Separation and quantitative determination of 2-acetyl-aminofluorene and its hydroxylated metabolites by high pressure liquid chromatography

A method utilizing high pressure liquid chromatography has been developed for the separation and quantitative estimation of all the major metabolites of the carcinogen 2-acetylaminofluorene in a single chromatographic determination. The method was used to separate 7-hydroxy-2-acetylaminofluorene, 5-hydroxy-2-acetylaminofluorene, 3-hydroxy-2-acetylaminofluorene, 1-hydroxy-2-acetyl-aminofluorene, 2-aminofluorene, N-hydroxy-2-acetylaminofluorene, and 2-acetylaminofluorene when 2-acetylaminofluorene was incubated with mouse liver microsomes and NADPH.This new high pressure liquid chromatography method for separating the metabolites arising from hydroxylations of 2-acetylaminofluorene should also prove useful in the isolation and quantitative analysis of metabolites from other N-acetylarylamines.     

Species variation in bladder cell and liver cell activation of acetylaminofluorene

The metabolism and mutagenicity of 2-acetylaminofluorene were measured using freshly prepared intact bladder and liver cells from the cow, dog and rat. High pressure liquid chromatography was used to separate 2-acetylaminofluorene metabolites, andSalmonella typhimurium, strain TA98, was used to detect mutagenic intermediates. Species differences as well as animal-to-animal variation within a species were observed. Mutagenic activity with 2-acetylaminofuorene was greater with cow bladder cells than with dog or rat bladder cells. However, dog bladder cells were most active in metabolizing 2-acetylaminofluorene, and rat bladder cells were least active. Liver cells from all three species metabolized 2-acetylaminofluorene to mutagens forSalmonella, with dog and cow cells being more active than rat liver cells. However, cow liver cells were the most active in metabolizing 2-acetylaminofuorene, followed by rat and dog cells. With all cell types studied, except rat bladder cells, aminofluorene was the major metabolite detected. Carbon and N-hydroxylated products were produced by liver and bladder cells of the three species and glucuronide and sulfate conjugates of the metabolites were detected from both cell types. Correlations between mutagenic activity and the level of metabolism or any individual metabolite were not apparent. The data suggest that the relative contribution of bladder cell metabolism in aromatic amine induced bladder cancer may vary with the species.Abbreviations AAF 2-acetylaminofluorene - 4-ABP 4-aminobiphenyl - AF aminofluorene - BZ benzidine - cytochrome P-450 a collective term for all forms of the cytochrome P-450 polysubstrate mono-oxygenases - FMO flavin mono-oxygenases - HPLC high pressure liquid chromatography - MNNG N-methyl-N-nitro-N-nitrosoguani-dine - 2-NA 2-naphthylamine - N-OH-AAF N-hydroxy-2-acetylaminofluorene - 1-OH-AAF 1-hydroxy-2-acetylaminofluorene - 5-OH-AAF 5-hydroxy-2-acetylaminofluorene - 7-OH-AAF 7-hydroxy-2-acetylaminofluorene - 8OH-AAF 8-hydroxy-2-acetylaminofluorene - 9-OH-AAF 9-hydroxy-2-acetylaminofluorene - UDS unscheduled DNA synthesis     

Human hepatocyte and kidney cell metabolism of 2-acetylaminofluorene and comparison to the respective rat cells

The metabolism and mutagenic activation of 2-acetylaminofluorene by human and rat hepatocytes and kidney cells were measured. High performance liquid chromatography was used to separate the 2-acetylaminofluorene metabolites, and a cell-mediated Salmonella typhimurium mutagenesis assay was used to detect mutagenic intermediates. Rat and human differences were observed with cells from both organs and levels of metabolism and mutagenesis were higher in human cells. Within a species, liver and kidney cell differences were also evident, with levels of hepatocyte-mediated metabolism and mutagenesis being greater than kidney cells. Human inter-individual variation was apparent with cells from both organs, but the variation observed was significantly greater in hepatocytes than kidney cells. A knowledge of such differences, including an understanding that they may vary with the chemical being studied, should be useful in the extrapolation of rodent carcinogenesis data to humans.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - DMSO dimethylsulfoxide - HPLC high performance liquid chromatography - N-OH-AAF N-hydroxy-2-acetylaminofluorene - 1-OH-AAF 1-hydroxy-2-acetylaminofluorene - 3-OH-AAF 3-hydroxy-2-acetylaminofluorene - 5/9-OH-AAF a combination of 5 and 9-hydroxy-2-acetylaminofluorene - 7-OH-AAF 7-hydroxy-2-acetylaminofluorene - 8-OH-AAF 8-hydroxy-2-acetylaminofluorene     

Peptization of the Gel of Konjac Mannan

The addition of 1,8-pyrenequinone into the assay system containing rat liver homogenates (S-9) caused an approximately 10-fold increase in the mutagenicity of 2-acetylaminofluorene (AAF) in the current Salmonella reversion assay system. Since no chemical reaction between 1,8-pyrenequinone and AAF was observed, the in vitro effects of 1,8-pyrenequinone on the metabolisms of AAF with S-9 mix were studied. The enhancement of mutagenicity by 1,8-pyrenequinone was not dependent on the dose of NADPH under the present assay condition. The mutagenicity of AAF was increased approximately 4-fold by the addition of 1,8-pyrenequinone into microsomes, whereas it remained at the spontaneous level in the presence of cytosol. However, by reconstituting microsomes with cytosol, the mutagenicity enhancing activity was recovered to the original level. Since 1,8-pyrenequinone inhibited the AAF hydroxylase activity, chemical analysis of the incubation mixture of AAF was tried. This indicated that a higher amount of unmetabolized AAF remained and higher amounts of 2-aminofluorene and N-hydroxy-2-acetylaminofluorene were accumulated in the presence of 1,8-pyrenequinone compared with those in the absence of 1,8-pyrenequinone. From these results, it seems probable that 1,8-pyrenequinone inhibits C-hydroxylation (the detoxifying pathway) and promotes N-hydroxylation (the activating pathway) as well as deacetylation in the AAF metabolism.     

Ellipticines and human liver microsomes: Spectral interaction with cytochrome P-450 and hydroxylation. Inhibition of aryl hydrocarbon metabolism and mutagenicity

Some pharmacological properties of ellipticine (E) and its derivatives linked to their interaction with cytochrome P-450 have been investigated with human liver microsomes. 9-Hydroxyellipticine (9-OHE) interacts with human liver cytochrome P-450 exhibiting a type II spectrum (λ_max: 428 nm, K_s = 1.1 μM). After incubation with human liver microsomes the E was converted to 9-OHE; 7-hydroxyellipticine was not produced. The cytotoxic effect of this biotransformation has been evaluated on leukemic L1210 cells, in vitro, and found to be equal to those elicited by liver microsomes of control or phenobarbital (PB) pretreated rats. Moreover, 9-OHE and 9-fluoroellipticine (9-FE) strongly inhibit the benzo[a]pyrene hydroxylase (AHH) activity of human liver microsomes (I₅₀ = 2.6 μM and 1.6 μM, respectively) as well as the mutagenesis induced by the polycyclic aromatic hydrocarbon 2-acetylaminofluorene (AAF); 1 μg/plate of each of these compounds is able to inhibit by more than 50% the mutagenicity of 5 μg/plate AAF.     

On the metabolic activation of the carcinogen N-hydroxy-N-2-acetylaminofluorene : III. Oxidation with horseradish peroxidase to yield 2-nitrosofluorene and N-acetoxy-N-2-acetylaminofluorene

Previous studies have shown that the carcinogen N-hydroxy-2-acetylaminofluorene is converted by one-electron oxidants to a free nitroxide radical which dismutates to N-acetoxy-2-acetylaminofluorene and 2-nitrosofluorene. The present study shows that the same oxidation can be achieved with horseradish peroxidase and H₂O₂. The free radical intermediate was detected by its ESR signal, and the yields of N-acetoxy-2-acetylaminofluorene and of 2-nitrosofluorene were determined under a number of conditions. Addition of tRNA to the reaction mixture containing N-acetoxy-N-2-acetyl[2′-³H]aminofluorene yielded tRNA-bound radioactivity; addition of guanosine yielded a reaction product which appears to be N-guanosin-8-yl)-2-acetylaminofluorene. The latter compound has previously been identified as a reaction product of N-acetoxy-2-acetylaminofluorene and guanosine. Preliminary attempts to demonstrate the formation of a nitroxide free radical or its dismutation products with rat liver mixed function oxidase systems were not successful.     

Stereoselective hydroxylation at the aliphatic carbons of 7,8- and 9,10-dihydrobenzo[a]pyrenes by rat liver microsomes

Optically active 7-hydroxy-7,8-dihydrobenzo[a]pyrene and 8-hydroxy-7,8-dihydrobenzo[a]pyrene were identified as two of the major metabolites formed by incubation of 7,8-dihydrobenzo[a]pyrene with rat liver microsomes. Optically active 9-hydroxy-9,10-dihydrobenzo[a]pyrene and 10-hydroxy-9,10-dihydrobenzo[a]pyrene were similarly identified as two of the minor metabolites of 9,10-dihydrobenzo[a]pyrene. The formation of these metabolites was abolished either by prior treatment of liver microsomes with carbon monoxide or the absence of NADPH, but was not inhibited by an epoxide hydrolase inhibitor. The results indicate that the aliphatic carbons of dihydro polycyclic aromatic hydrocarbons may undergo stereoselective hydroxylation reactions catalyzed by the cytochrome P-450 system of rat liver microsomes.     

N-Hydroxylation of arylamides by the rat and guinea pig: Evidence for substrate specificity and participation of cytochrome P1-450

The hypothesis that N-hydroxylation of arylamides is essential for carcinogenicity was examined in vivo and in vitro with N-2-fluorenylacetamide, a potent carcinogen, and with N-3-fluorenylacetamide, an isomer with marginal carcinogenicity. About 10–20% of 2-[9-¹⁴C]fluorenylacetamide administered intraperitoneally to the rat was excreted in the bile as the N-hydroxy-2-[9-¹⁴C]-derivative, whereas <0.1% of 3-[G-³H]fluorenylacetamide was found as the N-hydroxy metabolite in bile and urine. N-Hydroxylation of the 2- isomer by hepatic microsomes of untreated or 3-methylcholanthrene-treated rats was 40 to 50-fold greater than that of the 3- isomer. The role of cytochromes P-450 and P₁-450 in N-hydroxylation of arylamides by rat liver microsomes was shown by inhibition of the reaction with carbon monoxide and cobaltous chloride. Interaction of the arylamides with cytochrome P₁-450 was also demonstrated by binding spectra obtained on addition on 2- and 3-fluorenylacetamide to hepatic chromosomes of methylcholanthrene-treated rats. There appeared to be no correlation between the magnitude of the spectra and the extent of N-hydroxylation. N-Hydroxylation of the 2- isomer by hepatic microsomes of the guinea pig, a species resistant to the carcinogenecity of this compound, was markedly less than N-hydroxylation by rat liver microsomes, even though, as judged by the appearance of the binding spectra, both 2- and 3- isomers were bound by cytochrome P₁-450 of guinea pig-liver microsomes. The results are in agreement with the view that the microsomal N-hydroxylation of arylamides parallels their carcinogenicity.     

Regulation of human leukocyte microsomal hydroxymethylglutaryl-CoA reductase activity by a phosphorylation and dephosphorylation mechanism

The activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), obtained from cultured human IM-9 lymphoid cells or freshly isolated human peripheral blood leukocytes, is modulated by a phosphorylation/dephosphorylation mechanism. Addition of MgATP + ADP to IM-9 cell microsomal reductase leads to a time-dependent loss of enzyme activity. Inactivated reductase is reactivated by rat liver reductase phosphatase. Kinase-dependent IM-9 cell microsomal reductase, prepared by heating IM-9 microsomes for 15 min at 50°C, is inactivated in the presence of MgATP and ADP only after addition of cytosolic reductase kinase from either IM-9 cells, freshly isolated leukocytes or rat liver. Inactivation is time-dependent and dependent on the cytosolic protein concentration. Inactivated reductase is reactivated by rat liver reductase phosphatase. For cultured IM-9 cells and freshly isolated leukocytes incubated with culture medium for 2 h, the ratios of active (unphosphorylated) to total (phosphorylated + unphosphorylated) reductase activity are 0.22 and 0.43, respectively. Thus, in addition to its regulation by changes in the amount of total enzyme protein, human leukocyte reductase activity is also modulated by a phosphorylation/dephosphorylation mechanism.     

Hydroxymethylglutaryl CoA reductase and the modulation of microsomal cholesterol content by the nonspecific lipid transfer protein

The influence of membrane cholesterol content on 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase, EC 1.1.1.34) in rat liver microsomes was investigated. Microsomes were enriched in cholesterol by incubation with egg phosphatidylcholine-cholesterol vesicles and the nonspecific lipid transfer protein from rat liver. By this method, the microsomal cholesterol content was 2.5-fold enhanced up to final concentrations of 140 nmol cholesterol per mg microsomal protein. In another experiment, microsomes isolated from rats fed a cholesterol-rich diet were depleted of cholesterol by incubation with egg phosphatidylcholine vesicles and the transfer protein. Both cholesterol enrichment and depletion had virtually no effect on the microsomal HMG-CoA reductase activity. In another set of experiments, normal rat liver microsomes were incubated with human serum, resulting in a rise of microsomal cholesterol content. This was reflected in an increase of acyl-CoA:cholesterol acyltransferase activity but failed to have an effect on HMG-CoA reductase.     

Functional size of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase as determined by radiation inactivation

The functional molecular weight of rat liver 3-hydroxy-3-methylglutaryl-CoA reductase was determined by radiation inactivation. Both isolated hepatic microsomes and primary hepatocytes were irradiated with high energy electrons at -135 degrees C, and the residual microsomal enzyme activity was subsequently determined. The loss of enzyme activity in both irradiated microsomes and microsomes isolated from irradiated hepatocytes followed a single exponential decay which corresponded to a molecular mass of 200 kDa. This minimal molecular size of the functional enzyme was unaffected by either addition of cholestyramine to the rat diet or addition of 25-hydroxycholesterol plus mevalonate to the isolated rat hepatocytes. In addition, surviving enzyme protein was determined by immunoprecipitation of radiolabeled enzyme from hepatocytes that had been incubated with [35S]methionine before irradiation. The target size for loss of the monomer subunits was 98 kDa. The simplest interpretation of these results is that rat liver 3-hydroxy-3-methylglutaryl-CoA reductase in situ is a noncovalently linked dimer of the Mr = 97,200 enzyme subunit.     

AM1, a glycoprotein from the submandibular glands of the mouse, has esterolytic and amidolytic activities

The activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), obtained from cultured human IM-9 lymphoid cells or freshly isolated human peripheral blood leukocytes, is modulated by a phosphorylation/dephosphorylation mechanism. Addition of MgATP + ADP to IM-9 cell microsomal reductase leads to a time-dependent loss of enzyme activity. Inactivated reductase is reactivated by rat liver reductase phosphatase. Kinase-dependent IM-9 cell microsomal reductase, prepared by heating IM-9 microsomes for 15 min at 50 degrees C, is inactivated in the presence of MgATP and ADP only after addition of cytosolic reductase kinase from either IM-9 cells, freshly isolated leukocytes or rat liver. Inactivation is time-dependent and dependent on the cytosolic protein concentration. Inactivated reductase is reactivated by rat liver reductase phosphatase. For cultured IM-9 cells and freshly isolated leukocytes incubated with culture medium for 2 h, the ratios of active (unphosphorylated) to total (phosphorylated + unphosphorylated) reductase activity are 0.22 and 0.43, respectively. Thus, in addition to its regulation by changes in the amount of total enzyme protein, human leukocyte reductase activity is also modulated by a phosphorylation/dephosphorylation mechanism.     

Solubilization of 3-hydroxy-3-methylglutaryl coenzyme A reductase from rat and chicken liver microsomes

A simple, efficient, freeze-thaw procedure for the solubilization of liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase has been developed. Microsomes of chicken or rat liver were prepared by homogenization in buffer containing 100 mm sucrose, 50 mm KCl, 40 mm KH₂PO₄, 30 mm EDTA, and 2 mm DTT, pH 7.2 (buffer A). The homogenate was centrifuged at 12,000g (15 min), and the microsomes were separated from the supernatant by centrifugation at 100,000g (60 min). The isolated microsomes were frozen, either by dry ice-acetone or by storage in a freezer at ?20°C. The frozen microsomes were permitted to thaw at room temperature, homogenized in buffer A, and centrifuged at 100,000g (60 min). The extraction was repeated and the combined supernatants contained 70 to 90% of the microsomal HMG-CoA reductase activity. The yield of enzyme activity by the freeze-thaw technique is equal to or greater than previously reported methodologies and is significantly easier to perform. This procedure is particularly suited to the preparation of large quantities of solubilized enzyme for isolation and characterization of HMG-CoA reductase. In addition, this method does not require the use of detergents, sonification, or other procedures which might partially inactivate or alter the molecular properties of the enzyme.     

Formation of electrophilic N-acetoxyarylamines in cytosols from rat mammary gland and other tissues by trans-acetylation from the carcinogen N-hydroxy-4-acetylaminobiphenyl

The 105 000 × g supernatant fractions of various rat tissues catalyze the transfer of the N-acetyl group of certain carcinogenic aromatic acethydroxamic acids to the O atom of aromatic hydroxylamines. The resulting N-acetoxyhydroxylamines are strongly electrophilic and have been detected and analyzed through their reaction with N-acetylmethionine to yield methylmercaptoaminoarenes.Of the rat tissues studied the liver had the highest activity; kidney and small intestinal mucosa were about 15–20% as active. The transacetylase activities of these tissues were similar with respect to their ability to use either N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF or N-hydroxy-4-acetylaminobiphenyl (N-hydroxy-AABP) as acetyl donors, their stability on storage at 2–3°C, and their elution patterns from Sephadex G-100 columns. Low transacetylase activity was found in spleen and muscle.Mammary tissue from 16–21 day pregnant rats had 20% of the transacetylase activity of rat liver when N-hydroxy-AABP was used as acetyl donor and N-hydroxy-4-aminobiphenyl (N-hydroxy-ABP) was the acetyl acceptor. This enzyme system from mammary tissue did not utilize the fluorene derivatives as either acetyl donor or acetyl acceptor, was much more labile than the liver, kidney, or intestinal mucosa systems, and had a pH optimum at 7.5, as compared to pH 6.8 for liver. The mammary tissue system was similar to the hepatic system in being inhibited by sulfhydryl reagents; it required a source of reduced pyridine nucleotides for maximum activity.     

Activation of Polynuclear Aromatic Hydrocarbons to Mutagens by the Marine Ciliate Parauronema acutum

The marine ciliate Parauronema acutum converted 2-aminofluorene and 2-acetylaminofluorene to compounds with mutagenic activity in the Ames Salmonella test. The ciliate, however, did not activate benzo (α)pyrene or benzanthracene or destroy the mutagenic properties of nitrosoguanidine. Homogenates, when substituted for the liver S-9 fraction in the Salmonella/microsome test, activated 2-aminofluorene and 2-acetylaminofluorene to mutagens. Benzo(α)pyrene and benzanthracene were not activated, nor was nitrosoguanidine inactivated. Phenobarbitol did not induce or increase the amount of activating activity. The activation showed no requirement for the reduced nicotinamide adenine dinucleotide phosphate-regenerating system required by liver P-450 cytochromes. Upon differential sedimentation of a cell homogenate, the majority of the activity sedimented with a small-particle fraction with sedimentation properties like those of microsomes from higher eucaryotes. Benzo(α)pyrene, although not metabolized, was accumulated by cultures of P. acutum at a linear rate and was not appreciably released (10%) after removal of benzo(α)pyrene from the incubation medium. Hence, this ciliate could convert certain polynuclear aromatic hydrocarbons to mutagens and accumulate others.     

Oxidation pattern of the anticancer drug ellipticine by hepatic microsomes - similarity between human and rat systems

Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of DNA adducts mediated by cytochrome P450 (CYP). We investigated the ability of CYP enzymes in rat, rabbit and human hepatic microsomes to oxidize ellipticine and evaluated suitable animal models mimicking its oxidation in humans. Ellipticine is oxidized by microsomes of all species to 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and ellipticine N(2)-oxide. However, only rat microsomes generated the pattern of ellipticine metabolites reproducing that formed by human microsomes. While rabbit microsomes favored the production of ellipticine N(2)-oxide, human and rat microsomes predominantly formed 13-hydroxyellipticine. The species difference in expression and catalytic activities of individual CYPs in livers are the cause of these metabolic differences. Formation of 7-hydroxy- and 9-hydroxyellipticine was attributable to CYP1A in microsomes of all species. However, production of 13-hydroxy-, 12-hydroxyellipticine and ellipticine N(2)-oxide, the metabolites generating DNA adducts, was attributable to the orthologous CYPs only in rats and humans. CYP3A predominantly generates these metabolites in rat and human microsomes, while CYP2C3 activity prevails in microsomes of rabbits. The results underline the suitability of rat species as a model to evaluate human susceptibility to ellipticine.     

Induction of different isozymes of cytochrome P-450 and of microsomal epoxide hydrolase in rat liver by 2-acetylaminofluorene and structurally related compounds

The amounts of five different forms of cytochrome P-450 and of microsomal epoxide hydrolase were determined immunochemically in rat liver microsomes before and after treatment of the animals with 2-acetylaminofluorene and 15 structurally related compounds. The amount of cytochrome P-450c was found to be increased about 60-fold after treatment with 2-aminofluorene and 3-aminofluorene. Administration of 1-aminofluorene, 4-aminofluorene, 2-acetylaminofluorene and nitrofluorene increased this isozyme about 15-19 times. 2-Aminofluorene was found to inhibit the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to a cytoplasmic receptor 50% at a concentration of 3.12 microM, while no such inhibition could be detected with 2-acetylaminofluorene. Induction of ethoxyresorufin O-deethylase activity was found to be highly correlated (+0.95) with the induction of cytochrome P-450c. Also correlated with the induction of this form was the amount of cytochrome P-450d (+0.84), which could be maximally increased about fourfold. Cytochromes P-450b + e were induced by 2-acetylaminofluorene, 4-acetylaminofluorene and fluorene (about tenfold), while 4-aminofluorene and 4-acetylaminofluorene were found to elevate cytochrome P-450PB/PCN-E about threefold. Microsomal epoxide hydrolase was induced by many of the compounds tested, with 2,7-diaminofluorene, 2,7-diacetylaminofluorene, 2-acetylaminofluorene and 2-(N-hydroxy)acetylaminofluorene being the most potent. No correlation of the induction of this enzyme with the induction of any isozyme of cytochrome P-450 was observed.     

Estrogenic activity of an environmental pollutant, 2-nitrofluorene,after metabolic activation by rat liver microsomes

In this study, the metabolic activation of 2-nitrofluorene (NF) to estrogenic compounds was examined. NF was negative in estrogen reporter assays using estrogen-responsive yeast and human breast cancer cell line MCF-7. However, the compound exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH. Minor estrogenic activity was observed when liver microsomes of untreated or phenobarbital-treated rats were used instead of those from 3-methylcholanthrene-treated rats. When the compound was incubated with the liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH, 7-hydroxy-2-nitrofluorene (7-OH-NF) was formed as a major metabolite. However, little of the metabolite was formed by liver microsomes of untreated or phenobarbital-treated rats. Rat recombinant cytochrome P450 1A1 exhibited a significant oxidase activity toward NF, affording 7-OH-NF. Liver microsomes of phenobarbital-treated rats also enhanced oxidase activity toward NF. In this case, 9-hydroxy-2-nitrofluorene was formed. 7-OH-NF exhibited a significant estrogenic activity, while the activity of 9-hydroxy-2-nitrofluorene was much lower. These results suggest that the estrogenic activity of NF was due to formation of the 7-hydroxylated metabolite by liver microsomes.     

Biochemical basis for the resistance of guinea pigs to carcinogenesis by 2-acetylaminofluorene

Studies were made on why guinea pigs are resistant to carcinogenesis by 2-acetylaminofluorene. Cytochrome P-448 and arylhydrocarbon hydroxylase were not induced in either the microsomes and nuclei of guinea pigs by 3-methylcholanthrene treatment. 3-Methylcholanthrene treatment caused only 2-fold increase in the binding of 2-acetylaminofluorene to DNA in nuclei isolated from guinea pigs, while it caused 17-fold increase in the binding in rat nuclei. Microsomes from 3-methylcholanthrene treated rats had 5 times more effect than Microsomes from 3-methylcholanthrene treated guinea pigs on the binding of 2-acetylaminofluorene to DNA of nuclei from untreated guinea pigs. N-Hydroxy-2-acetylaminofluorene combined equally well with the DNA of rats and guinea pigs. In guinea pigs, there was a good correlation between the low inducibility of cytochrome P-448 and the low binding of 2-acetylaminofluorene to DNA. Our results clearly showed that guinea pigs are resistant to tumor induction by 2-acetylaminofluorene through inability to carry out the first step of activation of 2-acetylaminofluorene.     

Purification of a cytosolic enzyme from human liver with phospholipid hydroperoxide glutathione peroxidase activity

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein which inhibits peroxidation ofmicrosomes. The human enzyme, which may play an important role in protecting the cell from oxidative damage, has not been purified or characterized. PHGPx was isolated from human liver using ammonium sulphate fractionation, affinity chromatography on bromosulphophthalein-glutathione-agarose, gel filtration on Sephadex G-50, anion exchange chromatography on Mono Q resin and high resolution gel filtration on Superdex 75. The protein was purified about 112,000-fold, and 12 μg, was obtained from 140 g of human liver with a 9% yield. PHGPx was active on hydrogen peroxide, cumene hydroperoxide, linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide. The molecular weight, as estimated from non-denaturing gel filtration, was 16,100. The turnover number (37°C, pH 7.6) on (β-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl)-γ-palmitoyl)-l-α-phosphatidylcholine was 91 mol mo⁻¹ s⁻¹. As reported for pig PHGPx, activity of the enzyme from human liver on cumene hydroperoxide and on linoleic acid hydroperoxide was inhibited by deoxycholate. In the presence of glutathione, the enzyme was a potent inhibitor of ascorbate/Fe induced lipid peroxidation in microsomes derived from human B lymphoblastic AHH-1 TK ± CHol cells but not from human liver microsomes. Human cell line microsomes contained no detectable PHGPx activity. However, microsomes prepared from human liver contained 0.009 U/mg of endogenous PHGPx activity, which is 4–5 times the activity required for maximum inhibition of lipid peroxidation when pure PHGPx was added back to human lymphoblastic cell microsomes. PHGPx from human liver exhibits similar properties to previously described enzymes with PHGPx activity isolated from pig and rat tissues, but does not inhibit peroxidation of human liver microsomes owing to a high level of PHGPx activity already present in these microsomes.