Browsing by Author "Suthiram, Kimera Tamzin."

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An investigation into the biochemical effects of Kojic acid (KA) on human hepatocellular carcinoma (HepG2) cells.
(2020) Suthiram, Kimera Tamzin.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
Kojic acid (KA) is a secondary metabolite and divalent metal chelator that is widely used in the beauty industry as a skin lightener. However, KA toxicity is not well-established in humans. This study aimed to determine the toxicity of KA by assessing oxidative stress, nuclear factor kappa B (NFκB) signalling, and mitogen-activated protein kinase (MAPK) signalling in human hepatoma (HepG2) cells following 24 h exposure. Cell viability was assessed using the methylthiazol tetrazolium (MTT) and crystal violet assays. To confirm cell death, apoptosis (caspase -8, -9, -3/7 luminometry), and Lactate dehydrogenase (LDH) leakage were assessed. Oxidative stress (TBARS), DNA damage (8-OHdG), and protein oxidation (protein carbonyls assay) to determine macromoleculedamage. An assessment of inflammatory and oxidative stress markers were carried out using mRNA expression GPx, NFκB, actor erythroid-2 factor-2 (Nrf2), phospho-Nrf2 (ser40), catalase (CAT), c-Jun-N-terminal kinase (JNK), p38, phospho-Sirtuin 1 (ser47) (phospho-sirt1), NFκB, phospho-NFκB (ser536), and activator protein 1 (AP-1) were assessed using Western Blot in HepG2 cells. KA decreased cell viability in HepG2 cells and elevated the activities of caspase -9 (p < 0.0001), caspase -8 (p = 0.0003) and caspase 3/7 (p < 0.0001) at lower concentrations [4.22 & 8.02 mM] which served as confirmation of apoptosis. Necrosis at the higher concentration [12.67 mM] was confirmed by the presence of LDH leakage indicating membrane damage. Increased cell death was further correlated with increased miRNA-29b expression (p = 0.009), a miRNA responsible for elevated apoptotic activity. Adenosine Triphosphate (ATP) production was increased significantly at 12.67 mM (p < 0.0001), while oxidative stress (Malondialdehyde (MDA) levels) was increased significantly at 4.22 mM (p < 0.0001). Macromolecules are susceptible to damage in the presence of oxidative stress. Due the elevation of MDA levels, DNA damage and protein oxidation assays were carried out. Protein carbonyls were significantly decreased (p < 0.0001), suggesting a potential cytoprotective effect. Due to the presence of oxidative stress, Nrf2, is activated and is responsible for the transcription of antioxidant genes. This was illustrated by an increase in activated Nrf2 at lower concentrations (4.22 & 8.02 mM), whilst at higher a concentration (12.67 mM) decreased phospho- (p > 0.0001). CAT was decreased significantly (p = 0.0002) and GPx significantly increased at lower concentration [4.22 mM] (1.51-fold). A key function of the MAPK pathway is the initiation of stress-activated protein kinases, p38 and JNK, in response to oxidative stress. KA significantly increased, p38 at lower concentration (p = 0.0011) and significantly decreased JNK1 (p = 0.0039) and JNK2 (p < 0.0001) activity. Regulation of reactive oxygen species (OS) production by Sirt-1 occur via the alteration of immune responses through NFκB signalling and AP-1. Inflammatory ediators, phospho-Sirt1 was significantly decreased (p < 0.0001), while AP-1 expression was elevated (p 15 which is in agreement with repressed inflammatory responses reflected by decreased NFκB expression. KA treatment resulted in increased MDA levels and antioxidant responses. MAPK signalling was elevated in response to oxidative stress suggesting the involvement in cell death, whilst inflammation was suppressed. In conclusion, KA displayed low toxicity in HepG2 cells.