Role of mixed-function oxidases and non-protein sulfhydryl content in [14C]-2-chloro-4-acetotoluidide binding to liver and kidney in starlings

The compound 2-chloro-4-acetotoluidide (CAT) is highly toxic to many avian species, including the starling. In our earlier work, we demonstrated the covalent binding of radioactivity from [14C]-CAT to liver and kidneys of the starling. In the present study, the effects of inducers of mixed-function oxidase (MFO) and non-protein sulfhydryl (NPSH) depletor on the total and covalent binding of [14C]-CAT radioactivity to liver and kidney of the starling were examined. The total and covalently bound radioactivity from [14C]-CAT to liver and kidney were decreased significantly in the starling pretreated with the MFO inducer, 3-methylcholanthrene. However, pretreatment with phenobarbital, another inducer of MFO, had no effect. Pretreatment with the inhibitor of MFO, SKF 525-A, reduced the covalent binding of [14C]-CAT radioactivity to liver but not to kidney. There was no reduction in the NPSH content of liver or kidney following intravenous administration of CAT (3.5 mg kg-1). Reduction of NPSH levels in the liver or kidney following treatment with diethyl maleate caused a significant increase in the covalent binding of [14C]-CAT to kidney but not to liver.     

Metabolism and covalent binding of [14C]toluene by human and rat liver microsomal fractions and liver slices

The in vitro metabolism of [14C]toluene by liver microsomes and liver slices from male Fischer F344 rats and human subjects has been compared. Rat liver microsomes produced only benzyl alcohol from toluene. Liver microsomes from human subjects metabolized toluene to benzyl alcohol, benzaldehyde, and benzoic acid. Liver microsomes from one human donor also produced p-cresol and o-cresol. The overall rate of toluene metabolism by human liver microsomes was 9-fold greater than by rat liver microsomes. Human liver microsomal metabolism of benzyl alcohol to benzaldehyde required NADPH and was inhibited by carbon monoxide and high pH (pH 10). but was not inhibited by ADP-ribose or sodium azide. These results suggest that cytochrome P-450, rather than alcohol dehydrogenase, was responsible for the metabolism of benzyl alcohol to benzaldehyde. Human and rat liver slices metabolized toluene to hippuric acid and benzoic acid. The overall rate of toluene metabolism by human liver slices was 1.3-fold greater than by rat liver slices. Cresols and cresol conjugates were not detected in human or rat liver slice incubations. Covalent binding of [14C]toluene to human liver microsomes and slices was 21-fold and 4-fold greater than to the comparable rat liver preparations. Covalent binding did not occur in the absence of NADPH, was significantly decreased by coincubation with cysteine, glutathione, or superoxide dismutase, and was unaffected by coincubation with lysine. Protease and ribonuclease digestion decreased the amount of toluene covalently bound to human liver microsomes by 78% and 27% respectively. Acid washing of human liver microsomes had no effect on covalent binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro studies on the metabolism and covalent binding of [14C]1,1-dichloroethylene by mouse liver, kidney and lung

The metabolism and covalent binding of 1,1-dichloro[1,2-14C]ethylene (DCE) to subcellular fractions of liver, kidney and lung of C57BL/6N mice have been investigated in vitro. Covalent binding was NADPH- and cytochrome P-450-dependent. The microsomal fraction bound more radiolabel than any other subcellular fraction, and the levels of covalent binding in cell fractions correlated well with their cytochrome P-450 content. Covalent binding by mouse liver and lung microsomes also reflected their cytochrome P-450 content. However, although mouse kidney microsomes contained twice as much total cytochrome P-450 as the lung, no detectable covalent binding of DCE-derived radioactivity occurred in kidney. Omission of NADPH, heat inactivation of microsomes, carbon monoxide, addition of SKF-525A, piperonyl butoxide or reduced glutathione (GSH), all inhibited (40-90%) covalent binding of radiolabel to liver and lung microsomes. The absence of O2 (incubation under N2) did not greatly affect the metabolism and covalent binding. Pretreatment of mice with various inducers, phenobarbital (PB), beta-naphthoflavone (beta-NF), pregnenolone 16 alpha-carbonitrile (PCN) and 3-methylcholanthrene (3-MC), evoked increases in total liver microsomal cytochrome P-450 content (2-fold) and corresponding increases in covalent binding (3-fold). However, microsomes from PCN-treated mice showed only a 50% increase in DCE binding. Kidney microsomes from control, PB-, and beta-NF-pretreated mice were incapable of covalent binding of radiolabel but those from PCN- and 3-MC-pretreated mice showed levels of binding similar to untreated mouse lung microsomes. It is proposed that the nephrotoxicity of DCE may be due to translocation of reactive metabolites from the liver to the kidney.     

Characteristics of (+/-)-[14C]-oxprenolol and (+/-)-[14C]-propranolol incorporation by rat lung slices.

1 The uptake of (+/-)-[14C]-oxprenolol and (+/-)-[14C]propranolol has been studied in rat lung slices. The loss of these two radiolabelled beta-adrenoceptor antagonists from pre-loaded lung slices has also been studied. 2 Over the concentration range studied (3.3 x 10(-7)M to 1.7 x 10(-3)M) a biphasic uptake of both compounds was observed. At concentration below 1.5 x 10(-4)M approximately, there was some evidence of saturability, but at higher concentrations uptake appeared to be a linear function of drug concentration. 3 At concentrations of 6.6 x 10(-7)M and 1.7 x 10(-6)M respectively, the uptake of oxprenolol and propranolol was significantly reduced by low temperature, anaerobic conditions, incubation in NA+-free medium, and by the metabolic inhibitors potassium cyanide and 2,4-dinitrophenol. Ouabain had little or no effect. 4 At the same concentration, oxprenolol uptake was also inhibited in a concentration-dependent manner by propranolol, amphetamine, chlorpromazine and imipramine. Noradrenaline was without effect. 5 The loss of oxprenolol and propranolol from lung slices preloaded with the two compounds was fairly slow, with 60 to 70% of the drug originally taken up still remaining in the tissue after 30 min in fresh medium. 6 Possible mechanisms underlying the pulmonary accumulation of oxprenolol and propranolol are discussed.     

Comparative studies of the in vitro metabolism and covalent binding of 14C-benzene by liver slices and microsomal fraction of mouse, rat, and human

Metabolism of benzene by the liver has been suggested to play an important role in the hepatotoxicity of benzene. The role of the different benzene metabolites and the causes of species differences in benzene hepatotoxicity are, however, not known. The metabolism and covalent binding of 14C-benzene by liver microsomal fractions and liver slices from rat, mouse, and human subjects have been studied. Rat microsomal fraction formed phenol at a rate of 0.32 nmol/min/mg of protein; mouse microsomal fraction formed phenol at 0.64 nmol/min/mg and hydroquinone at 0.03 nmol/min/mg; and human microsomal fraction formed phenol at 0.46 nmol/min/mg and hydroquinone at 0.07 nmol/min/mg. Covalent binding of 14C-benzene metabolites to rat, mouse, and human liver microsomal protein was 29, 113, and 169 pmol/min/mg of protein, respectively. The rates of metabolite formation from benzene by liver slices in nmol/min/g of tissue were: rat, phenol 0.15, hydroquinone 0.26, and phenylsulfate 1.22; mouse: phenol 0.13, hydroquinone 0.29, phenylsulfate 1.37, and phenylglucuronide 1.34; and human: phenol 0.16, hydroquinone 0.27, phenylsulfate 0.83, and phenylglucuronide 0.52. trans,trans-Muconic acid formation was not detected with liver slices of any species. Covalent binding of 14C-benzene metabolites to rat, mouse, and human liver slices was 8.2, 79.7, and 27.3 pmol/min/g liver, respectively. There was no correlation between ascorbic acid levels in the human liver slices and covalent binding of 14C-benzene metabolites. The results show that phenol and hydroquinone found in extrahepatic tissues, including bone marrow, of animals exposed to benzene could originate from the liver. There was no evidence for the release of highly reactive benzene metabolites such as trans,trans-muconaldehyde or p-benzoquinone from liver cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further studies of the metabolic incorporation and covalent binding of inhaled [3H]- and [14C]formaldehyde in Fischer-344 rats: effects of glutathione depletion

Glutathione (GSH) is required for the oxidation of formaldehyde (HCHO) to formate catalyzed by formaldehyde dehydrogenase (FDH). The effects of GSH depletion on the mechanisms of labeling of macromolecules in the rat nasal mucosa and bone marrow by 3HCHO and H14CHO were investigated. Male rats were exposed for 3 hr to atmospheres containing 3HCHO and H14CHO at concentrations of 0.9, 2, 4, 6, or 10 ppm, 1 day after a single 3-hr preexposure to the same concentration of unlabeled HCHO. Two hours prior to the second exposure, the animals were injected either with phorone (300 mg/kg, ip) or with corn oil. The concentration of nonprotein sulfhydryls in the nasal respiratory mucosa of phorone-injected rats was decreased to 10% of that of corn oil-injected rats. The metabolic incorporation of 3HCHO and H14CHO into DNA, RNA, and proteins in the respiratory and olfactory mucosa and bone marrow (femur) was significantly decreased, and DNA-protein crosslinking was significantly increased in the respiratory mucosa of phorone-injected relative to corn oil-injected rats at all HCHO concentrations. DNA-protein crosslinks were not detected in the respiratory mucosa of corn oil-injected rats at 0.9 ppm. Evidence was obtained for the formation of adducts of HCHO with the RNA from the nasal respiratory mucosa of phorone-injected rats at concentrations above 0.9 ppm. Covalent binding of HCHO to macromolecules in the bone marrow was not detected. These results indicate that the GSH-dependent oxidation of HCHO catalyzed by FDH is an important defense mechanism against the covalent reactions of HCHO with nucleic acids in the respiratory mucosa. Experiments using phorone-injected rats exposed to 10 ppm of [3H]- and [14C]formaldehyde showed that the DNA from the respiratory mucosa was enriched in 3H relative to 14C in comparison to the inhaled vapor. The enrichment is explained by an isotope effect in the oxidation of 3HCHO and H14CHO (H. d'A, Heck and M. Casanova (1987). Toxicol. Appl. Pharmacol. 89, 122-134), which results in 3H enrichment of the residual (unoxidized) HCHO that binds to DNA. A non-linear pharmacokinetic model is proposed that depicts the potential effects of FDH saturation on the relative concentrations of intracellular to extracellular HCHO.     

Autoradiographic disposition of [1-methyl-14C]- and [2-14C]caffeine in mice

Male, C57B1/6J mice received either [1-methyl-¹⁴C]caffeine or [2-¹⁴C]caffeine via the tail vein at a dose of 0.7 or 11 mg/kg, respectively. At 0.1, 0.33, 1, 3, 9, and 24 hr after treatment, the mice were anesthetized with ether and frozen by immersion in dry ice/hexane. The mice were processed for whole-body autoradiography by the Ullberg technique; this procedure does not allow thawing or contact with solvents. All autoradiographs revealed some retention of radioactivity at early time intervals in the lacrimal glands, seminal vesicle fluid, nasal and olfactory epithelium, and retinal melanocytes. The remaining portion of the animal was densitometrically uniform except for the lower levels noted in the CNS and adipose tissues. Excretion of radioactivity by the liver and kidneys seems to be the major routes of elimination. Localization in the liver at late time intervals was confined principally to the centrilobular region. Late sites of retention, observed only after [1-methyl-¹⁴C]caffeine administration, included the pancreas, minor and major salivary glands, splenic red pulp, thymal cortex, bone marrow, and gastrointestinal epithelium. Sites of localization present in both studies included the olfactory epithelium, lacrimal glands, hair follicles, and retinal melanocytes. Further studies are needed to determine whether the localization at these various sites is due to metabolic degradation, active transport, or possibly a specific receptor interaction.     

Non-enzymatic covalent binding of radioactivity from [14C]thiourea to rat lung protein

In vitro covalent binding of radioactivity from [14C]thiourea (TU) to rat lung protein occurs in the presence of 100% CO2. Prior boiling of lung slices results in a subsequent increase in covalent binding. Covalently bound radioactivity was released from rat lung homogenates and acid-insoluble protein obtained from animals treated with [14C]TU. The release was achieved with ammonium ion. One possibility suggested by these results is that the bound species may involve binding of isothiocyanic acid via a thiocarbamylation reaction.     

Isotope effects and their implications for the covalent binding of inhaled [3H]- and [14C]formaldehyde in the rat nasal mucosa

DNA-protein crosslinks were formed in the nasal respiratory mucosa of Fischer-344 rats exposed for 3 hr to selected concentrations of [3H]- and [14C]formaldehyde (3HCHO and H14CHO) (M. Casanova and H. d'A. Heck (1987). Toxicol. Appl. Pharmacol. 89, 105-121). In rats depleted of glutathione (GSH) and exposed to 10 ppm of 3HCHO and H14CHO, the 3H/14C ratio of the fraction of the DNA that was crosslinked to proteins was significantly (39 +/- 6%) higher than that of the inhaled gas. This suggests an isotope effect, either on the formation of DNA-protein crosslinks by labeled HCHO or on the oxidation of labeled HCHO catalyzed by formaldehyde (FDH) or aldehyde dehydrogenase (AldDH). The possibility of an isotope effect on the formation of crosslinks was investigated using rat hepatic nuclei incubated with [3H]- and [14C]formaldehyde (0.1 mM, 37 degrees C). A small (3.4 +/- 0.9%) isotope effect was detected on this reaction, which slightly favored 3HCHO over H14CHO in binding to DNA. The magnitude of this isotope effect cannot account for the high isotope ratio observed in the crosslinked DNA in vivo. The possibility of an isotope effect on the oxidation of 3HCHO and H14CHO catalyzed by FDH was investigated using homogenates of the rat nasal mucosa incubated with [3H]- and [14C]formaldehyde at total formaldehyde concentrations ranging from 0.1 to 11 microM, NAD+ (1 mM), GSH (15 mM), and pyrazole (1 mM). The experiments showed that 3HCHO is oxidized significantly more slowly than H14CHO under these conditions (Vmax/Km (H14CHO) divided by Vmax/Km (3HCHO) = 1.82 +/- 0.11). A similar isotope effect was observed in the absence of GSH, presumably due to the oxidation of 3HCHO and H14CHO catalyzed by AldDH. These results suggest that the residual (unoxidized) formaldehyde present in the nasal mucosa of rats exposed to [3H]- and [14C]formaldehyde may be "enriched" in 3HCHO relative to H14CHO, which can bind to DNA resulting in an isotope ratio higher than that of the inhaled gas. The isotope effect on the oxidation of 3HCHO and H14CHO suggests that previous estimates of the amount of HCHO covalently bound to nasal mucosal DNA (M. Casanova-Schmitz, T. B. Starr, and H. d'A. Heck (1984). Toxicol. Appl. Pharmacol. 76, 26-44) may have been too large, especially at low airborne concentrations and that the shape of the concentration-response curve for DNA-protein crosslinking is more nonlinear than reported previously.     

Percutaneous absorption of co-administered N-methyl-2-[14C]pyrrolidinone and 2-[14C]pyrrolidinone in the rat.

The percutaneous absorption has been investigated in rats of a mixture (3:2, w/w) of N-methyl-2-pyrrolidinone (NMP) and 2-pyrrolidinone (2-P), a combination intended for use as a vehicle in the formulation of an antimycotic drug to enhance skin penetration on dermal application, following co-administration of the two 14C-radiolabelled compounds by the dermal and oral routes. Radioactivity was excreted predominantly in the urine after either route of administration, and comparison of the respective excretion profiles indicated that about three-quarters of the applied dose was absorbed through the skin. Plasma concentrations of each parent compound, as determined by radio-HPLC, reached peak values at 2 hr after oral dosing, and remained relatively uniform during 1-6 hr after application to the skin, suggesting constant percutaneous absorption during this period. NMP appeared to be absorbed through the skin more extensively and at a slightly faster rate than 2-P; total percutaneous absorption tended to be more extensive in female than in male rats. Together, these two 14C-compounds accounted for most of the plasma radioactivity up to 6-8 hr post-administration. However, by 12 hr (when plasma levels were relatively low), most of the radioactivity was associated with unknown polar metabolites. In view of the extensive percutaneous absorption and little first-pass metabolism of the two pyrrolidinones, the oral route was considered to represent a valid alternative to the dermal route for the assessment of the systemic toxicity of the two compounds.     

Human metabolism of [1-methyl-14C]- and [2-14C]caffeine after oral administration

Radiolabeled caffeine was administered orally at 5 mg/kg to adult, male volunteers. Blood, saliva, expired CO2, urine, and feces were collected and analyzed for total radiolabeled equivalents, caffeine, and its metabolites. High-performance liquid chromatography (HPLC) was the principal technique used to separate caffeine and the various metabolites with quantitation by liquid-scintillation counting. The half-life of caffeine in both serum and saliva was approximately 3 hr, with the concentration of caffeine in the saliva samples ranging from 65 to 85% of that found in the serum samples. The major metabolites found in serum and saliva were the dimethylxanthines. In the course of separating the urinary metabolites, our HPLC system partially resolved two unidentified polar metabolites arising from radiolabeled caffeine. The major component corresponded to 5-acetylamino-6-amino-3-methyluracil and in our subjects ranged from 7 to 35% of the administered dose. The other principal urinary metabolites were 1-methylxanthine at approximately 18% of the administered dose and 1-methyluric acid at 15%. The fecal samples contained approximately 5% of the dose, mainly as uric acid compounds which retained the 1-methyl group. In this study we accounted for approximately 90% of the administered radiolabeled dose and identified greater than 95% of the urinary radioactivity as specific metabolites.     

Structural specificity for inhibition of [14C]-5-hydroxytryptamine uptake by cerebral slices

Cerebral slices were prepared from mice pretreated with reserpine and nialamide. The slices were incubated for 40 min at 37° in a Krebs-Henseleit solution equilibrated with 5% carbon dioxide in oxygen and containing [¹⁴C]-5-hydroxytryptamine. An uptake, dependent on energy metabolism and temperature, was observed. The uptake was blocked by tricyclic antidepressants, the order of activity being chlorimipramine > imipramine > desipramine. For 50% inhibition 0.03 μg/ml of chlorimipramine was required, when added to the suspension medium, or 1 mg/kg, when injected intraperitoneally 20 min beforehand. Comparison with earlier experiments using different experimental techniques appears to justify the conclusion that the evidence obtained represents the uptake mechanism of cerebral 5-hydroxytryptamine neurons.     

The in-vitro metabolism of [14C]pentobarbitone and [14C]phenobarbitone by hamster liver microsomes

The metabolism of [14C]pentobarbitone and [14C]phenobarbitone has been reinvestigated using an in-vitro hepatic microsomal system (Syrian hamsters, Aroclor 1254 induction). The incubation system was routinely supplemented with EDTA (1 mM) and a substrate concentration study revealed the metabolism of [14C]pentobarbitone to be concentration-dependent, with the greatest overall metabolism (greater than 50%) occurring at 0.054 mumol per 3.5 mL. With [14C]phenobarbitone as substrate, overall metabolism was extremely low (3%) and independent of substrate concentration. Addition of further cofactors to the incubation mixture at 20 min intervals over an extended period resulted in almost complete metabolism of [14C]pentobarbitone (100 min), 3'-hydroxypentobarbitone and 3'-oxopentobarbitone being identified as metabolites together with many minor, unidentified products. With [14C]phenobarbitone as the substrate, cofactor addition up to 120 min resulted in 8% overall metabolism; p-hydroxyphenobarbitone was identified as a product of metabolism; other minor products were unidentified. The metabolism studies failed to produce a metabolite having the properties of the N-hydroxylated product of either [14C]pentobarbitone or [14C]phenobarbitone within the detection limits available (0.02% of 0.5 mumol per incubate).     

Synthesis of [2H4]oxymetazoline and [14C]oxymetazoline

An efficient synthesis of [²H₄] and [¹⁴C]oxymetazoline has been developed. Both compounds follow the same synthetic route with the introduction of the label occurring at different synthetic steps. The synthesis of [²H₄]oxymetazoline from [²H₄]ethylene diamine was achieved in one step with a 40% yield. The synthesis of [¹⁴C]oxymetazoline from potassium [¹⁴C]cyanide was achieved in two steps with an overall radiochemical yield of 67%. Copyright © 2010 John Wiley & Sons, Ltd.     

Covalent binding of [14C]-2,6-dimethylaniline to DNA of rat liver and ethmoid turbinate

The xylidide 2,6-dimethylaniline (2,6-DMA) has produced carcinomas and papillary adenomas in the nasal cavity of rats at high dietary doses (3000 ppm) in a 2-yr bioassay. The objective of the present study was to measure the covalent binding of 2,6-DMA to DNA of rat ethmoid turbinate tissues and, for comparison, to DNA of rat liver. The potent hepatocarcinogen 2-acetylaminofluorene (AAF) was studied as a positive control for adduct formation and covalent binding index (CBI) calculation. Both 2,6-DMA and AAF were administered as 14C-(ring)-labeled agents to naive rats and to rats pretreated for 9 d with unlabeled 2,6-DMA or AAF. The CBI value for 2,6-DMA adduct formation with ethmoid turbinate DNA was below the assay's sensitivity limit in nonpretreated rats, but increased to 41.9 in rats pretreated with unlabeled 2,6-DMA. It also increased from 0.6 in nonpretreated to 7.9 in liver of pretreated rats. The opposite pattern, however, was observed for AAF. In nonpretreated rats considerable adduct formation was observed in liver (CBI = 271.5) and modest values (CBI = 39.3) were calculated for ethmoid turbinate tissues. Pretreatment with unlabeled AAF caused a significant decrease in CBI values, to 18.3 for liver and less than 0.5 for ethmoid turbinate. The results suggest that there may be value in conducting DNA covalent binding assays in both naive animals and animals pretreated with the test article.     

Rat liver microsomal uptake and irreversible protein binding of [1,2-14C]vinyl bromide.

When rat liver microsomes are incubated in a [¹⁴C]vinyl bromide atmosphere, rapid equilibration of vinyl bromide occurs between the gas and the liquid incubation phases. The presence of NADPH in the incubation further increases the uptake of radioactivity into the incubation mixture which is related to microsomal metabolism of vinyl bromide. Saturation of metabolizing enzymes occurs at a partial pressure of vinyl bromide in the gas phase of 0.1 Torr, which is about one order of magnitude less than previously reported for vinyl chloride. The presence of 1,2,3-benzothiadiazole derivatives and SKF 525 A inhibits microsomal metabolism of vinyl bromide. Part of the vinyl bromide metabolites bind irreversibly to the microsomal protein. Irreversible binding depends on NADPH, with the V_max amounting to 6.6 pmol bound/mg of microsomal protein/min. The half-maximal binding occurs at a partial pressure of vinyl bromide in the gas phase of 0.022 Torr. If soluble proteins are added to the microsomal incubation, vinyl bromide metabolites also irreversibly bind to those proteins. Strain differences occur in metabolism of vinyl bromide by liver microsomes from Sprague-Dawley and Wistar rats.     

The binding of [14C]phenethylhydrazine to rat liver monoamine oxidase

A study has been made of the binding of the active site-directed inhibitor [¹⁴C]phenethylhydrazine to native rat liver monoamine oxidase (MAO), its multiple forms and sub-units. Inhibition of enzyme activity was time-dependent and was accompanied by irreversible binding of the drug to the enzyme protein. When fully inhibited, the ratio of moles inhibitor bound per 150.000 g of enzyme was in all cases approximately 1:1. It is concluded that each molecule of native enzyme and its multiple forms consists of two sub-units, only one of which possesses an active site. Other evidence presented suggests that the preparation contains two types of MAO.     

Metabolism of [14C]-5-chloro-1,3-benzodioxol-4-amine in male Wistar-derived rats following intraperitoneal administration

[¹⁴C]-5-chloro-1,3-benzodioxol-4-amine was administered intraperitoneally (i.p.) to bile duct-cannulated rats (Alpk:ApfSD, Wistar derived) at 25?mg?kg^?1 to determine the rates and routes of excretion of the compound and to investigate its metabolic fate. A total of 89.1% of the dose was excreted in the 48?h following administration, the majority being recovered in the urine during the first 12?h. The main metabolite in both urine and bile, detected by high-performance liquid chromatography (HPLC) with radioprofiling and mass spectrometry, was identified as a demethylenated monosulfate conjugate. Unchanged parent compound formed a major component of the radiolabel excreted in urine and, in addition to unchanged parent and demethylenated sulphate conjugate, a large number of minor metabolites were detected in urine and bile. The overall metabolic fate of 5-chloro-1,3-benzodioxol-4-amine in the rat was complex, with some similarities to previously studied methylenedioxyphenyl compounds.