Damage to protein synthesis concurrent with lipid peroxidation in rat liver slices: effect of halogenated compounds, peroxides, and vitamin E1

Protein synthesis and lipid peroxidation were evaluated in rat liver slices incubated in the presence of oxidants and protein synthesis inhibitors. Protein synthesis by rat liver slices was evaluated by [3H]leucine incorporation into the trichloroacetic acid (TCA)-insoluble material, and lipid peroxidation was evaluated by thiobarbituric acid-reactive substances (TBARS) released into the incubation medium. Protein synthesis inhibition by bromotrichloromethane (BrCCl3) or t-butyl hydroperoxide (t-BOOH) depended on the incubation time and oxidant concentration. [3H]Leucine incorporation was decreased to 20 and 47% of control values and TBARS were enhanced from the control value of 16.9 to 45.3 and 62.5 nmol/g of liver by incubation for 1 h with 1 mM BrCCl3 and t-BOOH, respectively. Following incubation, both protein synthesis damage and lipid peroxidation were decreased in control and oxidant-treated slices prepared from rats injected with 200 mg of DL-alpha-tocopherol/kg of body wt. Release of lactate dehydrogenase was not enhanced by oxidant treatment. Protein synthesis inhibitors reversibly decreased [3H]leucine incorporation, but the effect of oxidants on protein synthesis was irreversible. Cumene hydroperoxide and methyl ethyl ketone peroxide, but not hydrogen peroxide, damaged protein synthesis and induced lipid peroxidation. The ability of carbon tetrabromide, benzyl chloride, bromoform, bromobenzene, carbon tetrachloride, chloroform, dichloromethane, and bromochloromethane to inhibit protein synthesis was correlated with their ability to induce lipid peroxidation, and with their LD50. The results suggest that oxidant-induced lipid peroxidation and protein synthesis damage occurred concurrently, and that protein synthesis inhibition may be involved in cell injury or death mediated by free radicals.     

Lipid peroxidation measured as thiobarbituric acid-reactive substances in tissue slices: characterization and comparison with homogenates and microsomes

Liver slices were used to measure lipid peroxidation induced by bromotrichloromethane, tert-butyl hydroperoxide (t-BOOH), or ferrous iron. The responses of liver homogenates and microsomes to oxidative conditions were compared with the response of tissue slices. Lipid peroxidation was evaluated by the production of thiobarbituric acid-reactive substances (TBARS). As was observed in homogenates and microsomes, TBARS production by liver slices depended upon the amount of tissue, the incubation time, inducer, the amount of inducer, and the presence of antioxidant. Control liver slices incubated at 37 degrees C for 2 h produced 19 nmol of TBARS per g of liver. When slices were incubated in the presence of 1 mM BrCCl3, 1 mM t-BOOH, or 50 microM ferrous iron, TBARS production increased 4.6-, 8.2-, or 6.7-fold over the control value, respectively. Comparable induction of TBARS by liver homogenates and microsomes was observed when these preparations were incubated with the same inducers. Addition of 5 microM butylated hydroxytoluene (BHT) prevented the induction of TBARS by 50 microM ferrous iron by liver slices. The results indicate the usefulness of tissue slices to measure lipid peroxidation. The usefulness of tissue slices is emphasized when a number of compounds or tissues are studied and tissue integrity is desired as in toxicological, pharmacological, and nutritional studies where reduced numbers of experimental animals is a relevant issue.     

Damage to DNA concurrent with lipid peroxidation in rat liver slices.

Lipid peroxidation and DNA damage were evaluated in liver slices incubated for 2 h at 37 degrees C with 1 mM-t-butyl hydroperoxide (t-BOOH), 1 mM-BrCCl3 or 50 microM-ferrous iron. t-BOOH induced the greatest amount of damage to DNA and increased the production of thiobarbituric acid-reactive substances (TBARS). Both phenomena depended on the incubation time. Ferrous iron induced both DNA damage and TBARS production, and BrCCl3 did not induce significant DNA damage and was the weakest TBARS inducer. Butylated hydroxytoluene at 1 mM inhibited both DNA damage and TBARS production. DNA damage and lipid peroxidation in liver slices were correlated, indicating that these events were concurrent.     

Oxidant-increased proteolysis in rat liver slices: effect of bromotrichloromethane, antioxidants and effectors of proteolysis

Proteolysis and lipid peroxidation were evaluated in rat liver slices incubated in the presence of the oxidant bromotrichloromethane and effectors of proteolysis. Proteolysis was evaluated by S-amino acids and lipid peroxidation by thiobarbituric acid-reactive substances (TBARS) released into the incubation medium. The increased release of S-amino acids by BrCl3C depended on incubation time and oxidant concentration. S-Amino acid release increased 30% over control value and TBARS increased from 22 to 124 nmol/g liver by incubation for 120 min with 1 mM BrCl3C. Release of S-amino acids and TBARS was decreased when liver slices were treated with nor-dihydroguaiaretic acid (NDG), butylated hydroxyanisole (BHA), Trolox C, or N,N'-diphenyl-1,4-phenylenediamine (DPPD) immediately prior to addition of oxidant, suggesting participation of lipid-soluble free radicals. Oxidant-induced release of S-amino acids but not of TBARS was decreased by mannitol, suggesting participation of hydroxyl radical or a species with similar reactivity; and by superoxide dismutase and catalase, suggesting participation of superoxide and hydrogen peroxide, respectively. The decrease of S-amino acid release by sodium fluoride, sodium arsenate, 2,4-dinitrophenol, chloroquine, leupeptin, phenylmethylsulfonyl fluoride, EDTA and o-phenanthroline was variable, suggesting the presence in liver of several proteases to remove oxidatively-modified proteins.     

The role of phospholipase A2 in microsomal lipid peroxidation induced with t-butyl hydroperoxide

The role of phospholipase A2 (PlA2) in lipid peroxidation induced with t-butyl hydroperoxide was examined in rat liver microsomes. Exposure of microsomes to t-butyl hydroperoxide was associated with activation of endogenous PlA2. When PlA2 was inhibited with chlorpromazine, mepacrine, or p-bromphenacyl bromide, the accumulation of thiobarbituric acid reactive substances (TBARS) was reduced in a dose dependent manner. In contrast, the accumulation of conjugated dienes was not affected by chlorpromazine, and was slightly increased by mepacrine. When endogenous PlA2 was activated with mellitin prior to induction of peroxidation, accumulation of both TBARS and dienes was reduced. Analogously, pretreatment with exogenous PlA2 reduced both dienes and TBARS. In contrast, addition of mellitin following the induction of peroxidation did not alter either TBARS or dienes.     

Ethyl docosahexaenoate-associated decrease in fetal brain lipid peroxide production is mediated by activation of prostanoid and nitric oxide pathways

Previously we have shown that intraamniotic administration of ethyl docosahexaenoate (Et-DHA) to pregnant rats resulted in decreased lipid peroxidation in the fetal brain, under a variety of conditions (S. Glozman, P. Green, E. Yavin, J. Neurochem. 70 (1998) 2482-2491). In the present study we examine the potential mechanisms to explain this effect. This was done by a pharmacological approach, utilizing brain slice preparations from Et-DHA treated or control rats in the presence of various agents and examining the formation of products in the tissue slices or incubation medium. Et-DHA treated brains produced 2-3-fold more prostanoids (PN) than control brains, indicating cyclooxygenase (COX) activation. Indomethacin at 50 microM inhibited PN formation and also abolished Et-DHA induced decrease in lipid peroxides, as evident by the levels of thiobarbituric acid reactive substances (TBARS) released in the medium. The phospholipase A2 inhibitors quinacrine and p-bromophenacyl bromide added at 0.1 mM concentration each to either slices from controls or Et-DHA treated fetal brains, decreased TBARS production. Et-DHA treated brains released 2.2-fold more nitric oxide (NO) than control brains and NO synthase (NOS) inhibitors abolished this effect. Increasing the concentration of NO by the addition of an NO donor greatly decreased the concentration of the TBARS in the medium. These results suggest that at least some of the effect of Et-DHA on decreased lipid peroxidation may be explained by a shift of oxygen species utilization via enzymatically regulated, therefore metabolically controlled, COX and NOS activities.     

The effect of iron overload on urinary excretion of immunoreactive prostaglandin E2

The effect of in vivo lipid peroxidation on the excretion of immunoreactive prostaglandin E2 (PGE2) in the urine of rats was studied. Weanling, male Sprague-Dawley rats were fed a vitamin E-deficient diet containing 10% tocopherol-stripped corn oil (CO) or 5% cod liver oil (CLO) with or without 40 mg dl-alpha-tocopheryl acetate/kg. To induce a high, sustained level of lipid peroxidation, some rats were injected intraperitoneally with 100 mg of iron as iron dextran after 10 days of feeding. Iron overload stimulated in vivo lipid peroxidation in rats, as measured by the increase in expired ethane and pentane. Dietary vitamin E reversed this effect. Rats fed the CLO diet excreted 9.5-fold more urinary thiobarbituric acid-reactive substances (TBARS) than did rats fed the CO diet. Iron overload increased the excretion of TBARS in the urine of rats fed the CO diet, but not in urine of rats fed the CLO diet. Dietary vitamin E decreased TBARS in the urine of rats fed either the CO or the CLO diet. Iron overload decreased by 40% the urinary excretion of PGE2 by rats fed the CO diet, and dietary vitamin E did not reverse this effect. Iron overload had no statistically significant effect on urinary excretion of PGE2 by rats fed the CLO diet. A high level of lipid peroxidation occurred in iron-treated rats, as evidenced by an increase in alkane production and in TBARS in urine in this study, and by an increase in alkane production by slices of kidney from iron-treated rats in a previous study [V. C. Gavino, C. J. Dillard, and A. L. Tappel (1984) Arch. Biochem. Biophys. 233, 741-747]. Since PGE2 excretion in urine was not correlated with these effects, lipid peroxidation appears not to be a major factor in renal PGE2 flux.     

Inhibition of S-(1,2-dichlorovinyl)-L-cysteine-induced lipid peroxidation by antioxidants in rabbit renal cortical slices: Dissociation of lipid peroxidation and toxicity

Precision-cut, rabbit renal slices were used to examine the effects of three novel antioxidants (U-74006, U-74500, and U-78517) on S-(1,2-dichlorovinyl)-L-cysteine (DCVC)-induced lipid peroxidation and toxicity. Slices exposed to DCVC showed a dose- and time-dependent increase in lipid peroxidation (TBARS) and a decrease in cellular viability, as evidenced by the loss of intracellular potassium, during the course of a 3 hour incubation. Subsequent studies employed DCVC concentrations of 100 μM. Microemulsion formulations of U-78517, U-74500, and U-74006 (100 μM) inhibited DCVC-induced lipid peroxidation by 100±, 50±, and <5% (not significant), respectively. However, none of these antioxidants had a significant effect on DCVC-dependent cytotoxicity, as indicated by intracellular potassium release. The effects of U-78517, the most potent of the three antioxidants, were similar to those observed with two model antioxidants, diphenyl-p-phenylenedi-amine (DPPD) and the iron chelator, deferoxamine. Aminooxyacetic (AOAA), an inhibitor of renal cysteine conjugate β-lyase, had only a minimal effect on DCVC-induced lipid peroxidation, and no effect on toxicity. These data represent the first report of DCVC-induced lipid peroxidation in rabbit renal cortical slices, a system which has been widely used to investigate mechanisms of nephrotoxicity, including that induced by DCVC. Our results demonstrate that DCVC-induced lipid peroxidation in renal slices can be inhibited by a variety of antioxidant compounds operating by different mechanisms. Because inhibition of lipid peroxidation had minimal effect on DCVC-dependent cytotoxicity, the data suggest that DCVC-induced lipid peroxidation is not a major mechanism in the cytotoxicity induced by this compound.     

Effect of benzophenones from Hypericum annulatum on carbon tetrachloride-induced toxicity in freshly isolated rat hepatocytes

Five benzophenones and a xanthone, isolated from Hypericum annulatum Moris, were investigated for their protective effect against carbon tetrachloride toxicity in isolated rat hepatocytes. The benzophenones and the xanthone gentisein were administered alone (100 microM) and in combination with CCl4 (86 microM). CCl4 undergoes dehalogenation in the liver endoplasmic reticulum. This process leads to trichlormethyl radical (*CCl3) formation, initiation of lipid peroxidation, and measurable toxic effects on the hepatocytes. The levels of thiobarbituric acid reactive substances (TBARS) were assayed as an index of lipid peroxidation (LPO). Lactate dehydrogenase (LDH) leakage, cell viability and reduced glutathione (GSH) depletion were used as signs of cytotoxicity. CCl4 significantly decreased hepatocyte viability, GSH level and increased TBARS level and LDH leakage as compared to the control. Our data indicate that 2,3',5',6-tetrahydroxy-4-methoxybenzophenone, 2-O-alpha-L-arabinofuranosyl-3',5',6-trihydroxy-4-methoxybenzophenone and 2-O-alpha-L-3'-acetylarabinofuranosyl-3',5',6-trihydroxy-4-methoxybenzophenone showed weaker toxic effects compared to CCl4 and in combination showed statistically significant protection against the toxic agent.     

Damage of rat liver microsomal mixed function oxidase system by carbon tetrachloride. In vivo study with selective inhibitor of lipid peroxidation.

Pretreatment of rats with 4-[4-N-sodium-N-(5-ethyl-1-thia-3,4-diazol-2-yl)sulfophenylamin o]-5- methoxy-1,2-benzoquinone (Q) before carbon tetrachloride intoxication inhibited lipid peroxidation by 85% but did not prevent cytochrome P-450 destruction, decrease of hydroxylase activity, and loss of the capability to bioactivate carbon tetrachloride in rat liver microsomes. Also no influence of Q on 10-day lethality was found. We conclude that covalent binding of free radical products of metabolic cleavage to various cellular structures is apparently the main damage factor of carbon tetrachloride hepatotoxicity rather than lipid peroxidation.     

Release of ethane and pentane from rat tissue slices: effect of vitamin E, halogenated hydrocarbons, and iron overload

The effects of in vitro addition of halogenated hydrocarbons on the susceptibility of various rat tissues to lipid peroxidation, and of iron overload and dietary vitamin E in the intact rat on subsequent lipid peroxidation in rat tissue slices were examined. The ease and speed of tissue slice preparation allowed testing of multiple tissues from the same animals. Total ethane and pentane (TEP) released from the slices was as reliable as and more sensitive than thiobarbituric acid-reactive substances as an index of lipid peroxidation. TEP was released by tissues from vitamin E-deficient rats in the following order of magnitude:intestine = brain = kidney greater than liver = lung greater than heart greater than testes = diaphragm greater than skeletal muscle. The potency of halogenated hydrocarbons for causing increased TEP release from vitamin E-deficient rat liver slices was CBrCl3 greater than CCl4 = 1,1,2,2-tetrabromoethane = 1,1,2,2-tetrachloroethane greater than perchloroethylene. CBrCl3 also stimulated TEP release from kidney, intestine, and heart slices, thus identifying these as potential target organs for CBrCl3 toxicity. Dietary vitamin E decreased TEP release from liver and, to a lesser extent, from kidney. Iron overload in the rat increased TEP release by slices from all tissues tested except the brain.     

Halogenated hydrocarbon and hydroperoxide-induced peroxidation in rat tissue slices

Tissue slices were used to compare relative peroxidation capacity of bromotrichloromethane (BrCCl₃) and t-butyl hydroperoxide (BHP) by measurement of both peroxidation products and biochemical indices of damage. In liver and testes slices, BHP increased thiobarbituric acid reactive-substances (TBARS) and total aldehydes, measured as cyclohexanedione-reactive substances (CHDRS), to a greater extent than did an equimolar amount of BrCCl₃. GSH was decreased more by BHP than by BrCCl₃. Neither compound released lactate dehydrogenase or glutamic-pyruvicf transaminase from liver slices. Treatment of rats with cynamide, an aldehyde dehydrogenase inhibitor, increased the total CHDRS in liver slices and medium after incubation with BHP or BrCCl₃. HPLC of the CHDRS showed hexanal and propanal increased to the greatest extent. The hydroperoxide, BHP, which does not require metabolism to an active species, was a better initiator of peroxidation than the halogenated hydrocarbon, BrCCl₃, which must be metabolized to a radical species before it can initiate peroxidation.     

The Induction of Lipid Peroxidation in Rat Liver by Oral Intake of 9-Oxononanoic Acid Contained in Autoxidized Linoleic Acid

A component in the autoxidation products of linoleic acid (LA) which induced the endogenous lipid peroxidation was searched for. Secondary autoxidation products (SP) of LA induced the production of thiobarbituric acid reactive substances (TBARS) in liver as well as the LA hydroperoxides stimulated. SP were separated chromatographically into some fractions which were administered orally to rats. One fraction composed mainly of 9-oxononanoic acid (9-ONA) markedly stimulated the TBARS production in liver. Authentic 9-ONA also increased the lipid peroxide level as determined by a new test, as well as the activities of glutathione peroxidase and reductase in liver. Thus, hepatic lipid peroxidation was induced by orally given 9-ONA which was present in the autoxidation products of LA.     

Synthesis and evaluation of N-substituted indole-3-carboxamide derivatives as inhibitors of lipid peroxidation and superoxide anion formation

The in vitro antioxidant effects of novel N-substituted indole-3-carboxamides (I3CDs) 1-10 on rat liver microsomal NADPH-dependent lipid peroxidation (LP) levels and their free radicals scavenging properties were determined by the inhibition of superoxide anion formation (SOD). Among the synthesized compounds, 4, 5, 8 and 9 significantly inhibited SOD with an inhibition range at 84-100% at 10(-3) M concentration. The presence of halo substituents both ortho- and para- positions of these compounds resulted 100% inhibition of SOD. Comparison the activity results of halogenated and non-halogenated derivatives suggested that the halogenated compounds are more active than the non-halogenated compounds. On the other hand, the introduction of a para fluoro benzyl in the 1-position of indole (compounds 7, 8) has more impact on the SOD inhibition when the benzamide ring was mono halogenated. However, none of other compounds had a significant inhibitory effects on the level of lipid peroxidation.     

Inhibition of carbon tetrachloride-induced lipid peroxidation by novel antioxidants in rat hepatic microsomes: dissociation from hepatoprotective effects in vivo

The ability of two novel antioxidants, U-74,006F and U-78,517G, as well as the known antioxidant N,N'-diphenyl-p-phenylenediamine to inhibit lipid peroxidation induced by carbon tetrachloride (CCl4) was investigated in Aroclor 1254-induced rat hepatic microsomes. All three compounds completely inhibited lipid peroxidation in microsomes as measured by the formation of thiobarbituric acid reactive substances (TBARS). Inhibition of lipid peroxidation was not a function of decreased bioactivation of CCl4, as the compounds did not substantially inhibit benzphetamine N-demethylase activity or covalent binding of [14-C]CCl4 to lipid or protein. Parallel studies examined the hepatoprotective effects of the compounds in vivo. Rats were pretreated with antioxidant or vehicle prior to administration of CCl4 (300 or 600 microL/kg i.p.). Sera were collected 24 h postadministration of CCl4 and analyzed for alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities and total bilirubin. Administration of CCl4 produced elevations in ALT, moderate changes in bilirubin, and no change in ALP activities. Histological examination of CCl4-treated livers revealed lipidosis and centrilobular necrosis. The antioxidants partially improved the clinical chemistry parameters, but had minimal effects on the histological lesion. In contrast to the complete inhibition of lipid peroxidation observed in the in vitro studies, none of the antioxidants markedly protected against CCl4-induced toxicity in vivo.     

Evaluation of biological effects induced by diagnostic ultrasound in the rat foetal tissues

In recent years, there has been growing interest in estimating the degree of heating caused by the diagnostic ultrasound in clinical practice. Both theoretical and experimental methods have been suggested for estimating the heating potential, or thermal hazard, of diagnostic ultrasound. Aim of this study was to evaluate in vivo effects of ultrasound exposure of variable duration (from 10 up to 20 min) with commercially available imaging systems commonly used for diagnostic imaging. Numerical results related to the thermal effect are obtained by simulation program based on B-mode (scanning) and Doppler (non-scanning). To investigate the biological effects of the ultrasound exposure to the brain and liver tissues, the antioxidant enzyme activity and thiobarbituric acid reactive substances (TBARS) of the tissues were evaluated. In liver tissue, as a lipid peroxidation index, TBARS levels very significantly increase in Doppler group compared to control. However, in B-mode, TBARS levels are the same with the control group. Use of B-mode in foetal tissue is more reliable than Doppler mode because temperature rise is very small compared to the Doppler mode. On the other hand, the antioxidant enzyme activities tend to increase in B-mode and Doppler groups compared to the control group as a defensive mechanism. In the brain tissue, lipid peroxidation is increased slightly in B-mode compared to the control group. This situation is related to the molecular structure of the brain tissue because of its high lipid concentration. In brain tissue, the antioxidant enzyme activities and lipid peroxidation were significantly increased, such as liver tissue in Doppler groups. Doppler ultrasound may produce harmful effects in rat foetus liver and brain tissues as a result of the high temperature rises.     

Protective effect of fosfomycin on gentamicin-induced lipid peroxidation of rat renal tissue

Fosfomycin is clinically recognized to reduce the aminoglycoside antibiotics-induced nephrotoxicity. However, little has been clarified why fosfomycin protects the kidney from the aminoglycosides-induced nephrotoxicity. Gentamicin, a typical aminoglycoside, is reported to cause lipid peroxidation. We focused on lipid peroxidation induced by gentamicin as a mechanism for the aminoglycosides-induced nephrotoxicity. The aim of this study is to investigate the effect of fosfomycin on the gentamicin-induced lipid peroxidation. In rat renal cortex mitochondria, fosfomycin was shown to depress the gentamicin-induced lipid peroxidation, which was evaluated by formation of thiobarbituric acid reactive substances (TBARS). Interestingly, this effect was observed in rat renal cortex mitochondria, but not in rat liver microsomes. However, fosfomycin did not affect lipid peroxidation of arachidonic acid caused by gentamicin with iron. Fosfomycin inhibited the gentamicin-induced iron release from rat renal cortex mitochondria. These results indicated that fosfomycin inhibited the gentamicin-induced lipid peroxidation by depressing the iron release from mitochondria. This may possibly be one mechanism for the protection of fosfomycin against the gentamicin-induced nephrotoxicity.     

Study on lipid peroxidation potential in different tissues induced by ascorbate-Fe2+: Possible factors involved in their differential susceptibility

Susceptibility of four major rat tissues to oxidative damage in terms of lipid peroxidation induced by in vitro by ascorbate-Fe²⁺ in homogenates and mitochondria has been examined. Lipid peroxidation, as assessed by thiobarbituric acid reactive substances (TBARS) and conjugated dienes was maximum in brain followed by liver, kidney and heart. However, the time course of lipid peroxidation showed different patterns in tissues examined. The higher susceptibilities of brain and liver can be explained by substrate availability and to a lesser extent the level of antioxidants. The differences observed in the tissues studied may reflect their susceptibility to degenerative diseases and xenobiotic toxicity which are considered as a result of oxidative damage to membranes.     

Lignans from the Fruits of Forsythia suspensa (Thunb.) Vahl Protect High-Density Lipoprotein during Oxidative Stress

The objective of the present study was to investigate the beneficial properties lignan compounds obtained from the fruits of Forsythia suspensa (Thunb.) Vahl (Oleaceae) for protecting human high-density lipoprotein (HDL) against lipid peroxidation. The isolated compounds (1-8) inhibited the generation of thiobarbituric acid-reactive substances (TBARS) in a dose-dependent manner with IC50 values from 8.5 to 18.7 microM, since HDL oxidation mediated by catalytic Cu2+. They also exerted an inhibitory effect against thermo-labile radical initiator (AAPH)-induced lipid peroxidation of HDL with IC50 values from 12.1 to 51.1 microM. Compounds 1 and 5 exerted inhibitory effects against the Cu2+-induced lipid peroxidation of HDL, as shown by an extended lag time prolongation at the concentration of 3.0 microM. These results suggest that the antioxidative effects of F. suspensa are due to its lignans and that these constituents may be useful for preventing the oxidation of HDL.