Sodium monochloroacetate causes cytotoxic effects, an increased lactate and pyruvate level and induces ultra structural and cytoskeletal alterations in cultured kidney and liver epithelial cells.

1. Monochloroacetic acid (MCAA) and its sodium salt, sodium monochloroacetate (SMCA) are widely used in chemical industries as intermediates in the synthesis of carboxymethylcellulose, phenoxyacetic acid, thioglycolic acid, glycine, indigoid dyes and others. Moreover, MCAA has been found as a by-product of the chlorination disinfection of drinking water and as an environmental contaminant of the atmosphere from the photodechlorination reactions of chlorinated hydrocarbons. Little is known about the mode of action of both compounds on the cellular level. From cases of accidental poisoning of man it is known that MCAA accumulates in liver and kidney. 2. In this study, the cytotoxicity of SMCA on cultured liver (Chang liver cells) and kidney epithelial cells of the proximal tubule (Opossum kidney cells) was investigated and its effect on metabolism, ultrastructure and organization of cytoskeleton was examined. 3. Independent from the growth state of the cells (proliferating or quiescent), the results clearly show that SMCA causes a dose-dependent decrease in cell viability after an exposure period of 24 h. In all experiments, proliferating cells were more sensitive than quiescent and confluent cells. Liver cells were less sensitive against SMCA treatment than kidney epithelial cells. In contrast to liver cells, kidney cells exhibited a dose-dependent decrease in cell volume. The decrease in cell viability was accompanied by an increase of lactate and pyruvate concentrations released into the culture medium. In the case of Opossum kidney cells, lactate and pyruvate levels increased 5 - 6-fold, whereas in the case of Chang liver cells the increase was approximately twofold. While the ultrastructure of liver cells remained unaltered after drug treatment, kidney cells exhibited cytoplasmic vacuolization, membraneous disruption and especially mitochondrial alterations. In accordance with the changes in the ultrastructure of Opossum cells, was the reorganization of cytoskeletal elements with an increased stress fiber network at the basolateral surface as well as a partial depolymerization of microtubules and vimentin filaments. A cytoskeletal reorganization was not observed for Chang liver cells after SMCA treatment. 4. The results demonstrate that SMCA causes a dose-dependent cytotoxicity which is accompanied by metabolic, mitochondrial and cytoskeletal alterations in the cells.

Protective effect of Crataegus oxyacantha against reperfusion arrhythmias after global no-flow ischemia in the rat heart.

The protective effect against reperfusion arrhythmias of a 3-month oral pretreatment with a dried extract of Crataegus oxyacantha (LI 132)(standardized to 2.2% flavonoids) was studied with the Langendorff heart of the rat after global no-flow ischemia. The heart was perfused with a modified Krebs-Henseleit solution in which the K+ content was reduced to 3.4 mmol/l in order to lower the fibrillation threshold. According to pilot experiments which considered various durations of global no-flow ischemia ranging from 10 to 20 minutes, two durations were chosen for the present study: 20 minutes (group 20) in which ventricular fibrillation (VF) was the predominant form of arrhythmias, and 18 minutes (group 18) in which the prevalence of VF was markedly lower despite the small difference in the duration of ischemia. Crataegus pretreatment significantly (p = 0.02) reduced the average prevalence of malignant arrhythmias (VF + Flutter) as observed during the 20-min-period of reperfusion as follows: group 20: from 89% (control, n = 9) to 51% (LI 132, n = 7), group 18: from 48% (control, n = 8) to 8% (LI 132, n = 8). In group 20, ventricular tachycardia (VT) could be observed only in the treated group, because of the predominance of VF in the control group. LI 132 pretreatment reduced the average prevalence of VT in group 18 in spite of the identical percentage of occurrence (6 out of 8 rats, with and without treatment) due to a shorter duration of the VT episodes. Thus, under the conditions of our experiments, effective prevention against reperfusion arrhythmias by Crataegus pretreatment was evident.

Prolongation of life span in hypertensive rats by dietary interventions. Effects of garlic and linseed oil.

The effects of long-term dietary application of garlic (dried powder, 0.5% in weight of standard chow; G group) or linseed oil (2.5%; L group) as well as a combination of both interventions (L + G group) on the life span of hypertensive rats (SHR SP) was investigated. A further group fed with standard chow served as control (C). The dietary interventions were started at the age of three weeks. Besides regular measurements of the systolic arterial blood pressure (oscillometrically at the tail artery) as well as of heart rate and body weight, autopsy and histological investigations were performed. Both diets, and particularly their combination, prolonged life span significantly (mean values (days) C: 434.5 +/- 23.5; G: 453.2 +/- 16.2; L: 470.0 +/- 26.2; L + G: 494.8 +/- 39.2). There was no significant interaction of the factors garlic and linseed oil. Systolic blood pressure as measured during the compensatory stage (data used until the 39th week of life) was significantly lowered by both garlic (mean -5.8 mm Hg), linseed oil (mean -6.3 mm Hg), and their combination (mean -11.3 mm Hg). The animals died as a consequence of congestive left and right ventricular failure with ventricular hypertrophy, dilatation, myocardial fibrosis and cellular infiltration, left ventricular atrial thrombosis (in most cases), and terminal pneumonia. On the other hand, arteriosclerotic plaques and signs of cerebral stroke could not be detected. Except for the degree of hypertrophy, which was lower in the treated groups, no differences were obvious regarding the morphological findings at the time of death. There was a significant positive correlation between mean blood pressure and the degree of left ventricular hypertrophy. Furthermore, a significant negative correlation between mean blood pressure and ventricular hypertrophy on the one hand and survival on the other hand was obvious provided the total number of animals was considered, however, not within the individual groups. The same applies to the relation between the reduction of left ventricular hypertrophy and life span. The relatively slight hypotensive effect of both dietary interventions as well as the results of previous investigations speaks in favor of a substantial influence of factors independent of blood pressure. In view of controversial results and interpretations in international literature, the mechanisms involved need further study.

Myocardial protection by pretreatment with Crataegus oxyacantha: an assessment by means of the release of lactate dehydrogenase by the ischemic and reperfused Langendorff heart.

The effect of the pretreatment with the powder of crataegus oxyacantha on the release of lactate dehydrogenase (LDH) during ischemia and reperfusion was studied in an isolated rat heart model. Male Wistar rats were divided into control and crataegus group (for which the standard diet was mixed with a 2% crataegus powder standardized to 2.2% flavonoids). The investigations started 3 months after commencing the treatment. The hearts were isolated and a retrograde perfusion was performed at constant pressure according to the technique of Langendorff. The experimental protocol comprised 10 min equilibration, according to the technique of Langendorff. The experimental protocol comprised 10 min equilibration, according to the technique of Langendorff. The experimental protocol comprised 10 min equilibration, 110 min occlusion of the left anterior descending coronary artery, and 30 min reperfusion. The coronary effluent was sampled for the LDH determination after 5, 30, 90, 120 and 150 min. The LDH activity, which was initially very low in both groups (control, 16.5 +/- 4.3; crataegus, 26.0 +/- 8.8 mU/min) increased slightly during the ischemia, and very strongly as soon as the heart was reperfused. However, the increase in the crataegus group was significantly lower (1777.3 +/- 451.9 vs control 3795.3 +/- 511.9 mU/min, p = 0.01). At the end of the reperfusion period, LDH activity decreased markedly but did not reach the ischemic values. The attenuation of the LDH release by crataegus pretreatment suggests a preservation of the cell membrane and a protection from myocardial damage.

Myocardial protection by ischemic preconditioning: the influence of the composition of myocardial phospholipids.

It was the aim of this study to investigate (1) whether preconditioning modifies the fatty acid (FA) composition of myocardial phospholipids (PL), (2) whether a previous modification of membrane PL composition by the administration of coconut oil or fish oil influences the preconditioning, and (3) to compare the protective effects of preconditioning to those of dietary fish oil. To this end, three groups of rats were given during 10 weeks either a standard diet, or a standard diet + 10% coconut oil, or a standard diet + 10% fish oil. The preconditioning was performed in situ in the anesthetized open-chest rats by 2 cycles of 3 min left anterior descending coronary artery occlusion and 10 min reperfusion. It was followed by a 40 min ischemia and a 60 min reperfusion. ECG was recorded and used for the continuous count of the salves of extrasystoles, ventricular flutter and fibrillation. These rhythm disturbances were subsequently added and evaluated as total arrhythmias. The FA of tissue PL were analyzed in a sample of the ischemic zone the size of which was determined by means of malachite green. Coconut oil diet (rich in saturated FA) modified slightly the myocardial PL by increasing oleic acid and decreasing linoleic acid and resulted in the highest incidence of arrhythmias. Fish oil diet had the opposite effect in modifying drastically the PLFA (replacement of the n-6 FA by the n-3 FA) and minimizing significantly the arrhythmias in comparison with the standard diet group.(ABSTRACT TRUNCATED AT 250 WORDS)

n-3 versus n-6 fatty acid incorporation into the phospholipids of rat heart sarcolemma. A comparative study of four different oil diets.

The fatty acid (FA) composition of the phospholipids of rat heart sarcolemma was studied after a 10-week feeding period of diets containing 10% of each of coconut oil, corn oil, linseed oil, and fish oil in comparison to a low fat diet. The major modifications observed were the following: (1) a general increase of the n-6 FA family upon corn oil administration; (2) a dual effect of linseed oil in increasing 18:2 n-6 (more than did corn oil) and decreasing 20:4 n-6 and 22:4 n-6. At the same time n-3 FA increased significantly; and (3) a significant fall in the n-6 FA following fish oil, and a marked rise in the n-3 FA. The 10-fold increase in 22:6 n-3 supports the hypothesis of an inhibition of arachidonate synthesis, as well as that of the competition for the incorporation sites of phospholipids. In general, the n-3 FA diets produced the replacement of the n-6 FA by the n-3 FA in membrane phospholipids, and led to a significant increase of the double bond index (DBI).

Cardioprotective actions of wild garlic (allium ursinum) in ischemia and reperfusion.

The susceptibility to ventricular arrhythmias under the conditions of cardiac ischemia and reperfusion was investigated in the Langendorff heart preparation of rats fed for eight weeks a standard chow enriched with 2% of pulverized wild garlic leaves. The isolated hearts were perfused with a modified Krebs-Henseleit solution. The incidence of ventricular fibrillation (VF) during 20 min occlusion of the descending branch of the left coronary artery (LAD) was significantly reduced in the wild garlic group as compared to untreated controls (20% vs 88%). The same holds for the size of the ischemic zone (33.6% vs 40.9% of heart weight). In the reperfusion experiments (5 min after 10 min ischemia), ventricular tachycardia (VT) occurred in 70% of the wild garlic group vs 100% in untreated controls and VF in 50% vs 90%. The time until occurrence of extrasystoles, VT or VR was prolonged. No significant alterations in cardiac fatty acid composition could be observed. Although the prostacyclin production was slightly increased in hearts of the wild garlic group, inhibition of cyclooxygenase by acetylsalicylic acid (ASA; aspirin) could not completely prevent the cardioprotective effects suggesting that the prostaglandin system does not play a decisive role in the cardioprotective action of wild garlic. Furthermore, a moderate angiotensin converting enzyme (ACE) inhibiting action of wild garlic was found in vitro as well as in vivo that could contribute to the cardioprotective and blood pressure lowering action of wild garlic. Whether a free radical scavenging activity of wild garlic is involved in its cardioprotective effects remains to be established.

Effect of isoproterenol on the metabolism of myocardial fatty acids.

The effect of beta-adrenergic stimulation on myocardial fatty acid metabolism was studied in the anesthetized open-chest dog. Isoproterenol (1 microgram/kg/min) was infused; and samples of arterial blood and of left ventricular wall were taken at the 5th min for the determination of the following variables: in arterial blood: lactate and serial free fatty acids (FFA); in myocardial tissue: creatine phosphate (CP), ATP, lactate, carnitine, acylcarnitine, glycerol, and the fatty acid content of each of phospholipids (PL), cholesterol esters (CE), triglycerides (TG), diglycerides (DG), monoglycerides (MG), and FFA. A capillary gas chromatography was used for fatty acid assay. Isoproterenol decreased the content of creatine phosphate but not of ATP. Lactate increased in both arterial blood and myocardial tissue. The five-fold increase in arterial FFA (P less than 0.001) was accompanied with a significant increase in FFA serum/tissue ratio. Free carnitine decreased and acylcarnitine increased. Triglycerides content (which is expressed in terms of its total fatty acid content) was considerably reduced by beta-stimulation in comparison with control group (2694 +/- 689 vs. 7518 +/- 833 nmol/g wet wt, P less than 0.001), and tissue glycerol increased (P less than 0.01). The decrease in total MG content was significant, but not that of DG, nor that of CE. Total PL content did not change. The most marked individual changes (except in PL) were observed on monounsaturated fatty acids, 18: 1 omega 9, 18: 1 omega 7, and 16: 1 cis. The significant changes of monounsaturated/saturated and of 16:1 cis/16:1 trans ratios, in arterial FFA and tissue TG, suggested modifications in the distribution of fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Assay of lipids in dog myocardium using capillary gas chromatography and derivatization with boron trifluoride and methanol.

The fatty acids of three lipid classes (free fatty acids, triglycerides, and cholesteryl esters) from dog heart were analysed by gas chromatography. Samples of the left ventricle were homogenized and total lipids were extracted. After separation by thin-layer chromatography, the bands of the lipid classes studied were scraped off, transmethylated according to the boron trifluoride-methanol procedure, and the fatty acid methyl esters were extracted and analysed. The problems related to the quantitation of fatty acids were investigated, namely transmethylation procedure, thin-layer chromatography, and gas chromatographic conditions. Fatty acid methyl esters were separated on capillary columns coated in the laboratory with SP 2340 stationary phase. The high performance of the separation ensured the reliability and the precision of the analysis.

Effect of beta-adrenergic stimulation on free fatty acid content in subepicardial and subendocardial layers of the dog heart in situ. Separation and assay by capillary gas chromatography.

The effects of beta-adrenergic stimulation produced by an infusion of isoproterenol (1 microgram X kg-1 min-1, 30 min) were studied in situ in the anaesthetized dog placed under a total cardiopulmonary bypass. Samples of the subepicardial and the subendocardial layers were homogenized separately prior to the extraction and methylation of free fatty acids (FFA). Gas chromatography on Carbowax 20 M capillary columns was used for the quantitation of myristic (C 14:0), palmitic (C 16:0), palmitoleic (C 16:1), stearic (C 18:0), oleic (C 18:1), linoleic (C 18:2), and arachidonic (C 20:4) acids. Within 5 min, isoproterenol decreased the tissue content of FFA significantly. The decrease was more pronounced in the endocardial layer where the FFA concentration reached its minimum at the 5th or the 15th min. In the epicardial layer, all the FFA reached their minimal concentration at the 30th min of the isoproterenol infusion. In both layers, lactate content remained unchanged at 5 and 15 min and rose at the 30th min only and content in phosphorylated compounds (ATP, creatine-phosphate-CP) did not show any significant variation during the beta-stimulation period. A significant correlation was found between the chronotropic effect of isoproterenol and the reduction of FFA concentration.

Relationship between lactate arteriovenous gradient and tissue lactate content in subendocardial and subepicardial layers of the ischaemic in situ heart.

Myocardial lactate content and simultaneous evolution of a lactate arteriovenous gradient associated with incomplete obstruction of the left coronary bed were studied in dog hearts. Samples of blood were taken from a peripheral artery and the coronary sinus; transmural samples of myocardial tissue were obtained from the left ventricular wall by drill biopsy in animals under total cardiopulmonary by-pass. Lactate content was assessed in subendocardial and subepicardial layers separately. A 40 to 70% reduction in coronary flow induced a quick reduction and even an inversion of the positive lactate gradient while the tissue content, which was similar to the arterial content, rose considerably, chiefly in the subendocardial layer. Lactate accumulating in tissue was released into the blood approximately in proportion to the tissue concentration. The value of employing the lactate concentration difference between arterial blood and coronary sinus blood as an index of the severity and duration of myocardial ischaemia is discussed.

[Comparison of myocardial metabolic effects of in subendocardial and subepicardial layers ischaemia and beta-adrenergic stimulation (author's transl)]. / Comparison des effets métaboliques myocardiques de la stimulation beta-adrénergique et de l'ischémie effectuée dans les zones sous-endocardique et sous-épicardique.

The influence of beta-adrenergic stimulation on myocardium carbohydrate and lipid metabolism has been compared with the influence of ischemia in anesthetized dog heart in situ. Transmural samples necessary to the repeated determination of tissular substrates were taken from left ventricular wall according to the "drill biopsy" technique: they were made possible by a total cardiopulmonary by-pass system. The beta-adrenergic stimulation was obtained by infusion of isoproterenol (1 microgram/kg/min.) and the ischemia by injection into left coronary artery before its division of a viscous mixture, reducing coronary flow by 40 to 70%. In both subendocardial and subepicardial layers, but mainly in the former: --beta-adrenergic stimulation lowers glycogen content and raises lactate content immediately, lowers free fatty acid concentration more progressively without modifying triglyceride concentration significantly; --ischemia decreases glycogen content and increases lactate content in the same way, but raises free fatty acid and triglyceride concentration. Consequently, beta-adrenergic stimulation and ischemia are likely to add their effects on anaerobic glycolysis, whereas they exert an opposite influence on lipid metabolism.

Modifications by ischemia of carbohydrate and lipid metabolism of the dog heart in situ from circulating and endogenous substrates.

The uptake of circulating substrates, lactate, glucose and free fatty acids (FFA) has been investigated concurrently with the tissular contents of these principles and the glycogen and triglyceride stores in the dog heart in situ submitted to incomplete obstruction of left coronary bed. Transmural samples necessary for the repeated determination of tissular substrates were taken from left ventricular wall by means of a total cardiopulmonary by-pass system, then divided to allow the analysis separately in subendocardial and subepicardial layer. A 40 to 70% reduction in coronary blood flow gave rise to decrease or suppression of uptake of all the substrates or even to conversion of uptake into output. The modifications of uptake are chiefly related to the deficiency of breakdown by oxidation, though lessened in the case of FFA by incorporation into triglycerides and enhanced in the case of glucose by glycogenolysis. Glycogenolysis and consequent anaerobic glycolysis appear to be the main process available against the energy cellular defect linked with oxygen lack which affects notably more subendocardial than subepicardial layer.