Kg-1 Day-1 Research Articles

ACUTE AND SUBCHRONIC TOXICITY OF MULBERRY FRUITS

At present, the application of phytomedicine worldwide is substantial increase. The data of the acute and subchronic toxicity studies on medicinal plants or fruits and vegetables used for medicinal purposes should be obtained in order to increase the confidence in its safety to human, particularly for the application as functional food. Despite the widely consumption of mulberry (Morus alba L, Maraceae family) fruits both as fruit and as medicine, no systematic evaluation of the toxicity of mulberry fruits is available until now. This study aimed to determine the acute and subchronic oral toxicities of mulberry fruits in male and female Wistar rats. A single acute mulberry fruits extract dose of 2000 mg kg-1 dissolved in distilled water was administered by oral gavage for acute toxicity. Subchronic doses of 2, 10 and 500 mg kg-1 day-1 were also administered the same way for 90 days. The major toxicological endpoints examined included animal body weight, water and food intake, selected tissue weights and histopathological examinations. In addition, we also examined hematological and clinical chemistry values. The results showed no abnormalities in treated groups as compared to the controls. Although significantly different BUN and glucose were also observed, all of the values were within normal limits. Neither gross abnormalities nor histopathological changes were observed. The results suggest that mulberry fruits do not produce acute or subchronic toxicity in either female or male rats. Therefore mulberry fruits have the potential to be use as functional food. However, a chronic toxicity study should be further carried out to assure the safety for repetitive administration of this fruit before the application of functional food.

Can Soy Change Serum Estradiol Concentration in Neonate Ovariectomized Rat?

Problem statement: Phytoestrogens are plant derived compound found in a wide variety of foods, most notably soy. The purpose of this study was to assess the effect of soy isoflavones on circulating 17-β estradiol in neonate ovariectomized rat. Approach: Thirty, one day olds rats were used. Blood sample were collected after two months. 17-β estradiol was assayed by radioimmuno assay method. Data were analyzed by one way ANOVA and Duncan as post-hoc test. The level of significance was considered p<0.05. Results: Our data showed that ovariectomy significantly (p<0.05) reduced serum 17-β estradiol. Application of soy in doses 0.75 and 1.5 mL kg-1 day-1 following ovariectomy had not significant effect relative to ovariectomy without application of soy, but application of soy in dose 3 mL kg-1 day-1 significantly (p<0.05) increased 17-β estradiol relative to ovariectomy without application of soy. There was no significant difference between dose 3 mL kg-1 day-1 and control and sham group. Conclusion/Recommendation: Our results indicated that soy milk isoflavone can compensate the 17-β estradiol decrease in ovariectomized rat.

Histopathological effects of the food contaminant furan on some endocrine glands of prepubertal male rats

To evaluate the toxicity of furan on the thyroid gland, adrenal gland, and pancreas of prepubertal male rats. Materials and methods: Furan was administered orally to 3-4-week-old male rats at doses of 2, 4, and 8 mg kg-1 day-1 by dissolving in corn oil for 90 days. The rats were sacrificed by cervical dislocation at the end of the experiment. Their endocrine glands including the thyroid gland, adrenal gland, and pancreas were removed and weighed. Tissues were examined morphologically and histopathologically under a light microscope. Results: Organ and relative organ weights of rats did not change with furan administration. Histopathological changes were observed such as congestion in the pancreas and mononuclear cell infiltration, hyperplasia, and fibrosis in the adrenal gland. Conclusion: Furan administration caused histopathological changes in a dose-dependent manner in prepubertal male rats.

Parasitostatic effect of maslinic acid. II. Survival increase and immune protection in lethal Plasmodium yoelii-infected mice

BackgroundThe anti-malarial activity of maslinic acid (MA), a natural triterpene which has been previously shown to exert a parasitostatic action on Plasmodium falciparum cultures, was analysed in vivo by using the Plasmodium yoelii 17XL murine model.MethodsICR mice were infected with P. yoelii and treated with a single dose of MA by a intraperitoneal injection of MA (40 mg kg-1 day-1) followed by identical dose administration for the following three days. Parasitaemia and accumulation of intraerythrocytic stages was monitored microscopically. To assess protective immunity, cured mice were challenged with the same dose of parasites 40 days after recovery from the primary infection and parasitaemia was further monitored for 30 days. Humoral response was tested by ELISA and visualization of specific anti-P. yoelii antibodies was performed by Western-blotting.ResultsICR mice treated with MA increased the survival rate from 20% to 80%, showing an arrest of parasite maturation from day 3 to 7 after infection and leading to synchronization of the intraerythrocytic cycle and accumulation of schizonts by day 6, proving that MA also behaves as a parasitostatic agent in vivo. Mice which survived the primary infection displayed lower rates of parasitic growth, showing a decline of parasitaemia after day 15, and complete clearance at day 20. These mice remained immunoprotected, showing not malaria symptoms or detectable parasitaemia after rechallenge with the same lethal strain. The analysis of specific antibodies against P. yoelii, present in mice which survived the infection, showed a significant increase in the number and intensity of immunoreactive proteins, suggesting that the protected mice may trigger a strong humoral response.ConclusionThe survival increase observed in MA-treated mice can be explained considering that the parasitostatic effect exerted by this compound during the first days of infection increases the chances to develop effective innate and/or acquired immune responses. MA may represent a new class of anti-malarial compounds which, as a consequence of its parasitostatic action, favours the development of more effective sterilizing immune responses.

The disposition of the enantiomers of warfarin following chronic administration to rats: relationship to anticoagulant response.

A model of chronic anticoagulation has been used to investigate the whole liver and subcellular disposition of the individual enantiomers of warfarin in the Wistar rat in relation to anticoagulant response. Consistent pharmacodynamic responses were achieved by dosing daily with R-warfarin (0.4, or 0.8 mg kg-1 day-1 i.p.) or S-warfarin (0.1 mg kg-1 day-1 i.p.). After the administration of increasing doses of R-warfarin, prothrombin times were dose-dependent (16.3 +/- 0.5 s, 0.1 mg kg-1 day-1; 21.6 +/- 1.7 s, 0.4 mg kg-1 day-1; 55.1 +/- 9.0 s, 0.8 mg kg-1 day-1; results all measured 24 h after the final dose). Increasing doses of R-warfarin also produced increases in plasma, whole liver, and cytosolic concentrations of warfarin. However, there were no significant differences between the microsomal concentrations of R-warfarin in the three groups. The dose of S-warfarin required to produce a consistent and significant increase in the prothrombin time was four-fold lower than the dose of R-warfarin required to cause a similar effect. Plasma concentrations of S-warfarin were not significantly different from those seen after 0.4 mg kg-1 day-1 R-warfarin. Whole liver and cytosolic concentrations of the S-enantiomer were lower than those observed after a dose of 0.4 mg kg-1 day-1 R-warfarin. However, consistent with microsomal concentrations following increasing doses of R-warfarin, there was no significant difference between microsomal concentrations of R(+)- and S(-)-warfarin.(ABSTRACT TRUNCATED AT 250 WORDS)

Location of the mechanism of the clonidine withdrawal tachycardia in rats.

Withdrawal of chronic infusion of clonidine elicits severe tachycardia and short-lasting blood pressure elevations (upswings). Withdrawal of clonidine in low dosage (30 micrograms kg-1 day-1 i.c.v., 7 days) elicited a maximum of 10.9 +/- 0.5 upswings h-1. Cessation of s.c. infusion of clonidine (30 micrograms kg-1 day-1 7 days) evoked a maximum of 1.9 +/- 0.5 upswings h-1. After cessation of the two clonidine infusions no overshoot of heart rate occurred. Withdrawal of a higher dose of clonidine (300 micrograms kg-1 day-1 s.c., 7 days), however, induced tachycardia (from 302 +/- 8 to 433 +/- 8 beats min-1) and 7.6 +/- 1.4 upswings h-1. The administration of the alpha 2-adrenoceptor antagonist yohimbine precipitated withdrawal tachycardia in animals treated with oxymetazoline, a hydrophilic alpha-adrenoceptor agonist. Yohimbine (3 mg kg-1 i.p.) precipitated a severe rise in heart rate from 285 +/- 14 to 520 +/- 5 beats min-1 in oxymetazoline (300 micrograms kg-1 day-1 s.c., 7 days) treated rats and from 320 +/- 13 to 420 +/- 11 beats min-1 in saline-treated animals. Upswings were not induced by yohimbine treatment. It is concluded, that the blood pressure upswings after clonidine withdrawal are due to a central mechanism, whereas the mechanism of the overshoot of heart rate is located peripherally, probably at the cardiac presynaptic level.

Effect of the triazolobenzodiazepine estazolam on hepatic drug-metabolizing enzyme activity in rats.

Oral doses of the sedative/hypnotic estazolam (500 mg kg-1 day-1) to rats for 21 days caused statistically significant increases in liver weight, ascorbate excretion, cytochrome P-450 concentrations, and in aniline hydroxylase, ethylmorphine N-demethylase and glutathione S-transferase activities, as did approximately equivalent doses of flurazepam hydrochloride. Histologically, the centrilobular hepatocytes were enlarged. Some of these parameters were also increased after doses of estazolam of 100 mg kg-1 day-1, but not after 5 mg kg-1 day-1, which is about 50-fold greater than a clinical dose. Estazolam was a much less potent enzyme inducer than phenobarbitone under the conditions of these studies.

Effects of long-term oral administration of NZ-105, a novel calcium antagonist, with or without propranolol in spontaneously hypertensive rats.

A new calcium antagonist, NZ-105 ((+/-)-2-[benzyl(phenyl)amino]ethyl 1,4-dihydro-2,6-dimethyl-5-(5,5-dimethyl-2-oxo-1,3,2-dioxaphosphorina n-2-yl)-4- (3-nitrophenyl)-3-pyridinecarboxylate hydrochloride ethanol) (10 mg kg-1, p.o.), showed slow-onset hypotensive effect in spontaneously hypertensive rats (SHRs). The tachycardia evoked by NZ-105 was completely prevented when combined with a beta-adrenoceptor blocker, propranolol (20 mg kg-1), which did not affect the hypotensive response to NZ-105. In long-term administration experiments for 12 weeks with SHRs, the systolic blood pressure in the control group increased with age and the heart rate was stable throughout the period. NZ-105 (10 mg kg-1 day-1) alone and its combined treatment with propranolol (20 mg kg-1 day-1) maintained the systolic blood pressure and heart rate at a low level compared with the control group. The hypotensive action of NZ-105 were reproducible after repeated dosing for 12 weeks. Long-term administration of propranolol affected neither the elevation of the systolic blood pressure nor the heart rate substantially. The heart weight per body weight was significantly reduced after the chronic combination of both drugs, suggesting that the cardiac hypertrophy accompanying hypertension was prevented.

Effects of benzodiazepine administration on A1 adenosine receptor binding in-vivo and ex-vivo.

The adenosine receptor has been implicated in the central mechanism of action of benzodiazepines. The specific binding of an A1-selective adenosine antagonist radioligand, [3H]8-cyclopentyl-1,3-dipropylxanthine, was measured in-vivo in mice treated with alprazolam (2 mg kg-1, i.p.), lorazepam (2 mg kg-1, i.p.) and vehicle. Binding studies were performed in-vivo and ex-vivo in mice receiving continuous infusion of alprazolam (2 mg kg-1 day-1), lorazepam (2 mg kg-1 day-1) and vehicle by mini-osmotic pumps for 6 days. Continuous infusion of alprazolam and lorazepam significantly decreased specific binding by 34 and 53%, respectively, compared with vehicle treatment (P less than 0.01). Single doses of alprazolam and lorazepam induced a similar trend in specific binding in-vivo (P = 0.07). There were no alterations in A1-receptor density (Bmax) or affinity (Kd) in cortex, hippocampus or brainstem in ex-vivo studies. Benzodiazepine treatment may diminish A1- receptor binding in-vivo by inhibiting adenosine uptake or by direct occupancy of the A1 adenosine receptor recognition site.

Corticosterone and prolactin response to TFMPP in rats during repeated antidepressant administration

The corticosterone and prolactin response to acute administration of the 5-HT agonist 1-(m-trifluoromethylphenyl) piperazine (TFMPP) (10 mg kg-1) was assessed in rats treated for 10 days with either saline, amitriptyline (20 mg kg-1 day-1) or nialamide (40 mg kg-1 day-1). For all groups, TFMPP significantly increased both serum corticosterone and prolactin concentrations compared with control animals challenged with saline. However, the corticosterone response to TFMPP was attenuated significantly by nialamide pretreatment, while the prolactin response to TFMPP was enhanced significantly by amitriptyline pretreatment. These results support previous reports that antidepressants differentially affect 5-HT-ergic systems involved in the regulation of corticosterone and prolactin secretion.

Effect of chronic administration of nicotine or cocaine on steroidogenesis in rat adrenocortical cells

Rats were implanted subcutaneously with osmotic mini-pumps containing either 0.9% NaCl, nicotine (1.5 or 4.5 mg kg-1 day-1), or cocaine (30 mg kg-1 day-1), for 14 days. Neither nicotine nor cocaine treatment significantly altered the maximal rate of steroidogenesis in adrenocortical cell preparations from the animals. However, pretreatment with cocaine increased the sensitivity of the preparation to stimulation by ACTH, the ED50 was 5 pM compared with 10 pM from control animals. Addition of nicotine or cocaine at concentrations up to 100 microM to adrenal cell suspensions from naive rats did not stimulate steroidogenesis or increase the sensitivity of cells to ACTH stimulation. These results suggest that the primary chronic effect of nicotine on steroidogenesis is exerted at the level of the hypothalamus and/or pituitary and not directly on adrenocortical cells. On the contrary, pretreatment with cocaine causes persistent changes in adrenocortical cells.

The effects of 17 beta-oestradiol or testosterone on the response to S-warfarin in castrated male rats.

The effects of castration alone, or followed by administration of 17 beta-oestradiol or testosterone, on the action of S-warfarin have been investigated in male Wistar rats castrated at 4 weeks of age. Six to 10 weeks later, blood samples were taken before, and at 24 h and 7 days after onset of administration of either 17 beta-oestradiol (50 to 500 micrograms kg-1 day-1 i.p.), testosterone (500 micrograms kg-1 day-1 i.p.), or vehicle (polyethylene glycol 200 i.p.). On the seventh day of these interventions, S-warfarin (6.3 mg kg-1 i.p.) was given; prothrombin complex activity (PCA) was then measured over the next 72 h. In addition, a group of sham-operated rats were given S-warfarin, alone. The control prothrombin times were lower in castrated rats (17.9 +/- 0.4 s) than in sham-operated rats (19.1 +/- 0.6 s). Warfarin, over the first 24 h of its administration, caused a significant (P less than or equal to 0.01) fall in PCA in each group of rats, the fall in PCA in castrated rats pretreated with vehicle being significantly (P less than or equal to 0.05) less than that seen in the sham-operated rats. The fall was the same in castrated rats given vehicle or those given 17 beta-oestradiol. In contrast, warfarin caused a greater fall in PCA in the presence of testosterone than in the other groups of castrated rats.

A feasibility study of the multiple-peak approach for pharmacokinetic screening: population-based investigation of valproic acid relative clearance using routine clinical pharmacokinetic data.

The multiple-peak approach was used to evaluate the effect of age, body mass, dose and gender on the population estimates of valproic acid relative clearance. Routine clinical pharmacokinetic data (n = 474) was collected from 250 patients receiving valproic acid and no other drug. The data was analysed according to a simple steady-state pharmacokinetic model using NONMEM, a computer program designed for population pharmacokinetic analysis allowing pooling of data. The final regression model for relative clearance (CL, mL kg-1 h-1) was: CL = 18.9 x body weight(-0.276) x daily dose(0.142) x gender where gender = 1 for males, 0.877 for females. NONMEM estimates suggested that the rate of valproic acid clearance decreased nonlinearly with increasing body weight in the maturation process, and increased nonlinearly with increasing valproic acid dose (mg kg-1 day-1). The clearance in females was about 11% less than in males. These estimates were similar to those detected from previous studies.

Sex-related differences in rat liver microsomal enzymes and their induction by doxapram.

The effects of doxapram on the hepatic microsomal mono-oxygenase system of male and female rats were investigated. Male and female rats were administered doxapram (10-120 mg kg-1 day-1, i.p.) for 4 days. In female rats, administration of doxapram (20, 40, 60, 80, 100 and 120 mg kg-1) elevated the parameters in a dose-dependent manner while doxapram (100 and 120 mg kg-1) elevated the levels of cytochrome P450 and hexobarbitone hydroxylase in male rats. Doxapram (40 mg kg-1) caused induction of hepatic drug metabolism typified by an increase of hepatic microsomal cytochrome P450 content and activities of hexobarbitone hydroxylase, benzphetamine N-demethylase and ethylmorphine N-demethylase in female rats, but no change in male rats. These findings were supported by the results of SDS/polyacrylamide-gel electrophoresis. However, 7-ethoxycoumarin O-de-ethylase and arylhydrocarbon hydroxylase activities were significantly increased in male rats. NADPH-cytochrome c reductase and NADH-cytochrome c reductase activities, and cytochrome b5 content were unaffected in rats of both sexes. The sex-dependent cytochrome P450 species may be selectively sensitive to the action of doxapram.

Ambroxol Improves the Broncho-spasmolytic Activity of Clenbuterol in the Guinea-Pig

The effects of ambroxol on the spasmolytic action of clenbuterol were investigated on acetylcholine-induced bronchospasm in guinea-pigs. Ambroxol (50 mg kg-1 day-1) or vehicle was administered orally for 14 days. Approximately 45 min after the final dose on day 14, the animals were anaesthetized and the spasmolytic effects of clenbuterol (3, 6 or 12 micrograms kg-1 injected intravenously) were determined by use of acetylcholine (40 micrograms kg-1, i.v.)-induced bronchoconstriction. For both vehicle- and ambroxol-treated animals, a positive linear relationship was observed between the log-dose of clenbuterol and the percent inhibition of bronchospasm. The calculated ED25 of clenbuterol (i.e., the dose producing 25% inhibition of the acetylcholine-induced bronchospasm) was 3.98 micrograms kg-1 (3.29 to 4.82 micrograms kg-1, 95% confidence interval) in the presence of ambroxol and 5.81 micrograms kg-1 (4.98 to 6.79 micrograms kg-1) in the absence of ambroxol. The linear regressions with or without ambroxol differed from each other (P < 0.001) but ran parallel (covariance analysis), enabling us to calculate a relative potency, the value of which was 1.46 (1.16 to 1.84). These results demonstrate that the spasmolytic activity of clenbuterol is significantly improved in animals pretreated with ambroxol.

Cross-tolerance development to the prolongation of pentobarbitone-induced sleep by delta 8-tetrahydrocannabinol and 11-hydroxy-delta 8-tetrahydrocannabinol in mice.

Repeated administration (5 mg kg-1 day-1 i.v.) of delta 8-tetrahydrocannabinol and its active metabolite, 11-hydroxy-delta 8-tetrahydrocannabinol caused tolerance to develop to their prolonging effect on pentobarbitone-induced sleep in mice. Reciprocal cross-tolerance also developed after seven daily doses of these cannabinoids. The magnitude of the tolerance developed by the metabolite was greater than that by delta 8-tetrahydrocannabinol. The results suggest that 11-hydroxy-delta 8-tetrahydrocannabinol plays an important role both in the sleep-prolonging effect of delta 8-tetrahydrocannabinol and its tolerance development.

Influence of chronic bisacodyl treatment on the effect of acute bisacodyl on water and electrolyte transport in the rat colon.

Excessive or unnecessary taking of laxatives leads to a vicious circle with perpetuation of laxative abuse and increase of laxative dosage, accompanied by severe clinically evident disturbances. It was investigated in rats whether chronic administration of bisacodyl reduces the effects of acute bisacodyl administration on net water and electrolyte flux in the rat colon transport in-vivo. After chronic pretreatment with bisacodyl had been given for 21 days (3 mg and 10 mg kg-1 day-1), net water and sodium absorption was found to be decreased whereas potassium secretion was increased. After 21 days of pretreatment, the effect of acute bisacodyl (10 micrograms ml-1 intraluminal) on net water and sodium transport was reduced whereas the effect on potassium secretion remained unchanged. Serum and erythrocyte levels of sodium and potassium remained unchanged in chronically pretreated rats. Serum aldosterone levels were enhanced two fold. It is concluded that under the experimental conditions described, chronically administered bisacodyl leads to elevated serum aldosterone which counteracts the effect of bisacodyl on net water and sodium transport but acts synergistically with bisacodyl on the effect on potassium loss.

The effect of metyrapone on granuloma induced by carrageenan-impregnated sponges in normal and essential fatty acid deficient rats.

Treatment of normal rats with metyrapone (20 mg kg-1 day-1, s.c.) for 5 days, starting on the day before implantation, inhibited the production of granuloma, induced by carrageenan-impregnated sponges, determined 8 days after implantation. Exudate volume and prostaglandin (PG) production were unaffected. In essential fatty acid deficient (EFAD) rats, metyrapone did not alter the already existing adrenal hyperplasia due to EFAD and did not affect either granuloma formation or exudate production. The results are discussed in relation to earlier work using adrenalectomy and with regard to the effect of EFA deficiency on adrenal corticosteroid production. It is suggested that metyrapone is a more useful tool than adrenalectomy in studying the role of endogenous corticosteroids.

Effect of sodium valproate on midazolam distribution.

The displacement of midazolam, a new water-soluble, short acting benzodiazepine, from its plasma binding sites by sodium valproate, has been studied in man. An increase of its free fraction (ranging from 2.71 to 5.35%) in plasma from epileptic patients receiving sodium valproate was observed. A similar situation was created in rabbits by pretreatment with sodium valproate (600 mg kg-1 day-1) and posterior hypnosis with midazolam. Due to the interaction, sodium valproate-pretreated rabbits showed an increase in midazolam brain levels (130.91 micrograms g-1 in cortex vs 84.55 micrograms g-1 in control animals). Therefore, it seems likely that displacement of midazolam by sodium valproate in epileptic patients could lead to an increase of the midazolam response.

Amiodarone does not affect digoxin kinetics in the rabbit.

It has been reported that amiodarone may interact with digoxin in man. We investigated the effects of amiodarone pretreatment (35 mg kg-1 day-1) on the pharmacokinetics of a single dose of digoxin (50 micrograms kg-1) in 6 rabbits. Total body clearance of digoxin was 138.84 +/- 44.67 and 147.99 +/- 29.17 ml min-1, serum half life 187.9 +/- 60.9 and 181.34 +/- 25.57 min and volume of distribution 35.4 +/- 8.54 and 37.8 +/- 3.9 litres before and after amiodarone treatment, respectively. None of these changes were statistically significant. We conclude that the presence of an amiodarone-induced change in digoxin pharmacokinetics in the rabbit was not evident and that other animal models will be necessary for studying this interaction.