Differences in the In Vitro and In Vivo 5-Hydroxytryptamine Extraction Performance Among Three Common Microdialysis Membranes

The present study summarizes the results of an in vitro and in vivo comparison of the apparent 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid, and 3,4-dihydroxyphenylacetic acid dialysis performance of three types of membrane frequently used in intracerebral microdialysis experiments. The dialysis fiber types examined were a regenerated cellulose Cuprophan (GF), a proprietary polycarbonate ether (CMA), and a polyacrylonitrile/sodium methallylsulfonate copolymer (HOSPAL). The experiments unexpectedly revealed that the HOSPAL membrane-equipped probes displayed clearly aberrant 5-HT diffusion dynamics compared with GF and CMA probes, demonstrable not only in vitro, but also in in vivo experiments. In vitro, the GF and CMA membrane-equipped probes exhibited maximum relative recovery for 5-HT already in the first 20-min sample, whereas the 5-HT recovery of HOSPAL probes increased in a very slow and protracted manner over a period of a little less than 2 h. The GF and CMA probes further displayed an immediate washout of 5-HT when the probes were subsequently transferred to artificial CSF only-containing medium (no 5-HT), whereas approximately 2 h was required to yield near-total extinction of dialysate 5-HT with the standard HOSPAL probes. In vivo, the rat ventral hippocampal dialysate 5-HT output responses to K+ (100 mM) infusion, to Ca2+ omission, and to systemic 8-hydroxy-2-(di-n-propylamino)tetralin injection were all markedly retarded and blunted when HOSPAL instead of GF membrane-equipped probes were used. However, the 5-hydroxyindoleacetic acid and 3,4-dihydroxyphenylacetic acid extraction in vitro and in vivo were comparable using either of the membrane types.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,4-Dioxane: prediction of in vivo clastogenicity.

1,4-Dioxane was analyzed with the CASE program to determine the structural basis of its potential genotoxicity and carcinogenicity. These investigations led to the prediction that while 1,4-dioxane was not genotoxic in vitro, it was an inducer of micronuclei in the bone marrow of rats and a carcinogen for both rats and mice. If it is assumed that the induction of micronuclei is the result of DNA damage, then this potential and the previous report of the in vivo induction of DNA strand breaks in rat liver raise the possibility of a genotoxic action for 1,4-dioxane. However it is also conceivable that we have identified a structural feature which contributes to the induction of micronuclei by a non-genotoxic mechanism.     

Subtype of muscarinic receptor coupled to the attenuation of hormone-stimulated cAMP accumulation in NG108-15 neuroblastoma x glioma hybrid cells.

The subtype of muscarinic receptor which mediates cAMP attenuation is not established. Therefore, several selective muscarinic antagonists were used to characterize the subtype of muscarinic receptor coupled to the inhibition of hormone-stimulated cAMP accumulation using NG108-15 neuroblastoma x glioma hybrid cells. These cells were prelabeled with [2-3H]-adenine, washed, and resuspended in a culture medium containing the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM). The labeled cells were preincubated with the different antagonists 12-15 min. before they were challenged with agonists. The formation of [3H]-cAMP was activated by PGE1 (1 microM) or forskolin (1 microM). In all cases, [3H]-cAMP formed was separated and measured. Carbachol (100 microM) and McN-A343 (10 mM) were used as standard muscarinic agonists. These studies gave the following results: a) McN-A343 (10 mM), an M1 receptor agonist, was only a partial agonist causing 40% inhibition of cAMP accumulation indicating that this effect was not mediated by an M1 receptor; b) The M1-selective antagonist, pirenzepine, exhibited low affinity (pA2 6.2) further suggesting that an M1 receptor was not coupled to the attenuation of cAMP accumulation; c) Two selective M2 antagonists (AF-DX 116 and methoctramine) and M3 antagonist (HHSiD) were used to further characterize these muscarinic receptors. The order of all antagonists based on their affinities (pA2 values) could be arranged in the following order: atropine (9.0) > methoctramine (7.6) > HHSiD (6.9) > AF-DX 116 (6.6) > pirenzepine (6.2). HHSiD exhibits the same degree of affinity to M2 receptors of other tissues as it does to those of NG cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

A somatic cell hybrid panel and DNA probes for physical mapping of human chromosome 7p.

To identify by reverse genetics genes on the short arm of human chromosome 7 expected to be involved in the regulation of human craniofacial and limb development, we have set up a human mouse somatic cell hybrid panel that divides 7p into 9 fragments. The breakpoints are defined by deletions or translocations involving one chromosome 7 in the cells of the human cell fusion partners. Particularly densely covered with these cytogenetic anchor points is the proximal area of 7p within and around 7p13. The number of cytogenetic mapping points within proximal 7p could be increased by four, using two diploid human cell lines with small interstitial deletions in this region for dosage studies. We used Southern blots of this panel to assign to 7q or subregions of 7p more than 300 arbitrary DNA probes or genes that provide reference points for physical mapping of 7p. Three reciprocal translocations with one of the breakpoints in 7p13 mark the location of a gene involved in Greig cephalopolysyndactyly syndrome. To define an area in which we could identify candidates for this developmental gene, we established a macrorestriction map using probes flanking the putative gene region. The Greig translocations were found to be located within a 630-kb NotI restriction fragment.     

Enterohemolysin production is associated with a temperate bacteriophage in Escherichia coli serogroup O26 strains.

A temperate bacteriophage that determines the expression of enterohemolysin was isolated from Escherichia coli O26 strain C3888. The genetic determinant associated with enterohemolysin production (E-Hly determinant) was cloned from EcoRI-digested bacteriophage DNA in vector plasmid pUC8. pUC8 recombinant plasmid pEO19 carries a 3.7-kb EcoRI insert of phage DNA, and enterohemolysin was expressed in E. coli K-12 after transformation. Hemolysin-negative derivatives of pEO19 were generated by transposon mutagenesis with Tn1725. By subcloning, the phage E-Hly determinant was assigned to a 2,150-bp piece of DNA which is flanked by EcoRI and AccI restriction sites. The enterohemolysin-producing recombinant strains and wild-type strain C3888 express a 60-kDa protein which was detected in the bacterial outer membrane by Western immunoblotting. Biologically active enterohemolysin was detected only in bacteria grown to the stationary phase, and the hemolysin was not released into the culture medium. Lysis of erythrocytes was inhibited by 30 mM dextran 4, which functions as an osmotic protectant without destroying the enterohemolysin itself.     

Stimulation of dopamine synthesis and release by morphine and D-Ala2-D-Leu5-enkephalin in the mouse striatum in vivo

The effects of opiates on dopamine (DA) release and synthesis were assessed in the mouse striatum $\text{in vivo}$ by simultaneously measuring 3,4-dihydroxyphenylalanine (DOPA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels after inhibition of aromatic amino acid decarboxylase. This method was developed to assess stimulus-coupled changes in DA synthesis and release. Peripheral injections of morphine and intraventrcular injections of D-Ala²-Leu⁵-enkephalin elevated DOPAC levels, indicating that “opiates” stimulated DA release. Concomitantly, the rate of DA synthesis was increased. The effects were dose-dependent, saturable and antagonized by naloxone. When morphine and the enkephalin analog were given together in saturating doses, the effects of the two agents were not additive. Thus, the involvement of different receptors in the mediation of the effects of morphine and enkephalins could not be demonstrated.     

Endogenous opiates inhibit gastric acid secretion induced by central administration of Thyrotropin-Releasing Hormone (TRH)

Thyrotropin-releasing hormone (TRH) administered intraventricularly (ICV) to rats causes a dose-dependent increase in gastric acid secretion over a range of 0.01 μg to 10 μg in the pyloris ligated rat. The maximum increase in gastric acid secretion occurs in the first hour. This effect of TRH is not mediated by its metabolites, histidyl-proline diketopiperazine or pyroglutamyl-histidyl-proline (acid TRH). β-endorphin, D-alanine-methionine-enkephalin and the leucine-enkephalin precursor, dynorphin, all inhibit TRH-induced gastric acid secretion. Bombesin, which reduces basal gastric acid secretion had no effect on TRH-induced secretion.     

An evaluation of tests using DNA repair-deficient bacteria for predicting genotoxicity and carcinogenicity. A report of the U.S. EPA's Gene-TOX Program

The detection of DNA-damaging agents by repair-deficient bacterial assays is based on the differential inhibition of growth of repair-proficient and repair-deficient bacterial pairs. The various methodologies used are described and recommendations are made for their improved use. In a survey of the literature through April 1979, 91 of 276 papers evaluated contained usable data, resulting in an analysis of 611 compounds that had been assayed in 1 or more of 55 pairs of repair-proficient and repair-deficient strains. The results indicate that (1) a liquid suspension assay is more sensitive than a spot (diffusion) test. In a review of the Escherichia coli polA assay, 45 compounds that gave "No Test" in the spot test were clearly positive or negative in the liquid suspension assay. (2) Of the 21 compounds analyzed by the E. coli polA assay and by other E. coli repair-deficient strains (e.g., rec, uvr, hcr, and exr derivatives of WP2 and AB1157), 10 were in complete agreement in all strains except uvrA strains. This indicates that strains other than polA+/polA- are useful for detecting DNA-damaging agents. However, in selecting strains for use in these assays, care should be taken to consider repair pathway specificity for particular compounds. (3) There was a 78% correspondence between results obtained with E. coli polA and Bacillus subtilis (H17/M45, 17A/45T) rec assay and between E. coli polA and Proteus mirabilis. (4) In a comparison of test results with carcinogenicity data, 44 of 71 (62%) carcinogenic compounds assayed by the polA system were positive, 10 (14%) were negative, and 17 (24%) gave No Test or doubtful results. 7 carcinogens were assayed by other E. coli strains and all were positive. 56 carcinogens were assayed in B. subtilis: 24 (43%) were positive, 9 (16%) were negative, and 23 (41%) gave No Test or doubtful results. Of the 7 carcinogens assayed in P. mirabilis, 6 (86%) were positive and 1 (14%) was negative. (5) The results were analyzed with respect to chemical classes. E. coli polA detected the highest percentage of hydroxylamines and alkyl epoxides. The B. subtilis rec assay detected the highest percentage of nitrosamines and sulfur and nitrogen oxides. It is concluded that some of these test systems are effective tools for the detection of DNA-damaging and potentially carcinogenic compounds, especially if the assay is done in liquid suspension and if more than 1 pair of tester strains is used. Advantages and disadvantages of the assay are discussed and suggestions are made for improvements in the system.     

The influence of coal ash and thermal discharges upon the distribution and bioaccumulation of aquatic invertebrates

The distribution, density and uptake of twenty elements by aquatic invertebrates inhabiting a drainage system, that received excessive coal ash effluent (275 JTU of turbidity) at one end and thermal loading (44.5°C) at the other end, was studied for 15 months. The ash settling basin filled during the first eight months of sampling which resulted in the release of ash effluent directly into the receiving system. Density of invertebrates was lowest in the 300 m stream between the ash basin and swamp and highest 1200 m beyond the stream-swamp confluence where ash influence was minimal. Invertebrate density was lowest in the stations where turbidity from ash effluent was greatest. The most tolerant invertebrates to coal ash stress were odonates (Libellula sp. and Enallagma sp.), crayfish (Procambarus sp.), amphipods (Gammarus sp.) and gastropods (Physa. sp.), and midges (Chironomidae) when the basin was filling. During the period of ash overflow, all groups were either reduced in numbers or absent. In the thermally stressed station, Libellula sp. was the predominant invertebrate sampled when water temperature ranged from 25.5–45.5°C (257-1=28.7°C) all aquatic invertebrates were limited in numbers and density when temperature exceeded the lower and upper ranges of 10.0–38.0°C.This research was supported by AEC Contract AT (38-1-824)This research was supported by AEC Contract AT (38-1-824)     

Mutagenicity testing with Salmonella typhimurium strains. II. The effect of unusual phenotypes on the mutagenic response.

The enhanced sensitivity of some Salmonella typhimurium strains to the mutagenic action of a number of chemicals appears to be due to the defect in the uvrB gene product and not to an inability to produce H-2-S or to the absence of formic acid hydrogenlyase which also characterizes these strains.