Metabolism of α-naphthoflavone by rat, mouse, rabbit, and hamster liver microsomes

72,469-475(1984)

TOXICOLOGYANDAPPLIEDPHARh4ACOLOGY

Metabolism

of cr-Naphthoflavone by Rat, Mouse, Rabbit, and Hamster Liver Microsomes’

HINDA B. BERGMAN,* B. J. BRYANT,? AND STEPHENNESNOW*-2 *Carcinogenesis Laboratory,

and Metabolism Branch (MD-68), Genetic Toxicology Division, Health Efects U.S. Environmental Protection Agency, Research Triangle Park, North Carolina and fNorthrop Services, Inc., Research Triangle Park, North Carolina 27709

Received

June

7, 1983: accepted

September

Research 2771 I

20, I983

Metabolism of cu-Naphthoflavone by Rat, Mouse, Rabbit, and Hamster Liver Microsomes. H. B.,BRYANT, B. J., AND NESNOW, S. (1984). Toxicol Appl. Pharmacol. 72,469475. The metabolism of a-naphthoflavone (ANF) was studied in hepatic microsomes from rats, mice, rabbits, and hamsters, species in which ANF exerts its biological activities. The major metabolites produced by ail species were ANF-5,6-oxide, ANF-6-phenol, and ANF-7,8dihydrodiol. Minor metabolites produced by all species were ANF-5,6dihydrodiol, ANF-‘l-phenol, and ANF9-phenol. In general, the total rates of metabolism were similar within all species: 22-32 nmol ANF metabolized/l 5 min/mg protein. Mouse liver microsomes were approximately 1.7 to 2.9 times as active as the other species on a nanomole of cytochrome P-450 basis. The major sites of enzymatic oxidation were the $6 and 7,8 bonds of ANF where for all species, 49-71s and 15-46s of the total metabolism occurred, respectively. BERGMAN,

a-Naphthoflavone (ANF) is known to stimulate the enzymatic oxidation of benzoa)pyrene by uninduced rabbit, hamster, and mouse liver microsomes (Wiebel, 1980; Huang et al., 198 1) and by human liver homogenates (Kapitulnik et al., 1977; Huang et al., 198 1; Buening et al., 198 1). Liver microsomes from rats induced with phenobarbital also exhibit marked activation of benzo(a)pyrene oxidation by ANF, while liver microsomes from rats induced with 3-methylcholanthrene or ,&naphthoflavone exhibit inhibition of benzo(a)pyrene oxidation by ANF (Nesnow, 1979, 1983; Wiebel, 1980; Nesnow et al., 1982). ANF can stimulate, inhibit, or have no effect on benzo(a)pyrene hydroxylase in the ’ A preliminary report of part of this work has been presented at the Symposium on Organ and Species Specificity in Chemical Carcinogenesis, Raleigh, NC, March 2-4, 198 1 (Nesnow, 1983). * To whom requests for reprints should be addressed.

rat, these effects being related to age, sex, and nutritional status of the animals (Wiebel and Gelboin, 1975). It has been reported that ANF is metabolized by uninduced and phenobarbital, 3methycholanthrene, Aroclor- 1254, and Pnaphthoflavone induced rat liver microsomes (Nesnow et al.. 1980, 1982, 1983; Nesnow and Bergman, 198 1; Coombs et al., 198 1; Coombs, 1982; Nesnow, 1983; Vyas et al., 1983) by human liver preparations (Thakker et al., 198 1), by liver microsomes from the fish Stenotomus versicolor (Stegeman and Woodin, 1980) and by reconstituted cytochrome P-450 systems (Vyas et al., 1983). Based on the recent work of Nesnow et al. ( 1983) and Vyas et al. ( 1983) it is known that liver microsomes from uninduced rats or those induced by &naphthoflavone, 3-methylcholanthrene, phenobarbital, or Aroclor- 1254 metabolize ANF to the following metabolites: cw-naphthoflavone-5,6-oxide (ANF-5,6-oxide),

469

0041-008X/84

$3.00

Copyright 0 1984 by Academic Press. hc. All ngbts of reproduction in any form reserved

BERGMAN,

470

BRYANT,

56 - dihydro - $6 - dihydroxy - (Y- naphthofla vone (ANF-5,6-dihydrodiol), 6-hydroxy-crnaphthoflavone (ANF-6-phenol), 7,8dihydro7,8-dihydroxy-a-naphthoflavone (ANF-7,8dihydrodiol), 7-hydroxy-a-naphthoflavone (ANF-7-phenol), and 9-hydroxy-a-naphthoflavone (ANF-9-phenol). Human liver prep arations and liver microsomes from Stenotomus versicolor produced unidentified ANFdihydrodiol metabolites (Thakker et al., 198 1; Stegeman and Woodin, 1980). The diverse effects of ANF on mouse, rat, hamster, and rabbit liver mixed-function oxidase activity prompted an investigation into the metabolism of ANF by these species in which ANF exerts its effects. It was of interest to identify any similarity in metabolite profiles with the observed biochemical effects of ANF.

METHODS ANF was purchased from Aldrich Chemical Company (Milwaukee, WI), purified as previously described (Nesnow, 1979), generally labeled with tritium by Amersham Corporation (Arlington Heights, IL), and repurified by high pressure liquid chromatography (HPLC) as described by Nesnow and Bergman (1981). Glucose 6-phosphate, NADP, and glucosed-phosphate dehydrogenase were purchased from Sigma Chemical Company (St. Louis, MO). Acetone, ethyl acetate, and methanol were purchased from Burdick and Jackson (Muskegon, MI). All other chemicals used were of the highest purity commercially available. Dr. Robert Roth of Midwest Research Institute, (Kansas City, MO) prepared 5-hydroxy-cY-naphthoflavone (ANF-5-phenol), ANF-6-phenol, ANF-‘J-phenol, 8-hydroxy-cY-naphthoflavone (ANF-l-phenol), ANF9-phenol, and IO-hydroxy-o-naphthoflavone (ANF-lO-phenol) by the general methods of Mahal and Venkataraman (1934). These procedures have been described briefIy by Nesnow et al. (1983). Preparation of microsomes. Siityday-old male CD rats, 3Oday-old male CD- 1 mice, 60day-old male Syrian golden hamsters, and Cmonth-old male New Zealand white rabbits from Charles River Laboratory (Kingston, NY) were maintained on lab chow (Purina Lab Chow) ad libitum. Microsomes from individual uninduced animals were prepared by the following method: rats and hamsters were killed by decapitation and mice and rabbits by cervical dislocation. The liver from each animal was homogenized in 0.25 M sucrose and centrifuged at 9000g for 20 min at 4°C. The supematant fraction was removed and centrifuged at 100,OOOgfor 1 hr at 4°C. The microsomal

AND NESNOW pellet was suspended in 0.01 M potassium phosphate, 0.005 M ethylenediaminetetraacetic acid, 0.000 t M ditbiOthRitoi, and 20% glycerol at pH 7.5 (van der Hoeven et al. 1974). Microsomal protein was determined by the method of Lowry et al. (195 1). Cytochrome P-450 concentration was determined by the method of Omura and Sato (1964). Identification of ANF dihydrodiols. The identification of ANF dihydrodiols produced by hamster, mouse, and rabbit liver microsomes was accomplished by isolating the individual dihydrodiols by HPLC and subjecting them to mass spectral analysis and to acid dehydration for conversion to the corresponding phenol. These phenols were identified by chromatography with authentic standards and by their uv spectra. ANF metabolites were produced from microsomes and a NADPH-generating system according to the previously published procedures (Nesnow and Bergman, 198 1; Nesnow et al., 1983). The ethyl acetate:acetone (2: 1) soluble metabolite extracts were filtered (0.5 pm Teflon filter, Millipore Corp., Bedford, MA) and chromatographed by HPLC (DuPont Instruments Model 850, Wilmington, DE) with a uv detector at 254 nm on a 6.2-mm (id.) X 22.5cm Zorbax-ODS column with a methanohwater gradient mixture; Solvent A: water; Solvent B: methanol. A 20-min gradient program was used (DuPont program 9) at a flow rate of 1.7 ml/min with an initial solvent mixture of 70% B in A and a final solvent mixture of 100% B. Solutions of metabolites were collected and evaporated under dry nitrogen. Analytic composition of the mixtures was obtained by HPLC on the same column with a linear gradient: Solvent A, water; Solvent B, methanol. An initial solvent mixture of 15% B in A and a final solvent mixture of 100% B was employed, increasing B at l%/min at a flow rate of 2.0 ml/min as described by Nesnow et al. ( 1983). Mass spectra of the ANF dihydrodiols were obtained on a VG Micromass mass spectrometer at 70 eV.

Each dihydrodiol produced by each species was heated with 1 ml water and 4 ml of 5 N sulfuric acid on a steam bath for 2 hr (Coombs, 1982; Nesnow et al., 1983). The cooled solution was extracted twice with ethyl acetate (10 ml), mixed (2 min), and centrifuged. The organic layer was neutralized with 5% sodium bicarbonate, centrifuged, and evaporated under dry nitrogen. The acid dehydrated metabolites were reconstituted in methanol and chromatographed by HPLC on a 6.2 (i.d.) X 22.5-cm ZorbaxODS column with a gradient system: Solvent A, water:acetic acid (99:l); Solvent B, methanol:acetic acid (99: 1). An initial mixture of 70% B in A and a final mixture of 100% B with a 20-min gradient (DuPont program 9) was employed at a flow rate of 1.7 ml/min. A mixture of synthetic ANF phenols was used to identify the dehydration products and consisted oE ANF-S-phenol, ANF-6phenol, ANF-‘l-phenol, ANF-8-phenol, ANF-9-phenol, and ANF- lo-phenol. Quantitation of ANF metabolism. Metabolism of ANF by the microsomes of each species was determined by the method of Nesnow and Bergman (1981) with [3H]ANF and conditions previously determined to be optimal for

MAMMALIAN

LIVER

ANF metabolite formation. Incubations consisted of 1.OO mg microsomal protein, 50 mM potassium phosphate buffer, 3 mM magnesium chloride (pH 7.5), and an NADPH-generating system of 4.5 mM glucose 6-phosphate, 1 mM NADP, and 1.8 units/ml glucose-6-phosphate dehydrogenase in a 5-ml incubation volume. The reaction was started by the addition of 0.05 mM r3H]ANF (specific activity l-2 &i/rmol) and was carried out at 37°C for 15 min with agitation. After extraction with ethyl acetate:acetone (2: I), the organic layer was reduced in volume and chromatographed by HPLC on a Zorbax-ODS column. Metabolites were identified by chromatography with authentic standards and quantified by radiochemical methods described previously (Nesnow and Bergman, 1981).

RESULTS Identification

of ANF Metabolites

All of the ANF metabolites produced by rat, mouse, rabbit, and hamster liver microsomes were identified by cochromatography with authentic synthetic standards or with metabolic standards which had been previously identified by spectral analyses (Nesnow and Bergman, 198 1; Nesnow, 1983) except for the ANF dihydrodiols. The ANF dihydrodiols obtained by incubation of ANF with mouse, rabbit, and hamster liver microsomes were identified by collecting the individual dihydrodiols and subjecting them to the mass spectral and chemical degradation procedures, as had been performed by Nesnow et al. ( 1983) for ANF dihydrodiols formed by rat liver microsomes. Each microsome type produced two dihydrodiols, Dihydrodiol I (retention time, 42.8 min) and Dihydrodiol II (retention time, 50.4 min) [see Nesnow et al. (1983) for typical chromatograms]. Both Dihydrodiol I and II from all species produced a molecular ion of M/e 306 (C9Hi404) indicating an ANF dihydrodiol and each exhibited major fragmentation patterns of M/e 288 (M- 18 [HzO]) and M/e 186 (M-120 [H20, C,H&H]). In addition, all Dihydrodiol II metabolites exhibited a major fragmentation peak at M/e 260 (M46 [HzO, CO]) and all Dihydrodiol I metabolites exhibited a major fragmentation peak at M/e 277 (M-29 [CO,H]). Analysis of the

METABOLISM

OF ANF

471

metastable transitions by link-scan mass spectrometry (Haddon, 1979; Millington et al., 1979) indicated that for Dihydrodiol I, M/e 288 and M/e 277 were direct fragments of the molecular ion, while M/e 186 was a direct fragment of M/e 288. For Dihydrodiol II. IV/ e 288 was a direct fragment of the molecular ion while M/e 260 and M/e 186 were direct fragments of M/e 288. All ANF dihydrodiol metabolites were acid dehydrated to phenols which were then identified by chromatography with authentic standards. All Dihydrodiol I metabolites which were acid dehydrated chromatographed with a retention time of 24.6 min identical to that of ANF-6-phenol and each exhibited uv spectra at pH 7.0 and 14 identical to ANF-6-phenol. All Dihydrodiol II metabolites which were acid dehydrated chromatographed with a retention time of 2 1.2 min identical to that of ANF-7-phenol and each exhibited uv spectra at pH 7.0 and 14 identical to ANF-7-phenol. The HPLC chromatograms and uv spectra are similar to those reported by Nesnow et al. ( 1983) for the acid dehydrated ANF dihydrodiols isolated from rat liver microsomes. It is therefore concluded that all Dihydrodiol I metabolites are ANF-5,6-dihydrodiol while all Dihydrodiol II metabolites are ANF7,8-dihydrodiol. Quantitative

ANF Metabolism

All species produced significant amounts of ANF-5,6-oxide, ANF-7,8-dihydrodiol, and ANF-6-phenol although statistical differences were evident (Table 1). Mouse liver microsomes produced more ANF-5,6-oxide than liver microsomes from rabbits, rats, or hamsters based on equivalent amounts of microsomal protein in each incubation mixture (p =G0.05). It is recognized, however, that ANF5,6-oxide is quite sensitive to acid catalyzed rearrangement to ANF-6-phenol and that this rearrangement may occur during the extraction and chromatographic procedures. Mouse and hamster liver microsomes were more efficient at converting ANF to ANF-7,8-dihy-

BERGMAN,

BRYANT,

AND

NESNOW

drodiol than rat or rabbit liver microsomes, while rat, mouse, and hamster liver microsomes produced more ANF-6-phenol than rabbit liver microsomes. ANF-5,6dihydrodiol was produced by all species in small amounts, with rabbit microsomes producing slightly more than the other species. Hamster liver microsomes produced approximately four times the amount of ANF-7-phenol produced by rat, mouse, and rabbit liver microsomes. Hamster liver microsomes, however, produced the smallest quantity of ANF-9-phenol of all species. The total metabolism of ANF to organic soluble metabolites was greatest by mouse and hamster liver microsomes, and metabolism of ANF to water soluble (presumably polyhydroxylated) metabolites was greatest by hamster liver. It has been previously shown that the metabolism of ANF is cytochrome P-450 dependent (Nesnow and Bergman, 1981). Cytochrome P-450 activities were measured in the microsomes of each species and are presented in Table 2. Activity in mouse and rabbit liver microsomes was significantly less than hamster and rat (p < 0.05); however, the activity in the mouse and rabbit were not significantly different, and neither was the activity in hamster liver statistically different from the rat. When the metabolism of ANF by each species is expressed as total organic and water soluble ANF metabolites/ 15 min/nmol cytochrome P-450, the order of activity was mouse > rabbit > rat, hamster (Table 2). DISCUSSION Metabolism of ANF was measured in liver microsomes obtained from untreated mice, rats, hamsters, and rabbits. Qualitatively, the same metabolites were observed in all species. Quantitatively, the four species produced similar amounts (within an order of magnitude) of ANF metabolites on a per milligram protein basis. However, when based on cytochrome P-450 levels, mouse liver exhibited the highest specific ANF metabolizing activity: 130.9 nmol ANF metabolites/l5 min/nmol

MAMMALIAN

LIVER

METABOLISM

473

OF ANF

TABLE 2 HEPATIC

Species Rat Mouse Rabbit Hamster

MICROSOMAL

CYT~CHROME

P-450 AND a-NAPHTHOFLAVONE

Cytochrome P-450” (nmol/mg microsomal protein) 0.625 0.296 0.375 0.863

+ k * r

0.115 0.053 0.110 0.176

METABOLISM

RATE

Total ANF metabolism’ (nmol ANF metabolized/l 5 min/nmol cytochrome P-450) 46.8 130.8 77.9 45.9

u Cytochrome P-450 was determined by the method of Omura and Sato (1964). Values are means -t SD for five individual animals. * Total of organic and water soluble ANF metabolites formed after a 15min incubation.

cytochrome P-450. The high activity of mouse liver to metabolize xenobiotics based on cytochrome P-450 content has been observed in the hydroxylation of aniline, the demethylation of N-ethyl morphine, and the deethylation of 7-ethoxycoumarin when comparing mice, rats, and rabbits (Miranda and Chhabra, 1980). ANF was metabolized by all species at two major sites: the 5,6 bond and 7,8 bond, where 49-7 1% and 15-46% of the metabolism occurs, respectively, depending on species. Three metabolites are formed from 5,6-bond oxidation: ANF-5,6-oxide, ANF-5,6dihydrodiol, and ANF-6-phenol. ANF-5,6-oxide, a primary oxidation product of ANF, can rearrange to ANF-6-phenol, a process which is catalyzed by acid (Nesnow and Bergman, 1981). Although ANF-5,6-oxide can also be hydrated by epoxide hydrolase to ANF-S,(idihydrodiol, the majority of 5,6-bond oxidation is accounted for as ANF-5,6-oxide and ANF-6phenol (84-94% of total 5,6-bond oxidation). ANF-7,8-oxide (a presumed intermediate which has not been isolated) rearranges to form ANF-7-phenol or is hydrated to ANF7,8-dihydrodiol which is the major 7,8-bond oxidation product. Rat, mouse, and rabbit liver is more efficient at metabolizing ANF to 5,6-bond oxidation products (63-7 1% of total metabolites formed) than 7,8-bond products while hamster liver is equally as capable at metabolizing ANF at the 5,6 or 7,8 bond. Phenanthrene is similar in structure to

ANF. Oxidation at the 9,10 position of phenanthrene closely resembles 5,6bond oxidation of ANF. Chaturapit and Holder (1978) have shown that in Wistar rat liver microsomal incubations, 64% of metabolized phenanthrene is the 9,1 Odihydrodiol and less than 0.1% is the 9, I O-oxide. This value is similar in quantity (71%) to the extent of 5,6-bond oxidation of ANF observed in incubations of Charles River CD rat liver microsomes. Metabolism of phenanthrene by SW mouse liver microsomes resulted in 45% of the metabolized phenanthrene as 9,1 O-bond oxidation products, while DBA/U mouse liver microsomes produced 58% phenanthrene 9, IO-bond oxidation products. These results are comparable to ANF metabolism by Charles River CD-l mouse liver microsomes at the similar 5,6 bond (63%). Despite relatively high epoxide hydrolase activities in rodent liver microsomes as measured by the conversion of benzo(a)pyrene4,5-oxide to 4,5-dihydro-4,5-dihydroxybenzo(a)pyrene (rat, 7.5 nmol/min/mg; Bentley et al., 1976) (mouse, 1.5-2.1 nmol/min/ mg; Walker et al., 1978), (rabbit, 9.2 nmol/ min/mg; Miranda et al., 1979), most of the ANF-5,6-oxide remained unhydrated. This finding indicates that ANF-5,6-oxide is not a good substrate for microsomal epoxide hydrolase as reported earlier by Nesnow and Bergman (1981). Vyas et al. (1983) have recently reported that ANF-5,6-oxide was the poorest arene oxide substrate for purified ho-

474

BERGMAN,

BRYANT,

mogeneous epoxide hydrolase obtained from isosafrole-treated rats (1 nmol/min/mg protein). Hamster epoxide hydrolase has not been reported but would seem to be similar to the rat, mouse, and rabbit enzyme in its inability to hydrate ANF-5,6-oxide based on the ANF metabolism data. A multivariate analysis of variance was performed on the data in Table 1 to obtain correlation coefficients between the formation of metabolites. In considering only those correlations with p < 0.002 and a correlation coefficient > 0.52, several relationships were evident. The production of water soluble metabolites is related to the production of ANF5,6-dihydrodiol, ANF-7,8-dihydrodiol, and ANF-5,6-oxide. These results are consistent with the assumption that water soluble metabolites are polyhydroxylated ANF which are formed by the same enzyme process which form the ANF epoxides and dihydrodiols. As would be expected, the production of ANF6-phenol and ANF-5,6-dihydrodiol is related to the production of ANF-5,6-oxide. The concept that ANF might require metabolic activation to exert its inhibitory activity was proposed by Nesnow (1979). This concept has received support from the observation that the ANF metabolite, ANF-9-phenol, was more effective than ANF as an inhibitor of benzo(a)pyrene oxidation by /3-naphthoflavone induced rat liver microsomes. The amount of ANF-9-phenol formed during incubation of microsomes with ANF and benzo(a)pyrene could explain the major inhibitory activity exerted by ANF (Nesnow et al., 1982).

It has been reported that ANF stimulates the mixed-function oxidase activity from hepatic microsomes from rabbits, hamsters, and mice (Wiebel, 1980; Huang et al., 198 1). ANF exerts a range of effects in rat liver microsomes depending on pretreatment, sex, age, and nutritional status (Wiebel and Gelboin, 1975; Viswanathan and Alworth, 198 l), although ANF had no effect on benzo(a)pyrene metabolism by hepatic microsomes from rats used in this study (data not shown). The sim-

AND NESNOW

ilarity of the ANF metabolites formed by all four species and those from /3-naphthoflavone induced rat liver microsomes where ANF exerts an inhibitory effect indicates that no obvious relationship exists between ANF metabolism and ANF stimulation of mixed-function oxidase activity. This conclusion is supported in part by the observation that the synthetic ANF metabolites, ANF-6-phenol, ANF-7phenol, and ANF-9-phenol, did not stimulate benzo(a)pyrene metabolism as measured by benzo(a)pyrene monooxygenase in phenobarbital induced rat liver microsomes (Nesnow, 1983). ACKNOWLEDGMENTS The authors with to thank Dr. David Millington of the University of North Carolina at Chapel Hill for the mass spectra; Mr. Robert E. Easterling, Ms. Mary Morris, and Mr. Rodney Craven for their excellent technical assistance; Mr. Andrew Stead for the statistical analyses; and Mrs. Faye Gregory and Miss Joye Denning for their invaluable help in preparing this manuscript. This paper has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Mention oftrade names or commercial products does not constitute endorsement or recommendation for use.

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