Brief Report Differential oxidations of estradiol-17 by the chimpanzee in vivo.код для вставкиСкачать
American Journal of Primatoloxy 5971-275 (1983) BRIEF REPORT Differential Oxidations of Estradiol-176 by the Chimpanzee In Vivo P.I. MUSEY, D.C. COLLINS, K.G. GOULD, AND J.R.K. PREEDY Departmen&of Medicine and Yerkes Primate Center, Emory University School of MedicincNA Medical Center. Atlanta The metabolism of estradiol-170 is primarily a n oxidative process at either carbon-2 or carbon-16 in the human. The objective of this study was to determine the relative importance of these two oxygenation pathways in the chimpanzee. The rate of oxidation of estradiol-17fl at each position was determined by measuring the release of tritium into body water from carbon-2 or carbon-16. [2-3HJ-Estradiol-17flor [ 16-3H]-estradiol-17fiwas injected intravenously into three adult male chimpanzees, and blood samples were obtained at several time intervals between 1 and 48 hr. The blood was lyophilized, and the release of tritium from the specifically labeled estrogens into the body fluid pool was determined. The release of tritium from the 16a-position was very low and did not exceed 3% in any animal. The release of tritium from the carbon-2 was much faster, amounting to 2996, 34%,and 35%, respectively, by 24 hr. The ratio of tritium released from carbon2:carbon-16 was 5.0, 13.2 and 16.9, respectively, at 24 hr after injection of the specifically labeled estradiol-170. These results demonstrate clearly that the major pathway for oxidative metabolism of estradiol-170 in the chimpanzee is via oxygenation a t carbon-2, with the formation of catechol estrogens, as in the human Key words: chimpanzee, estradiol-Ii'P, metabolism, catechol estrogens, estriols, nonhuman primates, Pan troglodytes INTRODUCTION Close similarities in the metabolism of estrogens have previously been reported between human and nonhuman primates, especially the chimpanzee with respect to steroid conjugation and excretion [Musey et al, 1973,1976,19821. I n the human, the metabolism of estradiol-170 (biologically the most active estrogen) is characterized by a rapid initial oxidation to estrone. Thereafter, estrone, and to a lesser extent estradiol-170, may be oxidized at either carbon-2 or carbon-16 to form catechol estrogens or estriols, respectively [Fishman et al, 19601. The physiological consequence of oxygenation a t carbon-2 is a reduction in the estrogenicity of the molecule Received April 22, 1983; revision accepted June 30, 1983 Address reprint requests to Dr. P.I. Musey, Medical Research Service, VA Medical Center, 1670 Clairmont Road, Decatur, GA 30033. 0 1983 Alan R. I,iss, Inc. 272 Musey et a1 [Martucci & Fishman, 19771, whereas estrogens oxygenated a t carbon-16 retain considerable estrogenic activity [Clark et al, 1977; Fishman & Martucci, 19801. Alterations in the ratio of hydroxylation a t carbon-2 to hydroxylation at carbon-16 have been reported in a number of pathological conditions, such as thyroid dysfunction [Fishman et al, 19621, liver disease [Zumoff et al, 19681, and anorexia nervosa [Fishman et al, 19751. The rate of oxidation a t each position can be determined radiometrically by measuring the release of tritium from estradiol-17/3, specifically labeled at either carbon-2 or carbon-16, into body water [Fishman et al, 1970, 19801. This approach has been used to determine the site of oxidation in a number of physiological and pathophysiological conditions in the human [Fishman et al, 1962, 1975; Zumoff et al, 1968; Lahita et al, 19811. The present study was undertaken to determine the relative importance of the hydroxylation of estrogens a t carbon-2 and a t carbon-16 in the chimpanzee, using the radiometric approach described above. The results of this study suggest that hydroxylation a t carbon-2 is the major route for metabolism of estrogens in the chimpanzee, as well as in the human. MATERIALS AND METHODS Subjects Three healthy, adult male chimpanzees (Pan troglodytes) housed at Yerkes Regional Primate Research Center were fasted overnight prior to the injection of the radioactive steroid and collection of blood samples. The injection and blood collection procedures were conducted under ketamine hydrochloride anesthesia (610 mgkg). Radioactive Steroids. [2-3H]-Estradiol-17/3and [16a-3H]-estradiol-17Pwere provided by Dr. H.L. Bradlow, Rockefeller University, NY. Details of the synthesis, purification, and stability of these steroids have previously been described [Fishman et al, 1970, 19801. Briefly, [2-3H]-estradiol-17/3was prepared by catalytic 3H reduction of 2-iodoestradiol in methylene chloride in the presence of 5% palladiudcharcoal catalyst followed by NaBH4 reduction. [16a-3H]-Estradiol-17/3was similarly prepared by catalytic trituration of estrone enol diacetate followed by methanoliKHC03 refluxing to generate [ 16a,17a3H]-estradiol-17@. The latter product was then subjected to chromic acid oxidation to yield [16a3H]-estrone,which was then reduced to [ 16a3H]-estradiol-17P in methanol containing NaBH4. Tritiated water was purchased from New England Nuclear Corp., Boston, MA. Radioisotope Administration An initial control blood sample was collected from the chimpanzees. Then a known amount of [16a-3H]-17/3(5-7 pCi) in 0.1 ml propylene glycol diluted with 5 ml 10%ethanolic saline was injected into the femoral vein. Blood samples (5 ml) were taken in heparinized tubes a t 0.5, 1,2, 3,4,24, and 48 hr and stored until analyzed. After four days, the above protocol was repeated except that [2-3H]-estradiol-17fiwas injected. The plasma or whole blood samples were lyophilized to collect body water. Duplicate aliquots (1ml) of each lyophilate were analyzed for tritium. Total Body Water Volume The total body water volumes of the chimpanzees were determined by the body dilultion of ingested tritiated water, Seven days after the last steroid injection, 0.1 Estrogen Metabolism in the Chimpanzee 273 pCi of tritiated water was added to 200 cc of fruit juice and given to each animal by mouth. Blood samples were collected 2 and 3 hr after ingestion. The specific activity ( d p d m l ) of the blood lyophilate was used to calculate the volume of distribution by a simple dilution ratio. All radioactive samples were counted in a Packard 3255 Liquid Scintillation Spectrometer with automatic external standardization for dpm calculations [Collins & Preedy, 19751. Each ml of water was mixed with 5 ml of water-based commercial scintillation fluid (Hydrocount-Mallinckrodt Chemical Co., St. Louis, MO), and counted for a t least 90 min or until 10,000 counts were accumulated. RESULTS The amount of tritium released into the body water ( d p d m l blood) following intravenous administration of [2-3H]-estradiol-17~ or [16a-3H]-estradiol-17@ is shown in Table I. The release of the tritium label from the 16a-position was very low throughout the study period. The maximum count was achieved only a t 48 hr in one animal. Release of the tritium label from the 16a-position a t carbon-16 did not exceed 3% of dose in any animal. These results suggest that the oxidation of estrogens a t the 1601position is very slow in the adult male chimpanzee. Tritium released from the carbon-2 position of estradiol-170 reflects 2-hydroxylation and catechol estrogen formation. The release of tritium from this position was much faster than from the 16a-position (Table I). The release was somewhat faster in one animal (GR) than in the others. However, comparable results were attained in all animals by 24 hr following estrogen administration when 29%, 35%, and 34%, respectively, of the [2-3H]-estradiol-17/3dose had been released (Table 11). The ratio of tritium released from carbon-2:carbon-16varied between 2 and 32, with the higher ratios being achieved between 4 and 24 hr after injection. The rate of tritium release from the administered estrogens is affected by the rate of water turnover. Since the chimpanzees were sedated without any intravenous fluid infusion and did not void any urine a t least within the initial 4 hr, the results in Table I1 only approximate the real-time course of carbon-2 and carbon-16 oxidations for the first 4 hr. The release of tritium from [2-3H]-estradiol-170was relatively rapid during the initial 4 hr after injection, with 17% and 32% of the dose being metabolized a t 1 and 4 hr, respectively, in one subject (GR). Comparable values for the other subjects were 12% and 29%, respectively. T.4HLE I. The Concentration of Free Tritium Present in the Blood (dpm/ml hlood) at Different Times After Injection of [:'H]-Estradiol-l7PSpecifically Labeled at Carbon-:! or Carbon-1601 Subiect GR wo CH 'H (dpmiml) after Injection of: - [2-'H] "H Dose 8.42 ~. x 10-'((dpm) Time (hr) 1 2 3 4 24 48 73 110 132 142 157 128 [ 16a-"HI 9.81 5 6 6 5 7 15 l2-"H1 7.08 36 47 57 84 100 89 [ 16a-"HI 10.15 5 8 10 12 11 12 .~ [2-:'HI [16a-"HI 12.88 12.91 ~~~ 59 103 120 131 196 181 27 33 33 37 41 31 274 Musey et al TABLE 11. The Percent of Tritium Dose Keleased Into the Blood as "HzO at Different Times After Injection of ~'H]-EstradioI-l7PSpecifically Labeled at Carbon-:! and Carbon-ltb Time (hr) Subject GR % 12-"H] %' [16u-"H] Ratio 21161~ Subject CH r/r 12-:'H] 94 116a-'H] Ratio 2116cu Subject WO % 12-"H] 5% [ 1601-3H] Ratio 2116a 1 2~. 3 4 24 48 16.7 0.9 18.6 24.8 1.2 20.7 29.8 1.3 22.9 32.0 1.0 32.0 35.4 2.1 16.9 28.9 2.9 9.9 12.3 1.2 10.3 19.5 1.9 8.4 24.5 2.4 8.1 28.7 2.9 9.9 34.2 2.6 13.2 30.4 2.9 10.5 8.6 3.7 2.3 14.9 4.7 3.2 17.5 4.7 3.7 19.0 5.3 3.6 28.5 5.7 5.0 26.4 4.4 6.0 The ratio 2/16m was calculated from the ratio of t he percent of tritium released at each time. DISCUSSION The results of this study suggest that the chimpanzee oxidizes estradiol-170 predominantly a t carbon-2, leading to catechol estrogen formation as a major pathway for metabolism of estradiol-17P. The release of tritium from the 16a-position to form 16a-hydroxyestrone and estriols appears to be a minor pathway in this primate. These findings are similar to the metabolism of estrogens in the rhesus monkey [Musey et al, 19791,the pigmy chimpanzee [Musey et al, 19791, as well as the human [Kraychy & Gallagher, 1957; Fishman et al, 19801 in whom 2-hydroxy and 2methoxy estrogens are the major metabolites of estradiol. However, when compared to the human, nonhuman primates seem to convert a greater percentage of estrogens to catechol derivatives [Musey et al, 19821. The metabolism of estradiol-17p is primarily oxidative in nature, occurring almost exclusively a t carbon-2, carbon-16, and carbon-17 positions. Oxidation at carbon-17, resulting in estrone formation, is the most rapid pathway, with a halftime of less than 5 min [Fishman et al, 1970). Subsequent metabolism of estrone depends upon the competition between carbon-2 and carbon-16 hydroxylases. Oxidations at carbon-2 and carbon-16 are, to a n extent, mutually exclusive, leading to either carbon-2 oxygenated compounds (2-hydroxy and 2-methoxy estrogens) or to carbon-16 oxygenated products (16a-hydroxyestrone and estriol). In the human, oxidation of estradiol-17P at carbon-2 is reported to be complete within 6 hr, with a half-time of 2 hr, while the 16a-hydroxylation of estradiol-170 is fairly slow [Fishman et al, 19801. Our results suggest that a similar pattern of rapid oxidation a t carbon-2 and slow oxidation a t the 16a-position occurs in the male chimpanzee. The radiometric approach to estradiol-170 metabolism used in this study provides very useful information on the effects of metabolism on the possible physiologic consequences of circulating estrogens in a n individual. The 2- and 16-hydroxylated metabolites of estradiol-l7b have different physiological properties since estriols and 16a-hydroxyestrone are potent uterotrophic estrogens in vivo, while the catechol estrogens appear to be devoid of estrogenic activity [Martucci & Fishman, 19771. The catechol estrogens, however, are reported to have effects on the pituitaryhypothalamic axis [Schinfeld et al, 1980; Martucci & Fishman, 19791, and may also act a s anti-estrogens. Thus, the metabolism of estradiol-17P via these two competitive pathways may affect the physiological response of the animal. Estrogen Metabolism in the Chimpanzee 275 Our results clearly indicate that the major pathway for the metabolism of estradiol-17P in the chimpanzee is by oxidation at carbon-2. Thus, the chimpanzee is similar to the human and the rhesus monkey in the oxidation of estradiol-17P in that the catechol estrogen pathway also predominates the estriol pathway. It is clear that studies of estrogen biosynthesis and metabolism must include a measure of these important catechol estrogen metabolites which have been ignored in this species. ACKNOWLEDGMENTS Supported in part by NIH grants 5-RO1-CA-24616and RR-00165 and VA project 7176-001. Presented in part a t the 64th Annual Meeting of the Endocrine Society in San Francisco, 1982. REFERENCES Clark, J.H.; Paszko, Z.; Peck, E.J. Nuclear binding and retention of the receptor estrogen complex: Relation to the agonistic and antagonistic properties of estriol. ENDOCRINOLOGY 100:91-96,1977. Collins, D.C.; Preedy, J.R.K. Identification of some plasma metabolities of 6,7-3H-estrone glucosiduronate in male dogs. STEROIDS 25427-439, 1975. Fishman, J.; Boyar, R.M.; Hellman, L. Influence of body weight on estradiol-170 metabolism in young women. JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM 41:989-991, 1975. Fishman, J.; Bradlow, H.L.; Gallagher, T.F. Oxidative metabolism of estradiol. 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