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Hepatic 7-alkoxycoumarin O-dealkylase in mice: induction by beta-naphthoflavone of hepatic enzyme activity.

Hepatic 7-alkoxycoumarin O-dealkylation activities in control and beta-naphthoflavone-pretreated mice were determined. The O-demethylation and O-deethylation activities of 7-alkoxycoumarin in control mice were almost the same values, while the O-depropylation activity was lower than those of the other reactions. The O-dealkylase activity varied markedly among the 18 strains of mice surveyed, and strain-dependent differences in the cytochrome P-450 content were also detected among the strains. beta-Naphthoflavone induced O-dealkylation activity, especially O-deethylation and O-depropylation activities, only in ddY, DS and its substrains (A2-3, A3-1, C1-2 and Nh/+). C3H/He and C57BL/6J strains, but not in DBA/2, BALB/c and KYF/2 strains. The former strains of mice are thus classified as "responsive strains" to beta-naphthoflavone and the latter as "non-responsive strains". The O-dealkylase activity in other strains of mice were not clear in responsiveness to beta-naphthoflavone. The hepatic cytochrome P-450 content in responsive strains also increased upon pretreatment of the animals with beta-haphthoflavone. The results indicate marked strain differences in basal and beta-naphthoflavone-induced activity of hepatic 7-alkoxycoumarin O-dealkylase in mice.

Abstract-Hepatic 7-alkoxycoumarin 0-dealkylation activities in control and a9 naphthoflavone-pretreated mice were determined. The 0-demethylation and 0 deethylation activities of 7-alkoxycoumarin in control mice were almost the same values, while the 0-depropylation activity was lower than those of the other reactions. The 0-dealkylase activity varied markedly among the 18 strains of mice surveyed, and strain-dependent differences in the cytochrome P-450 content were also detected among the strains. 8-Naphthoflavone induced 0-dealkylation activity, especially 0-deethylation and 0-depropylation activities, only in ddY, DS and its substrains (A2-3, A3-1, C1-2 and Nh/+), C3H/He and C57BL/6J strains, but not in DBA/2, BALB/c and KYF/2 strains. The former strains of mice are thus classified as "responsive strains" to (3-naphthoflavone and the latter as "non-responsive strains".
The 0-dealkylase activity in other strains of mice were not clear in re sponsiveness to 8-naphthoflavone. The hepatic cytochrome P-450 content in responsive strains also increased upon pretreatment of the animals with 8-haph thoflavone.
The results indicate marked strain differences in basal and l3-naph thoflavone-induced activity of hepatic 7-alkoxycoumarin 0-dealkylase in mice.
The 0-dealkylation reaction of 7 alkoxycoumarin is known to be catalyzed by cytochrome P-450 (1,2). Recent studies have indicated a marked alteration in the substrate specificity and the specific activity of the microsomal 0-dealkylation reaction when rats are pretreated with specific inducers (3 6). Administration of 3-methylcholanthrene or (3-naphthoflavone markedly increases the 0-dealkylation activity of 7-ethoxy and 7 propoxycoumarins, but not of 7-methoxy coumarin, in both isolated microsomes and whole liver homogenate (3,5,6). The existence of multiple forms of cyto chrome P-450 in liver microsomes has been established by many investigators (7,8). Administration of various chemicals causes selective induction of one or more forms of the cytochrome, depending on the chemicals. When rats are employed as experimental animals, 3-methylcholanthrene or /3 naphthoflavone induces cytochrome P-448 (designated as P-448, P-450, MC-B, DE-II and so on by several investigators), which is preferentially active towards benzo(a)pyrene (8)(9)(10)(11)(12). Increase of 0-dealkylation activity of 7-ethoxy and 7-propoxy-coumarins by the administration of polycyclic hydrocarbons (3,5,6) probably indicates that the reaction is catalyzed by cytochrome P-448, similar to benzo(a)pyrene metabolism. Nebert and co workers have indicated the existence of strain differences in the inducibility of hepatic benzo(a)pyrene hydroxylase in mice by 3 methylcholanthrene (13)(14)(15). It is thus inter esting to determine the strain differences in the inducibility of 7-alkoxycoumarin 0 dealkylase in mouse liver for comparison with that of benzo (a) pyrene hydroxylase.
In the present study, the inducibility of hepatic 7-alkoxycoumarin 0-dealkylase was determined in 18 strains of mice. As described by Nebert et al. (13)(14)(15),  Group II: Animals classified in this group were established in the U.S.A. Relation ships among these strains have been described previously (16). Group III: Animals not classified in the above two groups. KYB, KYC and KYF/2 strains were recombinant inbred ones of dd, NC, A/J and CBA strains. animals (9-11 weeks old) of inbred strains were used for the experiments, unless otherwise stated. The following 18 strains were selected: ddY, DS, A2-3, A3-1, C1-2, Nh/+, DBA/2, CBA, C3H/He, C3HeB/Fe, BALB/c, A/J, C57BL/6J, SIc-ICR, Jcl-ICR, KYB, KYC and KYF/2" (Fig. 1 ). Three of them (ddY, SIc-ICR and JcI-ICR) were closed colony strains, and the animals from these strains were used without selection. Only for the A2-3 strain were female animals employed. Mice of ddY and SIc-ICR strains were obtained from Shizuoka Laboratory Center (Hamamatsu), Jcl-ICR, from Japan Clea Co. (Shiga), and KYF/2, from Funahashi Nojo (Chiba). Other strains were bred at the farm of Shionogi Research Laboratories. The animals were kept in an air-conditioned room (25+1 °C, 50-60% humidity) which was lighted 12 hr a day (8:00-20:00) and main tained on commercial rat chow (Ca-1, Japan Clea Co., Tokyo) and water ad libitum. All animals were allowed at least 7 days to become acclimatized to the housing con ditions prior to use in the experiments.
Some of the animals were given intra peritoneal administration of (9-naphthoflavone in sesame oil (80 mg/kg body weight), twice every two days, and used for the experiments 2 days after the last administration.
In some cases, mice received single intraperitoneal injections of various amounts of ,3-naphtho flavone and were killed one day later. Control animals received the same volume of sesame oil alone.
Preparation of enzyme samples: Non-fasted animals were killed by decapitation.
Livers were removed quickly, weighed and homo genized in 0.25 M sucrose containing 50 mM Tris-HCI buffer (pH 7.4). The concentration of the homogenate was adjusted to 100 mg wet weight equivalent of tissue/ml, and then it was filtered through two layers of coarse gauze to remove the undispersed connective tissue. The resulting filtrate was used for the enzyme assay at the homogenate level. When liver microsomes were required for the assay, they were prepared from pooled livers of ~> KYB , KYC and KYF/2 strains of mice were established by Dr. K. Tuchikawa of the National Institute of Genetics (Mishima).
three-four mice using a preparation medium containing 0.154 M KCI and 150 mM Tris HCI buffer, pH 7.4 (5). The protein concen tration of isolated microsomes was deter mined by the method of Lowry et al. (17) using bovine serum albumin as a standard.
Determination of 0-dealkylase activity and cytochrome content: 7-Alkoxycoumarin 0-dealkylase activity in liver homogenate and isolated microsomes was determined by the PCA-precipitation method (6). Cytochrome P-450 content in liver homogenate was detected as described previously (18), and its concentration was calculated using the molar extinction difference of 104 mM-1 cm-1 for the absorption difference between the peak position (at about 450 nm) and 490 nm (19). Cytochrome P-450 content in isolated microsomes was determined according to the method of Omura and Sato (20). Micro somal cytochrome b5 was determined as reported previously (21). Chemicals: 7-Hydroxycoumarin 0-methyl, 0-ethyl and 0-propyl derivatives were syn thesized as described previously (5). NADP, glucose 6-phosphate and glucose 6-phos phate dehydrogenase were purchased from Oriental Yeast Co. (Tokyo). Other chemicals of the purest grade available were obtained commercially and used for the experiments without further purification.

Results
Hepatic 7-alkoxycoumarin 0-dealkylase in various strains of mice: Cytochrome P-450 dependent monooxygenase activity in mouse liver was determined using the O-dealkylation reaction of 7-alkoxycoumarin as a model substrate. When mouse liver homogenate was employed as an enzyme source for the assay, almost the same activity was detected for both 0-demethylation and 0-deethylation reactions, while the O-depropylation activity was generally lower than those of the other reactions (Table 1 ). Interestingly, a remarkable difference in the specific activity of the 0 dealkylation reaction was detected in the 18 strains of mice surveyed. Cytochrome P-450 content in mouse liver also differed markedly in the various strains of animals. However, no correlation was found between the cyto chrome P-450 content and the specific activity of the 0-dealkylation reaction: the 0 dealkylation activity in the DS strain differed markedly from that of ddY, although the cytochrome P-450 content was almost the same ( Table 1). The results suggested that the composition of various forms of cyto chrome P-450 differed among the various strains of mice.
Induction of hepatic 7-alkoxycoumarin 0 dealkylase by /3-naphthoflavone: Nebert and co-workers have indicated a marked strain difference of mice in the inducibility of hepatic benzo(a)pyrene hydroxylase by 3-methyl cholanthrene (13)(14)(15). Thus, we studied the effect of r3-naphthoflavone administration on hepatic 7-alkoxycoumarin 0-dealkylase in mice to compare the characteristics of various strains of mice with the aforemen tioned results. Administration of a-naph thoflavone to mice in Group I (6 strains) led to a marked increase in the 0-deethylation and 0-depropylation activities, especially in the 0-depropylation activity, while the 0 demethylation activity showed only slight alteration ( Fig. 2A). As shown in the figure, cytochrome P-450 content also increased with administration of f3-naphthoflavone, although the extent of the increase was small compared with those in the 0-deethylase and 0-depropylase activities. When (3-naphthoflavone was administered to the mice in Group II, marked increases in the 0-deethylase and 0-depropylase activities and cytochrome P-450 content, as detected in the mice of Group I, were observed only in the C57BL/6J strain. Interestingly, slight alteration in the 0-dealkylase activity and cytochrome content by /3-naphthoflavone was observed in the DBA/2 strain, but no specific increases were found in the 0 deethylase and 0-depropylase activities. The 0-dealkylase activity and cytochroma P-450 content in other strains of mice in Group II were not affected or slightly stimulated by '3-naphthoflavone administration under the experimental conditions employed (Fig. 2B). Hepatic cytochrome P-450-dependent 7 alkoxycoumarin 0-dealkylase in most strains of mice in Group III was not stimulated clearly by 1;3-naphthoflavone treatment. Slight increase in the activity was detected only in KYB and KYC strains in Group III (Fig. 2C).
As shown in Fig. 2, f3-naphthoflavone induced activity of hepatic 7-alkoxycoumarin Fig. 2. Effect of (3-naphthoflavone pretreatment of various strains of mice upon hepatic 7-alkoxy coumarin 0-dealkylase activity and cytochrome P 450 content. The animals were given an intraperi toneal injection of 3-napthoflavone in sesame oil at the dose of 80 mg/kg, twice every 2 days, and livers were obtained 2 days after the last administration. Controls were given the same volume of sesame oil alone. Hepatic 0-dealkylase activity and cytochrome P-450 content were determined using liver homo genate as described in "Methods", and the relative activity or content in the (3-naphthoflavone pretreated mice against the control animals of the corresponding strain is shown. Number of mice used is shown in parenthesis. Figures (a), (b) and (c) represent the experimental data of Groups I, II and III, respectively. 0-dealkylase is strain-dependent in mice. Of the 18 strains of mice employed, seven strains (ddY, DS, A2-3, A3-1, C1-2, Nh/+ and C57BL/6J) were classified clearly as being responsive" to (3-naphthoflavone treatment, and the 0-dealkylase activity in these strains were stimulated by pretreating the mice with Q-naphthoflavone. On the other hand, the activity in three strains of mice (DBA/2, A/J and KYF/2) was not affected by 3-naph thoflavone administration, and thus, these strains of mice were classified as "non responsive".
Under the experimental con ditions employed, some other strains of mice seemed to be responsive, but to a lesser extent in the increase of the 0-dealkylase activity. In the case of closed-colony animals (ICR strain), where the population is he terogeneous, some mice were responsive but others were non-responsive. Administration of (3-naphthoflavone also increased liver weight, with a significant increase being observed only in responsive mice strains (data not shown).
Alteration of microsomal 7-alkoxycoumarin 0-dealkylase activity following administration of (9-naphthoflavone: Liver microsomal 7 alkoxycoumarin 0-dealkylase activity in con trol and ,3-naphthoflavone-pretreated mice was determined to obtain further information on the strain differences in the induction of hepatic 0-dealkylase by (3-naphthoflavone. Mice strains employed for this study were selected based on the results shown in Fig. 2. The 0-dealkylation activity and cytochrome content in liver microsomes prepared from control mice clearly showed strain differences as observed in the homogenate system. Pretreatment of mice with 8-naphthoflavone markedly increased microsomal 0-deethylase and 0-depropylase activities, but not 0 demethylase activity, in DS and C57BL/6J strains. Microsomal cytochrome P-450 con tent in these strains showed higher values. Similar effects of l3-naphthoflavone were observed in liver microsomes obtained from the C3H/He strain (Table 2). On the other hand, microsomal 0-dealkylase activity in DBA/2, BALB/c and SIc-ICR strains was not affected much by r3-naphthoflavone pre treatment. Cytochrome P-450 content in these strains was also not altered by this treatment ( Table 2).
Pretreatment of mice with various amounts of ,3-naphthoflavone: To compare the re sponsibility of hepatic 7-alkoxycoumarin 0 dealkylase to 1 -naphthoflavone, the animals were given single intraperitoneal injection of various amounts of ,3-naphthoflavone, and the hepatic 0-dealkylase activities were determined. As shown in Fig. 3, the activities of both 0-deethylation and 0-depropylation reactions in three strains (DS, C57BL/6J and C3H/He) increased gradually with increasing doses of (3-naphthoflavone. The most re markable increase in the 0-dealkylase activities were observed in the DS strain among the five strains surveyed. Increase of the activity by (3-naphthoflavone adminis tration was observed predominantly in the 0 depropylation reaction compared with that of 0-deethylation (Fig. 3). Hepatic cyto chrome P-450 (P-448) also showed dose dependent increase in these strains, but the 0-demethylation activity increased only slightly (data not shown).
On the other hand, hepatic 0-dealkylase activity and cytochrome P-450 content in non-responsive strains (DBA/2 and BALB/c) were unaffected or even slightly stimulated by the adminis tration of a high amount (1150 mg/kg) of the inducer (Fig. 3). Increases of both 0 deethylation and 0-depropylation activities in DS, C57BL/6J and C3H/He strains were statistically significant (data of statistical analyses are not shown in the figure).

Discussion
Species and strain differences in drug metabolism have been established by many investigators. The existence of species differ ences in the 7-alkoxycoumarin 0-dealkylation reaction was also demonstrated previously (5, 22). Thus, it was interesting to determine the 0-dealkylase activity in various strains of mice to clarify the strain differences in the 0-dealkylase system. As shown in Tables 1 and 2, the O-dealkylase activities and cyto chrome P-450 content in both liver homogenate and isolated microsomes pre pared from control animals varied markedly among the strains, indicating the existence of strain differences in hepatic 7-alkoxy coumarin 0-dealkylase.
On the other hand, the 0 dealkylase activity in DBA/2 and some strains were not affected by E3-naphthoflavone pretreatment (Figs. 2 and 3), and the animals in these strains are not correlated genetically to the responsive strains (Fig. 1). The results suggest genetic regulation of the respon sibility of hepatic 7-alkoxycoumarin 0 dealkylase to ;3-naphthoflavone.
Genetic re gulation of the responsibility of the hepatic benzo(a)pyrene hydroxylase system to 3 methylcholanthrene or other polycyclic hydro carbons has been studied in detail using various strains of mice (13-15, 25, 26 (15). The results suggested that the activities of both benzo(a)pyrene hydroxylation and 7 alkoxycoumarin 0-dealkylation increase by the administration of polycyclic hydro carbons only in the responsive strains of mice, and the present results agree with this. When hepatic 7-alkoxycoumarin 0 Fig. 3. Dose-dependent alterations of hepatic 7-alkoxycoumarin 0-dealkylase activity in various strains of mice. Animals were given single intraperitoneal administration of various amounts of (t-naphthoflavone and were killed 24 hr later. The 0-dealkylation activity of (A) 7-ethoxy and (B) 7-propoxy-coumarins were determined using liver homogenate as the enzyme source, and the relative activity is plotted as a function of (S-naphthoflavone dosage.
Data for each point came from 5 mice.
dealkylase activity was determined using rat samples, marked increases in the 0-deethy lation and 0-depropylation activities were observed by pretreatment of animals with 3 methylcholanthrene or l3-naphthoflavone (5, 6, 22). Similar results were obtained in responsive strains of mice (Figs. 2 and 3). The results suggest that /3-naphthoflavone inducible form(s) of cytochrome P-450 in responsive strains of mice has almost the same catalytic properties as that in rats. We do not presently know the properties of the cytochrome P-450's which catalyze the 0 dealkylation reaction of 7-alkoxycoumarin in mouse livers. Resolution and purification of the cytochrome P-450 monooxygenase system from responsive and non-responsive strains should provide valuable information on the regulation of 7-alkoxycoumarin 0 dealkylation and the oxidative metabolism of other drugs.