P. Di Simplicio

Actin carbonylation: from a simple marker of protein oxidation to relevant signs of severe functional impairment.

The number of protein-bound carbonyl groups is an established marker of protein oxidation. Recent evidence indicates a significant increase in actin carbonyl content in both Alzheimers disease brains and ischemic hearts. The enhancement of actin carbonylation, causing the disruption of the actin cytoskeleton and the loss of the barrier function, has also been found in human colonic cells after exposure to hypochlorous acid (HOCl). Here, the effects of oxidation induced by HOCl on purified actin are presented. Results show that HOCl causes a rapidly increasing yield of carbonyl groups. However, when carbonylation becomes evident, some Cys and Met residues have been already oxidized. Covalent intermolecular cross-linking as well as some noncovalent aggregation of carbonylated actin have been found. The covalent cross-linking, unaffected by reducing and denaturing agents, parallels an increase in dityrosine fluorescence. Moreover, HOCl-mediated oxidation induces the progressive disruption of actin filaments and the inhibition of F-actin formation. The molar ratios of HOCl to actin that lead to inhibition of actin polymerization seem to have effect only on cysteines and methionines. The process that involves oxidation of amino acid side chains with formation of a carbonyl group would occur at an extent of oxidative insult higher than that causing the oxidation of some critical amino acid residues. Therefore, the increase in actin content of carbonyl groups found in vivo would indicate drastic oxidative modification leading to drastic functional impairments.

Methionine oxidation as a major cause of the functional impairment of oxidized actin

A significant specific increase in the actin carbonyl content has been recently demonstrated in human brain regions severely affected by the Alzheimers disease pathology, in postischemic isolated rat hearts, and in human intestinal cell monolayers following incubation with hypochlorous acid (HOCl). We have very recently shown that exposure of actin to HOCl results in the immediate loss of Cys-374 thiol, oxidation of some methionine residues, and, at higher molar ratios of oxidant to protein, increase in protein carbonyl groups, associated with filament disruption and inhibition of filament formation. In the present work, we have studied the effect of methionine oxidation induced by chloramine-T (CT), which at neutral or slightly alkaline pH oxidizes preferentially Met and Cys residues, on actin filament formation and stability utilizing actin blocked at Cys-374. Methionines at positions 44, 47, and 355, which are the most solvent-exposed methionyl residues in the actin molecule, were found to be the most susceptible to oxidation to the sulfoxide derivative. Met-176, Met-190, Met-227, and Met-269 are the other sites of the oxidative modification. The increase in fluorescence associated with the binding of 8-anilino-1-naphtalene sulfonic acid to hydrophobic regions of the protein reveals that actin surface hydrophobicity increases with oxidation, indicating changes in protein conformation. Structural alterations were confirmed by the decreased susceptibility to proteolysis and by urea denaturation curves. Oxidation of some critical methionines (those at positions 176, 190, and 269) causes a complete inhibition of actin polymerization and severely affects the stability of actin filaments, which rapidly depolymerize. The present results would indicate that the oxidation of some critical methionines disrupts specific noncovalent interactions that normally stabilize the structure of actin filaments. We suggest that the process involving formation of actin carbonyl derivatives would occur at an extent of oxidative insult higher than that causing the oxidation of some critical methionine residues. Therefore, methionine oxidation could be a damaging event preceding the appearance of carbonyl groups on actin and a major cause for the functional impairment of the carbonylated protein recently observed both in vivo and in vitro.

Thiolation and nitrosation of cysteines in biological fluids and cells

Summary. Thiols (RSH) are potent nucleophilic agents, the rates of which depend on the pKa of the sulfhydryl. Unlike compounds having other nucleophile moieties (–OH or –NH2), RSH are involved in reactions, such as conjugations, redox and exchange reactions. Although protein SH groups (PSH) react like non-protein thiols (NPSH), the biochemistry of proteins is much more complex for reasons such as steric hindrance, charge distribution and accessibility of PSH to the solvent (protein conformation). The reaction rates and types of end-products of PSH vary a lot from protein to protein. The biological problem is even more complex because in all compartments and tissues, there may be specific competition between thiols (namely between GSH and PSH), regulated by the properties of antioxidant enzymes. Moreover, PSH are divided biologically into essential and non-essential and their respective influence in the various biological systems is unknown. It follows that during phenomena eliciting a prompt thiol response (oxidative stress), the antioxidant PSH response and reaction mechanisms vary considerably from case to case. For example, in spite of a relatively low pKa that should guarantee good antioxidant capacity, PSH of albumin has much less propensity to form adducts with conjugating agents than NPSH; moreover, the structural characteristics of the protein prevent albumin from forming protein disulfides when exposed to oxidants (whereas protein-thiol mixed disulfides are formed in relative abundance). On the other hand, proteins with a relatively high reactivity, such rat hemoglobin, have much greater antioxidant capacity than GSH, but although human hemoglobin has a pKa similar to GSH, for structural reasons it has less antioxidant capacity than GSH.When essential PSH are involved in S-thiolation and S-nitrosation reactions, a similar change in biological activity is observed. S-thiolated proteins are a recurrent phenomenon in oxidative stress elicited by reactive oxygen species (ROS). This event may be mediated by disulfides, that exchange with PSH, or by the protein intermediate sulfenic acid that reacts with thiols to form protein-mixed disulfides. During nitrosative stress elicited by reactive nitrogen species (RNS), depending on the oxygen concentration of the system, nitrosation reactions of thiols may also be accompanied by protein S-thiolation. In this review we discuss a number of cell processes and biochemical modifications of enzymes that indicate that S-thiolation and S-nitrosation may occur simultaneously in the same protein in the presence of appropriate interactions between ROS and RNS.

The Reactivity of the Sh Group of Bovine Serum Albumin with Free Radicals

The reactivity of the SH group of bovine serum albumin (BSA) towards free radicals generated by several different systems including gamma-radiolysis and hydrogen peroxide/metal salt mixtures was investigated. On exposure of BSA (1 mg/ml and 5 mg/ml) to HO. radicals generated radiolytically the protein-SH concentration was found to decrease in a dose-dependent manner. At 40 mg/ml albumin no loss of SH was observed. O2-. and HO2. radicals were much less aggressive towards the SH group. The effect of divalent metal salts (copper or iron) plus hydrogen peroxide was studied separately and in combination. H2O2 alone caused a decrease in SH group concentration the rate of which was not decreased by the presence of desferrioxamine and so was apparently not due to reactions catalysed by adventitious metal ions. Copper alone caused a dose-dependent decrease in SH group concentration and the mixture of the two agents caused a greater loss of SH than each separate component. However, this latter effect was again resistant to the effects of desferrioxamine. The SH group of BSA was only moderately sensitive to the presence of ferrous iron alone and in a system containing both ferrous iron and H2O2 rates of SH oxidation were obtained that were identical to those obtained with H2O2 alone. Desferrioxamine again did not alter the rate of SH oxidation in these experiments. We suggest that the highly reactive free radical HO. is not able to reach and to oxidize the SH group of BSA when generated by metal/H2O2 mixtures, in contrast to HO. generated radiolytically. Less reactive radicals and non-radical species such as H2O2 have more potential for doing so.

Antioxidant status in various tissues of the mouse after fasting and swimming stress

Abstract We studied the effect of fasting and swimming stress on a number of non-enzymatic and enzymatic antioxidant factors in various mouse tissues in order to see if their action was synergic. We examined levels of reduced (GSH), oxidized (GSSG) and total glutathione, total SH groups (TSH), sum of GSH and protein sulphydryl groups of cytosolic fractions, and the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase in adductor muscle, heart and liver. We also studied blood levels of GSH and glutathione bound to protein by mixed disulphides (GSSP). The case series consisted of four groups of animals (n=10 for each group), namely no swimming and no fast, no swimming and fast, swimming and no fast, and swimming and fast. Fasting (18 h) resulted in a significant GSH depletion in all of the organs studied (−39% in the liver, −30% in the adductor muscle, −21% in the heart); GSSG increased significantly in the heart (+19%). Swimming to exhaustion, which lasted 3.95 (0.18) min [mean (SD), n=10] with no significant difference between fast and no fast, resulted in a significant GSH depletion, to a percentage lower than that observed after fasting, in the adductor muscle and heart (−12% and −11%, respectively). In the blood of swimming mice, significant increases in GSH (+10%) and GSSG (+21%) levels were observed, whereas GSSP decreased (−15%). Enzyme activities after swimming were modified in only a few cases, and in a complex way. The findings of GSH depletion and a decrease in SOD activity in the adductor muscle seems to confirm the sensitivity of this organ to an overproduction of reactive oxygen species. At the same time, the GSSP decrease observed in blood was a new and unexpected finding, one that indicates a very prompt adaptation of red cells to increased oxidant states.

Sulfhydryl groups and peroxidase-like activity of albumin as scavenger of organic peroxides

The concentration, the reactivity of sulfhydryl (SH) groups and the peroxidase-like activity (PLA) of bovine serum albumin (BSA) have been determined in vitro after treatment with peroxides. Tert-butylhydroperoxide (tBOOH), cumene hydroperoxide (CuOOH), benzoyl hydroperoxide (BOOH) and hydrogen peroxide reacted with BSA, decreasing the titratable SH group concentration and increasing the value of the ratio between the reaction rate and the concentration of albumin SH groups in the sulfhydryl-disulfide exchange reaction. This value was defined as reaction constant (Kr). PLA of albumin was independent of the presence of the SH group, as SH depleted BSA maintained the same activity as the control. From our findings it derives that albumin may have two possibilities of scavenging peroxides: PLA and the SH group. The plasma SH concentration, Kr and PLA of albumin were also determined in carrageenan paw edema and in experimental adjuvant-arthritis in rats. A decrease in SH concentration, an increase in Kr and PLA of rat plasma albumin were observed in both inflammatory processes.

Studies on the biological effects of ozone: 9. Effects of ozone on human platelets

During the course of ozonated autohaemotherapy (O3-AHT) using heparin as an anticoagulant, it was occasionally observed that a few clots were retained in the filter during blood reinfusion. This observation prompted an investigation on the effect of ozone (O3) on human platelets. We have now shown, both by biochemical and morphological criteria, that heparin in the presence of O3 can promote platelet aggregation. In contrast, after Ca(2+) chelation with citrate, platelet aggregation is much reduced. The potential role of the transient formation of hydrogen peroxide (H2O2) in the presence of Ca2+ with the possible expression of adhesion molecules is briefly discussed.

Glutathione, glutathione utilizing enzymes and thioltransferase in platelets of insulin‐dependent diabetic patients: relation with platelet aggregation and with microangiopatic complications

Abstract. Reduced glutathione (GSH) and activity of GSH related enzymes play a key role in defence against oxygen free radicals, whose production is, as known, raised in patients affected by diabetes mellitus, and at the same time they may contribute to the process of platelet aggregation. The purpose of this study was to evaluate GSH levels and activity of glutathione peroxidase (GSH‐Px), glutathione reduc‐tase (GSSG‐Red), glutathione transferase (GSH‐Tr), glucose‐6‐phosphate‐dehydrogenase (G6PDH), and thioltransferase (TT) in platelets of insulin‐dependent diabetic patients in fair metabolic control (mean glycated haemoglobin: 6.5%), as related to presence of retinopathy, neuropathy or nephropathy and to platelet aggregation by arachidonic acid (AA)in vitro. Mean effective dose (ED50) of AA was on average significantly lower in the group of insulin‐dependent diabetic patients (0.41 ± 0.02mM (SEM),n= 46) as compared with that of control subjects strictly matched for age, sex and weight (0.77 ± 0.02, n= 51; P= 0.0001). Mean platelet GSH as well as the activity of GSH related enzymes expressed as geometric mean (95% confidence intervals) were similar in diabetic patients and in controls, except for GSSG‐Red whose activity was significantly higher in diabetic subjects (28.5 (14.4–57.5) mU 10‐9 platelets vs. 20.3 (8.7–56) mU 10‐9 platelets; P= 0.01). In the diabetic group TT was reduced when compared with healthy controls (3.8 (0.9–12.2) mU 10‐9 platelets vs. 6 (1.6–26.1) mU 10‐9 platelets; P = 004). Moreover TT was significantly reduced in diabetic patients with worse metabolic control or with increased urinary albumin excretion rate (AER ≤ 20 μg min‐1), while neither TT, GSH nor GSH related enzymes were significantly different in patients with retinopathy or neuropathy. In addition TT was significantly related to ED50 of AA (r= 0.51; P= 0.0003). In conclusion GSH is not modified in insulin‐dependent diabetic patients in fair metabolic control, probably due to an augmented GSSG‐Red activity. Diabetic status, increase in platelet aggregation, and raised urinary AER seem to be altogether associated with a selective reduction in platelet TT activity.

The relationship between gamma-glutamyl transpeptidase and Hg levels in Se/Hg antagonism in mouse liver and kidney

The gamma-glutamyl transpeptidase (gamma-GT) activity and Hg concentrations were studied in Se/Hg antagonism in mouse liver and kidney after treatment with methyl mercury (MM) (MM group) and MM + sodium selenite (SE) (SM group). In acute treatment (dietary doses: MM = 250 p.p.m.; SE = 90 p.p.m.; length of treatment 11 days), hepatic gamma-GT activity increased in both protected and unprotected animals with respect to controls and reached a peak after 3 days with respect to controls, its value being relatively greater in the MM group. On the contrary, renal gamma-GT decreased with time with respect to controls and was higher in the SM group at 3 and 7 days. Liver and kidney accumulation of Hg increased and decreased respectively with time, and was higher in SM groups in most cases. In chronic treatment (dietary doses: MM = 12.5 p.p.m.; SE = 9 p.p.m.; length of treatment 12 months) hepatic gamma-GT activity in the MM group was higher than in the SM group at 1.5 and 7 months, whereas the renal activity was lower at 7 months and unchanged at 1.5 and 12 months. In comparison with the acute treatment, the trend of Hg accumulation was similar in liver and different in kidney; Hg concentrations of the SM group were always greater than those of the MM group. Glutathione (GSH) in liver and non-protein SH groups (NPSH) in kidney were also measured in acutely treated animals. On the first day GSH was about 50% of the control value in both the MM and SM groups; it subsequently remained constant in the MM group, but increased to a peak at 7 days, without reaching the control value, in the SM group. Unlike the liver, renal NPSH increased in both groups on the first day, and then decreased with time without reaching the control value, SM group values always exceeding those of MM group. The modulation of gamma-GT activity in liver and kidney caused by SE suggests that the enzyme plays a role in Hg accumulation.

Profile of drug-metabolizing enzymes in the cortex and medulla of the human kidney.

The cortex and medulla were isolated from kidneys whose donors (5 men and 1 woman, aged between 44 and 68 years) were undergoing nephrectomy to remove a tumor. Kidneys with normal architecture for at least two thirds of the organ were included in the study. Tissue specimens used in our experiments were free from pathological changes. The activities of the following enzymes of phase I NADPH cytochrome c reductase, aminopyrine N-demethylase, ethoxycoumarin O-deethylase, ethoxyresorufin O-deethylase, microsomal and cytosolic epoxide hydrolases, glutathione reductase and glutathione peroxidase, and those of the following enzymes of phase II glutathione transferase, glucuronyl transferase, sulphotransferase, acetyltransferase, thiomethyltransferase, thiopurinemethyltransferase, thioltransferase and glyoxalase were measured. The activity in renal cortex was significantly higher than in medulla for NADPH cytochrome c reductase, cytosolic epoxide hydrolase, glutathione reductase and glutathione peroxidase (phase I enzymes), and glutathione transferase, acetyltransferase, thiomethyltransferase, thiopurinemethyltransferase, thioltransferase and glyoxalase (phase II enzymes). The other enzymes had similar activity in cortex and medulla. The distribution pattern of drug-metabolizing enzymes in the human kidney cannot be considered as a single pattern because of the observed enzyme-dependent differences between cortex and medulla.