Modulation of calcium by the carcinogenic process in the liver induced by a choline-deficient diet

A diet devoid of choline and low in methionine (CD), without any added carcinogen, has been shown to induce 100% preneoplastic nodules and more than 50% cancer in the rat liver. Attempts to understand the mechanism by which a CD diet induces liver cell cancer revealed that like chemical carcinogens, a CD diet also appears to cause alterations in DNA, perhaps mediated by free radicals. Indeed, a CD diet induces nuclear lipid peroxidation prior to the changes in DNA. The CD diet induced DNA alterations coupled with continuing liver cell proliferation may account for the induction of initiated hepatocytes by the CD diet. To gain insight into the nature of free radicals generated by the CD diet, experiments were designed to determine whether agents that modulate free radical effects influence the CD diet induced changes in the liver. We investigated the effect of Ca2+ in the modulation of CD diet induced alterations in the liver. The results show that extra Ca2+ when added to the CD diet prevented some of the early changes due to choline deficiency, such as nuclear lipid peroxidation and DNA damage, but had little or no effect on the triglyceride accumulation in the liver. Also, the same CD diet with extra Ca2+, when used as a promoter after initiation by diethylnitrosamine, decreased the number and size of early putative preneoplastic foci and nodules.     

New Insight into the Biochemical Pathology of Liver in Choline Deficiency

Abstract

A diet deficient in choline can cause liver cancer in rats. The previous work since 1932 emphasized the fat-removing ability of choline from the liver. There are other dietary factors, including methionine, which, like choline, can remove fat from the liver. These factors were termed as lipotropes. Since then, choline deficiency and lipotrope deficiency are used synonoumously. Recent work since 1980 has clearly demonstrated that choline deficiency (CD) and lipotrope deficiency (LD) are not the same. Generation of free radicals, DNA alterations, liver cell death, and liver cancer that occur due to CD are not generated by LD. Generation of free radicals due to CD diet and some of the agents that counteract free radical action also prevent CD effects except for lipid accumulation in the liver. Despite the recent observations on the role of phospholipase A, (PLA) as the protector of the membranes, it has been found that by preventing the rise of PLA, in the liver, cell death can be prevented. These new findings give choline a distinct role in liver cell death and cancer rather than the role of lipotrope. A new hypothesis linking dietary choline deficiency and liver cancer has been discussed.     

氟中毒大鼠肝肾自由基代谢及硒对其影响

为探讨硒对氟中毒大鼠肝、肾自由基代谢的影响,两组Wistar大鼠饮1.58 mmol/L和2.63 mmol/L高氟水;饮高氟水的同时加饲2.0 mg/kg硒饲料;饮氟水7个月后加饲硒饲料.实验14个月时用低温电子自旋共振(ESR)技术测其肝、肾活性氧自由基(FR)含量;同时测氟(F)、硒(Se)含量;谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)活性和脂质过氧化物(LPO)含量.结果：氟中毒大鼠在肝、肾氟升高的同时,FR和LPO上升,GSH-Px、SOD下降.在氟中毒不同时期投硒,大鼠肝、肾氟降低,FR和LPO减少,抗氧化酶活性恢复.表明硒不但可拮抗大鼠体内的高氟,还可纠正高氟造成的自由基代谢紊乱.     

Prevention by free radical scavenger AD5 of prooxidant effects of choline deficiency

This study was designed to explore the possible preventive effects of a novel radicophile, N-p-methoxyphenylacetyl-dehydroalanine (AD5) and three other antioxidants, N,N'-diphenyl-p-phenylenediamine (DPPD), butylated hydroxyanisole (BHA) and a water-soluble analogue of vitamin E, trolox C, on the acute effects of the liver of feeding a choline-deficient (CD) diet. It has been suggested that some of the acute effects of a CD diet are related to free radicals, the generation or metabolism of which is disturbed in this acute dietary model. AD5 was found to be very effective in preventing nuclear lipid peroxidation, DNA damage and cell death induced by a CD diet but to have little effect on triglyceride accumulation ("fatty liver"). DPPD, BHA, and trolox C were ineffective. These results add strength to the hypothesis that oxygen free radicals might be an important component in the early events during carcinogenesis induced by feeding a CD diet.     

Spin-trapping studies of hepatic free radicals formed following the acute administration of ethanol to rats: in vivo detection of 1-hydroxyethyl radicals with PBN.

The generation of free radicals in rat liver following the acute oral administration of ethanol was studied with the spin-trapping method, using a deuterated derivative of phenyl-N-tert-butylnitrone (PBN-d14) as the spin-trapping agent. After administration of ethanol and PBN-d14 to rats, organic extracts of the liver were prepared and subjected to ESR spectroscopy. In the case of ethanol-treated rats, the ESR spectra indicated that mixtures of radicals had been trapped, while spectra from control rats were essentially negative. The predominant spin adduct detected after ethanol treatment is proposed to be from a carbon-centered, primary alkyl radical, based on gamma-hydrogen hyperfine splitting patterns observed with PBN-d14. Oxygen-centered radicals also contributed to the ESR spectra. Liver extracts also contained low concentrations of the 1-hydroxyethyl radical spin adduct, which was indicated by weak spectral lines corresponding to those of the 1-13C-ethanol adduct. These data confirm previous suggestions that ethanol is metabolized to a free radical metabolite in rat liver. In addition, some information on types of lipid radicals generated during alcohol intoxication has been obtained.     

Free Radical-Induced Liver Injury. I. Effects of Dietary Vitamin E Deficiency on Triacylglycerol Level and its Fatty Acid Profile in Rat Liver

Effects of dietary vitamin E deficiency on the fatty acid compositions of total lipids and phospholipids were studied in several tissues of rats fed a vitamin E-deficient diet for 4, 6, and 9 months. No significant differences were observed between the vitamin E deficiency and controls except in the fatty acid profiles of liver total lipids. Triacylglycerol (TAG) accumulation was found in the liver of rats fed a vitamin E-deficient diet. The levels of TAG-palmitate and -oleate increased particularly in the liver from such animals. The fatty acid compositions of hepatic phospholipids were not affected by the diet. Increased TAG observed in the liver of rats fed a vitamin E-deficient diet was restored to normal when the diet was supplemented with 20 mg α-tocopheryl acetate/kg diet. These findings indicate that dietary vitamin E deficiency causes TAG accumulation in the liver and that the antioxidant, vitamin E, is capable of preventing free radical-induced liver injury.     

Polyphenols from Camellia sinenesis attenuate experimental cholestasis-induced liver fibrosis in rats

Accumulation of hydrophobic bile acids during cholestasis leads to generation of oxygen free radicals in the liver. Accordingly, this study investigated whether polyphenols from green tea Camellia sinenesis, which are potent free radical scavengers, decrease hepatic injury caused by experimental cholestasis. Rats were fed a standard chow or a diet containing 0.1% polyphenolic extracts from C. sinenesis starting 3 days before bile duct ligation. After bile duct ligation, serum alanine transaminase increased to 760 U/l after 1 day in rats fed a control diet. Focal necrosis and bile duct proliferation were also observed after 1-2 days, and fibrosis developed 2-3 wk after bile duct ligation. Additionally, procollagen-alpha1(I) mRNA increased 30-fold 3 wk after bile duct ligation, accompanied by increased expression of alpha-smooth muscle actin and transforming growth factor-beta and the accumulation of 4-hydroxynenonal, an end product of lipid peroxidation. Polyphenol feeding blocked or blunted all of these bile duct ligation-dependent changes by 45-73%. Together, the results indicate that cholestasis due to bile duct ligation causes liver injury by mechanisms involving oxidative stress. Polyphenols from C. sinenesis scavenge oxygen radicals and prevent activation of stellate cells, thereby minimizing liver fibrosis.     

Chronic ethanol feeding inhibits plasma levels of insulin-like growth factor-1

The purpose of this study was to determine whether the generalized catabolic effects of chronic ethanol may be associated with a decline in plasma levels of insulin-like growth factor-1 (IGF-1). Male Sprague-Dawley rats were fed a liquid diet containing 5% ethanol or pair-fed a diet made isocaloric with maltose-dextrin. Animals were maintained on this diet for either 12 days or 4.5 months. Another group of animals were fed control diet ad libitum for 2 weeks. After 12 days of feeding, plasma concentrations of IGF-1 in ad libitum fed rats were 771 +/- 41 ng/ml which was greater than concentrations in either pair-fed (595 +/- 23 ng/ml) or ethanol-fed (680 +/- 40 ng/ml) rats (P less than 0.05). After 4.5 months of feeding, plasma levels of IGF-1 in ad libitum and pair-fed rats were similar to the 12 day study (736 +/- 56 and 607 +/- 26 ng/ml, respectively). However, a significant decrease in plasma levels of IGF-1 was observed in ethanol-fed animals over the 4.5 month period (551 +/- 28 ng/ml, P less than 0.05). Results of a similar study in rats fed a high-fat diet for 4.5 months were similar to those found with the low-fat diet. These results indicate that 1) dietary restriction of the type routinely used in this pair-feeding regimen decreases plasma levels of IGF-1, 2) chronic ethanol feeding further decreases plasma IGF-1 levels compared to pair-fed rats, 3) the effects of ethanol on IGF-1 concentrations are not modified by dietary fat, and 4) the effects on IGF-1 are not directly dependent on elevated plasma ethanol concentrations. Our results suggest that IGF-1 secreting cells in the liver may be progressively damaged by chronic ethanol feeding.     

Effect of essential fatty acid deficiency on the lipid composition of the Yoshida ascites hepatoma (AH 130) and of the liver and blood plasma from host and normal rats.

In order to study the response of a poorly differentiated tumor to nutritional manipulation, the Yoshida ascites hepatoma (AH 130) was grown in rats fed an essential fatty acid (EFA)-deficient diet and in rats fed a control diet. Hepatomas, livers, and blood plasma from host rats and normal rats were studied as to the effects of EFA deficiency on the lipid composition. Normal rats fed an EFA-deficient diet showed an increased concentration of triglycerides and cholesteryl esters in the liver and a reduced level of total phospholipids in plasma. Host rats fed the EFA-deficient diet showed a lower concentration of triglycerides in the liver when compared with the host rats fed a control diet. In addition, EFA-deficient host rats had reduced levels of plasma free fatty acids and triglycerides. These latter were markedly high in host rats under normal dietetic conditions. As compared to the livers of either host rats or normal rats fed the control diet, the Yoshida hepatoma cells had a lower content of total phospholipids and free fatty acids as well as a higher level of free cholesterol; they also showed a typical fatty acid pattern in their phospholipids. The main characteristics of this pattern were a high content of oleic and palmitoleic acids and a low level of C20 and C22 polyunsaturated fatty acids. Exposure of Yoshida hepatoma cells to an EFA-deficient environment resulted in a decrease in the concentration of total phospholipids and free fatty acids and in changes in the fatty acid composition similar to those observed in the livers of normal and host rats. These changes suggest that, under the experimental conditions used, the Yoshida hepatoma cells are responsive to EFA deficiency.     

In vivo detection of free radicals induced by diethylnitrosamine in rat liver tissue

Diethylnitrosamine (DEN) is a well-known carcinogenic substance that requires microsomal activation before it can react with DNA to cause mutations and cancer. The aim of this study was to use in vivo spin trapping and spin probe techniques to investigate whether free radicals are generated in rat liver tissue during DEN activation. We used alpha-phenyl-n-tert-butylnitrone (PBN) as the spin trapping agent, which was delivered through an intraperitoneal injection before DEN administration. One hour after DEN administration, multicomponent PBN adducts in the bile were detected, and the intensities were diminished by the cytochrome P450 inhibitor SKF-525A. A computer simulation of the ESR signals revealed the presence of a lipid-derived radical. Using the in vivo spin probe/ESR technique, the signal decay rate of methoxycarbonyl-PROXYL was significantly increased in the DEN-treated group compared with the rate in the vehicle group. The enhanced signal decay rate was restored with PBN and/or SKF-525A pretreatment. These results suggested that lipid-derived free radicals were generated in the liver within 1 h after DEN administration.     

Effect of phytate in soy protein on the serum and liver cholesterol levels and liver fatty acid profile in rats

Dietary soy protein, in comparison with casein, generally lowers the serum cholesterol concentration in rats fed on a cholesterol-enriched diet, while mixed results were observed in rats fed on a diet free of cholesterol. Soy protein also suppresses the conversion of linoleic acid to arachidonic acid in the rat liver. The present study examines whether phytate, a minor component of a soy protein isolate, is responsible for these beneficial effects of soy protein. Weanling male rats were fed for 4 weeks on a purified diet containing a 20% level of protein (either casein (CAS), soy protein (SOY), phytate-depleted SOY (PDSOY) or phytate-replenished PDSOY (PRSOY)) and cholesterol (0 or 0.5%). The dietary protein source and phytate level only affected the serum and liver cholesterol concentrations when the animals were fed on the cholesterol-enriched diet, being significantly lower in those rats fed on the SOY and PRSOY diets than in those fed on the CAS diet, while the concentrations in the rats fed on the PDSOY diet were intermediate. When the animals were fed on the cholesterol-free diet, the ratio of (20:3n-6 + 20:4n-6)/18:2n-6 in liver phosphatidylcholine, a delta6 desaturation index, was significantly lower in the SOY diet group than in the CAS, PDSOY and PRSOY diet groups. Dietary cholesterol significantly depressed the ratio, but neither depletion nor replenishment of phytate affected the ratio. These results suggest that phytate in soy protein played a limited role in the cholesterol-lowering effect of soy protein and was not involved in the metabolism of linoleic acid.     

Caloric restriction increases free radicals and inducible nitric oxide synthase expression in mice infected with Salmonella Typhimurium

It is well known that CR (caloric restriction) reduces oxidative damage to proteins, lipids and DNA, although the underlying mechanism is unclear. However, information concerning the effect of CR on the host response to infection is sparse. In this study, 6-month-old mice that were fed AL (ad libitum) or with a CR diet were infected with Salmonella serovar Typhimurium. EPR (electron paramagnetic resonance; also known as ESR (electron spin resonance)) was used to identify FRs (free radicals). These results were subsequently correlated with SOD (superoxide dismutase) catalytic activity, iNOS [inducible NOS (nitric oxide synthase) or NOSII] expression and NO (nitric oxide) content. EPR analysis of liver samples demonstrated that there was a higher quantity of FRs and iron-nitrosyl complex in infected mice provided with a CR diet as compared with those on an AL diet, indicating that CR was beneficial by increasing the host response to Salmonella Typhimurium. Furthermore, in infected mice on the CR diet, NOSII expression was higher, NO content was greater and spleen colonization was lower, compared with mice on the AL diet. No changes in SOD activity were detected, indicating that the NO produced participated more in the formation of iron-nitrosyl complexes than peroxynitrite. These results suggest that CR exerts a protective effect against Salmonella Typhimurium infection by increasing NO production.     

The potentiating and protective effects of ascorbate on oxidative stress depend upon the concentration of dietary iron fed C3H mice

Ascorbic acid (AA) is an antioxidant that, in the presence of iron and hydrogen peroxide, increases the production of hydroxyl radicals in vitro. Whether AA has similar pro-oxidant properties in vivo may depend upon the relative balance of iron and AA concentrations. In this study, C3H mice were fed diets supplemented with 100 or 300 mg/kg iron, with or without AA (15 g/kg), for 12 months. Liver AA concentrations were greater in mice fed AA-supplemented diets with either low or high iron (P=.0001), while the high-iron diet was associated with a significantly lower liver AA concentration regardless of AA supplementation (P=.0001). Only mice fed the high-iron diet with AA had a significantly greater liver iron concentration (P=.05). In the high-iron group, AA reduced oxidative stress, as measured by greater activities of glutathione peroxidase, superoxide dismutase (SOD) and catalase and by significantly lower concentrations of 4-hydroxylalkenal (HAE) and malondialdehyde (MDA). In mice fed the low-iron diet, AA was associated with greater concentrations of HAE and MDA and with lower activities of SOD. However, AA did not increase the concentrations of modified DNA bases with the low-iron diet but was associated with significantly lower concentrations of modified DNA bases in mice fed the high-iron diet. In conclusion, dietary AA appears to have mild pro-oxidant properties at low-iron concentrations but has a strong antioxidant effect against oxidative stress and DNA damage induced by dietary iron in mouse liver.     

The effect of aging and dietary restriction on DNA repair

DNA repair was studied as a function of age in cells isolated from both the liver and the kidney of male Fischer F344 rats. DNA repair was measured by quantifying unscheduled DNA synthesis induced by UV irradiation. Unscheduled DNA synthesis decreased approximately 50% between the ages of 5 and 30 months in both hepatocytes and kidney cells. The age-related decline in unscheduled DNA synthesis in cells isolated from the liver and kidney was compared in rats fed ad libitum and rats fed a calorie-restricted diet; calorie restriction has been shown to increase the survival of rodents. The level of unscheduled DNA synthesis was significantly higher in hepatocytes and kidney cells isolated from the rats fed the restricted diet. Thus, calorie restriction appears to retard the age-related decline in DNA repair.     

Resveratrol, a red wine polyphenol, attenuates ethanol-induced oxidative stress in rat liver

The involvement of oxidative stress in the pathogenesis of alcoholic diseases in the liver has been repeatedly confirmed. Resveratrol, a natural phytoalexin present in grape skin and red wine possesses a variety of biological activities including antioxidant. This study was conducted to evaluate whether resveratrol has a preventive effect on the main indicators of hepatic oxidative status as an expression of the cellular damage caused by free radicals, and on antioxidant defence mechanism during chronic ethanol treatment. Wistar rats were treated daily with 35% ethanol solution (3 g/kg/day i.p.) during 6 weeks and fed basal diet or basal diet containing 5 g/kg resveratrol. Control rats were treated with i.p. saline and fed basal diet. Experimentally, chronic ethanol administration leads to hepatotoxicity as monitored by the increase in the level of hepatic marker enzymes and the appearance of fatty change, necrosis, fibrosis and inflammation in liver sections. Ethanol also enhanced the formation of MDA in the liver indicating an increase in lipid peroxidation, a major end-point of oxidative damage, and caused drastic alterations in antioxidant defence systems. Particularly the activities of hepatic superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were found reduced by ethanol treatment while glutathione reductase (GR) activity was unchanged. Dietary supplementation with resveratrol during ethanol treatment inhibited hepatic lipid peroxidation and ameliorated SOD, GPx and CAT activities in the liver. Conclusively, we can suggest that resveratrol could have a beneficial effect in inhibiting the oxidative damage induced by chronic ethanol administration, which was proved by the experiments that we conducted on rats.     

Lipid Metabolism in the Fatty Liver of Rats Fed a Threonine-deficient Diet

Feeding of a threonine-deficient diet to rats weighing approximately 53 g or 99 g caused a significant rise in liver lipids compared to the control diet containing 7% amino acid mixture. Whereas, when rats weighing approximately 155 g were fed either the control diet or the threonine-deficient diet, liver lipid content was essentially the same for both groups. Therefore, in the present paper, young rats were used to clarify the mechanism of liver lipid accumulation in threonine-deficiency. The increase in dietary fat content of the threonine-deficient diet did not prevent the lipid accumulation in rat liver. The rates of in vivo incorporation from radioactive acetate into liver lipids, body lipids and respiratory CO₂ of rats fed either the control diet or the threonine-deficient diet were measured. The threonine-deficient group tended to be lower in total activity of both the liver lipids and body lipids than those of the control group. Thus, these results suggest that the development of this type of fatty liver might not be due to the stimulation of lipid synthesis in the liver. In the serum of rats fed the threonine-deficient diet, the protein content of β-lipoproteins was significantly lower and free fatty acid level tended to be lower than the values of the control animals, respectively. From these results, decreased trasport of lipids from the liver may thus be considered a potential major factor responsible for the excessive lipid accumulation in the liver of rats fed the threonine-deficient diet.     

Immunofluorescent detection of 8-oxoguanine DNA lesions in liver cells from aging OXYS rats, a strain prone to overproduction of free radicals

Production of free radicals in animals is accompanied with a number of pathologic conditions, some of which may be manifested through DNA damage. Studies of mechanisms of oxidative DNA damage by free radicals in vivo are hindered by the lack of good animal models with significant overgeneration of or increased sensitivity to free radicals. An inbred rat strain (OXYS) is characterized by inherited overgeneration of free radicals, lipid peroxidation, protein oxidation, DNA rearrangements, and pathological conditions paralleling several human degenerative diseases. We have used monoclonal antibodies against a common pre-mutagenic base lesion 8-oxoguanine (8-oxoG) in combination with indirect immunofluorescence microscopy and image analysis to follow the relative age-dependent amounts and distribution of 8-oxoG in liver cells from OXYS and Wistar rats. 8-OxoG increased with age in both strains of rats, with OXYS rats always displaying statistically significantly higher levels of oxidative DNA damage than Wistar rats. Statistical analysis indicates that 8-oxoG does not uniformly accumulate in all cells with advancing age or increasing free radical load, but rather concentrates in a minor fraction of cells with a high damage level.     

Hepatic iron accumulation is not directly associated with induction of DNA strand breaks in the liver cells of Long-Evans Cinnamon (LEC) rats.

Effects of accumulation of copper and iron on induction of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) rats that spontaneously develop fulminant hepatitis. Copper and iron accumulated in the liver of LEC rats in an age-dependent manner from 4 to 15 weeks. Low-iron diet prevented the accumulation of iron in the liver, but did not prevent accumulation of copper. The amounts of DNA strand breaks that were estimated by comet assay in the liver cells of rats fed standard diet increased with age from 4 to 15 weeks. No significant differences were observed in the proportions of LEC rat liver cells without tail and the average lengths of tail momentum in the comet images between LEC rats that had been fed standard MF diet and low-iron diet. These results support the idea that accumulation of iron is not directly associated with the induction of DNA damage in the liver cells of LEC rats.