5-HT2A receptors stimulate ACTH, corticosterone, oxytocin, renin, and prolactin release and activate hypothalamic CRF and oxytocin-expressing cells.

The 5-HT(2A/2C) agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI) stimulates hypothalamic neurons to increase the secretion of several hormones. This study addressed two questions: 1) are the neuroendocrine effects of DOI mediated via activation of 5-HT(2A) receptors; and 2) which neurons are activated by 5-HT(2A) receptors. The 5-HT(2A) antagonist (+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol (MDL 100,907; 0.001, 0.01, or 0.1 mg/kg, s.c.) was administered before rats were challenged with DOI (2.5 mg/kg, i.p.). MDL 100,907 produced a dose-dependent inhibition (ED(50) congruent with 0.001 mg/kg) of the effect of DOI on plasma levels of ACTH, corticosterone, oxytocin, prolactin, and renin without altering basal hormone levels. Complete blockade of the effect of DOI was achieved for all hormones at MDL 100,907 doses of 0.01-0.1 mg/kg. In a parallel experiment, DOI was injected 2 hr before killing to determine its effects on the expression of Fos, the product of the immediate early gene c-fos. DOI induced an increase in Fos immunoreactivity in corticotropin-releasing factor (CRF) and in oxytocin-expressing neurons but not in vasopressin-containing neurons in the hypothalamic paraventricular nucleus or CRF cells in the amygdala. Pretreatment with MDL 100,907 (0.1 mg/kg, s.c.) blocked the DOI-induced increase in Fos expression in all regions including the hypothalamus, amygdala (central and corticomedial), bed nucleus of the stria terminalis, and prefrontal cortical regions. The combined neuroanatomical and pharmacological observations suggest that the neuroendocrine responses to DOI are mediated by activation of neurons in the hypothalamic paraventricular nucleus and associated circuitry. Furthermore, selective activation of 5-HT(2A) receptors mediates the hormonal and Fos-inducing effects of DOI.

Coadministration of 5-hydroxytryptamine(1A) antagonist WAY-100635 prevents fluoxetine-induced desensitization of postsynaptic 5-hydroxytryptamine(1A) receptors in hypothalamus.

Treatment with selective serotonin reuptake inhibitors induces a desensitization of hypothalamic postsynaptic 5-hydroxytryptamine (5-HT)(1A) receptors in humans and rats. This study investigated whether fluoxetine-induced desensitization is due to overactivation of postsynaptic 5-HT(1A) receptors; whether blockade of somatodendritic 5-HT(1A) autoreceptors accelerates this desensitization; and whether desensitization is associated with a reduction of Gz proteins, which couple to 5-HT(1A) receptors. WAY-100635 was tested at low doses (0.03-0.3 mg/kg), which antagonize somatodendritic 5-HT(1A) autoreceptors in the raphe nuclei, and at a higher dose (1 mg/kg), which completely blocks postsynaptic 5-HT(1A) receptors. Plasma levels of oxytocin and adrenal corticotrophic hormone (corticotropin) were measured as peripheral indicators of hypothalamic 5-HT(1A) receptor function. Daily injections of fluoxetine (10 mg/kg/day i.p.) for 2 days did not desensitize 5-HT(1A) receptors but three daily injections of fluoxetine produced a partial desensitization of the hormone responses to (+/-)-8-hydroxy-2-dipropylaminoetetralin (50 microg/kg s.c.). WAY-100635 (0.03-0.3 mg/kg) did not accelerate or potentiate the fluoxetine-induced desensitization of 5-HT(1A) receptors. However, WAY-100635 at a dose that completely blocks postsynaptic 5-HT(1A) receptors (1.0 mg/kg) completely prevented the fluoxetine-induced desensitization of 5-HT(1A) receptors. These data demonstrate that at least 3 days of fluoxetine exposure is required to produce a homologous desensitization of hypothalamic 5-HT(1A) receptors. Although previous studies indicate that injections of fluoxetine for 14 days produce a reduction of Gz protein levels in the hypothalamus, the levels of Gz proteins were not affected by either fluoxetine or WAY-100635. Alternative mechanisms mediating the initial stages of 5-HT(1A) receptor desensitization could involve post-translational modifications in the 5-HT(1A) receptor-Gz protein-signaling cascade.

Evidence that G(z)-proteins couple to hypothalamic 5-HT(1A) receptors in vivo.

Using in situ hybridization and immunoblot analysis, the present studies identified G(z) mRNA and G(z)-protein in the hypothalamic paraventricular nucleus. The role of G(z)-proteins in hypothalamic 5-HT(1A) receptor signaling was examined in vivo. Activation of 5-HT(1A) receptors increases the secretion of oxytocin and ACTH, but not prolactin. Intracerebroventricular infusion (3-4 d) of G(z) antisense oligodeoxynucleotides, with different sequences and different phosphorothioate modification patterns, reduced the levels of G(z)-protein in the hypothalamic paraventricular nucleus, whereas missense oligodeoxynucleotides had no effect. Neither antisense nor missense oligodeoxynucleotide treatment altered basal plasma levels of ACTH, oxytocin, or prolactin, when compared with untreated controls. An antisense-induced decrease in hypothalamic G(z)-protein levels was paralleled by a significant decrease in the oxytocin and ACTH responses to the 5-HT(1A) agonist 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT). In contrast, the prolactin response to 8-OH-DPAT (which cannot be blocked by 5-HT(1A) antagonists) was not inhibited by G(z) antisense oligodeoxynucleotides. G(z)-proteins are the only members of the G(i)/G(o)-protein family that are not inactivated by pertussis toxin. In a control experiment, pertussis toxin treatment (1 microgram/5 microliter, i.c.v.; 48 hr before the 8-OH-DPAT challenge) did not inhibit the ACTH response, potentiated the oxytocin response, and eliminated the prolactin response to 8-OH-DPAT. Thus, pertussis toxin-sensitive G(i)/G(o)-proteins do not mediate the 5-HT(1A) receptor-mediated increase in ACTH and oxytocin secretion. Combined, these studies provide the first in vivo evidence for a key role of G(z)-proteins in coupling hypothalamic 5-HT(1A) receptors to effector mechanisms.

[Plasma tryptophan bioavailability during chronic urticaria]. / Biodisponibilité plasmatique du tryptophane au cours de l'urticaire chronique.

OBJECTIVES: Chronic idiopathic urticaria is known to have psychogenic component with a triggering or favoring effect. Different tests or evaluation scales have been unable to identify a specific psychological profile. Erythrocyte-specific membrane transport of tyrptophan (TRP), the main plasma precursor of cerebral serotonin synthesis, controls, by a erythrocyte-specific storage and release mechanism, circulating TRP homeostasis. Bioavailability of circulating TRP is a factor controlling serotonin synthesis in the brain. An evaluation of the rate of TRP transfer could be a biochemical approach to chronic urticaria more informative than psychological tests. PATIENTS AND METHODS: A kinetic study of L-TRP influx into circulating erythrocytes was conducted in 17 patients with chronic urticaria with no detectable cause and in 35 healthy controls. Blood samples were marked with 3H-TRP. Maximum L-TRP-specific influx (Vmax) was expressed in mumol/cell/min. The urticaria patients also underwent psychological testing to determine anxiety and depression scores using standardized scales (Hamilton). RESULTS: Mean Vmax was not significantly difference between the two groups. Vmax values were quite similar in all the control subjects but showed wide dispersion in the urticaria group. Three subgroups were found in the urticaria patients depending on Vmax: those with Vmax equivalent in control levels (+2 SD), those with Vmax less then 2 SD (29% of the patients) and those with Vmax greater than 2 SD of control levels (23% of the patients). Thus more than 50% of the urticaria patients had perturbed erythrocyte-specific L-TRP influx. The anxiety and depression scores obtained from the psychological evaluation were not correlated with Vmax. DISCUSSION: Erythrocyte-specific TRP membrane transport, evaluated by Vmax. Would not appear to be perturbed in chronic urticaria. Even though the urticaria patients could be divided into three groups according to their Vmax, the mean value was not significantly different from that in controls. These findings do not allow a conclusion concerning a perturbation of bioavailability of plasmatic TRP and any possible central serotoninergic dysfunction in chronic urticaria.

Environmental monitoring for genotoxicity with plant systems. An introduction and study design.

Under the sponsorship of the International Programme on Chemical Safety (IPCS), 17 laboratories from diverse regions of the world participated in evaluating the utility of four plant bioassays for detecting genetic hazards of environmental chemicals. The bioassays included in this collaborative study were: Arabidopsis thaliana embryo and chlorophyll assay and Tradescantia stamen hair assay, Tradescantia paludosa micronucleus assay and Vicia faba root tip assay. Four to six laboratories participated in the performance of each of the bioassays. All laboratories participating in a particular bioassay were supplied with uniform plant material as well as standardized protocol. Five direct acting water soluble test chemicals, i.e. maleic hydrazide, methyl nitrosourea, ethyl methanesulfonate, sodium azide and azidoglycerol, were selected for this study. The study was designed to be completed in three phases. Ethyl methanesulfonate was used as a positive control and has already been reported earlier (Sandhu et al., 1991). The data from the remaining four chemicals used for the evaluation of four plant test systems in the first phase of the collaborative study are reported in this issue.

Environmental monitoring for genotoxicity with plant systems. Results and recommendations.

In the first phase of a collaborative study by the International Programme on Chemical Safety (IPCS), four coded chemicals, i.e. azidoglycerol (AG, 3-azido-1,2-propanediol), methyl nitrosourea (MNU), sodium azide (NaN3) and maleic hydrazide (MH), and ethyl methanesulfonate (EMS) as a positive control were tested in four plant bioassays, namely the Arabidopsis embryo and chlorophyll mutation assay, the Tradescantia stamen hair assay (Trad-SH assay), the Tradescantia micronucleus assay (Trad-MCN), and the Vicia faba root tip assay. Seventeen laboratories from diverse regions of the world participated with four to six laboratories each using one plant assay. For the Arabidopsis assay, laboratories were in agreement with MNU and AG giving positive responses and NaN3 giving a negative response. With the exception of one laboratory which reported MH as weakly mutagenic, no mutagenic response was reported for MH by the other laboratories. For the Vicia faba assay, all laboratories reported a positive response for MNU, AG, and MH, whereas two of the six laboratories reported a negative response for NaN3. For the Trad-SH assay, MH was reported as giving a positive response and a positive response was also observed for MNU with the exception of one laboratory. NaN3, which exhibited a relatively high degree of toxicity, elicited a positive response in three of the five laboratories. AG was found positive in only one of the two laboratories which tested this chemical. For the Trad-MCN assay, MNU and MH were reported as positive by all laboratories, while four out of five laboratories reported NaN3 to be positive. Only one of three laboratories reported AG to be positive. The major sources of variability were identified and considered to be in the same range as found in similar studies on other test systems. Recommendations were made for minor changes in methodology and for initiating the second phase of this study.

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. XII. Analysis of multiple-locus ad-3 mutations reveals a nonrandom distribution of the separate sites of recessive lethal damage throughout the genome.

Previous studies on X-ray-induced adenine-3 mutations induced in heterokaryon 12 of Neurospora crassa showed that they consisted of gene/point mutations, multilocus deletion mutations, and 3 different genotypic classes of multiple-locus mutations (designated [-3]IR + RLCL, ad-3R + RLCL, and ad-3R + RL). In the present paper, multiple-locus mutations consisting of gene/point mutations at the ad-3A or the ad-3B locus with sites of recessive lethal damage closely linked to the ad-3 region (designated ad-3R + RLCL) or with sites of recessive lethal damage elsewhere in the genome (designated ad-3R + RL) were analyzed to determine whether they resulted from mutations at the same sites or different sites throughout the genome. It was assumed that if the recessive lethal mutations in individual multiple-locus mutations showed complementation on adenine-supplemented medium, they resulted from mutations at different sites. Multiple-locus mutations from both major genotypic classes were combined, as forced heterokaryons, in all possible pairwise combinations and then were plated out on adenine-supplemented medium. These studies indicated that 89.3% (50/56) of the recessive lethal mutations in these 2 classes of multiple-locus mutations complement one another. Thus, they are presumed to have resulted predominantly from genetic damage at different sites throughout the genome. Within the group of 20 multiple-locus mutations that did not complement in various pairwise combinations, 90% (18/20) appear to map in a region, distal to the ad-3 region, defined by a series of overlapping multilocus deletion mutations in 6 mutations of genotype ad-3R + RLCL. The other 10% (2/20) are located elsewhere on Linkage Group I or elsewhere in the genome. The present data base on multiple-locus mutations is unique; such events either can not be detected, or can only be detected with difficulty, in other eukaryotic specific-locus assay systems such as mammalian cells in culture, Drosophila or mice. Our data on X-ray-induced ad-3 specific-locus mutations from the present and previous studies demonstrate the presence of additional sites of genetic damage, both closely linked with the ad-3 region or elsewhere in the genome, in ad-3 specific-locus mutations. Because the frequencies of each class of multiple-locus mutations is dose-dependent, they must be taken into account in genetic risk assessment exercises. Failure to acknowledge the presence of such additional sites of genetic damage in the utilization of specific-locus data could result in underestimation of the risk of human exposure to environmental mutagens.

Mechanisms to stimulate research on assay systems to detect aneuploidy.

The present database on the induction of aneuploidy is inadequate to make comparisons between effects on lower eukaryotic and higher eukaryotic organisms. There is also an urgent need to develop new assays to detect the induction of aneuploidy in animals, as well as assays in lower eukaryotic organisms which can be used in mass-screening programs. The development of chemical and data repositories of chemicals that induce aneuploidy with known activity is proposed as a mechanism to stimulate research in this area. An efficient mechanism to develop a comprehensive database on particular assay systems and a defined set of chemicals is an international Collaborative Trial developed under and sponsored by the International Program on Chemical Safety at the World Health Organization. IPCS is sponsored by voluntary contributions from those member countries who consider such collaborative studies of importance for the development of methodology that can be used to evaluate the mutagenic and carcinogenic potential of environmental chemicals. Exposure to natural and manmade chemicals can be unique, and member countries cannot necessarily assume that chemicals to which native populations will be exposed will be tested elsewhere. The development of an International Collaborative Trial on chemicals that induce aneuploidy and a wide range of eukaryotic assay systems provide an excellent mechanism for the development of a comprehensive database. Within 2-3 years these new data could help to resolve some of the numerous questions that have arisen during the course of the research presented in this Volume.

International programme for the evaluation of short-term tests for carcinogenicity.

A brief report of the design and initial results from the International Programme for the Evaluation of Short-Term Tests for Carcinogenicity is presented. A total of 42 chemicals, including 14 structurally-related, carcinogen/non-carcinogen pairs were coded and submitted for testing in 35 assays. Several of the assays provided good predictions of carcinogenic potential. The results revealed the advantages and disadvantages of the assays used and provide some indication of the applicability of the tests to screening for carcinogenicity.

Initial National Cancer Institute studies on mutagenesis as a prescreen for chemical carcinogens: an appraisal.

The efficacy of several in vitro and in vivo assays to detect carcinogens from a list of compounds was evaluated. Salmonella and polymerase A-deficient Escherichia coli in vitro were the most effective systems studied. Together they detected 82% of the organic carcinogens tested. Potential prescreening systems, which were thought to be currently insufficiently sensitive for the routine screening of potential carcinogens, included a) the development of resistance to thymidine overloading, methotrexate, and cytosine arabinoside by L5178y cells, b) Saccharomyces cerevisiae D3, c) the intraperitoneal host-mediated assay, and d) thymidine uptake as a reflection of DNA repair.

Strength and weaknesses of microbial test results for predicting human response.

Concern over the mutagenic activity of environmental chemicals is widespread as a result of exploratory work during the past ten years. Mutagens seem to exist in just about every category of chemicals in our environment, and many of these chemicals have been shown to have widespread human exposure. Probably the best example in recent times is the food additive AF-2, (2-[2-furyl]-3-[5-nitro-2-furyl]acrylamide) which was used as a food preservative in Japan for about ten years starting in the mid-60's. It was used to preserve soybean curd, fish, and meat sausage and many other foods which were considered staples in the diet of every person living in Japan during that period. In 1973, AF-2 was found by Japanese scientists to be mutagenic in newly developed microbial tests for mutagenicity as well as in mammalian cells in culture (de Serres, 1974, 1976). Subsequently, AF-2 was found to be mutagenic in numerous other assay systems by scientists in other parts of the world. There was great concern over potential mutagenic effects of AF-2 on the Japanese population early in 1974 as a result of the test data on mutagenicity from experiments on these laboratory organisms. However, it was not until AF-2 was found to produce cancer in the fore-stomach in mice (Sano et al., 1977) in August of 1974 that the use of AF-2 was finally banned by the Japanese Ministry of Health.