that apoptosis had occurred with the cells for all extracts used. Extract 1 showed a high caspase activity for caspase 8. This suggested that it followed the extrinsic pathway of apoptosis. Extracts 2 showed a high activity for caspase 9 which suggested that it followed the intrinsic pathway of apoptosis. The comet assay showed that the means of the control, extract 1 and extract 2 were 35.91 ± 21.93, 75.85 ± 11.43 and 60.48 ± 11.86, respectively. The extracts were significantly higher than the control (extract 1 and 2 p<0.0001). Extract 1 and 2 were compared to each other and had shown a significance between them (p<0.0001). The TBARS assay obtained the following MDA concentrations for the control, extract 1, extracts 2, negative and positive samples: 0,137, 0,132, 0,150, 0,088 and 20,502, respectively. The MDA concentration gives an indication of oxidative stress of the cells. From the cell viability assay, the secondary metabolites produced by B. cereus needed a lower concentration of extract to determine an IC50 value. This suggested that the secondary metabolites produced by B. cereus were more toxic than the secondary metabolites produced by V. alginolyticus. This was then further supported by assays such as mitochondrial depolarisation and the comet assay. The secondary metabolites that could be the reason why there were apoptosis and necrosis, are the toxins the bacteria produce. This is the enterotoxin or cereulide produced by B. cereus and TLH by V. alginolyticus. However, further studies need to be done to confirm if these toxins are the cause of cell death.
Antiproliferative effect of a novel synthesized carbazole compound on A549 lung cancer cell line.
(2014) Molatlhegi, Refilwe P.; Chuturgoon, Anil Amichund.
Increased death rates due to lung cancer have necessitated the search for potential novel anticancer compounds such as carbazole derivatives. Carbazoles are aromatic heterocyclic compounds with anticancer, antibacterial and anti-inflammatory activity. The study investigated the ability of the novel carbazole compound (Z)-4-[9-ethyl-9aH-carbazol-3-yl) amino] pent-3-en-2-one (ECAP) to inhibit the proliferation of lung cancer cells and its mechanism of action. ECAP was synthesized as a yellow powder with melting point of 240-247 °C. The 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lipid peroxidation and comet assays were used to assess the anti-proliferative effects of the compound on A549 lung cancer cell line. Protein expression was determined using western blots, apoptosis was measured by luminometry for caspase-3/7, -8 and -9 and flow cytometry was used to measure phosphatidylserine externalisation. ECAP induced a p53 mediated apoptosis of lung cancer cells by significantly down-regulating the expression of antioxidant defense proteins, Hsp70 (p < 0.02) and Bcl-2 (p < 0.0006), thereby up-regulating reactive oxygen species (ROS) production. This resulted in DNA damage (p < 0.0001) and subsequent up-regulation of Bax and caspase activity consequently inducing apoptosis of lung cancer cells. These results demonstrate the potential anticancer effects of ECAP on cultured lung cancer cells. However, further investigation and characterization is required to fully understand the possible use of carbazole compound (Z)-4-[9-ethyl-9aH-carbazol-3-yl) amino] pent-3- en-2-one as potential lung cancer treatment.
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.
Trigonella foenum-graecum seed and 4-hydroxyisoleucine mediates glucose uptake via proximal insulin signaling activation and related downstream gene expression in liver cells.
(2014) Naicker, Nikita.; Chuturgoon, Anil Amichund.; Phulukdaree, Alisa.
Fenugreek (Trigonella foenum-graecum) is one of the oldest medicinal plants used worldwide to treat a variety of ailments, including hyperglycaemia. The seed and active compound – 4-hydroxyisoleucine (4-OH-lle) is thought to aid in the treatment of insulin resistance. This study investigated the effects of fenugreek aqueous seed extract and 4-OH-lle, on human liver cells (HepG2) compared to insulin (100ng/ml) and metformin (2mM) controls. Cells were treated with fenugreek seed extract (FSE) and 4-OH-lle: 10 and 100ng/ml under normogylcaemic (5mM glucose) and hyperglycaemic (30mM) conditions for 72h. Tyrosine phosphorylation of insulin receptor-β (IR-β), protein kinase B (Akt) and glycogen synthase kinase-3α/β (GSK-3α/β) protein extracts was determined by western blotting. Gene expression of sterol regulatory element binding protein 1c (SREBP1c), glucose transporter 2 (GLUT2), glycogen synthase (GS) and glucokinase (GK) was evaluated by qPCR. Under normogylcaemic and hyperglycaemic conditions, FSE, 4-OH-lle and insulin at 100ng/ml and metformin (2mM) caused tyrosine phosphorylation of IR-β (p<0.0729; p<0.0121), Akt (p<0.0046; p<0.0005) and GSK-3α/β (p<0.0128; p<0.0048). However, FSE showed the greatest ability in positively controlling GS (*p<0.0262; *p<0.333) and GK (*p<0.333; *p<0.0213), which regulates glycogen synthesis. Also, FSE increased SREBP1c (*p<0.0157; *p<0.0012) which positively regulates GLUT2 (*p<0.0330, *p<0.0417), allowing glucose into the cell. The data suggests that FSE and 4-OH-lle causes an up-regulation of insulin signaling proteins at a proximal level and related downstream gene expression. Taken together, the study suggests that FSE has potential application in the management of chronic hyperglycaemia.
The phytoalexin, resveratrol ameliorates ochratoxin A genotoxicity in human embryonic kidney (HEK293) cells.
(2014) Raghubeer, Shanel.; Chuturgoon, Anil Amichund.; Phulukdaree, Alisa.
Background: Ochratoxin A (OTA) is a mycotoxin produced by fungal species of Aspergillus and Penicillium. OTA is nephrotoxic and carcinogenic in several animal models; it frequently contaminates human and animal food products. Chronic exposure is associated with progressive renal fibrosis in humans (Balkan endemic nephropathy). Resveratrol is a phytoalexin that possesses both anti-cancer and antioxidant properties. We investigated the mechanism of cellular oxidative stress induced by OTA in the human embryonic kidney (HEK293) cell line. Methods: An IC50 value of 1.5μM was determined from a dose-dependent cell viability curve using the methylthiazol tetrazolium (MTT) assay on HEK293 cells treated with a range of OTA concentrations (0.25μM–50μM) for 24hrs. Glutathione levels were quantified by luminometry and gene expression of Nrf2, OGG1, CAT, SOD and GPx was determined by qPCR. Protein expression of Nrf2 and phosphorylated SIRT1 (pSIRT1) was assessed by western blot, DNA damage was determined using the comet assay, and flow cytometry was employed for intracellular ROS detection. Results: Resveratrol decreased mRNA expression of OGG1 (p<0.05) and OTA significantly increased OGG1 expression (p<0.05). The comet assay proved that while OTA induced DNA damage, resveratrol protected the DNA against strand breaks. Both resveratrol and OTA significantly increased antioxidant defence gene expression (Nrf2, CAT, GPx and SOD) (p<0.05). OTA decreased intracellular ROS, while resveratrol-treated cells exhibited the lowest percentage of intracellular ROS. Luminometry analysis showed the OTA+Resveratrol co-treatment to have a synergistic effect on the concentration of GSH and GSSG. Western blot analysis of protein showed that resveratrol significantly increased the levels of pSIRT1 while concomitantly decreasing the protein levels of Nrf2 (p<0.05) and OTA significantly decreased pSIRT1 protein levels.
Biochemical, apoptotic and cellular stress studies in rheumatoid arthritis.
(2009) Moodley, Devapregasan.; Mody, Girish Mahasukhlal.; Chuturgoon, Anil Amichund.
Abstract available in PDF file.

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