High-affinity binding of [3H]doxepin to histamine H1-receptors in rat brain: Possible identification of a subclass of histamine H1-receptors

The binding of the radioactively labeled tricyclic antidepressant, [³H]doxepin, to rat brain tissie was examined. Scatchard plots of specific [³H]doxepin binding indicated the presence of two distinct binding sites. The equilibrium dissociation constant (K_D) of the high-affinity site was 0.020 nM with a maximal binding capacity (B_max) of 13.7 fmol/mg protein. The corresponding values for the low-affinity site were 3.6 nM and 740 fmol/mg protein, respectively. The high-affinity site was sensitive to competition by pharmacologically relevant concentrations of histamine H₁ antagonists such as pyrilamine (K_D = 1.0 nM), diphenhydramine (K_D = 20 nM), d-chlorpheniramine (K_D = 1.7 nM), and 1-chlorpheniramine (K_D = 97 nM). The B_max for [³H]doxepin binding in the high-affinity H₁-receptor, however, was approximately 10% of the B_max obtained using [³H]pyrilamine to label the H₁-receptor. Various tricyclic antidepressants were very potent inhibitors at the high-affinity [³H]doxepin site. Their potencies, however, did not correlated with their potencies previously reported for the H₁-receptor. The regional distribution of [³H]doxepin high-affinity sites correlated with the known distribution of H₁-receptors in the rat brain. These results suggest that [³H]doxepin is binding to a subclass of histamine H₁-receptors.     

Development of hepatic lesions in male Fischer-344 rats fed AIN-76A purified diet

The suitability of the AIN-76A diet for Fischer-344 rats was investigated. This diet, proposed by the American Institute of Nutrition for use when a purified diet composed of refined ingredients and added vitamins and minerals is required, was tested in Sprague-Dawley rats. Male weanling Fischer-344 rats were fed three different lots of the AIN-76A diet from two suppliers. Increase in body weights and food consumption were compared to animals fed a cereal-based control diet. Animals were sacrificed at various intervals and tissues were taken for histopathological observation. By 8 weeks moderate to marked periportal lipidosis developed in livers of all rats fed the AIN-76A diet. Liver-body weight ratios over the 8-week period were significantly higher in rats fed AIN-76A diets compared to rats fed the control diet. However, growth rates of rats fed the AIN-76A diet were similar to growth rates of controls. Some rats fed the AIN-76A diet developed severe hemorrhagic lesions. The AIN-76A diet in its present form is not suitable for use with male Fischer-344 rats.     

K+-evoked [3H]-5-HT release from rat frontal cortex slices: the effect of 5-HT agonists and antagonists

The pharmacological characteristics of a pre-junctional 5-HT autoreceptor have been studied by following the Ca²⁺-dependent, K⁺-evoked release of [³H]-5-HT from preloaded rat frontal cortex slices. Added 5-HT, in the presence of the 5-HT uptake inhibitor chlorimipramine. caused a dose related inhibition of the K²⁺-evoked release of [³H]-5-HT in this system as did the 5-HT analogues 5-methoxytryptamine, N-methyltryptamine, 5-methoxy-NN'-dimethyltryptainine, N-methyl 5-hydroxytryptamine and tryptamine.The inhibitory effect of 1 μM 5-HT on the K⁺evoked release of [³H]-5-HT was reversed in a doserelated manner by the 5-HT antagonist drug, methiothepin (pA₁₀ value 6.7). At a concentration of 1 μM, the 5-HT antagonists drugs cinanserin and mianserin produced a small but significant reversal of the 5-HT induced inhibition of K⁺-evoked [³H]-5-HT release, but methysergide, metergoline and cyproheptadine were completely without effect at this concentration. The results are interpreted as evidence for a pre-junctional autoreceptor for 5-HT in the frontal cortex of the rat with a different pharmacological specificity for 5-HT antagonists from previously studied 5-HT receptors.     

Fate of [3H]amezinium in sympathetically innervated rabbit tissues

Perfused rabbit hearts accumulated intra-aortically infused [³H]amezinium. At concentrations of 1 or 10 nM, the accumulation proceeded at a constant rate for at least 60 min. After 60 min, tissue medium ratios were between 6 (1 μM) and approx. 100 (1 or 10 nM [³H]amezinium). Cocaine or pretreatment with 6-hydroxydopamine abolished, and pretreatment with reserpine reduced the accumulation of [³H]amezinium (1nM). Kinetic analysis yielded a K_m value of 0.9 μM and a V_max of 1.2 nmole g^?1 min^?1. When hearts had been labelled with 1 or 10 nM [³H]amezinium, the fractional rate of the subsequent efflux was very low (0.001 min^?1). It was greatly increased when the animals had been pretreated with reserpine. Electrical stimulation of the sympathetic nerves released [³H] amezinium from pre-labelled rabbit pulmonary artery strips. The electrically evoked overflow was abolished by tetrodotoxin or omission of Ca²⁺; it was enhanced by cocaine, desipramine and yohimbine and decreased by clonidine. The results show that amezinium, at least at low concentrations, is selectively taken up into postganglionic sympathetic neurones, that it is partly sequestered in the vesicles, and that it is released by action potentials. Amezinium is a structurally novel substrate of both the noradrenaline transport mechanism of the axolemma and the transport mechanism of the noradrenaline-storing synaptic vesicles.     

Drug protein conjugates—I. A study of the covalent binding of [14C]captopril to plasma proteins in the rat

The metabolism of [¹⁴C]captopril has been investigated in vitro and in vivo in male Wistar rats. The formation of conjugates of [¹⁴C]captopril with plasma proteins was observed both in vitro and in vivo: 180 min after intravenous infusion of [¹⁴C]captopril 35 ± 5% of total radioactivity was covalently bound to plasma proteins. The fate of [¹⁴C]captopril-plasma protein conjugates was investigated in vivo. [¹⁴C]Captopril was incubated in vitro with rat and human plasma and the resulting captopril-protein conjugates were infused into male rats. The plasma concentration of [¹⁴C]captopril-rat plasma protein conjugates declined monoexponentially with a half-life of 71.1 ± 2.2 min. After 180 min 28 ± 3% of the radioactivity was excreted in urine, largely as [¹⁴C]captopril-cysteine mixed disulphide (67%). Thus although captopril readily forms covalent bonds with plasma proteins the resulting conjugates dissociate in vivo. The toxicological implications of these findings are discussed.     

Binding of adrenergic ligands ([3H]clonidine and [3H]WB-4101) to multiple sites in human brain

In order to identify alpha-adrenoceptors in post-mortem human brain and to detect the possible existence of multiple types of binding sites for adrenergic [³H]ligands, we studied the binding of [³H]clonidine and [³H]WB-4101 to human brain cerebral cortex, hippocampus, hypothalamus and striatum. Frontal cortex revealed two binding sites for [³H]clonidine (with K_D values of approximately 1 and 8 nM), as indicated by the biphasic Scatchard plot, the biphasic pattern of dissociation kinetics, and the biphasic inhibition by phentolamine on the binding of [³H]clonidine; the high-affinity site was heat-labile. Two high-affinity binding sites for [³H]WB-4101 were also detected in the human frontal cortex (with K_D values of about 0.09 and 1.5 nM), as revealed by a biphasic pattern of dissociation. A third site with low affinity binding for [³H]WB-4101 was detected by the biphasic inhibition by phentolamine (as well as by WB-4101 and prazosin) on the binding of [³H]WB-4101. The three other brain regions revealed very similar patterns exhibited by the frontal cortex, except that the density of the [³H]clonidine sites (of either high or low affinity) was highest in the hypothalamus, whereas the density of [³H]WB-4101 sites was highest in the hippocampus.     

Metabolism and excretion of 2,4-[14C]dinitrotoluene in conventional and axenic Fischer-344 rats

Comparisons of in vitro reduction of 2,4-dinitrotoluene (2,4-DNT) by cecal microflora and liver have indicated that microflora may play a large role in the in vivo metabolism of 2,4-DNT to reduced metabolites. Furthermore, reduction of 2,4-DNT by cecal microflora produces nitroso and, presumably, hydroxylamino intermediates which may account for the toxic actions of 2,4-DNT, including hepatocarcinogenesis. This study examines the metabolism, excretion, and hepatic covalent binding of 2,4-DNT in conventional, DNT-fed, and axenic Fischer-344 rats in order to define more precisely the role of DNT pretreatment and intestinal microflora in the disposition and toxicity of 2,4-DNT. No differences in 2,4-DNT disposition were produced by 30 days of feeding DNT (35 mg/kg/day) in the diet of male or female rats. Axenic males and females excreted less of a dose of 2,4-DNT in the urine than did conventional animals, and half-times for excretion of 4-(N-acetyl)amino-2-nitrobenzoic acid (4NAC2NBA), 2,4-dinitrobenzoic acid, 2-amino-4-nitrobenzoic acid (2A4NBA), and 2,4-dinitrobenzyl alcohol glucuronide were longer in axenic males than in conventional males. In axenic females half-times for excretion of only 4NAC2NBA and 2A4NBA were longer than in conventional females. Amounts of 4NAC2NBA and 2A4NBA excreted by axenic animals were 1/10th to 1/5th those excreted by conventional animals. Hepatic covalent binding was decreased by half in axenic animals. These data suggest that intestinal microflora play a major role in the appearance of reduced urinary metabolites and of covalently bound material after 2,4-DNT administration.     

Studies on the hepatic microsomal metabolism of [14C]phenanthrene

[¹⁴C]Phenanthrene was metabolished in vitro by hepatic microsomes obtained from untreated, sodium phenobarbital (PB), and 3-methylcholanthrene (MC) pretreated Wistar rats, guinea pigs, SW and DBA/2J mouse strains. Metabolite profiles were obtained by comparison of thinlayer radiochromatographs of the products with R_fs of authentic reference compounds, trans-dihydrodi-hydroxyphenanthrenes (dihydrodiols) were the major metabolites in all preparations. In every case, except with microsomes from MC pretreated guinea pigs, trans — 9,10 — dihydro — 9,10 — dihydroxyphenanthrene predominated. With microsomes from MC pretreated guinea pigs, the 1,2-dihydrodiol was the major metabolite. Addition of cyclohexene oxide (5.0 mM) to the incubation mixture inhibited dihydrodiol formation in rat and mouse preparations by 90 per cent, and in guinea pig preparations by 70 per cent. Phenols and phenanthrene — 9,10 — oxide were produced instead.     

Formation of poly(butyl 2-cyanoacrylate) microcapsules and the microencapsulation of aqueous solutions of [125I]-labelled proteins

Some features of the polymerization reaction of butyl 2-cyanoacrylate at different aqueous/organic solvent interfaces have been investigated. In particular, the effects of pH and the presence of protein on the formation of microcapsules by in situ interfacial polymerization of butyl 2-cyanoacrylate in w/o emulsions have been studied. [¹²⁵I]-labelled proteins have been used to study the procedure as a method of microencapsulating enzymes or other proteins within potentially biodegradable membranes. Preliminary in vitro degradation studies suggest that degradation of the microcapsules is inhibited by low levels of their breakdown products, thus allowing the storage of the microcapsules as aqueous suspensions for prolonged periods in sealed containers.     

Influence of elastase-induced emphysema and the inhalation of an irritant aerosol on deposition and retention of an inhaled insoluble aerosol in Fischer-344 rats

The purpose of this study was to assess the effects of elastase-induced pulmonary emphysema and the inhalation of an irritant aerosol (Triton X-100, a nonionic surfactant similar to those used in a number of pressurized consumer products) on pulmonary deposition and retention of an insoluble test aerosol, 59Fe-labeled Fe2O3. Untreated rats or rats pretreated by intratracheal instillation with elastase were exposed to an aerosol of 59Fe-labeled Fe2O3 either 18 hr or 7 days after exposure to aerosolized Triton X-100 which was administered in doses of 20, 100, or 200 micrograms/g of lung. Rats pretreated with elastase had significantly lower pulmonary deposition of 59Fe than the untreated controls (p less than 0.005). Pulmonary deposition of Fe2O3 was unaffected by pretreatment with Triton X-100. Elastase treatment alone had no effect on retention of Fe2O3. Triton X-100 administered 18 hr prior to exposure of rats to Fe2O3 aerosol resulted in dose-related increases in whole-body retention of 59Fe. When rats were exposed to Triton X-100 7 days before exposure to Fe2O3, increased retention of 59Fe was noted only in those treated at the highest Triton X-100 dose level (200 micrograms/g).     

Identification and characterisation of L-[3H]aspartate binding sites on rat spinal cord synaptic membranes

The binding of L-[3H] aspartate to extensively-washed rat spinal cord synaptic membranes was investigated. Specific binding was enriched in synaptic membranes and was optimal under physiological conditions of temperature and pH. Equilibrium binding was established relatively slowly over a period of 30 min, and was totally reversible within 40 min. Saturation analysis revealed complex binding patterns. Two sites were clearly demonstrable, only one of which was shown to be saturable over the ligand concentration range employed in the study (0.1-10 microM). There was also some indication of the presence of a higher affinity site, although this was not investigated in any detail. Saturable binding demonstrated a KD = 1.4 microM and Bmax = 105 pmole/mg protein. Structure-activity studies with a range of amino acid analogues indicated that binding was stereospecific and was inhibited by a very restricted range of compounds. The most potent inhibitors of binding were L-glutamate and L-aspartate. There was no evidence for the involvement of NMDA receptors. Effects of possible endogenous modulators, including ions and guanosine nucleotides were investigated, and the chemical nature of the binding site probed with a number of protein-modifying agents.     

Effects of long-term feeding of ammoniated, aflatoxin-contaminated corn to Fischer 344 rats

The effectiveness of ammonia treatment in reducing the chronic toxicity of aflatoxin-contaminated corn was determined. Fischer 344 rats were fed semi-purified rations containing 20% w/w corn that was either free of aflatoxin or naturally contaminated with 880 micrograms/kg total aflatoxin and was either treated with ammonia gas or was not treated. Therefore the rats that were fed the aflatoxin-contaminated diet received 176 ppb total aflatoxins. Body weight and food consumption were recorded throughout the study; hematological measurements were made after 87 weeks of feeding; and after 91 weeks the rats were killed and histopathological abnormalities were noted. Signs of chronic toxicosis in rats fed aflatoxin-contaminated corn included increased mortality, decreased hematocrit and hemoglobin levels, elevated serum alkaline phosphatase activities, and a 100% incidence of liver neoplasia. These signs did not occur in rats in the other dietary treatment groups, including those fed ammoniated, aflatoxin-contaminated corn. The results provide further evidence that the atmospheric ammoniation process effectively reduces the toxicity of aflatoxin-contaminated corn.     

Metabolism and macromolecular covalent binding of benzo[a]pyrene in cultured Fischer-344 rat lung type II epithelial cells

Pulmonary biotransformation of many xenobiotics may be important for the mutagenic, carcinogenic and/or toxic response of lung tissue to these compounds. Recently, a lung epithelial cell line (designated LEC), with morphological characteristics suggestive of type II cell origin, was developed in our laboratory. When LEC cells were co-cultivated with Chinese hamster ovary (CHO) cells in a cell-mediated mutagenesis assay, LEC metabolized promutagens to metabolites mutagenic to the CHO cells [A. P. Li, A. L. Brooks, J. M. Benson and F. F. Hahn, Environ. Mutagen. 4, 407 (1982)]. In the present investigation, rates of benzo[a]pyrene (BaP) metabolism in type II lung cells were determined, and the effects of various pollutants on rates of BaP metabolism and covalent binding of BaP to LEC macromolecules were measured. Cultures of LEC cells were incubated for 24 hr with 5 microM [14C]BaP, and the culture medium was analyzed for organic- and water-soluble metabolites. LEC cells metabolized BaP to BaP-7,8-diol and BaP-9,10-diol with total rates of formation of these metabolites measured at 500-600 pmoles per 10(6) cells per 24 hr. BaP-9,10-diol was the major metabolite accounting for about 80% of the total BaP metabolized. Enzyme hydrolysis studies revealed the presence of small quantities (less than 20% of BaP metabolized) of the glucuronide conjugates of BaP-7, 8-diol and 9-hydroxy-BaP. Pretreatment of LEC cells with benz[a]anthracene, coal gas condensate, or diesel exhaust particle extract (DEP) prior to incubation with BaP resulted in a 2- to 5-fold increase in overall rates of BaP metabolism. The largest increase in covalent binding of [14C]BaP equivalents to LEC macromolecules was seen after LEC cells were pretreated with DEP (3-fold). The data suggest that lung epithelial cells may play an important role in the biological fate of inhaled xenobiotics.     

3H]N-propylapomorphine and [3H]spiperone binding in brain indicate two states of the D2-dopamine receptor

[3H]N-propylapomorphine ([3H]NPA) a dopaminergic catecholamine derivative, labels a sub-set of D2-dopamine receptors in bovine caudate particulate preparation. [3H]Spiperone, a dopamine receptor antagonist, labels twice as many sites as [3H]NPA. Dopaminergic ergots and potent neuroleptics compete for both radioactive ligands with similar high affinities. Catecholamines and catecholamine derivatives compete more potently for [3H]NPA binding than for [3H]spiperone binding. Guanyl nucleotides reduce both [3H]NPA binding and the high affinity phase of catecholamine and catecholamine derivative competition for [3H]spiperone binding. These results are similar to binding results reported in studies of two-state receptors linked to adenylate cyclase such as the beta-adrenergic receptors. These observations indicate that the D2-dopamine receptor in the brain may exist in two states and may be inversely coupled to brain adenylate cyclase activity.