Chlordiazepoxide attenuates activity-induced anorexia and weight loss in rats.

In Experiment 1, the effect of repeated injections of 2.5, 5.0, or 10.0 mg/kg of chlordiazepoxide (CDP) on food intake and body weight was studied in rats on an activity anorexia (AA) regimen. For several days before CDP testing began, rats lived in activity wheels and had one 60-min meal per day. During CDP testing, this regimen continued except that each rat was injected with an appropriate dose of CDP or saline 30 min before each meal. CDP enhanced food intake; 5.0 mg/kg seemed most effective. However, the CDP-induced increase in eating did not noticeably stem weight loss. In Experiment 2, after several days of AA training, CDP (5.0 mg/kg) was tested under less severe conditions; food remained restricted, but access to the wheels was discontinued. Rats given CDP ate more and gained more weight than controls. These findings suggest that benzodiazepines such as CDP may help in treating anorexia nervosa and other anorectic conditions in humans.

Conformational changes in the crystal structure of rat glutathione transferase M1-1 with global substitution of 3-fluorotyrosine for tyrosine.

The structure of the tetradeca-(3-fluorotyrosyl) M1-1 GSH transferase (3-FTyr GSH transferase), a protein in which tyrosine residues are globally substituted by 3-fluorotyrosines has been determined at 2.2 A resolution. This variant was produced to study the effect on the enzymatic mechanism and the structure was undertaken to assess how the presence of the 3-fluorotyrosyl residue influences the protein conformation and hence its function. Although fluorinated amino acid residues have frequently been used in biochemical and NMR investigations of proteins, no structure of a protein that has been globally substituted with a fluorinated amino acid has previously been reported. Thus, this structure represents the first crystal structure of such a protein containing a library of 14 (28 crystallographically distinct) microenvironments from which the nature of the interactions of fluorine atoms with the rest of the protein can be evaluated. Numerous conformational changes are observed in the protein structure as a result of substitution of 3-fluorotyrosine for tyrosine. The results of the comparison of the crystal structure of the fluorinated protein with the native enzyme reveal that conformational changes are observed for most of the 3-fluorotyrosines. The largest differences are seen for residues where the fluorine, the OH, or both are directly involved in interactions with other regions of the protein or with a symmetry-related molecule. The fluorine atoms of the 3-fluorotyrosine interact primarily through hydrogen bonds with other residues and water molecules. In several cases, the conformation of a 3-fluorotyrosine is different in one of the monomers of the enzyme from that observed in the other, including different hydrogen-bonding patterns. Altered conformations can be related to differences in the crystal packing interactions of the two monomers in the asymmetric unit. The fluorine atom on the active-site Tyr6 is located near the S atom of the thioether product (9R,10R)-9-(S-glutathionyl)-10-hydroxy-9,10-dihydrophenanthrene and creates a different pattern of interactions between 3-fluorotyrosine 6 and the S atom. Studies of these interactions help explain why 3-FTyr GSH transferase exhibits spectral and kinetic properties distinct from the native GSH transferase.

Enzymes harboring unnatural amino acids: mechanistic and structural analysis of the enhanced catalytic activity of a glutathione transferase containing 5-fluorotryptophan.

The catalytic characteristics and structure of the M1-1 isoenzyme of rat glutathione (GSH) transferase in which all four tryptophan residues in each monomer are replaced with 5-fluorotryptophan are described. The fluorine-for-hydrogen substitution does not change the interaction of the enzyme with GSH even though two tryptophan residues (Trp7 and Trp45) are involved in direct hydrogen-bonding interactions with the substrate. The rate constants for association and dissociation of the peptide, measured by stopped-flow spectrometry, remain unchanged by the unnatural amino acid. The 5-FTrp-substituted enzyme exhibits a kcat of 73 s-1 as compared to 18 s-1 for the native enzyme toward 1-chloro-2,4-dinitrobenzene. That the increase in the turnover number is due to an enhanced rate of product release in the mutant is confirmed by the kinetics of the approach to equilibrium for binding of the product. The crystal structure of the 5-FTrp-containing enzyme was solved at a resolution of 2.0 A by difference Fourier techniques. The structure reveals local conformational changes in the structural elements that define the approach to the active site which are attributed to steric interactions of the fluorine atoms associated with 5-FTrp146 and 5-FTrp214 in domain II. These changes appear to result in the enhanced rate of product release. This structure represents the first of a protein substituted with 5-fluorotryptophan.

Structure and function of the xenobiotic substrate-binding site and location of a potential non-substrate-binding site in a class pi glutathione S-transferase.

Complex structures of a naturally occurring variant of human class pi glutathione S-transferase 1-1 (hGSTP1-1) with either S-hexylglutathione or (9R,10R)-9-(S-glutathionyl)-10-hydroxy-9, 10-dihydrophenanthrene [(9R,10R)-GSPhen] have been determined at resolutions of 1.8 and 1.9 A, respectively. The crystal structures reveal that the xenobiotic substrate-binding site (H-site) is located at a position similar to that observed in class mu GST 1-1 from rat liver (rGSTM1-1). In rGSTM1-1, the H-site is a hydrophobic cavity defined by the side chains of Y6, W7, V9, L12, I111, Y115, F208, and S209. In hGSTP1-1, the cavity is approximately half hydrophobic and half hydrophilic and is defined by the side chains of Y7, F8, V10, R13, V104, Y108, N204, and G205 and five water molecules. A hydrogen bond network connects the five water molecules and the side chains of R13 and N204. V104 is positioned such that the introduction of a methyl group (the result of the V104I mutation) disturbs the H-site water structure and alters the substrate-binding properties of the isozyme. The hydroxyl group of Y7 forms a hydrogen bond (3.2 A) with the sulfur atom of the product. There is a short hydrogen bond (2.5 A) between Y108 (OH) and (9R, 10R)-GSPhen (O5), indicating the hydroxyl group of Y108 as an electrophilic participant in the addition of glutathione to epoxides. An N-(2-hydroxethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) molecule is found in the cavity between beta2 and alphaI. The location and properties of this HEPES-binding site fit a possible non-substrate-binding site that is involved in noncompetitive inhibition of the enzyme.

First-sphere and second-sphere electrostatic effects in the active site of a class mu gluthathione transferase.

The activation of the thiol of glutathione (GSH) bound in the active site of the class mu glutathione transferase M1-1 from rat involves a hydrogen-bonding network that includes a direct (first-sphere) interaction between the hydroxyl group of Y6 and the sulfur of GSH and second-sphere interactions involving a hydrogen bond between the main-chain amide N-H of L12 and the hydroxyl group of Y6 and an on-face hydrogen bond between the hydroxyl group of T13 and the pi-electron cloud of Y6 (i.e., T13-OH---pi-Y6-OH--- -SG). The functions of these hydrogen bonds have been examined with a combination of site-specific mutagenesis and X-ray crystallography. The hydroxyl group of Y6 has a normal pKa of about 10 even though it is shielded from solvent and is in a largely hydrophobic environment. The apparent pKa of GSH in the binary Y6F.GSH complex is increased by 1.6 log units, and the reactivity of the enzyme-bound nucleophile is reduced. The catalytic properties of the Y6L mutant are identical to those of Y6F, suggesting that the weakly polar on-edge interaction between the aromatic ring and sulfur has no influence on catalysis. The refined three-dimensional structure of the Y6F mutant in complex with GSH shows no major structural perturbation of the protein other than a change in the coordination environment of the sulfur. Removal of the second-sphere influence of the on-face hydrogen bond between the hydroxyl groups T13 as in the T13V and T13A mutants elevates the pKa of enzyme-bound GSH by about 0.7 pKa units. Crystal structures of these mutants show that structural changes in the active site are minor and suggest that the changes in pKa of E.GSH are due to the presence or absence of the on-face hydrogen bond. The T13S mutant has a completely different side-chain hydrogen-bonding geometry than T13 in the native enzyme and catalytic properties similar to the T13A and T13V mutants consistent with the absence of an on-face hydrogen bond. The gamma-methyl group of T13 is essential in enforcing the on-face hydrogen bond geometry and preventing the hydroxyl group from forming more favorable conventional hydrogen bonds.

Further evaluation of a teratogenicity screen using an intravitelline injection technique.

A blind trial of 31 compounds was conducted in order to evaluate the intravitelline injection technique as a predictor of developmental toxicity. Rat conceptuses were explanted on day 9 of gestation and cultured until 11.5 days, at which time the compounds were injected into the vitelline circulation of the yolk sac. The embryos were then cultured for a further 24 hr after which time they were assessed on 14 parameters and assigned to one of three categories based on predetermined criteria. The results were then compared with the classifications previously allocated using data derived from in vitro studies. The compounds were examples of non-teratogens, non-cytotoxic teratogens and cytotoxic teratogens. Of the 31 compounds, 23 gave the predicted result, three non-teratogens being false positives and five teratogens having no effect in this system. The results therefore demonstrate the ability of the technique to distinguish between the three categories of compound, displaying a specificity of 75%, a sensitivity of 74% and an overall accuracy of 74.5%. Furthermore, it allowed the differentiation of active and inactive analogues of four paired compounds. This method may have applications as a teratogenicity screen or in the elucidation of the mechanism of action of teratogenic compounds.

Intravitelline injection of cultured rat embryos: An improved method for the identification of cytotoxic and non-cytotoxic teratogens.

A preliminary study of a novel developmental toxicity screen has been carried out. The technique involves the direct injection into the vitelline circulation of the 11.5-day rat conceptus, by-passing the metabolically active visceral yolk sac. The evaluation was performed blind using four coded model compounds: sulphanilamide (non-cytotoxic, non-teratogen), retinoic acid (teratogen) and methotrexate and cyclophosphamide (both cytotoxic teratogens). Seven parameters of teratogenicity and cytotoxicity were measured (yolk sac diameter, crown-rump length, somite number, yolk sac protein, yolk sac DNA, embryo protein, embryo DNA) and morphological abnormalities were also noted. The results showed that this technique successfully identified the developmental toxins and, moreover, differentiated between teratogens and cytotoxic teratogens. Additionally, the results show that methotrexate and cyclophosphamide produced an effect without prior exogenous activation as is necessary in other in vitro tests.

In vitro micromass teratogen test: Interpretation of results from a blind trial of 25 compounds using three separate criteria.

The results are presented on an interlaboratory study to validate the Micromass assay by testing compounds under code using a similar protocol, with an assessment of results for 25 compounds tested without S-9 mix. Four of these were co-tested with S-9 mix. Three separate sets of criteria have been proposed (Flint, 1986 and 1987; Flint and Orton, 1984) for interpreting the results for teratogenic hazard from in vitro data using IC(50) values: (i) the '< 500 mug/ml rule', (ii) the '< 50 mug/ml rule' and (iii) the 'specific inhibition of cell differentiation 2-fold rule'. The data were decoded and assessed using the criteria for their sensitivity, specificity and accuracy. Using the '< 500 mug/ml rule' a higher sensitivity was obtained but at the expense of a high false positive frequency (50%). Conversely, the '2-fold rule' gave a high specificity with only 10% false positives. Diphenhydramine and furazolidone gave false positive responses; the teratogens beta-aminopropionitrile and methotrexate were not detected. Selective inhibition of cell differentiation was related to high potential teratogenicity. In the application of the test, it is suggested that if there was an indication of teratogenic hazard using the '2-fold rule' the compound should be rejected without recourse to animal testing. In its present form the assay cannot be used to unequivocally identify non-teratogens.

Some effects of lignocaine on cultured mouse peritoneal macrophages.

Macrophages were exposed to lignocaine in perifused and non-perifused cultures using media with or without foetal calf serum. Effects on morphology, viability, phagocytosis and the release of enzymes were assessed. During the period of contact with lignocaine there was a selective release of beta-glucuronidase. After washing, enzyme release continued over a period of 7 hours and, in the absence of foetal calf serum, a decrease in the total beta-glucuronidase content was found in non-perifused cultures. Although lignocaine-treated cells phagocytosed particulates the rate of enzyme release was reduced compared with normal cells when subsequently exposed to quartz.

The effect and mode of action of zinc pyrithione on cell growth. I. In vitro studies.

The effects of zinc pyrithione (ZnPTO) were studied in a series of in vitro tests to determine whether its mode of action is primarily cytostatic or cytotoxic. Sodium pyrithione (NaPTO) was also studied, to check that pyrithione was the active moiety, and the known cytostatic chemical hydroxyurea was included for comparison. ZnPTO had a reversible inhibitory effect on the growth of BHK 21 cells at 0.1 microgram/ml, but had a rapid, irreversible inhibitory effect at 1 microgram/ml associated with cell rounding and detachment. NaPTO produced a similar effect but hydroxyurea produced an essentially reversible inhibition even at a dose well above that producing complete inhibition. ZnPTO and NaPTO both caused contraction, rounding and blebbing of BHK 21 cells in perfusion-chamber tests, at higher levels (1 and 10 micrograms/ml) than required for growth inhibition, but only 10 micrograms ZnPTO/ml caused lactate dehydrogenase (LDH) release. Hydroxyurea had no effects in these tests. ZnPTO and NaPTO also reduced the survival of Chinese hamster V79 cells sharply over a narrow dose range (0.01-0.03 microgram/ml), but the effect of hydroxyurea was not as sharp and occurred at much higher doses. All three showed elements of cytostasis and cytotoxicity as demonstrated by analysis of the relationship between survival and colony area. Of the three, only ZnPTO (at greater than or equal to 5 micrograms/ml) caused significant LDH release from the cells, though both ZnPTO and NaPTO (at 0.1-1 microgram/ml) inhibited cell growth as indicated by total LDH values. In studies with rat peritoneal mast cells, ZnPTO and NaPTO (at 10 ng/ml) both suppressed histamine release induced by 48/80 or Ca ionophore A23187, though neither caused histamine release directly. The combined results of these tests show that ZnPTO is primarily cytotoxic, rather than cytostatic.

Binding of a spin label analogue of compound 48/80 to rat peritoneal mast cells: correlation of binding properties with surface topography.

A spin label analogue of compound 48/80 has been synthesized and its binding to purified rat peritoneal mast cells has been studied by electron spin resonance spectroscopy. The spin label analogue (SL-48/80) had almost identical biological activity to unlabeled compound 48/80. SL-48/80 was used to estimate the number of binding sites per cell on normal mast cells (7.25 X 10(10)), on mast cells deactivated by sodium azide and 2-deoxyglucose or by heating to 46 degrees for 30 min (1 X 10(10)) and cells from animals actively-sensitized to ovalbumin (5.2 X 10(10)). SL-48/80 was also shown to bind to isolated mast cell granules. Differences in the binding properties of mast cells after the different treatments are related to their surface topography as seen by scanning electron microscopy, and the contribution of the granules to the number of binding sites is discussed.

In vitro cell perfusion. I. System for continuous observation of cells and analysis of perfusion fluid suitable for isolated mast cell and macrophage studies.

A system is described in which cells are perfused continuously with appropriate medium while simultaneously recording any change in cell morphology together with assay of released products (enzymes and mediators) following immunologic or non-immunologic perturbation. The released products in the perfusate are monitored continuously by automated fluorimetric assay procedures. The system is particularly applicable to investigate or toxicological studies of isolated mast cells or cultured macrophages.

An automated fluorimetric assay for the simultaneous determinations of histamine and 5-hydroxytryptamine released from rat mast cell preparations.

An automated assay suitable for the extraction and simultaneous fluorimetric determination of histamine and 5-hydroxytryptamine (5-HT) released from rat isolated peritoneal mast cells is described. The method gave good reproducibility and accuracy in use and required a sample volume of 0.5 ml for assay at a sampling rate of 30/h. The system was evaluated by assessment of its performance in the determination of histamine and 5-HT released from rat mast cells in response to compound 48/80. The detection limit for the procedure was 2 ng 5-HT base/ml and 20 ng histamine base/ml.