Study of the in-vivo antioestrogenic action of N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl (DPPE), a novel intracellular histamine antagonist and antioestrogen binding site ligand

N,N-Diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl (DPPE) binds with high affinity to the antioestrogen binding site (AEBS), but not to the oestrogen receptor. There is an association of AEBS with a novel intracellular histamine receptor (H1C) of micromolar affinity through which histamine acts as a second messenger. An optimal dose of 4 mg DPPE/kg antagonized the uterine growth-stimulating effects of oestradiol in immature oophorectomized rats. Unlike tamoxifen, DPPE alone was not a partial agonist, but decreased uterine size and weight below control values at concentrations between 0.1 and 75 mg/kg. DPPE also antagonized oestradiol-stimulated uterine growth at 72 h; the inhibition observed was not significantly different from that seen with tamoxifen. Oestradiol-treated animals receiving the combination of DPPE (4 mg/kg) + low dose tamoxifen (0.04 mg/kg) for 72 h had significantly smaller uteri than did those receiving the same dose of DPPE or tamoxifen alone. Histologically, either DPPE or tamoxifen antagonized oestradiol stimulation of eosinophil migration and glandular epithelial proliferation; the latter inhibition was significantly greater for DPPE + tamoxifen (0.04 mg/kg) than for the same dose of DPPE or tamoxifen alone. Unlike tamoxifen, DPPE did not antagonize oestradiol stimulation of luminal epithelial proliferation, but in the presence of oestradiol, DPPE significantly decreased tamoxifen (0.65 mg/kg)-induced hypertrophy of the luminal epithelium. Based on these findings, we suggest that binding to the AEBS/intracellular histamine receptor is important to the action of antioestrogens.     

Does intracellular histamine mediate mast cell histamine release?

Previously, we demonstrated that through binding a novel intracellular receptor of microM affinity (HIC), histamine mediates, and the HIC antagonist N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine. HCl (DPPE) inhibits, platelet aggregation and serotonin granule secretion; the latter response is dependent upon the same processes that mediate histamine release from mast cell granules. We now show that, as for platelet serotonin release, DPPE blocks concanavalin A-stimulated mast cell histamine release with a potency (IC50 = 30 microM) greater than the H1-antagonist, pyrilamine (IC50 = 150 microM) or the H2-antagonist cimetidine (IC50 = 5 mM), correlating with rank order of potency to inhibit 3H-histamine binding in rat brain membranes and liver microsomes. We postulate that histamine release from mast cells is mediated at HIC by second messenger intracellular histamine. However, unlike platelets, mast cells do not appear to rely on newly synthesized histamine. Rather, as for calcium, histamine may be mobilized from bound stores to mediate histamine secretion.     

New evidence that the antiestrogen binding site may be a novel growth-promoting histamine receptor (?H3) which mediates the antiestrogenic and antiproliferative effects of tamoxifen

Using as a probe [3H]-DPPE (N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine HCl), a novel compound selective for the antiestrogen binding site (AEBS), new evidence is presented that this site could be a growth-promoting histamine receptor of a type not previously described (?H3). In the rat uterus, DPPE alone at a concentration of 4 mg/kg acts as an estrogen antagonist, unlike TAM alone which is a partial estrogen agonist. In the presence of exogenous estradiol, both TAM and DPPE are partial antagonists. This suggests that the "antiestrogenic" effects of tamoxifen are mediated through AEBS/?H3 while the estrogenic effects are mediated through ER.     

Antiulcerogenic and antisecretory effects of a novel diphenylmethane derivative and antiestrogen binding site ligand

N,N-Diethyl-2-[4-(phenylmethyl)-phenoxy]-ethanamine hydrochloride (DPPE) is a para-diphenylmethane derivative that binds selectively and with high affinity to the microsomal antiestrogen binding site (AEBS). Recent studies with DPPE indicate that AEBS is closely related to a lower affinity non-H1, non-H2 histamine site that may be associated with calcium channels; the DPPE-AEBS site is different from that which verapamil binds, however, DPPE, but not verapamil, demonstrates antiproliferative effects in vitro and is antiestrogenic in vivo. We now show that DPPE profoundly inhibits restraint and cold stress and ethanol-induced gastric ulcer formation, accelerates ulcer healing, attenuates the stress-induced rise in plasma corticosterone level, and significantly reduces basal and H2 agonist (dimaprit)-stimulated and, to a lesser extent, bethanechol-stimulated gastric acid output in conscious rats. A nonulcerogenic but prostaglandin-depleting dose of indomethacin completely blocks the inhibitory effects of DPPE on stress ulcer formation. Conversely, verapamil only slightly attenuates dimaprit-stimulated gastric acid secretion and exacerbates ethanol-induced gastric ulcers; its anti-stress ulcer effects are only partially attenuated by indomethacin. These findings support the likelihood that the site of action of DPPE is different from that of verapamil, and that an effect on prostaglandins may, at least in part, contribute to its antiulcer and apparent cytoprotective effects.     

Synthesis, binding and structure-affinity studies of new ligands for the microsomal anti-estrogen binding site (AEBS)

New compounds have been synthesized based on the structure of the anti-tumoral drug tamoxifen and its diphenylmethane derivative, N,N-diethyl-2-[(4-phenyl-methyl)-phenoxy]-ethanamine, HCl (DPPE). These new compounds have no affinity for the estrogen receptor (ER) and bind with various affinity to the anti-estrogen binding site (AEBS). Compounds 2, 10, 12, 13, 20a, 20b, 23a, 23b, 29 exhibited 1.1-69.5 higher affinity than DPPE, and compounds 23a and 23b have 1.2 and 3.5 higher affinity than tamoxifen. Three-dimensional structure analysis, performed using the intersection of the van der Waals volume occupied by tamoxifen in its crystallographic state and the van der Waals volume of these new compounds in their calculated minimal energy conformation, correlated well with their pKi for AEBS (r = 0.84, P<0.0001, n = 18). This is the first structure-affinity relationship (SAR) ever reported for AEBS ligands. Moreover in this study we have reported the synthesis of new compounds of higher affinity than the lead compounds and that are highly specific for AEBS. Since these compounds do not bind ER they will be helpful to study AEBS mediated cytotoxicity. Moreover our study shows that our strategy is a new useful guide to design high affinity and selective ligands for AEBS.     

Evidence that tamoxifen is a histamine antagonist

Recently we reported that both the triphenylethylene antiestrogen tamoxifen, and the novel compound N,N-diethyl-2-[(4 phenylmethyl)-phenoxy]-ethanamine. HCl (DPPE), which is selective for the antiestrogen binding site, may be histamine antagonists and have suggested that the antiestrogen binding site may be a growth-promoting histamine receptor different from H1 and H2 (?H3). We now show that along with established H1-antagonists, tamoxifen and DPPE specifically block the histamine-induced (H1) contraction of canine tracheal smooth muscle in the order: pyrilamine = hydroxyzine greater than tamoxifen = 4-hydroxytamoxifen greater than DPPE. The H1-antagonist hydroxyzine, which competes about equally with DPPE for the antiestrogen binding site, is up to 10(3) times stronger than DPPE in blocking histamine-induced muscle contraction. This shows that H1 antagonism is distinct from binding to the antiestrogen binding site and suggests that if the latter is a histamine receptor, it is not H1; presumably tamoxifen and DPPE compete for this novel site in addition to, and with greater affinity than, H1.     

Factors affecting anti-estrogen binding site concentration in rat liver

It is known that synthetic anti-estrogens such as tamoxifen bind to specific high affinity anti-estrogen binding sites (AEBS), which are distinct from estrogen receptors. These binding sites are widely distributed in animal and human tissues, the highest concentrations being found in the liver. The physiological role of these intracellular binding sites, which are located predominantly in the microsomal fraction, is currently unknown, as is the nature and identity of their endogenous ligands. In an attempt to gain information which may provide clues to the possible physiological role of these binding sites, studies were carried out to determine whether the concentration of these binding sites in rat liver was affected by a number of physiological variables. The results of these studies indicated that in the rat (i) liver AEBS increased progressively with age; (ii) liver AEBS concentration tended to be higher among females than males after 100 days of age; (iii) there was no significant variation in liver AEBS level with different phases of the estrous cycle; (iv) liver AEBS level was not significantly affected by castration in both males and females or by estradiol replacement in castrated females; (v) liver AEBS concentration increased significantly with increases in ambient temperature; (vi) there was no clearly detectable alteration in liver AEBS levels with changes in the light:dark cycle; (vii) starvation for 24, 48, and 72 hr increased liver AEBS by approximately 1.5-, 3-, and 2-fold, respectively, while refeeding decreased its level; and (viii) liver AEBS was not affected by increasing dietary fat content from 0.5% to 20% (w/w), but was increased modestly by the addition of cholesterol (2% w/w) to the diet. These observations identify several physiological variables which are associated with changes in liver AEBS concentration and suggest possible avenues for future studies to define the physiological role of these binding sites.     

Evidence that the antiestrogen binding site is a histamine or histamine-like receptor

N,N-diethyl-2-[(4 phenylmethyl)-phenoxy]-ethanamine X HCl (DPPE), a compound selective for the antiestrogen binding site, is structurally similar to the aminoethyl ether group of antihistamines. Our studies now reveal that H1-, but not H2-antagonists, also compete for this site in the order: DPPE = hydroxyzine = perchlorperazine greater than phenyltoloxamine greater than pyrilamine greater than diphenhydramine. The affinity of these compounds for the antiestrogen binding site correlates with their in vitro cytotoxicity against MCF-7 and EVSA-T human breast cancer cells. Tamoxifen, DPPE and hydroxyzine also bind to H1 receptors present in digitonin-solubilized rat liver microsomes, but with less affinity than pyrilamine, which is selective for this site; the ratio of H1 to antiestrogen binding sites in this preparation is 4:1. The data suggest that the antiestrogen binding site may be, in whole or in part, a receptor for histamine different from H1 and H2.     

Roles of antiestrogen binding sites in human endometrial cancer cells

Estrogen-noncompatible antiestrogen binding sites (AEBS) as well as estrogen receptors (ER), and the growth-inhibitory effect of tamoxifen were investigated in two human endometrial cancer cell lines, IK-90 and HEC-IA cells. IK-90 cells contained specific AEBS, but no ER was found in these cells. Scatchard plot analysis of AEBS in 12,000 g supernatant from IK-90 cells showed a high affinity binding site for tamoxifen (Kd:5.6 +/- 1.0 nM) with the maximum binding site of 457 +/- 47 fmol/mg protein. However, no measurable ER or AEBS was found in HEC-IA cells. The effect of tamoxifen on the growth of cells was found to be identical in both cell lines; the addition of 10 microM tamoxifen to culture medium was cytocidal whereas tamoxifen at lower concentrations (1 nM-1 microM) did not significantly affect the growth of both IK-90 and HEC-IA cells. These results demonstrate for the first time the presence of AEBS in human endometrial cancer cells. The present results also suggest that AEBS does not play a fundamental role in mediating the growth-inhibitory effect of tamoxifen in endometrial cancer cells.     

Intracellular histamine and liver regeneration: high affinity binding of histamine to chromatin, low affinity binding to matrix, and depletion of a nuclear storage pool following partial hepatectomy.

We have demonstrated in rat hepatocytes that 3H-histamine binds specifically to novel low (microM) and high (nM) affinity sites, designated "HIC" to denote their intracellular location. Low affinity HIC sites are associated with microsomes, while both low and high affinity HIC sites are associated with the nucleus. A growth-regulatory action of intracellular histamine at HIC, independent of the rise in cytosolic calcium, has been demonstrated in mitogen-stimulated lymphocytes. We now report that the high affinity HIC sites in liver cell nuclei are associated exclusively with chromatin, while only low affinity sites are found in the residual material containing the nuclear matrix. Moreover, hepatocyte nuclei contain histamine (approximately 1 ng/mg protein), unaffected by incubation for up to 18 hours with the histidine decarboxylase inhibitor, alpha-FMH, suggesting a slow rate of turnover typical of a storage pool. A decrease in nuclear histamine parallels a rise in DNA synthesis in the first 24 hours after partial hepatectomy. Our findings support a role for a nuclear pool of pre-formed histamine in the mediation of liver regeneration.     

Murine and human hematopoietic colony formation: a possible regulatory role for intracellular histamine

Increasing number of data suggests that locally produced histamine is involved in regulation of hematopoiesis. In this study the granulocyte/macrophage (CFU-GM) colony formation by normal murine or human bone marrow cells, leukaemic colony formation (CFU-L) by a murine leukemia cell line (WEHI 3B), and colony formation by bone marrow cells from patients with chronic myeloid leukemia (CML) have been examined. We detected mRNA and protein expression of histidine decarboxylase (HDC), the only enzyme responsible for histamine synthesis both in normal bone marrow progenitor cells and in leukaemic progenitors. The significance of in situ generated histamine was shown on colony formation by inhibitory action of alphaFMH (blocking HDC activity, i.e. de novo histamine formation) and by N,N-diethyl-2-[4-(phenylmethyl)phenoxy]-ethanamine-HCl (DPPE) disturbing the interference of histamine with intracellular binding sites. These data provide further confirmation of the role of histamine in development and colony formation of bone marrow derived cells.     

Identification of two tamoxifen target proteins by photolabeling with 4-(2-morpholinoethoxy)benzophenone

Our quest to identify target proteins involved in the activity of tamoxifen led to the design of photoaffinity ligand analogues of tamoxifen able to cross-link such proteins. A new tritiated photoprobe, 4-(2-morpholinoethoxy)benzophenone (MBoPE), was synthesized and used to identify proteins involved in tamoxifen binding in rat liver. MBoPE, which has structural features in common with the potential antagonist of the intracellular histamine receptor (N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine HCl: DPPE) is unable to bind the estrogen receptor although it does compete with tamoxifen for an antiestrogen binding site (AEBS). This tritiated benzophenone derivative was obtained by metal-catalyzed halogen-tritium replacement reaction. Because of its high specific activity, four target proteins could be photolabeled, three of which were identified with M(r) of 60,000, 49,500, and 14,000, while the fourth at 27,500 was in too low an amount and could not be sequenced. The 49.5 kDa protein corresponded by mass spectrometry to the microsomal epoxide hydrolase already identified with an aryl azide photoprobe [Mesange, F., et al. (1998) Biochem. J. 334, 107-112]. The 60 and 14 kDa proteins were identified as the carboxylesterase (ES10) and the liver fatty acid binding protein (L-FABP), respectively. The inhibitory effect of tamoxifen on carboxylesterase activity and the competitive efficacy of oleic acid on [(3)H]tamoxifen binding suggest that both proteins are AEBS subunits. Moreover, treatment of hepatocytes with antisense mRNA directed against ES10 or L-FABP abolished both tamoxifen and MBoPE binding. On the basis of previous pharmacological arguments, the 27.5 kDa protein might correspond to the sigma I receptor. Altogether, these results confirm that the microsomal epoxide hydrolase is a target for tamoxifen and provide evidence of two new target proteins implicated in cell lipid metabolism.     

Antiestrogen binding within different pituitary cell populations. Comparison with androgen and estrogen receptors

Gonadotrope-enriched populations were prepared from 42-day old male rats by centrifugal elutriation. They contained 4.8 +/- 0.7% of the cells, 51 +/- 10% of the LH and less than 3% of the PRL (n = 4). Gonadotrope-depleted fractions were also obtained that contained most of PRL cells. Specific antiestrogen binding sites (AEBS) were quantitated in these populations after destruction of estrogen receptor. Results showed the presence of a distinct, specific high affinity binding site for antiestrogen in dispersed pituitary cells and in enriched fractions. However, AEBS are not specific of a pituitary cell type. Thus, AEBS appear different from estrogen receptors in pituitary gland: by the thermal stability of AEBS, by the localization of AEBS in particulate material, by the uniform distribution of AEBS in different populations which differ markedly for E2 binding sites. Whereas the ratio of binding AE/E2 averaged 11.4 in the initial cell suspension it reached only 2.9 in the gonadotropes. The dissociation constants for AEBS were in the same range (1.16 - 2.27 X 10(-9) M) for the different populations.     

Different h2 receptor antihistamines dissimilarly retard the growth of xenografted human melanoma cells in immunodeficient mice

Melanoma cells and tissues contain considerable amounts of histamine and express histamine receptors, suggesting the existence of autocrine and paracrine regulation by histamine. Our previous in vitro results suggested that histamine elevates melanoma cell growth through the H2 receptor. In this work we show that in vivo tumour proliferation in immunodeficient mice xenotransplanted with a human melanoma cell line is diminished by cimetidine, an H2 receptor antagonist, if combined with a tamoxifen derivate acting on cytochrome p450 molecules (DPPE). Ranitidine, another H2 receptor antagonist, has a weaker inhibitory effect, the kinetics and mechanism of which is probably dissimilar to that of the cimetidine/DPPE mixture.     

Importance of cholesterol and oxysterols metabolism in the pharmacology of tamoxifen and other AEBS ligands

Tamoxifen is one of the major drugs used for the hormonotherapy of estrogen receptor positive breast cancers. However, its therapeutic efficacy can be limited by acquired resistance and tumor recurrence can occur after several years of treatment. Tamoxifen is known as the prototypical modulator of estrogen receptors, but other targets have been identified that could account for its pharmacology. In particular, tamoxifen binds with high affinity to the microsomal antiestrogen binding site (AEBS) and inhibits cholesterol esterification at therapeutic doses. We have recently shown that the AEBS was a hetero-oligomeric complex composed of 3β-hydroxysterol-Δ(8)-Δ(7)-isomerase and 3β-hydroxysterol-Δ(7)-reductase, that binds different structural classes of ligands, including selective estrogen receptor modulators, several sigma receptor ligands, poly-unsaturated fatty acids and ring B oxysterols. We established a link between the modulation of cholesterol metabolism by tamoxifen and other AEBS ligands and their capacity to induce breast cancer cell differentiation, apoptosis and autophagy. Moreover, we showed that the AEBS carries out cholesterol-5,6-epoxide hydrolase activity and established that cholesterol-5,6-epoxide hydrolase is a new target for tamoxifen and other AEBS ligands. Finally in this review, we report on recent data from the literature showing how the modulation of cholesterol and oxysterol metabolism can be linked to the antitumor and chemopreventive properties of tamoxifen, and give new perspectives to improve the clinical outcome of the hormonotherapy of breast cancers.     

Novel ligands for the human histamine H1 receptor: synthesis, pharmacology, and comparative molecular field analysis studies of 2-dimethylamino-5-(6)-phenyl-1,2,3,4-tetrahydronaphthalenes

This paper reports the synthesis of a novel series of (+/-)-2-dimethylamino- 5- and 6-phenyl-1,2,3,4-tetrahydronaphthalene derivatives (5- and 6-APTs), and, corresponding affinity, functional activity, and, molecular modeling studies with regard to drug design targeting the human histamine H1 receptor. The 5-APTs have 2- to 4-fold higher H1 receptor affinity than the endogenous agonist histamine. The chemical nature of a meta-substituent on the 5-APT pendant phenyl moiety does not significantly affect H1 affinity. In contrast, analogous meta-substitution for the 6-APTs increases H1 affinity up to 100-fold. The new APTs do not activate H1 receptor-linked intracellular signaling and apparently are competitive H1 antagonists. A new model that establishes structural parameters for binding to the human H1 receptor by APTs and other ligands was developed using 3-D QSAR (CoMFA). The model predicts H1 ligand binding with a higher degree of external predictability compared to a previously reported model. The APTs also were examined for activity at human serotonin 5-HT2A and 5-HT2C receptors, which are phylogenetically closely related to the H1 receptor. 5-APT and m-Cl-6-APT were identified as novel agonists that selectively activate 5-HT2C receptors. It is concluded that the lipophilic (brain-penetrating) APT molecular scaffold may have pharmacotherapeutic potential in neuropsychiatric diseases.     

Binding of 4(5)-[2-(4-azido-2-nitroanilino)ethyl]imidazole to histamine receptors in guinea pig cerebral cortical membranes

The interaction of 4(5)-[2-(4-azido-2-nitroanilino)ethyl]imidazole (AAH), a photolabile histamine receptor antagonist, with the binding of histamine, mepyramine, and tiotidine to guinea pig cerebral cortical membranes was examined to evaluate the specificity of AAH for histamine H1 and H2 receptors. Saturable, specific binding of [3H]histamine, [3H]mepyramine, and [3H]tiotidine to the membranes was observed. Competition assays were used to assess the relative affinity of AAH for H1- and H2-receptors. The rank order of IC50 values obtained was (most to least potent) (i) for competing with [3H]histamine binding: histamine greater than AAH much greater than mepyramine approximately equal to tiotidine; (ii) for competing with [3H]mepyramine binding: mepyramine much greater than AAH greater than histamine greater than tiotidine; and (III) for competing with [3H]tiotidine binding: tiotidine much greater than mepyramine greater than histamine approximately equal to AAH. The affinity of AAH for H1 receptors was ca. 14-fold greater than for H2 receptors. These findings support previous evidence obtained in isolated smooth muscle preparations that AAH shows H1-receptor selectivity as an antagonist.     

Molecular characterization of the microsomal tamoxifen binding site

Tamoxifen is a selective estrogen receptor modulator widely used for the prophylactic treatment of breast cancer. In addition to the estrogen receptor (ER), tamoxifen binds with high affinity to the microsomal antiestrogen binding site (AEBS), which is involved in ER-independent effects of tamoxifen. In the present study, we investigate the modulation of the biosynthesis of cholesterol in tumor cell lines by AEBS ligands. As a consequence of the treatment with the antitumoral drugs tamoxifen or PBPE, a selective AEBS ligand, we show that tumor cells produced a significant concentration- and time-dependent accumulation of cholesterol precursors. Sterols have been purified by HPLC and gas chromatography, and their chemical structures determined by mass spectrometric analysis. The major metabolites identified were 5alpha-cholest-8-en-3beta-ol for tamoxifen treatment and 5alpha-cholest-8-en-3beta-ol and cholesta-5,7-dien-3beta-ol, for PBPE treatment, suggesting that these AEBS ligands affect at least two enzymatic steps: the 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase. Steroidal antiestrogens such as ICI 182,780 and RU 58,668 did not affect these enzymatic steps, because they do not bind to the AEBS. Transient co-expression of human 3beta-hydroxysterol-Delta8-Delta7-isomerase and 3beta-hydroxysterol-Delta7-reductase and immunoprecipitation experiments showed that both enzymes were required to reconstitute the AEBS in mammalian cells. Altogether, these data provide strong evidence that the AEBS is a hetero-oligomeric complex including 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase as subunits that are necessary and sufficient for tamoxifen binding in mammary cells. Furthermore, because selective AEBS ligands are antitumoral compounds, these data suggest a link between cholesterol metabolism at a post-lanosterol step and tumor growth control. These data afford both the identification of the AEBS and give new insight into a novel molecular mechanism of action for drugs of clinical value.     

Histamine formed in stimulated human platelets is cytoplasmic

The localization of histamine formed by human platelets in response to agonists was evaluated. 87 +/- 5% of the histamine in a suspension of platelets exposed to phorbol-12-myristate-13-acetate (PMA) was associated with the platelet pellet. Incubation of saponin-permeabilized platelets with the intracellular histamine antagonist, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine.HCl (DPPE), released 75 +/- 3.9% of the histamine into the supernatant. Under conditions where 90% of platelet serotonin was secreted into the supernatant, the majority (80%) of platelet histamine remained associated with the pellet. The results suggest that histamine synthesized in response to agonists is largely cytoplasmic.     

Interactive binding at cytochrome P-450 of cell growth regulatory bioamines, steroid hormones, antihormones, and drugs

The virtually universal family of P-450 isozymes contribute to the regulation of cell growth by modulating the levels of steroids and other lipid messengers for cytoplasmic and nuclear processes, including gene expression. In microsomes from rat liver cells, the concentration ( approximately 1 nmole/mg protein) of cytochromes P-450 approximates that of intracellular binding sites (K(d) 1.0-50 microM) for histamine. The potencies of certain therapeutic drugs to inhibit catalytic activity of, and histamine binding to, cytochromes P-450 in vitro were previously shown by us to be predictive of relative propensities to modulate tumor growth in rodents. Also, we demonstrated that growth-regulating polyamines potently interact with histamine at P-450. We now show that several classes of steroid hormones, antiestrogens, and antiandrogens, as well as various arylalkylamine drugs, all potently inhibit (3)H-histamine binding to cytochrome P-450 (K(i) values: testosterone 0.28 microM, progesterone 0.56 microM, flutamide 1.7 microM, tamoxifen 9.0 microM). Furthermore, all the various hormone and drug ligands are mutually inhibitory in their binding to cytochrome P-450; e.g., K(i) values of androstenedione and progesterone, to inhibit imipramine binding to P-450 (determined by spectral analysis), are 11 nM and 26 nM, respectively. The K(i) value of imiprimine to inhibit binding of androstenedione to P-450 is 3.5 microM. We estimate the total P-450 content in microsomes to be greater in male than in female rats and correlated with the number of binding sites for histamine, but not for steroids and drugs that appear to be more selective for P-450 isozymes. Thus, for at least some isozymes, the homeostatic role of the monooxygenases may be governed by histamine, modulated by endogenous ligands, and perturbed by many foreign molecules.