The estrogenic activity of DDT: Invivo and invitro induction of a specific estrogen inducible uterine protein by o,p'-DDT

The pesticide o,p'-DDT stimulates the production of a specific uterine protein, the so-called induced protein or IP, normally associated with an estrogenic response of the uterus. $\text{In}$$\text{vivo}$ stimulation of IP production is observed 1 hour after the administration of 250 mg/kg of o,p'-DDT to immature rats. $\text{In}$$\text{vitro}$ stimulation of IP production is observed after a 1 hour incubation of uteri with 100 μM o,p'-DDT. This $\text{in}$$\text{vitro}$ response is blocked by Actinomycin D. In contrast to o,p'-DDT, which binds to the cytoplasmic estrogen receptor and stimulates IP production, p,p'-DDT which does not bind well to the estrogen receptor does not stimulate IP production $\text{in}$$\text{vitro}$. These findings represent the first report of an estrogenic effect of o,p'-DDT in a completely $\text{in}$$\text{vitro}$ system.     

Preliminary characterization of a small intestinal binding component for retinol and fatty acids in the rat

A component which can bind retinol and fatty acids was detected in the rat's intestinal cell cytosol following intestinal perfusion $\text{in}$$\text{vivo}$ with ³H-all-trans retinol. Following Sephadex G-100 filtration of the cytosol, the void volume concentrate was treated with 2-mercaptoethanol and SDS. Sephadex G-100 filtration of the concentrate disclosed the presence of a cytosol binder of an approximate molecular weight of 12,000–17,000. The binder contained most of the ³H-retinol eluted off the column. $\text{In}$$\text{vitro}$ incubation experiments disclosed that ³H-retinol could be displaced from its bindinf cytosol fraction by the addition of nonradioactive retinol, retinyl acetate, and the fatty acids octanoic, linoleic, and linolenic. Butyric acid addition did not displace ³H-retinol from its binding fraction. The intestinal cytosol binding fraction may be involved in the trans-cytosol transport of lipid compounds from the lipid cell membrane to the intracellular organelles.     

Control of estrogen and androgen receptors in the rat pineal gland by catecholamine transmitter

Sucrose gradient studies of rat pineal cytosol incubated with ³H-estradiol (female pineals) or ³H-5 α -dihydrotestosterone (male pineals) revealed a radioactivity peak in the 8 S region which disappeared after superior cervical ganglionectomy or incubation with excess unlabeled hormone. Ganglionectomy decreased significantly estradiol and testosterone uptake by the pineal gland $\text{in vitro}$ as well as high affinity binding to pineal cytoplasmic and nuclear components. Norepinephrine treatment counteracted all the effects of ganglionectomy but was unable to modify hormone uptake and binding by the pineal gland of sham-operated controls. Pre-treatment with actinomycin D or propranolol but not with phentolamine impaired norepinephrine effects; propranolol blockage however was only partial. Administration of isoproterenol, L-dopa or phentolamine increased hormone uptake by denervated pineals. The effects of isoproterenol were also observed $\text{in vitro}$ and were blocked by propranolol. These results indicate that sex steroid receptors in the pinealocytes are controlled by norepinephrine via beta-adrenergic receptors and that depletion of neural norepinephrine enhanced responsiveness of pineal hormone receptors to exogenous catecholamines.     

A novel in vitro method for demonstrating proestrogens. Metabolism of methoxychlor and o,p'DDT by liver microsomes in the presence of uteri and effects on intracellular distribution of estrogen receptors.

A method for demonstrating proestrogens $\text{in}$$\text{vitro}$ has been developed. The method involves the incubation of the potential proestrogen with liver microsomes and NADPH in the presence of rat uteri, followed by examination of the effects of metabolism of the compound on the distribution of uterine estrogen receptor (R) in the cytosol (R_c) and in the nucleus (R_n). Thus, we examined whether DDT derivatives, which possess estrogenic activity $\text{in}$$\text{vivo}$, exhibit pro-estrogenic properties $\text{in}$$\text{vitro}$. Using this method, it appears that methoxychlor is a proestrogen, since the presence of microsomal enzymatic activity is required for methoxychlor to elicit translocation of uterine R_c into the nucleus, namely, the lowering of R_c and elevation of R_n. By contrast, o,p'DDT was active $\text{per}$$\text{se}$ in translocating R_c and did not require the presence of microsomal enzymes for activity.     

Partial purification and properties of a non-ligandin (3H) 3-methylcholanthrene binding protein from liver cytosol

(³H) 3-Methylcholanthrene binds $\text{in vivo}$ to a macromolecule in addition to the previously reported binding to ligandin in liver cytosol. The properties of this second molecule are identical to those of the glucocorticosteroid receptor (Binder II) through 400 fold purification over the cytosol proteins (elution position from DEAE-Sephadex A-50 columns, molecular weight by gel filtration and pI value by isoelectrofocusing). The carcinogen, probably a metabolite, binds very strongly or covalently to the macromolecule $\text{in vivo}$, but non-covalently $\text{in vitro}$ in the absence of microsomes. Large amounts of unlabeled carcinogen administered $\text{in vivo}$ do not compete significantly with subsequent (³H) dexamethasone binding to the hormone receptor fraction $\text{in vitro}$. Methylcholanthrene and dexamethasone do not compete for binding sites $\text{in vitro}$ on isolated unlabeled Binder II leading to the conclusion that the glucocorticosteroid receptor and the methylcholanthrene binding protein are distinct entities.     

Concepts related to salt resistant estradiol receptors in rat uterine nuclei: nuclear matrix

When ³H-estradiol (0.1 μg) is injected into immature female rats, virtually all of the label that is recovered with uterine nuclei can be solubilized by 0.6 M KCl. Salt resistant uterine nuclear estrogen binding sites do not become labeled within one hour after the injection of ³H-estradiol, but these sites do exist and can be revealed when isolated nuclei are subjected to an $\text{in vitro}$ estradiol exchange assay. These saturable, high affinity salt resistant sites appear to be associated with the uterine nuclear matrix, a residual structure of the nucleus.     

The role of cAMP on neoplastic cell growth and regression. III. Altered cAMP-binding in DBcAMP-unresponsive Walker 256 mammary carcinoma.

An exogenous supply of N⁶,O^2′-dibutyryl cyclic adenosine 3′,5′-monophosphate (DBcAMP) $\text{in vivo}$ produces regression of one type of Walker 256 mammary carcinoma cell population (DBcAMP-responsive); a second type of cell population continues to grow despite DBcAMP treatment (DBcAMP-unresponsive). A correlation was found between altered cAMP-binding of the tumor cytosol and DBcAMP-unresponsiveness. It was found that there was: a) a higher apparent dissociation constant (Kd) for cAMP-binding in unresponsive tumor cytosol $\text{in vitro}$, and b) unsaturability of cAMP-binding by unresponsive tumor cytosol in response to elevated cAMP levels $\text{in vivo}$. Cycloheximide abolished the saturation of cAMP binding $\text{in vivo}$ as well as tumor regression produced by DBcAMP.     

Irreversible protein binding of metabolites of ethynylestradiol in vivo and in vitro

During incubation of 6,7-³H-ethynylestradiol with rat liver microsomes up to 20 % of the radioactivity was bound irreversibly to the microsomal proteins. Incubations in presence of albumin resulted in a further radioactive labelling of the albumin. The irreversible nature of the steroid-protein bond was established by solvent extraction and charcoal treatment. Further evidence was obtained after hydrolyzing the microsomal protein with trypsin and submitting the labelled tryptic peptides to ion exchange chromatography and electrophoresis. The labelled albumin was applied to sephadex gel filtration which showed the association of the ethynylestradiol radioactivity to the albumin peak.The binding reaction required supply of NADPH, could be stimulated by pretreatment of the animals with phenobarbital and was inhibited by CO and SKF 525 A. On these characteristics the concept was based that, in analogy to the well known binding of estradiol and estrone, 2hydroxylation is also an essential prerequisite for the binding of ethynylestradiol. The concept was confirmed by trapping off the 2-hydroxy-ethynylestradiol with glutathione, which led to a decrease of the ethynylestradiol-protein binding.Further evidence resulted from experiments $\text{in vivo}$, dosing rats with 6,7-3H-ethynylestradiol and 6,7-3H-estradiol 48 hrs prior to sacrifice and examining the amount of radioactivity irreversibly bound to the liver endoplasmic reticulum. 3H-ethynylestradiol caused a radioactive labelling of microsomes twice as much as that after ³H-estradiol.     

Estrogen action in the mouse uterus: An additional nuclear event

An $\text{in vivo}$ injection of ³H-estradiol to an ovariectomized mouse resulted in the appearance of two increases of nuclear receptor: the first at 1 hr and the second occurring at approximately 7–8 hrs post-injection. These findings were substantiated by quantifying cytoplasmic and nuclear receptor levels with the exchange assay after injections of unlabeled estradiol. Cytosol receptor levels were depleted at 0.5–1 hr and replenished from 2–10 hr with values at 15–24 hr significantly greater than those at zero time. Nuclear receptors decreased precipitously to near control values after the initial translocation event. At 7–8 hr a second smaller nuclear increase occurred which then declined to near zero levels. Both of these nuclear events appear to be dose related. Similar experiments using cycloheximide did not abolish either of these events, but did significantly diminish the cytosol receptor replenishment.     

Identification of opiate receptor binding in intact animals.

After intravenous administration of ³H-naloxone to rats, particulate bound radioactivity accumulated in the brain is selectively associated with opiate receptor binding sites, providing a means of labeling the opiate receptor $\text{in vivo}$. The regional distribution of ³H-naloxone bound $\text{in vivo}$ closely parallels regional differences in opiate receptor binding $\text{in vitro}$ with highest levels in the corpus striatum, negligible receptor-associated binding in the cerebellum and intermediate levels in other regions. ³H-Naloxone binding $\text{in vivo}$ is saturable with the same total number of binding sites determined $\text{in vivo}$ as by $\text{in vitro}$ procedures. Nalorphine is markedly more potent than morphine in inhibiting ³H-naloxone binding $\text{in vivo}$ and non-opiates are ineffective. The half-life for dissociation of ³H-naloxone bound to particles $\text{in vivo}$ is the same as its dissociation rate after binding occurs $\text{in vitro}$, and sodium stabilizes ³H-naloxone bound $\text{in vivo}$ from initial rapid dissociation as predicted from the known properties of the opiate receptor $\text{in vitro}$.     

DDT inhibition of Ca-Mg ATPase from peripheral nerves and muscles of lobster, Homarus americanus

Sensitivity of CaMg ATPase from axonic plasma membrane (APM) and sarcoplasmic reticulum (SR) of lobster, $\text{Homarus}\text{,}\text{americanus}$, to DDT was studied. The CaMg ATPase found in SR with the high Ca²⁺ affinity is sensitive to DDT while the portion of ATPase related to the low Ca²⁺ affinity site is not inhibited by DDT. Also, DDT is more inhibitory against the CaMg ATPase prepared from APM than the one obtained from SR. The relationship between inhibition of the CaMg ATPase by DDT in the axonic nerve membrane and $\text{in}\text{,}\text{vivo}$ poisoning symptoms of the nervous system is discussed.     

In vitro and in vivo inhibition by zopiclone of benzodiazepine binding to rodent brain receptors.

Up to now the only drugs known to be able to inhibit the binding of benzodiazepines to rodent brain receptors are members of this chemical family.Zopiclone (RP 27 267), a new drug with a pharmacological profile similar to that of chlordiazepoxide and nitrazepam but entirely different chemically from benzodiazepines, has been tested for its ability to inhibit benzodiazepine binding. $\text{In vitro}$ and $\text{in vivo}$ studies have shown that zopiclone is able to inhibit the binding of [³H] diazepam and [³H] flunitrazepam to brain receptors. The potency of zopiclone is quite comparable to that of diazepam and nitrazepam $\text{in vitro}$ and to that of chlordiazepoxide $\text{in vivo}$.These results confirm the pharmacological similarities existing between zopiclone and the benzodiazepines.     

Differences in macromolecular binding between cyclic AMP and its dibutyryl derivative in vitro

Rat liver cytosol binds ³H-cAMP and ³H-DBcAMP $\text{in vitro}$. Fractionation of bound radioactivity by DEAE-Sephadex chromatography shows that ³H-cAMP is associated with a different cytosolic protein than is ³H-DBcAMP. The pI's of the cAMP-protein and the ³H-DBcAMP-protein complexes are 6.7 and 3.9, respectively. Competition studies between ³H-cAMP and its structural analogues have shown the following order of effectiveness in competing for binding sites in rat liver cytosol: cAMP > N⁶-MBcAMP > O²′-MBcAMP. No inhibition of ³H-cAMP binding was observed with 5′-AMP, adenosine, cGMP or DBcAMP. $\text{In vitro}$ binding experiments with rat serum has shown that only ³H-DBcAMP binds to any significant extent.     

Complex formation by 3 H-aldosterone in rat kidney and liver

Low molecular weight polar complexes were shown to be formed $\text{in vivo}$ from ³H-aldosterone in both kidney and liver subcellular fractions, the majority being present in the cytosol fractions. Significant differences were observed between the quantities of polar complexes present in kidney subcellular fractions from intact and adrenalectomized male rats and also between the quantities of these kidney polar complexes from spironolactone treated male rats. ³H-aldosterone macro-molecule complexes were shown to exist in appreciable quantities only in the kidney cytosol fractions of adrenalectomized male rats. These gel filtration studies also showed the ³H-aldosterone labeled macromolecule complexes to consist of two protein peaks; one of high molecular weight and the other of lower molecular weight (～50,000 mol. wt.). The amount of ³H-aldosterone labeled protein complexes in kidney cytosol was greatly reduced when adrenalectomized rats were pretreated $\text{in vivo}$ with spironolactone.     

Ethanol-induced inhibitions of testicular steroidogenesis in the male rat: Mechanisms of actions

Ethanol markedly inhibits the biosynthesis of testosterone in the male of several species. Since several $\text{in vitro}$ studies have suggested that ethanol $\text{per se}$ is not a gonadal toxin and that it must be metabolized to exert its effects, we examined this possibility under $\text{in vivo}$ conditions in the present studies. We found that the administration of the alcohol dehydrogenase inhibitor, pyrazole, to adult male rats significantly elevated blood ethanol levels. However, rather than resulting in a potentiation of the effects of ethanol on testicular steroidogenesis, pyrazole-induced elevations in blood ethanol concentrations produced a significant attenuation of ethanol's effects. In view of these observations, it is difficult to maintain that ethanol itself is responsible for inhibiting the production of testosterone. On the contrary, our results may provide the first $\text{in vivo}$ support for the hypothesis that ethanol must be metabolized to exert its effects on testicular steroidogenesis.     

Specific in vivo binding of 77Br-p-bromospiroperidol in rat brain: A potential tool for gamma ray imaging

The binding of the gamma labeled neuroleptic, ⁷⁷Br-$\text{p}$-bromosprioperidol, in the rat brain was examined $\text{in vivo}$. This binding parallels the binding of ³H-spiroperidol, in that binding is especially high in dopaminergically innervated areas, is saturable, and is displaced by high doses of unlabeled spiroperidol (1–5). Thus, ⁷⁷Br-$\text{p}$-bromospiroperidol is a suitable ligand for use in gamma ray imaging techniques for $\text{in vivo}$ monitoring of receptor binding.     

Characterization of a progestin-binding macromolecule in the amphibian oocyte cytosol.

A [³H]-progestin-binding macromolecule has been isolated from $\text{R}\text{.}\text{pipiens}$ oocyte cytosol and characterized using binding assays, gel filtration, DEAE-cellulose chromatography and ultracentrifugal techniques. Macromolecules present in the prophase oocyte cytosol have a high affinity and specificity for the synthetic progestin R5020 and the intact oocyte will concentrate both R5020 and progesterone 20–40 fold from the medium. This may be the first published case of a cytosol steroid binding macromolecule in a cell system in which the steroid appears to act at an extranuclear level.     

Studies on the possible role of 2-OH-estradiol in the control of sexual behavior in female rats

$\text{In vitro}$ studies have revealed that 2-OH-estradiol can be produced in the brain and that it can compete with estradiol for hypothalamic cytosol binding sites. In the present study ovariectomized female rats received 2-OH-estradiol via sc or iv injections and/or via direct intracerebral implantation either alone or in combination with vehicles, estradiol or testosterone. Behavioral tests after sc progesterone priming indicated that 2-OH-estradiol has relatively weak estrogenic, and no apparent anti-estrogenic or antiaromatization activity in the induction of sexual receptivity.     

Binding of [3H]methyltrienolone to androgen receptor in rat liver

The synthetic androgen methyltrienolone is superior to testosterone and androstenedione for the measurement of androgen receptor in tissues where the native ligands are metabolized into inactive derivatives. [³H]Methyltrienolone binds with a high affinity to androgen receptor in cytosol prepared from male rat livers, as the Scatchard analysis revealed that the K_d value was 3.3 · 10^?8 M and the number of binding sites was 35.5 fmol/mg protein. Since methyltrienolone also binds glucocorticoid receptor which exists in rat liver, the apparent binding of androgen receptor is faulty when measured in the presence of glucocorticoid receptor. The binding of methyltrienolone to glucocorticoid receptor can be blocked by the presence of a 100-fold molar excess of unlabeled synthetic glucocorticoid, triamcinolone acetonide, without interfering in its binding to androgen receptor, because triamcinolone does not bind to androgen receptor. Triamcinolone-blocked cytosol exhibited that the K_d value was 2.5 · 10^?8 M and the number of binding sites was 26.3 fmol/mg protein, indicating a reduction to $\text{3}\text{4}$ of that in the untreated cytosol. The profile of glycerol gradient centrifiguration indicated that [³H]methyltriemolone-bound receptor migrated in the 8–9 S region in both untreated and triamcinolone-blocked cytosols, but the 8–9 S peak in triamcinolone-blocked cytosol was reduced to about $\text{3}\text{4}$ of that of untreated cytosol.     

Demonstration of [3H] diazepam binding to benzodiazepine receptors in vivo.

Benzodiazepine receptors were labeled with [³H] diazepam following intravenous injection in rats. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ to rat forebrain membranes was displaceable by co-injection of clonazepam or the pharmacologically active enantiomers of two benzodiazepines, B9 and B10, but was not displaced by equal doses of the pharmacologically in-active enantiomers. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ was bserved in kidney, liver, and abdominal muscle, but was not stereospecifically diplaced in any peripheral tissue studied. The regional distribution of benzodiazepine receptors in brain was uneven, with specific [³H] diazepam binding being highest in the cerebral cortex and lowest in the ponsmedulla. Preliminary studies of the subcellular distribution of [³H] diazepam binding demonstrated highest specific binding to synaptosomal membranes. These data demonstrate the feasibility of labeling benzodiazepine receptors in rat brain $\text{in}$$\text{vivo}$.