Urinary excretion of 6 beta-hydroxycortisol in women during treatment with different oral contraceptive formulations.

The measurement of the urinary excretion ratio of 6 beta-hydroxycortisol (6 beta-OHC)/cortisol was used as a non-invasive method to investigate possible changes in the activity of drug-metabolizing enzymes in women receiving different oral contraceptive formulations for 1 up to 3 treatment cycles. The contraceptive preparations were either levonorgestrel, gestodene or cyproterone acetate, each in combination with ethinyl estradiol, or only the progestogens levonorgestrel or gestodene. There was either no or only a small decrease in the 6 beta-OHC/cortisol ratio. Thus, only a minor inhibitory effect, if any, can be ascribed to the investigated contraceptive steroids in vivo. Previously observed differences between selected contraceptive steroids in vitro were not observed in the same way in vivo. This may be due either to the absence of a marked inhibitory activity in vivo or to the insufficient sensitivity of the marker 6 beta-OHC/cortisol to detect these changes. Another possible reason may be the considerably higher drug concentrations used in the in vitro studies as compared to those present in the serum of women under oral contraceptive therapy.

Teratogenicity of the 13-cis and all-trans-isomers of the aromatic retinoid etretin: correlation to transplacental pharmacokinetics in mice during organogenesis after a single oral dose.

NMRI mice were treated on day 11 (day 0 = plug day) of gestation with a single oral dose of 100 mg/kg of either all-trans-etretin (acitretin) or 13-cis-etretin. For teratology studies mice were sacrificed on day 18 of gestation, and the fetuses were examined for malformations. For pharmacokinetic studies, groups of 5 mice were sacrificed after 5, 10, and 30 min and 1, 2, 4, and 8 h. The concentrations of retinoids in maternal plasma and in embryos were determined by a newly developed HPLC gradient method. All-trans-etretin induced malformations in 94% of the fetuses, mainly in fore and hind limbs and cleft palate. 13-cis-etretin did not show any teratogenic or embryo-lethal effects at the dose level used. These findings could be explained by a transplacental pharmacokinetic study. The maximum peak level and also the AUC (area under the concentration-time curve) value of all-trans-etretin in the embryos was 7-8 times higher than corresponding values for 13-cis-etretin, probably due to extensive transport of the trans-isomer and limited transport of the cis-isomer from maternal plasma to the embryos. The concentration quotient between embryo and the maternal plasma was between 0.43 and 1.10 for all-trans-etretin, and only 0.16-0.31 for 13-cis-etretin over the time period studied. An in vivo isomerization of the compounds was also observed which was more extensive for 13-cis-etretin than for all-trans-etretin. Our results indicate that the low teratogenic potency of 13-cis-etretin is due to a limited placental transfer of this compound; on the other hand, the potent teratogen all-trans-etretin is rapidly and extensively transferred to the embryo.

Teratogenicity of steady-state concentrations of etretinate and metabolite acitretin maintained in maternal plasma and embryo by intragastric infusion during organogenesis in the mouse: a possible model for the extended elimination phase in human therapy.

Etretinate (Tegison, Tigason), a retinoid used for the treatment of skin disorders such as psoriasis, was shown to teratogenic in the human. Because of the long terminal half-life of this drug (100 days), considerable plasma levels of etretinate and its main metabolite, etretin (acitretin), were observed for up to 2 years following discontinuation of therapy. We have therefore investigated, in a newly developed animal model, the potential teratogenic risk of such persisting levels of these aromatic retinoids. Etretinate was administered by intragastric infusion throughout organogenesis in the mouse (day 8-15) via subcutaneously implanted osmotic minipumps connected to external reservoirs containing oily solutions of the drug. Dose-dependent developmental effects were found, the fetal weight decreased and the resorption rate and incidence of major malformation increased. A dose of 0.84 mg/kg/day resulted in retinoid-specific defects, in particular shortening of the limbs and cleft palate. This low dose infused resulted in mean etretinate concentrations of 6.5 ng/ml maternal plasma and 12.5 ng/g embryo (measured on days 10 and 12 of gestation). The corresponding concentrations of the metabolite etretin were 38 ng/ml plasma and 95 ng/g embryo. Our results emphasize the high teratogenic risk of relatively low, persisting concentrations of etretinate and etretin such as those observed after discontinuation of human therapy, because the area of the concentration-time curve is likely to be the decisive parameter in regard to teratogenesis.

Teratogenicity and placental transfer of all-trans-, 13-cis-, 4-oxo-all-trans-, and 4-oxo-13-cis-retinoic acid after administration of a low oral dose during organogenesis in mice.

13-cis-Retinoic acid (isotretinoin) is teratogenic in humans at therapeutic doses (0.5-1.5 mg/kg) but only marginally teratogenic in the mouse at a high dose of 100 mg/kg. Previous results explained why the cis isomer of retinoic acid was much less teratogenic than the trans isomer in mice. It was found that the placental transfer of all-trans retinoic acid to the mouse embryo was far greater than that of the 13-cis isomer. Since our previous study had been performed with exceedingly high doses (100 mg/kg) of 13-cis-retinoic acid and all-trans-retinoic acid, we have now performed additional experiments with 10-fold lower doses. Studies were also done with the main metabolites of the two retinoids (the 4-oxo-derivatives) to elucidate the metabolism, pharmacokinetics, and teratogenicity of each single compound. It was shown that all-trans-retinoic acid and 4-oxo-all-trans-retinoic acid were extremely teratogenic, whereas their corresponding cis isomers caused only 2% cleft palate. Embryonic exposure to the trans isomers was likewise higher than that to the cis isomers, as shown by the far higher embryonic peak concentrations and by the 30-fold higher areas under the concentration-time curve values reached for the trans isomers compared with the cis isomers. At 8 hr, embryo/maternal plasma ratios were higher than 1 after administration of the all-trans compounds. Concentrations found in the placenta and yolk sac were higher for the trans forms than for the cis forms. We propose a model for a facilitated transport of the all-trans forms to the developing embryo and suggest that the conversion to the trans isomer and trans metabolite could play a major role in the teratogenicity of 13-cis-retinoic acid in humans.

Transplacental pharmacokinetics and teratogenicity of a single dose of retinol (vitamin A) during organogenesis in the mouse.

Pregnant mice received 10 or 100 mg retinol/kg body wt. by gavage on day 11 of gestation (plug day = day 0). One group of animals was used for a pharmacokinetic study. At various times after dosing, plasma and tissue samples were collected and analyzed by HPLC for retinyl esters, retinol, 13-cis- and all-trans-retinoic acid and 13-cis-4-oxo and all-trans-4-oxoretinoic acid. In the other group the fetuses were removed on day 18 and examined for malformations. After 10 mg/kg retinol, no teratogenic effect was observed. The pharmacokinetic investigation revealed a moderate increase of retinyl esters, retinol and all-trans-retinoic acid in plasma, embryonic tissue, placenta, yolk sac membranes and extraembryonic fluid. A high incidence of severe fetal malformations occurred after 100 mg/kg retinol. These malformations included limb defects (81% of fetuses) and cleft palate (55% of fetuses) which are characteristically found after administration of a single teratogenic dose of an active retinoid on day 11 of gestation. The concentration-time profile of retinoids after 100 mg/kg on day 11 showed a pronounced increase of retinyl esters and retinol in all compartments including the embryo and a massive generation of the polar metabolites all-trans-retinoic acid and all-trans-4-oxoretinoic acid. These polar metabolites were found in the embryo with peak concentrations of 327 +/- 115 and 143 +/- 20.7 ng/g (mean +/- SE) wet tissue, respectively. It is likely that all-trans-retinoic acid and all-trans-4-oxoretinoic acid, both well-known teratogens, largely contributed to the teratogenic outcome. The in-vivo oxidation of retinol may be an important factor in the teratogenic activity of high doses of vitamin A.

Extrahepatic sites of metabolism of halothane in the rat.

Rats were given 14C-halothane intravenously and whole-body autoradiography with freeze-dried sections, or with sections extracted in trichloroacetic acid, water, and organic solvents, was carried out to trace tissues accumulating halothane metabolites. In vitro incubations of tissue homogenates were performed to examine the capacity by the various organs to form tissue-bound 14C from the 14C-halothane. Autoradiography of isolated organs after incubation with 14C-halothane was performed to study the tissue localization of halothane metabolites formed under in vitro conditions. A localization of halothane metabolites was observed in several extrahepatic tissues in vivo, and the in vitro experiments showed a capacity by the same tissues to transform 14C-halothane to metabolites that bind strongly to tissue components. In addition to the liver, the other tissues shown to have a marked halothane-metabolizing capacity were the nasal mucosa, lateral nasal gland, mucosa of the tongue, cheek, soft palate (but not the hard palate), pharynx, larynx, oesophagus, and the tracheo-bronchial mucosa. The in vivo data obtained indicated a diffusion of the halothane over the walls of the large intestine and the caecum, followed by the formation of apparently reductive metabolites by intestinal microbes and a binding of the metabolites to the intestinal contents. The localization of halothane metabolites in the upper alimentary and respiratory pathways is correlated to the presence of cytochrome P-450 at these sites.

Transplacental pharmacokinetics of teratogenic doses of etretinate and other aromatic retinoids in mice.

The transplacental pharmacokinetics of single teratogenic doses of etretinate and motretinide were compared with particular emphasis on distribution and concentrations in the exposed embryos of the free acid metabolite, etretin. The three aromatic retinoids were also tested for their direct inhibitory effect on chondrogenesis in the limb bud mesenchymal cell "micromass" culture assay. After a standard dose of 100 mg/kg administered on day 11 of gestation in NMRI mice, all three compounds were teratogenic, but they differed from each other in potency. Etretinate was most active as a teratogen, equalling the potency of our standard all-trans-retinoic acid; every exposed fetus was deformed with severe shortening of all limb bones as well as cleft palate. Etretin was less potent than etretinate, and motretinide was considerably less active as a teratogen than the other two. In the in vitro assay, only etretin suppressed chondrogenesis and this activity was equivalent to that of all-trans-retinoic acid (IC50 of 12 ng/ml). Both etretinate and motretinide (which contain an ethyl ester and ethylamide terminal group, respectively) were essentially inactive in vitro, demonstrating the fact that a free carboxylic group may be a requirement for the in vitro suppression of chondrogenesis. These differences between the results obtained in vivo and in vitro could be resolved by pharmacokinetic investigations using HPLC methods. Both etretinate and motretinide were metabolized in vivo to etretin, their likely common teratogenic metabolite. The high teratogenic potency of etretinate was probably the result of high concentrations as well as AUC values of its metabolite etretin in the embryo. On the other hand, the comparatively low teratogenicity of motretinide could be related to approximately 5 x lower embryonic peak levels as well as AUC values of etretin. A comparison of these results with those previously obtained for all-trans- and 13-cis-retinoic acids confirms the correlation between embryonic exposure and teratogenic potency in the mouse. Our results indicate that pharmacokinetic studies are essential for the interpretation of relative teratogenic potencies of retinoids as well as apparent differences between in vivo and in vitro teratogenesis. A free carboxyl group at the terminal end of the tetraene chain was necessary for high activity of the retinoids studied.

Perinatal metabolism of N-nitrosodibutylamine in Syrian golden hamsters.

Whole-body autoradiography with 14C-labelled N-nitrosodibutylamine (NDBA) in pregnant Syrian golden hamsters on day 15 of gestation showed an uptake of radioactivity in the fetuses and a localization of metabolites in the tracheobronchial and nasal mucosa. Experiments in vitro showed a considerable capacity of these tissues of 15-day-old fetuses to form 14CO2 and tissue-bound 14C from the 14C-labelled NDBA, whereas in 12-day-old fetuses these transformations were insignificant. In infant hamsters (4-20 days old) the 14CO2-formation and the 14C-tissue-binding in the respiratory tissues were marked and in some instances even higher than in the tissues of adult animals. In hamsters the respiratory tissues are targets of transplacental carcinogenesis by NDBA and these tissues of the progeny may also undergo malignant transformations when the mothers are given NDBA during nursing. Our results indicate that a local bio-activation of the NDBA will occur in the respiratory tissues of fetuses at late gestation and in these tissues of infants and that this can be correlated to the carcinogenic effects.

Tracing tissues with chloroform-metabolizing capacity in rats.

Rats were injected i.v. or i.p. with [14C]chloroform and the localization and binding of metabolites in the tissues were studied by whole-body and microautoradiography. Based on the autoradiographic findings various tissues were tested for their capacity to form 14CO2 and to incorporate 14C into tissue-macromolecules from the [14C]chloroform. Autoradiography in vitro was used to localize the sites of [14C]chloroform metabolism under in vitro conditions. The results of the in vitro metabolism studies showed that several tissues had a capacity to metabolize the [14C]chloroform. Further, the results showed that there was a correlation between the ability of various tissues to accumulate metabolites in the rats injected with the [14C]chloroform and the ability of the same tissues to metabolize the [14C]chloroform in vitro. The in vitro autoradiography showed an accumulation of radioactivity at sites corresponding to the ones accumulating metabolites in vivo. It is concluded that many tissues have a capacity to metabolize chloroform in vivo and in vitro. The structures identified to have a marked chloroform-metabolizing capacity were, besides the liver, the kidney cortex, the mucosa of the bronchial tree, the tracheal mucosa, the olfactory and respiratory nasal mucosa, Bowman's glands in the olfactory lamina propria mucosae, Steno's gland (the lateral nasal gland), the mucosa of the oesophagus, the larynx, the tongue, the gingiva, the cheek, the naso-pharyngeal duct, the pharynx and the soft palate (but not the hard palate).

Tissue specificity of N-nitrosomorpholine metabolism in Sprague-Dawley rats.

The distribution and metabolism of N-nitroso[14C]morpholine ([14C]NMOR) in Sprague-Dawley rats were studied by autoradiographic and in vitro techniques. Low-temperature whole-body autoradiography indicated that the non-metabolized compound is able to pass freely through the cellular membranes and distribute evenly in most tissues of the body. Experiments in vitro showed that, in addition to the liver, the nasal mucosa had a high capacity to degrade the [14C]NMOR and a localization of metabolites was also observed in this tissue in vivo. Microautoradiography of the nasal olfactory mucosa showed the highest labelling over the subepithelial glands in the lamina propria mucosae. The nasal mucosa is, apart from the liver, the most prevalent site of NMOR carcinogenesis in the rat and the results indicate that a local bioactivation occurs in this tissue. Further experiments showed that NMOR metabolism in the nasal mucosa and the liver was inhibited by metyrapone and by a carbon monoxide atmosphere, indicating that the metabolism is cytochrome P-450 dependent. In addition to the nasal mucosa and the liver, a localization of metabolites was demonstrated in the oesophageal mucosa in vivo, while in vitro this tissue showed a capacity to degrade the [14C]NMOR. A low incidence of tumours of the oesophagus has been reported in rats treated with NMOR. It is concluded that NMOR is metabolized in the nasal and oesophageal mucosa as well as in the liver, and that local bioactivation at these sites may give rise to tumours.

Autoradiography of [14C]N-nitrosodiethanolamine in Sprague-Dawley rats.

Whole-body autoradiography in Sprague-Dawley rats injected i.v. with [14C]N-nitrosodiethanolamine ([14C]NDELA) showed a localization of tissue-bound radioactivity in the liver and the nasal olfactory mucosa. Microautoradiography of the nasal olfactory mucosa showed the highest labelling over the subepithelial glands (Bowman's glands) in the lamina propria mucosae. Experiments in vitro showed a capacity of the liver and the nasal mucosa to form 14CO2 from the [14C]NDELA. Most of the injected [14C]NDELA was recovered in the urine in a non-metabolized form. A small proportion of the dose was exhaled as 14CO2. The target tissues for the NDELA carcinogenesis in Sprague-Dawley rats are the liver and the nasal mucosa. Our results indicate that a bioactivation of the NDELA will take place in the nasal mucosa, as well as in the liver.

The disposition and metabolism of N-nitrosodiethylamine in adult, infant and foetal tissues of the Syrian golden hamster.

Low-temperature whole-body autoradiography in Syrian golden hamsters, 11 and 15 days pregnant, given N-14C-nitrosodiethylamine, indicated an ability of the non-metabolized substance to freely pass the cellular membranes of the maternal, placental and foetal tissues. Experiments in vitro with tissues of adult hamsters showed a capacity of the nasal olfactory and respiratory mucosa, the trachea, the lung, the lateral nasal gland, the liver and the kidney to degrade the N-nitrosodiethylamine. Autoradiography showed a localization of non-volatile N-14C-nitrosodiethylamine metabolites in these tissues in vivo. Since the mentioned tissues are the targets for the N-nitrosodiethylamine-induced neoplastic or preneoplastic lesions in the hamster, our results indicate that the ability of the sensitive tissues to perform the degradation of the substance is correlated to the tumour organotrophy. The main targets for the N-nitrosodiethylamine carcinogenicity - the nasal olfactory and respiratory mucosa and the trachea - were found to be very active in degrading the substance, and it was shown that cytochrome P450 is present in these tissues. Microautoradiography of the nasal mucosa showed the highest labelling over the subepithelial glands (Bowman's glands) in the olfactory region, but other areas were also labelled. In the 15 day pregnant hamster, autoradiography showed a marked in vivo localization of non-volatile metabolites in the foetal nasal and tracheo-bronchial mucosa and liver, which was not observed in the 11 day pregnant hamster. The in vivo data were shown to correlate to an in vitro capacity by the nasal mucosa, the lung and the liver of the 15 day old foetus to metabolize the substance. N-nitrosodiethylamine is a transplacental carcinogen only when administered on one of the last four days of pregnancy (day 12-15), and our results are in accord with the assumption that only at this late stage of pregnancy has the differentiation of the sensitive tissues reached a stage at which the substance can be metabolized to its ultimate carcinogen. In vitro experiments in 4, 10, and 20 day old infant hamsters showed a high capacity of the nasal mucosa, the lung and the liver to metabolize the N-nitrosodiethylamine at these early stages of life.

Extrahepatic sites of metabolism of carbon tetrachloride in rats.

Rats were injected i.v. and i.p. with [14C]carbon tetrachloride and the localization and binding of metabolites in the tissues were studied by autoradiography. Based on the autoradiographic findings, various tissues were tested for their capacity to form 14CO2 from [14C]carbon tetrachloride in vitro. Autoradiography in vitro was used to localize the sites of [14C]carbon tetrachloride metabolism under in vitro conditions. The results showed that several tissues accumulating metabolites in vivo had an ability to form 14CO2 in vitro, and accumulation of metabolites was observed also under the in vitro conditions. These results indicate that carbon tetrachloride is metabolized in many extrahepatic tissues in vivo. The structures identified to have a marked carbon tetrachloride-metabolizing capacity were, besides the liver, the mucosa of the bronchial tree, the tracheal mucosa, the olfactory and respiratory nasal mucosa, the oesophageal mucosa, the mucosa of the larynx, the tongue and the cheeks, the lateral nasal gland and the kidney cortex. It is well established that the degradation of carbon tetrachloride involves the cytochrome P-450 system, and the metabolism of the substance in the mentioned tissues is probably correlated to high concentrations of cytochrome P-450. The nasal olfactory mucosa was found to be the tissue with the highest capacity to form 14CO2 from the [14C]carbon tetrachloride and microautoradiography indicated that in this tissue the cells of the subepithelial glands of the lamina propria mucosae are most actively engaged in the metabolism. It was also shown that cytochrome P-450 is present in the nasal olfactory mucosa.

Localization and binding of N'-nitrosonornicotine metabolites in the nasal region and in some other tissues of Sprague-Dawley rats.

Microautoradiography of the nasal region of the Sprague-Dawley rat after N'-[14C]nitrosonornicotine administration showed the highest labeling in the subepithelial gland (Bowman's glands) beneath the olfactory epithelium. A lower but distinct radioactivity was also present in the olfactory and respiratory epithelia, covering various parts of the nasal cavity. An exception was the vomeronasal organ, in which neither the olfactory nor the respiratory epithelium was labeled. Determination of the radioactivity bound to DNA, RNA, and proteins isolated from the nasal mucosa and the liver and N'-[14C]nitrosonornicotine administration showed a higher binding of radioactivity to protein and RNA in the nasal mucosa than in the liver, whereas the binding to DNA was somewhat higher in the liver than in the nasal mucosa. Estimations of tissue-bound and non-tissue-bound radioactivity in various tissues of rats given N'-[14C]nitrosonornicotine showed high levels of non-bound radioactivity in the preputial, submaxillary, and Zymbal's glands, whereas a considerable level of tissue-bound radioactivity was present in the nasal mucosa and the liver and also in the esophagus, trachea, and lung. Microautoradiography of the latter tissues showed that this labeling was localized to the epithelia of the esophagus, trachea, and bronchial tree, respectively.

Sites of metabolism of N-nitrosodiethylamine in mice.

Low-temperature whole-body autoradiography and autoradiography with heated sections in C57Bl mice injected with N-[14C]nitrosodiethylamine showed a homogenously distributed volatile radioactivity in most tissues--indicating an ability of the non-metabolized substance to freely pass the biological membranes and distribute evenly in the intra- and extracellular tissue-water. A high level of non-volatile metabolites was found in several tissues: the nasal and tracheal mucosa, the mucosa of the bronchial tree, the salivary glands, the liver, the mucosa of the oesophagus and the tongue, and the lacrimal glands. Studies in vitro indicated that these tissues had a capacity to degrade N-[14C]nitrosodiethylamine (14CO2-production and incorporation of radioactivity in the acid-insoluble material of the tissues were used as indices of the metabolism), whereas several other tissues, which did not accumulate metabolites at short survival intervals in vivo, were devoid of significant metabolic capacity. The relationship between metabolic ability and carcinogenic response of the tissues for N-nitrosodiethylamine is discussed on basis of the obtained results.