Analyses of antioxidant status and nucleotide alterations in genes encoding antioxidant enzymes in patients with benign and malignant thyroid disorders.

BACKGROUND: Synthesis of thyroid hormones and regulation of their metabolism involve free radicals that may affect redox balance in the body. Thyroid disorders causing variations in the levels of thyroid hormones may alter cellular oxidative stress. The aim of this study was to measure the antioxidant activities and biomarkers of oxidative stress in serum and red blood cells (RBC) of patients with benign and malignant thyroid disorders and to investigate if changes in the antioxidant activities in these patients were linked to alterations in genes encoding the antioxidant enzymes. METHODS: Forty-one patients with thyroid disorders from University of Malaya Medical Centre were recruited. They were categorised into four groups: multinodular goitre (MNG) (n = 18), follicular thyroid adenoma (FTA) (n = 7), papillary thyroid cancer (PTC) (n = 10), and follicular thyroid cancer (FTC) (n = 6). Serum and RBC of patients were analysed for antioxidant activities, antioxidant enzymes, and biomarkers of oxidative stress. Alterations in genes encoding the antioxidant enzymes were analysed using whole exome sequencing and PCR-DNA sequencing. RESULTS: Patients with thyroid disorders had significantly higher serum superoxide dismutase (SOD) and catalase (CAT) activities compared to control, but had lower activities in RBC. There were no significant changes in serum glutathione peroxidase (GPx) activity. Meanwhile, GPx activity in RBC was reduced in PTC and FTC, compared to control and the respective benign groups. Antioxidant activities in serum were decreased in the thyroid disorder groups when compared to the control group. The levels of malondialdehyde (MDA) were elevated in the serum of FTA group when compared to controls, while in the RBC, only the MNG and PTC groups showed higher MDA equivalents than control. Serum reactive oxygen species (ROS) levels in PTC group of both serum and RBC were significantly higher than control group. Whole exome sequencing has resulted in identification of 49 single nucleotide polymorphisms (SNPs) in MNG and PTC patients and their genotypic and allelic frequencies were calculated. Analyses of the relationship between serum enzyme activities and the total SNPs identified in both groups revealed no correlation. DISCUSSION: Different forms of thyroid disorders influence the levels of antioxidant status in the serum and RBC of these patients, implying varying capability of preventing oxidative stress. A more comprehensive study with a larger target population should be done in order to further evaluate the relationships between antioxidant enzymes gene polymorphisms and thyroid disorders, as well as strengthening the minor evidences provided in literatures.

Polyphenols from the extract and fraction of T. indica seeds protected HepG2 cells against oxidative stress.

BACKGROUND: Tamarindus indica L. (T. indica) or locally known as "asam jawa" belongs to the family Leguminosae. T. indica seeds as by-products from the fruits were previously reported to contain high polyphenolic content. However, identification of their bioactive polyphenols using recent technologies is less well researched but nonetheless important. Hence, it was the aim of this study to provide further information on the polyphenolic content and antioxidant activities as well as to identify and quantify its bioactive polyphenols. METHODS: T. indica seeds were extracted with methanol and were then fractionated with different compositions of hexane, ethyl acetate and methanol. Polyphenolic contents were measured using Folin-Ciocalteu assay while antioxidant activities were measured using DPPH radical scavenging and ferric reducing (FRAP) activities. The cytotoxic activities of the crude extract and the active fraction were evaluated in HepG2 cells using MTT assay. The cells were then pre-treated with the IC20 concentrations and induced with H2O2 before measuring their cellular antioxidant activities including FRAP, DPPH, lipid peroxidation, ROS generation and antioxidant enzymes, SOD, GPx and CAT. Analyses of polyphenols in the crude extract and its active fraction were done using UHPLC and NMR. RESULTS: Amongst the 7 isolated fractions, fraction F3 showed the highest polyphenolic content and antioxidant activities. When HepG2 cells were treated with fraction F3 or the crude extract, the former demonstrated higher antioxidant activities. F3 also showed stronger inhibition of lipid peroxidation and ROS generation, and enhanced activities of SOD, GPx and CAT of HepG2 cells following H2O2-induced oxidative damage. UHPLC analyses revealed the presence of catechin, procyanidin B2, caffeic acid, ferulic acid, chloramphenicol, myricetin, morin, quercetin, apigenin and kaempferol, in the crude seed extract of T. indica. UHPLC and NMR analyses identified the presence of caffeic acid in fraction F3. Our studies were the first to report caffeic acid as the active polyphenol isolated from T. indica seeds which likely contributed to the potent antioxidant defense system of HepG2 cells. CONCLUSION: Results from this study indicate that caffeic acid together with other polyphenols in T. indica seeds can enhance the antioxidant activities of treated HepG2 cells which can provide protection against oxidative damage.