Medium-chain triglycerides inhibit free radical formation and TNF-alpha production in rats given enteral ethanol

This study determined whether free radical formation by the liver, tumor necrosis factor (TNF)-alpha production by isolated Kupffer cells, and plasma endotoxin are affected by dietary saturated fat. Rats were fed enteral ethanol and corn oil (E-CO) or medium-chain triglycerides (E-MCT) and control rats received corn oil (C-CO) or medium-chain triglycerides (C-MCT) for 2 wk. E-CO rats developed moderate fatty infiltration and slight inflammation; however, E-MCT prevented liver injury. Serum aspartate aminotransferase levels, gut permeability, and plasma endotoxin doubled with E-CO but were blunted approximately 50% with E-MCT. In Kupffer cells from E-CO rats, intracellular calcium was elevated by lipopolysaccharide (LPS) in a dose-dependent manner. In cells from E-MCT rats, increases were blunted by approximately 40-50% at all concentrations of LPS. The LPS-induced increase in TNF-alpha production by Kupffer cells was dose dependent and was blunted by 40% by MCT. E-CO increased radical adducts and was reduced approximately 50% by MCT. MCT prevent early alcohol-induced liver injury, in part, by inhibition of free radical formation and TNF-alpha production by inhibition of endotoxin-mediated activation of Kupffer cells.     

Cocoa extract protects against early alcohol-induced liver injury in the rat

Oxidants have been shown to be involved in alcohol-induced liver injury. This study was designed to determine whether cocoa flavonoid extract, composed mostly of epicatechin and epicatechin oligomers, protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-14 g/kg per day) and cocoa extract (400 mg/kg per day) continuously for 4 weeks using an enteral feeding protocol. Mean body weight gains ( approximately 4 g/day) were not significantly different between treatment groups. Cocoa extract did not affect average daily urine ethanol concentrations ( approximately 200mg/dL). After 4 weeks, serum alanine amino transferase levels of the ethanol group were increased nearly fourfold (110+/-16 IU/L) compared to control values (35+/-3 IU/L); this effect of ethanol was blocked by cocoa extract (60+/-6 IU/L). Additionally, enteral ethanol caused severe fat accumulation, mild inflammation, and necrosis in the liver; cocoa extract significantly blunted these changes. Increases in liver TNFalpha protein levels caused by ethanol were completely blocked by cocoa extract. Further, ethanol significantly increased the accumulation of protein adducts of 4-hydroxynonenal, a product of lipid peroxidation serving as an index of oxidative stress; again this was counteracted by the addition of cocoa extract. These results indicate that dietary flavanols such as those found in cocoa can prevent early alcohol-induced liver injury.     

Green tea extract protects against early alcohol-induced liver injury in rats

Oxidants have been shown to be involved in alcohol-induced liver injury. This study was designed to test the hypothesis that the antioxidant polyphenolic extract of green tea, comprised predominantly of epigallocatechin gallate, protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-14 g kg(-1) day(-1)) and green tea (300 mg kg(-1) day(-1)) continuously for 4 weeks using an intragastric enteral feeding protocol. Mean body weight gains (approximately 4 g/day) were not significantly different between treatment groups, and green tea extract did not the affect average concentration or the cycling of urine ethanol concentrations (0-550 mg dl(-1) day(-1)). After 4 weeks, serum ALT levels were increased significantly about 4-fold over control values (35+/-3 IU/l) by enteral ethanol (114+/-18); inclusion of green tea extract in the diet significantly blunted this increase (65+/-10). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver. While not affecting fat accumulation or inflammation, green tea extract significantly blunted increases in necrosis caused by ethanol. Furthermore, ethanol significantly increased the accumulation of protein adducts of 4-hydroxynonenal, a product of lipid peroxidation and an index of oxidative stress; green tea extract blocked this effect almost completely. TNFalpha protein levels were increased in liver by alcohol; this phenomenon was also blunted by green tea extract. These results indicate that simple dietary antioxidants, such as those found in green tea, prevent early alcohol-induced liver injury, most likely by preventing oxidative stress.     

Ebselen prevents early alcohol-induced liver injury in rats

Oxidants have been shown to be involved in alcohol-induced liver injury. Moreover, 2-phenyl-1,2-benzisoselenazole-3(2H)-one (ebselen), an organoselenium compound and glutathione peroxidase mimic, decreases oxidative stress and protects against stroke clinically. This study was designed to test the hypothesis that ebselen protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-16 g/kg/d) continuously for up to 4 weeks using the intragastric enteral feeding protocol developed by Tsukamoto and French. Ebselen (50 mg/kg twice daily, intragastrically) or vehicle (1% tylose) was administered throughout the experiment. Mean urine ethanol concentrations were not significantly different between treatment groups, and ebselen did not affect body weight gains or cyclic patterns of ethanol concentrations in urine. After 4 weeks, serum ALT levels were increased significantly about 4-fold over control values (37 +/- 5 IU/l) by enteral ethanol (112 +/- 7 IU/l); ebselen blunted this increase significantly (61 +/- 8 IU/l). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver (pathology score: 4.3 +/- 0.3). In contrast, these pathological changes were blunted significantly by ebselen (pathology score: 2.5 +/- 0.4). While there were no significant effects of either ethanol or ebselen on glutathione peroxidase activity in serum or liver tissue, ebselen blocked the increase in serum nitrate/nitrite caused by ethanol. Furthermore, ethanol increased the activity of NF-kappaB over 5-fold, the number of infiltrating neutrophils 4-fold, and the accumulation of 4-hydroxynonenal over 5-fold. Ebselen blunted all of these effects significantly. These results indicate that ebselen prevents early alcohol-induced liver injury, most likely by preventing oxidative stress, which decreases inflammation.     

Role of nitric oxide and nuclear factor-κB in the CYP2E1 potentiation of tumor necrosis factor α hepatotoxicity in mice

Induction of CYP2E1 by pyrazole (PY) potentiated the hepatotoxicity induced by TNFα in mice. We evaluated the role of nitrosative and oxidative stress and the NF-κB activation pathway in this liver injury. The iNOS inhibitor N-(3-aminomethyl)benzylacetamindine (1400W) or the antioxidant N-acetyl-l-cysteine (NAC) prevented this liver injury. TNFα plus PY treatment triggered radical stress in the liver with increased lipid peroxidation and decreased glutathione and caused mitochondrial damage as reflected by elevated membrane swelling and cytochrome c release. The radical stress and mitochondrial damage were prevented by 1400W and NAC. TNFα plus PY treatment elevated 3-nitrotyrosine adduct formation and induced NOS2 in the liver; 1400W and NAC blocked these changes. A lower extent of liver injury and oxidative stress was found in NOS2^?/? mice treated with TNFα plus PY compared with wild-type controls. Neither 1400W nor NAC modified CYP2E1 activity or protein. Activation of JNK and p38MAPK was weaker in TNFα plus PY-treated NOS2^?/? mice and 1400W and NAC blocked the activation of JNK and p38MAPK in wild-type mice. IKKα/β protein levels were decreased by TNFα plus PY treatment, whereas IκBα and IκBβ protein levels were elevated compared with saline, PY, or TNFα alone. NF-κB DNA binding activity was increased by TNFα alone but lowered by TNFα plus PY. All these changes were blocked by 1400W and NAC. NF-κB activation products such as Bcl-2, Bcl-X_L, cFLIP_S, cFLIP_L, and Mn-SOD were reduced by TNFα plus PY and restored by 1400W or NAC. We conclude that TNFα plus CYP2E1 induces oxidative/nitrosative stress, which plays a role in the activation of JNK or p38MAPK and mitochondrial damage. These effects combine with the blunting of the NF-κB activation pathways and the synthesis of protective factors to cause liver injury.     

Protective effect of glycine supplementation on the levels of lipid peroxidation and antioxidant enzymes in the erythrocyte of rats with alcohol-induced liver injury

We studied the effect of glycine supplementation on lipid peroxidation and antioxidants in the erythrocyte membrane, plasma and hepatocytes of rats with alcohol-induced hepatotoxicity. Administering ethanol (20%) for 60 days to male Wistar rats resulted in significantly elevated levels of erythrocyte membrane, plasma and hepatocyte thiobarbituric acid reactive substances (TBARS) as compared with those of the experimental control rats. Decreased activities of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), glutathione peroxidase (GPx) and glutathione reductase (GR) were also observed on alcohol supplementation as compared with those of the experimental control rats. Glycine was administered at a dose of 0.6 g kg(-1) body weight to rats with alcohol-induced liver injury, which significantly decreased the levels of TBARS and significantly elevated the activities of SOD, CAT, GSH, GPx and GR in the erythrocyte membrane, plasma and hepatocytes as compared to that of untreated alcohol supplemented rats. Thus, our data indicate that supplementation with glycine offers protection against free radical-mediated oxidative stress in the erythrocyte membrane, plasma and hepatocytes of animals with alcohol-induced liver injury.     

Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells

The current study examines the contribution of mitochondria-derived reactive oxygen species (ROS) in tert-butyl-hydroperoxide (TBH)-induced apoptotic signaling using clones of undifferentiated pheochromocytoma (PC-12) cells that stably overexpress the human mitochondrial or cytoplasmic forms of superoxide dismutase (SOD) (viz. Mn-SOD or CuZn-SOD, respectively). Exposure of wild type cells to TBH caused an early generation of ROS (30 min) that resulted in cell apoptosis at 24 h. These responses were attenuated with N-acetylcysteine pretreatment; however, N-acetylcysteine was ineffective in cytoprotection when added after TBH-induced ROS formation. Stable overexpression of SOD isoforms caused a 2- and 3.5-fold elevation in CuZn-SOD and Mn-SOD activities in the cytoplasm and mitochondria, respectively, and 3-fold increases in cellular GSH content. Accordingly, the stable overexpression of Mn-SOD attenuated TBH-induced mitochondrial ROS generation and cell apoptosis. Whereas transient Mn-SOD expression similarly prevented PC-12 apoptosis, this was associated with increases in SOD activity but not GSH, indicating that cytoprotection by Mn-SOD overexpression is related to mitochondrial ROS elimination and not due to increases in cellular GSH content per se. Stable or transient CuZn-SOD overexpression exacerbated cell apoptosis in conjunction with accelerated caspase-3 activation, regardless of cell GSH levels. Collectively, our results support a role for mitochondrial ROS in TBH-induced PC-12 apoptosis that is attenuated by Mn-SOD overexpression and is independent of cellular GSH levels per se.     

Replication properties of human adenovirus in vivo and in cultures of primary cells from different animal species

Oncolytic adenoviruses have emerged as a promising approach for the treatment of tumors resistant to other treatment modalities. However, preclinical safety studies are hampered by the lack of a permissive nonhuman host. Screening of a panel of primary cell cultures from seven different animal species revealed that porcine cells support productive replication of human adenovirus type 5 (Ad5) nearly as efficiently as human A549 cells, while release of infectious virus by cells from other animal species tested was diminished by several orders of magnitude. Restriction of productive Ad5 replication in rodent and rabbit cells seems to act primarily at a postentry step. Replication efficiency of adenoviral vectors harboring different E1 deletions or mutations in porcine cells was similar to that in A549 cells. Side-by-side comparison of the viral load kinetics in blood of swine and mice injected with Ad5 or a replication-deficient adenoviral vector failed to provide clear evidence for virus replication in mice. In contrast, evidence suggests that adenovirus replication occurs in swine, since adenoviral late gene expression produced a 13.5-fold increase in viral load in an individual swine from day 3 to day 7 and 100-fold increase in viral DNA levels in the Ad5-infected swine compared to the animal receiving a replication-deficient adenovirus. Lung histology of Ad5-infected swine revealed a severe interstitial pneumonia. Although the results in swine are based on a small number of animals and need to be confirmed, our data strongly suggest that infection of swine with human adenovirus or oncolytic adenoviral vectors is a more appropriate animal model to study adenoviral pathogenicity or pharmacodynamic and toxicity profiles of adenoviral vectors than infection of mice.     

Development of an animal model of chronic alcohol-induced pancreatitis in the rat

This study was designed to develop an animal model of alcoholic pancreatitis and to test the hypothesis that the dose of ethanol and the type of dietary fat affect free radical formation and pancreatic pathology. Female Wistar rats were fed liquid diets rich in corn oil (unsaturated fat), with or without a standard or high dose of ethanol, and medium-chain triglycerides (saturated fat) with a high dose of ethanol for 8 wk enterally. The dose of ethanol was increased as tolerance developed, which allowed approximately twice as much alcohol to be delivered in the high-dose group. Serum pancreatic enzymes and histology were normal after 4 wk of diets rich in unsaturated fat, with or without the standard dose of ethanol. In contrast, enzyme levels were elevated significantly by the high ethanol dose. Increases were blunted significantly by dietary saturated fat. Fibrosis and collagen alpha1(I) expression in the pancreas were not detectable after 4 wk of enteral ethanol feeding; however, they were enhanced significantly by the high dose after 8 wk. Furthermore, radical adducts detected by electron spin resonance were minimal with the standard dose; however, the high dose increased carbon-centered radical adducts as well as 4-hydroxynonenal, an index of lipid peroxidation, significantly. Radical adducts were also blunted by approximately 70% by dietary saturated fat. The animal model presented here is the first to demonstrate chronic alcohol-induced pancreatitis in a reproducible manner. The key factors responsible for pathology are the amount of ethanol administered and the type of dietary fat.     

Reduced early alcohol-induced liver injury in CD14-deficient mice

Activation of Kupffer cells by gut-derived endotoxin is associated with alcohol-induced liver injury. Recently, it was shown that CD14-deficient mice are more resistant to endotoxin-induced shock than wild-type controls. Therefore, this study was designed to investigate the role of CD14 receptors in early alcohol-induced liver injury using CD14 knockout and wild-type BALB/c mice in a model of enteral ethanol delivery. Animals were given a high-fat liquid diet continuously with ethanol or isocaloric maltose-dextrin as control for 4 wk. The liver to body weight ratio in wild-type mice (5.8 +/- 0.3%) was increased significantly by ethanol (7.3 +/- 0.2%) but was not altered by ethanol in CD14-deficient mice. Ethanol elevated serum alanine aminotransferase levels nearly 3-fold in wild-type mice, but not in CD14-deficient mice. Wild-type and knockout mice given the control high-fat diet had normal liver histology, whereas ethanol caused severe liver injury (steatosis, inflammation, and necrosis; pathology score = 3.8 +/- 0.4). In contrast, CD14-deficient mice given ethanol showed minimal hepatic changes (score = 1.6 +/- 0.3, p < 0.05). Additionally, NF-kappa B, TGF-beta, and TNF-alpha were increased significantly in wild-type mice fed ethanol but not in the CD14 knockout. Thus, chronic ethanol feeding caused more severe liver injury in wild-type than CD14 knockouts, supporting the hypothesis that endotoxin acting via CD14 plays a major role in the development of early alcohol-induced liver injury.     

Oxidative stress and antioxidant defenses in ethanol-induced cell injury

Although in the past several mechanisms and factors have been proposed to be responsible for alcoholic liver disease (ALD), at present the involvement of oxygen free radicals and consequently of oxidative stress has acquired remarkable credit. In numerous experimental studies it has been shown the occurrence of alcohol-induced generation of oxygen- and ethanol-derived free radicals through different pathways and from different sources. Mitochondria appear to be both an important source of reactive oxygen species (ROS) and also a primary target of ethanol-induced damage. The consistent induction of the mitochondrial antioxidant enzyme manganese superoxide dismutase (Mn-SOD) observed in experimental animals after acute and chronic ethanol administration has all the characteristics of a "stress response" to an oxidative insult.     

Formation of superoxide radicals in membranes of subcellular organelles in regenerating liver]

A correlation between the changes in the rates of superoxide radical generation, upsilon, in microsomes, mitochondria, and nuclei and the Cu, Zn- and Mn-SOD activities in rat liver during the first 5 days after partial hepatectomy, has been studied. Level of upsilon in microsomal and mitochondrial membranes in the regeneration process was reduced. The Cu, Zn- and Mn-SOD activities changed in an extreme and antibate manner: the former was characterized by a minimum, whereas the latter-by a maximum with an extreme on the 3rd day after surgery. Analysis of the correlation between the values of upsilon in the nuclear membranes and cell cycle stages (on a literary basis) revealed that the upsilon was decreased 2 times on the stage of DNA synthesis. When mitosis was at maximum, upsilon showed a 4-5-fold increase in comparison with the control, the Cu, Zn-SOD activity being essentially unchanged. A role of SOD and O2-. in cell division is postulated. O2-. is assumed to play a role in gene expression, disassembly, and regeneration of the nuclear membrane; that of SOD is thought to consist in regulation of the proliferative activity.     

Secretion of extracellular superoxide dismutase from muscle transduced with recombinant adenovirus inhibits the growth of B16 melanomas in mice

A number of reports have described the effects of oxidative stress on tumor growth. Therefore, these experiments were designed to test the hypothesis that overexpression of extracellular superoxide dismutase (ecSOD) would inhibit the growth of tumors arising from s.c. implantation of syngenic B16-F1 melanoma cells. C57BL/6 mice were infected i.m. with adenovirus containing either beta-galactosidase (Ad.lacZ) as control or the secreted extracellular isoform of SOD (Ad.ecSOD) 3 days before s.c. implantation of B16-F1 tumor cells. Serum SOD activity was elevated nearly approximately 5-fold over control animals. Two weeks after implantation, B16-F1 tumor size was 65% smaller in mice infected with Ad.ecSOD in comparison with mice infected with Ad.lacZ. However, the presence of SOD did not affect growth rates of B16-F1 cells in vitro. Consistent with smaller tumor volume, tumors from Ad.ecSOD-infected mice also expressed less vascular endothelial growth factor (VEGF). Moreover, in vitro studies using B16-F1 cells confirm that SOD blunts oxidant-dependent VEGF expression. Importantly, CD31 expression and vessel density were markedly reduced in tumors from Ad.ecSOD-infected mice compared with controls. These data suggest that tumor oxidative stress may facilitate tumor vascularization and thus promote tumor growth.     

Specific induction of metallothionein synthesis by mitochondrial oxidative stress.

Metallothionein (MT), a sulfhydryl-rich protein, may be increased by administration of a variety of agents, including metals, cytokines and oxidative stress agents. Mitochondria are a major source of reactive oxygen species, but antioxidant systems against mitochondrial free radicals are not fully understood. In this study, we examined the induction of MT synthesis by administration of mitochondrial-specific reactive oxygen generators such as antimycin A (AA), an electron transfer inhibitor, and 2,4-dinitrophenol (DNP), an uncoupling agent. Subcutaneous administration of AA to mice significantly increased the hepatic MT concentration in a dose- and time-dependent manner. AA slightly elevated glutathione peroxidase (GSHPx) activity, but the rate of increase in GSHPx (1.3-fold) was smaller than that in MT (11.8-fold). Other antioxidants such as catalase, manganese-superoxide dismutase (Mn-SOD), copper/zinc-superoxide dismutase (Cu/Zn-SOD) and GSHPx were not activated by AA treatment. Moreover, administration of DNP induced the synthesis of MT in the liver. Although DNP slightly elevated Mn-SOD activity, the rate of increase in Mn-SOD (1.3-fold) was smaller than that in MT (3.7-fold). Other antioxidants such as catalase, Cu/Zn-SOD and GSHPx were not activated by DNP treatment. These data suggest that MT plays a major role in protection against oxidative stress induced in mitochondria.     

Diphenyleneiodonium sulfate, an NADPH oxidase inhibitor, prevents early alcohol-induced liver injury in the rat

The oxidant source in alcohol-induced liver disease remains unclear. NADPH oxidase (mainly in liver Kupffer cells and infiltrating neutrophils) could be a potential free radical source. We aimed to determine if NADPH oxidase inhibitor diphenyleneiodonium sulfate (DPI) affects nuclear factor-kappaB (NF-kappaB) activation, liver tumor necrosis factor-alpha (TNF-alpha) mRNA expression, and early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-16 g. kg(-1). day(-1)) continuously for up to 4 wk, using the Tsukamoto-French intragastric enteral feeding protocol. DPI or saline vehicle was administered by subcutaneous injection for 4 wk. Mean urine ethanol concentrations were similar between the ethanol- and ethanol plus DPI-treated groups. Enteral ethanol feeding caused severe fat accumulation, mild inflammation, and necrosis in the liver (pathology score, 4.3 +/- 0.3). In contrast, DPI significantly blunted these changes (pathology score, 0.8 +/- 0.4). Enteral ethanol administration for 4 wk also significantly increased free radical adduct formation, NF-kappaB activity, and TNF-alpha expression in the liver. DPI almost completely blunted these parameters. These results indicate that DPI prevents early alcohol-induced liver injury, most likely by inhibiting free radical formation via NADPH oxidase, thereby preventing NF-kappaB activation and TNF-alpha mRNA expression in the liver.     

A spectrophotometric assay for superoxide dismutase activities in crude tissue fractions.

A sensitive and reliable assay method was developed to characterize crude cell homogenates and subcellular fractions with regard to their superoxide dismutase (SOD) activities. The determination of SOD activities was based on the well-known spectrophotometric assay introduced by McCord & Fridovich [(1969) J. Biol. Chem. 244, 6049-6055], with partially succinylated (3-carboxypropionylated) rather than native ferricytochrome c as indicating scavenger. Partial succinylation of cytochrome c resulted in minimization of interference associated with the interaction of cytochrome c with mitochondrial cytochrome c oxidase or cytochrome c reductases. The further increase in specificity, with regard to exclusion of cytochrome c oxidase interference, gained as a consequence of the high pH of 10 enabled the analysis of samples as rich in cytochrome c oxidase activity as the mitochondrial fraction in the presence or absence of membrane-disrupting detergents. Linear relationships for the dependence of the SOD activities with protein concentration were obtained with rat liver homogenate, mitochondrial and microsomal fractions, indicating negligible interference. Furthermore, by choosing a high pH for the assay medium, a 4-fold increase in sensitivity compared with the classical SOD assay, carried out at pH 7.8, was gained as well as a more precise resolution of Cu/Zn-SOD and Mn-SOD by 2 mM-KCN in samples with a high ratio of Mn-SOD to Cu/Zn-SOD, such as mitochondria. The complete trapping of the O2.- radicals, which was more feasible at pH 10 than at pH 7.8, enabled the application of a simple equation derived for the calculation of appropriately defined units of SOD activity from a single experiment.     

Identification of adenovirus serotype 5 hexon regions that interact with scavenger receptors

Most of an intravenous dose of species C adenovirus serotype 5 (Ad5) is destroyed by liver Kupffer cells. In contrast, another species C virus, Ad6, evades these cells to mediate more efficient liver gene delivery. Given that this difference in Kupffer cell interaction is mediated by the hypervariable (HVR) loops of the virus hexon protein, we genetically modified each of the seven HVRs of Ad5 with a cysteine residue to enable conditional blocking of these sites with polyethylene glycol (PEG). We show that these modifications do not affect in vitro virus transduction. In contrast, after intravenous injection, targeted PEGylation at HVRs 1, 2, 5, and 7 increased viral liver transduction up to 20-fold. Elimination or saturation of liver Kupffer cells did not significantly affect this increase in the liver transduction. In vitro, PEGylation blocked uptake of viruses via the Kupffer cell scavenger receptor SRA-II. These data suggest that HVRs 1, 2, 5, and 7 of Ad5 may be involved in Kupffer cell recognition and subsequent destruction. These data also demonstrate that this conditional genetic-chemical mutation strategy is a useful tool for investigating the interactions of viruses with host tissues.     

Subcellular distribution of superoxide dismutases (SOD) in rat liver: Cu,Zn-SOD in mitochondria

Rat liver was homogenized in isotonic buffer, fractionated by differential centrifugation, and then subfractionated by equilibrium sedimentation in Nycodenz gradients. Fractions were assayed for both Cu,Zn-superoxide dismutase (SOD) and Mn-SOD by exploiting the cyanide sensitivity of the former activity and by the use of specific antibodies. As expected, the cytosol and lysosomal fractions contained Cu,Zn-SOD; while the mitochondrial matrix contained Mn-SOD. In mitochondria, Cu,Zn-SOD was found in the intermembrane space and Mn-SOD in the matrix and also on the inner membrane. The Mn-SOD associated with the inner membrane was solubilized by 0.5 m NaCl. Surprisingly the intracellular membrane fraction (microsomes) contained bound Cu,Zn-SOD that could be solubilized with a detergent, and to lesser degree with 0.5 m NaCl. Both the cytosolic and mitochondrial Cu,Zn-SODs were isolated and compared. They have identical molecular mass, cyanide sensitivity, SDS sensitivity, heat stability, and chloroform + ethanol stability. Tissue from Cu,Zn-SOD knockout mice was entirely devoid of Cu,Zn-SOD; indicating that the cytosolic and the intermembrane space Cu,Zn-SODs are coded for by the same gene. The significance of this distribution of the SODs is discussed.