Inhibition of protein synthesis by carbonyl compounds (4-hydroxyalkenals) originating from the peroxidation of liver microsomal lipids

Carbonyl compounds released during the NADPH-Fe dependent peroxidation of liver microsomal lipids and identified as 4-hydroxyalkenals (almost entirely as 4-hydroxynonenal) inhibit protein synthesis in a rabbit reticulocyte lysate. The ID₅₀ was 0.48 mM. The inhibitory effect was reproduced by synthetic 4-hydroxynonenal. The inhibition was already evident at 1–2 min of incubation. The addition of ?SH groups to the incubation medium afforded a marked protection against the inhibition of protein synthesis. The inhibitory effect seems to be due to an interaction of the carbonyl compound with ?SH groups essential for the cellular protein synthetic machinery.     

Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids

During the NADPH-Fe induced peroxidation of liver microsomal lipids, products are formed which show various cytopathological effects including inhibition of microsomal glucose-6-phosphatase. The major cytotoxic substance has been isolated and identified as 4-hydroxy-2,3-trans-nonenal. The structure was ascertained by means of ultraviolet, infrared and mass spectrometry and high-pressure liquid chromatographic analysis. Moreover, 4-hydroxynonenal, prepared by chemical synthesis, was found to reproduce the biological effects brought about by the biogenic aldehyde. Preliminary investigations suggest that as compared to 4-hydroxynonenal very low amounts of other 4-hydroxyalkenals, namely 4-hydroxyoctenal, 4-hydroxydecenal and 4-hydroxyundecenal are also formed by actively peroxidizing liver microsomes. In the absence of NADPH-Fe liver microsomes produced only minute amounts of 4-hydroxyalkenals. The biochemical and biological effects of synthetic 4-hydroxyalkenals have been studied in great detail in the past. The results of these investigations together with the finding that 4-hydroxyalkenals, in particular 4-hydroxynonenal, are formed during NADPH-Fe stimulated peroxidation of liver microsomal lipids, may help to elucidate the mechanism by which lipid peroxidation causes deleterious effects on cells and cell constituents.     

Cytotoxic aldehydes originating from the peroxidation of liver microsomal lipids: Identification of 4,5-dihydroxydecenal

During the NADPH-Fe-induced peroxidation of liver microsomal lipids products are formed which are provided with cytopathological activities. In a previous study one of the major products was identified as an aldehyde of the 4-hydroxyalkenal class, namely 4-hydroxynonenal. In the present study another cytotoxic product has been isolated and identified as 4,5-dihydroxy-2,3-decenal. The isolation was performed by means of thin-layer chromatography and high-pressure liquid chromatography and the structure was ascertained mainly by means of mass spectroscopy of the free aldehyde and of its derivatives. In the absence of NADPH-Fe liver microsomes produced no 4,5-dihydroxydecenal. The inhibitory activity of 4,5-dihydroxydecenal on microsomal glucose-6-phosphatase is somewhat lower than that exhibited by 4-hydroxynonenal. This lower inhibitory activity correlates with the lower capacity to bind to the microsomal protein of 4,5-dihydroxydecenal as compared to 4-hydroxynonenal. The reactivities of the two aldehydes with cysteine were comparable. The production of toxic aldehydes may represent a mechanism by which lipid peroxidation causes deleterious effects on cellular functions.     

Cytotoxic aldehydes originating from the peroxidation of liver microsomal lipids. Identification of 4,5-dihydroxydecenal

During the NADPH-Fe-induced peroxidation of liver microsomal lipids products are formed which are provided with cytopathological activities. In a previous study one of the major products was identified as an aldehyde of the 4-hydroxyalkenal class, namely 4-hydroxynonenal. In the present study another cytotoxic product has been isolated and identified as 4,5-dihydroxy-2,3-decenal. The isolation was performed by means of thin-layer chromatography and high-pressure liquid chromatography and the structure was ascertained mainly by means of mass spectroscopy of the free aldehyde and of its derivatives. In the absence of NADPH-Fe liver microsomes produced no 4,5-dihydroxydecenal. The inhibitory activity of 4,5-dihydroxydecenal on microsomal glucose-6-phosphatase is somewhat lower than that exhibited by 4-hydroxynonenal. This lower inhibitory activity correlates with the lower capacity to bind to the microsomal protein of 4,5-dihydroxydecenal as compared to 4-hydroxynonenal. The reactivities of the two aldehydes with cysteine were comparable. The production of toxic aldehydes may represent a mechanism by which lipid peroxidation causes deleterious effects on cellular functions.     

Impaired calcium sequestration activity in liver microsomes from fasted rats

Calcium uptake activity was assayed in liver microsomal vesicles from fed and fasted rats. This activity required ATP and was stimulated by the calcium trapping agent oxalate. The most striking feature was the low rate of calcium accumulation in liver microsomes from fasted rats. Maximal rate was inhibited up to 66 and 82% after 1 and 3 days starvation, respectively. This defective microsomal calcium handling suggests its possible involvement in the massive glycogen breakdown during starvation.     

Binding of products originating from the peroxidation of liver microsomal lipids to the non-lipid constituents of the microsomal membrane.

The binding of products derived from the peroxidation of liver microsomal lipids to the non-lipid constituents of the microsomes was studied. To this end arachidonic acid labelled with tritium at the positions of the double bonds was given to rats and allowed to incorporate into the membrane lipids of the liver cell. When liver microsomes containing labelled arachidonic acid were incubated aerobically in the NADPH-dependent system, a marked production of malonic dialdehyde (MDA) occurred and, concomitantly, there was a consistent release of radioactivity from the microsomes into the incubation medium. The addition of EDTA to the incubation medium prevented, to a large extent, both the MDA formation and the release of radioactivity. Chromatographic studies showed that the bulk of the radioactivity released from the incubated microsomes is not MDA. In the incubated microsomes, the radioactivity decreased in total lipids, while it increased by about 15 times in the non-lipoidal residue. A similar increase in radioactivity was seen in microsomal protein, while no increase was observed in microsomal RNA (the radioactivity was negligible in both the incubated and the non-incubated samples). It seems therefore that products originating from lipoperoxidation of arachidonic acid covalently bind to the microsomal protein. In order to investigate whether alterations similar to those observed in the in vitro peroxidation of liver microsomes could be detected in the in vivo intoxication with carbon tetrachloride, rats given labelled arachidonic acid as above, were poisoned with CCl4. Sixty minutes after poisoning, the radioactivity present in the microsomal lipids was generally lower in the intoxicated rats than in the controls, while the labelling of the non-lipoidal residue and of the protein was higher in the CCl4-poisoned rats.     

Inhibition of liver microsomal lipid peroxidation by 13-cis-retinoic acid

The effects of 13-cis-retinoic acid on iron/ascorbate-dependent lipid peroxidation were investigated with rat liver microsomes. 13-cis-retinoic acid effectively inhibited malondialdehyde generation and molecular oxygen consumption associated with lipid peroxidation. Under the conditions employed, inhibition was complete at concentrations as low as 25 microM and the IC50 was 10 microM. Evidence for concomitant retinoid oxidation by microsomal unsaturated fatty acid-derived peroxyl radicals was demonstrated by detection of several retinoid-derived metabolites, including 5,8-oxy-13-cis-retinoic acid, generated during lipid peroxidation. The data indicate that 13-cis-retinoic acid inhibits lipid peroxidation by scavenging lipid peroxyl radicals with its conjugated polyene system. Its antioxidant properties may contribute to the pharmacological activities of this and related retinoids.     

Effect of t-butyl hydroperoxide on liver microsomal membranes and microsomal calcium sequestration

In vitro exposure of hepatocytes or liver microsomes to t-butyl hydroperoxide resulted in a marked decrease of liver microsomal calcium pump activity. Decreased calcium pump activity was dependent upon both concentration and time. Liver microsomes could be protected from this effect by glutathione or dithiothreitol. In addition to decreased calcium pump activity, exposure of liver microsomes to t-butyl hydroperoxide produced a concentration-dependent aggregation of microsomal membrane protein as determined by polyacrylamide gel electrophoresis. Inhibition of microsomal calcium pump activity was observed when intact hepatocytes were incubated, in vitro, with t-butyl hydroperoxide. However, aggregation of microsomal membrane protein was not observed when hepatocytes were incubated with t-butyl hydroperoxide. The effects produced by exposure of liver microsomes to this compound do not appear to be a complete model of actions of the compound on intact cells.     

Effects of diffusible products of peroxidation of rat liver microsomal lipids

The effects on cellular structures of products of peroxidation of rat liver microsomal lipids were investigated. A system containing actively peroxidizing liver microsomal fraction was separated from a revealing or target system by a dialysis membrane. The target system, contained in the dialysis tube, consisted of either intact cells (erythrocytes) or subcellular fractions (liver microsomal fraction). When liver microsomal fractions were incubated with NADPH (or an NADPH-generating system), lipid peroxidation, as measured by the amount of malonaldehyde formed, occurred very rapidly. The malon-aldehyde concentration tended to equilibrate across the dialysis membrane. When the target system consisted of erythrocytes, haemolysis occurred abruptly after a lag phase. The lysis was greatly accelerated when erythrocytes from vitamin E-deficient rats were used, but no haemolysis was observed when erythrocytes from vitamin E-treated rats were used. When, in the same system, freshly prepared liver microsomal fractions were exposed to diffusible factors produced by lipid peroxidation, the glucose 6-phosphatase activity markedly decreased. A similar decrease in glucose 6-phosphatase activity, as well as a smaller but significant decrease in cytochrome P-450, was observed when the target microsomal fractions were exposed to diffusible factors derived from the peroxidation of liver microsomal lipids in a separate preincubation step. These and additional experiments indicated that the toxicological activity is relatively stable. Experiments in which the hepatic microsomal fractions destined for lipid peroxidation contained radioactively labelled arachidonic acid, previously incorporated into the membranes, showed that part of the radioactivity released from the microsomal fraction into the incubation medium entered the dialysis tube and was recovered bound to the constituents of the microsomal fractions of the target system. These results indicate that during the course of the peroxidation of liver microsomal lipids toxic products are formed that are able to induce pathological effects at distant loci.     

Inhibition of microsomal calcium sequestration causes an impairment of initiation of protein synthesis in perfused rat liver

The present study examined the effect of 2,5-di-(tert-butyl)-hydroquinone (tBuHQ), an inhibitor of liver microsomal calcium sequestration, on initiation of protein synthesis in perfused rat liver. Perfusion of livers with a concentration of tBuHQ previously shown to completely inhibit microsomal calcium sequestration in isolated hepatocytes caused a 50% inhibition of protein synthesis. The inhibition was characterized by an increase in liver content of free ribosomal particles and a decrease in polysomes indicating that peptide-chain initiation was slowed relative to elongation. Furthermore, the inhibition was associated with a 7.5-fold increase in the proportion of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2) present in the phosphorylated form and a reduction in the activity of eukaryotic initiation factor 2B (eIF-2B) to 37% of the control value. The results suggest that protein synthesis in rat liver is regulated directly by changes in intracellular calcium concentration through a mechanism involving modulation of the phosphorylation state of eIF-2 alpha.     

Signaling properties of 4-hydroxyalkenals formed by lipid peroxidation in diabetes

Peroxidation of polyunsaturated fatty acids is intensified in cells subjected to oxidative stress and results in the generation of various bioactive compounds, of which 4-hydroxyalkenals are prominent. During the progression of type 2 diabetes mellitus, the ensuing hyperglycemia promotes the generation of reactive oxygen species (ROS) that contribute to the development of diabetic complications. It has been suggested that ROS-induced lipid peroxidation and the resulting 4-hydroxyalkenals markedly contribute to the development and progression of these pathologies. Recent findings, however, also suggest that noncytotoxic levels of 4-hydroxyalkenals play important signaling functions in the early phase of diabetes and act as hormetic factors to induce adaptive and protective responses in cells, enabling them to function in the hyperglycemic milieu. Our studies and others′ have proposed such regulatory functions for 4-hydroxynonenal and 4-hydroxydodecadienal in insulin secreting β-cells and vascular endothelial cells, respectively. This review presents and discusses the mechanisms regulating the generation of 4-hydroxyalkenals under high glucose conditions and the molecular interactions underlying the reciprocal transition from hormetic to cytotoxic agents.     

Active calcium sequestration by intestinal microsomes. Stimulation by increased calcium load

Calcium uptake by an endoplasmic reticulum-enriched membrane fraction isolated from rat small intestine was investigated using a rapid filtration technique. Calcium sequestration was stimulated by the presence of ATP and released by the calcium ionophore A23187. ATP stimulation of calcium uptake was dependent on the presence of magnesium, inhibited by vanadate, and refractory to calmodulin. Kinetic studies revealed a K0.5 for the ATP-stimulated uptake of 62.5 nM Ca and a Jmax of 1.4 nmol of Ca/mg protein X min. A high dietary calcium load stimulated maximal uptake by 80% with no change in affinity. The magnitude of maximal uptake and the high affinity of this transport system suggest that the endoplasmic reticulum may play a significant role in cytosolic calcium sequestration and that extracellular calcium leads to modulation of intracellular endoplasmic reticulum calcium buffering.     

Inhibition of free radical-induced peroxidation of rat liver microsomes by resveratrol and its analogues

Resveratrol (3,5,4'-trans-trihydroxystilbene) is a natural phytoalexin present in grapes and red wine, which possesses a variety of biological activities including antioxidative activity. To find more efficient antioxidants by structural modification, resveratrol analogues, that is, 3,4-dihydroxy-trans-stilbene (3,4-DHS), 4,4'-dihydroxy-trans-stilbene (4,4'-DHS), 4-hydroxy-trans-stilbene (4-HS) and 3,5-dihydroxy-trans-stilbene (3,5-DHS), were synthesized and their antioxidant activity studied for the free radical-induced peroxidation of rat liver microsomes in vitro. The peroxidation was initiated by either a water-soluble azo compound 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH) or Fe(2+)/ascorbate, and monitored by oxygen uptake and formation of thiobarbituric acid reactive substances (TBARS). It was found that all of these trans-stilbene derivatives are effective antioxidants against both AAPH- and iron-induced peroxidation of rat liver microsomes with an activity sequence of 3,4-DHS>4,4'-DHS>resveratrol>4-HS>3,5-DHS. The remarkably higher antioxidant activity of 3,4-DHS is discussed.     

NADPH-dependent reductase solubilized from microsomes by peroxidation and its activity

Isolated rat liver microsomes were subjected to enzymatic or non-enzymatic lipid peroxidation $\text{in vitro}$. NADPH-dependent cytochrome $\text{c}$ reductase activity was released from the microsomes into the media during peroxidation. This activity could be recovered from the media by DEAE-cellulose chromatography. The recovered enzyme retained high activity for the reduction of cytochrome $\text{c}$ and a lower level of activity for the reduction of cytochrome P-450. The active fractions were capable of enzymatically supporting the peroxidation of isolated mitochondria in the presence of organically complexed Fe⁺³ and NADPH, and in this respect the specific activity was found to be about ten times higher than in microsomes.     

Inhibition of protein carbonyl formation and lipid peroxidation by glutathione in rat liver microsomes.

The peroxidation of rat liver microsomal lipids is stimulated in the presence of iron by the addition of NADPH or ascorbate and is inhibited by the addition of glutathione (GSH). The fate of GSH and the oxidative modification of proteins under these conditions have not been well studied. Rat liver microsomes were incubated at 37 degrees C under 95% O2:5% CO2 in the presence of 10 microM ferric chloride, 400 microM ADP, and either 450 microM ascorbic acid or 400 microM NADPH. Lipid peroxidation was assessed in the presence 0, 0.2, 0.5, 1, or 5 mM GSH by measuring thiobarbituric acid reactive substance (TBARS) and oxidative modification of proteins by measuring protein thiol and carbonyl groups. GSH inhibited TBARS and protein carbonyl group formation in both ascorbate and NADPH systems in a dose-dependent manner. Heat denaturing of microsomes or treatment with trypsin resulted in the loss of this protection. The formation of protein carbonyl groups could be duplicated by incubating microsomes with 4-hydroxynonenal. Ascorbate-dependent peroxidation caused a loss of protein thiol groups which was diminished by GSH only in fresh microsomes. Both boiling and trypsin treatment significantly decreased the basal protein thiol content of microsomes and enhanced ascorbate-stimulated lipid peroxidation. Protection against protein carbonyl group formation by GSH correlated with the inhibition of lipid peroxidation and appeared not to be due to the formation of the GSH conjugate of 4-hydroxynonenal as only trace amounts of this conjugate were detected. Ninety percent of the GSH lost after 60 min of peroxidation was recoverable as borohydride reducible material in the supernatant fraction. The remaining 10% could be accounted for as GSH-bound protein mixed disulfides. However, only 75% of the GSH lost during peroxidation appeared as glutathione disulfide, suggesting that some was converted to other soluble borohydride reducible forms. These data support a role for protein thiol groups in the GSH-mediated protection of microsomes against lipid peroxidation.     

Inhibition of rat liver microsomal lipid peroxidation by N-acyldehydroalanines: an in vitro comparative study

Captodative substituted olefins are radical scavengers which react with free radicals to form stabilized radical adducts. One of those compounds, N-(paramethoxyphenylacetyl)dehydroalanine (AD-5), may react and scavenge both superoxide anion (O-2) and alk-oxyl radicals (RO.), and in this way prevent the appearance of their mediated biological effects. Nitrofurantoin and tert-butyl hydroperoxide were used as model compounds to stimulate free radical production and their mediated lipid peroxidation in rat liver microsomes. In addition, lipid peroxidation was also initiated by exposure of rat liver microsomal suspensions to ionizing radiation (gamma rays). The microsomal lipid peroxidation induced by these chemicals and physical agents was inhibited by the addition of AD-5. These effects were dose-dependent in a millimolar range of concentration. In addition, AD-5 has no effect on microsomal electron transport, showing that NADPH-cytochrome P450 reductase activity was not modified. These data, together with the comparisons of the effects of AD-5 and some antioxidant molecules such as superoxide dismutase, uric acid, and mannitol, support the conclusion that inhibition of lipid peroxidation by AD-5 is the result of its free radical scavenger activity. In addition, the inhibitory effect of AD-5 on microsomal lipid peroxidation was dependent of the nature of the free radical species involved in the initiation of the process, suggesting that O-2 is scavenged more efficiently than RO.     

Calcium sequestration activity in rat liver microsomes. Evidence for a cooperation of calcium transport with glucose-6-phosphatase

Mechanisms regulating the energy-dependent calcium sequestering activity of liver microsomes were studied. The possibility for a physiologic mechanism capable of entrapping the transported Ca2+ was investigated. It was found that the addition of glucose 6-phosphate to the incubation system for MgATP-dependent microsomal calcium transport results in a marked stimulation of Ca2+ uptake. The uptake at 30 min is about 50% of that obtained with oxalate when the incubation is carried out at pH 6.8, which is the pH optimum for oxalate-stimulated calcium uptake. However, at physiological pH values (7.2-7.4), the glucose 6-phosphate-stimulated calcium uptake is maximal and equals that obtained with oxalate at pH 6.8. The Vmax of the glucose 6-phosphate-stimulated transport is 22.3 nmol of calcium/mg protein per min. The apparent Km for calcium calculated from total calcium concentrations is 31.9 microM. After the incubation of the system for MgATP-dependent microsomal calcium transport in the presence of glucose 6-phosphate, inorganic phosphorus and calcium are found in equal concentrations, on a molar base, in the recovered microsomal fraction. In the system for the glucose 6-phosphate-stimulated calcium uptake, glucose 6-phosphate is actively hydrolyzed by the glucose-6-phosphatase activity of liver microsomes. The latter activity is not influenced by concomitant calcium uptake. Calcium uptake is maximal when the concentration of glucose 6-phosphate in the system is 1-3 mM, which is much lower than that necessary to saturate glucose-6-phosphatase. These results are interpreted in the light of a possible cooperative activity between the energy-dependent calcium pump of liver microsomes and the glucose-6-phosphatase multicomponent system. The physiological implications of such a cooperation are discussed.     

Hormone sensitive calcium uptake by liver microsomes

The effects of glucagon and insulin on hepatic microsomal calcium uptake were investigated. Microsomes isolated from perfused rat liver accumulated calcium in the presence of ATP and oxalate. Addition of glucagon to the perfusate significantly increased calcium uptake by microsomes subsequently isolated. In contrast, addition of insulin to the perfusate resulted in a decreased microsomal calcium uptake and inhibition of the glucagon effect. Because the effects of glucagon and insulin on hepatic microsomal calcium uptake are opposite, as are the metabolic effects of these hormones, it is likely that the observed differences are of physiological importance.     

Inhibition of lipid peroxidation by calcium ions and their protection of steroid hydroxylase activity from peroxidative damage

Lipid peroxidation of adrenocortical mitochondria and microsomes was greatly stimulated by addition of 1.0 mM or less ferric ions. In the presence of NADPH-yielding system, the formation of corticosterone from endogeneous cholesterol and exogeneous deoxycorticosterone was inhibited as the concentrations of iron increased. Of interest is the fact that 0.5 mM ferric ion-mediated lipid peroxidation was completely abroagated upon addition of 2 mM calcium ions. Accordingly, protected from the peroxidative damage.     

The role of phospholipase A activity in rat liver microsomal lipid peroxidation

The involvement of phospholipase(s) A in lipid peroxidation of rat liver microsomes was investigated by: (a) determining the effects of phospholipase A inhibitors (p-bromophenylacyl bromide, chlorpromazine, mepacrine) on the accumulation of thiobarbituric acid reactivity or on levels of oxidized phospholipids in response to selected oxidative stimuli and (b) measurement of phospholipase A activities in response to these agents. Lipid peroxidation in response to various peroxidation systems was inhibited completely by exposure of microsomes to p-bromophenylacyl bromide (250 microM). The effectiveness of p-bromophenylacyl bromide was dependent on the presence of glutathione (200 microM) in preincubation mixtures. Chlorpromazine (100 microM) and mepacrine (100 microM) also effectively inhibited peroxidation, and their potency was independent of glutathione. The accumulation of oxidized phospholipids in response to the potent peroxidation stimulus alloxan/ferrous ion was similarly inhibited by p-bromophenylacyl bromide, although the level of oxidized phospholipid in response to the initiator ADP/ferrous ion was not affected. Microsomal phospholipase A1 activity, assessed using a liposomal substrate, was substantially enhanced by promoters of lipid peroxidation. Phospholipase A2 activity was not detected using a liposomal substrate but was evident using radiolabeled microsomes as endogenous substrate and was enhanced by oxidative stimuli. We conclude that phospholipase A activity may play an integral role in the microsomal lipid peroxidation mechanism. Based on this study, we hypothesize a role for phospholipases in facilitating propagation reactions.