Tert-butyl Hydroperoxide-induced Oxidative Damage Research Articles

Propolis Protects GC-1spg Spermatogonial Cells against Tert-Butyl Hydroperoxide-Induced Oxidative Damage.

Propolis is a natural resin produced by honeybees with plenty of pharmacologic properties, including antioxidant activity. Oxidative stress disrupts germ cell development and sperm function, with demonstrated harmful effects on male reproduction. Several natural antioxidants have been shown to reduce oxidative damage and increase sperm fertility potential; however, little is known about the effects of propolis. This work evaluated the role of propolis in protecting spermatogonial cells from oxidative damage. Propolis' phytochemical composition and antioxidant potential were determined, and mouse GC-1spg spermatogonial cells were treated with 0.1-500 µg/mL propolis (12-48 h) in the presence or absence of an oxidant stimulus (tert-butyl hydroperoxide, TBHP, 0.005-3.6 µg/mL, 12 h). Cytotoxicity was assessed by MTT assays and proliferation by Ki-67 immunocytochemistry. Apoptosis, reactive oxygen species (ROS), and antioxidant defenses were evaluated colorimetrically. Propolis presented high phenolic and flavonoid content and moderate antioxidant activity, increasing the viability of GC-1spg cells and counteracting TBHP's effects on viability and proliferation. Additionally, propolis reduced ROS levels in GC-1spg, regardless of the presence of TBHP. Propolis decreased caspase-3 and increased glutathione peroxidase activity in TBHP-treated GC-1spg cells. The present study shows the protective action of propolis against oxidative damage in spermatogonia, opening the possibility of exploiting its benefits to male fertility.

Thiol antioxidants protect human lens epithelial (HLE B-3) cells against tert-butyl hydroperoxide-induced oxidative damage and cytotoxicity

Oxidative damage to lens epithelial cells plays an important role in the development of age-related cataract, and the health of the lens has important implications for overall ocular health. As a result, there is a need for effective therapeutic agents that prevent oxidative damage to the lens. Thiol antioxidants such as tiopronin or N-(2-mercaptopropionyl)glycine (MPG), N-acetylcysteine amide (NACA), N-acetylcysteine (NAC), and exogenous glutathione (GSH) may be promising candidates for this purpose, but their ability to protect lens epithelial cells is not well understood. The effectiveness of these compounds was compared by exposing human lens epithelial cells (HLE B-3) to the chemical oxidant tert-butyl hydroperoxide (tBHP) and treating the cells with each of the antioxidant compounds. MTT cell viability, apoptosis, reactive oxygen species (ROS), and levels of intracellular GSH, the most important antioxidant in the lens, were measured after treatment. All four compounds provided some degree of protection against tBHP-induced oxidative stress and cytotoxicity. Cells treated with NACA exhibited the highest viability after exposure to tBHP, as well as decreased ROS and increased intracellular GSH. Exogenous GSH also preserved viability and increased intracellular GSH levels. MPG scavenged significant amounts of ROS, and NAC increased intracellular GSH levels. Our results suggest that both scavenging ROS and increasing GSH may be necessary for effective protection of lens epithelial cells. Further, the compounds tested may be useful for the development of therapeutic strategies that aim to prevent oxidative damage to the lens.

Schisandrin B protects human keratinocyte-derived HaCaTcells from tert-butyl hydroperoxide-induced oxidative damage through activating the Nrf2 signaling pathway.

Schisandrin B (Sch B), an active extract of Schisandrachinensis, has demonstrated antioxidant activity in a number of invitro and invivo models. In the present study, the capacity of Sch B to protect against oxidative injury in keratinocytes using the human keratinocyte‑derived HaCaT cell line was investigated. To induce oxidative injury, tert‑Butyl hydroperoxide (tBHP) was employed. The results indicate that Sch B efficiently reduced tBHP‑induced cell death, reactive oxygen species (ROS) generation, protein oxidation, lipid peroxidation and DNA damage. Sch B also effectively attenuated the loss of mitochondrial membrane potential (MMP), and restored adenosine triphosphate (ATP) levels in tBHP‑injured HaCaT cells. Furthermore, Sch B enhanced the expression of key antioxidant enzymes, including catalase, heme oxygenase‑1, glutathione peroxidase, and superoxide dismutase, and further engaged the nuclear factor‑erythroid 2‑related factor 2 (Nrf2) signaling pathway by modulating its phosphorylation through activating multiple upstream kinases, including protein kinase B, adenosine monophosphate‑activated protein kinase and mitogen‑activated protein kinases (MAPKs). The present study suggests that Sch B provides a protective effect in keratinocytes in response to oxidative injury via reinforcing the endogenous antioxidant defense system. Therefore, it may be applied as an adjuvant therapy or in health foods to delay the skin aging process and the onset of skin diseases caused by oxidative stress.

Isotetrandrine ameliorates tert-butyl hydroperoxide-induced oxidative stress through upregulation of heme oxygenase-1 expression.

1R, 1'S-isotetrandrine, a naturally occurring plant alkaloid found in Mahonia of Berberidaceae, possesses anti-inflammatory, antibacterial, and antiviral properties, but the antioxidative activity and mechanism action remain unclear. In this study, we demonstrated the antioxidative effect and mechanism of 1R, 1'S-isotetrandrine against tert-butyl hydroperoxide-induced oxidative damage in HepG2 cells. We found that 1R, 1'S-isotetrandrine suppressed cytotoxicity, reactive oxygen species generation, and glutathione depletion. Additionally, our study confirmed that 1R, 1'S-isotetrandrine significantly increased the antioxidant enzyme heme oxygenase-1 expression and nuclear translocation of factor-erythroid 2 p45-related factor 2 (Nrf2). Specifically, the nuclear translocation of Nrf2 induced by 1R, 1'S-isotetrandrine was associated with Nrf2 negative regulatory protein Keap1 inactivation and phosphorylation of both extracellular signal-regulated protein kinase and c-Jun NH2-terminal kinase. Preincubation with thiol-reducing agents reduced 1R, 1'S-isotetrandrine-induced heme oxygenase-1 expression, and treatment with either extracellular signal-regulated protein kinase or c-Jun NH2-terminal kinase inhibitors attenuated the levels of 1R, 1'S-isotetrandrine-induced Nrf2 activation and heme oxygenase-1 expression. Furthermore, the cytoprotective effect of 1R, 1'S-isotetrandrine was abolished by heme oxygenase-1, extracellular signal-regulated protein kinase, and c-Jun NH2-terminal kinase inhibitors. These results indicated that the 1R, 1'S-isotetrandrine ameliorated tert-butyl hydroperoxide-induced oxidative damage through upregulation of heme oxygenase-1 expression by the dissociation of Nrf2 from Nrf2-Keap1 complex via extracellular signal-regulated protein kinase and c-Jun NH2-terminal kinase activation and Keap1 inactivation.

Lignan glycosides from sesame meal exhibit higher oral bioavailability and antioxidant activity in rat after nano/submicrosizing

Lignan glycosides are one of the major bioactive components in sesame meal. In the current study, we evaluated whether the absorption efficiency of lignan glycosides from sesame meal (LGSM) increases after wet milling. The current data show that oral bioavailability of LGSM significantly elevated from 0.18 to 0.26% after nano/submicro-sizing due to the smaller particle size and larger surface area. Oral administration of nano/submicro-sized LGSM (N-LGSM) significantly increased the concentrations of sesaminol triglucoside and sesaminol glucuronide in rat urine compared to administration of LGSM that was not nano/submicro-sized. More importantly, oral administration of N-LGSM increased the concentrations of enterolactone and enterodiol found in the plasma, liver, kidney, small intestine and urine of rats, suggesting that the nano/submicro-sizing process greatly improved the accessibility of intestinal microbiota to LGSM. In addition, oral administration of N-LGSM remarkably alleviated lipid peroxidation and increased antioxidant enzyme activities against tert-butyl hydroperoxide-induced oxidative damage in rats.

(Anti)mutagenic and immunomodulatory properties of quercetin glycosides.

Quercetin-3-O-β-D-glucopyranoside (isoquercitrin) and quercetin-3-O-rutinoside (rutin) are common components of a normal human diet and are increasingly used in food supplements. Here their effect on mutagenesis and immunity is shown. The in vitro (anti)mutagenic potential was compared with that of quercetin using the Ames test in Salmonella typhimurium His(-) strains TA100, TA98 and TA102. Isoquercitrin only slightly increased the number of revertants, while rutin was totally non-mutagenic. On the other hand, all compounds displayed dose-dependent protective activity against H2O2 - and tert-butyl hydroperoxide-induced oxidative damage to the TA102 strain and at 75 µmol L(-1) inhibited H2O2/Fe(2+)-induced formation of the open circular and linear forms of the DNA plasmid pBSIISK(-). In mice, none of the flavonols (0.86 µmol day(-1), 34 days) induced harmful effects. In immunized animals, all compounds enhanced ex vivo B cell proliferation; quercetin stimulated lymphocyte basal proliferation and increased the number of IgM-producing lymphocytes. Rutin promoted NK cytotoxic activity, supported T cells and enhanced gut epithelium renewal. No effect on IgG-forming cells was found. Isoquercitrin displayed negligible and rutin no mutagenicity, but both showed significant antimutagenic and DNA-protective effects against oxidative damage. In vivo, they supported the readiness of the immune system for specific humoral immune response.

Protective effect of carvacrol from Thymus quinquecostatus Celak against tert-butyl hydroperoxide-induced oxidative damage in Chang cells

Carvacrol is a monoterpenic phenol present in Thymus quinquecostatus Celak. The protective effects of carvacrol against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage were investigated in human Chang cells. Cells treated with carvacrol extracts promoted Chang cell survival and protection was associated with stabilization of the mitochondrial membrane potential (MMP), prevention of oxidative stress-triggered reactive oxygen species (ROS), and lipid peroxidation (MDA production). In addition, Annexin V/PI, observed using Hoechst staining, indicated that carvacrol inhibited t-BHP-induced cell damage and stimulated the antioxidant capability of Chang cells due to elevation of glutathione (GSH) levels, which were reduced by t-BHP treatment. Carvacrol prevented oxidative stress-induced Chang cell damage via suppression of ROS and MDA levels, and increased GSH levels.

Protective effects of oligomeric and polymeric procyanidin fractions from defatted grape seeds on tert-butyl hydroperoxide-induced oxidative damage in HepG2 cells

Oligomeric and polymeric procyanidins have been reported to possess different antioxidant capacities. However, the intracellular antioxidant mechanisms of oligomeric and polymeric procyanidins are still poorly understood. In this study, we evaluated the cytoprotective effects of the oligomeric procyanidin fraction (OPF) and the polymeric procyanidin fraction (PPF) from grape seeds against the oxidative damage induced by tert-butyl hydroperoxide (TBHP) in HepG2 cells. The levels of cellular reactive oxygen species (ROS), cellular lipid peroxidation, glutathione (GSH), and antioxidant enzyme activities were measured as biomarkers of cellular oxidative status. HepG2 cells were treated with different concentrations of procyanidin samples (0–20μg/ml) for 6h prior to treatment with TBHP for 3h. The incubation of HepG2 cells with TBHP led to an approximately 60% decrease in cell viability. However, pretreatment of the cells with the samples, at 5–20μg/ml, rescued cell viability in a dose-dependent manner. Cellular generation of ROS, formation of malondialdehyde (MDA), and depletion of GSH were reduced by OPF and PPF. Moreover, TBHP treatment increased the activities of glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD). However, a 6h pretreatment with each of the samples at 20μg/ml significantly decreased the activities of these enzymes. These results clearly showed that treatment with OPF and PPF protected against oxidative damage by modulating ROS production, GSH levels, MDA generation, and antioxidant enzyme activities in HepG2 cells.

Antioxidant activity of protein hydrolysates derived from threadfin bream surimi byproducts

Antioxidant activities of protein hydrolysates from threadfin bream surimi wastes, including frame, bone and skin (FBS) and refiner discharge (RD), were investigated. FBS and RD were rich in Lys, Glu, Gly, Pro, Asp, Leu, His, Tyr and Phe. FBS was hydrolysed to a greater extent than RD regardless of proteinases tested (Virgibacillus sp. SK33 proteinase, Alcalase, pepsin and trypsin). Pepsin-hydrolysed FBS, at a 5% degree of hydrolysis (DH), showed the highest antioxidant activity based on 2,2′-azinobis (3-ethyl-benzothiazoline-6-sulphonate) (ABTS) radical (0.455±0.054mg Trolox equivalents/mg leucine equivalents), ferric reducing antioxidant power (FRAP) (0.221±0.005mM Trolox equivalents) and inhibition of β-carotene bleaching assays. FBS hydrolysates showed higher antioxidant activity based on chemical assays than their RD counterparts. However, FBS and RD hydrolysates protected HepG2 cells against tert-butyl hydroperoxide-induced oxidative damage to a similar extent. Therefore, FBS and RD hydrolysates have a potential as antioxidative neutraceutical ingredients.

Protective mechanisms of 3-caffeoyl, 4-dihydrocaffeoyl quinic acid from Salicornia herbacea against tert-butyl hydroperoxide-induced oxidative damage

Salicornia herbacea has been used as a folk medicine for disorders such as constipation, obesity, diabetes, and cancer. Recent studies have shown that S. herbacea has antioxidative, anti-inflammatory, immunomodulatory, antihyperglycemic, and antihyperlipidemic activities. In the present work, we investigated the protective effects of the chlorogenic acid derivative, 3-caffeoyl, 4-dihydrocaffeoyl quinic acid (CDCQ), which was isolated from S. herbacea, against tert-butyl hydroperoxide ( t-BHP)-induced hepatotoxicity in Hepa1c1c7 cells. Pretreatment of Hepa1c1c7 cells with CDCQ significantly reduced t-BHP-induced generation of ROS, caspase-3 activation, and subsequent cell death. Also, CDCQ up-regulated heme oxygenase-1 (HO-1) expression, which conferred cytoprotection against oxidative injury induced by t-BHP. Moreover, CDCQ-induced nuclear translocation of the transcription factor NF-E2-related factor 2 (Nrf2), which is upstream of CDCQ-induced HO-1 expression, and PI3K/Akt activation, a pathway that is involved in induced Nrf2 nuclear translocation. Taken together, these results suggest that the protective effects of CDCQ against t-BHP-induced hepatotoxicity may be due, at least in part, to its ability to scavenge ROS and to regulate the antioxidant enzyme HO-1 via the PI3K/Akt-Nrf2 signaling pathways.

Protective activity of Hypericum androsaemum infusion against tert-butyl hydroperoxide-induced oxidative damage in isolated rat hepatocytes

Hypericum androsaemum L. (Guttiferae) is a medicinal plant with antioxidant activity. Increasing evidence regarding free radical generating agents suggests that hepatotoxic-related disorders may involve reactive oxygen species (ROS). The purpose of this study was to investigate the protective effect of Hypericum androsaemum infusion on isolated rat hepatocytes oxidative injury induced by tert-butyl hydroperoxide ( t-BHP). The results showed that pretreatment of the cells with this infusion (16, 62 and 250 μg/ml) prevented the leakage of lactate dehydrogenase (LDH) and lipid peroxidation caused by a 30-min treatment with t-BHP (1 mM). However, infusion-induced alterations on glutathione homeostasis were noticed, as it was observed by the increase in glutathione oxidised form (GSSG) and depletion in total glutathione levels, which indicates that plant-derived antioxidant extracts may not be considered a generalised way of treating pro-oxidant-related diseases.

Inhibitory effect of berberine on tert-butyl hydroperoxide-induced oxidative damage in rat liver.

Berberine, a main protoberberine component of Coptidis Rhizoma, was studied for the mechanism of its inhibitory effects on the tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity and lipid peroxidation in rat liver. In the preliminary study, berberine expressed an antioxidant property by its capacity for quenching the free radicals of 1,1-diphenyl-2-picrylhydrazyl (DPPH). Further investigations were conducted using t-BHP-induced cytotoxicity in rat primary hepatocytes and hepatotoxicity in rats to evaluate the antioxidative bioactivity of berberine. The results in rat primary hepatocytes demonstrated that berberine, at the concentrations of 0.01-1.0 mM, significantly decreased the leakage of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT), and the formation of malondialdehyde (MDA) induced by 30 min treatment of t-BHP (1.5 mM). Berberine also attenuated the t-BHP-induced depletion of glutathione (GSH) and induced a high level of DNA repair synthesis. The in vivo study showed that the intraperitoneal pretreatment with berberine (0.5 and 5 mg/kg) for 5 days before a single dose of t-BHP (0.1 mmol/kg) significantly lowered the serum levels of hepatic enzyme markers (ALT and aspartate aminotransferase) and reduced oxidative stress in the liver. The histopathological evaluation of the livers revealed that berberine reduced the incidence of liver lesions, including hepatocyte swelling, leukocyte infiltrations, and necrosis induced by t-BHP. These results lead us to speculate that berberine may play a chemopreventive role via reducing oxidative stress in living systems.

Nitric oxide protects cardiomyocytes against tert-butyl hydroperoxide-induced formation of alkoxyl and peroxyl radicals and peroxidation of phosphatidylserine.

We studied protective effects of nitric oxide against tert-butyl hydroperoxide-induced oxidative damage to cardiac myocytes. Two distinct free radicals species--alkoxyl radicals associated with non-heme iron catalytic sites and myoglobin protein-centered peroxyl radicals--were found in low-temperature EPR spectra of cardiac myocytes exposed to t-BuOOH. The t-BuOOH-induced radical formation was accompanied by site-specific oxidative stress in membrane phospholipids (peroxidation of phosphatidylserine) assayed by fluorescence HPLC after metabolic labeling of cell phospholipids with oxidation-sensitive cis-parinaric acid. An NO-donor, (Z)-1-[N-(3-ammonio-propyl)-N-(n-propyl) amino]-diazen-1-ium-1,2-diolate], protected cardiac myocytes against tert-butyl hydroperoxide-induced: (i) formation of non-protein- and protein-centered free radical species and (ii) concomitant peroxidation of phosphatidylserine. Thus nitric oxide can act as an effective antioxidant in live cardiomyocytes.