J. Lowy and J. R. Pasqualini
J. R. Pasqualini and E. Diczfalusy
S. Roy and J. R. Pasqualini
3H-Corticosterone was administered subcutaneously in vivo and in situ to 32 rat foetuses at 16–18 days of gestation. 30 min after the injection, 11% of the administered dose was localized in the liver tissue, 5% in the lungs, 3% in the intestines and 5% in the placenta. The foetal tissues were separated into different subcellular fractions: I) nucleo-fibrillar, II) mitochondrial-microsomal, and III) cytosol. It was observed that corticosterone was extensively metabolized in all three of these subcellular fractions. The following metabolites were identified in a radiochemically pure form: 11-dehydrocorticosterone, 5α-dihydrocorticosterone, tetrahydrocorticosterone, 5α-tetrahydrocorticosterone. Quantitative evaluation of the corticosterone metabolites showed that the greatest part of these steroids consisted of dihydro- and tetrahydrocorticosterone derivatives, principally with a 5α configuration.
In the liver tissues, 0.5 % of the radioactive material was found in a conjugated form, mainly as ester sulphates. The radioactive material liberated after solvolysis of these conjugates was tentatively identified as corticosterone, 5α-dihydrocorticosterone and 5α-tetrahydrocorticosterone.
J. R. Pasqualini, N. Wiqvist and E. Diczfalusy
In two cases undergoing surgical sterilization and interruption of pregnancy, tritium-labelled corticosterone sulphate was injected into the intact foeto-placental circulation at laparotomy and the metabolites recovered from the placenta and various foetal tissues were analyzed. Little hydrolysis of the injected corticosterone sulphate occurred in the placenta and in the various foetal tissues, the bulk of the administered tracer being metabolized in the form of 21-sulphate. No radioactive material was detected in the glucuronide fraction. After enzymic hydrolysis of the ester-sulphate fraction, the following compounds were isolated: 21-hydroxy-pregn-4-ene-3,11,20-trione (placenta, residual foetal tissues), 3α,11β,21-trihydroxy-5β-pregnan-20-one (liver, gastrointestinal tract and placenta), 3β,11β,21-trihydroxy-5α-pregnan-20-one (liver), and 6β,11β,21-trihydroxypregn-4-ene-3,20-dione (liver, residual foetal tissues and placenta).
A concept is presented describing the metabolism of corticosterone sulphate in the human foeto-placental unit at midpregnancy.
J. R. Pasqualini, C. Sumida and C. Gelly
The formation of [3H]oestradiol macromolecule complexes was studied in vivo and in vitro in the kidney, lung and liver of intact foetal guinea pig. Specific binding of [3H]oestradiol was demonstrated in the cytosol and nuclear fractions of foetal kidney. Intensive binding was found in the cytosol of foetal lung but most of the bound radioactive material (71 %) was [3H]oestrone. Some binding was found in the cytosol of foetal liver but not in the nucleus of this tissue.
In the in vivo experiments, the binding of radioactive material to plasma proteins was studied: 22 % of the plasma radioactivity was bound of which 67 % was identified as oestrone sulphate. Oestradiol sulphate represented 7 % and unconjugated oestradiol only 0.6 % (0.1 % of the total plasma radioactivity). On the other hand, 2–3 % of the foetal plasma radioactivity was found as unbound [3H]oestradiol.
In the kidney, the formation of [3H]oestradiol complexes in the cytosol fraction does not depend on temperature while nuclear [3H]oestradiol complexes increase with increasing temperature. Maximal formation of [3H]oestradiol complexes in the cytosol fraction and the 0.1 m TRIS and 0.3 m NaCl nuclear extracts was reached after 15 min but binding in the 1 m NaCl nuclear extract continued to increase up to 30 min.
After incubation of purified nuclei of foetal kidney with 1.1 × 10−7 m [3H]oestradiol, specific binding was found in the different nuclear fractions. Specific binding was also detected in isolated nuclei previously extracted by 0.1 m TRIS and 0.3 m NaCl before being incubated with 1.1 × 10−7 m [3H]oestradiol.
The Kd of binding of [3H]oestradiol in the renal cytosol fraction is 2.5 × 10−10 m with n = 4.5 × 10−14 moles/mg protein. Incubation of isolated 1 m NaCl nuclear extract from foetal kidney with [3H]oestradiol gives a Kd of 3.3 × 10−10 m with n = 2.5 × 10−14 moles/mg protein.
It is concluded that the nuclear complexes in the foetal kidney could be formed either through an intermediate cytosol complex or that the "two step" mechanism could take place in the nucleus. Furthermore, direct binding of [3H]oestradiol with high affinity was observed in the 1 m NaCl nuclear extract.
]. Lowy, T. Albepart and J. R. Pasqualini
3H-Corticosterone was administered intravenously into two rats. After 80 min the animals were sacrificed and the radioactive material of the different tissues (intestine, liver, kidneys, lungs, adrenals, spleen and the remainder of the animal) was analyzed. Most of this material was found in the intestine and in the liver in a conjugated form and identified as ester-sulphates. This material mainly consisted of hexahydro- and of tetrahydro-derivatives. Corticosterone 21-sulphate was identified in this fraction. 7 to 12% of the radioactivity corresponded to polar conjugated steroids non-hydrolyzed by β-glucuronidase, sulphatase or solvolysis treatment. After hydrolysis of this fraction by the enzymes of succus entericus of Helix pomatia, the radioactive material liberated mainly consisted of hexa- and tetrahydro-corticosterone metabolites.
J. R. Pasqualini, G. Mozere, N. Wiqvist and E. Diczfalusy
Two previable human foetuses were perfused at midpregnancy with corticosterone-1,2-3H and the metabolites formed were isolated from the various tissues and perfusates. With the exception of the adrenals, the bulk of the radioactive material present in the perfusates and various foetal tissues was in an unconjugated form.
The following compounds were isolated in a radiochemically homogeneous form: 21-hydroxy-pregn-4-ene-3,11,20-trione (liver, intestines, lungs, adrenals, residual foetal tissues and perfusates), 11β,20β,21-trihydroxy-pregn-4-en-3-one (liver, intestines, lungs, residual foetal tissues and perfusates), 3α,11β,21-trihydorxy-5β-pregnan-20-one (liver, intestines, lungs, residual foetal tissues and perfusates), 6β,11β,21-trihydroxy-pregn-4-ene-3,20-dione (liver, intestines, lungs, residual foetal tissues and perfusates), and 11β-hydroxy-pregn-4-ene-3,20-dion-21-yl sulphate (liver, intestines, lungs, adrenals, residual foetal tissues and perfusates). In addition, 20β,21-dihydroxy-pregn-4-ene-3,11-dione (liver, lungs, residual foetal tissues and perfusates) was identified by its chromatographic behaviour prior and subsequent to the formation of various derivatives. Finally, small quantities of corticosterone, 11β,20β,21-trihydroxy-pregn-4-en-3-one and 3α,1 1β,21-trihydroxy-5β-pregn-20-one were detected in the glucuronide fraction of the intestines, residual foetal tissues and perfusates.
A concept is presented describing the metabolism of corticosterone in the human foetus at midpregnancy.
F. Lecerf, B.-L. Nguyen and J. R. Pasqualini
Abstract. The biological and morphological effects of cis-tamoxifen, N-desmethyltamoxifen and 4-hydroxytamoxifen, administered sc alone (100 μg/animal) or combined with estradiol (20 μg/animal) were studied in the uterus and vagina of the guinea pig. After 2 days treatment the values of the uterine wet weights (mg ± sd of 6–10 animals in each study) were as follows: non-treated animals (control): 142 ± 15; animals treated with cis-tamoxifen: 119 ± 4; N-desmethyltamoxifen: 280 ± 20; 4-hydroxytamoxifen: 268 ± 25. The values after long treatment were: 177 ± 30; 490 ± 65; 394 ± 36 and 581 ± 60, respectively. After short treatment the weights of the vaginas were: control: 99 ± 20; cis-Tamoxifen: 67 ± 2; N-desmethyltamoxifen: 153 ± 25; 4-hydroxytamoxifen: 166 ± 7; and after the long treatment: 155 ± 40; 660 ± 41; 467 ± 38 and 502 ±61, respectively. N-desmethyltamoxifen and 4-hydroxytamoxifen increased the progesterone receptors in the uterus after short treatment (P < 0.01) but not after 12 days treatment. On the other hand, there was no effect on progesterone receptor in the vagina after the short treatment but a very stimulatory effect after the long treatment. The morphological alterations after 12-days treatment indicate that the three tamoxifen derivatives in the two tissues studied provoke intense alterations in different organelles. In conclusion, it is suggested that the tamoxifen derivatives can act as real agonists in the uterus and vagina of the newborn guinea pig, and they do not block the effect provoked by estradiol.
J. R. Pasqualini, M. Bedin and A. M. Cogneville
In vivo and in vitro transformation of labelled d-aldosterone (11β,21-dihydroxy-3,20-dioxo-4-pregnen-18-al) was studied in the foetal tissues and placenta of guinea pig (35–45 days of gestation). The principal metabolite found in all of these tissues was tetrahydroaldosterone (3α,18,21-trihydroxy-20-oxo-5β-pregnan-18-al). A large transformation into this metabolite was found in foetal liver (67–68 %) and in the placenta (69–82%). Incubation of [3H]- or [14C] aldosterone with the isolated placenta also showed a large conversion to the tetrahydroderivative suggesting that the reductase(s) of ring A of aldosterone is present in this tissue.
Most of the unmetabolized aldosterone and tetrahydroaldosterone was found in the unconjugated fraction. Very little of the radioactive material was present in the polar steroids extracted with n-butanol. Aldosterone and tetrahydroaldosterone were liberated from this fraction after enzymatic hydrolysis (β-glucuronidase and sulphatase) or by solvolysis, suggesting that part of these conjugates is present in the foetal compartment as sulphate esters and glucuronides. Moreover, in this fraction a polar, unconjugated aldosterone metabolite with the characteristics of an acidic compound was detected.
J. R. Pasqualini, B.-L. Nguyen, C. Mayrand and F. Lecerf
Abstract. Tamoxifen (TAM) alone or combined with oestradiol (E2) or progesterone (P) was administered to newborn guinea pigs (2 days old) for a short (2 days) or a long (12 days) treatment period. TAM alone provoked a great stimulatory effect on uterine growth and DNA content and the effect was particularly intense after the long treatment. These actions were markedly enhanced when TAM was administered together with E2. Following short treatment, the values of the uterine wet weights (mg ± sd) were as follows: control animals, 142 ± 15; animals treated with TAM, 298 ± 53; E2, 335 ± 15; (TAM + E2), 362 ± 16. The values after the long treatment were 177 ± 30, 555 ± 93, 709 ± 117 and 1263 ± 222, respectively.
Histological studies showed that TAM provoked morphological changes in both the endometrium and the myometrium. The effects were particularly great on the height of the luminal epithelial cells and on the uterine glands. After 2 days of treatment with E2, TAM and P, the thickness of the luminal epithelium, which was 13.5 μm ± 1.5 in the control animals, increased as follows: TAM, 19 μm ± 1.7; E2, 20.3 μm ± 3.3; (TAM + E2), 30.5 μm ± 5; P, 12 μm ± 0.9 and (P + TAM), 19.7 μm ± 1.2. The values after the 12 day treatment were: controls, 20.8 μm ± 1.8; TAM, 27.4 μm ±2.1; E2, 32 ± 3; (TAM + E2), 43 μm ± 5; P, 17.8 μm ± 1.2 and (P + TAM), 23.6 μm ± 1.5. After the short treatment TAM doubled the number of specific progesterone binding sites. It is concluded that TAM acts as a real oestrogen agonist in the uterus of newborn guinea pigs.