Irreversible binding of 14C-diphenyl ether-derived radioactivity to liver microsomes in vitro and tissue proteins in vivo

Following ip administration of 14C-diphenyl ether (5 mg/kg) to rats, a significant amount of radioactivity was found to bind irreversibly with tissue proteins of liver, lung and kidney, indicating the in vivo formation of reactive diphenyl ether metabolite(s). Irreversibly bound 14C-diphenyl ether-derived radioactivity to these organs peaked 2-4 h after the chemical was administered. Incubation of 14C-diphenyl ether in rat liver microsomes demonstrated that the hepatic mixed-function oxidase system mediated the formation of the chemically reactive metabolite(s) of 14C-diphenyl ether. The apparent Vmax and Km were, respectively, 23.3 pmol/mg microsomal protein/min and 1.33 x 10(-4)M.     

Irreversible in vivo and in vitro binding of thiabendazole to tissue protein of pregnant mice

In vivo experiments showed that [¹⁴C]thiabendazole is irreversibly bound to protein of the liver and embryo in pregnant mice, but not to DNA and RNA in these tissues. In experiments carried out in vitro, the binding of [¹⁴C]thiabendazole to microsomal protein of the liver was proportional to time and protein concentration, was dependent upon the presence of oxygen and NADPH and was inhibited by carbon monoxide or SKF-525A, indicating that it was mediated by the cytochrome P-450-dependent monooxygenase system. The binding capacity of microsomes in various tissues was examined, and that of the liver microsomes was found to be more pronounced than that of other tissues.     

Irreversible binding of 3-14C-antipyrine to hepatic protein in vivo and in metabolizing liver microsomes

Summary After i.p. injection of 3-¹⁴C-antipyrine (10 mole=1.9 mg with 10 Ci per 10 g of body weight) to mice radioactivity was irreversibly bound to liver proteins. The irreversible binding reached maximal values of 0.15 nmole/mg protein in liver microsomes after 30–60 min.During 60 min incubation with liver microsomes of mice and rabbits (phenobarbital pretreated) and a NADPH-regenerating system 3-¹⁴C-antipyrine was irreversibly bound to microsomal protein at a rate of 1.5 nmole/mg protein (mouse) and 3 nmole/mg protein (rabbit).In identical incubates with rabbit liver microsomes the 4-hydroxylation of antipyrine was 24 nmole/mg protein in 60 min and formaldehyde production from antipyrine 3 nmole/mg protein in 60 min.In incubates with rabbit liver microsomes the binding rate was 80–90% inhibited by 1mM metyrapone, SKF 525-A and trichloropropene epoxide respectively; 4-hydroxylation was 70–80% inhibited by the same substances. In the presence of 1 mM GSH, cysteine or ethylene diamine binding was 30–40% inhibited, whereas 4-hydroxylation showed no inhibition.Some of the results were presented at the 17th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Mainz, March 1976     

Irreversible protein binding of 14C-imipramine in rats in vivo

Forty-eight hours after a single dose of ¹⁴C-imipramine to rats, ¹⁴C-radioactivity could be measured in the following organs: liver > kidney > serum > fat > spleen > duodenum > lung > muscle and brain. Liver microsomes contained the main part of radioactivity derived from ¹⁴C-imipramine.After exhaustive extraction, only the proteins of liver, kidney, spleen, lung and serum contained measurable amounts of radioactive labeling. The greatest amount of ¹⁴C-imipramine irreversibly bound to proteins was detected in liver microsomes.The question, as to whether the irreversible protein binding of imipramine, if it occurs during therapy, results in toxic side-effects, is discussed.     

Irreversible in vivo binding of thiabendazole to macromolecules in pregnant mice and its relation to teratogenicity

The binding of [14C]thiabendazole ([14C]TBZ) to macromolecules in the liver, foetus and other tissues was investigated in Jcl:ICR mice on day 13 of gestation. TBZ suspended in olive oil was given orally in a dose of 1 g/kg body weight (5 microCi/mouse) and the mice (in groups of three) were killed 0.5, 1, 3, 6, 24 and 96 hr later. The bound radioactivity in the liver and foetus was at a maximum between 3 and 24 hr after treatment. The rate of decrease of the bound radioactivity was slower than that of total radioactivity. Bound radioactivity was also present in other tissues (including kidney, lung, heart, placenta and spleen). The level of bound radioactivity was measured in the liver and foetuses after oral administration of teratogenic doses of 200-1600 mg/kg. Disproportionate increases in bound radioactivity were observed in both tissues after administration of the highest dose.     

Metabolism of [14C]trichloroethylene to 14CO2 and interaction of a metabolite with liver DNA in rats and mice

Male Sprague-Dawley rats and male B6C3F1 mice excreted 5-15% of a tracer dose of [14C]trichloroethylene as 14CO2 within 24 h after ip injection of a single dose in a corn-oil vehicle. The proportion of the dose excreted as CO2 was greater in mice than in rats, but increased in the rats after starvation or pretreatment with phenobarbital. As the dose was increased toward the LD50 level, the proportion excreted as 14CO2 decreased slightly, but this was largely due to increased loss of unchanged trichloroethylene. The excretion of 14CO2 was thus correlated with the expected level of microsomal metabolism of trichloroethylene to an electrophilic intermediate capable of binding to glutathione or macromolecules. Liver protein labeling was observed to be relatively high (10,000-23,000 cpm/mg in the mouse), while DNA labeling was consistently observed to be very low, not allowing identification of any adducts by high-performance liquid chromatography (HPLC). Also, no effect on DNA fragmentation was seen by alkaline sucrose gradient centrifugation after injection of an LD50 dose of trichloroethylene. The ability of trichloroethylene to interact with DNA in vivo was thus observed to be very slight.     

Covalent binding of procainamide in vitro and in vivo to hepatic protein in mice

Procainamide (PA) formed reactive metabolites capable of covalently binding to protein both in vivo and in vitro. The in vitro covalent binding of PA to washed hepatic microsomal protein prepared from control male mice was dependent upon mixed-function oxidase activity. The binding was proportional with time and protein concentration. Glutathione and SKF 525-A inhibited the in vitro covalent binding by 88 and 51%, respectively. The addition of NaF to the incubation medium produced a concentration-dependent decrease in covalent binding. Covalent binding of N-acetylprocainamide in vitro was 90% less than that of procainamide and was not increased by NaF. The in vivo covalent binding of PA to hepatic protein in male mice was increased with phenobarbital and 3-methylcholanthrene pretreatment, resulting in increase in binding of 29 and 56%, respectively, compared to control mice. Pretreatment of mice with SKF 525-A inhibited binding by 39%. Depletion of hepatic glutathione with diethyl maleate pretreatment increased the amount of covalent binding in vivo. Bioactivation of PA by hepatic microsomal enzymes in the mouse produces a metabolite capable of covalent interactions with cellular macromolecules.     

In vivo and in vitro binding of alpha- and gamma-hexachlorocyclohexane to mouse liver macromolecules

alpha-Hexachlorocyclohexane (alpha-HCH) but not gamma-HCH, produces a strong neoplastic response in HPB strain mouse liver. In vivo and in vitro binding studies with 14C-labelled HCH isomers showed no preferential binding of the alpha-HCH isomer to protein or DNA. Moreover, the binding of both isomers to DNA in vivo or in vitro occurred only at very low levels, a result consistent with the lack of mutagenic activity associated with these compounds. The results suggest that the neoplastic response observed with alpha-HCH results from a non-genotoxic mechanism.     

Irreversible protein binding of [14-C]imipramine with rat and human liver microsomes.

After incubation of [¹⁴C]imipramine with rat liver microsomcs up to 0–7 mole/mg was irreversibly bound per mg of microsomal protein. If albumin was added to the microsomal incubations [¹⁴C]imiprámine was also irreversibly bound to this protein. The irreversible binding of imipramine to protein was determined by exhaustive solvent extraction and charcoal adsorption, and measurement of the remaining ¹⁴C-radioactivity in the protein. The binding reaction was dependent on oxygen, NADPH, microsomal protein content and substrate concentration. It was inhibited by CO and SKF 525-A. Pretreatment of rats with phenobarbital did not increase the amount of imipramine irreversibly bound to protein. Glutathione and other cysteine derivatives diminished the binding, whereas incubation with the epoxide hydrase inhibitor trichloropropene oxide resulted in an increase of imipramine irreversibly bound to protein. The results favour the concept that irreversible protein binding of imipramine is catalyzed by a cytochrome P-450-dependent hydroxylation via an epoxidation step. Irreversible protein binding of imipramine was also detectable with three samples of human liver microsomes.     

Irreversible binding and metabolism of propranolol by human liver microsomes--relationship to polymorphic oxidation

Studies were performed to investigate the irreversible binding and oxidative metabolism of propranolol in human liver microsomes and the relationship of binding and metabolism to the polymorphic oxidation of debrisoquine. Incubation of microsomes with 14C-labelled propranolol in the presence of a NADPH-generating system gave rise to irreversible binding which increased linearly with time and became saturated at high substrate concentrations. The extent of binding was decreased by the exclusion of cofactors, boiling, anaerobic conditions, and the addition of reduced glutathione and SKF-525A. Trichloropropene oxide had a negligible effect on cofactor-dependent binding. However, debrisoquine, antipyrine and phenacetin decreased binding to a considerable extent. The latter compound abolished cofactor-dependent binding completely at the concentration used (1 mM). Electrophoresis of microsomes which had been incubated with tritiated propranolol revealed that binding was probably occurring to a large number of proteins particularly in the 40,000-90,000 molecular weight range. Glutathione, debrisoquine and antipyrine did not inhibit the 4'-hydroxylation and N-deisopropylation of propranolol. In contrast, phenacetin exerted a very potent inhibitory action on both routes of metabolism. It is concluded that a product or products of propranolol oxidation bind irreversibly but non-selectively to human liver microsomal protein, the enzyme system responsible for the activation of propranolol appears to be related more closely to the cytochrome P-450 system which metabolizes phenacetin than to that metabolising debrisoquine, and radiolabelled propranolol is not a sufficiently specific probe for studying these cytochrome P-450 systems.     

Irreversible binding of 14C from 14CCl4 to liver microsomal lipids and proteins from rats pretreated with compounds altering microsomal mixed-function oxygenase activity

¹⁴C from ¹⁴CCl₄ irreversibly binds to lipid and protein components of different liver subcellular fractions. Microsomal lipids bind more ¹⁴C than does either mitochondria or 105,000 g supernatant. The proteins from the 105,000 g fraction bind more ¹⁴C than those from the other two fractions. The extent of the irreversible binding of ¹⁴C to microsomal lipids and proteins is decreased by the prior treatment of the rats with cystamine, pyrazole, 3-methylcholanthrene or metopirone while it is increased by pretreatment with phenobarbital. The prior treatment with 2-diethylaminoethyl 2,2-diphenyl valerate hydrochloride (SKF 525A) increased the irreversible binding of ¹⁴C to microsomal lipids, but not to microsomal proteins. The results suggest that the irreversible binding of ¹⁴C to microsomal lipids is a more reliable expression of the CCl₄-activation step than the one to microsomal proteins. The ¹⁴CCl₃ free radicals responsible for the binding of ¹⁴C to lipid would arise during the reduction of the CCl₄/cytochrome P-450 complex mediated by cytochrome P-450 reductase.     

Lack of covalent binding of peroxisome proliferators nafenopin and Wy-14 643 to DNA in vivo and in vitro

[3H][2-methyl-2-p-(1,2,3,4-tetrahydro-naphthyl)phenoxy] propionic acid (nafenopin), a hepatocarcinogenic peroxisome proliferator, when administered p.o. to normal intact and partially hepatectomized male F344 rats did not show any significant binding to DNA and RNA, but bound to proteins. The in vitro incubation of [3H]nafenopin and [3H]4-chloro-[6-(2,3-xylidino)pyrimidinylthio]acetic acid (Wy-14643), another peroxisome proliferator, with hepatic microsomes and calf thymus DNA also showed no significant binding of these chemicals to DNA.     

A note on the absorption and excretion of 14C-labelled thalidomide in pregnant mice

After oral administration of ¹⁴C-labelled thalidomide to pregnant mice, similar concentrations of the drug are found in several maternal tissues and in the placenta and foetus. Thalidomide and one of its hydrolysis products, α-(o-carboxybenzamido)-glutarimide, have been identified in the foetus.     

Pulmonary toxicity of trichloroethylene in mice. Covalent binding and morphological manifestations

We examined the time course of trichloroethylene (TCE)-induced pulmonary injury and focused on morphological changes and covalent binding of [14C]TCE soon after administration of a single dose of TCE (2000 mg/kg) to CD-1 male mice. At 1 hr after chemical treatment, Clara cells of the bronchiolar epithelium exhibited necrotic changes involving the mitochondria and endoplasmic reticulum. Dilatation of the endoplasmic reticulum became more severe at 2 hr after TCE administration and, by 4 hr, distended cisternae coalesced to form small vacuoles within the cytoplasmic matrix of the Clara cell. The severity of cellular damage increased progressively between 8 and 12 hr and, by 24 hr, the majority of Clara cells within an airway were severely vacuolated. Covalent binding of [14C]TCE to lung macromolecules was evident at 1 hr, peaked at 4 hr, declined thereafter, and reached a plateau between 12 and 24 hr. Peak binding (142.6 +/- 31.8 nmol/g of wet weight) represented approximately 20% of [14C]TCE distributed to the lung. Although the levels of binding in the liver were at all times greater than those in the lung, liver injury was relatively insignificant. The results demonstrate a positive correlation between the onset of Clara cell injury and the formation of reactive metabolites, as assessed by covalent binding of [14C]TCE.     

Evaluation of anaphylactoid constituents in vitro and in vivo

Natural medicine injections have been widely used in clinics, while adverse reaction reports also have increased rapidly in recent years. To examine the anaphylactoid constituents of natural medicine injections, RBL-2H3 cell degranulation and human serum complement activation models were used to screen the anaphylactoid constituents, and the BN rat model was used to explore the anaphylactoid mechanism of these constituents. The result of an in vitro study showed that the individual compounds of natural medicine injections (chlorogenic acid, hyodeoxycholic acid, cholalic acid, ginkgolic acid, phillyrin, schisandrin B, schisandrin A, puerarin, and tanshinone IIA) and polysaccharide could not induce RBL-2H3 to release histamine and β-hexosaminidase, while proteins Tween-80 and tannic acid were the main anaphylactoid constituents in the natural medicine injections. The in vivo study also indicated that > 10 kDa molecules (proteins) activated classical complement pathways through direct stimulation to cause an anaphylactoid reaction. Tween-80 activated direct stimulation and coagulation pathways through classical and alternative pathways; tannic acid induced anaphylactoid reaction through co-activation of the kallikrein-kinin system, coagulation, integrated, classical and alternative complement pathways. This is the first study to evaluate the anaphylactoid constituents systematically through in vitro and in vivo study. And tannic acid, > 10 kDa molecules (proteins), and injection additives such as Tween-80 are the main anaphylactoid constituents of natural medicine injections.     

Synthesis of 11C-labelled bis(phenylalkyl)amines and their in vitro and in vivo binding properties in rodent and monkey brains

Two new (11)C-labelled ligands, N-(3-(4-hydroxyphenyl)propyl)-3-(4-methoxyphenyl)propylamine ([(11)C]2) and N-(3-(4-hydroxyphenyl)butyl)-3-(4-methoxyphenyl)butylamine ([(11)C]3) were designed based on bis(phenylalkyl)amines (1) which have been reported as polyamine site antagonists with high-selectivity for NR1A/2B NMDA receptors, and radiolabelling of the corresponding phenol precursors with [(11)C]methyl iodide was readily accomplished. The in vitro inhibition experiments using rat brain slices showed that [(11)C]2 and [(11)C]3 share the binding sites with spermine and/or ifenprodil but not with CP-101,606, a highly potent NR2B-selective NMDA antagonist, and that divalent cations such as Zn(2+) produced significant inhibition of both [(11)C]2 and [(11)C]3 bindings. Intravenous injection of [(11)C]3 in mice showed almost homogeneous distribution throughout the brain. Attempts to block the tracer uptake of [(11)C]3 by pre-injection with the unlabelled 3 or spermine in rats were unsuccessful, but a small decrease in the cerebral uptake of [(11)C]3 by co-treatment with the unlabelled 3 was observed in a monkey PET study. The present findings indicate that none of these (11)C-labelled analogues have potential for PET study of binding sites on the N-methyl-D-aspartate (NMDA) receptors.