H. Uehleke

Binding of 14C-Carbon Tetrachloride to Microsomal Proteins in vitro and Formation of CHCl3 by Reduced Liver Microsomes

Abstract1. Addition of CCl4 to suspensions of reduced liver microsomes produces a difference spectrum with max. absorption at 454 nm. This absorption develops within approx. 5 min (37°).2. Dehalogenation of CCl4 to CHCl3 by NADPH reduced liver microsomes displays similar kinetics.3. The irreversible binding of 14CCl4 to microsomal proteins proceeds more slowly than CHCl3 formation.4. The first step in CCl4 liver toxicity is the very rapid formation of fast reacting intermediates. This step might occur between the binding of CCl4 to cytochrome P-450 and the release of CHCl3. The primary intermediates bind to liver lipids and proteins and also initiate lipid peroxidation.

Bildung von Chloroform aus Tetrachlorkohlenstoff in Lebermikrosomen, Lipidperoxidation und Zerstörung von Cytochrom P-450

en]Abstract1.In reduced and anaerobic suspensions of rabbit liver microsomes 2% of added CCl4 (1 mM) is converted to CHCl3 within 5 min. The CHCl3 formation is 50% inhibited by metyrapone, 75% by CO and 95% by O2.2.Oral application of 2.5 ml/kg CCl4 to rats produces a 30 to 40% loss of endoplasmic cytochrome P-450 within the first 3 h. This decline is not prevented by treatment of the rats with metyrapone, desipramine, piperonylbutoxide, disulfiram or silymarine.3.The decrease of cytochrome P-450 can be initiated in vitro by aerobic incubation of isolated liver microsomes with NADPH, and is accelerated further by CCl4. A good correlation was found between the rate of lipid peroxidation and the disappearance of cytochrome P-450. EDTA or GSH block lipid peroxidation and, consequently, the content of cytochrome P-450 remains at control levels even in the presence of CCl4. UV-irradiation of suspensions of microsomes effects lipid peroxidation and rapid destruction of cytochrome P-450.4.During the incubation of liver microsomes with linolenic acid and lipoxygenase from soybeans a similar correlation between cytochrome P-450 decline and lipid peroxidation was observed. Addition of either linolenic acid or lipoxygenase had no effects. Experiments and results indicate a coupled mechanism of lipidperoxidation with simultaneous oxidative breakdown of the haeme moities.Zusammenfassung1.In reduzierten und anaeroben Suspensionen von Kaninchenlebermikrosomen werden 2% von zugegebenem CCl4 (1 mM) innerhalb von 5 min zu CHCl3 umgewandelt. Die Bildung von CHCl3 wird durch Metyrapon um 50%, durch CO um 75% und durch O2 um 95% gehemmt.2.Orale Gaben von 2,5 ml/kg CCl4 an Ratten bewirken innerhalb der ersten 3 Std einen Abfall des endoplasmatischen Cytochroms P-450 um 30 bis 40%. Diese Verringerung wird durch Behandlung der Ratten mit Metyrapon, Desipramin, Piperonylbutoxid, Disulfiram oder Silymarin nicht verhindert.3.Die Abnahme von Cytochrom P-450 kann in vitro durch aerobes Inkubieren von isolierten Lebermikrosomen mit NADPH in Gang gesetzt werden und wird durch CCl4 weiter beschleunigt. Zwischen dem Ausmaß der Lipidperoxidation und der Abnahme von Cytochrom P-450 wurde eine gute Korrelation gefunden. ÄDTA oder GSH verhindern die Lipidperoxidation. Infolgedessen ändert sich der Gehalt an Cytochrom P-450 sogar in Gegenwart von CCl4 gegenüber den Kontrollen nicht. UV-Bestrahlung von Mikrosomensuspensionen bewirkt Lipidperoxidation und schnelle Zerstörung von Cytochrom P-450.4.Während der Inkubation von Lebermikrosomen mit Linolensäure und Lipoxygenase aus Sojabohnen wurde eine ähnliche Beziehung zwischen der Abnahme von Cytochrom P-450 und der Lipidperoxidation beobachtet. Zugabe von Linolensäure oder Lipoxygenase allein hatte keine Wirkungen. Die Untersuchungen und Befunde sprechen für einen gekoppelten Mechanismus der Lipidperoxidation mit gleichzeitiger oxidativer Zerstörung des Häm-Anteils.

A comparative study on the irreversible binding of labeled halothane trichlorofluoromethane, chloroform, and carbon tetrachloride to hepatic protein and lipids in vitro and in vivo.

Summary1)After intraperitoneal injection of labeled CCl4, CHCl3, and halothane in mice, 14C is preferentially bound to liver endoplasmic protein and lipid. A considerable activity is also associated with mitochondrial constituents. Maximal protein binding (nmol/mg): CCl4: 2.8 (0.5 hrs); CHCl3: 11.5 (6 hrs); halothane: 5 (6 hrs). Lipid binding: CCl4: 6.4 (5 min); CHCl3: 8 (4 hrs); halothane: 13.5 (2 hrs). The form of the binding curves in microsomal and mitochondrial protein and lipid differed with the individual haloalkanes.2)The irreversible (covalent) binding of 14C from labeled haloalkanes in anaerobic suspensions of isolated rabbit liver microsomes and NADPH after 30 min was for protein (lipid) (nmol/mg): CCl4: 15 (58); CHCl3: 3.4 (3.2); halothane: 2.3 (10); trichlorofluoromethane: 6.5 (30). Anaerobic incubation favored dehalogenation, but CHCl3 metabolism and irreversible binding requires oxygen. The greatest differences in the in vitro “covalent” binding rates were observed with CHCl3 in rat, mouse, and rabbit.3)Altered microsomal cytochrome P-450 concentrations in newborn animals, or produced by pretreatment of rats with phenobarbital, 3-methylcholanthrene (MC), or CoCl2 effected similar, but not proportional changes in the rates of irreversible protein and lipid binding. Upon addition of CCl4 the difference of light absorption of reduced liver microsomes from MC-pretreated rats containing cytochrome P-448 appeared at 452 nm. The irreversible binding rate in these microsomes was also increased. The small acceleration in irreversible binding in liver microsomes from rats pretreated with isopropanol is not proportional to the high increase of CCl4 toxicity.4)Practically no binding to added, soluble albumin or RNA was observed in microsomal incubates. However, 14C is bound to the nicotine-adenine dinucleotides of the NADPH system. All haloalkanes produced a similar increase of NADPH oxidation in incubates of rabbit liver microsomes and NADPH.Zusammenfassung1)Nach intraperitonealer Injektion von markiertem CCl4, CHCl3 und Halothan an Mäuse wird 14C bevorzugt an Protein und Lipid des endoplasmatischen Reticulums gebunden. Eine bemerkenswerte Aktivität befindet sich auch in Bestandteilen der Mitochondrien. Die höchste Proteinbindung betrug (nMol/mg): CCl4: 2,8 (0,5 hrs); CHCl3: 11,5 (6 hrs); Halothan: 5,0 (6 hrs). Bindung an Lipide: CCl4: 6,4 (5 min); CHCl3: 8,0 (4 hrs); Halothan: 13,5 (2 hrs). Der Verlauf der Bindung an mikrosomale und mitochondriale Proteine und Lipide war bei den einzelnen Haloalkanen unterschiedlich.2)Die irreversible Bindung von 14C aus markierten Haloalkanen in aeroben Suspensionen von isolierten Kaninchen-Lebermikrosomen und NADPH betrug nach 30 min für Protein (Lipid): CCl4: 15 (38); CHCl3: 3,4 (3,2); Halothan: 2,3 (10,0); Trichlorfluormethan: 6,5 (30). Inkubation unter anaeroben Bedingungen begünstigte die Enthalogenierung, jedoch ist Sauerstoff für den Stoffwechsel und für die irreversible Bindung erforderlich. Die größten Unterschiede der Bindungsgeschwindigkeiten wurden bei CHCl3 mit Lebermikrosomen von Ratten, Mäusen und Kaninchen gefunden.3)Abweichende Konzentrationen von Cytochrom P-450 in Mikrosomen von neugeborenen Tieren oder nach Vorbehandlung von Ratten mit Phenobarbital, Methylcholanthren (MC) oder CoCl2 bewirkten ähnliche, aber nicht proportionale Änderungen der covalenten Bindungen an Protein und Lipid. Die Absorptionsdifferenz von reduzierten Lebermikrosomen von MC-vorbehandelten Ratten mit Cytochrom P-448 lag nach Zugabe von CCl4 bei 452. Die covalenten Bindungen mit solchen Mikrosomen waren ebenfalls erhöht. Die geringe Beschleunigung derirreversiblen Bindungen mit Lebermikrosomen von Ratten nach Vorbehandlung mit Isopropanol steht jedoch in keinem Verhältnis zu dem starken Anstieg der Toxicität von CCl4.4)In Mikrosomenansätzen wurde praktisch keine Bindung an zugesetztes lösliches Albumin oder an RNS beobachtet. Jedoch wird 14C an Nicotin-Adenin-Dinucleotide des NADPH-Systems gebunden. Alle Haloalkane bewirkten eine ähnliche Beschleunigung der NADPH-Oxidation bei der Inkubation von Kaninchen-Lebermikrosomen und NADPH.

Irreversible binding of 14C-labelled trichloroethylene to mice liver constituents in vivo and in vitro

Abstract1.14C-labelled trichloroethylene was injected i.p. into male mice (10 μmole/g of b.w.). The radioactivity irreversibly bound to hepatic protein reached highest levels after 6 h: 2 nmole/mg in cytosol protein, 4.4 nmole/mg in mitochondrial protein, and 7.6 nmole/mg in microsomal protein.2.The commercial trichloroethylene contained radioactive impurities binding to proteins without metabolic activation. Purification by various extractions removed 60–70% of those materials. In aerobic incubates of mice hepatic microsomes and NADPH the covalent binding rate of the purified trichloroethylene was 1.4 nmole/mg protein in 60 min. The activity of rat liver microsomes was approximately 40% less. Covalent binding increased 2-fold with microsomes of mice pretreated with phenobarbital.

Spectral evidence for 2,2,3-trichloro-oxirane formation during microsomal trichloroethylene oxidation

During aerobic incubation of trichloroethylene with rabbit liver microsomes and NADPH a difference absorption peak appears at 451–452 nm. Trichloroethylene does not form a ligand absorption spectrum with hepatic microsomes reduced by dithionite, or in anaerobic incubates in the presence of NADPH.Addition of trichloroethylene epoxide (2,2,3-trichloro-oxirane) to reduced suspensions of rabbit liver microsomes produces high difference absorption at 452 nm, the optical Ks being approximately 2 mM. Of all possible metabolites of trichloroethylene only trichloroethanol forms absorption in the vicinity of 480 nm, and the broad absorption band reveals relatively low absorption near 450 nm. Dichloroacetyl chloride is the main thermal rearrangement product of trichloroethylene epoxide, and also produces 452 nm absorption in reduced microsomes. However, the difference absorption is 5 times smaller than the absorption produced by the intermediate formed during incubation of trichloroethylene in metabolising liver microsomes.These observations include strong evidence for epoxide formation during microsomal oxidation of trichloroethylene. 14C-labelled trichloroethylene binds irreversibly to hepatic macromolecules in vivo and in vitro. Possible rearrangement pathways of 2,2,3-trichloro-oxirane and reactive intermediates are presented.ZusammenfassungWährend der aeroben Inkubation von Trichloräthylen mit Lebermikrosomen von Kaninchen und NADPH erscheint eine Differenz-Absorption mit dem Maximum bei 451–452 nm. Trichloräthylen bildet kein Liganden-Absorptionsspektrum mit Dithionit-reduzierten Lebermikrosomen oder bei anaerober Inkubation in Gegenwart von NADPH.Die Zugabe von Trichloräthylen-Epoxid (2,2,3-Trichloroxiran) zu reduzierten Suspensionen von Lebermikrosomen von Kaninchen erzeugt eine hohe Differenzabsorption bei 452 nm; der optische Ks liegt ungefähr bei 2 mM. Von allen möglichen Metaboliten des Trichloräthylens erzeugt nur Trichloräthanol eine Absorption im Bereich von 480 nm, und die breite Absorptionsbande zeigt vergleichsweise geringe Absorption in der Nähe von 450 nm. Dichloracetylchlorid ist das hauptsächliche thermische Umlagerungsprodukt von Trichloräthylen-Epoxid und erzeugt ebenfalls eine Absorption bei 452 nm in reduzierten Mikrosomen. Die Differenzabsorption ist allerdings fünfmal kleiner als die während der Inkubation von Trichloräthylen in stoffwechselnden Lebermikrosomen durch gebildete Zwischenprodukte erzeugte Absorption.Die Befunde deuten stark auf die Bildung eines Epoxids bei der mikrosomalen Oxidation von Trichloräthylen hin. 14C-markiertes Trichloräthylen wird in vivo und in vitro irreversibel an Makromoleküle der Leber gebunden. Mögliche Umlagerungswege von 2,2,3-Trichloroxiran und reaktionsfähige Zwischenprodukte werden dargestellt.

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

SummaryAfter i.p. injection of 3-14C-antipyrine (10 μmole=1.9 mg with 10 μCi 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.

Kinetics and metabolism of 2,2-diethylallylacetamide in dog and man

SummaryIn dogs, slow intravenous injection of 100 mg of diethylallylacetamide (DA) resulted in maximal blood levels of 10–14 μg/ml, and a rapid phase of elimination during 90 min with a half life of 45 min, followed by a slower elimination rate with a half life of 80–90 min.After oral application of 30 mg DA/kg to female beagle dogs, maximal blood levels of 14 μg/ml were observed after 90–120 min. The blood concentrations declined with a mean half life of 5 h.In human volunteers, oral doses of 250 mg DA, or rectal application of 300 mg DA produced highest mean blood levels of 4.8 μg/ml (orally), and 5.4 μg/ml (rectally) after 180 min. The mean blood half life was 7.2 h (orally), and 9.2 h (rectal application). Undesirable effects such as nausea, vomiting, and disorientation began to appear at blood levels above 5 μg/ml.In the urine of dogs and human volunteers, only 2–3% of unchanged DA was recovered, and less than 1% of 2,2-diethyl-4,5-dihydroxypentanoic acid-γ-lactone (DA-lactone) was identified. Acid hydrolysis of the human urine liberated a total of 14–16% of DA-lactone. This percentage was not increased by splitting the urinary conjugates with glucuronidase and glusulase. Small amounts of 2,2-diethylallylacetic acid, 2,2-diethyl-4-one-pentanoic acid, and 2,2-diethyl-4,5-dihydroxypentanamide were detected.The new metabolites described were synthetized and fully characterized.

Inhibition of microsomal drug oxidations by aliphatic halohydrocarbons: correlation with vapour pressure

1. The effects of 12 halogenated aliphatic compounds on microsomal N-dealkylation, C-hydroxylation and N-oxidation of N-methylaniline and N-hydroxylation of p-chloroaniline were determined in closed reaction vessels. 2. There is no correlation between the volatility of the agents investigated and their binding characteristics to oxidized or reduced microsomal cytochrome P-450. 3. High correlation was observed between inhibiton of cytochrome P-450-mediated drug oxidations and the boiling points (vapour pressure) of the individual compounds. The N-oxidation of N-methylaniline, which is not catalysed by cytochrome P-450, did not show this correlation. 4. Other factors e.g. ligand formation with reduced cytochrome P-450, lipid peroxidation and haem destruction, production of carbon monoxide, and alterations of microsomal cofactors, did not produce significant inhibition. 5. Many artefacts occur when reaction velocities, inhibition constants and optical affinities to microsomal cytochromes are determined for volatile chemicals under uncontrolled conditions.