Effect of styrene oxide and diethyl maleate on expression of cytochrome P450 family 1 and glutathione store in mouse liver

Purpose: To determine the effect of the glutathione (GSH) suppressors styrene oxide (SO) and diethyl maleate (DEM) on the hepatic expression of cytochrome P450 family 1 (Cyp1) isoforms that are related to carcinogenesis including Cyp1a1, Cyp1a2, and Cyp1b1. Methods: Seven-week-old ICR mice were intraperitoneally injected with SO (150 and 300 mg/kg/day), DEM (175 and 350 mg/kg/day), or N-acetylcysteine (NAC; 300 and 600 mg/kg/day) for 7, 14, or 28 days. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, hepatic Cyp1 expression, total glutathione, reduced glutathione (GSH), and oxidized glutathione (GSSG) were determined. Results: ALT and AST levels were markedly increased by SO and DEM while GSH/GSSG ratio was decreased by SO in all treatments (p < 0.05), while high dose (350 mg/kg/day) DEM significantly suppressed GSH/GSSG ratio at 28 days (p < 0.05). The expressions of Cyp1a1, Cyp1a2, and Cyp1b1 were induced by SO and DEM, corresponding with induction of ethoxy/methoxy-resorufin O- dealkylase activities. Conclusion: The Cyp1 family metabolizes procarcinogens to carcinogenic DNA adducts; exposure to the industrial solvents, SO and DEM, raises the risk of carcinogenesis via GSH depletion coupled with Cyp1 induction.

Effect of Kaempferol and Its Glycoside Derivatives on Antioxidant Status of HL-60 Cells Treated with Etoposide

Kaempferol is a well-known antioxidant found in many plants and plant-based foods. In plants, kaempferol is present mainly in the form of glycoside derivatives. In this work, we focused on determining the effect of kaempferol and its glycoside derivatives on the expression level of genes related to the reduction of oxidative stress—NFE2L2, NQO1, SOD1, SOD2, and HO-1; the enzymatic activity of superoxide dismutases; and the level of glutathione. We used HL-60 acute promyelocytic leukemia cells, which were incubated with the anticancer drug etoposide and kaempferol or one of its three glycoside derivatives isolated from the aerial parts of Lens culinaris Medik.—kaempferol 3-O-[(6-O-E-caffeoyl)-β-d-glucopyranosyl-(1→2)]-β-d-galactopyranoside-7-O-β-d-glucuropyranoside (P2), kaempferol 3-O-[(6-O-E-p-coumaroyl)-β-d-glucopyranosyl-(1→2)]-β-d-galactopyranoside-7-O-β-d-glucuropyranoside (P5), and kaempferol 3-O-[(6-O-E-feruloyl)-β-d-glucopyranosyl-(1→2)]-β-d-galactopyranoside-7-O-β-d-glucuropyranoside (P7). We showed that none of the tested compounds affected NFE2L2 gene expression. Co-incubation with etoposide (1 µM) and kaempferol (10 and 50 µg/mL) leads to an increase in the expression of the HO-1 (9.49 and 9.33-fold at 10 µg/mL and 50 µg/mL, respectively), SOD1 (1.68-fold at 10 µg/mL), SOD2 (1.72-fold at 10–50 µg/mL), and NQO1 (1.84-fold at 50 µg/mL) genes in comparison to cells treated only with etoposide. The effect of kaempferol derivatives on gene expression differs depending on the derivative. All tested polyphenols increased the SOD activity in cells co-incubated with etoposide. We observed that the co-incubation of HL-60 cells with etoposide and kaempferol or derivative P7 increases the level of total glutathione in these cells. Taken together, our observations suggest that the antioxidant activity of kaempferol is related to the activation of antioxidant genes and proteins. Moreover, we observed that glycoside derivatives can have a different effect on the antioxidant cellular systems than kaempferol.

2-Deoxyglucose Glycolysis Inhibitor Augment Oncolytic Virotherapy to Induce Oxidative Stress and Apoptosis in Breast Cancer (Part Ⅲ)

One of the "hallmarks of cancer" is altered energy metabolism, which is increased glycolysis in cancer cells, the primary source of energy that uses this metabolic pathway to generate ATP. Oncolytic virotherapy with aerobic glycolysis inhibitor smart therapeutic approach to induce apoptosis in cancer cells. The current study aimed to use the 2-Deoxyglucose (2DG), a specific glycolysis inhibitor, to enhance the Newcastle disease virus (NDV). In this study, a mouse model of breast cancer allograft with mammary adenocarcinoma tumor cells (AN3) was used and treated with 2DG, NDV, and a combination of both. Anti-tumor efficacy and glycolysis analysis (hexokinase -1 (HK-1), pyruvate, and ATP) were determined. The induction of oxidative stress was investigated by reactive oxygen species (ROS) and total glutathione assay examination. Apoptosis induction was investigated using immunohistochemistry (cleaved Caspase-3) and histopathology. The result showed that combination therapy enhances anti-tumor efficacy (decrease in relative tumor volume and increase in tumor growth inhibition) of NDV against breast cancer. This effect was accompanied by a reduction in HK-1 concentration, pyruvate, and ATP (glycolysis products). Moreover, NDV+2DG therapy induces oxidative stress (decreases total glutathione and increases ROS). Immunohistochemistry and histopathological examination showed the apoptotic area in tumor tissues in treated groups. In conclusion, the present study found that the combination therapy could be considered as an effective cancer therapy through induction of glycolysis inhibition, oxidative stress, and apoptosis selectively in cancer cells.

Commiphora myrrh Supplementation Protects and Cures Ethanol-Induced Oxidative Alterations of Gastric Ulceration in Rats

Gastric ulceration is a multifactorial disease defined as a defect in the gastric wall that extends through the muscularis mucosae into the deeper layers of the wall. The most common cause of gastric ulceration is alcohol consumption. In the current study, rats were gavaged by ethanol to investigate the protective (before ethanol) and curative (after ethanol) ability of Commiphora myrrh (myrrh) oil and extract against gastric ulcer oxidative alterations induced by ethanol. Myrrh significantly improved ulcer index, histomorphology, and periodic acid Schiff (PAS) impaired by ethanol. In addition, myrrh improved the antioxidant potential of gastric mucosa through enhancement of nuclear factor related to erythroid 2 (Nrf2), total glutathione (GSH), reduced GSH, and oxidized glutathione (GSSG), along with significant reduction in malondialdehyde (MDA) levels. Amelioration of gastric oxidative stress by myrrh enables gastric mucosa to counteract the ethanol’s inflammatory and apoptotic processes leading to improved gastric proliferation and healing. Interestingly, myrrh extract showed better protective and curative effects than myrrh oil against gastric ulceration.

Protofibril formation: decreased total glutathione concentration as an early indicator of neuron damage in the brainstems of Wistar rats treated with rotenone

Background: Rotenone treatment causes oxidative stress in neurons and forms the basis of animal models of Parkinson's disease. The reduced form of glutathione is predicted to detoxify rotenone from neurons in the brainstem. This study aims to measure the concentration of total glutathione and analyze the formation of protofibril in the brainstem of Wistar rats treated with rotenone. Methods: Seventy-two male Wistar rats aged 8–9 weeks weighing 200–250 g were divided into two investigations: total glutathione determination and protofibril analysis. The independent variables were treatment group, observation time, and location in the brainstem. The dependent variables were the concentration of total glutathione and protofibril density. Results: The concentration of total glutathione was not significantly different among treatment groups (p: 0.084), observation time (p: 0.608), or the location in the brainstem (p: 0.372). Protofibril density was different in the treatment groups (p: 0.001), observation time (p: 0.001), and between the upper and lower brainstem (p: 0.001). Rotenone treatment subcortically induced the concentration of total glutathione in the brainstem to decrease, but protofibril density tended to increase. Conclusions: The total glutathione concentration is inversely proportional to protofibril density. Total glutathione might be an early marker of neuronal damage.

1,25(OH)2D3 Inhibited Ferroptosis in Zebrafish Liver Cells (ZFL) by Regulating Keap1-Nrf2-GPx4 and NF-κB-hepcidin Axis

Ferroptosis is a kind of iron-dependent programed cell death. Vitamin D has been shown to be an antioxidant and a regulator of iron metabolism, but the relationship between vitamin D and ferroptosis is poorly studied in fish. This study used zebrafish liver cells (ZFL) to establish a ferroptosis model to explore the effect of 1,25(OH)2D3 on cell ferroptosis and its mechanism of action. The results showed that different incubation patterns of 1,25(OH)2D3 improved the survival rate of ZFL, mitigated mitochondrial damage, enhanced total glutathione peroxidase (GPx) activity, and reduced intracellular reactive oxygen species (ROS), lipid peroxidation (LPO), and malondialdehyde (MDA), as well as iron ion levels, with the best effect at 200 pM 1,25(OH)2D3 preincubation for 72 h. Preincubation of ZFL at 200 pM 1,25(OH)2D3 for 72 h downgraded keap1 and ptgs2 gene expression, increased nrf2, ho-1, fth1, gpx4a,b expression, and lowered the expression of the nf-κb p65,il-6,il-1β gene, thus reducing the expression of hamp1. The above results indicate that different incubation patterns of 1,25(OH)2D3 have protective effects on ferroptosis of ZFL induced by ferroptosis activator RSL3 and 1,25(OH)2D3 can inhibit ferroptosis of ZFL by regulating Keap1–Nrf2–GPx4 and NF-κB–hepcidin axis.

Gastroprotective Effect of Myricetin on Ethanol-Induced Acute Gastric Injury in Rats

The flavonoid myricetin is abundant in vegetables and has various bioactive properties, including anti-inflammatory and antioxidative activities. In the present study, we explored the effects of myricetin on alcohol-induced gastric ulcer in a rat model. To induce gastric ulcer, absolute ethanol (5 mL/kg body weight) was orally administrated to each rat. The positive control and myricetin-treated groups were given oral doses of omeprazole (20 mg/kg) or myricetin (12 mg/kg), respectively, 1 hour prior to the administration of absolute alcohol. We found that pretreatment with myricetin significantly decreased alcohol-induced gastric ulcer, hemorrhage, hyperemia, and epithelial cell loss in the gastric mucosa. Myricetin pretreatment reduced the level of malondialdehyde (MDA) and increased that of total glutathione (GSSG/GSH) and superoxide dismutase (SOD) in gastric tissues. In addition, it elevated the expression levels of cyclooxygenase-1 (COX-1) and prostaglandin E2 (PGE2) and decreased the phosphorylation of nuclear factor kappa B (NF-κB). Together, these results indicate that myricetin effectively inhibits ethanol-induced acute gastric injury by preventing oxidative damage, stimulating PGE2 production, and inhibiting NF-κB activation. We suggest that myricetin may be an alternative treatment for gastric injury caused by alcohol intake.

Fluorometric Optimized Determination of Total Glutathione in Erythrocytes

Glutathione is a tripeptide natural product characterized by a non-canonical peptide bond with an amide moiety linking the nitrogen of cysteine to the γ-carboxyl of glutamate, and is found ubiquitously in nature, in animals, plants and microorganisms. One of the most abundant biological matrices is represented by erythrocytes, being glutathione the only sulfur-containing mechanism for the red blood cell oxidative protection. Several analytical methods for glutathione determination from different samples are described in the literature and most of these methods are based on the use of high-performance liquid chromatography. HPLC equipment is not available in all the biochemical laboratories, and, moreover, displays lot of economic and ecological limitations, including organic solvent consumption and time-consuming analysis. Here, an organic-free high-throughput fluorometric methodology for the analysis of total glutathione in erythrocytes is reported, avoiding the use of time-consuming and not-sustainable techniques.

Optimized models of xenobiotic-induced oxidative stress in HepG2 cells

Purpose: To evaluate the molecular impact of ethanol, sodium selenite, and tert-butyl hydroperoxide (TBHP) on oxidant-antioxidant balance in HepG2 cells to establish an optimized oxidative stress model of HepG2 cells. Methods: HepG2 cells were treated with ethanol (10 - 500 mM) and sodium selenite (1 - 10 µM) for 24 and 48 h and with TBHP (50 - 200 µM) for 3 and 24 h, respectively. Biomarkers for cellular injury, ie, lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA), and for antioxidant system, i.e., superoxide dismutase (SOD), catalase (CAT), and total glutathione content, were determined. Results: All treatments increased the levels of LDH, AST, ALT, and MDA but decreased SOD and CAT activities and the total glutathione content in HepG2 cells. Oxidative stress was induced by these oxidative stressors in HepG2 cells via oxidant-antioxidant imbalance, with TBHP (100 µM, 3 h) acting as a powerful oxidant based on the minimal time to induce oxidative stress. The antioxidants, ascorbic acid and gallic acid, improved oxidant-antioxidant imbalance against xenobiotic-induced oxidative stress in HepG2 cells. Conclusion: These oxidative stress models are suitable for investigating the antioxidant and/or hepatoprotective potential of chemicals, including natural compounds.

Multi-Omics Analysis of Anti-Inflammatory Action of Alkaline Extract of the Leaves of Sasa sp.

Efficient utilization of alkaline extracts of several plants for the treatment of oral diseases has been reported. To investigate the mechanism of anti-inflammatory activity of alkaline extract of the leaves of Sasa sp. (SE), multi-omics analysis using metabolomics and DNA array was performed. Human gingival fibroblasts (HGFs) were treated for IL-1β to induce inflammation (detected by PGE2 production in culture medium) in the presence or absence of SE. Both IL-1β and SE showed slight hormetic growth stimulation against HGF. SE inhibited PGE2 production dose- and time-dependently. Its inhibitory action was more pronounced by first treating the cells with SE, rather than with IL-1β. At 3 h after IL-1β treatment, 18 amino acids (except cysteine and glutamic acid), total glutathione (GSH, GSSG, Cys-GSH disulfide), Met-sulfoxide, 5-oxoproline, and SAM declined, whereas DNA expressions of AKT, CASP3, and CXCL3 were elevated. These changes were reversed by simultaneous treatment with SE. The present study suggests that the anti-inflammatory action of SE is mediated via various metabolic pathways for cell survival, apoptosis, and leukocyte recruitment.