Rat Liver Microsomal System Research Articles

Effects of perinatal exposure to benzo(a)pyrene on the aryl hydrocarbon hydroxylase system of adult rat liver.

Early exposure to benzo(a)pyrene, B(a(P, produces long-lasting effects on the cytochrome P-450 dependent monooxygenase system of rat liver microsomes. Adult male offspring of rats given B(a(P, 20 mg/kg i.p. during late pregnancy, showed either a small but significant depression of basal aryl hydrocarbon hydroxylase acitivity or an impaired induction response to B(a)P. Few significant changes were found in the relative amounts of the individual metabolites formed from B(a)P by microsomes from perinatally exposed rats, either in the basal or B(a)P-induced state. In addition, perinatal exposure to B(a)P tended to decrease the binding of B(a)P to calf thymus DNA in in vitro incubations.

The metabolism of the 10,11-dihydrodiol of benz[a]-anthracene to a vicinal diol-epoxide that is not involved in metabolic activation.

Benz[a]anthracene-10,11-diol, a major metabolite of benz[a]anthracene, is metabolized by a rat-liver microsomal system to form anti-BA-10,11-diol 8, 9-oxide (t-10,r-11-dihydroxy-t-8,9-oxy-8,9,10,11 -tetrahydrobenz[a]anthracene) and, to a lesser extent, syn-BA-10,11-diol 8,9-oxide (t-10,r-11-dihydroxy-c-8,9-oxy-8,9,10,11-tetrahydrobenz[a] anthracene). However, when benz[a]anthracene is incubated with DNA in a rat-liver microsomal system, anti-BA-10,11-diol 8,9-oxide does not contribute to the covalent binding of this hydrocarbon to DNA.

4-Hydroxy-5-oxo-4,5-dihydrobenzo(a)pyrene, a new metabolite of benzo(a)pyrene.

The detection and the isolation in phenobarbital-induced rat liver microsomal incubation systems of a new metabolite of benzo(a)pyrene are described. The structure of this metabolite and the way in which it is formed are described.

Effect of detergents on the microsomal cytochrome P-450-dependent monooxygenases from frog and rat liver

Abstract 1. 1. A frog liver microsomal hydroxylase system was more susceptible to inhibition by five detergents (sodium cholate, sodium deoxycholate, Triton X-100, Emulgen 913 and Tween 20) in the assay for laurate hydroxylation than the rat liver microsomal hydroxylase system. This suggests that liver microsomal cytochrome P-450-dependent monooxygenases from different sources may not have the same response to detergents. 2. 2. Emulgen 913 was the most effective detergent for the solubilization of cytochrome P-450 from frog and rat liver microsomes among the five detergents. 3. 3. The average level of cytochrome P-450 as determined in the frog ( 0.401 ± 0.023 nmol/mg protein ) is lower than that in the rat (0.60 or 1.19 ± 0.10 ), rabbit ( 1.45 ± 0.35 ), swine ( 0.59 ± 0.16 ), and approximately the same as in the human ( 0.39 ± 0.14 ) and the cat ( 0.380 ± 0.085 ).

Fluorescence spectral evidence that benzo[a]pyrene is activated by metabolism in mouse skin to a diol-epoxide and a phenol-epoxide

Hydrolysates of DNA that had been isolated from mouse skin treated with 3H-labelled benzo[a]pyrene were subjected to chromatography on Sephadex LH20. Two major products were eluted in the region expected for deoxyribonucleoside-hydrocarbon adducts and these were purified further by h.p.l.c. The fluorescence emission and excitation spectra of one of the adducts were identical to that of the adduct obtained from DNA that was treated with BP-7,8-diol 9,10-oxide (r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a] pyrene). The fluorescence emission and excitation spectra of the other adducts were identical to the published spectra of 9-OHBP-4,5-diol (4,5-dihydro-4,5,9-trihydroxy-benzo[a]pyrene) and of the deoxyribonucleoside-hydrocarbon adduct obtained from DNA that had been incubated with 9-OHBP (9-hydroxybenzo[a] pyrene) in the presence of a rat-liver microsomal system. The metabolic activation of benzo[a]pyrene in mouse skin, a target tissue for carcinogenesis by this hydrocarbon, thus appears to involve the formation of adducts derived from both BP-7,8-diol 9,10-oxide and 9-OHBP 4,5-oxide (9-hydroxybenzo-[a]pyrene 4,5-oxide), although quantitatively, the adduct derived from 9-OHBP 4,5-oxide is a minor product.

Characteristics of the rat liver microsomal enzyme system converting cholecalciferol into 25-hydroxycholecalciferol. Evidence for the participation of cytochrome p-450

Properties of the rat hepatic cholecalciferol 25-hydroxylase have been studied. An assay system has been developed in which 25-hydroxycholecalciferol production is linear for at least 2h in both homogenates and microsomal fraction. Furthermore, the initial reaction velocity is linearly related to the amount of liver tissue or microsomal fraction. This enzyme system also metabolizes an analogue of cholecalciferol, namely dihydrotachysterol 3, into 25-hydroxydihydrotachysterol 3. The 25-hydroxylase is in the microsomal fraction and not in mitochondria. It has a Km of 44 nM for cholecalciferol and 360 nM for dihydrotachysterol 3. Its activity is not altered by dietary concentrations of calcium and phosphorus. Vitamin D-deficient rats have higher activities of the hepatic 25-hydroxylase than those receiving 25 ng of cholecalciferol daily. The 25-hydroxylase is inhibited by metyrapone. An atmosphere of CO/O2 (9:1, v/v) inhibits the reaction by 87%. This inhibition is partially reversed by white light. Additionally, cholecalciferol and 25-hydroxycholecalciferol competitively inhibit aminopyrine demethylase. These results support the idea that the cholecalciferol 25-hydroxylase is a cytochrome P-450-dependent mono-oxygenase.

The involvement of a non-‘bay-region’ diol-epoxide in the formation of benz(a)anthracene-DNA adducts in a rat-liver microsomal system

The metabolic activation of benz(a)anthracene was investigated by incubating [ 3H]-benz(a)anthracene with DNA, a NADPH-generating system and rat-liver microsomes. When hydrolysates of the DNA were chromatographed on Sephadex LH20 columns, three hydrocarbon-nucleoside adduct peaks were resolved and these were further examined using HPLC. One adduct probably results from the reaction of the non-bay-region diol-epoxide r -8, t -9-dihydroxy- t -10,11-oxy-8,9,10,11-tetrahydrobenz(a)anthracene ( anti -BA-8,9-diol 10,11-oxide) with DNA. The other two adducts did not co-chromatograph with adducts formed from any of the four possible isomeric diolepoxides that can be formed in the 8,9,10,11-ring of benz(a)anthracene.

Mutagenic effects of styrene and styrene oxide

Styrene and its presumed metabolite styrene oxide were tested in the Salmonella/liver-microsome test on the tester strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium. Styrene oxide, in the concentration range 1–10 μmoles per plate, was mutagenic on TA1535 and TA100, both in the absence and the presence of a rat-liver microsomal system. Styrene, on the other hand, in the concentration range 10 −3 to 15 μmoles per plate, was not mutagenic in any of the tester strains, regardless of the presence or absence of a 9000 × g supernatant of rat-liver homogenate. Two differently induced microsomal fractions were used, one induced with Aroclor 1254 and one with another PCB mixture, Clophen C. Styrene was negative in both systems. Styrene is presumed to be metabolized to styrene oxide, and in an attempt to potentiate the possible mutagenicity of styrene, two inhibitors of the metabolic breakdown of styrene oxide were added to the homogenates. They were 1,1,1-trichloropropene 2,3-oxide, an inhibitor of epoxide hydratase and diethyl maleate, an inhibitor of the glutathione conjugation pathway. This did not result in any mutagenic effect of styrene in the test. Our conclusion is that in the Salmonella/liver-microsome test, styrene oxide is mutagenic whereas its presumed precursor in the presence of a metabolizing system is not. This might indicate that when styrene is tested together with a rat-liver microsomal fraction, the concentration of styrene oxide is too low to be detected as mutagenic.

The microsomal activation of aflatoxin B 1 and 2-( N-ethylcarbamoyloxymethyl)furan in vitro using a novel diffusion apparatus

The activation by rat liver microsomal systems in vitro of a naturally occurring and a synthetic furan-containing toxin, aflatoxin B 1 and 2-( N-ethylcarbamoyloxymethyl)furan (CMF) has been examined. Both compounds are metabolised to form products which bind covalently to DNA and microsomal protein, Using a specially designed two-chamber diffusion apparatus it has been demonstrated that the active metabolite of CMF is able to bind covalently to DNA separated by a membrane barrier from the microsomal site of activation. In the case of aflatoxin B 1 the DNA must be in physical contact with the microsomal system for the active metabolite of aflatoxin B 1 to bind covalently. Differences between the activation of the two compounds have also been found with regard to their relative efficiencies in binding to DNA and also the effects of the nucleophile GSH. These results have suggested that if the molecular mechanisms of activation of the two compounds be similar, other factors, for example differences in lipid solubility, may play important roles in determining the relative biological activaties of the compounds. The results suggested that the subcellular site of activation of aflatoxin B 1, unlike that of CMF, may need to be adjacent to the target DNA. It is proposed that this site might be the outer nuclear membrane. Alternatively a carrier molecular might exist for the activated aflatoxin B 1 metabolite in vivo.

Sulfur oxyacid production as a consequence of parathion desulfuration

Parathion desulfuration in vitro by the rat liver microsomal oxidase system gives rise to reactive sulfur atoms which either covalently bind to microsomal macromolecules, most likely as hydrodisulfides (-SSH) or appear as water-soluble metabolites. The latter were tentatively identified as sulfate, thiosulfate, and a minor amount of sulfite. Production of sulfate was enhanced by glutathione. Moreover, glutathione and other thiols removed covalently bound sulfur from the microsomal membranes and partially restored activity to enzymes known to be inactivated during parathion metabolism. A semilogarithmic plot of sulfur removal with time indicated at least two populations of covalently bound sulfur differing in the stability of the hydrodisulfide bond or accessibility to thiol compounds. The significance of transient hydrodisulfide formation with regard to the production of sulfur oxyacids is discussed. Both macromolecular and low molecular weight endogenous sulfhy dryl-containing biochemicals may play a role in preventing sulfur binding in vivo since only low level sulfur binding in liver homogenates was observed after the in vivo administration of parathion.

Isothermal gas chromatography with wall-coated glass capillary columns, electron-capture detection and a solid injector : II. Application to the assay of 2-fluorenylacetamide n-hydroxylase activity in a rat-liver microsomal system

The presumed superiority of wali-coated (WCOT) glass capillary columns with electron-capture detection (ECD) over packed columns and ECD was tested for the assay of N-hydroxy-2-fluorenylacetamide after a two-step derivatization into N-chloro-2-fluorenyltrifluoroacetamide or N-chloro-2-fluorenylmonochloroacetamide. Co-derivatization of N-hydroxy-4-biphenylacetamide yielded the internal standard. A commercial glass solid injector, short WCOT columns coated with a film of medium thickness of a non-polar phase and a macro-ECD cell were used isothermally with helium as the carrier gas. Repetitive analyses were possible with both derivatives at a speed unattainable with packed columns.

Effects of soluble fractions of livers from neonatal, mature and hepatoma-bearing rats on microsomal lipid peroxidation

AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 R.E. Talcott, H. Denk, R. Eckerstorfer, J.B. Schenkman, Chem. -Biol. Interact., 12, (1976), 355– 361. 2 T. Kamataki, O. Sugita, M. Kitada, S. Naminohira, H. Kitagawa, Res. Comm. Chem. Pathol. Pharmacol., 17, (1977), 265– 273. 3 T.J. Player, D.J. Mills, A.A. Horton, Biochem. Biophys. Res. Commun., 78, (1977), 1397– 1402. 4 T.J. Player, D.J. Mills, A.A. Horton, Biochem. Soc. Trans., 5, (1977), 1506– 1508. 5 R.W. Baldwin, C.R. Barker, Int. J. Cancer, 2, (1967), 355– 364. 6 F.E. Hunter, J.M. Gebicki, P.E. Hoffsten, J. Weinstein, A. Scott, J. Biol. Chem., 238, (1963), 828– 835. 7 J.M. McCord, I. Fridovich, J. Biol. Chem., 244, (1969), 6049– 6055. 8 A.G. Gornall, C.J. Bardawill, M.M. David, J. Biol. Chem., 177, (1949), 751– 766. 9 T.C. Pederson, S.D. Aust, Biochem. Biophys. Res. Commun., 48, (1972), 789– 795. 10 J.L. Poyer, P.B. McCay, J. Biol. Chem., 246, (1971), 263– 269. 11 K. Utsumi, T. Yoshioka, N. Yamanaka, T. Nakazawa, FEBS Lett., 79, (1977), 1– 3. 12 O.P. Sharma, Biochem. Biophys. Res. Commun., 78, (1977), 469– 475. 13 E.D. Wills, Biochem. J., 113, (1969), 324– 332. 14 K. Fong, P.B. McCay, J.L. Poyer, J. Biol. Chem., 243, (1973), 7792– 7797. Citing Literature Volume89, Issue1May 01, 1978Pages 103-106 ReferencesRelatedInformation

Identification of the principal aflatoxin B1-DNA adduct formed in vivo in rat liver.

The products of in vivo covalent binding of activated aflatoxin B1 (AFB1) to DNA have been investigated in rats. The principal covalent product formed in liver DNA of rats treated with AFB1 has been identified as 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1. This compound was isolated from the liver DNA of rats dosed with AFB1 (2.0 mg/kg) in sufficient quantity for characterization by physicochemical techniques, including field-desorption mass spectrometry. This information together with results of chemical methylation of the compound proved that the major adduct formed between DNA and AFB1 in vivo is identical to that produced in vitro when AFB1 is incubated with DNA in the presence of a rat liver microsomal activating system. Quantitative studies of formation of this compound revealed a dose-dependent relationship between the level of its occurence in liver DNA and AFB1 doses over the range 0.125-1.0 mg/kg.

Differences between products of binding of 7,12-dimethylbenz[a]anthracene to DNA in mouse skin and in a rat liver microsomal system

Hydrocarbon-deoxyribonucleoside products from the DNA of mouse skin exposed in vivo to 7,12-dimethylbenz[a]anthracene are chromatographically the same as the products formed in mouse embryo cell cultures. These products, which are known to arise through the generation of a diol-epoxide in the 1,2,3,4-ring of the hydrocarbon, are chromatographically separable from products that result from reaction of the K-region oxide of this hydrocarbon with DNA. However, when 7,12-dimethylbenz[a]anthracene is bound to DNA in the presence of a microsomal system analogous to those used in various carcinogen detection systems, the hydrocarbon-deoxyribonucleoside products co-chromatograph with the K-region oxide products. Differences in the profiles of metabolites formed in mouse embryo cell cultures and rat liver microsomal systems are consistent with the differences between the DNA-bound products in these two systems.

Metabolism of Halogenated Ethylenes

The metabolism of the chlorinated ethylenes may be explained by the formation of chloroethylene epoxides as the first intermediate products. The evidence indicates that these epoxides rearrange with migration of chlorine to form chloroacetaldehydes and chloroacetyl chlorides. Thus, monochloroacetic acid, chloral hydrate, and trichloroacetic acid have been found in reaction mixtures of 1,1-dichloroethylene, trichloroethylene, and tetrachloroethylene, respectively, with rat liver microsomal systems. Rearrangements of the chloroethylene, and glycols formed from the epoxides by hydration may also take place, but would appear, at least in the case of 1,1-dichloroethylene, to be quantitatively less important. The literature on the metabolism of chlorinated ethylenes and its relationship to their toxicity is reviewed.

Metabolism of halogenated ethylenes.

The metabolism of the chlorinated ethylenes may be explained by the formation of chloroethylene epoxides as the first intermediate products. The evidence indicates that these epoxides rearrange with migration of chlorine to form chloroacetaldehydes and chloroacetyl chlorides. Thus, monochloroacetic acid, chloral hydrate, and trichloroacetic acid have been found in reaction mixtures of 1,1-dichloroethylene, trichloroethylene, and tetrachloroethylene, respectively, with rat liver microsomal systems. Rearrangements of the chloroethylene, and glycols formed from the epoxides by hydration may also take place, but would appear, at least in the case of 1,1-dichloroethylene, to be quantitatively less important. The literature on the metabolism of chlorinated ethylenes and its relationship to their toxicity is reviewed.

Action on mitochondria and toxicity of metabolites of tri- n-butyltin derivatives

Tri- n-butyltin derivatives are metabolized by a cytochrome P450-dependent rat liver microsomal monooxygenase system and by mice to yield carbon-hydroxylated metabolites, i.e. the α-, β-, γ- and δ-hydroxybutyl-dibutyltin derivatives, as well as the corresponding γ-ketone. The latter three metabolites are sufficiently stable at physiological pH for comparisons with tributyltin chloride with regard to their action on mitochondrial functions and intraperitoneal toxicity to mice. The δ-hydroxy compound differs most greatly from the other metabolites in potency for altering mitochondrial functions possibly because of its greater polarity or its lower propensity for intramolecular coordination of the introduced oxygen with the tin atom. The γ-hydroxy, δ-hydroxy and γ-keto compounds are less toxic to mice than tributyltin derivatives and do not increase the water content of the brain under conditions where triethyltin bromide does.

Interaction between 2,6-dimethyl-3,5-dicarbethoxy-1,4-dihydropyridine and enzymes of the NADPH-specific electron transport chain of rat liver microsomes

2,6-Dimethyl-3,5-dicarboethoxy-1,4-dihydropyridine (DHP) interacts with NADPH-dependent electron transfer system of rat liver microsomes: it forms a complex with the terminal oxidase-cytochrome P-450, according to type I, and inhibits clearly the activity of NADPH-cytochromes c-reductase and mitindione dimethylesterase. DHP repeatedly administered in vivo rendered no inducing influence upon the microsomal enzymes.

Mutagenicity of smoke condensates from cigarettes, cigars and pipe tobacco

Smoke condensates from cigarettes, cigars and pipe tobacco were mutagenic on Salmonella typhimurium TA100 and TA98 when activated with rat liver microsomal system. Mutagenicity of a unit weight of smoke condensate was rather high in cigars, low in pipe tobacco and intermediate in cigarettes. Specific mutagenic activity was almost comparable among smoke condensates from low- to high-tar cigarettes, although some variations were observed depending upon the country producing the cigarettes. Marked mutagenicity of cigarette smoke condensate could not be explained by the benzo (a) pyrene or nitroso compounds it actually contained, suggesting the presence of other very potent mutagens in tobacco smoke condensates.

Mutagenic evaluation of ronidazole

Ronidazole was evaluated for mutagenic potential using in vitro microbial tests and in vivo studies in mice. The microbial test used the histidine requiring mutants of Salmonella typhimurium with and without a rat liver microsomal activation system (Ames test). The studies in mice included the dominant lethal test, micronucleus test and cytogenetic assays. Ronidazole was given orally in doses of 50, 100 and 200 mg/kg/day in the in vivo studies. In the dominant lethal test, groups of male mice were treated for five consecutive days before being mated with untreated females. In the micronucleus test, the mice were administered the compound for 2 or 5 consecutive days; they were killed 6 h after the last dose and bone marrow was examined for the presence of micronuclei in developing erythrocytes. In the cytogenetic assays, bone marrow cells in metaphase were examined for chromosome aberrations, 6, 24 and 48 h after mice were treated acutely with the test compound. In addition, similar examinations of chromosomes were made on mice given five consecutive dosages of ronidazole and killed 6 h after the last dose. The results of the various in vivo studies did not suggest that ronidazole would be mutagenic for the mammal. Ronidazole at concentrations of 10 and 50 μg/plate was found to increase the number of back mutations of missense mutants in the in vitro bacterial test. This finding confirms the results of Voogd et al. [19]. Incorporation of the microsomal activation system had no effect on the mutagenic capability of the test compound. In conclusion, although ronidazole was shown to be mutagenic in in vitro bacterial systems, the in vivo systems did not suggest that the compound would be mutagenic for the mammal.