key: cord-0007318-5y15tf5v
authors: Graichen, M. Elizabeth; Dent, John G.
title: Elevation of hepatic microsomal epoxide hydrolase activity by 2-acetylaminofluorene: strain and species differences
date: 1984-01-03
journal: Carcinogenesis
DOI: 10.1093/carcin/5.1.23
sha: 4686ccda4bd99b0cce854a67ab539958c1c09743
doc_id: 7318
cord_uid: 5y15tf5v

Hepatocarcinogens have been shown to cause marked elevation of hepatic microsomal epoxide hydrobase activity in the rat at short intervals after administration. The present studies were designed to characterize 2-acetylaminofluorene (AAF) mediated epoxide hydrolase elevation and to investigate the relationship between epoxide hydrolase increases, AAF metabolism, and hepatocarcinogenicity. Oral or i.p. administration of AAF to F-344 rats produced log-linear doseresponse curves for epoxide hydrolase elevation, measured with either benzo[a]pyrene-4, 5-oxide or styrene oxide substrate. Following a single dose of AAF (35 mg/kg), epoxide hydrolase activity was maximally increased (560% of control) within 48 h, and the activity declined slowly, with a halflife of 17.5 days. Co-treatment with actinomycin D effectively blocked the AAF dependent increase in epoxide hydrolase, suggesblng that de novo protein synthesis is associated with the increase in enzyme activity. Dose-response curves for epoxide hydrolase induction by AAF, N-hydroxy-2-acetylaminofluorene (N-OH-AAF), and aminofluorene were compared, and the potencies for increasing epoxide hydrolase activity reflected the relative hepatocarcinogenic potentials of these agents. In mice, which are resistant to the hepatocarcinogenic action of AAF and deficient in AAF-N-hydroxylase activity, AAF caused no significant increase in hepatic microsomal epoxide hydrolase activity. Similarly, in Cotton rats and guinea pigs, which are lacking in ability to form the sulfate conjugate of N-OH-AAF, neither i.p. nor dietary administration of AAF elicited increases in epoxide hydrolase activity at doses which were maximally effective in F-344 rats. These results support the hypothesis that the ability of compounds to increase epoxide hydrolase activity is related to their carcinogenic potency. Furthermore, the results suggest that increases in epoxide hydrolase activity are associated with metabolism of AAF to the putative proximate carcinogen N-OH-AAF, and the subsequent conversion of this compound to the N-O-sulfate conjugate.

Short-term exposure of rats to hepatocarcinogens has been shown to result in a marked increase in hepatic microsomal epoxide hydrolase (EC 3.3.2.3) activity, while having little effect on cytochrome P^45O-dependent mixed-function oxidase activities (1 -5) . The elevation of epoxide hydrolase activity has been observed following treatment with a wide variety of hepatocarcinogens and is dose-related. Comparison of the slopes of the log-linear dose response curves for increases in epoxide hydrolase elicited by several hepatocarcinogens of diverse structure showed them to be similar, suggesting that the increases in epoxide hydrolase occur through a common mechanism. Furthermore, a correlation has been suggested between heaptocarcinogenic potency and potency in increasing epoxide hydrolase activity (1) .

Okita and co-workers (6) demonstrated the presence of an antigenic component, termed preneoplastic or PN-antigen*, in phenotypically altered foci of liver cells induced by chemical carcinogens. Purification of PN-antigen led to its identification as epoxide hydrolase (7, 8) . More recently, Lin et al. (9) suggested that PN-antigen and epoxide hydrolase are different proteins. While the relationship between PN-antigen and epoxide hydrolase remains to be resolved, it is evident that phenotypically altered foci of hepatocytes induced by chemical carcinogens contain elevated quantities of epoxide hydrolase (8) . Elevated quantities of epoxide hydrolase have been demonstrated immunocytochemically in hyperplastic nodules (10, 11) , and increased epoxide hydrolase activity has been observed in rats subjected to various promoting regimens for induction of hyperplastic nodules (12) and in several hepatomas (8, 13) .

The metabolic activation and carcinogenicity of 2-acetylaminofluorene (AAF) have been extensively studied (for reviews see [14] [15] [16] . The putative proximate carcinogenic metabolite N-hydroxy-2-acetylaminofluorene (N-OH-AAF) is formed by the hepatic cytochrome P-450 mixed-function oxidase system (17) . Several enzymes are known to further metabolize N-OH-AAF, including microsomal deacetylase and cytosolic N-O acetyltransferase and sulfotransferase (18 -20) . Species and strain differences in the metabolic activation of AAF have been reported, and are correlated with susceptibilty to its carcinogenic effects (14,21 -23) .

The objectives of the studies described here were to utilize the known species and strain differences in metabolism of AAF and sensitivity to AAF-induced hepatocarcinogenicity to investigate the mechanism of hepatic microsomal epoxide hydrolase elevation by this compound. All animals were acclimated for 2 weeks prior to use, by which time the mice, guinea pigs, and F-344 rats were 9-10 weeks of age. Due to the difficulties involved in raising Cotton rats, these animals were between 8 and 30 weeks of age when treatments began. Older and younger rats were mixed within treatment groups, and no age-related variations in enzymatic activity were noted in control or treated animals.

The majority of treatments were administered by i.p. injection (1 or 2 ml/kg in DMSO) or by gavage (10 mlAg in corn oil). Control animals received the vehicle alone. Cotton rats were lightly anesthetized with CO 2 before injection. For dietary administration, animals were fed a semisynthetic diet (24) containing 0.02% AAF, purchased from Bio-Serve, Inc., Frenchtown, NJ. Control animals received the semisynthetic basal diet.

Animals were killed by cervical dislocation or by exsanguination under methoxyflurane anesthesia. Livers were quickly removed and washed in icecold 1.15% KC1. After weighing, the livers were coarsely chopped, washed in ice-cold 1.15% KC1, 20 mM Tris-HCl, pH 7.4, and homogenized in 3 volumes of this buffer in a teflon-glass Potter-Elvehjem homogenizer. Postmitochondrial supernatant (PMS) was prepared by centrifugation of the liver homogenate at 10 000 g for 20 min. In some studies, microsomes were isolated by centrifugation of the PMS for 100 000 g-h, followed by resuspension in 20 mM Tris-HCl (pH 7.4) containing 250 mM sucrose and 5.4 mM EDTA. Subcellular fractions were stored at -80°C.

Epoxide hydrolase activity was measured using BPO or SO substrate. After incubation, the reaction products were separated by extraction (mice) (25, 26) or by t.l.c. (all other experiments) (27) . DT-diaphorase (EC 1.6.99.2) activity was assayed by measuring the rate of reduction of methyl red (28) . Protein concentrations were determined by the biuret method (29) using American Monitors Total Protein Kit (American Monitor Co., Indianapolis, IN) with bovine serum albumin as a standard.

Where appropriate, data were evaluated by a one-way analysis of variance. Where significant F values were obtained, tests for differences between treatment means were performed using the Student-Newman-Keuls procedure (30) .

Following administration of a single oral dose of AAF (35 mg/kg) to male F-344 rats, hepatic microsomal epoxide hydrolase activity reached a peak level (560% of control) at 48 h ( Figure 1 ). The enzyme activity declined very slowly, in a log-linear fashion, with a half-life of 17.5 days. At 32 days following treatment, the epoxide hydrolase activity was still nearly twice control levels.

A single i.p. injection of F-344 rats with AAF resulted in a log-linear dose-related increase in epoxide hydrolase activity measured 48 h later, to a maximum of 450% control at a dose of 100 mg/kg ( Figure 2 ). The pattern of increased en- of AAF, epoxide hydrolase activity measured 48 h after the last dose was again increased in a log-linear, dose-related manner ( Figure 3 ). Oral administration of AAF produced a slightly greater maximal increase in epoxide hydrolase than intraperitoneal administration (570% and 380% control, respectively); however the slope and shape of the doseresponse curves were independent of the route of administration. The ED[50] for epoxide hydrolase elevation following three oral or i.p. doses was 12 mg/kg/day. Cytosolic DT-diaphorase activity was also increased in a dose-related manner after three treatments with AAF, but the maximal activity attained was only 156% of control at a dose of 100 mg/kg (Figure 4) .

Co-administration of AD with AAF inhibited the AAF dependent increase in epoxide hydrolase activity in F-344 rats (Table I) . A single dose of 35 mg/kg AAF elicited a 250% increase in epoxide hydrolase activity, but in animals treated with AAF plus AD, epoxide hydrolase activity was not significantly different from control. AD alone had no significant effect on hepatic epoxide hydrolase levels.

Both N-OH-AAF and AF increased hepatic epoxide hydrolase activity in F-344 rats ( Figure 2 ). N-OH-AAF was a more potent inducer than AAF, with a single dose ED[50] of 12 mg/kg, assuming a maximal response equal to that obtained with AAF. The maximal response to N-OH-AAF could not be determined as doses above 20 mg/kg were lethal. In contrast, AF at a dose of 240 mg/kg produced only a 2-fold increase in epoxide hydrolase activity.

Treatment of guinea pigs with three daily doses of AAF as high as 240 mg/kg/day, or with 10 mg/kg N-OH-AAF failed to elicit any significant changes in epoxide hydrolase activity Bars represent mean ± SEM, n = 3, except for the Cotton 100 mgAg group for which n = 2 and the range is displayed. Data for F-344 rats are reproduced from Figure 2 to facilitate comparisons.

•Significantly different from corresponding control, p <0.05.

( Table II) . As noted by other workers (31) , control epoxide hydrolase activity was extremely high in the guinea pig. Similarly, five days' dietary administration of 0.02% AAF to C57B16 or DBA2 mice caused no significant changes in epoxide hydrolase activity (Table III) . Three daily i.p. injections of AAF (35 mgAg) also failed to elicit an increase in hepatic Cotton Fig. 6 . Effects of dietary administration of AAF on EH activity in Cotton and F-344 rats. Animals received 0.02% AAF in the diet for 2 weeks. EH activity was measured in PMS with BPO substrate. Control (•); AAF (0). Bars represent mean ± SEM, n = 3 or 4 animals, except in the case of the Cotton rat controls, for which n = 2 and the range is indicated.

•Significantly different from corresponding control, p <0.05. epoxide hydrolase in these two strains (data not shown).

The epoxide hydrolase activity in Cotton rats was not increased in response to three daily treatments ( Figure 5 ) or two weeks of dietary administration ( Figure 6 ) of AAF at doses that caused 4-to 7-fold increases in F-344 rats. AFB and PB were also administered to Cotton rats as positive controls for epoxide hydrolase increase. Three daily doses of 80 mg/kg PB caused a 230% elevation of epoxide hydrolase activity (data not shown). However, it is of interest to note that AFB did not elicit any significant changes in epoxide hydrolase activity in this strain either when measured 24 -96 h after a single dose of 0.32 mgAg, or when measured 48 h after three daily doses of 0.16 or 0.32 mgAg (data not shown). By comparison, the ED[50] for AFB-dependent epoxide hydrolase elevation in F-344 rats measured 48 h after three daily doses, is0.18mg/kg/day (1).

Treatment of male F-344 rats with AAF elicited a dramatic and dose-related increase in hepatic microsomal epoxide hydrolase activity. The 5-to 6-fold increases measured after only 1 or 3 doses are similar in magnitude to those reported by others (1-4) .

The pattern of elevation observed was independent of the substrate used to assay the enzyme or the route of administration. The same pattern of activity was evident when epoxide hydrolase was measured in microsomes or PMS. A cytosolic epoxide hydrolase (31) and a membrane-bound form of the cytosolic enzyme (32) have recently been reported; however these enzymes display no detectable activity with BPO or SO substrate under the assay conditions (pH 9.0) employed in this study (31, 32) , and thus the epoxide hydrolase activities reported in this study are solely due to the microsomal enzyme.

A dose-related increase in cytosolic DT-diaphorase activity was observed in F-344 rats following AAF treatment, but the maximal increase seen was relatively slight (156% of control). This is in accord with previous observations on the changes in DT-diaphorase activity elicited by other hepatocarcinogens (1) . Some workers, however, have noted larger AAF-dependent increases in DT-diaphorase in Sprague-Dawley rats (3, 33) , possibly indicating a strain difference in response to this compound.

Following a single oral dose of AAF, epoxide hydrolase activity increased rapidly and then declined slowly, with a halflife of 17.5 days. The persistence of the altered enzyme activity suggests that this change is not the result of enzyme activation. These Findings are at variance with those of Astrom and DePierre (3), who reported a decline of elevated epoxide hydrolase activity to approximately twice control level by 7 days after the last of 5 daily doses of AAF in Sprague-Dawley rats. This discrepancy may again be a reflection of a strain difference.

Co-treatment of rats with AD, an inhibitor of RNA synthesis (34) , effectively blocked the epoxide hydrolase elevation in response to AAF. This result is in accord with the work of Gonzalez et al. (35) , who demonstrated that both AAF and N-OH-AAF cause an increase in intracellular levels of mRNA coding for epoxide hydrolase in rats. This finding, considered together with the magnitude and persistence of the elevation observed, strongly suggests that the increase in epoxide hydrolase activity is the result of enhanced enzyme synthesis.

The potencies of the structurally related compounds AAF, N-OH-AAF, and AF for inducing epoxide hydrolase activity reflect their hepatocarcinogenic potencies. N-OH-AAF, which is more hepatocarcinogenic than AAF in the rat (36) yielded an ED[50] of ~ 12 mg/kg versus 20 mg/kg for AF. The less potent hepatocarcinogen AF (37,38) elicited a relatively small increase in epoxide hydrolase activity (240% of control), and no dose-response relationship over the range of doses employed.

Hepatic epoxide hydrolase was not inducible by AAF in guinea pigs, mice, or Cotton rats, or by N-OH-AAF in guinea pigs. This reflects the hepatocarcinogenic potency of these compounds in these species (21, 23) . The strain and species differences in susceptibility to AAF-induced hepatocarcinogenesis have been attributed to variations in the metabolic activation of this compound. Guinea pigs are lacking in sulfotransferase activity, and also readily convert N-OH-AAF to 7-OH-2-acetylaminofluorene, which is inactive as a carcinogen (39) . Cotton rats possess 5 times the AAF-N-hydroxylase activity, 3 times the N-OH-AAF-deacetylase activity, but only 1/3 the N-OH-AAF-sulfotransferase activity of Sprague-Dawley rats (23) . It is probable that the lack of AAFdependent epoxide hydrolase induction observed in these animals is related to this altered pattern of AAF metabolism. The results are consistent with the hypothesis that formation of the N-O-sulfate conjugate of N-OH-AAF is necessary for epoxide hydrolase elevation by this compound. Mice possess very low levels of AAF-N-hydroxylase activity (22, 40) . The lack of epoxide hydrolase increase in response to AAF in this species is likely a reflection of the low rate of formation of N-OH-AAF.

The results presented support the hypothesis that AAF dependent increases in epoxide hydrolase activity are the result of enzyme induction. The relative potencies of AAF, N-OH-AAF, and AF for inducing epoxide hydrolase reflect the hepatocarcinogenicity of these compounds. The extent of epoxide hydrolase elevation by AAF is related to its hepatocarcinogenic potency in several species and strains. These observations provide further support for the hypothesis that a relationship exists between hepatocarcinogenic potency and increases in epoxide hydrolase. Furthermore, these data, considered together with subsequent studies involving modulation of AAF metabolism (Graichen and Dent, manuscript in preparation), support the suggestion that metabolic activation of AAF to N-OH-AAF and the subsequent conjugation of N-OH-AAF with sulfate are required for AAF to elicit increases in epoxide hydrolase activity.

Effect of hepatocarcinogens on epoxide hydrolase and other xenobiotic metabolizing enzymes, Carcinogenesis

Alterations in the enzyme activity and polypeptide composition of rat hepatic endoplasmic reticulum during acute exposure to 2-acetylaminofluorene

Characterization of the induction of drug-metabolizing enzymes by 2-acetylaminofluorene

Effects of hepatocarcinogens and hepatocarcinogenesis on the activity of rat liver microsomal epoxide hydrolase and observations on the electrophoretic behavior of this enzyme

Epoxide hydrolase activity in small biopsy samples of preneoplastic and neoplastic rat liver

A new common marker for premalignant hepatocytes induced in the rat by chemical carcinogens

Purification and quantitation of preneoplastic antigen from hyperplastic nodules and normal liver

Identification of epoxide hydrolase as the preneoplastic antigen in rat liver hyperplastic nodules

Purification and partial characterization of a preneoplastic antigen in liver carcinogenesis

Immunocytochemical localization of epoxide hydrase in hyperplastic nodules induced in rat liver of 2-acetylaminofluorene

Characterization of microsomal epoxide hydrolase in hyperplastic liver nodules of rats

Comparison of hepatic initiation-promotion systems

Quantitation of epoxide hydrolase released from hyperplastic nodules and hepatoma microsomes

Species and tissue variations in the metabolic activation of aromatic amines

The metabolic activation of carcinogenic aromatic amines and amides

Biochemical formation and pharmacological, toxicological, and pathological properties of hydroxylamines and hydroxamic acids

The role of cytochrome P-450in N-hydroxylation of 2-acetylaminofluorene

Electrophilk N-acetoxyaminoarenes derived from carcinogenic N-hydroxy-Nacetylaminoarenes by enzymatic deacetylation and transacetylation in liver

N-hydroxy-2-acetyiaminofluorene sulfotransferase: its probable role in carcinogenesis and in protein-(methion-S-yl) binding in rat liver

Mechanism of reaction, tissue distribution, and inhibition of arylhydroxamic acid acetyltransferase

The comparative carcinogenicities of 2-acetylaminofluorene and its N-hydroxy metabolite in mice, hamsters, and guinea pigs

Genetic differences in the aromatic hydrocarbon-inducible N-hydroxylation of 2-acetylaminofluorene and acetominophen-produced hepatotoxicity in mice

In vitro metabolism and mutagenic activation of 2-acetylaminofluorene by subcellular liver fractions from Cotton rats

Rapid emergence of carcinogen-induced hyperplastic lesions in a new model for the sequential analysis of liver carcinogenesis

A rapid assay for epoxide hydratase activity with benzo[a]pyrene-4,5-(K-region)-oxide as substrate

A radiometric assay for hepatic epoxide hydratase activity

Hepatic microsomal epoxide hydrase: a sensitive radiometric assay for hydration of arene oxides of carcinogenic aromatic hydrocarbons

A radiometric assay for methyl red azoreductase-DT diaphorase

Determination of serum proteins by means of the Biuret reaction

Principles and Procedures of Statistics

Cytyosolic and microsomal epoxide hydrolases: differential properties in mammalian liver

A new microsomal epoxide hydrolase, 77ie Pharmacologist

Rat liver post-mkrosomal D-T diaphorase: activation of the enzymes by two carcinogens

Trans-stilbene oxide: a selective inducer of rat liver epoxide hydratase

Effects of 2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorerie on cellular levels of epoxide hydratase, cytochrome P-450 b, and NADPH-cytochrome c (P-450) oxidoreductase messenger ribonucleic acids

N-hydroxy-2-acetylaminofluorene: a metabolite of 2-acetyl-aminofluorene with increased carcinogenic activity in the rat

Studies of the carcinogenic action in the rat of 2-nitro-, 2-amino-, 2-acetylamino-, and 2-diacetylaminofluorene after ingestion and after painting

The carcinogenicity of compounds related to 2-acetylaminofluorene. II. Variations in the bridges and the 2-substituent

Metabolism of N-hydroxy-2-acetylaminofluorene and N-hydroxy-2-aminofluorene by guinea pig liver microsomes

Genetic differences in the enzymic properties of the aromatic hydrocarbon inducible N-hydroxylation of 2-acetylaminonuorene in mouse liver

We wish to express our appreciation to Stephanie Schnell and Doug Neptun for excellent technical assistance and Drs. James Popp and Thomas Leonard for many useful discussions.