Experimental studies on the mechanism of action of 4-hydroxy-2,3-trans-nonenal, a lipid peroxidation product displaying chemotactic activity toward rat neutrophils.

The effects of 4-hydroxy-2,3-trans-nonenal (HNE) and nonanal on the activity of phosphoinositide-specific phospholipase C of rat neutrophils have been studied in parallel with their action on neutrophil oriented migration. Concentrations of HNE ranging from 10(-7) to 10(-5) M significantly stimulated the oriented migration of rat polymorphonuclear leukocytes. HNE stimulated both the basal and GTP gamma S-induced phospholipase C activity when used at concentrations between 10(-8) and 10(-6) M. Nonanal was devoid both of chemotactic activity and of any action on phospholipase C activity. The effect of GTP gamma S on the stimulation of phospholipase C induced by HNE was higher when the lowest dose of the aldehyde was used; the finding of an additive effect between 10(-8) M HNE and 2 x 10(-5) M GTP gamma S suggests that the two compounds may share a final common pathway of action. These results suggest that the chemotactic activity of HNE might be mediated, like that of other more well-known chemoattractants, by the stimulation of phosphoinositide-specific phospholipase C.

Changes of serum iron transferrin and copper ceruloplasmin in rats given Cu(II)2 (acetylsalicylate)4 during acute inflammation.

Modifications of serum levels of iron transferrin and copper ceruloplasmin after acute inflammation by carrageenan and treatment with acetyl salicylic acid [ASA] or Cu(II)2(acetylsalicylate)4 [Cu(II)2(AS)4] were studied in the rat by EPR spectroscopy. Furthermore, the ulcerogenic potential of the two drugs was investigated after a single high oral dose. Our results indicate that Cu(II)2(AS)4 is more effective than ASA in limiting the inflammation provoked by the phlogogen. In these conditions the iron(III) non-heme and copper(II) ceruloplasmin concentration in serum was modified either during inflammation or after treatment with antiphlogistic agents: in carrageenan-injected rats the level of serum iron(III) non-heme was found to be very low, while the copper(II) ceruloplasmin concentration was partially reduced. On the other hand, after the pharmacological treatments, no changes of iron transferrin were observed and the concentration of copper ceruloplasmin was increased. With regard to their ulcerogenic effect, ASA appeared to be more irritating for gastric mucosa than Cu(II)2(AS)4.

Biological properties of pancreatic elastase in relation to atherosclerotic disease.

The inhibitory effect of elastase on experimental atherosclerosis has been reported in numerous studies. In our investigation, performed in the rat, a pancreatic extract provided with elastolytic activity has been shown to possess an anti-aggregative effect in vitro and ex vivo and anti-thrombotic properties. In addition, the elastase was capable of inhibiting endothelial exfoliation induced by the desquamatory agent sodium citrate. This agent was tested for its microhaemorrhoeological activity in acute and subacute experiments. In both these conditions, elastase was able to increase the flexibility of red blood cells and their resistance to lysis provoked by hypotonic solutions. In animals fed on an atherogenic diet, this substance limited the lipoprotein accumulation in the aorta wall. Moreover, it reduced the enhanced calcium content, induced by vitamin D administration, in the tissue of arteries. These data indicate that elastase can counteract some pathobiological aspects that characterize atherosclerotic events.

Effect of teomorfolin [N-(7'-theophylline acetyl)morpholine], a new drug, on dislipidaemic conditions induced in the rat.

A study of a new compound with an original structure, teomorfolin [N-(7'-theophylline acetyl)morpholine], was performed in the rat to investigate interaction with experimentally induced dislipidaemic disturbance related to atherosclerotic disorders. The drug succeeded in normalizing the cholesterol and triglyceride serum levels in acute as well as in chronic hyperlipaemia, and, in this connection, it provoked a significant increase of serum alpha-lipoproteins and a decrease of serum beta-lipoproteins, with a consequent improvement of the beta/alpha ratio. In addition, it significantly limited the lipolytic effect produced by adrenaline. Moreover, the drug demonstrated in vitro and in vivo platelet anti-aggregant activity and was able to increase erythrocyte flexibility. Finally, the drug inhibited prostacyclin biosynthesis; this action could explain the anti-aggregant activity demonstrated by teomorfolin. In conclusion, the chemical under investigation showed a wide spectrum of biological properties that interfere with metabolic disturbances involved in atherosclerotic conditions.

Non-steroidal anti-inflammatory agents and hepatic lipid peroxidation in normal- and carrageenin-treated rats.

A single oral dose of indomethacin (3 mg/kg b.w.) and phenylbutazone (200 mg/kg b.w.) induces an enhancement of thio-barbituric acid reacting substances (TBArs) formation by total liver homogenate of male rats. On the contrary, treatment with ibuprofen (200 mg/kg b.w., os) does not influence the susceptibility of hepatic tissue to "in vitro" lipid peroxidation. The former two chemicals do not interfere with the pro-oxidant action of carbon tetrachloride (1.0 ml/kg b.w., os) whereas ibuprofen limits the extension of TBArs production provoked by the haloalkane. Further, ibuprofen does not affect the level of glutathione in liver tissue, while indomethacin and phenylbutazone do. Similar results fatty acids was studied "in vivo" by monitoring conjugated diene absorption spectrum of hepatic microsomal lipids. Oedema produced acutely by injection of carrageenin into the plantar region of rat hind paw does not influence the peroxidative decomposition of hepatic lipids as found "in vitro" and/or "in vivo" neither in the presence nor in the absence of all the mentioned chemicals. Carbon tetrachloride administered orally (1.0 ml/kg b.w.) appears to be able to depress significantly the paw oedema induced by carrageenin but does not potentiate the anti-inflammatory effect of the tested non-steroid agents.

Behaviour of cyclic nucleotides and Ca2+ levels in liver tissue of rats poisoned by white phosphorus and trichlorobromomethane.

The content of hepatic cyclic AMP was increased soon after intoxication by white phosphorus. Its level reached a maximum 4 h after poisoning, but in subsequent phases tended to return to normal. In contrast, the cyclic GMP concentration was altered only 24 and 36 h after treatment with the same hepatotoxin. Similar modifications of cAMP and cGMP content were also detected after poisoning by trichlorobromomethane (CBrCl3). As a consequence, an altered cGMP/cAMP ratio was found in both experimental conditions. Further, the modification of cAMP content after white phosphorus was detected prior to liver damage (steatosis and necrosis), while the highest concentration of the cyclic nucleotide in CBrCl3-poisoned rats was found when fatty liver was already evident. In addition, in phosphorus-poisoned rats, the hepatic content of Ca2+ was found to be unmodified during all phases of the intoxication, while after CBrCl3 a phasic increase of the Ca2+ level was observed at 4, 24 and 36 h.

Neutrophil chemotactic responses to aldehydes.

The influence of 4-hydroxynonenal (HNE), 4-hydroxytetradecenal (HTDE) and methylglyoxal (MGL) on rat polymorphonuclear leukocyte locomotion has been studied by means of boyden chambers. The two alkenals have been found to exert a chemotactic activity in a range of concentrations between 10-9 and 10-5 M, the most potent chemical being HTDE. The chemotactic effect, however, appeared influenced by the concentration of serum albumin in the medium. In addition, chemotactic doses of the same aldehydes were unable to affect the cell random migration. Higher concentrations of these alkenals depressed both the directed and random locomotion. Methylglyoxal appeared deprived of chemotactic power when assayed at the doses that were active with the above mentioned alkenals and it did not interfere with random migration. 5 X 10-4 M Methylglyoxal exerted a slight, but significant, inhibition of the locomotion events. These results suggest that aldehydes, present at the inflammatory sites, as final products of lipid peroxidation, might play a role in modulating the granulocyte cell locomotion, therefore interfering with the development of phlogosis.

[Preliminary data on the influence of 4-hydroxyalkenals on the phagocytic activity polymorphonuclear leukocytes in the rat]. / Dati preliminari sull'influenza di 4-idrossialchenali sull'attività fagocitaria di polimorfonucleati di ratto.

The effect of low concentrations of 4-hydroxy-2-trans-pentenal, 4-hydroxy-2-trans-nonenal and 4-hydroxy-2-trans-tetradecenal (4-hydroxyalkenals produced during lipid peroxidation) was evaluated on the phagocytic activity of polymorphonuclear cells obtained from rat peritoneum. Our results show that the above compounds can influence, at different degrees, the endocytic powers, as measured "in vitro", of leukocytes through an inhibition that appears, to some extent, dose-related. Since lipoperoxidative processes can take place at the inflammed area, aldehydes originating from the peroxidative derangement of fatty acids may play a role in interfering with the cellular reactions at the inflammatory site.

[Chemotaxis and chemokinesis of rat polymorphonuclear leukocytes in response to 4-hydroxy-2-tetradecenal and 4-hydroxy-2-nonenal]. / Chemiotassi e chemiocinesi di polimorfunucleati di ratto in risposta al 4-idrossi-2-tetradecenale e al 4-idrossi-2-nonenale.

Since a number of experimental evidences suggests that some lipoperoxidation products can affect leukocyte migration "in vitro", we have investigated the chemotactic and chemokinetic properties of two of these products (4-hydroxy-2,3-trans-tetradecenal and 4-hydroxy-2,3-trans-nonenal) using rat neutrophils. The cells were obtained from the pleural cavity after injection of 1.0 ml isologous serum. The granulocytes were suspended in Hanks' plus BSA 2% and the motility determined by means of a modified Boyden chamber. For evaluating the chemotactic properties, the aldehyde were added into the lower compartment, while for detecting the chemokinetic power, the compounds were placed in both the compartments. Our results show that both the chemicals (in a range between nano- and micromolar concentrations) are able to exert -at different degree- a chemotactic activity. In this connection, the more active aldehyde appeared to be the tetradecenal. On the contrary, the same compounds seem uneffective in stimulating the random migration of polymorphonuclear cells.

The influence of experimental hypo- and hyperthyroid states on acute and chronic inflammatory reactions: modified response to non-steroidal anti-inflammatory agents.

Hyperthyroid and hypothyroid states were induced in rats by administration of triiodothyronine or surgical thyroparathyroidectomy. The anti-inflammatory activity of Indomethacin, Oxametacine and Phenylbutazone was evaluated in these animals using paw oedema provoked by carrageenan granulomas induced by cotton pellets and polyarthritis induced by Freund complete adjuvant. Our results indicate that the hyperthyroid state leads to a significant inhibition of the acute inflammatory response to carrageenan, while hypothyroidism has no effect. There was a marked increase in the anti-inflammatory activity of Indomethacin and Phenylbutazone in hyperthyroid rats. By contrast, in thyroparathyroidectomised animals the anti-inflammatory effect of these drugs appeared less than in euthyroid rats. The hyperthyroid state slightly inhibited the development of the cotton pellet-induced granuloma, while hypothyroidism enhanced it. Treatment of hypothyroid animals with the anti-inflammatory drugs resulted in a significant decrease in the size of the granuloma. In the hyperthyroid state, the activity of the compounds appeared similar to that detected in euthyroid rats. Neither hyperthyroidism, nor hypothyroidism affected the inflammatory response to mycobacterial adjuvant, or the effect of anti-inflammatory drugs on the response. These results suggest that thyroid hormones may influence, to various degrees the development of acute inflammation due to carrageenan, and chronic inflammation due to implanted cotton pellets. Our results indicate also that hyper- and hypothyroid states can modify the response of the rat to some non-steroidal anti-inflammatory drugs.

[Lipid peroxidation in the rat liver after acute inflammation induced by carrageenan. I. Influence of non-steroidal anti-inflammatory agents]. / Perossidazione lipidica nel fegato di ratto dopo flogosi acuta indotta da carragenina. 1. Influenza di alcuni farmaci anti-inflammatori non steroidei.

Oral administration of Indomethacin (3 mg/Kg) and Phenylbutazone (200 mg/Kg) induces an increase of TBA reacting substances (TBArs) by total liver homogenates, while treatment with Ibuprofen(200 mg/Kg, os) does not affect the susceptibility of liver tissue to lipid peroxidation. The former compounds do not influence the pro-oxidant action of CCl4 (1,0 ml/Kg, os) as evaluated "in vitro", whereas Ibuprofen appears to limit the extension of the TBArs production induced by the halomethane. The acute inflammatory state determined by carrageenan injection in the rat hind paw does not interfere with the peroxidative derangement found "in vitro" neither in the presence or in the absence of the all mentioned chemicals. Carbon tetrachloride (1,0 ml/Kg, os) is able in depressing significantly the rat paw oedema provoked by carrageenan, but does not potentiates the anti-inflammatory action of non-steroid agents.

[Influence of thyroid states on PGE2 biosynthesis]. / Influenza degli stati tiroidei sulla biosintesi di PGE2.

Hyperthyroid and hypothyroid states were induced in the rat by daily injection of 3,3',5-triiodothyronine (T3; 33 microgram/100 g b. wt., ip for 6 days) or by thyroparathyroidectomy, respectively. Control animals underwent only a sham operation or received the T3 vehicle. In these experimental conditions the biosynthesis of PGE2-like substance was determined "in vitro" in the fraction obtained after centrifugation at 80,000xg for 60 min of pooled spleens (5). Assays of PGE2 activity were carried out on rat stomach strip preparations according to Vane (8). In a series of additional experiments the influence of thyroid states on the inhibitory activity exerted by Indomethacin, Oxametacine and Phenylbutazone on PGE2 biosynthesis was also evaluated. The results obtained indicate that the hyperthyroidism induces a significant increase of PGE2 production "in vitro" from added arachidonic acid (100% enhancement above the control values) while thyroparathyroidectomy is unable to interfere with such biosynthetic activity. In addition, the inhibitory effect of Indomethacin, Oxametacine and Phenylbutazone on PGE2 production is not affected by hyperthyroid state whereas hypothyroidism significantly limits the action of the mentioned non-steroid anti-inflammatory agents.

[Susceptibility of the liver to lipid peroxidation after treatment with paracetamol]. / Suscettibilita' del fegato alla perossidazione pipidica dopo trattamento con paracetamolo.

Intoxication of rats with paracetamol (2.0 g/kg, b. wt.,os) is not followed by peroxidative decomposition of liver microsomal lipids "in vivo" but seems to interfere with ATPase and 5'-Nucleotidase activity in isolated plasmamembranes. Treatment with reduced glutathione, cys=teine and 2. mercaptopropionylglycine results in partial protection against liver injury provoked by the toxin. However, these sulphydryl compounds are not able to prevent the fall of liver GSH content occurring after paracetamol.

[Influence of propyl-gallate and 2-mercaptopropionylglycine on the development of acute inflammatory reactions and on biosynthesis of PGE2]. / Influenze del propil gallato e della 2-mercaptopropionilglicina sullo sviluppo di reazioni infiammatorie acute e sulla biosinte si di PGE2.

Anti-inflammatory activity of Propyl Gallate and 2.mercaptopropionylglycine, administered intraperitoneally to the rat, was evaluated against paw edema induced by Carrageenan, Bradykinin, Serotonin and Dextran. In addition, the influence of these chemicals on PGE2 formation from added arachidonic acid to spleen microsomal fraction incubated "in vitro" was assayed. Our results indicate Propyl Gallate and 2.mercaptopropionylglycine depress significantly the development of acute inflammatory reactions provoked by the above mentioned phlogogens and are able to limit the biosynthesis of PGE2. The last inhibitory activity appears to be less potent than that exerted by some non-steroidal anti-inflammatory agents, namely Indomethacin and Oxametacine, which act primarily by interfering with cyclo-oxigenase activity. In our opinion, the anti-inflammatory effect of Propyl Gallate and 2.mercaptopropionylglycine might be dependent on both the scavenger properties of the two compounds against some final products of lipid peroxides (aldehydes) originated at the inflammation site and the partial inhibition of the formation of PGE2 by acting on the cyclo-oxigenase system.

[Influence of pretreatment with carbon tetrachloride on paracetamol-induced hepatotoxicity]. / Influenza del pretrattamento con tetracloruro di carbonio sull'epatotossicita' del paracetamolo.

The influence of preventive treatment with a low dose of carbon tetrachloride on paracetamol-induced hepatotoxicity was evaluated in the rat. The haloalkane was given intraperitoneally (200 microliter/kg) 48 hours prior to paracetamol (PRCT; 2000 mg/kg, os). In parallel groups of rats were treated with CCl4 or PRCT alone. Twelve hours after paracetamol all the animals were killed. Liver damage was determined by evaluating total lipid and triglyceride accumulation in hepatic tissue and the serum activity of alanine-amino transferase (S.GPT). In addition, both the hepatic concentration of reduced glutathione (GSH) and the production "in vitro" of TBA-reacting compounds by liver homogenate were assayed. The results obtained indicate CCl4 "per se" induces a significant triglyceride accumulation but does not influence either the hepatic GSH level or the leakage of GPT into the blood stream. In addition, the haloalkane does not stimulate the production of TBA-reacting substances by hepatic tissue. Paracetamol, alone, produces a slight increase of hepatic triglycerides while induces a significant (+ 108%) enhancement of S.GPT activity. The drug is also able to stimulate the lipid peroxidation "in vitro", whereas provokes a marked decrease of GSH in liver tissue. Combined treatment with the two poisons results in a minor alteration of hepatocyte function as shown by the lack of GPT in serum and by the reduced fall of hepatic GSH as well as by a decreased production of TBA-reacting compounds. In our opinion, CCl4 partially protects against paracetamol-induced liver injury by interacting with enzymes which are responsible for the biotransformation of PRCT to a reactive arylating species that bind to cell molecules.

[Liver damage after paracetamol and carbon tetrachloride administration]. / Osservazioni sperimentali sul danno epatico conseguente alla somministrazione di paracetamolo e tetracloruro di carbonio.

Preliminary data on the liver damage following combined treatment with paracetamol and carbon tetrachloride in the rat are reported. Administration of a single dose of paracetamol (2000 mg/kg, os) was followed after 1 hour by an intraperitoneal injection of CCl4 (1.0 ml/Kg). Experiments in parallel were performed in rat given paracetamol or CCl4 alone. Our results indicate paracetamol induces a drastic decrease of hepatic GSH that appears in relation with a marked production of TBA-reacting compounds in liver tissue, while CCl4 does not modify the hepatic content of GSH and provokes a slight increase of TBA-reacting substances. Preventive treatment with paracetamol of rats intoxicated after 1 hour with carbon tetrachloride results in a partial protection against fatty liver and necrosis following haloalkane poisoning. On the other hand, the combined treatment with both the hepatotoxins was followed by a minor decrease of GSH. These data are discussed in considering a possible interaction of the two chemicals at the site of their activation.

[Relationship between GSH depletion and "in vitro" lipoperoxidative processes in liver of rats intoxicated with paracetamol (author's transl)]. / Rapporto fra la caduta del livello di GSH ed i processi lipoperossidativi "in vitro" nel fegato di ratti intossicati con paracetamol.

The time-course of reduced glutathione depletion in total liver homogenates from rats intoxicated with a single toxic dose of paracetamol (2000 mg/kg,os) was determined in parallel with the rate of production of TBA-reacting compounds. In addition, liver damage, as assayed by evaluation of leakage into blood stream of alanine-amino transferase and of the concentration of total hepatic lipids and triglycerides, was analysed. Our data indicate that at 4 and 6 hours after paracetamol giving GSH content of liver tissue appears reduced to about 27 and 38% of the control values, respectively. In the latter phases (8 and 24 hours) the concentration of GSH gradually tends to regain the values as estimated in untreated rats. At the same time the production of TBA-reacting substances is markedly increased. In spite of these biochemical disturbances not signs of both liver necrosis and hepatocellular degeneration was found. In fact, only at 24 hours after hepatotoxin a slight increase (P 0.05) of serum activity of alanine-amino transferase and of triglyceride content in the liver was revealed. The relationship between the hepatic GSH depletion and the increased production of TBA-reacting substances is discussed.

[Interference of antioxidants E/O of some free radical "scavengers" with the activity of glucose-6-phosphatase after administration of carbon tetrachloride]. / Inferenza di antiossidanti E/O di taluni "scavengers" di radicali liberi con l'attivita' della glucoso-6-fosfatasi dopo somministrazione di tetracloruro di carbonio.

G-6-Pase activity was investigated in the microsomal fraction from rat liver in the presence of carbon tetrachloride and/or propyl gallate (PG), reduced glutathione (GSH) and superoxide dismutase. Results obtained "in vitro" demonstrated that CCl4 induced a 60% inhibition of the microsomal enzyme activity. Moreover, a marked inhibition of G-6-Pase activity was found also when propyl gallate and reduced glutathione were added, at different concentrations, to incubation mixture. In addition, these drugs were unable to interfere with the dangerous effect exerted on the enzymatic activity by the haloalkane. Additional experiments carried out "in vivo" with propyl gallate produced evidence that intraperitoneal administration of the antioxidant was followed by a significant inhibition of G-6-Pase activity, while the damaging action of CCl4 was unaffected. Some possible explanations of these results are reported.

[The content of reduced glutathione in rat liver after poisoning with white phosphorus]. / Contenuto di glutatione ridotto nel fegato di ratto dopo intossicazione con fosforo bianco.

The content of hepatic GSH was evaluated in rats after poisoning with white phosphorus. In addition, liver damage following the administration of the hepatotoxin was assessed by determining hepatic triglyceride accumulation. Experiments in parallel were carried out in an attempt to evaluate the enhanced susceptibility of hepatic tissue to peroxidative decomposition of unsaturated lipids 'in vitro', as measured by the production of TBA-reacting substances. Our data indicate that only in the early stage of intoxication is it possible to detect a slight decrease of GSH content in the liver, while during the subsequent stages the concentration of GSH was unaffected. At 6 hours of intoxication the level of hepatic triglycerides was significantly increased. Pretreatment with GSH was followed by an amelioration of fatty infiltration, but the content of hepatic GSH was unchanged. The production of TBA-reacting products was found enhanced only at 6 hours of intoxication. These results are discussed in relation to the role of lipid peroxidation in liver injury by white phosphorus.

[Modifications of liver content of cyclic nucleotides in the rat poisoned with white phosphorus]. / Modificazioni del contenuto epatico di nucleotidi ciclici nel ratto intossicato con fosforo bianco.

In rat liver following white phorphorus poisoning a biphasic increase in cyclic AMP concentration was observed. After a lag period of 1 hour the cyclic AMP content rose to a first peak at 4 hours and to a second peak at 12 hours of intoxication. The cyclic AMP level fell to normal after 24 hours, by which time the cyclic nucleotide concentration was approaching control values. On the contrary, cyclic GMP content was found to the normal level during the different stages of intoxication. Only at 36 hours the cyclic GMP amount appeared significantly increased above the control values. Serum activity of alanine- and aspartate-amino transferases was found changed from 8 hours to 24 hours after poisoning. The serum level of the two enzymes was overlapping the control values after 36 hours. These results are discussed in relation to hepatocyte necrosis following white phosphorus intoxication.