Extrahepatic sites of metabolism of carbon tetrachloride in rats

Rats were injected i.v. and i.p. with [14C]carbon tetrachloride and the localization and binding of metabolites in the tissues were studied by autoradiography. Based on the autoradiographic findings, various tissues were tested for their capacity to form 14CO2 from [14C]carbon tetrachloride in vitro. Autoradiography in vitro was used to localize the sites of [14C]carbon tetrachloride metabolism under in vitro conditions. The results showed that several tissues accumulating metabolites in vivo had an ability to form 14CO2 in vitro, and accumulation of metabolites was observed also under the in vitro conditions. These results indicate that carbon tetrachloride is metabolized in many extrahepatic tissues in vivo. The structures identified to have a marked carbon tetrachloride-metabolizing capacity were, besides the liver, the mucosa of the bronchial tree, the tracheal mucosa, the olfactory and respiratory nasal mucosa, the oesophageal mucosa, the mucosa of the larynx, the tongue and the cheeks, the lateral nasal gland and the kidney cortex. It is well established that the degradation of carbon tetrachloride involves the cytochrome P-450 system, and the metabolism of the substance in the mentioned tissues is probably correlated to high concentrations of cytochrome P-450. The nasal olfactory mucosa was found to be the tissue with the highest capacity to form 14CO2 from the [14C]carbon tetrachloride and microautoradiography indicated that in this tissue the cells of the subepithelial glands of the lamina propria mucosae are most actively engaged in the metabolism. It was also shown that cytochrome P-450 is present in the nasal olfactory mucosa.     

Metabolic activation and toxicity of a DDT-metabolite, 3-methylsulphonyl-DDE, in the adrenal zona fasciculata in mice

Whole-body autoradiography of 14C-labelled 3-methylsulphonyl-DDE (3-MeSO2-DDE) in female C57BL mice revealed a heavy accumulation in the adrenal cortex. Fairly high radioactivity appeared in the nasal mucosa and fat, while the labelling of the liver was intermediate. The adrenal radioactivity remained largely unextracted in tissue-sections treated with organic solvents. In the liver and intestinal contents the radioactivity was partly extracted, whereas in all other tissues almost completely extracted. According to light microscopic autoradiography, the tissue-bound adrenal radioactivity was confined to the zona fasciculata, leaving the other adrenal zones devoid of bound material. Incubation of 3-MeSO2-DDE with adrenal tissue (300 X g supernatant) revealed a dose- and time-dependent covalent binding to protein and formation of water-soluble metabolites. The cytochrome P-450 inhibitors metyrapone and carbon monoxide inhibited both covalent binding and polar metabolite formation. Addition of reduced glutathione decreased binding, while polar metabolite formation was increased. Histopathological examination of adrenals from 3-MeSO2-DDE-treated mice revealed extensive vacuolation and necrosis of the zona fasciculata 1-12 days after single doses down to 25 mg/kg. Degenerative changes were observed at 12.5 mg/kg. In contrast to 3-MeSO2-DDE, 14C-labelled 3,3'-bis(methylsulphonyl)-DDE was not accumulated in the adrenal cortex. 3-MeSO2-DDE is thus a persistent environmental pollutant with a unique ability to produce acute toxicity subsequent to metabolic activation in a mammalian tissue.     

Comparison of dietary and waterborne exposure to benzo a pyrene: bioavailability,tissue disposition and CYP1A1 induction in rainbow trout Oncorhynchus mykiss

Absorption and tissue distribution of benzo\[ a ]pyrene (BaP)-derived radioactivity were studied in juvenile rainbow trout following dietary or waterborne exposure. In order to compare the bioavailability of BaP, the fish were exposed to 1.5 mCi 3H-BaP kg-1 fish, either in the diet or in the water as a 2 days static exposure. Furthermore, tissue levels of BaP-derived radioactivity bound to macromolecules in different tissues were studied in non-induced fish, and in fish induced by additional treatment with unlabelled BaP (corresponding to 5 mg kg-1 fish) in the water. Absorption and tissue distribution of 3H BaP were studied by liquid scintillation counting and whole-body autoradiography. BaPderived radioactivity bound to macromolecules in different tissues was studied by autoradiography of solvent-extracted whole-body sections. The hepatic CYP1A induction was measured as EROD activity. Exposure to unlabelled BaP resulted in a marked induction of hepatic EROD activity in rainbow trout 2 days after the start of the exposure. Significant higher concentrations of radiolabelled compound were observed in waterborne-exposed fish, in contrast to dietary-exposed fish. High concentrations of radiolabelling were observed in the gills, liver, bile, intestines, olfactory organ, kidney and the skin of the waterborne-exposed fish. In the dietary-exposed fish, high levels of radioactivity were observed in the intestines and the bile, whereas lower concentrations were present in the liver. Only traces of radioactive compound were observed in the gills. In contrast to waterborne-exposed fish, no radioactivity was detected in the olfactory organ or skin. In autoradiograms of sections extracted with a series of polar and non-polar solvents, a large fraction of radioactivity was still present in the gills, olfactory organ, liver, kidney, skin and intestinal mucosa of the waterborne-exposed fish, indicating that reactive BaP intermediates formed by CYP1A-mediated metabolism were bound to macromolecules in these tissues.     

Disposition and cellular binding of 3H-benzo(a)pyrene at subzero temperatures: studies in an aglomerular arctic teleost fish – the polar cod (Boreogadus saida)

Autoradiography at different levels of resolution was used to study the disposition of the polycyclic aromatic hydrocarbon benzo(a)pyrene (³H-BaP) in juvenile and sexually mature polar cod (Boreogadus saida). Exposure took place via the water or after intragastric administration at subzero temperatures. In water-exposed fish, high total tissue levels were found in the gills, olfactory organ, anterior kidney, liver, skin and intestinal wall. Only traces of radioactivity were present in the muscle, brain and gonads. No major differences in tissue levels or in general distribution pattern between males, females or juvenile fish were observed. The gills appeared to be the absorption site for exposure via water. After oral administration, tissue levels of ³H-BaP-derived radioactivity were negligible. Following both administration routes, levels of radioactivity were highest in the bile and intestinal contents while only traces were observed in the urine, indicating biliary excretion as the major excretory pathway in this aglomerular species. Tape-section autoradiography of fish exposed via water revealed tissue-bound residues of ³H-BaP in the olfactory organs, gills, kidney, liver, skin and intestinal mucosa. Light-microscopy autoradiography demonstrated that the bound residues in the olfactory organ, gills and anterior kidney were localized in epithelial cells, while those in liver and intestinal mucosa were evenly distributed. In conclusion, the present study shows that BaP is absorbed from the water via the gills at subzero temperatures, that tissue levels are considerably higher after water exposure than after dietary exposure, that biliary excretion is predominant and, finally, that site-specific tissue binding in the olfactory organs, gills and anterior kidney is confined to epithelial cells. Accepted: 3 January 2000     

Covalent binding of benzo[a]pyrene metabolites to DNA, RNA and chromatin proteins in the AKR mouse embryo cell-line

A specific fraction from the nuclei of the AKR mouse embryo cell-line (fraction I) displayed a much greater localization of radioactivity compared to fraction II and III when the chemical carcinogen, [3H]benzo[a]pyrene (B[a]P) was incubated with the cells for 24 h. The radioactivity in fraction I consisted of both covalently and non-covalently bound metabolites. Isolation of the DNA, RNA and protein of fraction I revealed that 94% of the covalently bound radioactivity was to protein, 5% to RNA and 1% to DNA. Analysis of the fraction I proteins by SDS gel electrophoresis revealed that there was more radioactivity covalently bound to the larger proteins than to smaller proteins. Isoelectric focusing (IEF) of the purified proteins displayed two peaks of radioactivity, one at a pH of 5 and the other at 11. The former proteins bound more radioactivity per mass of protein than the latter proteins. Analysis of fraction I histones on acid urea polyacrylamide gels showed that the radioactivity coincided with histones H3 and H2B and low levels of radioactivity associated with histones H1, H2A and H4. Two significant peaks of radioactivity closely migrated near but did not co-migrate with histone H1. The distribution of the bound radioactivity is probably a reflection of the availability of the proteins to the reactive carcinogen metabolites. The possible binding of B[a]P metabolites to phosphorylated histones and to the high mobility of group (HMG) proteins 1 and 2 is discussed.     

o,p'-DDD in the mouse lung: selective uptake, covalent binding and effect on drug metabolism

By means of autoradiography a high and selective accumulation was observed in the lung alveolar region of C57Bl mice injected with o,p'-[14C]DDD. Exhaustive extraction of lung tissue showed that a large fraction of the radioactivity was covalently bound to protein. Covalent binding in liver was 20-30 times lower and represented a smaller fraction of the total radioactivity present in this tissue. Formation of a cytochrome P-450 catalysed reactive metabolite in lung and liver was indicated by a decreased covalent binding in these tissues in mice pretreated with metyrapone. Both beta-naphthoflavone (beta NF) and phenobarbital (PB) pretreatment decreased binding of o,p'-DDD in lung tissue, while binding in the liver was induced by PB but remained unaffected by beta NF. Pretreatment with high doses of o,p'-DDD and p,p'-DDT gave a significantly decreased binding of o,p'-[14C]DDD in lung, whereas binding in liver remained unchanged. Conjugation with glutathione does not appear to be a major inactivation pathway for the reactive lung metabolite, since a high dose of o,p'-DDD did not deplete non-protein thiols (NPSH) in lung tissue. Pretreatment with o,p'-DDD decreased the N-demethylation of [dimethyl-14C]aminopyrine in both lung and liver in a dose-dependent manner, suggesting that the drug-metabolizing enzyme system may be a target for o,p'-DDD in vivo.     

The covalent binding of 3-methylindole metabolites to bovine tissue

Tritiated 3-methylindole (3MI) was administered intravenously to calves. Total and covalent bound radioactivity were measured in different tissues. Pulmonary tissue showed the highest concentration of covalent bound radioactivity. (G-³H) or (methyl-¹⁴C) 3MI became covalently bound to microsomal protein when incubated with bovine lung microsomes. This covalent binding was dependent on time, temperature, oxygen and NADPH and was inhibited by SKF-525A, cytochrome c, a carbon monoxide enriched atmosphere and cysteine. The microsomal enzyme system catalysing covalent binding of 3MI has the classical characteristics of a cytochrome P-450 dependent mixed function oxidase. Metabolic activation of 3MI to a highly electrophilic intermediate, may be fundamental in the pathogenesis of 3MI induced pulmonary damage.     

Comparison of ratios of covalent binding to total metabolism of the pulmonary toxin, 4-ipomeanol, In vitro in pulmonary and hepatic microsomes,and the effects of pretreatments with phenobarbital or 3-methylcholanthrene

Studies of the ratios of the amounts of 4-ipomeanol covalently bound to the total amounts metabolized support the view that the high rates of $\text{in}\text{vitro}$ pulmonary microsomal alkylation by 4-ipomeanol reflect high rates of NADPH-mediated metabolic activation of the compound rather than a relative deficiency of a microsomal detoxication pathway. Moreover, the ability of 3-methylcholanthene pretreatment, but not phenobarbital pretreatment, to shift the $\text{in}\text{vivo}$ target organ alkylation and toxicity of 4-ipomeanol from the lung to the liver in rats could not be explained by a major alteration in the balances between microsomal toxication and detoxication pathways measurable in the $\text{in}\text{vitro}$ systems examined, nor upon a major change in the nature of the reactive 4-ipomeanol metabolites produced in the lungs or livers of the pretreated animals.     

In vitro and in vivo binding of toremifene and its metabolites in rat uterus

The in vitro binding affinities of toremifene (TOR), 4-hydroxy toremifene (4-OH-TOR) and several other metabolites for the rat uterine cytosolic estrogen receptor were compared with those of tamoxifen (TAM) and 4-hydroxy tamoxifen (4-OH-TAM). Only small differences were observed and the binding affinities of both 4-hydroxy metabolites were similar to that of estradiol (E2). Uterine uptake and subcellular distribution of [3H]TOR and [3H]TAM were then compared at 1, 8 and 72 h after administration to castrated rats. The uptake and retention of both antiestrogens were similar at all times. In each case the amount of nuclear bound radioactivity declined to low levels at 8 and 72 h but the ratios of 4-OH-TAM/TAM and 4-OH-TOR/TOR determined by HPLC analysis increased dramatically at 72 h. The level of radioactivity in both plasma and uterine cytosol at 72 h was significantly higher following [3H]TAM administration. However, most of the radioactivity appeared to be in a conjugated form since it was not extractable with solvent. Finally, the ability of prior administration of each antiestrogen (100 mg/kg) to block uterine [3H]estradiol uptake was examined at 3 and 7 days. It was found that uterine wet weights were higher than control one week after administration of both compounds. Prior administration of TOR increased nuclear uptake of [3H]E2 whereas TAM had no effect. The results of these experiments suggest that toremifene and tamoxifen have very similar in vitro and in vivo binding properties but differences in metabolism exist that may be important.     

Metabolism of [11-3H]retinyl acetate in liver tissues of vitamin A-sufficient, -deficient and retinoic acid-supplemented rats

A study was conducted on the incorporation of [11-3H]retinyl acetate into various retinyl esters in liver tissues of rats either vitamin A-sufficient, vitamin A-deficient or vitamin A-deficient and maintained on retinoic acid. Further, the metabolism of [11-3H]retinyl acetate to polar metabolites in liver tissues of these three groups of animals was investigated. Retinol metabolites were analyzed by high-performance liquid chromatography. In vitamin A-sufficient rat liver, the incorporation of radioactivity into retinyl palmitate and stearate was observed at 0.25 h after the injection of the label. The label was further detected in retinyl laurate, myristate, palmitoleate, linoleate, pentadecanoate and heptadecanoate 3 h after the injection. The specific radioactivities (dpm/nmol) of all retinyl esters increased with time. However, the rate of increase in the specific radioactivity of retinyl laurate was found to be significantly higher (66-fold) than that of retinyl palmitate 24 h after the injection of the label. 7 days after the injection of the label, the specific radioactivity between different retinyl esters were found to be similar, indicating that newly dosed labelled vitamin A had now mixed uniformly with the endogenous pool of vitamin A in the liver. The esterification of labelled retinol was not detected in liver tissues of vitamin A-deficient or retinoic acid-supplemented rats at any of the time point studied. Among the polar metabolites analyzed, the formation of [3H]retinoic acid from [3H]retinyl acetate was found only in vitamin A-deficient rat liver 24 h after the injection of the label. A new polar metabolite of retinol (RM) was detected in liver of the three groups of animals. The formation of 3H-labelled metabolite RM from [3H]retinyl acetate was not detected until 7 days after the injection of the label in the vitamin A-sufficient rat liver, suggesting that metabolite RM could be derived from a more stable pool of vitamin A.     

Identification of radioactivity in cell nuclei from brain, pituitary gland and genital tract of male rhesus monkeys after the administration of [3H]testosterone

Enzymes are present in the primate brain that convert testosterone into 17 beta-hydroxy-5 alpha-androstan-3-one (dihydrotestosterone), estradiol-17 beta and 4-androstene-3,17-dione. To identify the metabolites of testosterone that accumulate in cell nuclei obtained from different regions of the brain, 9 adult castrated male rhesus monkeys were injected with 5 mCi [3H]testosterone as an intravenous bolus. After 1 h, brains were rapidly removed and the left halves were used for autoradiography while the right halves were dissected to provide 14 samples. Radioactive metabolites in cell nuclei were identified by high-performance liquid chromatography (HPLC) and by repeated recrystallization. In autoradiograms of brain, most of the labeled neurons were in the hypothalamus, preoptic area and amygdala. These three regions also had the highest levels of radioactivity. The major form of this radioactivity was [3H]estradiol-17 beta (Type I tissues) and the major radioactive androgen present was [3H]testosterone. In all other brain regions and pituitary gland, the major form of radioactivity was unchanged [3H]testosterone (Type II tissues). In genital tract structures, [3H]dihydrotestosterone predominated (Type III tissues). These results suggested that, in contrast to its actions on genital tract structures, testosterone acts on neuronal nuclei mainly in unmetabolized form or after conversion to estradiol-17 beta.     

Ubiquitous binding of benzo[a]pyrene metabolites to DNA and protein in tissues of the mouse and rabbit

The in vivo formation of benzo[alpha]pyrene (BP) metabolite-DNA adducts in several tissues of mice and rabbits was examined. Included were tissues with widely divergent xenobiotic metabolizing capabilities such as liver and brain. The major adduct identified in each tissue was the (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDEI)-deoxyguanosine adduct. A 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydro-BP (BPDEII)-deoxyguanosine adduct, a (-)-BPDEI-deoxyguanosine adduct and an unidentified adduct were also observed. These adducts were present in all of the tissues of the mice and in the lungs of the rabbits; only BPDEI and BPDEII were seen in the rest of the rabbit tissues. In all of the tissues studied, the DNA adduct levels were unexpectedly similar. For example, the BPDEI-DNA adduct levels in muscle and brain of mice were approx. 50% of those in lung and liver at each oral BP dose examined. After an i.v. dose of BP in rabbits, the BPDEI adduct levels in lung were three times those in brain or liver and twice those in muscle. The binding of BP metabolites to protein was also determined in these tissues. The tissue-to-tissue variation in protein binding levels of BP metabolites was greater than that for BPDEI-DNA adducts. There are several possible explanations for the in vivo binding of BP metabolites to DNA and protein of various tissues. First, oxidative metabolism of BP in each of the examined tissues might account for the observed binding. Second, reactive metabolites could be formed in tissues such as liver and lung and be transported to cells in tissues such as muscle and brain where they bind to DNA and protein. In any case, the tissue-to-tissue variations in protein and DNA binding of BP-derived radioactivity do not correlate with differences in cytochrome P-450 activity.     

Drug protein conjugates--III. Inhibition of the irreversible binding of ethinylestradiol to rat liver microsomal protein by mixed-function oxidase inhibitors, ascorbic acid and thiols

The metabolism of [6,7-3H]ethinylestradiol [( 3H]EE2) by rat liver microsomes was studied in vitro. After incubation of [3H]EE2 with rat liver microsomes for 20 min, 90% of the substrate was metabolised and 18% of the 3H-labelled material irreversibly bound to microsomal protein. Ascorbic acid (1 mM) decreased irreversible binding of 3H and produced an accumulation of 2-hydroxyethinylestradiol (2OH-EE2), while mixed-function oxidase inhibitors (0.5 mM) decreased binding of 3H to protein by inhibiting EE2 2-hydroxylation. Addition of thiols gave water-soluble metabolites which were characterised as 1(4)-thioether derivatives of 2OH-EE2 by co-chromatography with synthetic products. The results are consistent with the hypothesis that the chemically reactive metabolite of EE2 formed in vitro is either a quinone or o-semiquinone derived from 2OH-EE2 [1].     

Movement to bark and metabolism of xylem cytokinins in stems of Lupinus angustifolius

Following uptake of [(3)H]zeatin riboside and [(3)H]dihydrozeatin riboside by girdled lupin (Lupinus angustifolius L.) stems via the transpiration stream, rapid lateral movement of the radioactivity from xylem to bark was observed. Short-term studies with intact stems, and other studies with excised stem tissues, revealed that the ribosides and/or the corresponding nucleotides were the cytokinin forms which actually moved into the bark tissues. Relative to cytokinin metabolism in xylem plus pith, metabolism in bark was both more rapid and more complex. Riboside cleavage and formation of the O-acetylzeatin and O-acetyldihydrozeatin ribosides and nucleotides were almost completely confined to bark tissues. Exogenous (3)H-labelled O-acetylzeatin riboside was converted to zeatin riboside in bark tissue, but the presence of the acetyl group suppressed degradation to adenine metabolites. The sequestration and modification of xylem cytokinins by stem tissues probably contributes significantly to the cytokinin status of the shoot. New cytokinins identified by mass spectrometry in lupin were: O-acetyldihydrozeatin 9-riboside, a metabolite of exogenous dihydrozeatin riboside in stem bark; O-methylzeatin nucleotide and O-methyldihydrozeatin 9-riboside, metabolites of endogenous cytokinins in stem bark; O-methylzeatin nucleotide and O-methylzeatin 9-riboside, metabolites of exogenous zeatin riboside in excised pod walls.     

Thymus cell differentiation and in vivo T-cell migration. I. Migration of lectin-selected thymocytes

The in vivo quantitative distribution and tissue positioning of mouse thymocytes selected in vitro by Lyt phenotype and lectin binding properties were examined. Lyt 1+2- thymocytes were selected for by cytotoxic elimination; peanut agglutinin (PNA) and soybean agglutinin (SBA) binding and nonbinding thymocyte fractions were separated by an agglutinin technique. Selected cell suspensions were labelled in vitro with 51chromium (51Cr) or [3H]adenosine. Labeled washed cells were injected intravenously into syngeneic recipients which were killed at 1, 24 or 48 hr. In recipients of 51Cr-labeled cells, tissues were collected for gamma counting, and the overall percentage recovery of injected radiolabel from the various tissues was assessed. Tissues collected from recipients of [3H]adenosine-labeled cells were fixed, sectioned, and processed for autoradiography; the positioning of labeled cells within the tissues was determined. Selected Lyt 1+2-, PNA-, and SBA- sets all showed significantly enhanced entry into lymph nodes and intestinal lymphoid tissues. Entry of SBA+ cells into these tissues was comparable to that of peripheral T cells. PNA- and SBA- selected sets, but not Lyt 1+2- selected cells, also showed increased localization to the spleen and lungs, and decreased localization to the liver. By autoradiography, PNA- cells entered lymphoid tissues much more than PNA+ cells, and at 1 hr fewer PNA+ cells in spleen were associated with lymphoid follicles. At 24 and 48 hr almost all labeled cells in lymphoid tissues were positioned in T-dependent areas. These results suggest that enrichment for thymocyte subpopulations described as "mature" also enriches for cells with the ability to enter lymphoid tissue. They also suggest that interactions at other tissue sites are important in the determination of in vivo migration, and that surface carbohydrate composition is an important factor in this determination.     

Evidence that 1-naphthol is not an obligate intermediate in the covalent binding and the pulmonary bronchiolar necrosis by naphthalene

Recent studies of a number of volatile aromatic hydrocarbons have suggested that the formation of covalently bound metabolites arises solely through the intermediate formation of phenols. This study further examines the involvement of 1-naphthol in the in vivo and in vitro formation of covalently bound metabolites and pulmonary bronchiolar necrosis by naphthalene. Marked differences were observed in the rate of 1-naphthol formation in lung and liver microsomal incubations without correspondingly large differences between the rates of formation of covalently bound metabolites from naphthalene and 1-naphthol. Glutathione decreased covalent binding in hepatic microsomal incubations containing 14[C]1-naphthol but did not result in the formation of any of the glutathione adducts isolated from identical incubations containing 14[C]naphthalene. Tissue levels of covalently bound radioactivity in mice treated with 14[C]1-naphthol or 14[C]naphthalene were similar; however, in contrast to studies with naphthalene, 1-naphthol administration did not deplete tissue glutathione nor result in detectable tissue injury. These studies indicate that 1-naphthol is not an obligate intermediate in the formation of covalently bound metabolites from naphthalene nor does it appear to be a more proximate lung toxic metabolite.     

The binding of radioactive label from labelled phenacetin and related compounds to rat tissues in vivo and to nucleic acids and bovine plasma albumin in vitro

1. acetyl-(3)H- and ethyl-(14)C-labelled derivatives of phenacetin and related compounds are described. 2. Radioactive label from the ethyl-(14)C-labelled derivatives of 4-nitrophenetole, 4-phenetidine and phenacetin binds in vitro to various extents to bovine plasma albumin, salmon sperm DNA and yeast RNA; the extent of binding is increased in the presence of a rat liver microsomal hydroxylating system and further increased when the microsomal enymes are induced by prior treatment of rats with 3-methylcholanthrene. 3. The ratios of the bound radioactive labels in vitro from [ethyl-(14)C]phenacetin, N-acetoxy[ethyl-(14)C]phenacetin, [acetyl-(3)H]phenacetin and [diacetyl-(3)H]N-acetoxyphenacetin per g-atom of DNA P, RNA P and per mol of protein in the absence of the microsomal system are approximately 1:60:11:863, 1:68:41:1835 and 1:88:713:2399 respectively. 4. Radioactive label from labelled phenacetin binds in vitro to all tissues examined, including the spleen, intestines, kidney and bladder; about 80% of the radioactivity bound to the liver is concentrated in the RNA and proteins. 5. Comparison of the relative extents of binding of radioactive label derived from equimolar amounts of labelled phenacetin, ethanol or acetate shows that the incorporation of labelled C(2) units into tissues and biological macromolecules in vivo and in vitro may account for only a part of the total bound radioactive label derived from phenacetin and not at all from the incorporation of radioactive acetate into nucleic acids. 6. Some implications of these findings are discussed.     

High-density lipoprotein subpopulations as substrates for the transfer of cholesteryl esters to very-low-density lipoproteins.

1. Human total HDL (high-density lipoprotein), HDL2 and HDL3 were labelled in vitro by incubation with lipoprotein-deficient serum (LPDS) which contained either [3H]cholesteryl oleate or [14C]cholesterol under different conditions. The lipoproteins were then subfractionated by heparin-Sepharose column chromatography, and three subfractions (A, B and C) were successively eluted from each preparation of HDL, HDL2 and HDL3. When the labelling was done at 37 degrees C for 17 h, the subfractions were homogeneously labelled with [3H]cholesteryl oleate. However, when it was performed for only 30 min at 4 degrees C, the subfractions showed marked differences in the 3H specific radioactivity, which was much higher in the C fractions than in the others. 2. 3H-labelled HDL2 and HDL3 subfractions behaved differently under the precipitant action of heparin-Mn2+; fraction C (the richest in apolipoprotein E) produced the largest amount of radioactive and chemical precipitate. More 3H radioactivity, but not the cholesterol, was precipitated from HDL2 or HDL3 by the reagent, demonstrating that 3H-labelled HDL2 and HDL3 behave like their fraction C, which becomes labelled to the highest specific radioactivity despite having the smallest mass. 3. The incubation of 3H-labelled HDL subfractions with human LPDS and very-low-density lipoprotein (VLDL) at 37 degrees C increased the quantity of 3H radioactivity that was precipitated, in proportion to the amount of VLDL present in the media. These changes were attributable to the action of cholesterol ester transfer protein, since they did not occur at 4 degrees C or when human LPDS was replaced with rat LPDS. 4. Kinetics of the transfer of HDL [3H]cholesteryl oleate to VLDL showed a greater apparent Vmax for fractions A than for fractions B from either HDL2 or HDL3, whereas the apparent Km values were very similar, which suggest that this transfer process is influenced by the apoprotein composition of the donor lipoprotein.     

Concentration of 3-Methylindole (3MI) and distribution of radioactivity from 14C3MI in goat tissues associated with acute pulmonary edema

3-Methylindole (3MI) can cause acute pulmonary edema in goats. Because of known lipophilic properties and direct effects on biological membranes, the concentration of 3MI and distribution of radioactivity from ¹⁴C3MI in tissues was investigated during development of 3MI-induced APE. Goats were given a 2 hr jugular infusion of 3MI containing ¹⁴C3MI using propylene glycol as the vehicle. Groups of 3 goats were killed at 0, .5, 2 and 4 hr and 2 goats were killed at 8 and 24 hr. Plasma, lung, liver, kidney and other selected tissues were collected. 3MI was rapidly cleared from blood plasma and tissues after infusion, and 81% of the radioactivity was excreted in the urine by 24 hr. Maximum concentrations of unmetabolized 3MI in the tissues ranged from 2.6 to 15 μg 3MI/g, including 7.5 μg 3MI/g in the lung. The ratios of equivalent radioactivity to unmetabolized 3MI indicated rapid metabolism and the presence of metabolites in all tissues studied. The lung contained the highest proportion of metabolites with ratios of radioactivity to unmetabolized 3MI of about 50, 10, 250, 150 and 80 at 0.5, 2, 4, 8 and 24 hr. The data demonstrate that 3MI does not selectively concentrate in the lung and that the concentrations are lower than those usually associated with direct membrane damage. They also indicate that 3MI is rapidly metabolized and that metabolites are present in tissues, especially the lung. These results suggest that direct effects of 3MI on biological membranes are not primarily responsible for lung injury in goats.     

Synthesis and biodistribution of [(11)C]R116301, a promising PET ligand for central NK(1) receptors

N1-(2,6-Dimethylphenyl)-2-(4-{(2R,4S)-2-benzyl-1-[3,5-di(trifluoromethyl)[carbonyl-(11)C]benzoyl]hexahydro-4-pyridinyl}piperazino)acetamide ([(11)C]R116301) was prepared and evaluated as a potential positron emission tomography (PET) ligand for investigation of central neurokinin(1) (NK(1)) receptors. 1-Bromo-3,5-di(trifluoromethyl)benzene was converted in three steps into 3,5-di(trifluoromethyl)[carbonyl-(11)C]benzoyl chloride, which was reacted with N1-(2,6-dimethylphenyl)-2-{4-[(2R,4S)-2-benzylhexahydro-4-pyridinyl]piperazino}acetamide providing [(11)C]R116301 in 45-57% decay-corrected radiochemical yield. The total synthesis time, from end of bombardment (EOB) to the formulated product, was 35 min. Specific activity (SA) was 82-172 GBq/micromol (n=10) at the end of synthesis. N1-([4-(3)H]-2,6-Dimethylphenyl)-2-(4-{(2R,4S)-2-benzyl-1-[3,5-di(trifluoromethyl)benzoyl]hexahydro-4-pyridinyl}piperazino)acetamide ([(3)H]R116301) was also synthesized (SA: 467 GBq/mmol). The B(max) for [(3)H]R116301 measured in vitro on Chinese hamster ovary cell membranes stably transfected with the human NK(1) receptor was 19.10+/-1.02 pmol/mg protein with an apparent dissociation constant of 0.08+/-0.01 nM. Ex vivo, in vivo and in vitro autoradiography studies with [(3)H]R116301 in gerbils demonstrated a preferential accumulation of the radioactivity in the striatum, olfactory tubercule, olfactory bulb and locus coeruleus. In vivo, the biodistribution of [(11)C]R116301 in gerbils revealed that the highest initial uptake is in the lung, followed by the liver and kidney. In the brain, maximum accumulation was found in the olfactory tubercules (1.10+/-0.08 injected dose (ID)/g 20 min post injection (p.i.)) and the nucleus accumbens (1.00+/-0.12ID/g 10 min p.i.). Tissue/cerebellum concentration ratios for striatum and nucleus accumbens increased with time due to rapid uptake followed by a slow wash out (1.29 and 1.64, respectively, 30 min p.i.). A tissue to cerebellum ratio of 1.33 and 1.62 was also observed for olfactory bulb and olfactory tubercules, respectively (20 min p.i.). In summary, [(11)C]R116301 appears to be a promising radioligand suitable for the visualization of NK(1) receptors in vivo using PET.