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Hippocampal cell loss during aging has been related to the toxic effects of corticosterone on this cell population. It is not known which receptor mediates corticosterone cytotoxicity. At least two types of receptors for corticosterone have been recognized in the rat brain, type I (corticosterone preferring receptor, CR) and type II (glucocorticoid receptor, GR). In the present study the possible changes in GR immunoreactivity (IR) in various tel- and diencephalic regions of the aged rat have been investigated using immunocytochemistry coupled with computer-assisted image analysis. Male Sprague-Dawley rats of 3, 12 and 24 months of age were used (n = 5/group). A selective decrease of GR-IR was observed in the CA1 hippocampal field and central amygdaloid nucleus of the 24-month-old with respect to both 3- and 12-month-old rats. While in the former region GR-IR decrease was paralleled by a decrease of IR field area, no age-related decrease of GR-IR profile number was detected in central amygdaloid nucleus. A significant decrease of GR-IR and IR field area was also observed in the dentate gyrus of 24- vs 12-month-old rats but not vs 3-month-old rats. The analysis of adjacent sections stained with Cresyl violet showed a pattern of age-related changes (decrease of neuronal profiles in CA1 field pyramidal layer and dentate gyrus granular layer, and no change in the central amygdaloid nucleus of aged rats) which paralleled the observed changes in GR-IR in the same areas. This study provides evidence that GR are selectively decreased in the hippocampal formation and in the central amygdaloid nucleus of the aged rat.(ABSTRACT TRUNCATED AT 250 WORDS)

By means of double immunolabeling procedures it has been possible to demonstrate glucocorticoid receptor (GR) immunoreactivity (IR) in large numbers of various peptidergic neurons of the brain including neurons containing gastrointestinal peptides, opioid peptides, and peptides with a hypothalamic hormone function. For each peptide system, however, marked heterogeneities exist among brain regions. Thus, in the neocortex and the hippocampal formation most of the brain peptide neurons lack GR IR, while the same types of peptide neurons in the arcuate and paraventricular nucleus [e.g. neuropeptide Y (NPY), somatostatin (SRIF) and the cholecystokinin (CCK) neurons] possess strong GR IR. Furthermore, in the arcuate, parvocellular part of the paraventricular nuclei and the central amygdaloid nucleus practically all the peptidergic neurons are strongly GR IR, while in the lateral hypothalamus, mainly the neurotensin (NT) and galanin (GAL) IR neurons are GR IR. These marked differences among areas probably reflect functional differences dependent upon their participation in stress regulated circuits. All the paraventricular NT, corticotropin-releasing factor (CRF), growth hormone-releasing factor (GRF), thyrotropin-releasing hormone (TRH) and SRIF IR neurons appear to contain GR IR, while the luteinizing hormone-releasing hormone (LHRH) IR neurons lack GR IR, underlying the importance of glucocorticoids (GC) in controlling endocrine function. Finally, the GC may influence pain and mood control mainly via effects on enkephalin (ENK) neurons especially in the basal ganglia (mood) and on all beta-endorphin (beta-END) neurons of the arcuate nucleus, while most of the dynorphin neurons are not directly controlled by GC.

Partially purified preparations of the human progestin receptor and the human and rat glucocorticoid receptor proteins were covalently charged with the synthetic progestin, [3H]promegestone, by photoaffinity labeling. After labeling, the denaturated protein was cleaved and the mixture of peptides subjected to radiosequence analysis as previously described for the rat glucocorticoid receptor protein (Carlstedt-Duke, J., Strömstedt, P.-E., Persson, B., Cederlund, E., Gustafsson, J.-A., and Jörnvall, H. (1988) J. Biol. Chem. 263, 6842-6846). The radioactivity labels identified, corresponded to Met-759 and Met-909 after photoaffinity labeling of the human progestin receptor, and Met-622 and Cys-754 after labeling of the rat glucocorticoid receptor. The residues labeled in the glucocorticoid receptor are the same as those previously reported to bind triamcinolone actonide. The corresponding residues were also labeled in the human glucocorticoid receptor. Met-759 of the progestin receptor and Met-622 of the rat glucocorticoid receptor are positioned within a segment with an overall high degree of sequence similarity and are equivalent. However, Met-909 (progestin receptor) and Cys-754 (glucocorticoid receptor) do not occur within equivalent segments of the two proteins. Thus, although the two classes of steroid hormone share a common structure within the A-ring, there are subtle differences in their interaction with the two separate receptor proteins.

Estrogens are important for the development and function of the normal mammary gland as well as for development of mammary cancer. The frontline therapy for treatment of estrogen receptor (ER alpha) positive breast cancer is antiestrogens. A second estrogen receptor (ER beta) is also expressed in the breast but it has not been measured because it is not detected by the immunoassays used to detect ER alpha. In many cell systems ER beta has actions which are opposite to those of ER alpha and this finding has raised questions about the role of ER beta in the development and treatment of breast cancer.

Androgens and estrogens influence voiding. In this study their target sites in the lower urinary tract of the male rat were identified.Cryosections of the bladder body, bladder neck, prostatic urethra, mid proximal urethra and prostatic autonomic ganglia of adult male rats were immunostained with specific estrogen receptor alpha, estrogen receptor beta (ERbeta) or androgen receptor (AR) antibodies. The sections were then examined under conventional, fluorescence or confocal fluorescence microscopy.Co-expression of AR and ERbeta in the urothelium, bladder smooth muscle cells, proximal urethra striated muscle cells and neurons in the autonomic ganglia of the prostatic plexus suggests that estrogen and androgen have direct effects in the lower urinary tract.The local interaction of AR and ERbeta in the hormonal control of voiding is an intriguing possibility.

Thioredoxins are a class of small redox-regulating proteins that have been implicated in the control of various aspects of cellular functions and seem to be one of the key regulators of signalling in the cellular responses to various stresses. Thioredoxin-2 (Trx2) is a novel mammalian thioredoxin which, in contrast to previously known cytosolic thioredoxin (Trx1), has been localized to the mitochondria. Trx2 is abundantly expressed in skeletal muscle, heart and adrenal gland, as well as in some other peripheral tissues with high metabolic activity. Using in situ hybridization and immunohistochemistry, we have studied the distribution and regulation of Trx2 expression in the rat brain. Trx2 mRNA and protein are highly expressed in the neurons in several brain regions, including the olfactory bulb, frontal cortex, hippocampus, some hypothalamic and thalamic nuclei, cerebellum and numerous brainstem nuclei. In addition, the Trx2 mRNA expression in paraventricular hypothalamic nucleus and reticular thalamic nucleus was found to be sensitive to peripheral glucocorticoids, as dexamethasone treatment caused significant elevation of Trx2 mRNA level in this area. No changes in other brain areas were observed after dexamethasone treatment. These findings implicate a significant regulatory and/or protective function of Trx2 in the nervous system.

Pulmonary expression of several cytochrome P450 (CYP) monooxygenases is detected late in gestation. Little is known of the factors involved in this differentiation-dependent expression. C/EBP factors are known regulators of differentiation and differentiation-dependent gene expression in several tissues. In this study we demonstrate the importance of C/EBP alpha and C/EBP delta in pulmonary epithelial CYP2B1 gene expression. A 1.3 kb CYP2B1 promoter fragment which recently has been shown to confer lung tissue- and cell-specific expression of CYP2B1 in transgenic mice was used in transient transfection studies. Both C/EBP alpha and C/EBP delta transactivated the CYP2B1 promoter in the lung epithelial cell lines A549 and NCI-H441. C/EBP alpha in nuclear extracts from isolated rat primary bronchiolar Clara cells was capable of interacting with a C/EBP-binding site in the proximal CYP2B1 promoter. Site-directed mutagenesis studies showed that this proximal C/EBP-binding site is necessary for transactivation of the CYP2B1 gene by C/EBP alpha and C/EBP delta. Thi study shows that C/EBP factors have a role in pulmonary CYP2B1 expression and suggests that these transcription factors may be important for the differentiation-dependent expression of CYP2B1 in the lung.

An obligatory role for estrogen in growth, development, and functions of the mammary gland is well established, but the roles of the two estrogen receptors remain unclear. With the use of specific antibodies, it was found that both estrogen receptors, ERalpha and ERbeta, are expressed in the rat mammary gland but the presence and cellular distribution of the two receptors are distinct. In prepubertal rats, ERalpha was detected in 40% of the epithelial cell nuclei. This decreased to 30% at puberty and continued to decrease throughout pregnancy to a low of 5% at day 14. During lactation there was a large induction of ERalpha with up to 70% of the nuclei positive at day 21. Approximately 60-70% of epithelial cells expressed ERbeta at all stages of breast development. Cells coexpressing ERalpha and ERbeta were rare during pregnancy, a proliferative phase, but they represented up to 60% of the epithelial cells during lactation, a postproliferative phase. Western blot analysis and sucrose gradient centrifugation confirmed this pattern of expression. During pregnancy, the proliferating cell nuclear antigen was not expressed in ERalpha-positive cells but was observed in 3-7% of ERbeta-containing cells. Because more than 90% of ERbeta-bearing cells do not proliferate, and 55-70% of the dividing cells have neither ERalpha nor ERbeta, it is clear that the presence of these receptors in epithelial cells is not a prerequisite for estrogen-mediated proliferation.

Transcriptional activation by nuclear receptors (NRs) involves the concerted action of coactivators, chromatin components, and the basal transcription machinery. Crucial NR coactivators, which target primarily the conserved ligand-regulated activation (AF-2) domain, include p160 family members, such as TIF2, as well as p160-associated coactivators, such as CBP/p300. Because these coactivators possess intrinsic histone acetyltransferase activity, they are believed to function mainly by regulating chromatin-dependent transcriptional activation. Recent evidence suggests the existence of an additional NR coactivator complex, referred to as the thyroid hormone receptor-associated protein (TRAP) complex, which may function more directly as a bridging complex to the basal transcription machinery. TRAP220, the 220-kDa NR-binding subunit of the complex, has been identified in independent studies using both biochemical and genetic approaches. In light of the functional differences identified between p160 and TRAP coactivator complexes in NR activation, we have attempted to compare interaction and functional characteristics of TIF 2 and TRAP220. Our findings imply that competition between the NR-binding subunits of distinct coactivator complexes may act as a putative regulatory step in establishing either a sequential activation cascade or the formation of independent coactivator complexes.

The recently discovered estrogen receptor-beta (ERbeta) is expressed in rodent and human testes. To obtain insight in the physiological role of ERbeta we have investigated the cell type-specific expression pattern of ERbeta messenger RNA (mRNA) and protein in the testis of rats of various ages by in situ hybridization and immunohistochemistry. In fetal testes of rats 16 days postcoitum and testes of 4-day-old animals, fetal germ cells (gonocytes) reveal the ERbeta mRNA in their cytoplasm and the ERbeta protein in their nucleus. In testes of 11- and 15-day-old rats, ERbeta mRNA and protein were detected in Sertoli cells and type A spermatogonia. No signal was found in other types of germ cells. In the adult testes, expression of ERbeta mRNA as well as ERbeta protein was found in pachytene spermatocytes from epithelial stages VII-XIV and in round spermatids from stages I-VIII. Low ERbeta expression was observed in all type A spermatogonia, including undifferentiated A spermatogonia, whereas no expression was found in In and type B spermatogonia and early spermatocytes. At all ages, Sertoli cells showed a weak hybridization signal as well as weak immunoreactivity for ERbeta. In adult testes, no ERbeta mRNA or protein was detected in the interstitial tissue, indicating that Leydig cells and peritubular myoid cells do not express ERbeta. The expression of ERbeta in fetal and late male germ cells as well as in Sertoli cells suggests that estrogens directly affect germ cells during testicular development and spermatogenesis.

Cooking of protein-rich food generates procarcinogenic heterocyclic aromatic amines (HCA) in amounts that range in the part per billion levels. HCA have been reported to induce xenobiotic metabolizing enzymes of the cytochrome P450 1A (CYP1A) subfamily, most notably the CYP1A2 isoform. The regulator mechanism of the CYP1A induction by HCA is, however, unclear. Studies in vivo in rats and in primary hepatocyte cultures revealed "that MelQx induced both CYP1A1/1A2 proteins and the corresponding catalytic activities. In contrast to previous studies, no preferential induction of CYP1A2 was observed in the present study. CYP1A1 and CYP1A2 are target genes of the intracellular dioxin receptor. This receptor interacts with xenobiotic response elements (XREs) of target promoters upon binding the environmental pollutant dioxin or related compounds. HCA exhibited capacity to activate the dioxin receptor to a form which interacts with XRE in vitro. Taken together, these results suggest that MelQx regulates both CYP1A isozymes by the same mechanism involving the dioxin receptor. Another group of putative dioxin receptor ligands of dietary orgin are the indolocarbazoles, which are produced in vivo from precursor molecules in cruciferous plants. Indolocarbazoles potently regulated gene expression of a reporter gene driven by a minimal XRE in both mouse and human hepatoma cells. The indolocarbazole-induced human receptor appeared to form more stable complexes with XRE in vitro relative to those generated by the dioxin-activated receptor. This study indicates that the HCA and the indolocarbazoles both represent distinct classes of dietary dioxin receptor agonists.

Synthetic estrogens act as tumor promoters in rat liver. Because estrogen treatment markedly increases the secretion of pituitary prolactin, also shown to be a tumor promoter in rat liver, the possibility of a pituitary influence in estrogen promotion was investigated in Wistar rats. In diethylnitrosamine (DEN)-initiated hypophysectomized (hx) female rats, 24 weeks of ethinyl estradiol (EE) administration (500 microg/kg/d, intraperitoneally) did not increase the number of hepatocyte nodules and did not induce hepatocellular carcinoma (HCC) in a 2-year study. Very few placental forms of glutathione-S-transferase (GST-P)-positive foci were observed at the end of EE administration. Estrogen receptor (ER) messenger RNA (mRNA) levels in hx females were 20% of the levels in intact females. EE administration (range, 160-210 microg/kg/d, subcutaneous release pellets) to DEN-initiated intact males and females increased the number and size of hepatocyte foci. A significant increase in HCC frequency was observed in EE-treated females compared with females receiving sham-release pellets, and the latency period for HCC induction was decreased by EE in both males and females. Inhibition of prolactin (PRL) secretion by bromocriptine (Brc) (ParlodelLAR, slow intramuscular release vehicles) during EE treatment decreased the number of foci without affecting their size and markedly prolonged the latency period in both sexes. EE treatment also significantly increased the expression of c-myc, and c-jun, enhanced the levels of growth hormone receptor (GHr) mRNA in females and the levels of ER mRNA in males and "feminized" the expression of the GH-regulated genes cytochrome P450 (CYP), 2C11, CYP 2C12, and GHr in male liver. Brc administration decreased the mRNA levels of the female-predominant CYP 2C12 in EE-treated males but otherwise had no effects. In conclusion, a decreased promotive effect of EE was obtained by decreasing the PRL levels, indicating that estrogens exert at least part of their promotion effects indirectly, by increasing the levels of pituitary PRL.

Cytochrome P450 was partially purified from rat breast tissue from 1-, 2-, 3-, 6-, 9-, and 15-week-old pregnant, lactating, or 3-week postlactation rats. The detergent-solubilized P450 was spectrally quantified, and the P450 isozyme pattern in the different samples was characterized by Western blot analysis with antibodies against cytochromes P450 1A1, 1A2, 2A, 2B, 2D4, 3A, 4A, 2E, and 19. The yield of P450 was 5-60 pmol/g wet weight tissue, with the highest yields in 1- and 2-week-old pups and lactating rats. The cytochromes P450 expressed in the breast can be divided into six main groups on the basis of their pattern of regulation: (a) those present in all samples (4A, 2E1, and 2D4), (b) those highly expressed in 1- to 3-week-old rats (2D4 and 3A), (c) those expressed only after 9 weeks of age [P450 19 (aromatase)], (d) those induced in pregnancy and maintained during lactation (1A1), (e) those induced in pregnancy and not maintained during lactation (2A), and (f) those induced 3 weeks after lactation (2B, 2A, and 3A). Reverse transcription-polymerase reaction amplification was used to confirm the presence of P450 isoforms in the breast. The mRNAs of cytochromes P450 1A1, 2A1, 2B1-3, 2D1, 2D3, 2D4, 2E1, and 4A3 were detected on analysis of total breast RNA. The mRNA of CYP 3A1 was not convincingly detected in untreated rat breast but was inducible by treatment with pregnenolone-16-alpha-carbonitrile. The presence of these various forms of P450 in the breast and their regulation by age and endocrine status may have implications for in situ metabolism of steroids and steroid antagonists and for activation of procarcinogens.

The possible relationship between cytochrome P450 (P450) in brain and degenerative diseases of the central nervous system prompted an investigation into the members of the 2D subfamily in rat brain. The mRNA for P450 2D4 was much more abundant than those for 2D1 and 2D5, which are major hepatic forms. 2D2 and 2D3 mRNAs, which are also abundant forms in liver, were not detectable in brain. To evaluate the quantitative significance of 2D4 protein in brain, specific antibodies were raised. The full length 2D4 cDNA was cloned from brain mRNA by reverse transcription-polymerase chain reaction amplification and was translated, in a reticulocyte lysate system, into a protein of approximately 50 kDa. A [35S]methionine-labeled protein of 50 kDa could be immunoprecipitated from in vitro translated 2D4 mRNA. In Western blots, no signals were obtained with brain microsomes. However, with P450 extracted from brain a band of 50 kDa could be detected when 60 pmol or more were loaded in each lane. There was no detectable developmental regulation of the 2D4 mRNA and no change during pregnancy, during lactation, or after treatment with ethanol, conditions under which the P450 content of brain increases. In Dark Agouti rats, which do not express P450 2D1 mRNA in liver, 2D4 mRNA in brain was as abundant as in Wistar rats. We conclude that 2D4 is expressed as a stable protein in the brains of untreated rats, where it represents5% of the total P450.

Humans frequently inhale as well as ingest cooked-food mutagens, among which the heterocyclic amines are the quantitatively most important. An extensive systemic distribution of these mutagens implies that most tissues in the body are exposed. Tissues containing cytochrome P450 (CYP) may be particularly susceptible to DNA damage. Accordingly, animal experiments have shown that oral exposure to heterocyclic amines leads to tumor formation at multiple sites. CYP1A2, which has only been demonstrated in the liver, seems to be the isozyme most efficient in metabolically activating the heterocyclic amines. In extrahepatic tissues, however, other CYP forms are likely to be important. Using Salmonella mutagenicity as an endpoint, we have studied the metabolic activation of 2-amino-3-methylimidazo[4,5,f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5,f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by isolated lung microsomes from rats and mice. Our studies show that CYP2A3, an isozyme that has hitherto not been investigated with regard to its capacity to activate heterocyclic amines, catalyses a major part of the IQ activating reactions in the uninduced lung. The formation of mutagens during cooking of meat is highly temperature dependent and meat extracts heated at 200 degrees C show a strong mutagenic activity in the Ames Salmonella assay. These extracts caused mutations at the HPRT locus in normal human fibroblasts as well as a pronounced decrease in survival of the cells. Furthermore, the heated meat extracts caused a decreased proliferative activity in primary cultures of normal mouse colonic epithelial cells as measured by autoradiography.

The cytochrome P-4501A1 and P-4501A2 enzymes are involved in the metabolic activation of a number of environmental precarcinogens including the food-derived heterocyclic amines. These compounds also induce CYP1A activity in rats, a feature they have in common with several of the xenobiotics (most notably benzo[a]pyrene) which are metabolically activated by the cytochrome P-4501A isozymes. Using an in vitro DNA binding assay and an in vivo functional (transactivating) assay, we have found that the cytochrome P-4501A1 induction response produced by the heterocyclic amines is mediated by the intracellular dioxin receptor. In the absence of ligand, the receptor is inactive and does not bind DNA. In this report we demonstrate a correlation between activation of DNA binding activity of the receptor by the heterocyclic amines and their ability to induce transcription from a minimal dioxin-response element-driven promoter construct. However, relatively high doses of these compounds are required to produce these effects. Therefore, despite a relatively high dietary intake of heterocyclic amines as compared to other cytochrome P-4501A1-inducing substances, the heterocyclic amines most probably only play a minor role in the induction of this enzyme activity. In contrast to the heterocyclic amines, indolo derivatives found in cruciferous vegetables exhibit high affinity for the dioxin receptor and represent potent activators of the DNA binding activity of the receptor. The indolo derivatives are therefore likely to have a larger impact than the heterocyclic amines on human CYP1A1 activity.

We have used a panel of monoclonal antibodies and a cell-free transcription assay to study the function of the tau 1 transactivation domain of the human glucocorticoid receptor. Three antibodies (monoclonal antibodies 250, 275, and 286) specifically inhibited tau 1-dependent transcription, but had little or no effect on either basal transcription or the activity of an unrelated yeast transcription factor. This inhibition was not due to interference of DNA binding activity, as all three antibodies super shifted tau 1-containing protein-DNA complexes. Epitopes for all three antibodies were localized to a region between amino acids 190 and 200, which lies within the recently defined 41-amino acid core region of tau 1 that is required for transactivation (Dahlman-Wright, K., Almlöf, T., McEwan, I.J., Gustafsson, J-A., and Wright, A. P.H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1619-1623). In contrast to the effect on tau 1-dependent transcription none of the antibodies tested antagonized the squelching ability of the tau 1 domain, suggesting that tau 1-mediated transactivation involves interactions in addition to those identified by the squelching assay. Consistent with this, a comparison of the kinetics of tau 1 squelching and inhibition of transactivation by monoclonal antibodies suggested a role for tau 1 mediated transcriptional induction at two or more steps during transcription initiation by RNA polymerase II.

By means of a monoclonal mouse immunoglobulin G2a antibody against the rat liver glucocorticoid receptor and the indirect immunoperoxidase technique, the distribution of glucocorticoid receptors in neuronal and glial cell populations was mapped in the central nervous system of the male rat. The mapping was complemented by computer-assisted morphometric and microdensitometric evaluation of glucocorticoid receptor immunoreactivity in many brain regions. The quantitative analysis allowed us to achieve for the first time an objective characterization of glucocorticoid receptor distribution in the CNS, thus avoiding the ambiguities of previous mapping studies based on subjective evaluations. In addition, a taxonomic analysis of central nervous system regions containing glucocorticoid receptor immunoreactivity was carried out utilizing the quantitative parameters obtained in the morphometric evaluation. Nuclei of neuronal and glial cells containing glucocorticoid receptor immunoreactivity were detected in a widespread, but still highly heterogeneous, fashion in the central nervous system, underlining the view that glucocorticoids can control a large number of central nervous system target cells via effects on gene expression. Many nerve cell populations have been shown to contain substantial amounts of nuclear glucocorticoid receptor immunoreactivity, whereas only a low density of glial cells, in both gray and white matter, show nuclear glucocorticoid receptor immunoreactivity. Thus, in most brain areas, the major target for glucocorticoids appears to be the nerve cells. Interestingly, an inverse correlation was found in the regional density of glucocorticoid receptor-immunoreactive nerve and glial cells, suggesting that glucocorticoids may influence a brain area either via glial cells or, more frequently, via nerve cells. The results on mapping highlight the impact of glucocorticoids in areas both traditionally and not traditionally involved in stress responses. The distribution of glucocorticoid receptor immunoreactivity also emphasizes a role of glucocorticoids in the regulation of the afferent regions of the basal ganglia and the cerebellar cortex, and of both afferent and efferent layers of the cerebral cortex. Glucocorticoid receptor immunoreactivity is widely distributed over the thalamus, probably leading to modulation of activity in the various thalamocortical pathways transmittinginter alia specific sensory information to the cerebral cortex. Many unspecific afferents to the cerebral cortex are potentially regulated by glucocortoid receptors such as the noradrenaline and 5-hydroxytryptamine afferents, since their nerve cells of origin contain strong glucocorticoid receptor immunoreactivity. Eight brain regions involving sensory, motor and limbic areas were shown to have a similarity with regard to glucocorticoid receptor-immunoreactive parameters at the level of 95%. The density of glucocorticoid receptor-immunoreactive nerve cells appeared to be the main factor in determining such a very high level of similarity. Overall, our results emphasize that glucocorticoids may appropriately tune networks of different areas to obtain optimal integration and in this way improve survival of the animal under challenging conditions.

The effect of aging on the hippocampal formation of the male Brown Norway rat was studied by immunohistochemistry and measurements of the immunoreactive hippocampal cells using stereological techniques. The total estimated number of glucocorticoid receptor (GR) immunoreactive neurons of the CA1-CA2 area did not differ in the 3- and the 36-month-old rat. However, the intensity of the GR immunoreactivity was decreased in the aged animals. A gradual decrease of the immunoreactivity for the mineralocorticoid receptor was also observed in the CA1-CA2 area. In the stratum oriens and the stratum radiatum of the CA1-CA2 area the immunoreactivity for basic fibroblast growth factor (bFGF) present in the glia was found to be reduced [20,000 ± 2100 ( n = 6)] in the 36-month-old rat vs the 3-month-old rat [ 28,500 ± 4500 (n = 4) ( ∗ P = 0.05) ]. However, there was no difference in the number of glial fibrillary acidic protein immunoreactive cells of this area in these two age groups. The present findings give evidence that in the Brown Norway rat there is no loss of the neuronal population containing glucocorticoid receptors of the CA1-CA2 area during aging but suggest that aging is characterized by deficits of glially derived growth factors, such as bFGF.

The basic region/helix-loop-helix dioxin receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin). Upon ligand binding the dioxin receptor is converted from a latent, non-DNA binding form to a form that directly interacts with target genes by binding to dioxin-responsive transcriptional control elements. We have purified by conventional and DNA affinity chromatographic procedures the ligand-activated, DNA binding form of dioxin receptor to examine its architecture and functional properties. We observed that the DNA binding activity of the receptor was labile. Most notably, this activity was lost following DNA affinity purification. In complementation experiments we have identified an auxiliary factor(s) that exhibited very poor, if any, intrinsic affinity for the DNA target sequence in vitro but strongly increased the DNA binding activity of the purified receptor-containing material identified by immunoblot analysis. In a similar fashion the in vitro expressed basic region/helix-loop-helix factor Arnt (that has been postulated to modulate the nuclear translocation function of the receptor) reconstituted the DNA binding function of the purified receptor, and the purified auxiliary factor reconstituted receptor activity upon addition to an extract from mutant, Arnt-deficient hepatoma cells. Conversely, purified dioxin receptor reconstituted DNA binding activity in extracts from receptor-deficient hepatoma cells which express bona fide levels of Arnt. Interestingly, UV cross-linking studies using a BrdU-substituted DNA target sequence indicated that primarily the receptor protein was bound to DNA. Moreover, we demonstrate that purified receptor or Arnt exhibited virtually no detectable affinity for the target sequence individually but, in the presence of one another, showed a strong synergy in DNA binding activity in vitro. Importantly, simultaneous expression of the receptor and Arnt resulted in synergistic induction of gene expression in vivo. These data demonstrate that Arnt plays a central role in control of dioxin receptor function by cooperatively modulating the DNA binding activity of the receptor in vitro and dioxin-dependent transactivation in vivo.

Neurotoxicology 15(3): 621-624, 1994. In the present study we have investigated the effects of toluene exposure (80 ppm, 4 weeks, 5 day/week, 6 h/day) on the serum levels of prolactin, and elaborated our earlier findings about persistent effects of toluene exposure on apomorphine-induced (1 mg/kg, s.c.) locomotor activity. We found that the serum levels of prolactin were increased by 67% in the toluene-exposed rats, as analyzed 17 days after the last exposure. The locomotor activity counts of the control rats were not normally distributed before log-transformation, since most rats showed a low level of activity and only a few showed a very high activity level. The toluene-exposed rats showed a higher level of apomorphine-induced locomotion and motility but not rearing, as analyzed 17 days after the last exposure, whereas spontaneous locomotor activity was unaffected. These results indicate that subacute exposure to 80 ppm of toluene causes persistent impairments in dopamine-mediated neurotransmission.

The role of brain cytochrome P450 (P450) in regulating the levels of the potent anesthetic steroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP) has been investigated. By analogy with the elimination of androgen from its target tissues, we present evidence that it is 3 beta-hydroxy-5 alpha-pregnan-20-one (3 beta-OH-DHP) and not 3 alpha-OH-DHP that represents the major pathway for the formation of more polar metabolites and thus the elimination of the 5 alpha-reduced metabolites of progesterone from target tissues. No polar metabolites were formed when 3 alpha-OH-DHP was incubated with microsomal fractions prepared from rat brain, but 3 beta-OH-DHP was hydroxylated at the 6 alpha- and 7 alpha-positions. These 3 beta-diols were not formed to any detectable extent in the liver or kidney but were formed in prostate, pituitary, brain, and breast. The highest catalytic activity, 512 nmol of products formed/g of tissue/hr, was found in the prostate. The corresponding rates in the pituitary, brain, and breast were 71.9, 28.1, and 6.7 nmol/g/hr, respectively. These hydroxylations were confirmed to be P450-catalyzed reactions by solubilization of the P450 from prostate, brain, and breast microsomes and reconstitution of the catalytic activity with NADPH-P450 reductase (EC 1.6.2.4) and lipid. Because 5 alpha-androstane-3 beta,17 beta-diol (3 beta-Adiol) has been shown to be a good substrate for prostate and brain P450, competition experiments were performed to determine whether the same form of P450 is involved in the elimination of 3 beta-Adiol and 3 beta-OH-DHP in the brain. These two substrates competed with each other for metabolism in microsomal fractions and in reconstitution experiments with P450 extracted from the brain or prostate. To test the hypothesis that the hydroxylation of 3 beta-OH-DHP represents a pathway for regulation of the level of 3 alpha-OH-DHP in the brain, the effect of inhibition of the hydroxylation of 3 beta-OH-DHP on the duration of 3 alpha-OH-DHP-induced anesthesia was examined. The nonanesthetic steroid 3 beta-Adiol was used as a competitive inhibitor of the metabolism of 3 beta-OH-DHP. The duration of anesthesia upon intravenous administration of 3 alpha-OH-DHP was increased by 33% when 3 beta-Adiol was coadministered.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of growth hormone (GH) and retinoids on P4502C7 mRNA levels were investigated in cultured primary hepatocytes from normal female rats. Northern blot analysis of total nucleic acids from hepatocytes maintained in culture for 90 hr showed low basal levels of P4502C7 mRNA, which were marginally increased after continuous treatment with GH. Retinal treatment gave a slightly higher induction than GH, whereas treatment with all-trans retinoic acid alone or with GH in combination with retinol induced P4502C7 mRNA to levels about one-third of those in normal female rat liver. The effects of retinoids on P4502C7 mRNA were dependent on both the dose and type of retinoid used. All-trans retinoic acid produced a saturable dose-response curve with a 50% maximal induction of P4502C7 mRNA at 1.5 microM. The isomer 9-cis retinoic acid showed a dose-dependent activation of P4502C7 mRNA similar to that of all-trans retinoic acid. Retinol gave a 50% maximal response at approximately 5 microM. In the presence of GH, the induction of P4502C7 mRNA appeared additive to the effect of retinol at all concentrations used and to all-trans retinoic acid at concentrations up to 1 microM. As determined by a quantitative solution hybridization assay, P4502C7 mRNA levels were induced 3-fold by GH, 5-fold by retinol, and 19-fold by all-trans retinoic acid. In the presence of GH, P4502C7 was induced 8-fold by retinol, whereas the induction by saturating concentration of all-trans retinoic acid showed no significant additional effect of GH. The importance of vitamin A for the expression of P4502C7 in vivo was confirmed by the low abundance of P4502C7 mRNA in vitamin A-deficient animals as compared with vitamin A-adequate control rats. Nuclear run-on experiments performed in cultured primary hepatocytes showed that both GH and retinoic acid exert their effects at the transcriptional level. We conclude that both GH and retinoids can induce P4502C7 mRNA in rat liver hepatocytes, retinoic acid being the dominant inducer.

In the present study we have investigated the capacity of various compounds sterically related to indolo[3,2-b]carbazole to inhibit specific 2,3,7,8-tetrachloro[1,6-3H]dibenzo-p-dioxin binding in rat liver cytosol, as analyzed by electrofocusing in polyacrylamide gels. When the two nitrogen atoms of indolo[3,2-b]carbazole (IC50 = 3.6 nM) were replaced with sulfur atoms, the affinity for the specific binding sites (IC50 = 3.3 nM) was similar to that of the parent compound, whereas the affinity decreased when the two nitrogen atoms were replaced with oxygen atoms (IC50 = 29 nM). Substitution with methyl groups at positions 5 and 11 (on the nitrogens) of indolo[3,2-b]carbazole resulted in increased affinity (IC50 = 1.2 nM), compared with that of the parent compound, whereas dimethylation at the 4,10- or 2,8-positions decreased the affinity (IC50 = 19 nM and IC50150 nM, respectively). Substitution at positions 5 and 11 of indolo[3,2-b]carbazole with substituents larger than methyl, as in 5,11-diethylindolo[3,2-b]carbazole (IC50 = 8.9 nM), diacetylindolo[3,2-b]carbazole (IC50 = 11.2 nM), 5,11-dibutylindolo[3,2-b]carbazole (IC50150 nM), and 5,11-di(N,N-dimethylaminoethyl)indolo[3,2-b]carbazole (IC501500 nM), also decreased the affinity. Introduction of oxygen in, or hydroxylation of, the middle ring of indolo[3,2-b]carbazole, giving indolo[3,2-b]carbazole-6,12-quinone (IC50150 nM) or 6,12-dihydroxyindolo[3,2-b]carbazole (IC501500 nM), respectively, also lowered the affinity. We calculated the Gibbs free energy of solvation of the analogue isoquino[3,4-b]phenanthridine (IC50 = 137 nM), relative to that of dibenz[a,h]anthracene (IC50 = 2.5 nM), in water to be -6 kcal/mol by free energy perturbation, which indicates that the most important explanation for the observed difference in binding affinity is the smaller difference in relative free energy of binding at the binding sites, compared with the Gibbs free energy of solvation of the two compounds.

Glucocorticoids stimulate the transcription of rat alpha 2u-globulin (RUG) genes. Because this induction occurs after a time lag of several hours and is blocked by inhibitors of protein synthesis, it exemplifies a delayed secondary response to steroid hormones. In this report, we show that a region of RUG-transcribed DNA (approximately +1800 to +2174) contains multiple footprint sites for glucocorticoid receptor that are, apparently, organized into at least three independent binding clusters. The DNA sequences bound by the receptor and the location of binding sites were determined. A family of sequences related to half-sites of the consensus primary glucocorticoid response element (GRE) is discernible at each cluster of sites. Compared to the consensus GRE, which contains two pseudo-palindromic hexanucleotides arranged in a tail-to-tail fashion and separated by three bases, the arrangements of hexanucleotides within this segment of RUG DNA are unusual and heterogeneous. Methylation interference of a binding cluster containing three receptor footprints demonstrates that certain guanines of the GRE-like hexanucleotides are essential for efficient receptor binding. A synthetic 29-base pair (bp) RUG element, containing one receptor footprint from this cluster, selectively binds the receptor. Within this 29-bp element, six nucleotides separate two directly repeated copies of GRE-like hexanucleotides. RUG DNA fragments containing all or part of the three binding clusters, including the 29-bp element, confer a delayed secondary hormone responsiveness upon a linked heterologous promoter and reporter gene in stably transfected cell lines. We speculate that the unusual DNA sequence motifs of the receptor-binding sites are crucial for the generation of certain delayed secondary responses.

Two protein fragments containing the DNA-binding domain (DBD) of the glucocorticoid receptor (GR) have been studied by two-dimensional 1H NMR spectroscopy. The two peptides (93 and 115 residues, respectively) contain a common segment corresponding to residues C440-I519 of the rat GR or residues C421-I500 of the human GR and include two Zn-binding "finger" domains. The structures of this segment are almost identical in the two protein fragments, as judged from chemical shifts and sequential NOE connectivities. More than 90% of all observable 1H resonances within a 71-residue segment encompassing C440-R510 (rat GR) could be sequentially assigned by standard techniques, and stereospecific assignments could be made for the methyl groups in four valine residues within this segment. Sequential NOE connectivities indicate several elements of secondary structure including two alpha-helical segments consisting of residues S459-E469 and P493-G504, a type I reverse turn between residues R479 and C482, a type II reverse turn between residues L475 and G478, and several regions of extended peptide conformation. No evidence for alpha-helical conformation was found within the two putative zinc-finger domains, indicating that the structures of these domains differ from that of TFIIIA-type zinc fingers. The observation of some very slowly exchanging amide protons in the N-terminal (CI) domain of the DBD in combination with slow rotation of the Y452 aromatic ring indicates that this domain has a restricted conformational flexibility compared to the C-terminal (CII) domain. We also observe several long-range NOE connectivities within C440-R510, suggesting that the sequential assignments presented here will provide a basis for a complete structure determination of this segment of the GR.

Glucocorticoid receptor-immunoreactive nerve cells have been analysed in the dorsal striatum and nucleus accumbens of the rat by means of a monoclonal antibody against rat liver glucocorticoid receptor. Glucocorticoid receptor immunoreactivity was present in the nuclei of the vast majority of the striatal nerve cells. The analysis of sections stained with glucocorticoid receptor antibody and cresyl violet showed that around 90% of the entire striatal neuronal population contained glucocorticoid receptor immunoreactivity. By means of the double immunoperoxidase technique evidence was provided that somatostatin- and choline acetyltransferase-immunoreactive nerve cells in the striatum do not contain glucocorticoid receptor immunoreactivity. The density of glucocorticoid receptor-immunoreactive nerve cells in the grey matter and the presence of clusters of glucocorticoid receptor-immunoreactive nerve cells have been investigated in three fields located in the medial and central dorsal striatum and nucleus accumbens at the coronal level A 8620 microns according to the König and Klippel atlas using computer-assisted image analysis. Every aggregate containing three or more glucocorticoid receptor-immunoreactive nerve cells, which had an intercenter distance less than the mean diameter (10-11 microns) of the striatal cells, was considered an island. A higher density of both glucocorticoid receptor-immunoreactive nerve cell nuclei and islands was found in the nucleus accumbens with respect to dorsal striatal areas. The most frequent island formed consisted of three to ten nerve cells both in dorsal striatum and nucleus accumbens. Furthermore, some nucleus accumbens islands contained up to 100 nerve cells, whereas in the dorsal striatum the maximum number of glucocorticoid receptor-immunoreactive nerve cells per island ranged from 50 to 60. The present procedure proved to be a sensitive method to reveal clusters of chemically identified structures and provided evidence for a basic cytoarchitectonic organization of the dorsal striatum and nucleus accumbens of the rat. This paper also demonstrated that the vast majority, but not all, striatal nerve cells contained glucocorticoid receptor immunoreactivity, and thus may be under the control of circulating glucocorticoids. In fact, only small transmitter-identified neuronal populations, such as somatostatin- and choline acetyltransferase-immunoreactive nerve cells, were devoid of glucocorticoid receptor immunoreactivity.

Certain metabolites of polychlorinated biphenyls (PCBs) are retained in the Clara cells and in the airway lumen of rodent lung due to their interaction with a secretory 13-kDa protein. Here, we report the isolation of a cDNA encoding the rat lung PCB-binding protein. The identity of the PCB-binding protein is supported by expression of the cDNA in Cos-1 cells where the homogenates from transfected cells show specific binding of 4,4'-bis([ 3H]methylsulfonyl)-2,2',5,5'-tetrachlorobiphenyl, a high affinity ligand for the PCB-binding protein. Also a monospecific antiserum to the PCB-binding protein recognizes a 13-kDa protein in the homogenates of transfected cells but not in the corresponding fraction of mock-transfected cells. Northern blot analysis of total RNA from different rat tissues demonstrates that the cDNA detects a approximately 600-base pair mRNA which appears to be solely expressed in lung. Interestingly, DNA sequence analysis and prediction of the amino acid sequence reveals that the PCB-binding protein shares 53% positional amino acid identity with uteroglobin, a progesterone-binding protein found in rabbit uterus and lung. Furthermore, amino acids shown by x-ray crystallography to delineate the central cavity of uteroglobin, which fits progesterone, are highly conserved in the two proteins.

Marked sex differences in the growth of enzyme-altered hepatic foci have been observed in rats treated according to the "resistant hepatocyte model." The present study was performed to investigate the effect of neonatal and adult castration of male rats, with or without testosterone substitution, on the growth rate of foci during selection of initiated cells with 2-acetylaminofluorene and partial hepatectomy. Neonatal castration of male rats decreased focal growth to the same level as in female rats. Castration of adult male rats 2 wk before initiation with diethylnitrosamine also decreased the growth rate of foci, but less markedly than in neonatally castrated rats. Testosterone substitution of male rats castrated as neonates or as adults, from 10 days after initiation with diethylnitrosamine, restored focal growth to that of sham-castrated controls. Previous investigations concerning the role of gonadal hormones in sex differentiation of various liver functions indicated a role of the hypothalamo-pituitary-liver axis in mediating the effects of androgens. It is therefore also suggested that the effects of androgens on early steps of hepatocarcinogenesis observed in the present study are mediated by similar mechanisms, possibly through an influence on the metabolism of 2-acetylaminofluorene.

The activation in vitro of dioxin and glucocorticoid receptors from a non-DNA binding to a DNA binding state was characterized. Ligand-free dioxin and glucocorticoid receptors were partially co-purified from rat liver cytosol, and both receptors sedimented at 9 S following labeling with the respective ligand. The 9 S forms of the dioxin and glucocorticoid receptors have previously been shown to represent heteromeric complexes containing the Mr approximately equal to 90,000 heat shock protein. The 9 S ligand-free or ligand-bound glucocorticoid receptor was converted to the monomeric 4-5 S form upon exposure to 0.4 M NaCl even in the presence of the stabilizing agent molybdate. Under identical conditions, the 9 S ligand-free and ligand-bound dioxin receptor forms remained essentially intact. However, in the absence of molybdate, the dioxin receptor could be converted to a 4-5 S form upon exposure to high concentrations of salt. These results indicate that the glucocorticoid receptor readily dissociates from the 9 S to the 4-5 S form even in the absence of hormone, whereas both the ligand-free and ligand-occupied 9 S dioxin receptor forms represent more stable species. Gel mobility shift experiments revealed that the 4-5 S glucocorticoid receptor interacted with a glucocorticoid response element both in the absence and presence of ligand. On the other hand, occupation of the dioxin receptor by ligand greatly enhanced the ability of the receptor to be activated to a form that binds to its target enhancer element. Once dissociated, the monomeric form of the dioxin receptor was also able to interact with its DNA target sequences even in the absence of ligand. Thus, ligand binding efficiently facilitates subunit dissociation of the dioxin receptor but is not a prerequisite for DNA binding per se. Given the apparent stability of its non-DNA binding 9 S form, the dioxin receptor system might be a useful model for the investigation of the mechanism of activation of soluble receptor proteins.

The glucocorticoid receptor belongs to a family of ligand activated nuclear receptors. In addition to steroid hormone receptors, this family also includes receptors for thyroid hormone, retinoic acid and 1,25-dihydroxyvitamin D3 as well as some receptors with as yet unknown ligands, the so called orphan receptors. It is possible that the dioxin receptor is a member of this family as well although this cannot be definitely assessed until the receptor has been cloned. Interestingly, the clofibrate receptor, believed to be involved in clofibrate induction of certain isozymes of cytochrome P-450, has recently been shown to be a member of the steroid receptor supergene family and it is conceivable that other P-450 inducers might act via as yet uncharacterized receptors belonging to the same gene family. Therefore, we should learn significantly about principles of P-450 regulation by considering basic mechanisms of glucocorticoid action; the glucocorticoid receptor probably represents the most extensively studied member of the nuclear receptor family. Our laboratory has been particularly interested in structural aspects of this receptor and has used gene technology to overexpress its different domains, particularly the DNA-binding domain, the structure of which has been resolved using NMR.

Subacute treatment with toluene (80-1500 p.p.m.) produces a dose-dependent reduction of affinity and increase in density of the beta-adrenergic antagonist [3H]dihydroalprenolol binding sites in the frontoparietal cortex of the male rat, while the binding characteristics of alpha 1-adrenergic ([3H]WB 4101) and alpha 2-adrenergic ([3H]p-aminoclonidine) binding sites in the same region is unaffected by this treatment as evaluated in vitro. Therefore, it is suggested that the cortical beta-adrenergic receptors are particularly vulnerable to the action of toluene in vivo. It is speculated that as a result cortical beta-adrenergic neurotransmission may be altered following exposure to low concentrations of toluene, possibly related to the physico-chemical properties of toluene, leading to changes in membrane fluidity.

The activated glucocorticoid receptor (GR) from rat liver cytosol was purified by sequential chromatography on DNA-cellulose and DEAE-Sepharose. Analysis by sodium dodecyl sulfate-gel electrophoresis demonstrated a main band with Mr = 94,000 (94K band). Two minor bands with Mr = 79,000 (79K band) and 72,000 (72K band) were also seen in this preparation. Photoaffinity labeling showed that the hormone is bound to the 94K and 79K components but not to the 72K component. Immunoblotting using antibodies raised against the 94K protein demonstrated cross-reactivity between the 94K and 79K components but not with the 72K species. The 72K species could be partially separated from the 94K and 79K components by density gradient centrifugation. Limited proteolysis of the purified GR with trypsin or alpha-chymotrypsin led to degradation of the 94K and 79K components and appearance of a 39K fragment which still retained the hormone and could be bound to DNA-cellulose. The 72K component was not affected by digestion with trypsin or alpha-chymotrypsin. However, chromatography on DNA-cellulose of the alpha-chymotrypsin-treated GR resulted in elution of the 72K component in the flow-through of the column while the 39K fragment was retained on the column and eluted with 0.18 M NaCl. In the control experiment where no alpha-chymotrypsin treatment was performed, the 72K component could not be detected in the flow-through fraction but was eluted together with the 94K and 79K components at 0.18 M NaCl. These results suggest that the 72K protein might be bound to the 94K and/or 79K component. The 39K fragment did not bind antibodies raised against the 94K protein. The 39K fragment was further degraded by trypsin but not by alpha-chymotrypsin to a 27K and a 25K fragment while both still retained the ligand. These data obtained with limited proteolysis of the purified GR are in agreement with previous findings on proteolysis of the GR in crude cytosol (Wrange, O., and Gustafsson, J.-A. (1978) J. Biol. Chem. 253, 856-865; Carlstedt-Duke, J., Okret, S., Wrange, O., and Gustafsson, J.-A. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4260-4264).

By means of monoclonal antibodies against the rat liver glucocorticoid receptor (GR) and the human estrogen receptor (ER), in combination with an immunocytochemical analysis, it has been possible to map out GR and ER immunoreactive (IR) neurons in the rat central nervous system and GR IR glial cells in the white matter. The GR IR is located in the cytoplasm and especially in the nucleus while the ER IR is only demonstrated in the nuclei of the neurons. Upon adrenalectomy the GR IR appears to be present exclusively in the cytoplasm, while after castration the ER IR is still exclusively present in the nuclei. Upon corticosterone treatment of the adrenalectomized rat the GR IR is again predominantly found in the nuclei of the neurons. These results indicate that the occupied GR and the unoccupied and occupied ER are located in the nuclei and the unoccupied GR in the cytoplasm. Evidence has been presented that large numbers of monoamine and peptide nerve cell bodies contain GR IR. Furthermore, neuronal GR IR is found in neuronal populations all over the central nervous system, especially in the cerebral cortex, the thalamus and the hypothalamus, indicating a major role of GR in regulating the metabolic and synaptic functions of the brain. The ER IR is instead limited to certain neuronal populations, mainly those of the preoptic area, the bed nucleus of the striae terminalis and the arcuate nucleus, suggesting a specific role in control of LHRH secretion and reproductive behaviour.

Subacute exposure to high concentrations (500--1000 ppm) of toluene vapour led to an increase of noradrenaline levels in the subependymal layer of the median eminence (SEL) and to an increase of noradrenaline turnover within the subependymal layer, the paraventricular hypothalamic nucleus and the anterior periventricular hypothalamic nucleus (PV I). Increased dopamine levels in the lateral palisade zone of the median eminence (LPZ) and increased catecholamine turnover within the medial palisade zone (MPZ) were also produced. Measurement of the anterior pituitary hormone secretion showed a significant increase of FSH and delayed increase of corticosterone secretion following toluene exposure.

After a single oral dose of 2-nitro[9-14C]fluorene (NF) to germfree and conventional rats, radioactivity with associated mutagenic activity was rapidly excreted in both urine and feces. The mutagenicity excreted from germfree animals exceeded the mutagenicity excreted from conventional animals. Absence of the microflora resulted in lack of nitroreduction and excreted metabolites from germfree rats were mono- or dihydroxylated nitrofluorenes, as assessed by liquid chromatography/mass spectrometry (LC/MS). These hydroxy-NFs were associated with the high direct-acting mutagenicity that was excreted in germfree animals. Only traces of NF were found in urine from germfree rats and no NF was detected in urine from conventional animals. Thus, in contrast to a number of reports on other mutagenic compounds, these results would tend to indicate that the intestinal microflora causes a reduced excretion of mutagenic metabolites of NF.

The effects of chronic toluene exposure (CTE) (80 ppm, 6 h/day, 5 days/week, 3 months) were studied on neuropeptide and 5-hydroxytryptamine receptors, on protein phosphorylation levels and on catecholamine levels in various brain regions in the 15-month-old male rat. Behavioral parameters and serum levels of hypophyseal hormones and corticosterone were also analyzed. CTE selectively reduced [ 3 H]neurotensin ([ 3 H]NT) binding in the basal layers of the orbital cortex. Instead, CTE increased the binding of [ 3 H]etorphine in the nucleus accumbens and of [ 125 I]vasoactive intestinal polypeptide ([ 125 I]VIP) in the area postrema and hypoglossal nucleus. Acute treatment with the irreversible monoamine receptor antagonist N -ethoxycarboxyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) increased the binding of [ 3 H]NT in the orbital cortex in toluene exposed rats as compared with the reduced [ 3 H]NT binding obtained in air exposed rats treated with EEDQ. Furthermore, the EEDQ induced increase in [ 125 I]VIP binding in the area postrema and the hypoglossal nucleus was replaced by a reduced binding of [ 125 I]VIP in EEDQ-treated CTE rats. CTE produced an overall increase in calcium-induced back phosphorylation and an overall decrease in cyclic adenosine monophosphate-induced back phosphorylation in the frontoparietal cortex. Noradrenaline stores tended to be reduced within various hypothalamic subnuclei and the serum prolactin levels were increased following CTE. However, no marked effects of CTE were seen on the behavioral parameters. In conclusion, the regional selectivity of CTE in disturbing [ 3 H]NT and [ 125 I]VIP binding may be due to the demonstrated vulnerability of monoamine-neuropeptide interactions to toluene.

2-Nitrofluorene (NF), a model substance for nitrated polycyclic aromatic hydrocarbons, is present in exhaust from diesel- and petrol-driven vehicles, in exhaust from kerosene heaters, and in urban air and river sediments. Therefore the possible consequences of human exposure are important to elucidate. In the present study the initiating and promoting activity of NF was studied in a liver model for chemical carcinogenesis in the rat. Furthermore, the in vivo metabolism and formation of genotoxic metabolites of NF were investigated. NF was found to be a moderate initiator and a weak promoter when compared to diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) respectively, both of which are potent carcinogens. Animals given NF excreted more urinary mutagenicity when compared to animals given AAF or DEN.

Anterior hypothalamic deafferentation and infusion of human GH (hGH) in the normal male rat caused a marked reduction in the hepatic concentration of α2u-globulin, an androgen-dependent protein. Although s.c. injections of hGH (twice-daily) resulted in more than a 50% reduction in the hepatic level of α2u-globulin, the same dose of hGH when administered continuously through osmotic minipumps caused a threefold greater inhibition. The decreased hepatic concentration of α2u-globulin after hGH administration was associated with corresponding changes in the hepatic level of translatable α2u-globulin messenger RNA. Continuous infusion of hGH through osmotic minipumps and removal of the anterior hypothalamic influence on GH secretion by deafferentation also caused a marked reduction in the cytoplasmic androgen-binding activity of the rat liver. These results suggest that alterations in the level and pattern of GH secretion may influence hepatic androgen-binding activity and α2u-globulin synthesis. J. Endocr. (1986) 108, 351–355

Subacute exposure of male rats to various concentrations (70-1000 ppm) of dichloromethane (DCM) produces a selective reduction of dopamine (DA) levels without a change of DA turnover in certain types of forebrain DA nerve terminal systems. In the low concentration (70 ppm) a selective reduction of DA turnover was observed in the medial palisade zone (MPZ) of the median eminence. This chlorinated organic solvent also produced a discrete dose-dependent increase of noradrenaline (NA) turnover within the anterior periventricular hypothalamic area and with the highest concentration an increase of NA turnover in the anteromedial frontal cortex. DCM reduced NA levels dose-dependently in the posterior periventricular hypothalamic area and also reduced NA levels in the dorsomedial hypothalamic nucleus (1000 ppm). Following tyrosine hydroxylase inhibition DCM produced an inversely dose-related increase of serum LH levels and at the highest concentration an increase of ACTH secretion was observed. It is suggested that DCM can produce discrete changes in amine storage and turnover in catecholamine (CA) nerve terminal systems of the tel- and diencephalon, some of which may contribute to the DCM induced disturbances of the secretion of anterior pituitary hormones but actions on other transmitter-identified neurons involved in neuroendocrine regulation must also be considered.

The tissue distribution of [3H]estramustine, the dephosphorylated metabolite of estramustine phosphate (Estracyt), in the male rat was compared to that of [3H]estradiol 30 min and 2 hr following i.p. administration. In contrast to estradiol, estramustine was found to be efficiently concentrated in the ventral prostate gland by a soluble protein. The binding characteristics of this protein were studied in vitro using cytosol preparations of the gland. With a dextran-coated charcoal technique, the protein was found to bind estramustine with a broad pH optimum between pH 7 and pH 8.5, with an apparent Kd of 10 to 30 nM, and with a binding capacity of about 5 nmol/mg cytosol protein. The estramustine/protein complex was not retained by DNA-cellulose. None of the natural steroids tested inhibited the binding of 10 nM [3H]estramustine by more than 35% (progesterone), even when added in 4500-fold excess. The presence of a nitrogen mustard moiety at position 3 of the steroid was necessary for high-affinity binding to the protein. The protein was calculated to constitute about 20% of the total cytosol protein content.

The mechanism of regulation of glucocorticoid receptor (GR) expression by cAMP was investigated in rat hepatoma cells (HTC). Incubation of HTC cells with the cAMP-inducing agent, forskolin, caused a significant increase in the levels of both [3H]dexamethasone binding capacity and GR mRNA by about 2- to 2.4-fold within 4 h. Incubation of HTC cells with the cAMP analogue, 8-bromo-cAMP, also increased the GR mRNA level to a similar degree in a concentration-dependent manner. The increase in GR mRNA did not require ongoing translation or transcription. Determination of GR mRNA stability in 8-bromo-cAMP-induced cells showed that the message had a half-life of approximately 10 h, which is about 2.5 times longer than the GR mRNA half-life in nontreated cells (t1/2 = 4 h). These results indicate that the increased steady state level of GR mRNA induced by cAMP analogue is, at least in part, caused by increased GR mRNA stability. In both forskolin-pretreated and nontreated HTC cells, dexamethasone caused an approximately 70% down-regulation of GR protein levels. However, since forskolin induced the GR level 2- to 2.4-fold, the relative amount of GR protein remaining in cells treated with both forskolin and dexamethasone was about 2- to 2.4-fold higher compared to cells treated with dexamethasone alone. This increased GR level correlated well with the increase in inducibility of two glucocorticoid regulated genes, the endogenous tyrosine aminotransferase and the stably integrated mouse mammary tumor virus. These data suggest that relatively small changes in GR levels are reflected in parallel changes in cellular response to glucocorticoid hormones. This also implicates a limiting nature of the GR protein in determining the biological response.

The effects of ganglioside GM1 treatment (10 mg/kg, i.p., 3 days, once daily) were analyzed on N-[3H]propylnorapomorphine ([3H]NPA) binding characteristics in striatal membrane preparations from air or toluene (80 ppm, 3 days, 6 h/day) exposed adult male rats. It was found that toluene exposure induced increased Kd and Bmax values for [3H]NPA binding. These changes could be blocked by GM1 treatment, which by itself had no significant effects on the binding characteristics of [3H]NPA. The addition of toluene (9.3 mumol/ml) in vitro had similar effects on [3H]NPA binding characteristics as toluene exposure in vivo. These results indicate that the effects of toluene exposure on dopamine receptors are due to an alteration of membrane fluidity which is prevented by GM1 treatment.

Nicotine treatment of normal male rats and of ovariectomized female rats results in increases of dopamine (DA), noradrenaline (NA) and adrenaline (A) turnover in the hypothalamus, especially within the median eminence. These effects may in part mediate the inhibition of gonadotrophin and TSH secretion produced by nicotine.

Sex differences exist in the metabolism of histidine, drugs and steroids by the liver. These differences are controlled, at least in the rat, by the hypothalamo-pituitary axis. Histidase is controlled by growth hormone, giving an increase in the male to the female level. Drug and steroid metabolism are controlled directly from the pituitary in the female rat by means of an unidientified hormone designated "feminizing factor". In the male rat, androgens derived from the testes and adrenals are involved in the regulation of liver function, probably by a feedback action on the pituitary secretion of feminizing factor. The thyroid glands are involved in the control of liver function in both males and females. Regulation of lactogenic receptors in rat liver is very similar to that of drug and steroid metabolism. The secretion of feminizing factor is regulated by the hypothalamus and probably involves higher centres, notably the amygdala.