Amount Of Covalent Binding Research Articles

Species and Strain Differences in the Hepatic Cytochrome P450-Mediated Biotransformation of 1,4-Dichlorobenzene

Our goal was to characterize possible species and strain differences in the hepatic microsomal biotransformation of 1,4-dichlorobenzene (1,4-DCB). Experiments compared extent of labeled 1,4-DCB conversion to oxidized metabolites, glutathione conjugates, and covalently bound metabolites by hepatic microsomes from humans, from male B6C3F1 mice, and from males of three rat strains (Fischer 344, Sprague–Dawley (SD), and Wistar). These rodents were selected for comparison because of their dissimilar responses to 1,4-DCB, notably, hepatocarcinogenicity in the B6C3F1 mouse but not the Wistar or Fischer rat, and nephrotoxicity and carcinogenicity in the Fischer rat. The species rank order for totalin vitroconversion of 1,4-DCB was mouse > rat >> human. Conversion by microsomes from Fischer and Wistar rats was similar, whereas SD rats showed less biotransformation than the other two strains. Microsomes from the mouse produced most of the reactive metabolites as indicated by covalent binding to macromolecules (>20% of total metabolites formed). This covalent binding by mouse microsomes was extensively inhibited by ascorbic acid (AA), with a concomitant increase in hydroquinone formation, indicating an important role for benzoquinones as reactive metabolites. Phenobarbital pretreatment of rats enhanced thein vitroconversion of 1,4-DCB and the amount of covalent binding. Covalent binding for all rat microsomes was partly (33–79%) inhibited by AA. Addition of glutathione (GSH) plus AA further diminished the covalent binding with concomitant increased formation of the GSH-conjugated epoxide. Human microsomes produced the least reactive metabolites, with the majority (>70%) of this covalent binding prevented by GSH addition. The observed species differences, notably the more pronounced biotransformation of 1,4-DCB to reactive species including benzoquinones, could be factors in this compound's liver carcinogenicity in B6C3F1 mice but not other rodent species.

Metabolism of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) in isolated liver cells from guinea pig, hamster, mouse, and rat.

The metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant compound of the aminoimidazoazaarens (AIA) group of mutagens/carcinogens isolated from the crust of fried and broiled meat, was examined in freshly isolated hepatocytes from untreated rat, mouse, hamster, and guinea pig. Activation was evaluated by the total level of covalent binding of PhIP to macromolecules. Rat hepatocytes had the lowest rate of metabolism, both to reactive and detoxified metabolites. The products were identified as 4'-PhIP-sulfate, PhIP-glucuronide, and N(OH)-PhIP-glucuronide. The ring hydroxylation rate was much greater in mouse hepatocytes, the main products being 4'-PhIP-sulfate and 4-hydroxy-PhIP. The level of covalent binding in the mouse hepatocytes exceeded those of the rat and guinea pig at high doses of PhIP. An extensive metabolism was seen in guinea pig hepatocytes, the major products being 4'-PhIP-sulfate, 4'-O-PhIP glucuronide, PhIP-glucuronide, and N(OH)-PhIP-glucuronide. In addition, several other unknown metabolites were formed. However, the amount of covalent binding in guinea pig hepatocytes was similar to that in rat hepatocytes. Covalent binding of PhIP metabolites was highest in hamster hepatocytes. Three of the main metabolites were identified as 4'-PhIP-sulfate, 4'-O-PhIP-glucuronide, and PhIP-glucuronide, but several unknown PhIP metabolites also were formed. Only minor amounts of N(OH)-PhIP-glucuronide were produced in the hamster. The present study shows that both the direct detoxification of PhIP and further conjugation of the 2-hydroxylamino-PhIP to reactive and/or detoxified metabolites are important for the resulting covalent binding.

Dose-response study on covalent binding to forestomach protein from male F344 rats following oral administration of [14C]3-BHA.

A dose-response study on covalent binding to forestomach protein was performed using male F344 rats following oral administration of [14C]3-tert-butyl-4-hydroxyanisole (3-BHA). The order of tissue distribution of radioactivity 6 h after oral administration of 1% [14C]3-BHA was forestomach greater than glandular stomach greater than liver greater than kidney greater than plasma. The covalent binding levels to forestomach protein were very low until 0.1% 3-BHA, but rapidly increased at concentrations of 1% and 2% 3-BHA. The dose-response relations of 3-BHA levels to the covalent binding to protein coincided well with the incidence of forestomach papilloma reported previously. The binding levels of forestomach and glandular stomach were compared. In case of i.v. administration, both binding levels were almost the same, however, in case of 0.1% p.o. administration, the forestomach level was approximately 8-fold higher than the glandular stomach level. The binding level of forestomach protein by p.o. administration was approximately 54-fold higher than that by i.v. administration. Although the amount of tert-butylhydroquinone (BHQ) was very low compared with the amount of covalent binding, the BHQ levels in forestomach were dependent upon the dose levels of 3-BHA. Our study indicates that the dose-response study on covalent binding to target tissue protein is an efficient method for the quantitative estimation of the active metabolites coming from the chemicals which form the quinone metabolites.

In Vitro Covalent Binding of New Brain Tracer, Para125I-Amphetamine, to Rat Liver and Lung Microsomes

p-125I-amphetamine (I-Amp) is retained significantly in liver and lung during brain tomoscintigraphy. To attempt to explain this clinical observation, we have investigated the interaction of I-Amp with rat liver and lung microsomal proteins. Studies using spectral shift technique indicate that low concentration of I-Amp gives a type I complex and high concentration appears very stable type II complex with cytochrome P-450 Fe III. In the presence of NADPH, I-Amp gives rise to a 455 nm absorbing complex with similar properties to the Fe-RNO complexes. This complex formation was greatly enhanced with phenobarbital treated liver microsomes. The in vitro binding study shows that I-Amp and/or its metabolites was covalently bound to macromolecules in the presence of the molecular oxygen and NADPH-generating system. Incubation in the presence of glutathione, cystein and radical scavengers decreases binding. Mixed function oxydase (MFO) inhibitors diminish the amount of covalent binding and alter the extent of metabolite formation. The total covalent binding level increased with liver microsomes from PB pretreated rats as it was observed with the 455nm complex formation. The radioactivity distribution on microsomal proteins was examinated with SDS polyacrylamide gel electrophoresis and autoradiography. This experiment proves that the radiolabelled compounds are bound on the cytochrome P-450. The radioactivity bound increased when the PB induced rat liver microsomes were used. All these results indicate that I-Amp was activated by an oxydative process dependent on the MFO system which suggests a N-oxydation of I-Amp and the formation of reactive entities which covalently bind to proteins.

Selective protein arylation and the age dependency of acetaminophen hepatotoxicity in mice

Male CD-1 mice 1, 1.5, 2, and 3 months old were given 600 mg of acetaminophen (APAP)/kg, po, and liver damage was assessed 12 hr later. The most severe hepatotoxicity was in 3-month-old mice, while the other age groups exhibited little damage. The onset of susceptibility to APAP hepatotoxicity did not correlate with the level of activity of the mixed-function oxidase system as assessed in vitro, since drug metabolizing capability was similar between 2- and 3-month-old mice. Through 4 hr after administration of APAP to 2- and 3-month-old mice in vivo, glutathione (GSH) depletion and both plasma and liver APAP concentrations were similar between ages. Additionally, 24 hr after dosing, 3-month-old mice excreted marginally more APAP-glucuronide conjugate and parent compound in urine than 2-month-old animals, while both age groups excreted similar amounts of the APAP-sulfate and GSH-derived conjugates. Even though the extent of binding of radioactive APAP to macromolecules at 4 hr was similar between 2- and 3-month-old animals, the pattern of immunochemically targetted cytosolic and microsomal proteins was different. Thus, in APAP exposure the extent of binding to specific proteins rather than the overall amount of covalent binding may be the critical determinant of the hepatotoxic response. In the present study, the age-related differences in susceptibility to APAP-induced hepatotoxicity were related to the differences in selective protein arylation.

THE BINDING OF ACETALDEHYDE TO THE ACTIVE SITE OF RIBONUCLEASE: ALTERATIONS IN CATALYTIC ACTIVITY AND EFFECTS OF PHOSPHATE

Ribonuclease A was reacted with [1-13C,1,2-14C]acetaldehyde and sodium cyanoborohydride in the presence or absence of 0.2 M phosphate. After several hours of incubation at 4 degrees C (pH 7.4) stable acetaldehyde-RNase adducts were formed, and the extent of their formation was similar regardless of the presence of phosphate. Although the total amount of covalent binding was comparable in the absence or presence of phosphate, this active site ligand prevented the inhibition of enzymatic activity seen in its absence. This protective action of phosphate diminished with progressive ethylation of RNase, indicating that the reversible association of phosphate with the active site lysyl residue was overcome by the irreversible process of reductive ethylation. Modified RNase was analysed using 13C proton decoupled NMR spectroscopy. Peaks arising from the covalent binding of enriched acetaldehyde to free amino groups in the absence of phosphate were as follows: NH2-terminal alpha amino group, 47.3 ppm; bulk ethylation at epsilon amino groups of nonessential lysyl residues, 43.0 ppm; and the epsilon amino group of lysine-41 at the active site, 47.4 ppm. In the spectrum of RNase ethylated in the presence of phosphate, the peak at 47.4 ppm was absent. When RNase was selectively premethylated in the presence of phosphate, to block all but the active site lysyl residues and then ethylated in its absence, the signal at 43.0 ppm was greatly diminished, and that arising from the active site lysyl residue at 47.4 ppm was enhanced. These results indicate that phosphate specifically protected the active site lysine from reaction with acetaldehyde, and that modification of this lysine by acetaldehyde adduct formation resulted in inhibition of catalytic activity.

Lack of correlation between formation of reactive metabolites and thymic atrophy caused by 3, 4, 3', 4'-tetrachlorobiphenyl in C57BL/6N mice.

The possible role of active metabolites of 3, 4, 3', 4'-tetrachlorobiphenyl (TCB) in causing thymic atrophy was investigated using inbred strains of mice. The generation of reactive species which bind covalently to cellular proteins was used to monitor the formation of active TCB metabolites. The amount of in vitro covalent binding of TCB to proteins by liver microsomes was increased markedly by pretreatment of AHH-responsive C57BL/6N mice with either 3-methylcholanthrene (MC) or TCB itself, although these two inducers were not effective in AHH-nonresponsive DBA/2N mice. MC treatment also caused an induction of microsomal TCB-binding activity in all of the (C57BL/6N X DBA/2N) F1 mice. Moreover, among 38 individuals of [(C57BL/6N) (DBA/2N) F1 X DBA/2N] backcross, 23 mice responded to MC with respect to microsomal TCB-binding activity while others did not. These results suggest that the conversion of TCB to protein-bound metabolites is mediated by particular form(s) of cytochrome P-450 which is (are) induced by an Ah receptor mechanism. In order to ascertain whether the active TCB metabolites play a role in causing thymic atrophy, 14C-labeled TCB was administered IP to C57BL/6N mice and the amount of covalent binding of radioactive metabolites to tissue proteins was determined. The in vivo binding was evident in the liver, particularly in the microsomal fraction, on the basis of protein content. In contrast, the thymic proteins contained no measurable amounts of bound radioactivity even when the mice showed marked thymic atrophy. These data suggest that thymic atrophy caused by TCB is not likely to result from the generation of reactive metabolites.

Development of a novel method for measuring covalent binding and its application to investigations of bromobenzene hepatotoxicity.

The covalent binding of chemicals or metabolites to cellular tissue macromolecules may represent an important mechanism for chemical toxicity. Covalent interactions have been suggested to be involved in hepatic, renal, pulmonary and bone marrow toxicity as well as carcinogenicity (Mitchell et al., 1976; Mitchell et al., 1977; Hinson, 1980; Gillette et al., 1974; Boyd, 1980; Irons et al., 1980; Miller, 1970). More recently it has become apparent that the amount of covalent binding measured in a biological system may not be the most toxicologically relevant parameter. Gillette (1974) emphasized that reactive metabolites may bind to noncritical macromolecules and such interaction may be of little or no functional significance. To further our understanding of the role of covalent binding in chemical toxicity it is necessary to study the specificity of covalent binding.

Effect of lipid peroxidation on covalent binding of active metabolite of acetaminophen to liver microsomal macromolecules in several animal species.

There was good correlation of lipid peroxidation and covalent binding of acetaminophen (AAP) to liver microsomal protein in mice, guinea pigs and rabbits, but not in rats. By increasing concentrations of EDTA which is an inhibitor of lipid peroxidation, the degree of microsomal lipid peroxidation decreased in mice, guinea pigs, rabbits and rats, and the amount of covalent binding of AAP metabolite to liver microsomal protein increased in mice, guinea pigs and rabbits, but no consistent results were observed in rats. GSH depletion caused by AAP metabolite was increased in accordance with increase in the concentration of EDTA in mice and rats. In rats, the metabolism of AAP increased with increasing concentrations of EDTA and, in contrast to liver microsomes, the radioactivity of enzyme-mediated 14C-AAP metabolite in liver microsomal lipid was not observed. These results indicated that the decrease in covalent binding for AAP metabolite to liver microsomal protein with increasing concentrations of EDTA was not due to the decrease in the amount of AAP metabolite and the binding of 14C-AAP metabolite to liver microsomal lipid layer.

The metabolism of benzo(a)pyrene, 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene and 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene by short-term organ cultures of hamster lung

The metabolism of benzo(a)pyrene and two of its metabolites 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene (7,8-dihydrodiol) and 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene (9,10-dihydrodiol) to both ethyl acetate-soluble and water-soluble metabolites has been studied using short-term organ cultures of hamster lung. Benzo(a)pyrene is metabolised to ethyl acetate-soluble metabolites which co-chromatograph with 9,10-dihydrodiol, 7,8-dihydrodiol and benzo(a)pyren-3-yl hydrogen sulphate but little or no 3-hydroxybenzo(a)pyrene and 4,5-dihydro-4,5-dihydroxybenzo(a)pyrene (4,5-dihydrodiol) are detected. After culture with benzo(a)pyrene, the amount of 9,10-dihydrodiol in the medium is 9-fold greater than the amount of 7,8-dihydrodiol. Benzo(a)pyrene is also metabolised by short-term organ cultures of hamster lung to water-soluble metabolites, which on hydrolysis with β-glucuronidase yield metabolites co-chromatographing with 3-hydroxybenzo(a)pyrene, quinones, 4,5-dihydrodiol and 7,8-dihydrodiol. However little or no 9,10-dihydrodiol is detected. Both 7,8- and 9,10-dihydrodiols are metabolised by cultures of hamster lung to an ethyl acetate-soluble metabolite which co-chromatographs and has similar fluorescence excitation and emission spectra to 7,8,9,10-tetrahydro-7,8,9,10-tetrahydroxybenzo(a)pyrene (7,8,9,10-tetrahydrotetrol). More 7,8,9,10-tetrahydrotetrol is formed from 7,8-than 9,10-dihydrodiol. A major route for metabolism of 7,8-dihydrodiol is conversion into water-soluble metabolites, which on hydrolysis with β-glucuronidase yield an ethyl acetate-soluble metabolite co-chromatographing with 7,8-dihydrodiol. However only small amounts of water-soluble metabolites are observed after short-term organ culture with 9,10-dihydrodiol. The amount of covalent binding after short-term organ culture with 7,8-dihydrodiol was greater than that with 9,10-dihydrodiol and benzo(a)pyrene. This was in agreement with the many observations showing the high biological activity of the further metabolite of 7,8-dihydrodiol, i.e. 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide. These results however, also raise the possibility of a role for 9,10-dihydrodiol as a precursor of active metabolites.

Centrolobular hepatic necrosis related to covalent binding of metabolites of halogenated aromatic hydrocarbons

The mechanism of centrolobular hepatic necrosis produced by treating rats or mice with 14C-labeled halogenated aromatic hydrocarbons was studied as an animal model of drug-induced tissue lesions. The development of hepatic necrosis was associated with the covalent binding of substantial amounts of radiolabeled material to liver proteins, and autoradiograms revealed that most of the covalently bound material was localized within the necrotic centrolobular hepatocytes. Prior induction of hepatic microsomal enzymes by phenobarbital administration potentiated the covalent binding and necrosis, whereas prior inhibition of hydrocarbon metabolism had the opposite effect, suggesting that the binding and necrosis are caused by toxic metabolites of the hydrocarbons. A positive correlation between the amount of covalent binding and the severity of centrolobular necrosis was obtained after various drug treatments and with several different halogenated benzene derivatives of varying hepatotoxicity. These results suggest that covalent binding of toxic metabolites may be an important mechanism in the pathogenesis of tissue lesions elicited by a variety of foreign compounds.