IV. Synthesis and assay of analogs of adenosine 3',5'-diphosphate as inhibitors of bovine adrenal estrogen sulfotranferase.

Analogs of adenosine 3',5'-diphosphate are described which aid in the characterization of the inhibition of estrone sulfurylation by 3'-phosphoadenosine 5'-phosphosulfate as mediated by bovine adrenal estrogen sulfotransferase (3'-5'-phosphosulfate:estrone 3-sulphotransferase, EC 2.8.2.4). The facile conversion of ribonucleosides to 2',3'-cyclic phosphate 5'-phosphate in neat pyrophosphoryl chloride is utilized to provide a reliable route to the requisite intermediates for enzymatic regiospecific conversion to ribonucleoside 3',5'- and 2',5'-diphosphates. The importance of the 3'-phosphate ester to inhibition of estrone sulfurylation is confirmed by Ki measurements. Replacement of the 6-amino group by hydrogen or oxygen leads to considerable loss in affinity for the enzyme as does also dimethylation of the exocylic amino group. Alterations in the pyrimidine ring are not well tolerated by the sulfotransferase but modifications in the imidazole ring as in tubercidin (7 -deazaadenosine) and 8-bromoadenosine 3',5'-diphosphate lead to an enhanced affinity. The latter findings are discussed in terms of an hypothesis of stacking of the aromatic ring of the estrogen substrate and the purine moiety and its analogs.

Role of sulfate conjugation in estrogen metabolism and activity.

There has been an increasing interest in the sulfate conjugates of estrogens as important metabolites in steroid hormone homeostasis and activity. In women estrogen sulfates have been known as major components of plasma originating from ovarian secretion and hepatic metabolism. However, only recently has the capacity to sulfurylate estrogens been demonstrated in estrogen target tissues. Porcine uterus estrogen sulfotransferase appears only after the first complete estrous cycle. Following puberty, gilt uterine sulfurylation of estrogens is extremely active during diestrus, whereas estrogen sulfotransferase is not present during estrus. This cycling of estrogen sulfurylation in porcine and human uteri can be related directly to plasma progesterone levels. Rodent and human mammary tumors are also highly active in both steroid alcohol and estrogen sulfotransferases. Unlike uterine sulfotransferases, these enzymes are apparently stimulated by factors that appear following ovariectomy. The function of estrogen sulfurylation by target tissues remains obscure. However, recent investigations have indicated that the cyclic variation in endometrial estrogen sulfurylation may control the availability of 17 beta-estradiol to the cytoplasmic receptor. This premise is supported by the continued high estrogen sulfurylation activity and low nuclear receptor levels during implantation in fertilized gilts and sows. Utilizing purified bovine adrenal sulfotransferase, the substrate and inhibitor requirements were determined for this enzymes. It was also possible to design a specific inhibitor that will block estrogen sulfurylation without interfering with the receptor binding and nuclear migration of physiological levels of 17 beta-estradiol. This inhibitor, 3-methoxy-4-nitroestrone, will help in establishing the role of uterine and mammary estrogen sulfurylation.

GLC determination for ftorafur in biological fluids.

A GLC analysis for free ftorafur was developed to follow in drug disposition in body fluids of patients. The free drug was extracted from aqueous biological samples with chloroform, derivatized by methylation, and chromatographed on 1% HI-EFF 8BP using flame-ionization detection. The analysis is sensitive (0.25 microgram/ml of plasma) and specific for the intact molecule, and it does not interfere with subsequent fluorouracil analysis of the same sample.

The steroid alcohol and estrogen sulfotransferases in rodent and human mammary tumors.

Rodent and human mammary tumor systems were investigated to relate the steroid alcohol and estrogen sulfotransferase activities to the hormoanl dependency of the tumor as determined by estrogen receptor content. Unlike the normal mammary gland or the hyperplastic alveolar nodule, rodent mammary neoplasms displayed significant levels of these two sulfotransferases. In the hormone-independent mouse tumors produced from out-growth lines D1, D2, and D8, high dehydroepiandrosterone sulfotransferase activity was characteristic of the rapidity with which hyperplastic alveolar nodules developed into a neoplasms (V-max = 52.8 versus 1.8 fmoles/min/mg protein) while estrone sulfotransferase activity was either not detectable or low (V-max = 5.5 fmoles). After oophorectomy of mice bearing slowly developing tumors, both sulfotransferases in the nonregressing neoplasms showed marked increases in activity (V-max dehydroepiandrosterone = 30.0 fmoles; V-max estrone = 18.5 fmoles). Strain differences not the estrogen receptor content of hormone-dependent rat mammary tumors. In Wistar-Lewis rats the steroid alcohol sulfotransferase activity was at least 35 times higher than in the Sprague-Dawley strain. As was observed in the mouse mammary tumor, Sprague-Dawley rat neoplasms that grew in the absence of ovarian hormones contained significantly greater levels of the steroid alcohol sulfotransferase. Possible correlaion between presence of the steroid alcohol sulfotransferase and the estrogen receptor protein was observed in a limited number of human breast carcinomas.

Specific sites of fatty acid sterol synthesis in isolated skin components.

Metabolic studies on isolated mouse skin components were undertaken to determine the specific sites of fatty acid and sterol synthesis. The concentrations of long-chain fatty acids and sterols and the incorporation of radioactivity from acetate-1-(14)C into these lipids are reported for various skin components and intact whole skin. Only fatty acids having chain lengths of 18 carbons or less were produced by the connective tissue cells of the dermis, while fatty acids containing 20 carbons or more, as well as the acids of 18 carbons or less, were synthesized in the upper dermis (papillary reticulum). The upper dermis also produced significant quantities of eicosenoic acid and of an octadecadienoic acid (not linoleic acid), and incorporated labeled acetate into fatty acids containing an odd number of carbons. Removal of the epidermis and adnexa diminished sterol synthesis. However, the upper region of the dermis was capable of synthesizing, from acetate, large quantities of unidentified nonsaponifiable lipids which were neither sterols nor squalene.