Determination of Thioredoxin Reductase Activity in Rat Liver Supernatant

Hill, Kristina E.; McCollum, Gary W.; Burk, Raymond F.

doi:10.1006/abio.1997.2373

Cited by 108 publications

(57 citation statements)

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“…The absorbance was measured by a spectrophotometer at a wavelength of 412 nm (UN2CO-WFT2100; Aoyi Co. Ltd., Shanghai, China). TrxR activity was assayed using the dithio-bis-nitrobenzoic acid (DTNB) method, in which the reduction of DTNB was monitored at 412 nm (Hill et al 1997). SelP levels were measured by a radioimmunoassay (Hill et al 1996).…”

Section: Methodsmentioning

confidence: 99%

Vitamin A affects the expression of antioxidant genes in bovine mammary epithelial cells with oxidative stress induced by diethylene triamine-nitric oxide

Shi¹,

Yan²,

Jin³

et al. 2016

CZECH J ANIM SCI

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ABSTRACT:The considerable increase in oxygen requirements due to the high metabolic rate of the bovine mammary epithelial cells (BMEC) during lactation results in an augmented production of reactive nitrogen species (RNS), such as nitric oxide (NO), which may expose cows to increased oxidative stress. Vitamin A (VA) has been shown in several studies to enhance the antioxidant defence system against oxidative stress, but whether the reason is related to a reduced NO production remains unclear. Diethylene triamine-nitric oxide polymer (NOp) is a type of NO-generating compound, which is safe, efficacious, and releases NO over a long period. The current study was conducted to investigate the effect of VA on the antioxidant function in BMEC and the underlying mechanism by discussing the protection of VA on NO-induced oxidative stress of BMEC. The experiment was conducted using a single-factor completely randomized arrangement. Primary BMEC were isolated from the mammary glands of Holstein dairy cows. The third generation cells were randomly divided into four equal groups with six replicates each. Each group received different combinations of VA and NOp treatment as follows: controls (without VA and NOp), NOp treatment alone, VA treatment alone, and VA and NOp treatment together. The lysates were collected to evaluate the activities of glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) and the contents of reactive oxygen species (ROS) and malondialdehyde (MDA), and the cell-free supernatants were collected to analyze selenoprotein P (SelP) content, inducible nitric oxide synthase (iNOS) activities and nitric oxide (NO), interleukin-1 (IL-1), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α) contents. The results suggested that compared to the control, the cell proliferation, the activity of the antioxidants GPx and TrxR, the content of SelP and the antioxidant gene expressions of GPx1, GPx4, and TrxR1 were significantly decreased (P < 0.05), and the contents of ROS and MDA, the activity of iNOS, the contents of NO and IL-1, IL-6, TNF-α, and their mRNA expressions were increased dramatically in the NOp treatment alone group (P < 0.05), but the opposite changes were observed in the VA treatment alone group. Compared to the NOp treatment alone, the VA and NOp treatment together significantly improved cell proliferation, the activities of the antioxidants GPx and TrxR, and the gene expressions of GPx1 and TrxR1, and dramatically decreased the contents of ROS and MDA, the activity of iNOS, the contents of NO and IL-1, IL-6, TNF-α and their mRNA expression levels (P < 0.05). The present research suggests that VA can improve the antioxidant function of BMEC and protect the cells from experiencing the NOp-induced oxidative stress by regulating antioxidant gene expression. The probable mechanism is that VA can reduce the activity of iNOS and its mRNA expression by down-regulating of the expression of IL-1, IL-6, and TNF-α to reduce NO production. However, the exact mechanism warrants future exploration. Keyword...

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Section: Methodsmentioning

confidence: 99%

Vitamin A affects the expression of antioxidant genes in bovine mammary epithelial cells with oxidative stress induced by diethylene triamine-nitric oxide

Shi¹,

Yan²,

Jin³

et al. 2016

CZECH J ANIM SCI

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“…The retentate was washed thrice with PE buffer (50 mmol/L potassium phosphate and 2 mmol/L EDTA, pH 7.5) and used as crude cell extract. Thioredoxin reductase activity in the cell extract was measured by the modified 5,5 ¶-dithiobis(2-nitrobenzoic acid) reduction assay (37). Protein levels of the cell extracts were determined using the Bio-Rad Protein Assay kit.…”

Section: Methodsmentioning

confidence: 99%

Heat Shock Protein 27 Protects L929 Cells from Cisplatin-Induced Apoptosis by Enhancing Akt Activation and Abating Suppression of Thioredoxin Reductase Activity

Zhang

Shen

2007

Clinical Cancer Research

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Purpose: Heat shock protein 27 (Hsp27) is up-regulated in multiple malignancies and implicated in cisplatin resistance. It is attempted to know how Hsp27 endues cell with cisplatin resistance by interfering with upstream of both apoptosis signal–regulating kinase 1 (ASK1)/p38 mitogen-activated protein kinase–activated apoptotic signaling and serine/threonine kinase Akt-dependent survival signaling. Experimental Design: The mouse L929 cells stably transfected with human Hsp27 or its dominant-negative mutant and the human cervical cancer HeLa cells transfected with Hsp27 siRNA were used. The cisplatin-induced apoptosis and activation of ASK1, p38, and Akt were compared in control cells, cells overexpressing Hsp27, and cells with their endogenous Hsp27 knocked down. Results: Hsp27 effectively protected the cells from cisplatin-induced DNA fragmentation. The p38 inhibitors obviously decreased whereas Akt inhibitors markedly increased the apoptotic fraction in cisplatin-treated cells. Overexpression of Hsp27 doubly enhanced the drug-induced Akt activation while substantially depressing the drug-induced activation of ASK1 and p38. Knockdown of the endogenous Hsp27 in HeLa cells resulted in the effects opposite to that observed in the Hsp27-overexpressing cells. Enhancement of Akt activation is associated with complex formation between Akt and Hsp27, whereas depression of ASK1/p38 activation is attributed to a reversion of the drug-induced inhibition of thioredoxin reductase activity and subsequent oxidation of thioredoxin. Conclusions: Hsp27 endues cells with cisplatin resistance via depression of the drug-induced ASK1/p38 activation and enhancement of the drug-induced Akt activation. This study revealed the intervention of Hsp27 in upstream of both ASK1/p38 apoptotic signaling and phosphatidylinositol 3-kinase/Akt survival signaling. Therefore, the inhibition of Hsp27 may be a novel strategy of cancer chemotherapy.

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“…Mitochondrial thioredoxin reductase activity was determined based on the methods of Hill and co-workers [18]. Mitochondria (10-40 µg protein) were incubated in potassium phosphate buffer (100 mmol/l; pH 7.0) in the presence of 5 mmol/l 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), 20 mmol/l EDTA, and 0.2 mg/ml BSA for 5 min.…”

Section: Mitochondrial Antioxidant Enzyme Assaysmentioning

confidence: 99%

Two subpopulations of mitochondria in the aging rat heart display heterogenous levels of oxidative stress

Suh

Heath

Hagen

2003

Free Radical Biology and Medicine

115

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Cardiac mitochondria are composed of two distinct subpopulations: one beneath the sarcolemma (subsarcolemmal mitochondria: SSM), and another along the myofilaments (interfibrillary mitochondria: IFM). Previous studies suggest a preferential loss of IFM function with age; however, the age-related changes in oxidative stress in these mitochondrial subpopulations have not been examined. To this end, the changes in mitochondrial antioxidant capacity, oxidant output, and oxidative damage to Complex IV in IFM and SSM from young and old rats were studied. Results show no apparent differences in any parameters examined between IFM and SSM from young rats. However, relative to young, only IFM from old rats had a significantly higher rate of oxidant production and a decline in mitochondrial ascorbate levels and GSH redox status. The age-related decline in mitochondrial antioxidant capacity in IFM was accompanied by a marked loss in glutaredoxin and GSSG reductase activities, suggesting a diminished reductive capacity in IFM with age. Moreover, the loss in Complex IV activity was limited to the IFM of old rats, which was accompanied by a 4-fold increase in 4-hydroxynonenal-modified Complex IV. Thus, mitochondrial decay is not uniform and further indicates that myofibrils may be uniquely under oxidative stress in the aging heart.

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Determination of Thioredoxin Reductase Activity in Rat Liver Supernatant

Cited by 108 publications

References 4 publications

Vitamin A affects the expression of antioxidant genes in bovine mammary epithelial cells with oxidative stress induced by diethylene triamine-nitric oxide

Vitamin A affects the expression of antioxidant genes in bovine mammary epithelial cells with oxidative stress induced by diethylene triamine-nitric oxide

Heat Shock Protein 27 Protects L929 Cells from Cisplatin-Induced Apoptosis by Enhancing Akt Activation and Abating Suppression of Thioredoxin Reductase Activity

Two subpopulations of mitochondria in the aging rat heart display heterogenous levels of oxidative stress

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