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Medical Biochemistry

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Browsing Medical Biochemistry by Author "Anderson, Samantha Mary."

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    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.
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    An investigation of stress-responses in pregnant women exposed to ambient air pollution in Durban, South Africa.
    (2017) Anderson, Samantha Mary.; Chuturgoon, Anil Amichund.
    Living or working within an unhealthy environment is attributed to 12.6 million deaths worldwide and 2.2 million deaths in Africa. Ambient air pollution (AAP) exposure is amongst the major contributors of environmental and air quality decay. Durban South Africa (SA) is a rapidly developing city that requires increased infrastructure, transportation, and energy production to support the growing urban population. This leads to air quality degradation, in addition to the heavy burden of human immunodeficiency virus (HIV) and obesity SA faces increase the susceptibility of pathological conditions including respiratory diseases and adverse birth outcomes. Infants in utero are particularly vulnerable to adverse AAP effects, attributed to oxidative stress (OS), inflammation and genetic susceptibility, due to their biological vulnerability, sensitivity to their environment and rapid differentiation and growth. South Durban (SD) comprises a complex mix of dense residential settlements and heavily industrialised areas with high levels of air pollution (AP). This makes SD an ideal location to investigate the effects of AAP, in particular, traffic-related AP (atmospheric oxides of nitrogen (NOx)), on OS and endoplasmic reticulum (ER) stress responses within third trimester pregnant women. A comparison sample of pregnant women, located within north Durban (ND) of similar socio-economic status were used for this study. The susceptibility of OS markers, including 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-OHdG) DNA adducts, lipid peroxidation (LP) and nitric oxide (NO) levels, on adverse birth outcomes, including low birthweight (LBW) and pre-term birth (PTB), were also determined. Additional risk factors such as HIV, obesity and single nucleotide polymorphisms (SNP) within genes of the antioxidant response pathway were investigated for OS and adverse birth outcome susceptibility. Atmospheric NOx pollution data were obtained from land use regression modelling that was previously reported. Atmospheric NOx and maternal serum 8-OHdG adducts were significantly elevated within SD living pregnant women. This induction of DNA damage was found to be the direct consequence of NOx exposure. Pregnant women carrying the variant and wild-type (wt) genotypes of glutathione S transferase (GST) P1 and M1 SNPs, respectively, increased the susceptibility of NOx induced OS. Exposure to increased NOx levels significantly reduced the gestational age (GA) of these pregnant women, with increased susceptibility for mothers carrying male neonates. The wt 8-oxoguanine glycosylase 1 (OGG1) Ser326Cys genotype was found to be associated with both HIV and obesity. Therefore pregnant women infected with HIV (HIV+) and carrying the wt genotype significantly increased the risk for HIV associated LBW and PTB. In addition, living within SD and being exposed to higher levels of AAP significantly increased the susceptibility for PTB. Comorbid HIV and obesity were identified as additional risk factors for birthweight (BW) reduction. Increased maternal serum NO levels were observed within HIV+ women, with reciprocal activity on malondialdehyde (MDA) levels. Increased levels of NO directly reduced BW, especially for HIV+ and SD living women. This suggests NO may play a key role in LBW aetiology as a consequence of HIV infection and traffic-related AP. HIV was shown to differentially modulate MDA’s effect on neonatal BW. Exposure to increased levels of NOx and HIV infection induced the expression of microRNA (miR)-144, which was shown to negatively regulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This transcription factor, Nrf2, was shown to significantly increase antioxidant gene expressions. Therefore the induction of miR-144 was implicated as a mechanism for increased OS due to HIV and NOx exposure. In addition, elevated ER stress genes were observed within HIV negative SD living patients. Hence, exposure to higher levels of AAP within SD led to increased ER stress, which may act reciprocally on the induction of ROS leading to increased OS. These findings indicate that exposure to atmospheric NOx, elevated AAP levels within SD and exposure to HIV infection resulted in increased OS with increased susceptibility towards adverse birth outcomes within pregnant women. Further studies into the mechanisms proposed within a larger population including multiple pollutants and gene interactions may give additional insight into the aetiology of adverse birth outcomes as a consequence of AAP exposure.