beta-adrenergic responsiveness of rats treated chronically with isoproterenol

The effect of chronic administration of isoproterenol on isoproterenol-induced thirst and isoproterenol-induced changes in heart rate and selected organ weights of male rats was studied. Administration of 25 micrograms isoproterenol/kg, s.c., in saline daily for 10 days was accompanied by a significant attenuation of the characteristic increase in water intake following a challenging dose of isoproterenol (25 micrograms/kg, s.c.) on the 11th day. Administration of 25 micrograms isoproterenol/kg, s.c., every 2nd, 3rd or 4th day for 10 days was without significant effect on water intake following isoproterenol (25 micrograms/kg, s.c.) on the 11th day. Administration of 25 micrograms isoproterenol/kg, s.c., every day for 10 days led to a slight increase in cardiac responsiveness to a challenging dose of isoproterenol (25 micrograms/kg) on the 11th day. Chronic treatment with this low dose of isoproterenol for 10 days was also accompanied by a significant increase in the ratio of heart weight to body weight but no significant changes in the ratio of kidney, adrenal, thyroid, spleen, or interscapular brown fat to body weight. Thus, daily administration of the beta-adrenergic agonist isoproterenol for 10 days can alter beta-adrenergic responsiveness in the rat with beta 1 (heart rate) and beta 2 (thirst) mediated responses showing opposite effects. In addition, the results suggest that tests of beta-adrenergic responsiveness must be assessed in terms of the frequency of administration of the agonist.     

Dopaminergic control of aldosterone secretion in hypertensive patients chronically treated with an angiotensin converting enzyme inhibitor

To investigate whether dopamine plays a role in the regulation of aldosterone secretion during long-term blockade of the renin-angiotensin system, we studied the effect of metoclopramide, a competitive antagonist of dopamine, in 6 patients with essential hypertension chronically treated with the angiotensin converting enzyme inhibitor enalapril. All but one of these patients received a diuretic in addition to enalapril. Six hours after the daily morning dose of enalapril (10-40 mg p.o.) a 10 mg bolus dose of metoclopramide was injected intravenously. In one patient a hypotensive episode developed following metoclopramide administration. In the 5 other patients plasma aldosterone significantly rose within 30 min after metoclopramide from 51 +/- 8.7 to 128.2 +/- 29.2 pg/ml. This metoclopramide-induced release of aldosterone occurred in the absence of concomitant changes in circulating angiotensin 11, potassium and ACTH levels. Metoclopramide given during chronic blockade of the renin-angiotensin system caused anxiety and agitation in 2 patients. The increase in plasma aldosterone following competitive dopamine blockade in the face of chronic angiotensin converting enzyme inhibition, unchanged plasma potassium and ACTH levels strongly suggests that in hypertensive patients, dopamine exerts a direct inhibitory effect on aldosterone secretion.     

High-performance liquid chromatography of the aromatase inhibitor, letrozole, and its metabolite in biological fluids with automated liquid-solid extraction and fluorescence detection

An analytical method for the determination of letrozole (CGS 20 267) in plasma and of letrozole and its metabolite, CGP 44 645, in urine is described. Automated liquid-solid extraction of compounds from plasma and urine was performed on disposable 100-mg C₈ columns using the ASPEC system. The separation was achieved on an ODS Hypersil C₁₈ column using acetonitrile-phosphate buffer, pH 7, as the mobile phase at a flow-rate of 1.5 ml/min. A fluorescence detector was used for the quantitation. The excitation and emission wavelengths were 230 and 295 nm, respectively. The limits of quantitation (LOQ) of letrozole in plasma and in urine were 1.40 nmol/l (0.4 ng/ml) and 2.80 nmol/l, respectively. The respective mean recoveries and coefficient of variation (C.V.) were 96.5% (9.8%) in plasma and 104% (7.7%) in urine. The LOQ of CGP 44 645 in urine was 8.54 nmol/l (2 ng/ml). The mean recovery was 108% (6.3%). The compounds were well separated from co-extracted endogenous components and no interferences were observed at the retention times of compounds. The sensitivity of this method for letrozole in plasma should be sufficient for kinetic studies in humans with single doses of 0.5 mg and possibly less.     

Lipid peroxidation in liver, plasma, and erythrocytes of rats chronically treated with ethanol

The effect of ingestion of water containing 20% ethanol for 1-2 months on lipid peroxide levels of liver, plasma, and erythrocyte was investigated in rats. Our results show that elevated plasma lipid peroxide levels and erythrocyte susceptibility to lipid peroxidation may reflect stimulated lipid peroxidation in rat liver following chronic ethanol ingestion.     

Decreased cardiac lipoprotein lipase activity in rats treated chronically with adriamycin

Rats treated chronically with the anticancer agent adriamycin (1.5 mg/kg/week X 14 weeks) exhibited cardiac and renal lesions typical of anthracycline toxicity, and had serum hyperlipidemia characterized by 4 to 10 fold elevations in all lipoprotein classes. Heparin-releasable lipoprotein lipase activity measured in perfused heart preparations was decreased 69% in adriamycin-treated rats compared to saline-treated controls. Residual (non-heparin-releasable) activity was not significantly different after adriamycin treatment. The decrease in functional cardiac lipoprotein lipase may account, at least in part, for the serum hyperlipidemia observed in adriamycin-treated rats, and might play a role in the development of heart muscle disease.     

Aminoglutethimide, an inhibitor of aromatase from chick embryo ovary

The gonads from 13-day-old female chick embryos were cultured in vitro on TC medium 199, and oestradiol production was measured by radioimmunoassay. In the presence of dehydroepiandrosterone as substrate, oestradiol synthesis was markedly increased, but when aminoglutethimide was also present, it was greatly reduced, depending on the concentration of the drug. This result demonstrates inhibition of the aromatizing enzyme system of the chick embryo ovary by aminoglutethimide in vitro. However, sex differentiation of the female gonads was not modified after in vivo treatment. Since it is not known whether their production is completely suppressed in vivo, the hypothesis cannot be dismissed that oestrogens play a role in ovarian differentiation.     

Toxicity of the aromatase inhibitor letrozole to Japanese medaka (Oryzias latipes) eggs, larvae and breeding adults

Letrozole is a synthetic aromatase inhibitor and interferes in the committed step in the synthesis of endogenous estrogens from androgens. To evaluate potential effects on the early life stages of Japanese medaka, larvae and fertilized eggs were exposed to letrozole for 96 h and 14 days, respectively. No larvae died and no adverse effects were found on embryonic development at concentrations up to 3125 microg/L. Reproductive effects were assessed by exposing adults to 1, 5, 25, 125 and 625 microg/L letrozole for 21 days. A dose-dependent decrease in fecundity (>25 microg/L) and fertility (>5 microg/L) accompanied by histological changes suggested the inhibition of oocyte growth and possibly maturation. At 625 microg/L, the fish ceased spawning during the last week of exposure. Letrozole (>5 microg/L) reduced plasma vitellogenin levels in females in a dose-dependent manner. Transgenerational effects were evaluated by removing freshly-laid F1 eggs from letrozole-contaminated water and raising them to 15 days post-hatching in control water. Hatchability and time to hatching were detrimentally affected (>5 microg/L), but no morphological deformities were observed. Furthermore, a dose-dependent increase in the proportion of genotypic F1 males was found (>5 microg/L).     

Vitamin A metabolism in rats chronically treated with 3,3',4,4',5,5'-hexabromobiphenyl

Chronic dietary administration of 3,3',4,4',5,5'-hexabromobiphenyl (HBB), 1 mg/kg diet, caused a decrease in retinol (20-fold) and retinyl esters (23-fold) in the livers of female rats, but resulted in a 6.4-fold increase in retinol and 7.4-fold increase in retinyl esters in the kidneys. Liver acyl-CoA:retinol acyltransferase and retinyl palmitate hydrolase activities were reduced while serum concentration of retinol was unaffected by HBB feeding. Metabolism of a physiological dose of [11-3H]retinyl acetate (10 micrograms), was examined in rats fed either vitamin A-adequate diet, or marginal amounts of vitamin A, or vitamin A-adequate diet containing HBB. A 13-fold greater amount of the administered vitamin A was found in kidneys of HBB-treated rats. In rats fed adequate or low amounts of vitamin A, kidney radioactivity was primarily in the retinol fraction, while in HBB-fed rats the radioactivity was associated mostly with retinyl esters. Fecal and urinary excretion of radioactivity was greatly increased in HBB-treated rats. Chronic HBB feeding results in a loss of ability of liver to store vitamin A, and severely alters the uptake and metabolism of vitamin A in the kidneys. We conclude that HBB causes major disturbances in the regulation of vitamin A metabolism.     

Selective 19-hydroxylase inhibition by an aromatase inhibitor, 4-hydroxyandrostenedione

19-Nor-deoxycorticosterone is a newly recognized mineralocorticoid which has been associated with some forms of genetic, experimental, and human hypertension. To further examine this relationship, specific inhibitors of 19-nor-deoxycorticosterone biosynthesis must be developed. Since 19-hydroxylation is the pivotal step in both 19-nor-deoxycorticosterone biosynthesis and aromatization of androgens to estrogens, we evaluated an aromatase inhibitor, 4-hydroxyandrost-4-ene-3,17-dione on the inhibition of 19-hydroxylation in both rat and human adrenal mitochondria in vitro and 19-nor-deoxycorticosterone production and blood pressure in spontaneously hypertensive rats in vivo. Adrenal mitochondria from 48 male Sprague-Dawley rats and 1 patient with an aldosterone-producing adenoma were incubated in the presence of deoxycorticosterone substrate both with and without 4-hydroxyandrost-4-ene-3,17-dione. 4-Hydroxyandrost-4-ene-3,17-dione produced significant inhibition of 19-hydroxy-deoxycorticosterone production in both rat and human adrenal mitochondria, with a smaller and not significant inhibition of corticosterone and 18-hydroxy-corticosterone. 4-Hydroxyandrost-4-ene-3,17-dione given subcutaneously to spontaneously hypertensive rats lowered 19-nor-deoxycorticosterone by 69% and completely abolished hypertension compared to Wistar-Kyoto controls. These data demonstrate that 4-hydroxyandrost-4-ene-3,17-dione is a specific inhibitor of 19-hydroxylase, that it lowers 19-nor-deoxycorticosterone production and prevents hypertension in the spontaneously hypertensive rat. These studies reinforce the possible pathogenic significance of 19-nor-deoxycorticosterone in hypertension in spontaneously hypertensive rats.     

NMR-based metabonomic evaluation of livers from rats chronically treated with tamoxifen,mestranol, and phenobarbital

In this study, we look at the metabolic effects of long-term dosing with tamoxifen, mestranol or phenobarbital on the liver. Tamoxifen, mestranol and phenobarbital have all been reported to act as promoters of hepatic tumors. While tamoxifen and mestranol are known to have estrogenic activity, in the liver phenobarbital is a non-estrogenic compound. Aqueous and lipophilic liver extracts from control and chronically treated Fisher 344 rats were evaluated by nuclear magnetic resonance spectroscopy (NMR). In both the aqueous and lipophilic sample sets, the estrogenic action of mestranol appears to be responsible for the clustering of these samples with those animals treated with tamoxifen. Phenobarbital does not have estrogenic activity and, therefore, clusters away from the estrogenic and control groups. In the lipophilic samples, the fatty acid peak (CH₂)_n was higher in tamoxifen-treated rats than in control, phenobarbital- or mestranol-treated rats. In the aqueous samples, serine and choline levels were higher in phenobarbital-treated rats than controls, which may be an indication that the folate–homocysteine metabolic pathways were altered.     

Effect of adrenaline on insulin secretion in rats treated chronically with adrenaline.

To determine whether rats could adapt to a chronic exogenous supply of adrenaline by a decrease in the well-known inhibitory effect of adrenaline on insulin secretion, plasma glucose and insulin levels were measured in unanesthetized control and adrenaline-treated rats (300 mug/kg twice a day for 28 days) during an adrenaline infusion (0.75 mug kg-1 min-1), after an acute glucose load (0.5 g/kg), and during the simultaneous administration of both agents. Chronic treatment with adrenaline did not modify the initial glucose levels but it greatly diminished the basal insulin values (21.57+/-2.48 vs. 44.69+/-3.3muU/ml, p less than 0.01). In the control rats, despite the elevated glucose concentrations, a significant drop in plasma insulin levels was observed within the first 15 min of adrenaline infusion, followed by a period of recovery. In the adrenaline-treated group, in which plasma glucose levels were lower than in control animals, plasma insulin levels did not drop as in control rats, but a significant increase was found after 30 min of infusion. During the intravenous glucose tolerance test, the plasma glucose and insulin responses showed similar patterns; however, during the concomitant adrenaline infusion, the treated rats showed a better glucose tolerance than their controls. These results indicate that rats chronically treated with adrenaline adapt to the diabetogenic effect of an infusion of adrenaline by have a lower inhibition of insulin release, although the lower basal insulin levels may indicate a greater sensitivity to endogenous insulin.     

Neurochemical changes in rats chronically treated with a high concentration of manganese chloride

Several neurochemical parameters were studied in brain regions of rats chronically treated with a high concentration of manganese chloride (20 mg MnCl₂.4H₂O per ml. of drinking water) throughout development until adulthood. Large increases in Mn accumulation were found in all brain regions (hypothalamus, +530%; striatum, +479%; other regions, +152 to +250%) of Mn-treated adult rats. In these animals, Ca levels were decreased (–20 to –46%) in cerebellum, hypothalamus, and cerebral cortex but were increased (+186%) in midbrain. Mg levels were decreased (–12 to –32%) in pons and medulla, midbrain, and cerebellum. Fe levels were increased (+95%) in striatum but were decreased (–28%) in cerebral cortex. Cu levels were increased (+43 to +100%) in pons and medulla and striatum but Zn levels were decreased (–30%) in pons and medulla. Na levels were increased (+22%) in striatum but those of K and Cl remained unchanged. Type A monoamine oxidase activities were decreased (–13 to –16%) in midbrain, striatum, and cerebral cortex, but type B monoamine oxidase activities decreased (–13%) only in hypothalamus. Acetylcholinesterase activities were increased (+20 to +22%) in striatum and cerebellum. The results are consistent with out hypothesis that chronic manganese encephalopathy not only affects brain metabolism of Mn but also that of other metals.We dedicate this paper to Professor Alan N. Davison. Professor Davison has conducted pioneering research in several important areas including: brain development and myelination, aging and Alzheimer's disease, and multiple sclerosis. He encouraged us to investigate the neurochemical mechanisms of neurotoxicity of metal ions, particularly in connection with neurological diseases. His encouragement and continued support facilitated the launching of our multidisciplinary research program in the long-term effects of manganese toxicity on brain development and aging.     

Effect of a dopamine D-1 agonist in rats treated chronically with zuclopenthixol

We have previously demonstrated long-lasting increases in vacuous chewing movements (VCM) and tongue protrusions in rats treated discontinuously (DISC), but not continuously (CONT), with neuroleptics. To test whether this increase in mouth movements could be a result of exaggerated activity at the D-1 site, 34 rats were divided into three groups receiving the neuroleptic zuclopenthixol (ZU) DISC or CONT for 15 weeks, or no treatment. After withdrawal DISC treated animals showed an increase in oral activity compared to CONT treated. Two weeks after termination of medication the animals were tested with the D-1 agonist SK&F 38393. The increases in VCM after SK&F 38393 did not differ among the groups, but in contrast to control (CTRL) rats, treated rats showed a significant increase in tongue protrusions. There were no significant differences in the densities of D-1 and D-2 receptors in the striatum between the groups. The increase in tongue protrusions after SK&F 38393 in neuroleptic treated animals implies behavioural D-1 receptor supersensitivity. No significant differences in the rise in tongue protrusions and VCM after SK&F 38393 were seen between DISC and CONT treated animals. Our results thus do not indicate that increased D-1 receptor responsiveness is significant for the rise in spontaneous oral activity found after neuroleptic withdrawal.     

The effects of an aromatase inhibitor and a 5 alpha-reductase inhibitor upon the occurrence of polyovular follicles, persistent anovulation, and permanent vaginal stratification in mice treated neonatally with testosterone

Female C57BL/Tw mice were given 5 daily injection of 20 micrograms testosterone (T), 100 micrograms 4-hydroxy-4-androstene-3, 17-dione (4-HA), 100 micrograms 6-methylene-4-pregnene-3, 20-dione (6-MP), 4-HA + 6-MP, T + 4-HA, T + 6-MP, and T + 4-HA + 6-MP starting on the day of birth. The animals were ovariectomized at 30 days or 90 days of age and were killed at 150 days. The incidence of polyovular follicles (PF) at 30 days of age was significantly increased by neonatal treatment with T. By contrast, the PF incidence was significantly reduced by injections of 4-HA given simultaneously with T. Neonatally T- or T + 6-MP-treated 90-day-old mice had ovaries containing follicles and hypertrophied interstitial cells but no corpora lutea. By contrast, T + 4-HA (64%)- and T + 4-HA + 6-MP (82%)-treated mice had ovaries with corpora lutea. In the T + 4-HA + 6-MP (82%)-treated mice had ovaries with corpora lutea. In the T + 4-HA + 6-MP-treated, 150-day-old, ovariectomized mice, the number of mice showing vaginal epithelial stratification was significantly decreased as compared with T-treated mice. There were no significant differences in the number of layers, thickness, and mitotic rate of vaginal epithelium of T-treated mice compared with mice treated with T + 4-HA, T + 6-MP, or T + 4-HA + 6-MP. The present results indicate that development of PF and persistent anovulation are due to the direct action of estrogen (E) derived from T upon neonatal ovarian follicles and the neonatal hypothalamo-hypophysial system, and that T itself can induce ovary-independent vaginal changes, although 5 alpha-reduced androgen and estrogen derived from T seem to be more effective in this regard.     

Impaired reproduction in adult male, but not female, rats following juvenile treatment with the aromatase inhibitor, exemestane

BACKGROUND: Exemestane is an irreversible steroidal inhibitor of cytochrome‐P450 aromatase required for estrogen synthesis. The safety of the drug in the pediatric population, particularly in males, has not previously been evaluated. Given the increased interest in treating children with aromatase inhibitors, we undertook a study in rats to assess the potential for exemestane to alter reproductive development and function when administered to juveniles. METHODS: Male and female rats were treated with exemestane at doses anticipated to produce exposures approximately 2‐ and 35‐fold the expected clinical plasma exposure in young adult males during the period of reproductive maturation. After maturation, treated rats were mated to evaluate the potential impact on reproductive function. RESULTS AND CONCLUSION: There were no effects on sexual maturation in either sex or on female reproductive function. Treatment of juvenile male rats caused increased cohabitation time and decreased copulation rates; pregnancy rates and litter size were not affected in rats that mated. Decreased testis (10–15%) and epididymis (20–30%) weights, and decreased Sertoli cell numbers were noted at all doses. This indicates that exemestane can reduce Sertoli cell proliferation during maturation. The sensitive window for this effect is expected to be limited to the period of Sertoli cell proliferation, which is completed by around postnatal day 15 in rats and before puberty in humans. Treatment beginning at a later time relative to the window for Sertoli cell proliferation or for a longer duration is not expected to have additional adverse effect as the effect was not shown to be degenerative. Birth Defects Res (Part B) 92:304–313, 2011. © 2011 Wiley‐Liss, Inc.     

Effects of the nonsteroidal aromatase inhibitor, Fadrozole, on sexual behavior in male rats.

The new nonsteroidal aromatase inhibitor, Fadrozole (CGS 16949A, CIBA-Geigy Corp.), was tested for its ability (i) to inhibit the conversion of testosterone (T) to estradiol (E2) in brain and (ii) to suppress male sexual activity. Sprague-Dawley rats were castrated and immediately given sc Silastic T-implants and osmotic minipumps delivering 2.5 mg/kg/day Fadrozole (N = 4), 0.25 mg/kg/day Fadrozole (N = 4), or water (N = 4 controls). T-implants were removed after 6 days and, 3 days later, 3H-T (1 microCi/g) was given as an iv bolus. No 3H-E2 was detected in hypothalamic or amygdaloid nuclear pellets from Fadrozole-treated males but this metabolite predominated in controls. However, nuclear concentrations of 3H-T and [3H]dihydrotestosterone were similar in all groups. In another group of males (N = 18), brain aromatase activity was reduced by more than 96% at the 0.25 mg/kg dose level. Additional castrated, T-implanted males received minipumps delivering 0.25 mg/kg/day Fadrozole (six males) or water (six behaviorally matched controls) and were tested weekly with receptive females. After 2 weeks, ejaculations were reduced by 77% compared with controls (P less than 0.01) and, after 4 weeks, intromissions were also significantly reduced (P less than 0.05) but less so (48%). Radioenzymatic estimates of plasma aromatase inhibitor levels remained elevated throughout Fadrozole treatment. These males were then given Silastic E2 implants: intromissions increased significantly in 1 week (P less than 0.01), but ejaculations remained below control values. Results supported the view that aromatization is important for sexual behavior in male rats and suggested that Fadrozole has utility for studying the mechanisms by which testosterone affects behavior.     

Histochemical staining of cadmium thiolate clusters in livers of rats treated chronically with cadmium

Summary In livers of rats exposed to varying doses of CdCl₂ 80–90% of the cadmium content present in the fresh tissue is retained if these livers are fixed with a neutral or acid formalin fixative.Cadmium assays during different stages of the staining procedure for protein bound disulphides show the ability of this staining to demonstrate cadmium thiolate clusters next to disulphides. The methods described may also be useful in gaining more insight in the mechanism involved in fixation and staining procedure of some other metals.