Effects of atipamezole, detomidine and medetomidine on release of steroid hormones by porcine adrenocortical cells in vitro.

The 4-substituted imidazole type alpha2-adrenoceptor ligands atipamezole, detomidine, and medetomidine were screened for actions on the release of aldosterone by a suspension of porcine adrenocortical cells with deoxycorticosterone (1 microM) as substrate. Progesterone, pregnenolone or corticosterone (all at 1 microM) were also used as substrates. With pregnenolone as substrate, drug-induced effects on the output of nine steroids (aldosterone, corticosterone, cortisol, deoxycortisol, testosterone, progesterone, 17alpha-hydroxyprogesterone, androstenedione, dehydroepiandrosterone) were monitored simultaneously. The alpha2-adrenoceptor antagonist atipamezole was a potent inhibitor of aldosterone release (range 10-1000 nM). The sedative alpha2-adrenoceptor agonists medetomidine and detomidine also inhibited aldosterone release (range 10-1000 nM). With pregnenolone as substrate, the inhibition induced by 4-substituted imidazoles of the release of corticosterone and cortisol was more pronounced than that of aldosterone. Androstenedione and deoxycortisol release was enhanced. The 4-substituted imidazoles atipamezole, detomidine, and medetomidine inhibited mitochondrial cytochrome P450(11beta/18) in vitro. This inhibition was unrelated to their alpha2-adrenoceptor actions. The 4-substituted imidazole type alpha2-adrenoceptor ligands used to control sedation/anaesthesia can alter the steroid-based defence mechanisms of the body.

Effects of alpha1-antagonists on production and release of aldosterone and other steroid hormones by porcine adrenocortical cells in vitro.

Quinazoline type alpha1-adrenoceptor antagonists (range 10-100 microM) inhibited aldosterone release of a cell suspension of porcine adrenocortical cells, potency order: doxazosin > prazosin > trimazosin. Phenoxybenzamine also inhibited the aldosterone release at a concentration of 100 microM. Alpha1-adrenoceptor antagonists from other chemical classes had no measurable effect on the aldosterone output from adrenocortical cells in vitro. Agonists selective for either alpha1- or beta-adrenoceptors did not affect the aldosterone release. The inhibition of the aldosterone release induced by quinazolines was similar with different substrates. The small differences between the drug-induced inhibitions could be ranked as corticosterone = progesterone > pregnenolone = deoxycorticosterone. The doxazosin (10 microM)-induced changes in the release of nine steroids indicated that quinazoline-type alpha1-antagonists interfere with enzymes of the aldosterone biogenesis pathway involved in C18-oxidation and C21beta-hydroxylation, reducing the release of both aldosterone and corticosterone. At higher concentrations (100 microM), the C21beta-hydroxylation in the cortisol biogenesis pathway is also affected, decreasing the output of cortisol and deoxycortisol, but increasing the output of progesterone and OH-progesterone. Simultaneously, the C17-oxidation and side-chain cleavage is also inhibited, decreasing the output of androstenedione. The rank order of phenoxybenzamine (100 microM)-induced inhibition of the aldosterone release with different substrates is pregnenolone > corticosterone = progesterone > deoxycorticosterone. With pregnenolone as substrate, the output of aldosterone, corticosterone, and cortisol was reduced to the same extent. The dehydroepiandrosterone, androstenedione, and progesterone release was enhanced. It seems that phenoxybenzamine is a rather selective inhibitor of the mitochondrial P450(11beta/18) enzymes.

Differential effects of nitrofurans on the production/release of steroid hormones by porcine adrenocortical cells in vitro.

Changes in the biogenesis of corticosteroids caused by nitrofurans were studied. The three nitrofurans used: furazolidone, furaltadone and nitrofurantoin, altered the steroid production/release by porcine adrenocortical cells in vitro during 1 h incubations. With pregnenolone as a substrate the nitrofurans inhibited aldosterone production/release. Although the nitrofurans differed in potency (nitrofurantoin > furazolidone > furaltadone) maximum inhibition occurred at 100 microM. In this concentration the nitrofurans changed also the release/production of other corticosteroids. The output of corticosterone and cortisol decreased by 50%. The production/release of deoxycortisol stayed the same. In contrast the output of progesterone and 17alpha-hydroxyprogesterone increased to more than 200% of control. The nitrofurans slightly reduced the output of androstenedione. No significant increases of the production/release of other steroids (testosterone, dehydroepiandrosterone, estradiol-17beta and estrone) by the cell suspension could be observed. The profile of the nitrofuran-induced changes lead to the conclusion that nitrofurans interfere with mitochondrial enzymes. These enzymes, presumably cytochrome P450(11,18) mediate the hydroxylation and the oxidation at C11 and C18, the final steps in the biogenesis of aldosterone, corticosterone and cortisol. The rapid and reversible fall in the output of these steroids occurs in vitro at concentrations which are below therapeutic blood concentrations seen in vivo. At higher concentrations the nitrofurans hinder the biogenesis of androgens. Thus nitrofurans can also affect steps in the steroid biogenesis located in the endoplasmatic reticulum.

Screening for drug-induced alterations in the production and release of steroid hormones by porcine adrenocortical cells in vitro.

To screen drugs rapidly and at minimal expense for their potential to alter steroidogenesis, an in vitro model using porcine adrenocortical cells was developed. Pregnenolone, progesterone, deoxycorticosterone or corticosterone (all at 1 muM) were used as substrates. Drug-induced changes in the production/release of aldosterone were measured after 1-hr incubation. With pregnenolone, drug-induced effects on the release of nine steroids (aldosterone, corticosterone, cortisol, deoxycortisol, testosterone, progesterone, HO-progesterone, androstenedione, dehydroepiandrosterone) were monitored simultaneously. For assessment of cell viability and the amount of steroids produced/released, a cheap, simple modified Krebs solution was at least comparable to an elaborate cell culture medium. Within the conditions adopted, the cell suspension reacted to varying potassium concentrations as expected. ACTH stimulated steroid production/release only without added substrate. 11 agents known to interfere with steroid biogenesis were tested at 0.1-100 muM. Although all known points of action of the test compounds were located, several showed additional activity. Spironolactone shifted steroid biogenesis from aldosterone and cortisol towards androgenic steroids. Aminoglutethimide inhibited the release of aldosterone with corticosterone as substrate, but not with deoxycorticosterone or progesterone as substrate, revealing an alternative pathway in the biogenesis of aldosterone by-passing corticosterone. Trilostane (0.1-1 muM) completely blocked conversion of pregnenolone to progesterone and OH-progesterone; the release of androstenedione was at most only halved, whereas the release of dehydroepiandrosterone and testosterone was greatly enhanced. This implies isoenzymes of 3beta-hydroxysteroid dehydrogenase/isomerase with different sensitivities towards trilostane. Mitotane, metyrapone, ketoconazole and etomidate all inhibited the mitochondrial P450 (11beta 18 ) enzymes. In addition, mitotane and ketoconazole also inhibited (albeit to a lesser extent) endoplasmic enzymes involved in transformations at C21 and at C17, respectively. Cyproheptadine blocks all transformations with progesterone or HO-progesterone as starting point. Finasteride reduced the release of most steroids, except the androgens, presumably by inhibition of transformations at C3 and at C11. Carbadox and related quinoxalines inhibited not only C18 oxidation but also C21 hydroxylation. Steroidogenesis in these porcine adrenocortical cells in vitro could be described as similar to that in other mammals. A notable feature was that inhibition of the release/production of a steroid hormone was usually accompanied by an increased release of other steroid hormones. This screening model also yields information about the point of action of drugs interfering with steroidogenesis.

Effects of feed additives and veterinary drugs on aldosterone production and release by porcine adrenal cells in vitro.

The preparation of suspensions of porcine adrenocortical cells is described. Within the conditions adopted, the cell suspension responded to various agents as expected. It was possible to screen drugs (standard range 0.3-100 microM, incubation period 1 h) for actions on the production/release of aldosterone by the cortical cells using 1 microM deoxycorticosterone as substrate. Progesterone, pregnenolone or corticosterone were also used as substrates. Feed additives of the quinoxaline type induced a slowly developing inhibition of aldosterone production/release by the cell suspension, which was virtually irreversible. During the standard 1 h incubation period inhibitions of up to 22 +/- 2% of control were observed, which increased upon prolongation of the incubation by 2 h. With 100 microM cyadox the inhibition increased from 19 +/- 2% to 35 +/- 2% with prolonged incubation. Ten nitrofuran compounds exerted a more rapidly developing inhibition (by up to 79 +/- 1% of control) of aldosterone production/release, which was reversible. A submaximal inhibition with 10 microM furazolidone of 21 +/- 5% increased to 40 +/- 1% with longer incubation. The concentrations at which these compounds exerted this effect in vitro were well below the peak blood plasma concentrations encountered after administration of the drugs in therapeutic doses. Polyether-antibacterials/ionophores rapidly inhibited aldosterone production/release (to 26 +/- 1% of control) and this effect was completely reversible. The nitroimidazole compounds tested did not affect aldosterone production/release when deoxycorticosterone or progesterone were used as substrates. With use of corticosterone and to a lesser extent with pregnenolone as substrates a clear inhibition (to 73 +/- 3% of control) of aldosterone production was obtained. Amprolium in concentrations up to 100 microM, with deoxycorticosterone as substrate, did not induce a significant change in aldosterone production/release by the suspension of adrenocortical cells. In the same dose range tylosin and roxarsone induced a small but significant inhibition (by up to 10 +/- 3% of control) of aldosterone production/release, which was not dose-dependent. It is concluded that a wide range of growth-promoting drugs may be able to change aldosterone production/release in the animal.

Clinical signs and performance of pigs treated with different doses of carbadox, cyadox and olaquindox.

An experiment was designed to study the clinical effects of different levels of carbadox, cyadox and olaquindox in the ration on health, weekly weight gain and feed conversion in pigs. Four different carbadox and olaquindox (25, 50, 100 and 200 ppm) levels and five different cyadox (25, 50, 100, 200 and 400 ppm) levels were tested in groups of 6 pigs during 6 weeks. The 13 groups were compared with a control group fed on the same diet with only vehicle. After one week the first clinical sign, a high faecal dry matter (FDM) content, was observed in the 200 ppm carbadox group, followed by the 100 and 50 ppm carbadox, the 400 and 100 ppm cyadox, and the 200 and 100 ppm olaquindox groups two weeks later. A second clinical sign, urine drinking from the floor or from pen-mates, was observed in the same pens, occurring in the same sequence. The third important clinical sign, a decreased abdominal volume, was also observed in almost the same sequence, however, in the 50 ppm olaquindox and cyadox groups this clinical sign was not observed. Average weekly weight gain was significantly decreased in the higher carbadox and olaquindox groups. Weight gain was significantly increased in the 200 ppm cyadox group. Hematocrit values were significantly increased in the 200 and 100 ppm carbadox groups only. From this study one may conclude that, within the dosages used, carbadox is more harmful than olaquindox for pigs, and it seems that cyadox is harmless for pigs in dosages up to 400 ppm.

Comparative study of the effect of the effect of carbadox, olaquindox and cyadox on aldosterone, sodium and potassium plasma levels in weaned pigs.

To study the effects of olaquindox and cyadox on aldosterone, sodium and potassium in the blood in comparison with the effects of carbadox, weaned pigs were fed these compounds in different doses. Pigs treated with 100 and 200 ppm carbadox showed a significant decline of aldosterone after five and three weeks, respectively, compared with control values. In the 200 ppm group treatment was interrupted at week 4. With olaquindox a continuous, significant decline was found from 50 ppm and above after five weeks, and from 25 ppm and above (but excluding the 100 ppm group), after six weeks. In the cyadox groups a significant decline was measured after six weeks in the 50, 200 and 400 ppm groups. Only the 200 ppm group had an earlier response at three and five weeks. A decrease of sodium to hyponatraemic levels in the carbadox groups was seen after three weeks in the 200, and after five weeks in the 100 ppm group. In the olaquindox groups only the 200 ppm dosage showed a consistent decrease to hyponatraemic levels from four weeks treatment. In the cyadox groups the 200 ppm dosage reached a hyponatraemic level after six weeks. An increase of potassium to hyperkalaemic levels occurred at 100 and 200 ppm carbadox dosage after four and three weeks, respectively, and at 200 ppm olaquindox dosage after four weeks. No hyperkalaemic levels were seen in the cyadox groups. It is concluded that the toxic effect of olaquindox, despite minor differences, is comparable with that of carbadox but that cyadox is less toxic.

Persistence of carbadox-induced adrenal lesions in pigs following drug withdrawal and recovery of aldosterone plasma concentrations.

This study was performed to investigate the persistence of carbadox-induced adrenal lesions in pigs after withdrawal of the drug. Six groups (N = 13) received 0 (control group), 25, 50, 100 and 200 ppm carbadox. After 10 weeks, carbadox was withdrawn from the feed. Five and 11 weeks after withdrawal, two pigs per group were necropsied and the adrenals were examined histologically. Five weeks after withdrawal, recovery of lesions was seen in the 25 and 50 ppm groups. In the 100 and 150 ppm groups, adrenal changes were still present. After 11 weeks an incomplete recovery occurred in the 100 ppm group and in one of the pigs from the 150 ppm group; the second pig of this group still demonstrated moderate changes. Pigs from the 200 ppm group showed severe changes and absence of a clear zonal differentiation. Plasma aldosterone values started to recover 2 weeks after withdrawal of carbadox. Histological examination suggested stimulation of the aldosterone-producing glomerular zone, eventually resulting in regressive changes. The mechanisms that possibly induced this continuous stimulation are discussed.

Some pharmacokinetic observations of carbadox medication in pigs.

Concentrations of carbadox and a first metabolite, desoxycarbadox, were measured in contents of the porcine gastrointestinal tract after in-feed administration of carbadox in therapeutic dosages (100-150 ppm). The levels of carbadox in the relevant parts of the gastrointestinal tract were found to be lower than the MIC-values reported for enteropathogenic microorganisms at their sites of action. The presented observations do not provide a pharmacological rationale for the therapeutic use of carbadox in the treatment of dysentery and diarrhoea in swine. The carbadox levels encountered in the proximal part of the gut (stomach, duodenum) however, seem to indicate that in-feed administration of 50 ppm carbadox can provide an effective prophylaxis against Treponema hyodysenteriae, a causative agent in swine dysentery. The timecourse of the blood levels of carbadox and desoxycarbadox after in-feed administration of carbadox (50 ppm) and the concentration profiles in the gastrointestinal tract are discussed with regard to the disposition of this drug in pigs.

Carbadox-induced inhibition of aldosterone production in porcine adrenals in vitro.

Carbadox, a growth promoter used in pig husbandry, inhibited aldosterone production in sliced porcine adrenals in vitro. The adrenal slices, obtained from 3-5-wk-old piglets, were maintained in a Krebs solution and exposed to 1-40 mug carbadox/ml for 1 hr. Aldosterone was estimated (using a solid-phase radioimmunoassay kit) in samples taken hourly for up to 5 hr after carbadox exposure. The results showed a dose-dependent inhibition of aldosterone production by carbadox, with 25-35% inhibition by a concentration of 40 mug/ml. The findings support the hypothesis, based on clinical and histopathological evidence from in vivo experiments in pigs, that carbadox interferes with aldosterone production in the adrenal zona glomerulosa.

Cadmium, zinc, lead, and copper in livers and kidneys of cattle in the neighbourhood of zinc refineries.

Cattle in the Kempen area (in the province North-Brabant, the Netherlands) were investigated for cadmium, lead, zinc, and copper in livers and kidneys. The animals originated from farms located within a 20 km radius around several zinc refinery plants. The local soil is polluted with zinc and cadmium because of a thermal refining process used in the past.Mean cadmium organ concentrations were 2.5 times, and mean lead organ concentrations were 1.5 times higher than the concentrations found in controls. Copper levels tended to be decreased, but zinc levels did not differ from controls.The observed cadmium and lead organ concentrations did not indicate intoxication of the animals, but 22% of the kidneys and 3% of the livers investigated trespassed the maximum tolerance limit of cadmium with regard to human consumption.Continuous control of cadmium organ contents in organs from slaughtercattle kept in cadmium-polluted areas is recommended.

Hypoaldosteronism in piglets induced by carbadox.

An exploratory study was made of the mechanisms underlying the toxic action of carbadox in young pigs: dehydration, loss of appetite and at autopsy seemingly specific and selective structural alterations of the glomerular zone of the adrenal cortex. Administration of carbadox in the feed, in dosages of 150 ppm (approximately 6 mg X kg-1 b. wt X day-1) caused a rapid decline in the plasma aldosterone levels (to 10% of control) followed by significant changes in the sodium and potassium levels in blood. Characteristic for the toxic action of carbadox are the rapid and seemingly selective and specific alterations in the aldosterone-releasing zona glomerulosa of the adrenals. Our results indicate that with carbadox a functional and possibly reversible extirpation of the adrenal zona glomerulosa can be achieved in pigs.

Metal pollution in a salt marsh and its effect on sheep.

In a salt marsh in the Westerschelde, samples were taken from soil and vegetation during 15 months. Concentrations of Cd, Pb, Cu, Zn, Mn, and Fe were measured and compared with levels of these metals in livers, kidneys, and faeces of sheep grazing there. The contamination extent of soil ranges from 2 (Cu) to 8 (Cd); in spring some plants exceed the advised maximum tolerable level of dietary Cd for sheep. However, levels of metals in organs from sheep do not indicate intoxication, nor did clinical inspections and autopsies. It is suggested that sheep exhibit a selective consumption behaviour and that the bioavailability of the metals in this marsh is low.

Liquid chromatographic determination of carbadox and desoxycarbadox in medicated feeds and in porcine gastrointestinal tract.

A liquid chromatographic method for the assay of carbadox and desoxycarbadox in medicated feeds and porcine stomach and intestinal contents is described. Samples were extracted with dimethylformamide-water and cleaned up on an alumina column. The eluate was chromatographed using either gradient elution for simultaneous assay of both compounds or isocratic elution for carbadox only. Detection of carbadox by its native fluorescence yielded a sensitive and specific assay without interferences by metabolites or matrix components. The optimal UV absorption of desoxycarbadox was at 280 nm. Mean recoveries of carbadox in spiked feed and stomach contents were 104 and 97%, respectively; mean recovery of desoxycarbadox in stomach contents was 106%. The day-to-day reproducibility for carbadox in different feed samples and stomach contents samples had a coefficient of variation of 6-13%.

A case of chronic zinc poisoning in calves fed with zinc-contaminated roughage.

A case of zinc intoxication in young female cattle is described. The clinical signs consisted of reduced appetite, emaciation, submandibular oedema and diarrhoea. The source of zinc proved to be roughage harvested in the vicinity of a factory galvanizing steel tubes. In this roughage zinc levels between 3000 and 7300 mg/kg dry weight were found. In the liver of four animals zinc levels varied between 420 and 1600 mg/kg, and between 910 and 1680 mg/kg dry weight in the kidneys.