Reduction of paraquat and related bipyridylium compounds to free radical metabolites by rat hepatocytes.

The toxicity of paraquat is due to the oxygen-derived radicals formed by the reaction of oxygen with bipyridylium radical cations. Although paraquat is known to cause lung toxicity, the related bipyridylium compounds such as diquat and morfamquat do not affect the lung as seriously, but rather cause liver toxicity. Paraquat, diquat, morfamquat, and benzyl viologen are reduced by rat hepatocytes to their respective radical cations. An intracellular component of the signal was detected from diquat and benzyl viologen radical cations. These radical cations generated inside the cell can cross the plasma membrane. Generation of the diquat radical cation by hepatocytes is not affected by the inhibition of cytochrome P-450 by carbon monoxide or metyrapone, suggesting that this enzyme is probably not involved in the reduction of diquat as had been proposed previously. The reduction of paraquat is generally attributed to NADPH-cytochrome P-450 reductase, and presumably diquat is also reduced by this flavoprotein. Some transition metal chelates such as ferric diethylenetriaminepentaacetic acid delay the detection of the diquat radical cation. This may be due to the reduction of the ferric chelate by the diquat radical cation resulting in the formation of the ferrous chelate and the parent bipyridylium dication. When all the ferric chelate has been reduced to the ferrous chelate, then the bipyridylium radical can be detected. Alternatively, if the ferric chelate enters the cell, it can compete with the parent bipyridylium dication for the reductase, which would also lead to delayed detection.     

Peroxyl radical trapping activity of anthocyanins and generation of free radical intermediates

The inhibition by anthocyanins of the free radical-mediated peroxidation of linoleic acid in a SDS micelle system was studied at pH 7.4 and at 37°C, by oxygraphic and ESR tecniques. The number of peroxyl radicals trapped by anthocyanins and the efficiency of these molecules in the trapping reaction, which are two fundamental aspects of the antioxidant action, were measured and discussed in the light of the molecular structure. In particular the contribution of the substituents to the efficiency is –OH>–OCH₃>–H. By ESR we found that the free radicals of anthocyanins are generated in the inhibition of the peroxidation of linoleic acid. The life time of these radical intermediates, the concentration of which ranges from 7 to 59 nM under our experimental conditions, is strictly correlated with the anthocyanin efficiency and with the heat of formation of the radical, as calculated by a semiempirical molecular orbital approach.     

Peroxyl radical trapping activity of anthocyanins and generation of free radical intermediates

The inhibition by anthocyanins of the free radical-mediated peroxidation of linoleic acid in a SDS micelle system was studied at pH 7.4 and at 37 degrees C, by oxygraphic and ESR tecniques. The number of peroxyl radicals trapped by anthocyanins and the efficiency of these molecules in the trapping reaction, which are two fundamental aspects of the antioxidant action, were measured and discussed in the light of the molecular structure. In particular the contribution of the substituents to the efficiency is -OH>-OCH(3)>-H. By ESR we found that the free radicals of anthocyanins are generated in the inhibition of the peroxidation of linoleic acid. The life time of these radical intermediates, the concentration of which ranges from 7 to 59 nM under our experimental conditions, is strictly correlated with the anthocyanin efficiency and with the heat of formation of the radical, as calculated by a semiempirical molecular orbital approach.     

Metabolic activation of 1,2-dibromoethane to a free radical intermediate by rat liver microsomes and isolated hepatocytes

A one-electron reductive metabolism of 1,2-dibromoethane (DBE) is described that gives rise to a free radical intermediate, which can be stabilized by a spin trapping agent and detected by electron spin resonance spectroscopy. Using rat liver microsomes or isolated hepatocytes from phenobarbitone pretreated animals, under hypoxic conditions, it has been possible to trap a free radical intermediate and identify it by using ¹³C-DBE. Inhibition experiments have demonstrated that the site of activation is the microsomal drug metabolizing system.     

Activation and deactivation of aflatoxin B1 in isolated rat hepatocytes

To characterize the true substrate for aldolase from Clostridium perfringens (optimum pH = 7.2) several experiments were carried out wherein the substrate Neu5Ac was generated in situ at pH 5.4 by the action of sialidase on its substrate Neu5Ac(alpha, 2 leads to 3) lactose. The alpha-anomer formed in this reaction was found to be split by aldolase at this pH into ManNAc and pyruvate. beta-Neu5Ac as such was not converted at pH 5.4. However, when it was first mutarotated until the equilibrium mixture alpha:beta = 7.2:92.8 was obtained, it could be split. Inhibition experiments suggested that Neu5Ac was bound to the enzyme in a conformation that strongly resembled that of its alpha-anomer. The open chain form of ManNAc which arose after the action of aldolase preferentially formed the alpha-anomer followed by a fast mutarotation.     

Rapid stimulation of free glucuronate formation by non-glucuronidable xenobiotics in isolated rat hepatocytes

Vitamin C synthesis in rat liver is enhanced by several xenobiotics, including aminopyrine and chloretone. The effect of these agents has been linked to induction of enzymes potentially involved in the formation of glucuronate, a precursor of vitamin C. Using isolated rat hepatocytes as a model, we show that a series of agents (aminopyrine, antipyrine, chloretone, clotrimazole, metyrapone, proadifen, and barbital) induced in a few minutes an up to 15-fold increase in the formation of glucuronate, which was best observed in the presence of sorbinil, an inhibitor of glucuronate reductase. They also caused an approximately 2-fold decrease in the concentration of UDP-glucuronate but little if any change in the concentration of UDP-glucose. Depletion of UDP-glucuronate with resorcinol or d-galactosamine markedly decreased the formation of glucuronate both in the presence and in the absence of aminopyrine, confirming the precursor-product relationship between UDP-glucuronate and free glucuronate. Most of the agents did not induce the formation of detectable amounts of glucuronides, indicating that the formation of glucuronate is not due to a glucuronidation-deglucuronidation cycle. With the exception of barbital (which inhibits glucuronate reductase), all of the above mentioned agents also caused an increase in the concentration of ascorbic acid. They had little effect on glutathione concentration, and their effect on glucuronate and vitamin C formation was not mimicked by glutathione-depleting agents such as diamide and buthionine sulfoximine. It is concluded that the stimulation of vitamin C synthesis exerted by some xenobiotics is mediated through a rapid increase in the conversion of UDP-glucuronate to glucuronate, which does not apparently involve a glucuronidation-deglucuronidation cycle.     

The metabolism of halothane by hepatocytes: a comparison between free radical spin trapping and lipid peroxidation in relation to cell damage

The technique of free radical spin trapping has been applied to demonstrate the formation of free radicals produced during the metabolism of halothane by rat liver hepatocytes under hypoxic conditions. The results obtained support previous findings that reported sex differences in the metabolic activation of halothane by rats in vivo. Cell viability under hypoxic conditions, as judged by trypan blue staining and lactate dehydrogenase release, shows a correlation with the extent of metabolism of halothane as measured by electron spin resonance spectroscopy. The extent of lipid peroxidation was measured by diene conjugation, malondialdehyde production and chemiluminescence. The latter technique allowed the demonstration of lipid peroxidation during incubations of hepatocytes under aerobic conditions. The magnitude of the aerobic chemiluminescence showed a similar sex dependency to the extent of free radical formation under hypoxic conditions. Cell viability measurements show that halothane metabolism in both hypoxic and aerobic conditions can lead to cell death. Consequently, oxidative lipid damage could be a cause of cell damage, as judged by cell viability, additional to covalent binding.     

PGE1 protection against apoptosis induced by D-galactosamine is not related to the modulation of intracellular free radical production in primary culture of rat hepatocytes

d -galactosamine ( d -GalN) toxicity is a useful experimental model of liver failure in human. It has been previously observed that PGE 1 treatment reduced necrosis and apoptosis induced by d -GalN in rats. Primary cultured rat hepatocytes were used to evaluate if intracellular oxidative stress was involved during the induction of apoptosis and necrosis by d -GalN (0-40 mM). Also, the present study investigated if PGE 1 (1 μM) was equally potent reducing both types of cell death. The presence of hypodiploid cells, DNA fragmentation and caspase-3 activation were used as a marker of hepatocyte apoptosis. Necrosis was measured by lactate dehydrogenase (LDH) release. Oxidative stress was evaluated by the intracellular production of hydrogen peroxide (H 2 O 2 ), the disturbances on the mitochondrial transmembrane potential (MTP), thiobarbituric-reacting substances (TBARS) release and the GSH/GSSG ratio. Data showed that intermediate range of d -GalN concentrations (2.5-10 mM) induced apoptosis in association with a moderate oxidative stress. High d -GalN concentration (40 mM) induced a reduction of all parameters associated with apoptosis and enhanced all those related to necrosis and intracellular oxidative stress, including a reduction of GSH/GSSG ratio and MTP in comparison with d -GalN (2.5-10 mM)-treated cells. Although PGE 1 reduced apoptosis induced by d -GalN, it was not able to reduce the oxidative stress and cell necrosis induced by the hepatotoxin in spite to its ability to abolish the GSH depletion.     

Regulation of free and bound magnesium in rat hepatocytes and isolated mitochondria

Null point titration techniques have been developed for measurements of cytosolic free Mg2+ in isolated cells and matrix free Mg2+ in isolated mitochondria using antipyrylazo III as a spectrophotometric Mg2+ indicator. A cytosolic free Mg2+ of 0.37 +/- 0.02 mM was obtained with hepatocytes. This represented about 6% of the total cytosolic magnesium content (activity coefficient of 5.8 X 10(-2). Nondiffusable Mg2+-binding sites in the cytosol were equal to 11.1 nmol/mg cell dry weight with an apparent dissociation constant of 0.71 mM and accounted for binding of 32% of the cytosolic magnesium. The null point method gave a value of 0.35 +/- 0.01 mM for the mitochondrial matrix free Mg2+ concentration (activity coefficient of 8.8 X 10(-3). Nondiffusable Mg2+ binding sites in the mitochondria were estimated at 25.7 nmol/mg mitochondrial protein with an apparent dissociation constant of 0.22 mM, compared with an apparent dissociation constant of 1.66 microM for bound calcium. These data demonstrate the absence of a significant gradient of free Mg2+ between the cytosolic and mitochondrial compartments. They also demonstrate a high ligand binding capacity for magnesium in both compartments with relatively low affinity resulting in a constant value for free Mg2+ when total cell magnesium is constant. This maintains a ratio between free Mg2+ and free Ca2+ of about 2000 in the cytosol and 100 in the mitochondria. The high concentration and low affinity of Mg2+ binding sites results in rather large changes of free Mg2+ with small variations in total cell magnesium. This is apparent in hepatocytes isolated from streptozotocin diabetic rats which had a decreased total magnesium content and a cytosolic free Mg2+ of 0.16 +/- 0.02 mM.     

Salicylate as an in vivo free radical trap: studies on ischemic insult to the rat intestine.

Ischemia of rat intestine was induced in vivo by occlusion of the superior mesenteric artery (SMA) for 15 min. Sodium salicylate, 100 mg/kg, given IP, 30 min prior to the ischemic event served as a specific trap for hydroxyl radicals. Portions of the bowel were sequentially isolated and removed--2 min prior to ischemia, 2 min prior to declamping of the SMA, and 10 min following reperfusion. The bowel segments were homogenized in 3% TCA. The homogenate was centrifuged and filtrated through a 0.22 mu filter. The hydroxylation products of salicylate, dihydroxybenzoic acid (DHBA) derivatives, were isolated, identified, and quantified by HPLC coupled with electrochemical detection (ECD). The level of 2,5-DHBA (M +/- SE, ng/g tissue) in the preischemic bowel (N = 21) was 241.8 +/- 10.0. In the ischemic specimen the level of 2,5-DHBA increased significantly to 313.3 +/- 15.5 (p = 0.0129), and remained unchanged in the reperfusion period (322.8 +/- 15.5). The histological examination correlated well with these levels: mild villi damage in the ischemic period with no further exacerbation during the reperfusion period. This study in an in vivo animal model of intestinal ischemia-reperfusion provides direct evidence for the involvement of free radicals during the ischemic insult.     

Hydroxyl radical formation in chondrocytes and cartilage as detected by electron paramagnetic resonance spectroscopy using spin trapping reagents

Chondrocytes have been shown to produce superoxide and hydrogen peroxide, suggesting possible formation of hydroxyl radical in these cells. In this study, we used electron spin resonance/spin trapping technique to detect hydroxyl radicals in chondrocytes. We found that hydroxyl radicals could be detected as α-hydroxyethyl spin trapped adduct of 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN) in chondrocytes stimulated with phorbol 12-myristate 13-acetate in the presence of ferrous ion. The formation of hydroxyl radical appears to be mediated by the transition metal-catalyzed Haber-Weiss reaction since no hydroxyl radical was detected in the absence of exogenous iron. The hydroxyl radical formation was inhibited by catalase but not by superoxide dismutase, suggesting that the hydrogen peroxide is the precursor. Cytokines, IL-1 and TNF enhanced the hydroxyl radical formation in phorbol 12-myristate 13-acetate treated chondrocytes. Interestingly, hydroxyl radical could be detected in unstimulated fresh human and rabbit cartilage tissue pieces in the presence of iron. These results suggest that the formation of hydroxyl radical in cartilage could play a role in cartilage matrix degradation.     

Confirmation of assignment of the trichloromethyl radical spin adduct detected by spin trapping during 13C-carbon tetrachloride metabolism in vitro and in vivo

Rat liver microsomal incubation systems containing the free radical spin trap, phenyl-t-butyl nitrone, as well as an NADPH generating system and [¹³C]CCl₄ (90 atom % ¹³C) produce electron spin resonance spectra consistent with that expected for a trichloromethyl-phenyl-t-butyl nitrone adduct. This same spectrum is observed in a lipid extract of the liver from a rat orally administered [¹³C]CCl₄ as well as in a solution of phenyl-t-butyl nitrone and [¹³C]CCl₄ irradiated with ultraviolet light.     

Activation of phospholipid methyltransferase by glucagon in rat hepatocytes

Glucagon produces a time- and dose-dependent activation of phospholipid methyltransferase activity in isolated rat hepatocytes. Half-maximal effect is caused by a dose of glucagon of 1 x 10(-10) M. This activation is due to an increase of the Vmax value of the enzyme, without affecting the Km value for S-adenosylmethionine. Exogenous cyclic AMP added to isolated rat hepatocytes mimics the effect of glucagon, and the activation of phospholipid methyltransferase by a nonsaturating concentration of glucagon is spontaneously reversible within 40 min of incubation.     

Apoptosis induced in rat hepatocytes by in vivo exposure to taurochenodeoxycholate

Enzymatic and molecular cytochemistry was used to detect and follow the hepatotoxic effects caused in overnight-fasted Sprague--Dawley rats by a 1-h continuous intrafemoral infusion of taurochenodeoxycholate at 0.4 and 0.8 μmol^?1 min^?1 100 g^?1 body weight dose levels. Rats were killed at 0, 1 and 24 h from the end of perfusion. Their livers were examined for morphology, DNA fragmentation (by a TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labelling assay), cell regeneration (by in vivo bromodeoxydurine incorporation), reduced glutathione, calcium and several enzyme cytochemical activities. Isolated injured hepatocytes randomly scattered throughout the liver were already evident at the end of perfusion. DNA fragmentation and cytoplasm shrink age were prominent and early features of injured hepatocytes, which later showed calcium loading and chromatin clumping. Preserved cytochemical enzymatic activities indicated that plasma and mitochondria membranes were not severely damaged. Inflammatory response was absent. These observations indicate that an acute exposure to taurochenodeoxycholate induces a cell death process with apoptotic features     

Age and sex related changes of plasma membrane fluidity in isolated rat hepatocytes

The influence of sex and age on membrane fluidity, has been investigated in 6, 12, 18 weeks old Sprague-Dawley rats. Fluorescence polarization (P) was determined at 37 degrees C with a Perkin Elmer MPF 44A fluorescence spectrophotometer. The fluorescent probe TMA-DPH was added to isolated hepatocytes prepared by collagenase method. The membrane fluidity was constantly lower in males than in females, but the difference was statistically significant only in the 12 weeks old group. Major differences appeared related to aging with a significant age-related decrease in fluidity in all animals.     

Apoptosis induced in rat hepatocytes by in vivo exposure to taurochenodeoxycholate

Enzymatic and molecular cytochemistry was used to detect and follow the hepatotoxic effects caused in overnight-fasted Sprague--Dawley rats by a 1-h continuous intrafemoral infusion of taurochenodeoxycholate at 0.4 and 0.8 μmol⁻¹ min⁻¹ 100 g⁻¹ body weight dose levels. Rats were killed at 0, 1 and 24 h from the end of perfusion. Their livers were examined for morphology, DNA fragmentation (by a TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labelling assay), cell regeneration (by in vivo bromodeoxydurine incorporation), reduced glutathione, calcium and several enzyme cytochemical activities. Isolated injured hepatocytes randomly scattered throughout the liver were already evident at the end of perfusion. DNA fragmentation and cytoplasm shrink age were prominent and early features of injured hepatocytes, which later showed calcium loading and chromatin clumping. Preserved cytochemical enzymatic activities indicated that plasma and mitochondria membranes were not severely damaged. Inflammatory response was absent. These observations indicate that an acute exposure to taurochenodeoxycholate induces a cell death process with apoptotic features This revised version was published online in November 2006 with corrections to the Cover Date.     

Cholesterogenesis and related enzymes in isolated rat hepatocytes during pre- and postnatal life

Cholesterogenesis pathway during pre- and postnatal development was studied in isolated rat hepatocytes. No modified activity of cytosol acetoacetyl coenzyme A (CoA), thiolase, or 3-hydroxy-3-methylglutaryl CoA (HMGCoA) synthase was detectable at the different stages examined. Minimal levels of 1(14)C-acetate incorporation into cholesterol and HMGCoA reductase activity were present at 16 days of fetal development in newborn and suckling rats, whereas both parameters increased rapidly before birth. The pattern of NaF nonsuppressible reductase activity showed a different activation state of the enzyme, suggesting the appearance of a modulation state, probably related to the development of some short-term regulatory mechanisms.     

Activation of branched-chain alpha-ketoacid dehydrogenase in isolated hepatocytes by branched-chain alpha-ketoacids

The effects of branched-chain alpha-ketoacids on flux through and activity state of the branched-chain alpha-ketoacid dehydrogenase complex were studied in hepatocytes prepared from chow-fed, starved, and low-protein-diet-fed rats. Very low concentrations of alpha-ketoisocaproate caused a dramatic stimulation (50% activation at 20 microM) of alpha-ketoisovalerate decarboxylation in hepatocytes from low-protein-fed rats. alpha-Keto-beta-methylvalerate was also effective, but less so than alpha-ketoisocaproate. alpha-Ketoisocaproate did not stimulate alpha-ketoisovalerate decarboxylation by hepatocytes from chow-fed or starved rats. To a smaller degree, alpha-keto-beta-methylvalerate and alpha-ketoisovalerate stimulated alpha-ketoisocaproate decarboxylation by hepatocytes from low-protein-fed rats. The implied order of potency of stimulation of flux through branched-chain alpha-ketoacid dehydrogenase was alpha-ketoisocaproate greater than alpha-keto-beta-methylvalerate greater than alpha-ketoisovalerate, i.e., the same order of potency of these compounds as branched-chain alpha-ketoacid dehydrogenase kinase inhibitors. Fluoride, known to inhibit branched-chain alpha-ketoacid dehydrogenase phosphatase, largely prevented alpha-ketoisocaproate and alpha-chloroisocaproate activation of flux through the branched-chain alpha-ketoacid dehydrogenase. Assay of the branched-chain alpha-ketoacid complex in cell-free extracts of hepatocytes isolated from low-protein-diet-fed rats confirmed that alpha-ketoacids affected the activity state of the complex. Branched-chain alpha-ketoacids failed to activate flux in hepatocytes prepared from chow-fed and starved rats because essentially all of the complex was already in the dephosphorylated, active state. These findings indicate that inhibition of branched-chain alpha-ketoacid dehydrogenase kinase activity by branched-chain alpha-ketoacids is important for regulation of the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase.     

Aziridine biotransformation by microsomes and lethality to hepatocytes isolated from rat

To clarify the relationship of aziridine biotransformation to their cytotoxic activities, the metabolism of optical isomers of typical cytotoxic and non-cytotoxic aziridines was studied in isolated hepatocytes, rat liver microsomes, mitochondria and L-1210 mouse leukemia cells. Cytotoxic 1-methyl-2-beta-naphthylaziridine (NAZ) gave nitrosomethane as one of the bioactivation products in isolated hepatocytes and simultaneously induced a marked decrease in cellular ATP followed by cell lethality. NAZ itself did not directly affect the respiratory function of mitochondria in isolated hepatocytes or in buffer solution, however, it inhibited the mitochondrial activity in the presence of microsomes in the buffer solution. Nitroso-t-butane or nitrosomethane dimer, used as a substitute for extremely labile nitrosomethane, strongly inhibited the respiration of mitochondria. On the other hand, optical isomers of 2-aziridinecarboxylic acid (AZC) which did not give nitrosomethane in isolated hepatocytes or microsomes also did not show cytotoxicity. Thus, the cytotoxicity of NAZ seems to be induced by bioactivation via cellular oxidases with the nitrosomethane generated being a major toxic component. This may occur with most of the cytotoxic aziridine derivatives.     

Alkyl free radicals from the beta-scission of fatty acid alkoxyl radicals as detected by spin trapping in a lipoxygenase system

2-Methyl-2-nitrosopropane (tNB)-radical adducts from incubation mixtures of fatty acids and soybean lipoxygenase in borate buffer (pH 9.0) were measured by electron paramagnetic resonance (EPR). In addition to the previously reported six-line signal of secondary carbon-centered radicals (RCHR'), a weak signal submerged in the baseline was detected after the peroxidation phase was finished. We propose that this radical is a decomposition product formed via beta-scission of fatty acid alkoxyl radicals. EPR spectra of tNB-radical adducts formed in mixtures of either linoleic acid, arachidonic acid, or 15-hydroperoxyeicosatetraenoic acid with lipoxygenase exhibited hyperfine structure characteristic of tNB/.CH2CH2-R with hyperfine coupling constants: aN = 17.1 G; aH beta = 11.2 G (2H); and aH gamma = 0.6 G (2H). In the case of linolenic acid, this radical tNB/.CH=CH-R' with hyperfine coupling constants: aN = 17.1 G; aH beta = 10.9 G (2H); aH gamma = 1.1 G; and aH delta = 0.5 G. In accord with the decomposition scheme of hydroperoxides derived from unsaturated fatty acids, the radical adducts tNB/.CH2CH2-R and tNB/.CH2-CH=CH-R' were assigned as the pentyl and 2-pentenyl radicals, respectively.