Effect of physical restraint on oxidative stress in mice fed a selenium and vitamin E deficient diet

Physical restraint has been associated with increased oxidative damage to lipid, protein, and DNA. The purpose of this experiment was to determine whether physical restraint would further exacerbate oxidative stress in mice fed a selenium (Se) and vitamin E (VE) deficient diet. Three-week-old mice were fed a Torula yeast diet containing adequate or deficient Se and VE. Menhaden oil was added to the deficient diet to impose an additional oxidative stress. After 4 wk feeding, half the mice in each group were restrained for 5 d in well-ventilated conical tubes for 8 h daily. Mice fed the Se and VE deficient diets had increased liver thiobarbituric acid-reactive substance (TBARS) levels and decreased liver glutathione peroxidase (GPX1) activity and α-tocopherol levels. Plasma corticosterone levels were elevated in restrained mice fed the deficient diet compared to unrestrained mice fed the adequate diet. Restraint had no effect on liver TBARS or α-tocopherol levels. Liver GPX1 activity, however, was lower in restrained mice fed the adequate diet. In addition, liver superoxide dismutase (SOD) activity was lower in the restrained mice fed the adequate or deficient diet. Thus, under our conditions, Se and VE deficient diet, but not restraint, increased lipid peroxidation in mice. Restraint, however, decreased antioxidant protection in mice due to decreased activities of GPX1 and SOD enzymes.     

Using MT(-/-) mice to study metallothionein and oxidative stress

Mice with null mutations for metallothionein genes MT-1 and MT-2 were used to study the role that metallothionein plays in protecting cellular targets in vivo from oxidative stress. Wild-type (MT(+/+)) and MT-null (MT(-/-)) mice were treated with either saline or zinc and exposed to two types of oxidative stress: gamma-irradiation or 2-nitropropane. There was no alteration in the antioxidant defense system (superoxide dismutase, catalase, or glutathione peroxidase and glutathione levels) to compensate for the lack of the metallothionein in the MT(-/-) mice. The amount of oxidative damage to liver DNA, lipids, and proteins were similar for the MT(-/-) and MT(+/+) mice even though the levels of metallothionein in the livers of the saline- or zinc-pretreated MT(+/+) mice were 5- to 100-fold greater than found in the MT(-/-) mice. To determine if metallothionein can protect mice from the lethal effects of ionizing radiation, the mean survivals of MT(-/-) and MT(+/+) mice exposed to whole body gamma-irradiation were measured and found to be similar. However, the mean survival increased significantly after zinc pretreatment for both the MT(-/-) and MT(+/+) mice. These results demonstrate that tissue levels of metallothionein do not protect mice in vivo against oxidative stress.     

Mitochondrial DNA Damage and Dysfunction,and Oxidative Stress Are Associated with Endoplasmic Reticulum Stress,Protein Degradation and Apoptosis in High Fat Diet-Induced Insulin Resistance Mice

Background

Recent studies showed a link between a high fat diet (HFD)-induced obesity and lipid accumulation in non-adipose tissues, such as skeletal muscle and liver, and insulin resistance (IR). Although the mechanisms responsible for IR in those tissues are different, oxidative stress and mitochondrial dysfunction have been implicated in the disease process. We tested the hypothesis that HFD induced mitochondrial DNA (mtDNA) damage and that this damage is associated with mitochondrial dysfunction, oxidative stress, and induction of markers of endoplasmic reticulum (ER) stress, protein degradation and apoptosis in skeletal muscle and liver in a mouse model of obesity-induced IR.

Methodology/Principal Findings

C57BL/6J male mice were fed either a HFD (60% fat) or normal chow (NC) (10% fat) for 16 weeks. We found that HFD-induced IR correlated with increased mtDNA damage, mitochondrial dysfunction and markers of oxidative stress in skeletal muscle and liver. Also, a HFD causes a change in the expression level of DNA repair enzymes in both nuclei and mitochondria in skeletal muscle and liver. Furthermore, a HFD leads to activation of ER stress, protein degradation and apoptosis in skeletal muscle and liver, and significantly reduced the content of two major proteins involved in insulin signaling, Akt and IRS-1 in skeletal muscle, and Akt in liver. Basal p-Akt level was not significantly influenced by HFD feeding in skeletal muscle and liver.

Conclusions/Significance

This study provides new evidence that HFD-induced mtDNA damage correlates with mitochondrial dysfunction and increased oxidative stress in skeletal muscle and liver, which is associated with the induction of markers of ER stress, protein degradation and apoptosis.     

Activation of Kupffer Cells Is Associated with a Specific Dysbiosis Induced by Fructose or High Fat Diet in Mice

The increase consumption of fructose in diet is associated with liver inflammation. As a specific fructan substrate, fructose may modify the gut microbiota which is involved in obesity-induced liver disease. Here, we aimed to assess whether fructose-induced liver damage was associated with a specific dysbiosis, especially in mice fed a high fat diet (HFD). To this end, four groups of mice were fed with normal and HFD added or not with fructose. Body weight and glucose sensitivity, liver inflammation, dysbiosis and the phenotype of Kupffer cells were determined after 16 weeks of diet. Food intake was increased in the two groups of mice fed with the HFD. Mice fed with HFD and fructose showed a higher infiltration of lymphocytes into the liver and a lower inflammatory profile of Kupffer cells than mice fed with the HFD without fructose. The dysbiosis associated with diets showed that fructose specifically prevented the decrease of Mouse intestinal bacteria in HFD fed mice and increased Erysipelotrichi in mice fed with fructose, independently of the amount of fat. In conclusion, fructose, used as a sweetener, induced a dysbiosis which is different in presence of fat in the diet. Consequently, the activation of Kupffer cells involved in mice model of HFD-induced liver inflammation was not observed in an HFD/fructose combined diet. These data highlight that the complexity of diet composition could highly impact the development of liver lesions during obesity. Specific dysbiosis associated with the diet could explain that the progressions of liver damage are different.     

Pulmonary toxicity caused by acute exposure to mercury vapor is enhanced in metallothionein-null mice.

This study examined the protective role of metallothionein (MT) against pulmonary damage caused by acute exposure to metallic mercury (Hg0) vapor using MT-null and wild-type mice. Both strains of mice were exposed to Hg0 at 6.6 to 7.5 mg/m3 for 4 hr each day for 3 consecutive days. This dosing protocol was lethal to over 60% of MT-null mice but did not kill any wild-type mice. More severe pulmonary damage was found by histopathological observation in MT-null mice than in wild-type mice. MT levels in the lung were elevated in wild-type mice after Hg0 vapor exposure, and gel filtration of the lung cytosol revealed that most of the mercury was associated with MT. In MT-null mice, MT levels were below the limit of detection (0.2 microg/g tissue) for the MT assay even after exposure. After exposure to Hg0 vapor for 3 consecutive days, the pulmonary mercury levels in wild-type mice were significantly higher than in MT-null mice. These findings suggest that MT plays a protective role against the acute pulmonary toxicity of Hg0 vapor.     

Changes in the histostructure of functional activity of the immunocompetent organs in damaged portal blood supply of the liver

In CBA mice calibrated stenosis of the portal vein was produced. Liver and immunocompetent organs were morphologically analyzed. The total number of hemopoietic stem cells in the bone marrow was estimated by the colony-forming cells and in the spleen after immunization with sheep red cells by the plaque forming method. It is established that stenosis of the portal vein (on the average by 45% and 58%) produced the histostructural changes in the liver and in the immunocompetent organs. Expression of morphological changes depended on the time elapsed after operation and the degree of the portal vein stenosis. These changes were the most pronounced on the 16-17th day when stenosis of the portal vein was 58%. The character of the changes in the number of the hemopoietic stem cells in the bone marrow and in that of antibody-forming cells in the spleen depended on the degree of the liver damage. These changes increased with the degree of the liver histostructure damage. The maximal liver damage was accompanied by a decrease of these indices.     

DNA damage in tissues and organs of mice treated with diphenyl diselenide

Diphenyl diselenide (DPDS) is an organoselenium compound with interesting pharmacological activities and various toxic effects. In previous reports, we demonstrated the pro-oxidant action and the mutagenic properties of this molecule in bacteria, yeast and cultured mammalian cells. This study investigated the genotoxic effects of DPDS in multiple organs (brain, kidney, liver, spleen, testes and urinary bladder) and tissues (bone marrow, lymphocytes) of mice using in vivo comet assay, in order to determine the threshold of dose at which it has beneficial or toxic effects. We assessed the mechanism underlying the genotoxicity through the measurement of GSH content and thiobarbituric acid reactive species, two oxidative stress biomarkers. Male CF-1 mice were given 0.2-200 micromol/kg BW DPDS intraperitonially. DPDS induced DNA damage in brain, liver, kidney and testes in a dose response manner, in a broad dose range at 75-200 micromol/kg with the brain showing the highest level of damage. Overall, our analysis demonstrated a high correlation among decreased levels of GSH content and an increase in lipid peroxidation and DNA damage. This finding establishes an interrelationship between pro-oxidant and genotoxic effects. In addition, DPDS was not genotoxic and did not increase lipid peroxidation levels in any organs at doses < 50 micromol/kg. Finally, pre-treatment with N-acetyl-cysteine completely prevented DPDS-induced oxidative damage by the maintenance of cellular GSH levels, reinforcing the positive relationship of DPDS-induced GSH depletion and DNA damage. In summary, DPDS induces systemic genotoxicity in mammals as it causes DNA damage in vital organs like brain, liver, kidney and testes.     

Cypermethrin-induced DNA damage in organs and tissues of the mouse: evidence from the comet assay

Cypermethrin is the most widely used Type II pyrethroid pesticide because of its high effectiveness against target species and its low mammalian toxicity reported so far. It is a fast-acting neurotoxin and is known to cause free radical-mediated tissue damage. The present study investigates the genotoxic effects of cypermethrin in multiple organs (brain, kidney, liver, spleen) and tissues (bone marrow, lymphocytes) of the mouse, using the alkaline comet assay. Male Swiss albino mice were given 12.5, 25, 50, 100, 200 mg/kg BW of cypermethrin intraperitoneally, daily for 5 consecutive days. A statistically significant (p<0.05) dose-dependent increase in DNA damage was observed in all the organs assessed, as evident from the comet-assay parameters, viz., Olive tail moment (OTM; arbitrary unit), tail DNA (%) and tail length (microm). Brain showed maximum DNA damage followed by spleen>kidney>bone marrow>liver>lymphocytes, as evident by the OTM. Our data demonstrate that cypermethrin induces systemic genotoxicity in mammals as it causes DNA damage in vital organs like brain, liver, kidney, apart from that in the hematopoietic system.     

Intervention of astaxanthin against cyclophosphamide-induced oxidative stress and DNA damage: A study in mice

Astaxanthin, a natural and nutritional red carotenoid pigment, is used as a dietary supplement. The intention of the present study was to investigate the beneficial effects of dietary pigment astaxanthin, against cyclophosphamide-induced oxidative stress and DNA damage. The end points of evaluation of the study included: (a) malondialdehyde, glutathione and superoxide dismutase concentration in liver to detect oxidative stress; (b) normal and modified alkaline comet assays (the latter includes lesion-specific enzymes formamidopyrimidine-DNA glycosylase and endonuclease-III) to detect normal and oxidative stress-induced DNA damage by cyclophosphamide in the mouse bone marrow and the peripheral blood lymphocytes. In addition, micronucleus assay and chromosomal aberration test capable of detecting the DNA damage were also carried out in peripheral blood and bone marrow of mice. Cyclophosphamide (100 mg/kg intra-peritoneal) treatment led to significant increase in liver malondialdehyde and decreased the antioxidant enzymes glutathione and superoxide dismutase. Further, cyclophosphamide also significantly increased the DNA damage as observed from normal and modified comet assays as well as micronucleus and chromosomal aberration assay. Pre-treatment with astaxanthin (12.5, 25 and 50 mg/kg/day for 5 days per oral) resulted in the restoration of oxidative stress markers such as malondialdehyde, glutathione and superoxide dismutase in liver. The amelioration of oxidative stress with astaxanthin pre-treatment correlated well with the decreased DNA damage as evident from normal and modified alkaline comet assays of bone marrow cells and peripheral blood lymphocytes. Further astaxanthin pre-treatment also reduced the frequency of chromosomal breakage and micronucleus formation in the mouse bone marrow cells and peripheral blood reticulocytes. It is thus concluded that pre-treatment with astaxanthin attenuates cyclophosphamide-induced oxidative stress and subsequent DNA damage in mice and it can be used as a chemoprotective agent against the toxicity of anticancer drug cyclophosphamide.     

Postnatal essential fatty acid deficiency in mice affects lipoproteins, hepatic lipids, fatty acids and mRNA expression

We have previously reported that essential fatty acid deficiency (EFAD) during suckling in mice resulted in an adult lean phenotype and a resistance to diet-induced obesity. We now hypothesized that postnatal EFAD would cause long-term effects on lipid metabolism. C57BL/6 mice were fed an EFAD or a control diet from the 16th day of gestation and throughout lactation. The pups were weaned to standard diet (STD) and at 15 weeks of age given either high fat diet (HFD) or STD. Lipoprotein profiles, hepatic lipids, fatty acids and mRNA expression were analyzed in 3-week-old and 25-week-old offspring. At weaning, the EFAD pups had higher cholesterol levels in both plasma and liver and 6-fold higher concentrations of hepatic cholesterol esters than control pups. Adult EFAD offspring had higher levels of hepatic cholesterol and linoleic acid, but lower levels of dihomo-γ-linolenic acid and Pparg mRNA expression in the liver. In addition, HFD fed EFAD offspring had lower plasma total cholesterol, lower hepatic triglycerides and lower liver weight compared to controls fed HFD. In conclusion, early postnatal EFAD resulted in short-term alterations with increased hepatic cholesterol accumulation and long-term protection against diet-induced liver steatosis and hypercholesterolemia.     

Impaired hepatic regeneration in metallothionein-I/II knockout mice after partial hepatectomy

Although the translocation of metallothionein (MT) from cytoplasm to nucleus has been demonstrated in liver during times of high requirement for zinc (fetal development and the neonatal period), the role of MT in cellular growth is not well understood. In this study, a potential role of MT in liver regeneration was investigated in wild type (WT) and MT-I and MT-II gene knockout (MT-null) mice after 35% partial hepatectomy (PH) or sham laparotomy. Hepatic MT levels and proliferation index were measured at 0, 5, 15, 24, 36, 48, and 60 hrs after PH and 48 hrs after sham laparotomy (control). MT levels were increased in WT mice (peak at 24 hrs after PH) and declined to normal levels by 60 hrs after PH. Immunohistochemical staining for MT in WT mice indicated the presence of MT in both nucleus and cytoplasm of hepatocytes at 24 hrs after PH, whereas MT was present mainly in the cytoplasm at 36-60 hrs after PH and 48 hrs after sham laparotomy. Hepatic proliferation index in both WT and MT-null mice, as determined by argyrophilic nucleolar organizing region staining and proliferating cell nuclear antigen immunohistochemical staining, reached a peak at 48 hrs and declined by 60 hrs after PH. Cell proliferation was significantly less in MT-null mice as compared to WT mice during liver regeneration after PH. These results suggest that MT may play a positive role in hepatic regeneration after PH.     

In vivo genotoxicity of heterocyclic amines detected by a modified alkaline single cell gel electrophoresis assay in a multiple organ study in the mouse

We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of 6 heterocyclic amines, Trp-P-1 (25 mg/kg), Trp-P-2 (13 mg/kg), IQ (13 mg/kg), MeIQ (13 mg/kg), MeIQx (13 mg/kg) and PhIP (40 mg/kg), in mouse liver, lung, kidney, brain, spleen, bone marrow and stomach mucosa. Mice were sacrificed 1, 3, and 24 h after intraperitoneal injection. Trp-P-2, IQ, MeIQ, and MeIQx yielded statistically significant DNA damage in the stomach, liver, kidney, lung and brain; Trp-P-1 in the stomach, liver and lung; and PhIP in the liver, kidney and brain. None of the heterocyclic amines induced DNA damage in the spleen and bone marrow. Our results suggest that the alkaline SCG assay applied to multiple organs is a good way to detect organ-specific genotoxicity of heterocyclic amines in mammals.     

敲除Nrf2促进高脂饮食诱导小鼠肝脏NF-kB的活化

目的：氧化应激和炎症反应是NASH进展的关键因素,同时二者之间存在着密切关系,而转录因子Nrf2和NF-kB分别是氧化应激和炎症信号通路的关键调控靶点,因此,研究Nrf2对高脂饮食诱导小鼠肝脏NF-kB信号通路的影响,对探讨NASH进展具有重要的意义。方法：雄性野生型（WT）和Nrf2基因敲除（Nrf2-/-）ICR小鼠各10只,随机分为WT对照组（Control）、Nrf2-/-对照组（KO）、WT高脂饮食组（HFD）和Nrf2-/-高脂饮食组（KOHFD）（n=5）。喂养8周后,观察肝脏光镜下改变,检测肝脏GSH、MDA、TNFα和IL-6水平。Western-Blot检测肝脏NF-kB蛋白表达水平,观察敲除Nrf2对肝脏NF-kB活性作用的影响。结果：1.光镜下观察,Control组与KO组小鼠肝脏结构无明显变化,HFD组小鼠肝脏呈现大片脂肪沉积和炎症细胞浸润,KOHFD组小鼠肝脏则呈现明显的大泡性变性,且炎症细胞浸润较HFD组明显加重;2.与Control组相比,KO组小鼠肝脏MDA轻度升高,GSH轻度降低,但无明显差异,而HFD组和KOHFD组小鼠肝脏MDA显著升高（P〈0.05）,GSH显著降低（P〈0.05）,且KOHFD组MDA明显高于HFD组（P〈0.05）,GSH明显低于HFD组（P〈0.05）。3.ELISA结果显示,与Control组相比,KO组小鼠肝脏TNFα和IL-6分泌轻度增加,而HFD组和KOHFD组小鼠肝脏TNFα与IL-6水平显著升高（P〈0.05）,且KOHFD组小鼠肝脏TNFα与IL-6显著高于HFD组（P〈0.05）;4.Western-Blot结果显示,Control组和KO组之间无明显差异,而KOHFD组和HFD组小鼠肝脏胞核NF-kB蛋白表达水平显著升高,且KOHFD组高于HFD组。结论：敲除Nrf2可以显著加重高脂饮食诱导的小鼠肝脏氧化应激水平,进而促进NF-kB的活化,从而为通过以Nrf2为靶点治疗NASH提供重要的实验依据。     

A model of Ph' positive chronic myeloid leukemia-blast crisis cell line growth in immunodeficient SCID mice.

A human Philadelphia-chromosome positive chronic myeloid leukemia-blast crisis (CML-BC) cell line BV173 proliferated in the hematopoietic tissues, infiltrated various organs and caused the death of immunodeficient SCID mice. Leukemia spreading was assessed with diminished number of bone marrow cells and caused splenomegaly. The leukemic colonies grew from single cell suspension of bone marrow, spleen and peripheral blood. Bcr-abl m-RNA was detectable in bone marrow, spleen, peripheral blood, liver, lungs and brain. Dying mice demonstrated severely hypoplastic bone marrow, splenomegaly and massive metastases in the liver and kidneys. The survival time of animals was dependent on the number of inoculated leukemia cells.     

Long-Term Administration of High-Fat Diet Corrects Abnormal Bone Remodeling in the Tibiae of Interleukin-6-Deficient Mice

In this study, we aimed to evaluate the influence of diet-induced obesity on IL-6 deficiency-induced bone remodeling abnormality. Seven-week-old IL-6^-/- mice and their wild type (WT) littermates were fed a standard diet (SD) or high-fat diet (HFD) for 25 weeks. Lipid formation and bone metabolism in mice tibiae were investigated by histochemical analysis. Both IL-6^-/- and WT mice fed the HFD showed notable body weight gain, thickened cortical bones, and adipose accumulation in the bone marrow. Notably, the HFD normalized the bone phenotype of IL-6^-/- mice to that of their WT counterpart, as characterized by a decrease in bone mass and the presence of an obliquely arranged, plate-like morphology in the trabecular bone. Alkaline phosphatase and osteocalcin expressions were attenuated in both genotypes after HFD feeding, especially for the IL-6^-/- mice. Meanwhile, tartrate-resistant acid phosphatase staining was inhibited, osteoclast apoptosis rate down-regulated (revealed by TUNEL assay), and the proportion of cathepsin K (CK)-positive osteoclasts significantly increased in IL-6^-/- mice on a HFD as compared with IL-6^-/- mice on standard chow. Our results demonstrate that HFD-induced obesity reverses IL-6 deficiency-associated bone metabolic disorders by suppressing osteoblast activity, upregulating osteoclastic activity, and inhibiting osteoclast apoptosis.     

Ebselen attenuates cyclophosphamide-induced oxidative stress and DNA damage in mice

The role of selenium, a trace element in the human diet, has been extensively studied against cancer, immunity and infectious/inflammatory diseases. The purpose of the present study was to investigate the beneficial effect of ebselen, an organo-selenium compound, against cyclophosphamide-induced oxidative stress and DNA damage in mice. Malondialdehyde and total glutathione were estimated in the liver to detect the oxidative stress induced by cyclophosphamide. Standard and modified comet assays (the latter incorporated lesion-specific enzymes, formamidopyrimidine-DNA glycosylase and endonuclease-III) were used to detect the normal and oxidative stress-induced DNA damage by cyclophosphamide in the mouse bone marrow and the peripheral blood lymphocytes. In addition, a micronucleus assay capable of detecting DNA damage was also carried out in the mouse bone marrow and the peripheral blood reticulocytes induced by cyclophosphamide. The results confirm that pre-treatment with ebselen (2.5, 5 and 10 mg/kg) for 5 consecutive days decreased the oxidative stress induced by cyclophosphamide (100 mg/kg) based on the restoration in concentration of malondialdehyde and glutathione in the liver and decreased DNA damage and micronuclei count in the bone marrow and the peripheral blood. It is concluded that pre-treatment with ebselen attenuates cyclophosphamide-induced oxidative stress and the resultant DNA damage in mice.     

Administration of glucocorticoids markedly increases the numbers of granulocytes and extrathymic T cells in the bone marrow.

Glucocorticoids, steroid hormones, are widely used as an anti-inflammatory drug. However, clinicians have sometimes encountered adverse drug reactions such as ulcers and tissue damage. In this study, we investigated how such adverse reactions of glucocorticoids are evoked, using an experimental mice model. When hydrocortisone (0.5 or 1.0 mg/day/mouse) was administered daily for 2 weeks, severe leukocytopenia was induced in all immune system organs. However, granulocytes (Gr-1(+)Mac-1(+)) were increased in number in the bone marrow and peripheral blood. This seemed to be due to an elevated level of myelopoiesis in the bone marrow. As well as increasing in number, granulocytes were functionally activated as estimated by the Ca2+ influx and superoxide production. The proportion of primordial T cells (CD3(int)IL-2Rbeta+) in the thymus and the number of primordial T cells in the bone marrow also increased. Mice administered hydrocortisone became susceptible to stress. Thus, these mice showed gastric ulcers when they were exposed to restraint stress for 12 h. These results suggest that activated granulocytes and primordial T cells might provide a mechanism involved in steroid ulcers and tissue damage, possibly through the superoxide production of granulocytes and the autoreactivity of primordial T cells.     

Dantrolene prevents hepatic steatosis by reducing cytoplasmic Ca2+ level and ER stress

IntroductionOur previous studies demonstrated that dantrolene, a ryanodine receptor stabilizer, prevents endoplasmic reticulum (ER) stress in the heart. ER stress is a strong mediator of impaired lipid metabolism in the liver, thereby contributing to fatty liver disease. In this study, we investigated the effects of dantrolene on fatty liver disease in mice and ER stress in hepatocytes.Methods and resultsEight weeks old C57BL/6 mice were fed high-fat diet (HFD) for 8 weeks with or without the oral administration of dantrolene (100 mg/kg/day). The livers of mice without dantrolene (HFD group) showed severe fatty liver, whereas the livers of the mice treated with dantrolene (HFD + DAN group) only showed slightly fatty liver. To address the preventive effects of dantrolene, primary hepatocytes were cultured with palmitate in the presence or absence of dantrolene. Dantrolene reduced lipid load and prevents palmitate-induced increase in cytoplasmic Ca²⁺ and ER stress. Based on these findings, we propose that dantrolene is a potential new therapeutic agent against fatty liver disease.