Low normal level of protein C or of antithrombin increases risk for recurrent cardiovascular events.

The relationship between haemostatic factors and recurrent cardiovascular events was investigated in patients enrolled with acute coronary syndrome (acute non-Q myocardial infarction or unstable angina pectoris). One hundred and fifteen patients, aged 64 +/- 10 years, were included in the study. Haemostatic parameters [prothrombin time, activities of factor VII, factor VIII, factor X, antithrombin (AT) and protein C (PC), and concentrations of free protein S, fibrinogen, D-dimer, prothrombin fragment 1+2, and thrombin-antithrombin complex] were measured four times: within 48 h of hospitalization, at discharge (days 5-8), at 3 months and after 1 year. Screening for factor V Leiden mutation was also performed. Patients were followed for cardiovascular endpoints (new or refractory unstable angina pectoris, non-fatal myocardial infarction, stroke, or death) for an average of 555 days. Of all patients, 35 had an endpoint during the follow-up ("endpoint" group) and 80 patients did not ("no endpoint" group). Analysing the whole follow-up period, PC (P < 0.01) and AT (P < 0.01) were lower in the "endpoint" than in the "no endpoint" group. With 50% percentiles at enrollment, the odds ratio for getting an endpoint in the low (cut-off value < 100%) versus high PC group was 2.72 (95% confidence interval, 1.18-6.29; P < 0.05). Lower levels of AT (P < 0.05) and PC (P < 0.05) during the whole follow-up were associated with a shorter event-free time. In conclusion, lower PC and AT values, even within the normal range, seem to be associated with elevated risk for recurrent cardiovascular events and shorter event-free time in acute coronary syndrome patients.

Cytotoxicity and biotransformation inducing activity of rodent beddings: a global survey using the Hepa-1 assay.

Unknown and uncontrolled toxic materials in the environment of experimental animals endanger the reliability of research results. In this study, the cytotoxic and biotransformation inducer activity of beddings obtained from a number of institutes in Africa, Americas, Asia, Australia and Europe were compared using the Hepa-1 in vitro-assay. A great variation in both cytotoxicity and inducer activity was observed. Pine shavings beddings, commonly used all over the world appeared in general to be highly cytotoxic. Extracts of corn cob, rice hulls and straws, used in a few laboratories, were practically non-toxic. Some softwood extracts, especially from pine, were also potent inducers of cytochrome P4501A1. Grass as well as hardwood materials were much less active. Due to great variation in toxicity and inducer activity in the beddings it is possible that, in spite of standardization of feed and other environmental factors, different biological responses to chemicals and drugs in even genetically identical animal strains in different laboratories, may be obtained. Control of bedding, one of the animals' closest environmental factors, for its toxicity and biotransformation enzyme induction is strongly recommended.

Hepa-1 enzyme induction assay as an in vitro indicator of the CYP1A1-inducing potencies of laboratory rodent diets in vivo.

The Hepa-1 enzyme induction assay (assay of the induction of CYP1A1 catalytic activities in the Hepa-1 mouse hepatoma cell line by various compounds or mixtures) was evaluated as an in vitro indicator of the CYP1A1-inducing potencies of laboratory rodent diets in vivo. C57BL/6J mice were fed for three weeks four selected commercially available diets (one semisynthetic and three standard natural ingredient diets) exhibiting different enzyme-inducing effects in the Hepa-1 assay. beta-Naphthoflavone mixed in a semisynthetic diet (33 and 330 mg/kg of diet) was used as a model inducer. CYP1A1-dependent enzyme activities (aryl hydrocarbon hydroxylase and 7-ethoxyresorufin O-deethylase) were measured in the small intestinal mucosa and liver. There was good agreement between the induction of CYP1A1 in vitro and in vivo: the rank order of the enzyme activities elicited by the diets was the same in the mice as in the Hepa-1 cells. The standard diets were less effective inducers than beta-naphthoflavone in the Hepa-1 cells and in the mice, especially in the small intestinal mucosa. The Hepa-1 enzyme induction assay thus seems to be a mechanistically sound, reliable and sensitive in vitro indicator of the CYP1A1-inducing potencies of laboratory rodent diets in vivo.

Effects of oat-bran concentrate on rat serum lipids and liver fat infiltration.

Oat bran concentrated with respect to both soluble and insoluble dietary fibre was fed to adult rats, and its effects on serum cholesterol and liver fat infiltration were studied. The feeds contained 15, 30 or 45 g beta-glucan/kg, except in control groups where the fibre of the feed was cellulose. Half the experimental groups received the feeds with an addition of 10 g cholesterol and 2 g cholic acid/kg in order to create an hypercholesterolaemic condition. In normocholesterolaemic rats the concentrated oat bran did not cause any significant changes in serum cholesterol concentrations, but reduced liver weight compared with control group rats fed on the cellulose-containing diet. In hypercholesterolaemic rats the concentrated oat bran reduced serum total cholesterol and increased high-density-lipoprotein cholesterol concentration. The effect was seen already at the 15 g/kg concentration of beta-glucan, the higher doses tested did not significantly improve the effect. Infiltration of fat into liver cells, especially in the periportal areas, was observed only in hypercholesterolaemic animals. The fat infiltration was accentuated in proportion to the amount of oat-bran concentrate fed.

Cytotoxic and aryl hydrocarbon hydroxylase-inducing effects of laboratory rodent diets. A cell culture study.

Extracts of several rodent diets were studied for their cytotoxic and aryl hydrocarbon hydroxylase-inducing properties by an in vitro method. The cell culture system based on a mouse hepatoma cell line (Hepa-1) was shown to be a convenient and sensitive method for screening of diets for these parameters implying the presence of compounds potentially harmful in vivo. Considerable differences among diets and batches were detected. Smallest effects were observed with a semipurified diet and with the unrefined diet which - contrary to other four unrefined diets - contained no fish.

Absorption of oral enalapril in germ-free and microbially-associated rats.

The effect of microbial status and presence of food in the gastrointestinal (GI) tract on absorption of an orally administered antihypertensive drug [14C]-enalapril (1 mg/10 microCi/kg body weight) was studied in non-fasted and fasted germ-free (GF) and microbially-associated (MA) rats. The absorption was evaluated from blood samples taken 0.5, 2 and 4 h after the administration of enalapril. Drug levels in the lungs, liver, and duodenal mucosa were also measured by counting [14C]-radioactivities at 4 h after the drug administration. During the 4 h monitoring period enalapril was poorly absorbed in non-fasted rats as compared to fasted animals. In non-fasted rats, the absorption of enalapril was slightly slower in GF than in MA rats. In fasted rats, no difference was found between GF and MA animals. The results show that food in the GI tract substantially lowers or retards the absorption of enalapril in rats. The GF status of the GI tract, however, did not substantially affect the absorption of the ester-type drug enalapril.

Enzyme-inducing and cytotoxic effects of wood-based materials used as bedding for laboratory animals. Comparison by a cell culture study.

Enzyme-inducing and cytotoxic effects of wood-based materials used as bedding for laboratory animals were studied in a cell culture system. Mouse hepatoma cell line, Hepa-1, was exposed to acetone extracts of hardwoods (alder and aspen), softwoods (pine and a mixture of pine and spruce) and cellulose materials. Cytotoxicity and induction of cytochrome P450IA1 (aryl hydrocarbon hydroxylase) and aldehyde dehydrogenase were measured. Both softwood and hardwood extracts were shown to contain inducers of these enzymes. Pine appeared to be the most potent inducer and softwoods more potent than hardwoods. The softwoods and alder were clearly more cytotoxic than aspen. The two bleached cellulose materials were found to contain inducers of aryl hydrocarbon hydroxylase. Unlike the wood beddings, the extracts of the cellulose materials were not found to be toxic to the cells. Hepa-1 cell culture system was found to be a rapid and sensitive method for screening and comparative purposes.

Role of gut in xenobiotic metabolism.

The gastrointestinal tract forms the first line of defense in the body against the main load of xenobiotics. The gastrointestinal mucosa has several mechanisms through which the xenobiotics are modified. The monooxygenase activities in most species are relatively low in the mucosa as compared to the liver, but conjugation, for example, via glucuronide formation proceeds efficiently. UDP-glucuronosyltransferase activities can exceed those in the liver. Glutathione S-transferase activity is also high. The biotransformation activities are readily inducible in the mucosa and this is, at least partly, responsible for the oral-aboral gradient seen in enzyme activities. In rainbow trout glutathione S-transferase is, however, significantly higher at the aboral third than in two oral segments, although in rats the intestinal glutathione S-transferase shows a clear oral-aboral gradient. The gradient is independent of the presence of microflora at least in the case of carboxylesterase and glutathione S-transferase. A similar gradient can also be found from the gut lumen, in both germ-free and specific pathogen-free rats. The cells in the middle of the villi appear to be most responsive under the influence of inducers. The readily occurring induction in the mucosa provides a suitable model for studies on biological effects to defined compounds and mixtures.

Enzyme activities of myocardial energy metabolism during prolonged digoxin treatment in rats.

The effect of prolonged digoxin treatment (1 mg/kg day for 8 days) on the activity levels of some enzymes of energy metabolism (phosphofructokinase, lactate dehydrogenase, citrate synthase, succinate dehydrogenase) in rat myocardium was studied. In the control animals receiving the solvent mixture (glycerol:ethanol:water in 1:1:1) a transient decrease in the lactate dehydrogenase and citrate synthase activity levels was observed. In the hearts of digoxin treated rats the level of activity of phosphofructokinase was permanently lowered by the fourth day and the level of activity of citrate synthase permanently increased after the first day of treatment. A transient increase in the activity level of succinate dehydrogenase in the myocardium of digoxin treated animals was seen between days 1 and 6. In this study a permanent decrease in phosphofructokinase and an increase in citrate synthase activity levels in rat heart muscle was noted during prolonged digoxin treatment.