Selective synergistic anticancer effects of cisplatin and oridonin against human p53-mutant esophageal squamous carcinoma cells.

Oridonin (ORI) is known to pose anticancer activity against cancer, which could induce the therapeutic impact of chemotherapy drugs. However, such simple combinations have numerous side effects such as higher toxicity to normal cells and tissues. To enhance the therapeutic effects with minimal side effects, here we used ORI in combination with cisplitin (CIS) against different esophageal squamous cell carcinoma (ESCC) cell lines in vitro, to investigate the synergistic anticancer effects of the two drugs against ESCC. Calcusyn Graphing Software was used to assess the synergistic effect. Apoptosis, wound healing and cell invasion assay were conducted to further confirm the synergistic effects of ORI and CIS. Intracellular glutathione (GSH) and reactive oxygen species assay, immunofluorescence staining and western blot were used to verify the mechanism of synergistic cytotoxicity. ORI and CIS pose selective synergistic effects on ESCC cells with p53 mutations. Moreover, we found that the synergistic effects of these drugs are mediated by GSH/ROS systems, such that intracellular GSH production was inhibited, whereas the ROS generation was induced following ORI and CIS application. In addition, we noted that DNA damage was induced as in response to ORI and CIS treatment. Overall, these results suggest that ORI can synergistically enhance the effect of CIS, and GSH deficiency and p53 mutation, might be biomarkers for the combinational usage of ORI and CIS.

The effect of diclofenac sodium intoxication on the cardiovascular system in rats.

OBJECTIVES: Diclofenac sodium (DS) is a widely used nonsteroidal anti-inflammatory drug. Although its high doses are known to cause toxic effects in many tissues including liver and kidney, the effects on the cardiovascular system (CVS) have not been fully elucidated yet. Therefore, this study aimed to investigate the effect of DS on CVS. METHODS: The Control group did not receive medication; however, a single dose of 240 mg/kg DS was administered orally to the DS group. Electrocardiography (ECG) measurements were performed in all animals before (0thhour) and after (1st,6th,12th,24thhour) intoxication. After 24 h, All animals were sacrificed. Biochemical (malondialdehyde [MDA], and glutathione (GSH), Apelin, Elabela, Meteorin, Endoglin, Keap1, and Nrf2) and histopathological analyzes were performed on heart tissue samples. RESULTS: ECG results showed that there was a statistically significant increase in QTc, QRS, and heart rate at the 12th and 24th hours in the DS group. The biochemical analysis showed that GSH, Apelin, Keap1, and NRF2 values decreased significantly while Meteorin and Endoglin levels increased in the DS group. When histopathological results were evaluated, distinct lesions were observed in the DS group. CONCLUSION: In conclusion, high doses of DS intake can cause adverse effects on and damage to CVS.

Topotecan induces hepatocellular injury via ASCT2 mediated oxidative stress.

BACKGROUND: Topotecan is an anti-cancer chemotherapy drug with common side effects, including hepatotoxicity. In this study, we aim to investigate the mechanisms of topotecan-induced hepatocellular injury beyond conventional DNA damage. MATERIALS AND METHODS: Methyl Thiazolyl Tetrazolium (MTT) assay was used to detect the inhibitory effect of topotecan on cell proliferation. Western blot was used to detect protein expression. Flow cytometry assay was performed to determine apoptosis rate under topotecan treatment. ASCT2 overexpression was addressed using adenovirus vector. qRT-PCR and western blot assay were used to detect the expression of ASCT2. Glutamine uptake, intracellular glutathione (GSH) and reactive oxygen species (ROS) level were detected by glutamine detection kit, GSH detection kit and ROS detection kit respectively. RESULTS: MTT results showed that topotecan had an inhibitory effect on cell proliferation and induced apoptosis in both L02 and HepG2 cell lines. Topotecan inhibited the expression of glutamine transporter ASCT2 and the uptake of glutamine in both L02 and HepG2 cell lines. The uptake of glutamine and the GSH level was increased in both L02 and HepG2 cell lines after ASCT2 overexpression. The ROS level was inhibited by ASCT2 overexpression upon topotecan treatment in both L02 and HepG2 cell lines. Topotecan-induced hepatocellular apoptosis and proliferation inhibition were attenuated by ASCT2 overexpression in both L02 and HepG2 cell lines. CONCLUSION: Topotecan-induced hepatocytes death is dependent on ASCT2 down-regulation, which causes oxidative stress via inhibiting GSH production.

Assessment of ameliorative effect of Trigonella foenum-graecum against CuO-NPs induced toxicity in Oreochromis mossambicus.

The current study assessed the ameliorative effect of Trigonella foenum graceum extract against copper oxide nanoparticles (CuO-NPs) induced toxicity in Oreochromis mossambicus. For this purpose 100 healthy fish weighing 20±2.34g were randomly divided into five different groups in duplicates and designated as control (C) no treatment, positive control (G*) treated with 0.12mg/L of CuO-NPs, experimental co-treated groups G1, G2 and G3 were treated with Trigonella foenum-graecum extract @ 18, 26 and 52mg/L along with 0.12 mg/L of CuO-NPs, respectively. In this study significant (P<0.05) changes were observed in the antioxidant activity of enzymes and histological alterations in the liver and intestine of fish in G*, G1 and G2 groups while a good ameliorative response of Trigonella foenum-graecum was observed in G3. Dose dependent alterations in glutathione, lipid peroxides, catalase, and malondialdehyde as well as histological architecture of liver and intestine were observed in treated groups, where more alterations were observed in positive control and low dose treated groups of Trigonella foenum-graecum. Moreover, more ameliorative effect was observed in high dose of Trigonella foenum-graecum treated group (G3). This study is novel as no previous data is available on the amelioration of Trigonella foenum-graecum extract against CuO-NPs induced toxicity in fish.

In vivo antioxidant potential of fruit mucilage from two varieties of Cucumis melo subsp. agrestis against oxidative stress induced toxicity.

To evaluate in-vivo antioxidant potential of fruit mucilage from Cucumis melo variety momordica (PM) and variety agrestis (KM) using rats as experimental animals, the fruits were collected, identified, dried and pulverized. Mucilages were isolated from the fruit powders by microwave-assisted method. Aqueous extracts obtained were filtered to remove fruit pulp. Each filtrate was centrifuged at 4000xg rpm for 15 min. Each supernatant was precipitated with 3 volumes of 95% ethanol and maintained overnight at 4°C. These precipitates were filtered and lyophilized. In vivo antioxidant activity was determined using rats for 14 days. Paracetamol (75mg/Kg, i.p.) for inducing oxidative stress and Vitamin C & Vitamin E (200mg/Kg each, p.o.) as standard treatment were used. PM and KM were given in 500mg/Kg and 1000mg/Kg, p.o. doses in separate groups. SOD, MDA, GSH and CAT levels were estimated in organs (liver, kidney, heart, brain) of all groups using standard procedures. Toxic control showed prominent toxicity in the liver. The levels of GSH, CAT and SOD were raised and MDA levels were reduced in all organs of test and standard groups. The levels of antioxidant biomarkers varied in all remaining groups. The overall results are significant suggesting strong antioxidant potential of PM and KM.

Protective effects of chitosan and chitosan oligosaccharide against oxidative damage in liver tissue of rats with fluorine poisoning.

Fluorine toxicity has negative effects on soft tissue besides skeletal and dental tissues. In the present study, we have investigated the protective effect of chitosan (CS) and chitosan oligosaccharide (COS) on liver tissue of fluorine-intoxicated rats taking the antioxidant characteristics of chitosan and its derivatives into consideration. In this study, 42 male Wistar albino rats were randomly selected to determine the control and experimental fluorosis groups. Our study lasted for 12 weeks. As a consequence of the study, MDA significantly increased in the liver tissue of NaF group while some antioxidant values significantly decreased. It was detected that serum AST and LDH levels increased significantly while ALB and TP values significantly decreased in NaF group. The degenerations were identified in the liver histopathology of all fluoride-treated groups. We have concluded according to the results that chitosan oligosaccharide can be more effective compared with chitosan.

Effect of Polygonatum odoratum ethanol extract on high glucose-induced tubular epithelial cell apoptosis and oxidative stress.

This work aims to analyze the effect of the ethanol extract from Polygonatum odoratum on high glucose-induced tubular epithelial cell apoptosis and oxidative stress. HK-2 injury of tubular epithelial cells was induced by high glucose, and the ethanol extract from Polygonatum odoratum was given. HK-2 cell activity and apoptosis were detected by MTT method and flow cytometry, respectively. Western blot was performed to analyze Cleaved-caspase3, Pro-caspase3, Nrf2, HO-1 protein expression. The levels of MDA, GSH, SOD were evaluated using commercial Kit. si-Nrf2 was transfected into HK-2 cells and high-glucose induction and ethanol extract from Polygonatum odoratum were given to observe the changes of cell apoptosis and oxidative stress. Ethanol extract from Polygonatum odoratum increased the high glucose-induced HK-2 cell activity, Pro-caspase3, Nrf2, HO-1 protein, GSH, SOD levels and decreased its apoptosis rate, Cleaved-caspase3 protein and MDA levels, showing statistically significant difference (p<0.05). After Nrf2 interference, high glucose-induced HK-2 cell activity, Pro-caspase3 protein, GSH, and SOD levels were decreased under the action of ethanol extract from Polygonatum odoratum, while the apoptosis rate, Cleaved-caspase3 protein, and MDA levels were increased significantly (p<0.05). The ethanol extract from Polygonatum odoratum can inhibit high glucose-induced tubular epithelial cell apoptosis and reduce oxidative stress by activating the Nrf2-ARE signaling pathway.

Evaluation of efficiency omega 3 fatty acid improves the behavioural phenotype and protects against oxidative stress against MPP+ induces Parkinson's disease in mice.

This experiment proposed to study the efficiency omega 3 fatty acid on behavioural phenotype of Parkinson's disease (PD) in mice. Totally 7 groups (each group 6 mice) were used in this assessment, each groups were treated with saline (control), MPP+, L-DOPA, Omega 3 oil, Omega 3 oil (three different concentrations) +MPP+ separately. The behavioral assessments such as bar test, open field test, maze test, hang test were noted on 7th, 14th, 21st and 28th day. After the examination period, the tested animals' midbrains and frontal cortex were dissected to analyze TBARS, GSH, Catalase, Superoxide Dismutase and Glutathione Peroxidase assay. In the bar test, 500mg omega 3 fatty acid administrated mice showed a high cataleptic scores. In open field Test, significant reductions in behavior analysis were observed from the tested mice group. Maze test and hang test doesn't show much difference. In biochemical test, tested groups showed promising results compared to control group. The result strongly proved that the omega 3 fatty acid has remarkable abilities to control the neurodegenerative diseases.

Neuroprotective activity of ursodeoxycholic acid in CHMP2BIntron5 models of frontotemporal dementia.

Frontotemporal dementia (FTD) is one of the most prevalent forms of early-onset dementia. It represents part of the FTD-Amyotrophic Lateral Sclerosis (ALS) spectrum, a continuum of genetically and pathologically overlapping disorders. FTD-causing mutations in CHMP2B, a gene encoding a core component of the heteromeric ESCRT-III Complex, lead to perturbed endosomal-lysosomal and autophagic trafficking with impaired proteostasis. While CHMP2B mutations are rare, dysfunctional endosomal-lysosomal signalling is common across the FTD-ALS spectrum. Using our established Drosophila and mammalian models of CHMP2BIntron5 induced FTD we demonstrate that the FDA-approved compound Ursodeoxycholic Acid (UDCA) conveys neuroprotection, downstream of endosomal-lysosomal dysfunction in both Drosophila and primary mammalian neurons. UDCA exhibited a dose dependent rescue of neuronal structure and function in Drosophila pan-neuronally expressing CHMP2BIntron5. Rescue of CHMP2BIntron5 dependent dendritic collapse and apoptosis with UDCA in rat primary neurons was also observed. UDCA failed to ameliorate aberrant accumulation of endosomal and autophagic organelles or ubiquitinated neuronal inclusions in both models. We demonstrate the neuroprotective activity of UDCA downstream of endosomal-lysosomal and autophagic dysfunction, delineating the molecular mode of action of UDCA and highlighting its potential as a therapeutic for the treatment of FTD-ALS spectrum disorders.

Oxidative Stress Experienced during Early Development Influences the Offspring Phenotype.

AbstractOxidative stress (OS) experienced early in life can affect an individual's phenotype. However, its consequences for the next generation remain largely unexplored. We manipulated the OS level endured by zebra finches (Taeniopygia guttata) during their development by transitorily inhibiting the synthesis of the key antioxidant glutathione ("early-high-OS"). The offspring of these birds and control parents were cross fostered at hatching to enlarge or reduce its brood size. Independent of parents' early-life OS levels, the chicks raised in enlarged broods showed lower erythrocyte glutathione levels, revealing glutathione sensitivity to environmental conditions. Control biological mothers produced females, not males, that attained a higher body mass when raised in a benign environment (i.e., the reduced brood). In contrast, biological mothers exposed to early-life OS produced heavier males, not females, when allocated in reduced broods. Early-life OS also affected the parental rearing capacity because 12-day-old nestlings raised by a foster pair with both early-high-OS members grew shorter legs (tarsus) than chicks from other groups. The results indicate that environmental conditions during development can affect early glutathione levels, which may in turn influence the next generation through both pre- and postnatal parental effects. The results also demonstrate that early-life OS can constrain the offspring phenotype.

Endogenous Cys-Assisted GSH@AgNCs-rGO Nanoprobe for Real-Time Monitoring of Dynamic Change in GSH Levels Regulated by Natural Drug.

Glutathione (GSH) levels are closely related to the homeostasis of redox state which directly affects human disease occurrence by regulating cell apoptosis. Hence, real-time monitoring of dynamic changes in intracellular GSH levels is urgently needed for disease early diagnosis and evaluation of therapy efficiency. In this study, an endogenous cysteine (Cys)-assisted detection system based on GSH@AgNCs and reduced graphene oxide (rGO) with high sensitivity and specificity was developed for GSH detection. Compared with GSH, GSH@AgNCs with weaker affinity and bonding force was quite easier to extrude from the rGO surface when competing against GSH, leading to the obvious change in fluorescence signal. This phenomenon was termed as "a crowding out effect". Furthermore, the presence of Cys can improve GSH assay sensitivity by enhancing the quenching efficiency of rGO on the GSH@AgNCs. In vitro assay indicated that the efficiency of fluorescence recovery was positively related with GSH concentration in the range from 0 to 10 mM. In addition, the method was employed for real-time monitoring of the dynamic changes in GSH levels regulated by natural drugs. The imaging results showed that the natural compound 3 (C3) can downregulate GSH levels in HepG2 cells, which was accompanied by reactive oxygen species (ROS) release and apoptosis induction. Finally, the method was used to monitor the change of GSH levels in serum samples with chronic hepatitis B (CHB) infection. The results demonstrated that the occurrence and development of CHB may be positively correlated with GSH levels to some extent. Overall, the above results demonstrate the potential application of this new nanosystem in anticancer natural drug screening and clinical assay regarding GSH levels.

Chronic exposure of adult male Wistar rats to bisphenol A causes testicular oxidative stress: Role of gallic acid.

OBJECTIVES: Bisphenol A (BPA) has been reported that among other male reproductive dys-functions, it can cause marked estrogenic effects including alteration in serum hormones as well as testicular lesions in exposed animals. This work sought to study the role of gallic acid (GA), a known antioxidant, on the BPA-induced testicular oxidative stress in adult male Wistar rats using serum hormone analysis, histopathology, and biochemical assays. METHODS: Adult male rats were divided into four groups (n=10) including control (0.2 ml of corn oil), GA (20 mg/kg/day), BPA (10 mg/kg/day), BPA+GA (BPA, 10 mg/kg/day + GA, 20 mg/kg/day). All medications were given by oral gavage for 45 consecutive days. The body and testicular weights were measured. Blood and organ samples were collected for the serum hormonal assay: testosterone (T), luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL), and tissue biochemistry analysis: superoxide dismutase (SOD), reduced glutathione (GSH), glutathione-S-transferase (GST), malondialdehyde (MDA), hydrogen peroxide (H2O2), respectively. RESULTS: The BPA-treated rats showed significant reduction in the gonadosomatic index. BPA also caused significant decrease in the levels of the serum testosterone and prolactin. Furthermore, BPA induced testicular oxidative stress by decreasing the activities of antioxidant enzymes and increasing reactive oxygen species. However, co-treatment with GA protected against these alterations. CONCLUSION: Findings from the present study confirmed the previously reported data and show that the ability of GA, as a potent antioxidant, may protect against BPA-induced alterations in the male reproductive function. Hence, GA protects against testicular oxidative stress in adult male Wistar rats following chronic exposure to BPA.

Gonadoprotective ability of Vincetoxicum arnottianum extract against bisphenol A-induced testicular toxicity and hormonal imbalance in male Sprague Dawley rats.

Vincetoxicum arnottianum (Wight) of family Apocynaceae is a rich source of therapeutic alkaloids, phenolics and flavonoids. Study aims to evaluate the protective potential of methanol extract of Vincetoxicum arnottianum (VAM) on bisphenol A (BPA)-induced testicular toxicity in male Sprague Dawley rat. Quantitative analysis of VAM for total phenolic (TPC), total flavonoid (TFC) and total alkaloid content (TAC) along with HPLC analysis for polyphenolics was carried out. BPA-induced testicular toxicity was determined through analysis of antioxidant enzymes, DNA damages and testicular histopathology along with reproductive hormones in serum of rat. VAM was constituted of TFC (382.50 ± 1.67 µg GAE/mg), TPC (291.17 ± 0.82 µg RE/mg), TAC (16.5 ± 0.5%), ferulic acid (2.2433 µg/mg) and vanillic acid (2.1249 µg/mg). VAM co-administration to BPA-treated rats attenuated the toxic effects of BPA and restored the body and testis weights. Altered level of luteinizing hormone (LH), testosterone and follicle-stimulating hormone (FSH) in serum, and level of antioxidants (GSH, POD, CAT and SOD) and nitric oxide in testis tissues of BPA-induced toxicity were significantly restored by VAM. Histological and comet assay studies also sanctioned the protective potential of VAM in BPA-intoxicated rats. The presence of polyphenols and alkaloids might contribute towards the scavenging and ameliorative potential of VAM in testicular toxicity induced by BPA.

Effects of curcumin on sperm quality, lipid profile, antioxidant activity and histopathological changes in streptozotocin-induced diabetes in rats.

In this study, the effect of low-dose curcumin on sperm parameters, reproductive hormones, lipid profile, biochemical antioxidant parameters and the histopathological structure of the testis in diabetic male rats were evaluated. In the study, 28 male Wistar albino rats weighing 300-370 g and aged 8-10 weeks were used. Four groups of equal numbers have been created. Diabetes mellitus was induced with 45 mg/kg streptozotocin (STZ) in seven rats. Curcumin was administered to the rats in curcumin and the diabetes + curcumin group by gavage for 15 days at a dose of 10 mg/kg. Then, the rats were sacrificed. Blood samples and testis tissues were obtained, while the rats were under anaesthesia. Glucose, lipid profile, reproductive hormones, sperm parameters, biochemical antioxidant parameters and histopathological examination of the testis were performed. Abnormal sperm ratio, malondialdehyde, glucose, cholesterol, low-density lipoprotein, and triglyceride levels and caspase-3 expression were increased in diabetic rats, while the sperm motility and intensity and reduced glutathione, catalase and testosterone levels were decreased. When low-dose curcumin (10 mg/kg) was administered to diabetic rats, we found that curcumin significantly increased sperm motility and density, and decreased abnormal sperm rate according to the diabetic group. Moreover, curcumin significantly suppressed the lipid profile and increased follicle-stimulating hormone (FSH) and testosterone levels compared to the diabetic group. On testicular damage and decreased reproductive hormones caused by diabetes, curcumin may have a protective effect with indirect effect of glycaemic control by curcumin.

Paeonol protects against testicular ischaemia-reperfusion injury in rats through inhibition of oxidative stress and inflammation.

Ischaemia-reperfusion (IR) is the most common form of testicular injury that results in oxidative damage and inflammation ending by subinfertility. Paeonol, a natural phenolic compound, exhibits antioxidant and anti-inflammatory effects. Thus, the present study investigated the role of paeonol in rat testicular IR injury. Thirty adult Wistar rats were randomly divided into five groups; sham, sham treated with paeonol, IR injury, and IR pre-treated with paeonol at low and high doses. Serum testosterone and testicular levels of malondialdehyde and reduced glutathione (GSH) besides superoxide dismutase (SOD) activity were determined. Gene quantifications for tumour necrosis factor-α (TNF-α), hypoxia-inducible factor-1α (HIF-1α) and heat shock protein 70 (HSP70) were also assessed. Histopathological pictures and the immunohistochemical expression of testicular nuclear factor erythroid 2-related factor 2 (Nrf2), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) were shown. Pre-treatment with paeonol prevented the drop in serum testosterone, alongside with improvement of testicular malondialdehyde and GSH levels plus SOD activity. Paeonol regained the normal spermatogenesis with prevention of IR-induced increase in TNF-α, HIF-1α and HSP70 gene expression besides IL-1ß and IL-6 immunostaining and reduction in Nrf2 protein expression. Paeonol exerted a dose-dependent beneficial effect on testicular IR injury. This effect was achieved by its antioxidant and anti-inflammatory effects.

Celastrol Alleviates Gamma Irradiation-Induced Damage by Modulating Diverse Inflammatory Mediators.

The present study aimed to explore the possible radioprotective effects of celastrol and relevant molecular mechanisms in an in vitro cell and in vivo mouse models exposed to gamma radiation. Human keratinocytes (HaCaT) and foreskin fibroblast (BJ) cells were exposed to gamma radiation of 20Gy, followed by treatment with celastrol for 24 h. Cell viability, reactive oxygen species (ROS), nitric oxide (NO) and glutathione (GSH) production, lipid peroxidation, DNA damage, inflammatory cytokine levels, and NF-κB pathway activation were examined. The survival rate, levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in blood, and p65 and phospho-p65 expression were also evaluated in mice after exposure to gamma radiation and celastrol treatment. The gamma irradiation of HaCaT cells induced decreased cell viability, but treatment with celastrol significantly blocked this cytotoxicity. Gamma irradiation also increased free radical production (e.g., ROS and NO), decreased the level of GSH, and enhanced oxidative DNA damage and lipid peroxidation in cells, which were effectively reversed by celastrol treatment. Moreover, inflammatory responses induced by gamma irradiation, as demonstrated by increased levels of IL-6, TNF-α, and IL-1ß, were also blocked by celastrol. The increased activity of NF-κB DNA binding following gamma radiation was significantly attenuated after celastrol treatment. In the irradiated mice, treatment with celastrol significantly improved overall survival rate, reduced the excessive inflammatory responses, and decreased NF-κB activity. As a NF-κB pathway blocker and antioxidant, celastrol may represent a promising pharmacological agent with protective effects against gamma irradiation-induced injury.

Cardioprotective Effect of Tangeretin by Inhibiting PTEN/AKT/mTOR Axis in Experimental Sepsis-Induced Myocardial Dysfunction.

Sepsis aggregates undesirable immune response causing depression of ventricular myocardium and diastolic dysfunction. This present study examined the effect of a plant-derived flavone tangeretin (TG) on autophagy and reduction in myocardial dysfunction. The sepsis was induced by cecum ligation and puncture (CLP) in male Sprague-Dawley rats. Abnormal changes were seen in the heart after the sepsis induction. These abnormalities were analyzed based on the cardiac markers, namely Cardiac myosin light chain-1 (cMLC1) and Cardiac troponin I (cTnl), echocardiography, and plasma parameters, like Lactate dehydrogenase (LDH) and Creatinine kinase (CK). Microanatomy of the heart was studied using hematoxylin and eosin stained histopathological samples of cardiac tissue. Western blot technique was used to detect the nature and extent of protein with the amount of a specific RNA (gene expression) in the cardiac homogenate. Oxidative damage was analyzed using redox marker, reduced glutathione. This study successfully showed that TG attenuated sepsis-induced myocardial dysfunction by inhibiting myocardial autophagy via silencing the Phosphatase and tensin homolog (PTEN) expression and acting on the AKT/mTOR pathway. The present findings supported that TG is a novel cardioprotective therapeutic target for sepsis induced myocardial dysfunction.

Multitissue analysis of exercise and metformin on doxorubicin-induced iron dysregulation.

Doxorubicin (DOX) is an effective chemotherapeutic treatment with lasting side effects in heart and skeletal muscle. DOX is known to bind with iron, contributing to oxidative damage resulting in cardiac and skeletal muscle toxicity. However, major cellular changes to iron regulation in response to DOX are poorly understood in liver, heart, and skeletal muscle. Additionally, two cotreatments, exercise (EX) and metformin (MET), were studied for their effectiveness in reducing DOX toxicity by ameliorating iron dysregulation and preventing oxidative stress. The purposes of this study were to 1) characterize the DOX-induced changes of the major iron regulation pathway in liver, heart, and skeletal muscle and 2) to determine whether EX and MET exert their benefits by minimizing DOX-induced iron dysregulation. Mice were assigned to receive saline or DOX (15 mg/kg) treatments, paired with either EX (5 days) or MET (500 mg/kg), and were euthanized 3 days after DOX treatment. Results suggest that the cellular response to DOX is protective against oxidative stress by reducing iron availability. DOX increased iron storage capacity through elevated ferritin levels in liver, heart, and skeletal muscle. DOX reduced iron transport capacity through reduced transferrin receptor levels in heart and skeletal muscle. EX and MET cotreatments had protective effects in the liver through reduced transferrin receptor levels. At 3 days after DOX, oxidative stress was mild, as shown by normal glutathione and lipid peroxidation levels. Together these results suggest that the cellular response to reduce iron availability in response to DOX treatment is sufficient to match oxidative stress.

Exposure of Cultured Astrocytes to Menadione Triggers Rapid Radical Formation, Glutathione Oxidation and Mrp1-Mediated Export of Glutathione Disulfide.

Menadione (2-methyl-1,4-naphthoquinone) is a synthetic derivative of vitamin K that allows rapid redox cycling in cells and thereby generates reactive oxygen species (ROS). To test for the consequences of a treatment of brain astrocytes with menadione, we incubated primary astrocyte cultures with this compound. Incubation with menadione in concentrations of up to 30 µM did not affect cell viability. In contrast, exposure of astrocytes to 100 µM menadione caused a time-dependent impairment of cellular metabolism and cell functions as demonstrated by impaired glycolytic lactate production and strong increases in the activity of extracellular lactate dehydrogenase and in the number of propidium iodide-positive cells within 4 h of incubation. In addition, already 5 min after exposure of astrocytes to menadione a concentration-dependent increase in the number of ROS-positive cells as well as a concentration-dependent and transient accumulation of cellular glutathione disulfide (GSSG) were observed. The rapid intracellular GSSG accumulation was followed by an export of GSSG that was prevented in the presence of MK571, an inhibitor of the multidrug resistance protein 1 (Mrp1). Menadione-induced glutathione (GSH) oxidation and ROS formation were found accelerated after glucose-deprivation, while the presence of dicoumarol, an inhibitor of the menadione-reducing enzyme NQO1, did not affect the menadione-dependent GSSG accumulation. Our study demonstrates that menadione rapidly depletes cultured astrocytes of GSH via ROS-induced oxidation to GSSG that is subsequently exported via Mrp1.

Identification of DNA and glutathione adducts in male Sprague-Dawley rats exposed to 1-bromopropane.

Occupational exposure of workers to 1-bromopropane (1-BP) has raised concerns in industry for many years. Despite the known toxicity of this chemical, molecular events attributed to exposure to 1-BP have not been extensively studied. The aim of the present study was to examine the effects of 1-BP exposure on adduct formation with DNA and glutathione (GSH) in male Sprague-Dawley rats in an attempt to determine the early stages of toxicity. Following 6 h after either single or daily exposure to 1-BP for 3 days, N7-propyl guanine and S-propyl GSH were quantified in several organs by using liquid chromatography-mass spectrometry (LC-MS/MS). The results showed that N7-propyl guanine was maximally formed in liver followed by spleen, testes, and lung in both dose- and time-dependent manners. However, DNA adduct was not detected in cardiac tissue. In the case of S-propyl GSH, this compound was formed in the following order in various organs: liver > testes > spleen > kidney > lung > heart. In a subsequent in vitro study, formation of N7-propyl guanine initiated by 1-BP in calf thymus DNA was not markedly affected by addition of liver homogenates, which indicated that this chemical may be acting as a direct alkylating agent. In contrast, an in vitro study with free GSH demonstrated that 1-BP reduced GSH and elevated production of S-propyl GSH, and that the production of this adduct was significantly higher in the presence of active liver homogenates. Data indicated that formation of GSH adducts initiated by 1-BP might be associated with an enzyme-driven process. Although further characterization is necessary, it would appear that N7-propyl guanine and S-propyl GSH might serve as useful markers in cases of exposure assessment of 1-BP.