Studies in porphyria. I. A defect in the reductive transformation of natural steroid hormones in the hereditary liver disease, acute intermittent porphyria.

A variety of 5beta steroid metabolites derived from hormones natural to man are potent inducers experimentally of delta-aminolevulinate synthetase, the rate-limiting enzyme in porphyrin-heme formation. This mitochondrial enzyme is found at high levels of activity in the livers of patients with the genetic disease, acute intermittent porphyria (AIP). In this study the metabolism of (14)C-labeled testosterone was examined in AIP patients to determine whether there was a disproportionate conversion of the hormone to its 5beta, compared to its 5alpha metabolite. The results indicate that AIP subjects do generate a substantially greater than normal fraction of 5beta metabolite from this steroid; the excessive degree of ring A reduction of testosterone taking place via the 5beta pathway in the porphyric patients averages 350% greater than in the nonporphyric subjects. In one asymptomatic AIP patient the disproportionate generation of 5beta metabolite from the hormone reached a level 10 times the normal mean. Studies with a second (14)C-labeled hormone, dehydroisoandrosterone, whose metabolism in man resembles that of testosterone, confirmed the derangement in reductive transformation of steroids found in the individuals carrying the genetic lesion of AIP. These findings define a new endocrine abnormality in AIP patients and raise the possibility that endogenously derived 5beta steroids may contribute by an induction mechanism to the increased levels of hepatic delta-aminolevulinate synthetase activity found in AIP patients.

Studies in porphyria. II. Evidence for a deficiency of steroid delta-4-5-alpha-reductase activity in acute intermittent porphyria.

Patients with the genetic liver disease, acute intermittent porphyria (AIP), have a defect in the reductive transformation of steroid hormones that is manifest by the disproportionate generation of 5beta-steroid metabolites from precursor hormones. 5beta-steroid metabolites were earlier shown to be potent inducers experimentally of delta-aminolevulinate synthetase (ALAS), the mitochondrial enzyme that is rate-limiting in porphyrin synthesis, and that is found at high levels of activity in the livers of AIP patients. In this report, the basis for the defective steroid metabolism in AIP has been shown, through studies with the (14)C-labeled adrenal hormone 11beta-hydroxy-Delta(4)-androstenedione, to reside in a substantial deficiency of hepatic steroid Delta(4)-5alpha-reductase activity. This enzymic deficiency was found in all seven AIP patients studied, and ranged from 34% to as much as 70% below the mean enzyme activity characterizing normal subjects. The functional consequence of the low levels of 5alpha-reductase activity in AIP is to divert the reductive transformation of certain natural hormones from the 5alpha- to the 5beta-pathway; the latter is the metabolic route through which endogenous steroids having the potential for inducing hepatic ALAS are generated. It is not presently known whether the 5alpha-reductase deficiency in AIP is acquired in some fashion or whether it has partial genetic determinants. It seems probable, however, that this enzymatic abnormality, coupled with the dramatic increase in hormone synthesis that occurs at puberty, may be of major importance in determining clinical expression of the latent gene defect for AIP in many individuals. The 5alpha-reductases for steroid hormones are known to be localized in the endoplasmic reticulum of hepatic cells and the present findings in AIP thus represent the first demonstration that an enzymic component of these membranous structures is functionally abnormal in this hereditary liver disease.

Diindolylmethane (DIM) spontaneously forms from indole-3-carbinol (I3C) during cell culture experiments.

UNLABELLED: Indole-3-carbinol (I3C) when given orally is converted to diindolylmethane (DIM) and other oligomers catalyzed by stomach acid. This suggests that DIM is the predominant active agent and that I3C is a precursor, 'pro-drug' in vivo. However, in cell culture studies carried out in neutral solutions, I3C has been considered fully active. MATERIALS AND METHODS: The stability of I3C in cell culture media was studied. RESULTS: In the 8 different cell culture media tested, greater than 50% dimerization of I3C into DIM occurred in 24 hours. At 48 hour, greater than 60% conversion was found. When neutral synthetic cerebrospinal fluid (CSF) or peritoneal fluid (PF) was studied, a large peak, tentitively identified as I3C's linear trimer (LTR) conversion product by mass spectra, and two smaller peaks, were seen. When CSF or PF was diluted 1:1 with media, the formation of these additional peaks was diminished. CONCLUSION: Because of the greater biologic potency of DIM when studied in parallel with I3C in vitro, this extent of dimerization shows that DIM rather than I3C is the active agent in cell culture studies.

Cortisol metabolism in cirrhosis.

The production and peripheral metabolism of cortisol have been studied in 10 cirrhotics and 11 controls after i.v. tracers of cortisol-(14)C. The findings were as follows: (a) Total urinary excretion of radioactivity was normal (81% of the dose) but a decreased fraction was present as glucosiduronates: 18-47% of the dose (average 34%) compared to a normal average of 54%. (b) There was a distinctively abnormal pattern of cortisol metabolites, not previously observed in other illnesses: tetrahydrocortisone was decreased to 14% of the enzyme hydrolysate (normal 26%); cortolones were increased to 34% (normal 19%), owing entirely to an increase in cortolone (20alpha) formation, since beta-cortolone (20beta) was not significantly increased; Reichstein's substances U and epi-U were increased, averaging 2.6% for the former and 4.7% for the latter; tetrahydrocortisol, allotetrahydrocortisol and cortols were normal. This pattern was independent of the degree of decreased glucosiduronate formation and also independent of the presence or absence of a portacaval shunt. (c) Cortisol production, determined by isotope dilution, was normal in each of six cirrhotic patients. From these data, taken in conjunction with our previously reported findings concerning the influence of norethandrolone on cortisol metabolism, the following conclusions were drawn: (a) Cirrhotic patients have decreased A-ring reduction of cortisone to tetrahydrocortisone and correspondingly increased 20-ketone reduction of cortisone to Reichstein's substances U and epi-U and then to the cortolones. (b) Intrahepatic cholestasis, a regular pathophysiological feature of cirrhosis, may be responsible for the observed abnormal cortisol metabolite pattern in this disease. (c) The slowed metabolic turnover rate of cortisol in cirrhosis may be due to decreased transport and/or binding of cortisol to its intracellular metabolic sites rather than to abnormalities of any specific metabolizing enzymes.

Induction of a deficiency of steroid delta 4-5 alpha-reductase activity in liver by a porphyrinogenic drug.

The hepatic enzymes that catalyze drug oxidations and the reductive metabolism of steroid hormones to 5alpha-derivatives are localized in membranes of the endoplasmic reticulum. Phenobarbital, which exacerbates acute intermittent porphyria in man, induces drug-oxidizing enzymes in liver. Additionally, patients in whome the primary gene defect (uroporphyrinogen-I-synthetase deficiency) of acute intermittent porphyria has become clinically expressed have low levels of hepatic steroid delta4-5alpha-reductase activity. This 5alpha-reductase deficiency in acute intermittent porphyria leads to the disproportionate generation of 5beta-steroid metabolites from precursor hormones; such steroid metabolites have significant porphyria-inducing action experimentally. In this study the effects of phenobarbital on drug oxidation and steroid 5alpha-reduction in man were examined to determine if this drug could produce changes in steroid 5alpha-reductase activity which mimicked those seen in patients with acute intermittent porphyria. Metabolic studies with [14C]-testosterone and 11beta-[3H]hydroxyandrostenedione were carried out in five normal volunteers. In all five subjects phenobarbital administration (2 mg/kg/per day for 21 days) enhanced plasma removal of the test drugs antipyrine and phenylbutazone as expected; but in four subjects phenobarbital also substantially depressed 5alpha-metabolite formation from [14C]testosterone and resulted in a pattern of hormone biotransformation characterized by a high ratio of 5beta/5alpha-metabolite formation. Studies with 11beta-[3H]hydroxy-androstenedione in three subjects confirmed that phenobarbital produced this high 5beta/5alpha ratio of steroid metabolism by depressing 5alpha-reductase activity for steroid hormones in liver. The high ratio of 5beta/5alpha-metabolites formed in normals after drug treatment mimicks the high 5beta/5alpha-steroid metabolite ratio formed from endogenous hormones in acute intermittent porphyria. The proximate mechanism by which phenobarbital induces reciprocal changes in activities of the microsomal enzymes which catalyze drug oxidations and steroid 5alpha-reductions is not known. This action of phenobarbital raises the possibility, however, that certain drugs which provoke exacerbations of human porphyria may do so, in part, by producing deleterious shifts in the patterns of endogenous steroid hormone metabolism.

Induction of estradiol metabolism by dietary indole-3-carbinol in humans.

Dietary indoles in cruciferous vegetables induce cytochrome P450 enzymes and have prevented tumors in various animal models. Because estradiol metabolism is also cytochrome P450 mediated and linked to breast cancer risk, indoles may similarly reduce estrogen-responsive tumors in humans. We examined several indoles in female Sprague-Dawley rats for induction of hepatic estradiol 2-hydroxylation. The most potent inducer, indole-3-carbinol, was administered to humans (500 mg daily for 1 wk). It significantly increased the extent (mean +/- SEM) of estradiol 2-hydroxylation from 29.3% +/- 2.1% to 45.6% +/- 2.1% (P less than .001). These results indicate that indole-3-carbinol strongly influences estradiol metabolism in humans and may provide a new chemopreventive approach to estrogen-dependent diseases.

Changes in levels of urinary estrogen metabolites after oral indole-3-carbinol treatment in humans.

BACKGROUND: The oxidative metabolism of estrogens in humans is mediated primarily by cytochrome P450, many isoenzymes of which are inducible by dietary and pharmacologic agents. One major pathway, 2-hydroxylation, is induced by dietary indole-3-carbinol (I3C), which is present in cruciferous vegetables (e.g., cabbage and broccoli). PURPOSE: Because the pool of available estrogen substrates for all pathways is limited, we hypothesized that increased 2-hydroxylation of estrogens would lead to decreased activity in competing metabolic pathways. METHODS: Urine samples were collected from subjects before and after oral ingestion of I3C (6-7 mg/kg per day). In the first study, seven men received I3C for 1 week; in the second study, 10 women received I3C for 2 months. A profile of 13 estrogens was measured in each sample by gas chromatography-mass spectrometry. RESULTS: In both men and women, I3C significantly increased the urinary excretion of C-2 estrogens. The urinary concentrations of nearly all other estrogen metabolites, including levels of estradiol, estrone, estriol, and 16alpha-hydroxyestrone, were lower after I3C treatment. CONCLUSIONS: These findings support the hypothesis that I3C-induced estrogen 2-hydroxylation results in decreased concentrations of several metabolites known to activate the estrogen receptor. This effect may lower estrogenic stimulation in women. IMPLICATIONS: I3C may have chemopreventive activity against breast cancer in humans, although the long-term effects of higher catechol estrogen levels in women require further investigation.

Upregulation of estradiol C16 alpha-hydroxylation in human breast tissue: a potential biomarker of breast cancer risk.

BACKGROUND: The biotransformation of the natural estrogen 17 beta-estradiol (E2) via the C16 alpha-hydroxylation pathway is elevated in patients with breast cancer, in subjects at increased risk for developing breast cancer, and in c-Ha-ras-initiated mouse mammary epithelial cells. PURPOSE: To determine whether differences in the extent of E2 C16 alpha-hydroxylation are related to the risk of developing breast cancer, we examined the extent of biotransformation of E2 via the C16 alpha-hydroxylation pathway in the mammary terminal duct lobular units (TDLUs), epithelial organoids that are a presumptive target site of human breast carcinogenesis, and in nontarget component mammary fat tissue. METHODS: Noninvolved mammary tissue was obtained from four patients undergoing reduction mammoplasty and from four undergoing mastectomy for breast cancer. A radiometric assay that measures 3H2O formation caused by stoichiometric 3H exchange from [C16 alpha-3H]E2 was utilized to compare the relative extent of C16 alpha-hydroxylation in explant cultures of TDLUs and mammary fat. RESULTS: The extent of E2 C16 alpha-hydroxylation was 1.83-fold higher (95% confidence interval [CI] = 1.71-1.97) in the TDLUs from reduction mammoplasty (i.e., "low-risk") patients and 7.96-fold higher (95% CI = 6.38-10.55) in the TDLUs from mastectomy (i.e., "high-risk") patients than in the corresponding values observed in the mammary fat. In the TDLUs obtained from the patients undergoing mastectomy for cancer, the extent of this metabolism was 4.56-fold higher (95% CI = 3.97-5.33) than that observed in TDLUs obtained from reduction mammoplasty patients who did not have cancer. CONCLUSION: The increase in the extent of C16 alpha-hydroxylation of E2 in the epithelial organoids of the human breast, TDLUs in particular, may be an important factor for breast cancer induction. This upregulation may represent an endocrine biomarker for the risk of developing breast cancer. IMPLICATION: A larger prospective study is required to confirm the clinical significance of this endocrine biomarker.

Induction by estrogen metabolite 16 alpha-hydroxyestrone of genotoxic damage and aberrant proliferation in mouse mammary epithelial cells.

BACKGROUND: Estrogens are potent mammary tumor promoters influencing post-initiation events via epigenetic mechanisms. The upregulation (i.e., induction) of the C16 alpha-hydroxylation pathway during 17 beta-estradiol (E2) biotransformation has been associated with mammary cell transformation. The action of E2 metabolites on tumorigenic transformation, however, is poorly understood. PURPOSE: The newly established mammary epithelial cell line C57/MG, derived from the C57BL mouse strain, was used to examine whether E2 or its metabolites, 16-hydroxyestrone (16 alpha-OHE1) and estriol (E3), function as initiators of mammary cell transformation. METHODS: DNA repair (hydroxyurea-insensitive thymidine uptake), estrogen metabolism (3H exchange to form 3H2O), hyperproliferation (increased cell number), and acquisition of anchorage-independent growth (soft-agar colonies) were used as quantitative end points to measure the relative extent of transformation. RESULTS: Treatment of cells with 200 ng/mL 16 alpha-OHE1 resulted in a 55.2% increase in DNA repair synthesis, a 23.09% increase in proliferative activity, and a 18-fold increase in the number of soft-agar colonies, relative to the solvent controls (P less than .0001). The extent of upregulation of the three end points was similar to that induced by the genotoxic mammary carcinogen 7, 12-dimethylbenz[a]anthracene (DMBA, positive control). DMBA treatment also upregulated the ratio of 16 alpha/C2 hydroxylation of E2 leading to increased formation of 16 alpha-OHE1. E2 and E3 were not effective in upregulating these markers for transformation. CONCLUSION: These results demonstrate that in nontransformed C57/MG cells, 16 alpha-OHE1 may function as an initiator, perturbing the intermediate biomarkers for preneoplastic transformation.

Selective responsiveness of human breast cancer cells to indole-3-carbinol, a chemopreventive agent.

BACKGROUND: Indole-3-carbinol (I3C), a compound found in cabbage, broccoli, and brussels sprouts, inhibits the growth of mammary tumors when fed to certain strains of mice. The chemopreventive and antitumor effects of I3C depend on the species and tissue type. The mechanism of action and specific human cell types that respond to I3C are not known. PURPOSE: Our purpose was to study the mechanism of action of I3C in estrogen-responsive (MCF-7) and estrogen-nonresponsive (MDA-MB-231) human breast cancer cell lines. METHODS: Estrogen responsiveness was determined by the ability of estradiol to stimulate the growth of breast cancer cells deprived of estrogen. The effect of I3C was measured on cell growth, estradiol metabolism, and level of cytochrome P-4501A1. Growth was measured by cell counts and soft-agar assay, estrogen metabolism was examined by a radiometric assay, and the level of cytochrome P-4501A1 was measured by Western blots with a polyclonal antibody. RESULTS: I3C inhibits the growth of estrogen-responsive cell line MCF-7 but has little effect on estrogen-nonresponsive cell line MDA-MB-231. Specific C-2 hydroxylation of estrogen and induction of cytochrome P-4501A1 was enhanced by I3C in the MCF-7 but not in the MDA-MB-231 cells. CONCLUSION: I3C has specific antigrowth effects in human breast cancer cells. The inhibitory effects of I3C may involve selective induction of estradiol metabolism and the related cytochrome P-450 system that may be limited to estrogen-sensitive cells.

A reassessment of the role of breast tumor aromatization.

While the role of estrogens in the maintenance of human breast carcinoma has been firmly established for many years, the sources of this estrogen remain unresolved. Input-output analysis of steroid uptake by breast carcinomas showed no specific uptake of estrogens from blood. Based on the detection of the necessary enzyme systems. Adams and Wong proposed that breast tumors could function as "paraendocrine organs" capable of producing sufficient estradiol (E2) to stimulate their own growth (Adams, J. B., and Wong, M. S. F. Lancet, 2: 1163, 1968). Using the reported values for the concentration of C19 steroid precursors and the rho TTC19-E2 values one can estimate that the contribution of in situ aromatization to the tumor estrogen pool is quite small.

Hormone profiles in hormone-dependent cancers.

Studies on the relationship of urinary excretion of androgen metabolites and estrogens to the natural history of breast cancer are reviewed. The importance of distinguishing between "within-population" studies (i.e., cancer patients versus normal controls) and "between populations" studies (i.e., low-risk versus high-risk populations) is emphasized, and it is pointed out that "qualitative" agreement (i.e. the same direction of differences) between the two types of studies must be present in order to implicate a hormonal parameter as a determinant of the natural history of breast cancer. For reasons detailed in this paper, it is concluded that the reported relationship of low urinary androgen metabolite excretion to increased risk of developing breast cancer and poor response to adrenalectomy or hypophysectomy and the validity of the "estriol hypothesis," namely, that a high urinary ratio of estriol to estrone-plus-estradiol in early life is protective against subsequent development of breast cancer, are both dubious. A new hypothesis concerning the relationship of estrogens to breast cancer risk is presented: "A period of of time, prior to age 30, during which the amount of biological availability of active estrogens' (i.e., estrone and estradiol) is diminished, protects against subsequent development of cancer." This hypothesis is shown to be compatible with the epidemiological and biochemical data. Reports concerning the influence of nutrition on endocrine parameters are reviewed. Inanition and obesity have been shown to alter steroid metabolism but it is not known whether nutritional "microdifferences" (i.e., differences between populations or individuals that are due to cultural, geographic, or socioeconomic factors, but that fall within the range of "normal" or adequate nutrition) can also alter steroid metabolism.

Proteases in cyst fluid from human gross cyst breast disease.

Cyst fluid from women with gross cystic breast disease was found to contain protease activity when assayed against [14C]albumin. At least six different proteases were detected when the fluid was fractionated by a combination of S-300 Sephacel, hydroxylapatite, and DEAE-Sephacel chromatographic techniques. The distribution of the proteases appeared to be related to the ionic composition of the fluids. A major protease component, found in both high Na and high K fluids, was isolated. It showed chymotryptic cleavage characteristics against the beta-chain of insulin. It was partially inhibited by alpha 2-macroglobulin, N-tosyl-L-phenylalanine chloromethyl ketone, and benzamidine but not by leupeptin, pepstatin, N-tosyl-L-lysine chloromethyl ketone, or alpha 1-protease inhibitor. The protease has an apparent molecular weight of 110,000 with Mr 24,000 subunits. This protease may be identical or closely associated with Haagensen's GCDFP-24 progesterone binding protein which was isolated in a similar manner. An imbalance between protease and protease inhibitors in cyst fluid may account for gross cyst formation and may be involved in the tumorigenic process. The accumulation of poorly diffusible peptide fragments, as a result of protease activity, would increase the oncotic pressure leading to enlargement of the cyst cavity as water enters to reestablish osmotic equilibrium.

Steroid hormone accumulation in human breast cyst fluid.

Elevated concentrations of peptide hormones have been described previously in human breast fluid. In the current study, the levels of cortisol, progesterone, testosterone, dihydrotestosterone, androsterone, androsterone sulfate, dehydroisoandrosterone, dehydroisoandrosterone sulfate, estradiol, estrone, estradiol sulfate, and estrone sulfate were measured. The levels of the four 17-ketosteroids and the two estrogen sulfates were markedly elevated over the plasma level, while that of the other compounds was the same or only slightly higher than the plasma levels of the same compounds.

PIP: As part of a program to explore whether a relationship existed between the compounds present in breast cyst fluid and the risk for breast cancer, concentrations of a wide variety of hormones, enzymes, tumor-associated antigens, and ions in breast cyst fluid were determined. In this report, the concentrations of steroid hormones in cyst fluid samples from the same population used for previously published studies of peptide hormone concentrations were measured. This study determined the levels of cortisol, progesterone, testosterone, dihydrotestosterone, androsterone, androsterone sulfate, dehydroisoandrosterone, dehydroisoandrosterone sulfate, estradiol, estrone, estradiol sulfate, and estrone sulfate. In previous studies, levels of peptide hormones were shown to be elevated in breast cyst fluid, and in this study, the levels of the 4 17-ketosteroids and the 2 estrogen sulfates were markedly elevated in the breast cyst fluid over the plasma level. The concentrations of the other compounds were either the same or only slightly higher than plasma levels of the same compounds. To date, however, no correlation has been obtained between the occurrence of breast cancer and levels of any of these assayed hormones.

Abnormal estrogen conjugation in women at risk for familial breast cancer at the periovulatory stage of the menstrual cycle.

The present study was designed to establish whether women with a family history of breast cancer exhibit endocrine abnormalities which could be responsible for their increased risk for the disease. Plasma hormone levels were measured every second day throughout the menstrual cycle in 30 women at risk for familial breast cancer and in an equal number of matched controls. Thirteen of the 14 substances measured exhibited no differences between the two populations, but plasma androsterone sulfate was significantly lower in the high-risk subjects. Thirteen urinary hormones were measured every day throughout the cycle with only the mean estrone and estradiol glucuronide but not estriol glucuronide content being significantly lower in the high-risk subjects. A compensatory increase in the urinary estrogen sulfates was observed. Daily analysis of these differences showed that they were most pronounced in thry day throughout the cycle with only the mean estrone and estradiol glucuronide but not estriol glucuronide content being significantly lower in the high-risk subjects. A compensatory increase in the urinary estrogen sulfates was observed. Daily analysis of these differences showed that they were most pronounced in thry day throughout the cycle with only the mean estrone and estradiol glucuronide but not estriol glucuronide content being significantly lower in the high-risk subjects. A compensatory increase in the urinary estrogen sulfates was observed. Daily analysis of these differences showed that they were most pronounced in the periovulatory period of the cycle. These results suggest that the genetic risk for breast cancer is associated with an abnormality in estrogen conjugation at a specific time of the ovulatory cycle.

Reciprocal expression of ERalpha and ERbeta is associated with estrogen-mediated modulation of intestinal tumorigenesis.

Menopausal hormone replacement therapy has been widely used to alleviate the symptoms of menopause and to decrease the detrimental effects of ovarian hormone loss on bone density and cardiovascular health. Multiple studies of colorectal cancer epidemiology also support a role for hormone replacement therapy in prevention of colorectal cancer. We studied the effect of ovariectomy and estrogen replacement on tumor formation in C57BL/6J-Min/+ (Min/+) mice, animals that bear a germline mutation in murine Apc. These mice develop multiple intestinal tumors that show loss of wild-type Apc protein. After ovariectomy, intestinal adenomas in Min/+ mice increased by 77% (P = 0.0004). Ovariectomized Min/+ mice that were treated with a replacement dose of 17beta-estradiol had the same number of tumors as Min/+ mice that were neither castrated nor treated with estrogen replacement (P = 0.85). Examination of estrogen receptor (ER) levels in intestinal tissue by immunoblot showed changes in relative expression levels of ERalpha and ERbeta, with highest ERalpha and lowest ERbeta expression in the normal-appearing intestine of Min/+ mice, and lowest ERalpha and highest ERbeta expression in the enterocytes of animals that received 17beta-estradiol. These results suggest that endogenous estrogens protect against Apc-associated tumor formation and that tumor prevention by 17beta-estradiol is associated with an increase in ERbeta and a decrease in ERalpha expression in the target tissue.

Glucocorticoid effects on contact hypersensitivity and on the cutaneous response to ultraviolet light in the mouse.

A single exposure to 254 nm ultraviolet irradiation (UV) can systemically suppress experimental sensitization to the simple allergen 2,4-dinitro, 1-chlorobenzene (DNCB) in the mouse. We show here that topical application at the site of irradiation of the 21-oic acid methyl ester derivative of the synthetic glucocorticoid triamcinolone acetonide (TAme) prevents UV suppression of sensitization. That is, mice painted with TAme at the site of UV exposure developed normal contact hypersensitivity (CH); mice exposed to UV only, like mice treated with the parent compound triamcinolone acetonide (TA), failed to be sensitized by DNCB applied to a distal site. TAme is inactivated rapidly by plasma esterases, so its effect is thought to be confined to the skin. Apparently, TAme blocked the cutaneous signal(s) for systemic suppression of CH. Histologically, irradiated skin exhibited mild inflammation and hyperproliferation, but these effects were greatly exaggerated and prolonged in the UV + TAme-treated skin, independent of sensitization at the distal site. The infiltrate consisted mostly of neutrophils and lacked the round cells characteristic of cell-mediated immunity. Apparently, normal immune suppression by UV prevented this vigorous reaction to irradiated skin. Applied together with DNCB. TAme blocked sensitization. It also prevented response to challenge by DNCB in previously sensitized animals. However, unlike the parent compound triamcinolone acetonide (TA), Budesonide or Beclomethasone diproprionate, each of which can penetrate the epidermis in active form, TAme had no effect on sensitization when applied at a distal site. Likewise, TAme did not affect plasma B (17-desoxycortisol) levels, whereas the other three compounds reduced plasma B tenfold, as expected of compounds causing adrenal-pituitary suppression. The results as a whole show that glucocorticoids can specifically inhibit cutaneous steps in induction of cell-mediated immunity or its suppression, and can, at the site of challenge, prevent its expression in CH.

Triamcinolone acetonide 21-oic acid methyl ester: a potent local antiinflammatory steroid without detectable systemic effects.

There have been many previous attempts to prepare glucocorticoid analogs that would have high antiinflammatory activity at the site of application but minimal systemic side effects. In principle, esters of cortoic acids could fulfill these criteria, if they had sufficient affinity for the glucocorticoid receptors but were rapidly hydrolyzed to the inactive acids in the circulation. With this rationale, we have synthesized esters of the 21-oic acid of triamcinolone acetonide (TA, 9 alpha-fluoro-11 beta, 16 alpha, 17 alpha, 21-tetrahydroxy-pregna-1, 4-diene-3,20-dione 16,17-acetonide), a potent synthetic glucocorticoid, in both tritiated and unlabeled forms. The synthesis involves 1) oxidation to the 21-dehydro compound with methanolic cupric acetate, 2) further oxidation to the acid with methylene blue in the presence of KCN at pH 6.5, 3) esterification with diazomethane in the presence of methanol or ethanol, to produce the methyl ester of TA (TAme) or ethyl ester, respectively, and 4) purification of the products by TLC and HPLC. The molecular weights and structures of the esters were established by mass spectrometry and nuclear magnetic resonance. The binding of [3H]TAme to steroid receptors or serum steroid-binding proteins and the in vitro hydrolysis of the ester were evaluated simultaneously, by chromatography on Sephadex LH-20 columns in aqueous buffer. [3H]TAme is bound with high affinity by receptors from human leukemic cells and rat liver. The pattern of competition for this binding is characteristic of glucocorticoid receptors: TA approximately equal to TAme greater than R5020 (a synthetic progestin) approximately equal to aldosterone greater than 5 alpha-dihydrotestosterone. [3H]TAme is not bound detectably by serum steroid-binding proteins and is rapidly hydrolyzed during incubation with serum at 37 C. The acidic product has a very low affinity for the glucocorticoid receptor. Complexes of [3H]TAme with human and rat receptors have sedimentation coefficients of 9-10S in hypotonic buffer containing 20 mM Na2MoO4 and approximately 4S in hypertonic, molybdate-free buffer. These values of s20,w are similar to those of the oligomeric and monomeric forms, respectively, of the same receptors labeled with [3H]TA, and of mammalian steroid receptors, in general. The antiinflammatory activity of TAme in rats is comparable to that of prednisolone, but the ester is devoid of the side effects associated with prednisolone treatment (suppression of thymic weight and of serum corticosterone concentration). These bioassay data and the high affinity of the ester for human glucocorticoid receptors suggest that TAme may eventually be useful clinically, as a loc

Differential effects of estradiol and 16 alpha-hydroxyestrone on pituitary and preoptic estrogen receptor regulation.

16 alpha-Hydroxyestrone (16OHE1), an endogenous metabolite of estradiol (E2), binds to the estrogen receptor (ER) with low affinity, but is estrogenic in various bioassay systems. 16OHE1 binds covalently to the ER in vitro, exhibits prolonged estrogenic bioactivity in vivo, and has been implicated in several estrogen-dependent diseases. This study examined the effects of 13 days of continuous infusion of E2 or 16OHE1 on lordotic behavior, pituitary growth, and ER regulation in the cytosolic and nuclear fractions of the pituitary and preoptic area of both sexes. Finally, simultaneous pituitary nuclear exchange assays and enzyme immunoassays were performed to search for covalent 16OHE1-ER complexes in vivo. E2 induced lordosis and pituitary growth in both sexes, while 16OHE1 was only slightly less effective. While E2 treatment increased nuclear ER concentrations 2-fold vs. control values, it decreased both cytosolic and total (cytosolic plus nuclear) ER concentrations in pituitary and preoptic area by approximately 3-fold vs. control values in both sexes by exchange assay. In contrast, 16OHE1 did not decrease total pituitary ER concentrations and only minimally decreased total preoptic ER concentrations. Simultaneous exchange assay and immunoassay of pituitary nuclear extracts demonstrated proportionate increases in ER levels in female vs. male and in E2-treated vs. 16OHE1-treated rats. The ratios of (ER enzyme immunoassay divided by ER-exchange) for each rat were similar regardless of metabolite administration. The correlation of individual measurements implied that ER localized to the nuclear fraction by either E2 or 16OHE1 retained both exchangeability and immunoassayability to similar extents, but did not support the presence of 16OHE1-ER covalent complexes. The results of this study suggest that 16OHE1 has significant estrogenic bioactivity, as manifest by its effects on lordosis and pituitary growth, but, in contrast to E2, does not decrease pituitary ER concentrations and only minimally decreases preoptic ER concentrations. This property may be important in the proposed pathogenetic action of 16OHE1 in estrogen-dependent disease.

Sex difference in the metabolism of dehydroisoandrosterone sulfate.

The metabolism of a tracer of [3H]dehydroisoandrosterone sulfate ([3H]DHAS) was studied in five normal women and six normal men. Two major sex differences were found. 1) The total urinary recovery of radioactivity was considerably greater in women (73 +/- 5.5% of the dose vs. 51 +/- 3.5; P less than 0.01), principally due to higher excretion of nonglucuronide, nonsulfate conjugates (35 +/- 2.2% of the dose vs. 21 +/- 3.9%; P less than 0.025). This finding is analogous to the sex difference in estradiol metabolism we have reported and is hypothesized to represent diversion of excretion of these conjugates in women from the biliary to the urinary excretion route because of the known lower biliary excretory capacity of women, which is a consequence of their higher estrogen levels. 2) The recovery of androsterone and etiocholanolone was considerably higher in the women (15.4 +/- 1.4% of the dose vs. 10 +/- 1.8%; P less than 0.05). Since conversion of DHAS to etiocholanolone and androsterone in vivo entails prior cleavage of DHAS to DHA, this sex difference in the recovery of androsterone and etiocholanolone is interpreted to mean that DHAS leads to DHA cleavage is considerably greater in women than in men. It is predicted that the plasma DHA to DHAS ratio should be substantially greater in women then in men.