Lipid peroxidation in hepatomas of different degrees of deviation

Lipid peroxidation rate in four different hepatomas is quite different and seems to be related to their degree of deviation, low deviation tumours displaying higher peroxidative ability. Moreover, the supernatant of the highly anaplastic Yoshida hepatoma is able to decrease the peroxidation rate in normal liver microsomes. This antioxidant ability is not dependent upon an increased level of glutathione. The concentration of reduced glutathione (GSH) declines strongly during incubation in conditions favouring lipid peroxidation. Unlike normal liver homogenates, this decline of GSH in hepatomas is not due to the transformation of GSH into oxidized glutathione (GSSG) but mostly to the increased activity of the γ-glutamyl-transpeptidase pathway.     

Studies on ornithine decarboxylase from liver, kidney and tumoral tissues during activity decay following cycloheximide administration

We have studied the activity, thiol-dependency and Km of ornithine decarboxylase (ODC) from the following sources: liver of rats subjected to partial hepatectomy or administered thioacetamide, the rat 3924A Morris hepatoma, the rat AH130 Yoshida ascites hepatoma, a mouse transplantable mammary carcinoma and kidney of rats administered testosterone propionate. In order to detect possible changes occurring during in vivo ageing of this enzyme we inhibited protein synthesis with cycloheximide. A gradual decrease of Km during ageing was observed in ODC from liver.     

Lipid peroxidation and physical properties of smooth and rough hepatoma microsomes

Smooth and rough endoplasmic reticulum of two Morris hepatomas, the slow growing 9618A and the fast growing 3924A, have been isolated, and their biochemical composition, supramolecular organization, and response to the action of peroxidative agents have been studied. Cytochrome P₄₅₀ content and lipid availability are the limiting factors of their peroxidizability. The hemoprotein content is reduced about 80% in hepatoma 9618A and is virtually absent in hepatoma 3924A. The peroxidizability decreases with increasing growth rate of the tumor. The protein, phospholipid, and cholesterol content, the fatty acid composition as well as the double bond index, and the saturated and unsaturated fatty acid content are reported. Differences have been found between normal liver and tumors and between the fractions within a given tumoral tissue. The molecular order, as determined by fluorescence anisotrophy decay of DPH, increases in total microsomes and in the smooth fraction going from liver 9618A to 3924A, whereas for the rough fraction it is the same in liver and hepatoma 9618A; in 3924A it increases of about 30%. Fluidity decreases in total microsomes going from liver to 3924A, to 9618A. In both the purified fractions it decreases with increasing deviation of the tumor.     

Lipid peroxidation and physical properties of smooth and rough hepatoma microsomes

Smooth and rough endoplasmic reticulum of two Morris hepatomas, the slow growing 9618A and the fast growing 3924A, have been isolated, and their biochemical composition, supramolecular organization, and response to the action of peroxidative agents have been studied. Cytochrome P450 content and lipid availability are the limiting factors of their peroxidizability. The hemoprotein content is reduced about 80% in hepatoma 9618A and is virtually absent in hepatoma 3924A. The peroxidizability decreases with increasing growth rate of the tumor. The protein, phospholipid, and cholesterol content, the fatty acid composition as well as the double bond index, and the saturated and unsaturated fatty acid content are reported. Differences have been found between normal liver and tumors and between the fractions within a given tumoral tissue. The molecular order, as determined by fluorescence anisotrophy decay of DPH, increases in total microsomes and in the smooth fraction going from liver 9618A to 3924A, whereas for the rough fraction it is the same in liver and hepatoma 9618A; in 3924A it increases of about 30%. Fluidity decreases in total microsomes going from liver to 3924A, to 9618A. In both the purified fractions it decreases with increasing deviation of the tumor.     

Age-dependent changes in rat liver lipid peroxidation and glutathione content induced by acute ethanol ingestion

The study of the influence of the age of animals (13 to 53 weeks) on total liver thiobarbituric acid reactive substances (TBAR) content showed an increase which is maximal in rats of 39 weeks of age compared to young animals (13 weeks), followed by a dimunition in the 53 weeks old group. In this situation, the content of hepatic GSH and total GSH equivalents as well as the GSH/GSSG ratio were decreased with ageing, while GSSG levels were enhanced in the oldest group studied. Acute ethanol intoxication resulted in a marked increase in liver TBAR content in young animals, together with a decline in GSH, total GSH equivalents and GSH/GSSG ratio, and an enhancement in GSSG. These changes elicited by ethanol intake were reduced with ageing. It is concluded that ethanol-induced oxidative stress in the liver is diminished during ageing, despite the progressive decrease in the glutathione content of the tissue observed in control animals.     

Superoxide-dependent lipid peroxidation and vitamin E content of microsomes from hepatomas with different growth rates

Lipid peroxidation of microsomal membranes isolated from rat liver, and Morris hepatomas 9618A (slow-growing) and 3924A (fast-growing) was induced by superoxide radicals generated by the action of xanthine oxidase on xanthine. The peroxidation, measured as malondialdehyde and lipid hydroperoxide formation, was optimized with regard to iron concentration and chelation of iron by ADP. In such conditions hepatoma microsomes catalyze lower rates of lipid peroxidation than the normal counterpart. However, while microsomes from hepatoma 3924A show a marked decrease in both the malondialdehyde and hydroperoxide production rates, microsomes from hepatoma 9618A differ moderately from the control, mainly in the long-term production of hydroperoxides. It is also reported here that the 9618A microsomes partially lack cytochrome P-450 (about 40% deficiency), but they have a fatty acid composition similar to that of control. No differences were found in the content of vitamin E between normal and hepatoma 3924A microsomes. Moreover, induction of vitamin E deficiency in hepatoma 3924A microsomes does not influence the rate of either malondialdehyde or lipid hydroperoxide production. On the basis of these results and previous data on the lipid composition of hepatoma 3924A microsomes it is proposed that the high resistance to superoxide-dependent lipid peroxidation of hepatoma 3924A microsomes is related to the low substrate availability rather than the content of membrane antioxidants; and a limitation only in the propagation phase characterizes the hepatoma 9618A microsomal lipid peroxidation and would be due to the partial deficiency of the endogenous propagating agent, cytochrome P-450.     

Glutathione disulfide reduction in tumor mitochondria after t-butyl hydroperoxide treatment.

Treatment of isolated mitochondria from rat hepatoma tumor cells (AS-30D) with the oxidant, t-butyl hydroperoxide (tBuOOH, 1 or 5 mumol/ml) resulted in the oxidation of glutathione (GSH to GSSG) and the formation of protein-glutathione mixed disulfides (ProSSG). The GSSG was retained inside of the hepatoma mitochondria. In the presence of ADP+succinate (5 or 10 mM), or ketoglutarate (10 mM) or malate (5 mM), the GSSG was reduced to GSH, but the amount of ProSSG stayed constant. With saline or ADP+glutamate (10 mM)/malate (0.1 mm) no reduction of GSSG to GSH occurred. The presence of antimycin (5 micrograms/ml) with ADP+succinate inhibited reduction. At a concentration of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, 0.5 mM) which inhibited a major portion of the glutathione reductase activity, the reduction of GSSG to replenish GSH was also inhibited. NADPH may play a critical role as well, for the addition of 2.4 mM NADPH to permeabilized hepatoma mitochondria fostered the reduction of GSSG after tBuOOH treatment. Therefore, hepatoma mitochondria possess a glutathione reductase-dependent system to reduce GSSG to GSH. The reaction only occurs with actively respiring mitochondria.     

Decreased activity of cytidine 3':5'-monophosphate (cyclic CMP) phosphodiesterase in the fast-growing Morris hepatoma 3924A, but not in the slow-growing Morris hepatoma 9618A.

The activity level of the newly-identified cyclic CMP phosphodiesterase in the fast-growing Morris hepatoma 3924A was found to be much lower than the control (normal or host) liver. Its level in the slow-growing Morris hepatoma 9618A (a minimal deviation tumor), on the other hand, was the same as the host liver. The level of cyclic AMP phosphodiesterase was higher, whereas that of cyclic GMP phosphodiesterase was lower, in hepatoma 3924A than the control liver. In comparison, the levels of the two enzymes were both depressed in hepatoma 9618A. These findings suggest that depression of cyclic CMP phosphodiesterase may be related to the process and the rate of malignant growth, and that metabolism of cyclic CMP may be more crucial than that of cyclic AMP or cyclic GMP in the neoplastic cell proliferation.     

Administration of glutathione and lipid peroxidation induced during fasting

It is well known that lipid peroxidation may be initiated or exaggerated by conditions leading to hepatic GSH depletion or altered GSH/GSSG ratio. In our study we evaluated the effects of GSH administration on hepatic, bile and plasma GSH, GSSG and MDA in rats depleted of the tripeptide by a prolonged. fasting. An exteriorized biliary-duodenal fistula was established and GSH or saline solution was administered i.p. for a period of 6h. Rats treated with GSH exhibited an increased GSH and decreased GSSG biliary excretion. Whereas in control rats an opposite pattern was observed, namely enhanced GSSG and decreased GSH biliary excretion. While hepatic GSH and GSSG concentrations were comparable in the two groups, a significant increase in liver and plasma MDA production was found in controls compared to GSH treated rats. Our data suggest a protective role of GSH against the production of lipoperoxidation as evidenced by the decrease of hepatic, biliary and plasma MDA levels and by a decreased percentage of biliary GSSG. In addition, the significant increase of biliary GSH excretion, observed in rats treated with GSH compared to controls, may be due to an increased supply of the tripeptide which is known to be preferentially excreted into bile in the reduced form.     

"In vitro" effect of CCl4, PG and EDTA on GSH levels at different time of incubation of rat liver homogenates

During CCl4-induced lipid peroxidation GSH content in total homogenate from rat liver falls very rapidly in the first 30 min. of incubation "in vitro". CCl4 does not enhance the decrease in total glutathione (TG) during the incubation time, so GSH loss is mainly due to its oxidation to GSSG. On the contrary PG and EDTA, two substances decreasing lipid peroxidation rate, are able to decrease GSH oxidation, without affecting TG content. At 25 degrees C EDTA and PG completely prevent GSH decrease at pH 7.4, while at pH 6 PG affords only a partial prevention. At 37 degrees C both compounds are able to limit GSH decrease at a large extent. Lipid peroxidation seems to have a great importance in the kinetics of GSH decrease and GSSG formation, at least "in vitro". It is noteworthy that PG which inhibits lipid peroxidation stimulated by CCl4 is also able to limit the high GSH loss observed in the homogenates incubated in the presence of halogeno-alkane.     

Prepubertal children with suitable fitness and physical activity present reduced risk of oxidative stress

To assess the impact of fitness status and physical activity on oxidative stress in prepubertal children, we measured selected biomarkers such as protein carbonyls (PC), lipid peroxidation products, and total nitrites, as well as the antioxidant system: total glutathione (TG), oxidized glutathione (GSSG), reduced glutathione (GSH), superoxide dismutase activity, and glutathione peroxidase. A total of 132 healthy children ages 7-12, at prepubertal stage, were classified into two groups according to their fitness level: low fitness (LF) and high fitness (HF). They were observed while engaged in an after-school exercise program, and a questionnaire was created to obtain information on their physical activity or sedentary habits. Plasma and red blood cells were obtained to analyze biomarkers. Regarding oxidative stress markers, the LF group and the sedentary group showed higher levels of TG and GSSG and a lower GSH/GSSG ratio than the HF group and the children engaged in physical activity. A negative association was found between PC and GSSG and TG and between TG and the GSH/GSSG ratio. Moreover, a negative correlation was found between GSSG and fitness, with a positive correlation with the GSH/GSSG ratio. TG, GSSG, and the GSH/GSSG ratio seem to be reliable markers of oxidative stress in healthy prepubertal children with low fitness or sedentary habits. This research contributes to the recognition that an adequate level of fitness and recreational physical activity in childhood leads to better health and oxidative status.     

Inhibition of glutathione disulfide reductase by glutathione

Rat-liver glutathione disulfide reductase is significantly inhibited by physiological concentrations of the product, glutathione. GSH is a noncompetitive inhibitor against GSSG and an uncompetitive inhibitor against NADPH at saturating concentrations of the fixed substrate. In both cases, the inhibition by GSH is parabolic, consistent with the requirement for 2 eq. of GSH in the reverse reaction. The inhibition of GSSG reduction by physiological levels of the product, GSH, would result in a significantly more oxidizing intracellular environment than would be realized in the absence of inhibition. Considering inhibition by the high intracellular concentration of GSH, the steady-state concentration of GSSG required to maintain a basal glutathione peroxidase flux of 300 nmol/min/g in rat liver is estimated at 8-9 microM, about 1000-fold higher than the concentration of GSSG predicted from the equilibrium constant for glutathione reductase. The kinetic properties of glutathione reductase also provide a rationale for the increased glutathione (GSSG) efflux observed when cells are exposed to oxidative stress. The resulting decrease in intracellular GSH relieves the noncompetitive inhibition of glutathione reductase and results in an increased capacity (Vmax) and decreased Km for GSSG.     

Effect of chlorpyrifos on thiobarbituric acid reactive substances, scavenging enzymes and glutathione in rat tissues

The present study showed that exposure of chlorpyrifos, O,O'-diethyl-O-3,5,6-trichloro-2-pyridyl phosphorothionate (CPF), a widely used pesticide in rats caused significant inhibition of acetylcholinesterase (AChE) activity in different tissues viz., liver, kidney and spleen. CPF exposure also generated oxidative stress in the body, as evidenced by increase in thiobarbituric acid reactive substances (TBARS), decrease in the levels of superoxide scavenging enzymes viz., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in liver, kidney and spleen at all doses. Malondialdehyde levels were increased by 14%, 31% and 76% in liver, 11%, 31% and 64% in kidney and 32%, 75% and 99.9% in spleen when 50 mg, 100 mg and 200 mg/kg body wt. CPF was administered for three days. SOD and CAT activities were decreased in liver, kidney and spleen, while GPx activity showed slight increase in kidney at 50 mg and 100 mg dose, and decreased on further increase in dose of CPF. Liver and spleen showed dose-dependent decrease in GPx activity. The levels of reduced glutathione (GSH) was decreased, while oxidized glutathione (GSSG) was increased, thus a marked fall in GSH/GSSG ratio was observed in all tissues. A maximum decrease of 83% was observed in liver, followed by kidney and spleen, which showed 78% and 57% decrease, respectively in group given 200 mg/kg CPF. The levels of glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) were also decreased in liver and kidney, while spleen showed increase at lower doses, but decrease at high dose of CPF. The data provide evidence for induction of oxidative stress on CPF exposure.     

Hepatic response to the oxidative stress induced by E. coli endotoxin: Glutathione as an index of the acute phase during the endotoxic shock

Reactive oxygen species are important mediators of cellular damage during endotoxic shock. In order to investigate the hepatic response to the oxidative stress induced by endotoxin, hepatic and plasma glutathione (total, GSH and GSSG), GSSG/GSH ratio as well as Mn-superoxide dismutase and catalase activities were determined during the acute and recovery phases of reversible endotoxic shock in the rat. A significant increase in liver and plasma total glutathione content was observed 5 h after endotoxin treatment (acute phase), followed by a diminution of these parameters below control values at 48 h (recovery phase). The significant increases of GSSG levels and GSSG/GSH ratio are indicative of oxidative stress occurring during the acute phase. Liver Mn-SOD activity showed a similar time dependency as the GSSG/GSH ratio; however, a marked decrease in the liver catalase activity was observed during the process. These results indicate the participation of liver glutathione in the response to endotoxin and the possible use of plasma glutathione levels and GSSG/GSH ratio as indicators of the acute phase during the endotoxic process. (Mol Cell Biochem 159: 115-121, 1996)     

Glutathione oxidation and activation of pentose phosphate cycle during hydroperoxide metabolism. A comparison of livers from fed and fasted rats

Perfusion of livers from fed and fasted rats with 0.07--0.1 mM t-butyl hydroperoxide for 15 min decreased the levels of reduced glutathione (GSH) by 1.5 mumol/g liver in both nutritional states. Glutathione disulfide (GSSG) was increased by 70 and 140 nmol/g liver and glutathione mixed disulfides enhanced by 45 and 150 nmol/g liver in livers from fed and fasted animals, respectively. The ratio of GSH/GSSG was decreased from 243 to 58 in fed animals, and from 122 to 8 in fasted animals. The increase of GSSG and the mixed disulfides was nearly parallel until an apparently critical low GSH content of 1.5 mumol/g was reached. Only in livers from fasted rats 14CO2-production from [1-14C]glucose was stimulated upon t-butyl hydroperoxide infusion at the employed rates. Flux of glucose through pentose phosphate cycle rose from 8 to 12% of glucose utilization via glycolysis, whereas in livers from fed animals this portion remained unchanged at 8% Dithio-erythritol reversed pentose phosphate cycle activity as well as GSSG and protein-bound glutathione contents to the original levels. In livers from fasted rats the activity of glucose-6-phosphate dehydrogenase was increased by 34% by t-butyl hydroperoxide infusion.     

Factors influencing the inhibition of biliary glutathione efflux induced by biliary obstruction

The aim of this study was to investigate mechanisms responsible for the inhibition of biliary glutathione efflux in rats with secondary biliary cirrhosis. Rats were studied after bile duct obstruction for 28 days. The biliary secretion of reduced glutathione (GSH), oxidised glutathione (GSSG) and cysteine were completely inhibited in biliary obstructed rats. Hepatic gamma glutamyltranspeptidase (gamma-GT) activity increased significantly, but following its inhibition by acivicin administration GSH, GSSG and cysteine were still absent in bile. Biliary obstruction resulted in a significant increase of the permeability of the paracellular pathway, as shown by the higher bile/plasma ratio and hepatic clearance of [14C]sucrose. GSH and GSSG were, however, significantly lower in the carotid artery and hepatic vein of obstructed animals and the arteriovenous difference across the liver was reduced. The concentration of GSH was significantly reduced and that of GSSG increased in the liver of obstructed rats. Biliary obstruction induced an increase in the hepatic concentration of cysteine and an inhibition of both gamma glutamylcysteine synthetase and methionine adenosyl transferase activities. Dichlorofluorescein (DCF) and the GSSG/GSH ratio and thiobarbituric acid reactive substances (TBARS) concentration, markers of reactive oxygen species production and lipid peroxidation, respectively, were significantly increased. Our data indicate that increased degradation or blood reflux of glutathione do not participate in the disruption of its secretion into bile and support the view that impairment of glutathione synthesis and oxidative stress could contribute to the decline in biliary glutathione output.     

Role of S-adenosylmethionine on the hepatobiliary homeostasis of glutathione during cyclosporine A treatment

The effects of cyclosporine A (CyA) treatment on the hepatic content and biliary output of reduced (GSH) and oxidized (GSSG) glutathione and lipid peroxidation in the liver, and the ability of S-adenosylmethionine (SAMe) to antagonize the CyA-induced alterations were studied in male Wistar rats. To evaluate the efficacy of SAMe, three CyA and SAMe protocols were used: cotreatment with SAMe plus CyA, pretreatment with SAMe before starting cotreatment, and post-treatment with SAMe after beginning treatment with CyA alone. CyA treatment for one and four weeks depleted liver GSH, decreased the GSH/GSSG ratio and significantly reduced GSH and GSSG biliary concentrations and secretion rates. Additionally, long-term treatment enhanced lipid peroxidation. By contrast, when the rats were treated with CyA plus SAMe using any of the administration protocols, SAMe was seen to be efficient in antagonizing the GSH hepatic depletion, the changes in hepatic GSH/GSSG ratio and the increase induced by CyA in lipid peroxidation. Furthermore, SAMe also abolished the effects of CyA on the biliary secretion rates of GSH and GSSG. The efficacy of SAMe was similar, regardless of the administration protocols used. In conclusion, our results clearly demonstrate that SAMe is good for preventing, antagonizing and reversing the CyA-induced alterations in the hepatobiliary homeostasis of glutathione.     

Study of carbohydrate metabolism in glycogen storing cell lines derived from cultured rat hepatocytes

Some aspects of carbohydrate metabolism were investigated in three non-malignant, glycogen storing, cell lines derived from a primary culture of rat hepatocytes, and in the Morris hepatoma 3924 cells. The three cell lines show biochemical alterations which are, to a large extent, similar to those found in the hepatoma cells: increased activity of glycolytic enzymes and decreased activity of gluconeogenetic enzymes. An increase of glucose-6-phosphate dehydrogenase activity is also found. The three cell lines, as the Morris hepatoma cells, actively convert glucose into lactate under the in vitro conditions of culture. Fructose is not taken up as quickly as glucose and galactose is not metabolized. As compared with normal hepatocytes, the three cell lines have altered metabolism and growth behaviour. They largely resemble the preneoplastic cells appearing in rat liver at the early stages of experimental carcinogenesis.     

DNA polymerase alpha from normal rat liver is different than DNA polymerases alpha from Morris hepatoma strains

To investigate whether DNA replication in rat hepatoma cells is altered compared with that in normal rat liver, the main replicative enzyme, i.e. the DNA polymerase alpha complex, was partially purified from a slow-growing (TC5123) and a fast-growing (MH3924) Morris hepatoma cell strain as well as from normal rat liver. The purified DNA polymerase alpha complexes contained RNA primase. DNA polymerase alpha activities of these complexes were characterized with regard to both their molecular properties and their dNTP and DNA binding sites. The latter were probed with competitive inhibitors of dNTP binding, resulting in Ki values, and with DNA templates, yielding Km values. The sedimentation coefficients of native DNA polymerases alpha from Morris hepatoma cells were found to be lower than that of polymerase alpha from normal rat liver. Consequently, when following the procedure of Siegel and Monty for determination of molecular mass considerably smaller molecular masses were calculated for polymerases of hepatoma strains (TC5123, 127 kDa; MH3924, 138 kDa; rat liver, 168 kDa). Similar differences were found when the dNTP binding site was probed with inhibitors. Ki values obtained with butylphenyl-dGTP were higher for polymerases of the hepatoma strains than for that of normal rat liver. However, Ki values measured with aphidicolin and butylanilino-dATP were lower for DNA polymerase alpha from the fast-growing hepatoma cell strain than for that from normal rat liver, indicating a reduced affinity of the dNTP binding sites for dATP and dCTP. This reduced affinity could be responsible for lowered specificity of nucleotide selection in the base-pairing process which in turn may cause an enhanced error rate in DNA replication in malignant cells. Furthermore, when the DNA binding site was characterized by Michaelis-Menten constants using gapped DNA as a template, Km values were similar for all three DNA polymerases. In contrast, the Km value measured with single-stranded DNA as a template was found to be lower for DNA polymerase alpha from the fast-growing hepatoma MH3924 than for that from normal rat liver. Thus, the DNA-polymerizing complex from MH3924 combines both higher binding strength to single-stranded DNA templates and decreased nucleotide selection, properties which may enhance replication velocity and may lower fidelity.     

Glutathione contents of tissues in the aging mouse.

1. Previous results from this laboratory demonstrated that the erythrocyte content of reduced glutathione (GSH) decreased as a function of both increasing cell age and mouse age [Abraham, Taylor & Lang (1978) Biochem. J. 174, 819-825]. In the present investigation glutathione concentrations were determined in other tissues of the C57BL/6J mouse of different ages (6--31 months) throughout the life-span. 2. At all ages the total glutathione and the GSH concentrations in liver were 3 times that in kidney and 10 times that in heart. In the old (31 months) mouse the GSH contents were lower by 30% in the liver, 34% in the kidney and 20% in the heart than in the mature (17--23 months) animals. 3. The oxidized glutathione (GSSG) concentrations of the tissues did not vary with age and constituted less than 3% of the total glutathione. 4. The decreased in GSH concentrations were not due to changes in organ weights, which were constant from 10 to 36 months of age. 5. These findings extend our previous results and indicate that a general characteristic of aging tissues may be a decrease in GSH concentrations. Further, this is consistent with our hypothesis that the reducing potential of tissues decreases in senescence.