Prosaposin is a novel coenzyme Q10-binding protein.

Coenzyme Q10 (CoQ10) is essential for mitochondrial ATP production and functions as an important antioxidant in every biomembrane and lipoprotein. Due to its hydrophobicity, a binding and transfer protein for CoQ10 is plausible, and we previously described saposin B as a CoQ10-binding and transfer protein. Here, we report that prosaposin, the precursor of saposin B, also binds CoQ10. As prosaposin is both a secretory protein and integral membrane protein, it is ubiquitous in the body. Prosaposin was isolated from human seminal plasma, and CoQ10 was extracted from hexane solution into the water phase. It was additionally found that immunoprecipitates of mouse brain cytosol generated using two different anti-prosaposin antibodies contained coenzyme Q9. Furthermore, mouse liver cytosol and mouse kidney cytosol also contained prosaposin-coenzyme Q9 complex. These results suggest that prosaposin binds CoQ10 in human cells and body fluids. The significance and role of the Psap-CoQ10 complex in vivo is also discussed.

Riboflavin compounds show NAD(P)H dependent quinone oxidoreductase-like quinone reducing activity.

NAD(P)H-dependent quinone oxidoreductase (NQO) is an essential enzyme in living organisms and cells protecting them from oxidative stress. NQO reduces coenzyme Q (CoQ) using NAD(P)H as an electron donor. In the present study, we searched for coenzyme Q10 reducing activity from fractions of gel filtration-fractionated rat liver homogenate. In addition to the large-molecular-weight fraction containing NQO, CoQ10 reducing activity was also detected in a low-molecular-weight fraction. Furthermore, dicumarol, a conventional inhibitor of NQO1 (DT diaphorase), did not inhibit the reduction but quercetin did, suggesting that the activity was not due to NQO1. After further purification, the NADH-dependent CoQ10-reducing compound was identified as riboflavin. Riboflavin is an active substituent of other flavin compounds such as FAD and FMN. These flavin compounds also reduced not only CoQ homologues but also vitamin K homologues in the presence of NADH. The mechanism was speculated to work as follows: NADH reduces flavin compounds to the corresponding reduced forms, and subsequently, the reduced flavin compounds immediately reduce bio-quinones. Furthermore, the flavin-NADH system reduces CoQ10 bound with saposin B, which is believed to function as a CoQ transfer protein in vivo. This flavin-dependent CoQ10 reduction, therefore, may function in aqueous phases such as the cell cytosol and bodily fluids.

Method for detecting CoQ10 incorporation in the mitochondrial respiratory chain supercomplex.

Coenzyme Q10 is an important component of the mitochondrial electron transfer chain. A supercomplex of mitochondrial electron transfer system proteins exists. This complex also contains coenzyme Q10. The concentrations of coenzyme Q10 in tissues decrease with age and pathology. Coenzyme Q10 is given as a supplement. It is unknown whether coenzyme Q10 is transported to the supercomplex. We develop a method for measuring coenzyme Q10 in the mitochondrial respiratory chain supercomplex in this study. Blue native electrophoresis was used to separate mitochondrial membranes. Electrophoresis gels were cut into 3 mm slices. Hexane was used to extract coenzyme Q10 from this slice, and HPLC-ECD was used to analyze coenzyme Q10. Coenzyme Q10 was found in the gel at the same site as the supercomplex. Coenzyme Q10 at this location was thought to be coenzyme Q10 in the supercomplex. We discovered that 4-nitrobenzoate, a coenzyme Q10 biosynthesis inhibitor, reduced the amount of coenzyme Q10 both within and outside the supercomplex. We also observed that the addition of coenzyme Q10 to cells increased the amount of coenzyme Q10 in the supercomplex. It is expected to analysis coenzyme Q10 level in supercomplex in various samples by using this novel method.

Transferrin, insulin, and progesterone modulate intracellular concentrations of coenzyme Q and cholesterol, products of the mevalonate pathway, in undifferentiated PC12 cells.

Coenzyme Q (CoQ) is important not only as an essential lipid for the mitochondrial electron transport system, but also as an antioxidant. CoQ levels decrease during aging and in various diseases. Orally administered CoQ is not readily taken up in the brain, so it is necessary to develop a method to increase the amount of CoQ in neurons. CoQ is synthesized via mevalonate pathway, like cholesterol. Transferrin, insulin, and progesterone are factors used in the culture of neurons. In this study, we determined the effect of these reagents on cellular CoQ and cholesterol levels. The administration of transferrin, insulin, and progesterone increased cellular CoQ levels in undifferentiated PC12 cells. When serum was removed and only insulin was administered, intracellular CoQ levels increased. This increase was even more pronounced with concurrent administration of transferrin, insulin, and progesterone. Cholesterol level decreased by the administration of transferrin, insulin, and progesterone. Progesterone treatment lowered intracellular cholesterol levels in a concentration-dependent manner. Our findings suggest that transferrin, insulin, and progesterone may be useful in regulating CoQ levels and cholesterol levels, which are products of the mevalonate pathway.

Cellular level of coenzyme Q increases with neuronal differentiation, playing an important role in neural elongations.

Deficiency of coenzyme Q has been reported in various neuro-logical diseases, and the behavior of this lipid in neurons has attracted attention. However, the behavior of this lipid in normal neurons remains unclear. In this study, we analyzed the concen-tration of coenzyme Q before and after neuronal differentiation. Nerve growth factor treatment of PC12 cells caused neurite outgrowth and neuronal differentiation, and the amount of intra-cellular coenzyme Q increased dramatically during this process. In addition, when the serum was removed from the culture medium of N1E-115 cells and the neurite outgrowth was confirmed, the intracellular coenzyme Q level also increased. To elucidate the role of the increased coenzyme Q, we administered nerve growth factor to PC12 cells with coenzyme Q synthesis inhibitors and found that coenzyme Q levels decreased, neurite outgrowth was impaired, and differentiation markers were reduced. These results indicate that coenzyme Q levels increase during neuronal differentiation and that this increase is important for neurite outgrowth.

Coenzyme Q10 levels increase with embryonic development in medaka.

Coenzyme Q10 is an important molecule for mitochondrial respiration and as an antioxidant. Maintenance of the ovum in a good condition is considered to be important for successful fertilization and development, which has been reported to be promoted by coenzyme Q10. In this study, we investigated the level of coenzyme Q10 during ovum fertilization and maturation. We attempted to analyze coenzyme Q10 levels during ovum development in species that use coenzyme Q10 but not coenzyme Q9. It was shown that medaka produces coenzyme Q10. We then measured the amount of coenzyme Q10 after fertilization of medaka ovum and found that it increased. The amount of free cholesterol biosynthesized from acetyl CoA as well as coenzyme Q10 increased during development, but the increase in coenzyme Q10 was more pronounced. The mRNA expression level of coq9 also increased during embryonic development, but the mRNA expression levels of other coenzyme Q10 synthases did not. These results suggest that the coq9 gene is upregulated during the development of medaka ovum after fertilization, resulting in an increase in the amount of coenzyme Q10 in the ovum. Medaka, which like humans has coenzyme Q10, is expected to become a model animal for coenzyme Q10 research.

Reduced prosaposin levels in HepG2 cells with long-term coenzyme Q10 deficiency.

Glycosphingolipids are involved in intercellular signaling, adhe-sion, proliferation, and differentiation. Saposins A, B, C, and D are cofactors required for glycosphingolipid hydrolysis. Saposins A-D are present in series in a common precursor protein, prosaposin. Thus, glycosphingolipids amounts depend on prosaposin cellular levels. We previously reported that prosaposin and saposin B bind coenzyme Q10 in human cells. Coenzyme Q10 is an essential lipid of the mitochondrial electron transport system, and its reduced form is an important antioxidant. Coenzyme Q10 level decrease in aging and in various progressive diseases. Therefore, it is interesting to understand the cellular response to long-term coenzyme Q10 deficiency. We established a long-term coenzyme Q10 deficient cell model by using the coenzyme Q10 biosynthesis inhibitor, 4-nitrobenzoate. The levels of coenzyme Q10 were reduced by 4-nitrobenzoate in HepG2 cells. Administration of 4-nitrobenzoate also decreased prosaposin protein and mRNA levels. The cellular levels of coenzyme Q10 and prosaposin were recovered by treatment with 4-hydroxybenzoquinone, a substrate for coenzyme Q10 synthesis that counteracts the effect of 4-nitrobenzoate. Furthermore, the ganglioside levels were altered in 4-nitrobenzoate treated cells. These results imply that long-term coenzyme Q10 deficiency reduces cellular prosaposin levels and disturbs glycosphingolipid metabolism.

Differentiation of THP-1 monocytes to macrophages increased mitochondrial DNA copy number but did not increase expression of mitochondrial respiratory proteins or mitochondrial transcription factor A.

Monocytes are differentiated into macrophages. In this study, mitochondrial DNA copy number (mtDNAcn) levels and downstream events such as the expression of respiratory chain mRNAs were investigated during the phorbol 12-myristate 13-acetate (PMA)-induced differentiation of monocytes. Although PMA treatment increased mtDNAcn, the expression levels of mRNAs encoded in mtDNA were decreased. The levels of mitochondrial transcription factor A mRNA and protein were also decreased. The levels of coenzyme Q10 remained unchanged. These results imply that, although mtDNAcn is considered as a health marker, the levels of mtDNAcn may not always be consistent with the parameters of mitochondrial functions.

Prosaposin knockdown in Caco-2 cells decreases cellular levels of coenzyme Q10 and ATP, and results in the loss of tight junction barriers.

Coenzyme Q10 (CoQ10) is a key component of the mitochondrial electron transfer chain and is one of the most important antioxidants. We previously found that glycoprotein prosaposin (Psap) binds CoQ10 in human cells. Although Psap is expressed in the intestines, its role in the gastrointestinal tract is not clear. To elucidate the role of Psap in the intestines, we established Psap knockdown (KD) Caco-2 cells, which are an intestinal epithelial cell line. Cellular CoQ10 levels decreased significantly in Psap KD Caco-2 cells as compared to parental Caco-2 cells. Cellular ATP levels also decreased significantly in Psap KD Caco-2 cells as compared to parental Caco-2 cells. Lower ATP levels in the intestines have been reported to result in the failure of tight junction formation. As expected, Psap KD Caco-2 monolayers did not produce transepithelial electrical resistance, while parental Caco-2 monolayers did. Moreover, a fluorescent dye, lucifer yellow, leaked out through Psap KD Caco-2 monolayers, whereas it did not through parental Caco-2 monolayers. These results indicate that Psap is essential to maintain cellular levels of CoQ10 and ATP, and consequently to form tight junctions in the gastrointestinal tract.

Prosaposin regulates coenzyme Q10 levels in HepG2 cells, especially those in mitochondria.

Coenzyme Q10 (CoQ10) is a key component of the mitochondrial electron transfer chain and is one of the most important cellular antioxidants. We previously reported that glycoprotein saposin B (SapB) binds CoQ10 in human cells. To elucidate the physiological role of SapB and its precursor, prosaposin (Psap), we prepared stable transfectants of HepG2 that overexpress wild-type human Psap (Wt-Tf). We also established a SapB domain mutated Psap (Mt-Tf) in which cysteine(198) was replaced with serine to disrupt three dimensional protein structure by the loss of S-S bridging. Psap knockdown (KD) strains were also examined. Western blotting analysis confirmed overexpression or knockdown of Psap in these HepG2 cells. The cellular ratios of CoQ10 to free cholesterol (FC) significantly decreased in the order of Wt-Tf>parental>Mt-Tf>KD. Additionally, the ratios of CoQ10/FC in mitochondrial fractions decreased in the order of Wt-Tf>parental>KD. These data indicate that Psap and/or SapB regulate CoQ10 levels in HepG2 cells, especially in their mitochondria.

AI-based Apoptosis Cell Classification Using Phase-contrast Images of K562 Cells.

BACKGROUND/AIM: This study aimed to automate the classification of cells, particularly in identifying apoptosis, using artificial intelligence (AI) in conjunction with phase-contrast microscopy. The objective was to reduce reliance on manual observation, which is often time-consuming and subject to human error. MATERIALS AND METHODS: K562 cells were used as a model system and apoptosis was induced following administration of gamma-secretase inhibitors. Fluorescence staining was applied to detect DNA fragmentation and caspase activity. Cell images were obtained using both phase-contrast and fluorescence microscopy. Two AI models, Lobe(R) and a server-based ResNet50, were trained using these images and evaluated using F-values through five-fold cross-validation. RESULTS: Both AI models demonstrated effectively categorized individual cells into three groups: caspase-negative/no DNA fragmentation, caspase-positive/no DNA fragmentation, and caspase-positive/DNA fragmentation. Notably, the AI models' ability to differentiate cells relied on subtle variations in phase-contrast images, potentially linked to changes in refractive indices during apoptosis progression. Both AI models exhibited high accuracy, with the server-based ResNet50 model showing improved performance through repeated training. CONCLUSION: This study demonstrates the potential of AI-assisted phase-contrast microscopy as a powerful tool for automating cell classification, especially in the context of apoptosis research and the discovery of anticancer substances. By reducing the need for manual labor and enhancing classification accuracy, this approach holds promise for expediting high-throughput cell screening, significantly contributing to advancements in medical diagnostics and drug development.

Dynamic aspects of ascorbic acid metabolism in the circulation: analysis by ascorbate oxidase with a prolonged in vivo half-life.

Because AA (L-ascorbic acid) scavenges various types of free radicals to form MDAA (monodehydroascorbic acid) and DAA (dehydroascorbic acid), its regeneration from the oxidized metabolites is critically important for humans and other animals that lack the ability to synthesize this antioxidant. To study the dynamic aspects of AA metabolism in the circulation, a long acting AOase (ascorbate oxidase) derivative was synthesized by covalently linking PEG [poly(ethylene glycol)] to the enzyme. Fairly low concentrations of the modified enzyme (PEG-AOase) rapidly decreased AA levels in isolated fresh plasma and blood samples with a concomitant increase in their levels of MDAA and DAA. In contrast, relatively high doses of PEG-AOase were required to decrease the circulating plasma AA levels of both normal rats and ODS (osteogenic disorder Shionogi) rats that lack the ability to synthesize AA. Administration of 50 units of PEG-AOase/kg of body weight rapidly decreased AA levels in plasma and the kidney without affecting the levels in other tissues, such as the liver, brain, lung, adrenal grand and skeletal muscles. PEG-AOase slightly, but significantly, decreased glutathione (GSH) levels in the liver without affecting those in other tissues. Suppression of hepatic synthesis of GSH by administration of BSO [L-buthionin-(S,R)-sulfoximine] enhanced the PEG-AOase-induced decrease in plasma AA levels. These and other results suggest that the circulating AA is reductively regenerated from MDAA extremely rapidly and that hepatic GSH plays important roles in the regeneration of this antioxidant.

Coenzyme Q10-Binding/Transfer Protein Saposin B also Binds gamma-Tocopherol.

gamma-Tocopherol, the major form of dietary vitamin E, is absorbed in the intestine and is secreted in chylomicrons, which are then transferred to liver lysosomes. Most gamma-tocopherol is transferred to liver microsomes and is catabolized by cytochrome p450. Due to the hydrophobicity of gamma-tocopherol, a binding and transfer protein is plausible, but none have yet been isolated and characterized. We recently found that a ubiquitous cytosolic protein, saposin B, binds and transfers coenzyme Q10 (CoQ10), which is an essential factor for ATP production and an important antioxidant. Here, we report that saposin B also binds gamma-tocopherol, but not alpha-tocopherol, as efficiently as CoQ10 at pH 7.4. At acidic pH, saposin B binds gamma-tocopherol preferentially to CoQ10 and alpha-tocopherol. Furthermore, we confirmed that saposin B selectively binds gamma-tocopherol instead of CoQ10 and alpha-tocopherol at every pH between 5.4 and 8.0 when all three lipids are competing for binding. We detected gamma-tocopherol in human saposin B monoclonal antibody-induced immunoprecipitates from human urine, although the amount of gamma-tocopherol was much smaller than that of CoQ10. These results suggest that saposin B binds and transports gamma-tocopherol in human cells.

Saposin B is a human coenzyme q10-binding/transfer protein.

Coenzyme Q10 (CoQ10) is essential for ATP production in the mitochondria, and is an important antioxidant in every biomembrane and lipoprotein. Due to its hydrophobicity, a binding and transfer protein for CoQ10 is plausible, but none have yet been isolated and characterized. Here we purified a CoQ10-binding protein from human urine and identified it to be saposin B, a housekeeping protein necessary for sphingolipid hydrolysis in lysosomes. We confirmed that cellular saposin B binds CoQ10 in human sperm and the hepatoma cell line HepG2 by using saposin B monoclonal antibody. The molar ratios of CoQ10 to saposin B were estimated to be 0.22 in urine, 0.003 in HepG2, and 0.12 in sperm. We then confirmed that aqueous saposin B extracts CoQ10 from hexane to form a saposin B-CoQ10 complex. Lipid binding affinity to saposin B decreased in the following order: CoQ10>CoQ9>CoQ7>>alpha-tocopherol>>cholesterol (no binding). The CoQ10-binding affinity to saposin B increased with pH, with maximal binding seen at pH 7.4. On the other hand, the CoQ10-donating activity of the saposin B-CoQ10 complex to erythrocyte ghost membranes increased with decreasing pH. These results suggest that saposin B binds and transports CoQ10 in human cells.

Erythrocytes with T-state-stabilized hemoglobin as a therapeutic tool for postischemic liver dysfunction.

This study aimed to examine if T-state stabilization of hemoglobin in erythrocytes could protect against postischemic organ injury. Human erythrocytes containing three different states of Hb allostery were prepared: control Hb (hRBC), CO-Hb that is stabilized under R-state with the 6-coordinated prosthetic heme (CO-hRBC), and alpha-NO-deoxyHb stabilized under T-state (alpha-NO-hRBC). To prepare alpha-NO-RBC, deoxygenated RBC was treated with FK409, a thiol-free NO donor, at its half molar concentration to that of Hb; this procedure resulted in the 5-coordinated NO binding on the alpha-subunit heme, as judged by electron spin resonance spectrometry. Rats were subject to 20 min systemic hemorrhage to maintain mean arterial pressure at 40 mm Hg, and reperfused with one of hRBCs. This protocol for ischemia, followed by 60 min reperfusion with physiological saline, caused modest metabolic acidosis and cholestasis. Administration of hRBC or COhRBC significantly attenuated cholestasis and improved acidosis. Rats treated with alpha-NO-hRBC exhibited greater recovery of metabolic acidosis and bile excretion than those treated with hRBC or CO-hRBC, displaying the best outcome of local oxygen utilization in hepatic lobules. Half-life time of alpha-NO-RBC administered in vivo was approximately 60 min. These results suggest that T-state Hb stabilization by NO serves as a stratagem to treat postischemic organ dysfunction.

Cadmium exposure alters metabolomics of sulfur-containing amino acids in rat testes.

This study aimed to examine distribution of cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE), the hydrogen sulfide (H(2)S)-generating enzymes, and metabolomic alterations in sulfur-containing amino acids in rat testes exposed to stressors. Immunohistochemistry revealed distinct distribution of the two enzymes: CBS occurred mainly in Leydig cells and was also detectable in Sertoli cells and germ cells, whereas CSE was evident in Sertoli cells and immature germ cells involving spermatogonia. The amounts of CSE and CBS in testes did not alter in response to administration of cadmium chloride, an antispermatogenic stressor leading to apoptosis. Metabolome analyses assisted by liquid chromatography equipped with mass spectrometry revealed marked alterations in sulfur-containing amino acid metabolism: amounts of methionine and cysteine were significantly elevated concurrently with a decrease in the ratio between S-adenosylhomocysteine and Sadenosylmethionine, suggesting expansion of the remethylation cycle and acceleration of methyl donation. Despite a marked increase in cysteine, amounts of H(2)S were unchanged, leading to a remarkable decline of the H(2)S/cysteine ratio in the cadmium-treated rats. Under such circumstances, oxidized glutathione (GSSG) was significantly reduced, whereas reduced glutathione (GSH) was well maintained, and the GSH/GSSG ratio was consequently elevated. These results collectively showed that cadmium induces metabolomic remodeling of sulfur-containing amino acids even when the protein expression of CBS or CSE is not evident. Although detailed mechanisms for such a remodeling event remain unknown, our study suggests that metabolomic analyses serve as a powerful tool to pinpoint a critical enzymatic reaction that regulates metabolic systems as a whole.

Hydrogen sulfide as an endogenous modulator of biliary bicarbonate excretion in the rat liver.

Cystathionine gamma-lyase (CSE) is an enzyme catalyzing cystathionine and cysteine to yield cysteine and hydrogen sulfide (H(2)S), respectively. This study aimed to examine if H(2)S generated from the enzyme could serve as an endogenous regulator of hepatobiliary function. Gas chromatographic analyses indicated that, among rat organs herein examined, liver constituted one of the greatest components of H(2)S generation in the body, at 100 mumol/g of tissue, comparable to that in kidney and 1.5-fold greater than that in brain, where roles of the gas in the regulation of neurotransmission were reported previously. At least half of the gas amount in the liver appeared to be derived from CSE, because blockade of the enzyme by propargylglycine suppressed it by 50%. Immunohistochemistry revealed that CSE occurs not only in hepatocytes, but also in bile duct. In livers in vivo, as well as in those perfused ex vivo, treatment with the CSE inhibitor induced choleresis by stimulating the basal excretion of bicarbonate in bile samples. Transportal supplementation of NaHS at 30 mumol/L, but not that of N-acetylcysteine as a cysteine donor, abolished these changes elicited by the CSE inhibitor in the perfused liver. The changes elicited by the CSE blockade did not coincide with alterations in hepatic vascular resistance, showing little involvement of vasodilatory effects of the gas in these events, if any. These results first provided evidence that H(2)S generated through CSE modulates biliary bicarbonate excretion and is thus a determinant of bile salt-independent bile formation in the rat liver.

Impaired ascorbic acid metabolism in streptozotocin-induced diabetic rats.

Ascorbic acid (AA) metabolism in streptozotocin (STZ)-induced diabetic rats was determined by examining urinary excretion, renal reabsorption, reductive regeneration, and biosynthesis of AA at 3 and 14 days after STZ administration. AA concentrations in the plasma, liver, and kidney of the diabetic rats were significantly lower than those of controls on d 3, and decreased further as the diabetic state continued. Hepatic AA regeneration significantly decreased in the diabetic rats on d 3 in spite of increased gene expressions of AA regenerating enzymes and was further reduced on d 14. Hepatic activity of L-gulono-gamma-lactone oxidase, a terminal enzyme of hepatic AA biosynthesis, also decreased significantly on d 3 and decreased further on d 14. Urinary excretion of AA was significantly increased on d 3, with an increase in urine volume but no change in gene expressions of renal AA transporters (SVCT1 and SVCT2). Urinary excretion of AA was normalized on d 14. The results suggest that impaired hepatic and renal regeneration, as well as increased urinary excretion and impaired hepatic biosynthesis of AA, contributed to the decrease in AA in plasma and tissues of STZ-induced diabetic rats.

From O2 to H2S: a landscape view of gas biology.

The majority of molecular oxygen (O2) consumed in the body is used as a substrate of cytochrome c oxidase to maintain oxidative phosphorylation for ATP synthesis. Rest of the O2 is used for oxidative biosynthesis including synthesis of vasoactive substances such as prostaglandins and secondary gaseous mediators such as nitric oxide (NO) and carbon monoxide (CO). Thus, O2 is not only used for maintenance of energy supply but also for regulating blood supply into tissues. Nitrous oxide (N2O), laughing gas for anesthesia, is generated endogenously through NO reductase in bacteria and fungi, and has recently been shown to modulate N-methyl-D-aspartic acid (NMDA) receptor function. A number of other biologically active gases could participate in regulation of cell and tissue functions. Carbon dioxide (CO2) is generated mainly through the Krebs cycle as a result of glucose oxidation and serves as a potent vasodilator, and hydrogen sulfide (H2S) synthesized through degradation of cysteine has recently been postulated to be a neuromodulator, although their receptor proteins for signaling have not been verified as a discernible molecular entity. Easy penetration allow these gases to access the inner space of receptor proteins and to execute their biological actions. These gases are generated and consumed in anaerobic bacteria through varied reactions distinct from those in mammals. This review summarizes recent information on mechanisms for gas generation and reception in biological systems.

Vitamin E prevents increase in oxidative damage to lipids and DNA in liver of ODS rats given total body X-ray irradiation.

We examined the effects of dietary vitamin E (VE) on oxidative damage to DNA and lipids in the liver a few days after total body irradiation (TBI). ODS rats, which lack vitamin C synthesis, were fed either a low VE diet (4.3 mg VE/kg) or a basal VE diet (75.6 mg VE/kg) for 5 weeks while vitamin C was supplied in the drinking water. The VE level in the liver of the low VE group was lower and the levels of lipid peroxides were higher compared to those of the basal VE group: the relative levels in the two groups were 1:30 for VE, 18:1 for 4-hydroxynonenal (HNE), and 10:1 for hexanal (HA). The level of 8-hydroxydeoxyguanosine (8OHdG), a marker of oxidative DNA damage, did not differ between the low VE and the basal VE groups. When the rats received TBI at the dose of 3 Gy and were killed on day 6, the levels of HNE, HA and 8OHdG increased by 2.2-, 2-, and 1.5-times, respectively, in the low VE group, but TBI did not cause such increases in the basal VE group. Changes in antioxidative enzymes (glutathione peroxidase, catalase, and Cu/Zn-SOD) in the liver could not explain the different responses of the two diet groups to TBI-induced oxidative damage. The concentrations of vitamin C and glutathione in the liver did not differ between the two groups. These results suggest that dietary VE can prevent the oxidative damage to DNA and lipids in the liver which appear a few days after TBI at dose of 3 Gy.