XENOBIOTICA,

1992, VOL. 22,

NO.

11, 1329-1 337

Effects of oxygen concentration on the metabolism of anisole homologues by rat liver microsomes H. OHIT, E. TAKAHARA, S. O H T A and M. HIROBES Faculty of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 1 1 3, Japan

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Received 16 December 1991; accepted 22 May 1992

1 . The effects of oxygen concentration were studied on the metabolic pathways of anisole homologues (anisole, phenetole and isopropoxybenzene) catalysed by liver microsomes from phenobarbital-treated rats. 2. With increase of oxygen concentration, the rate of anisole o-hydroxylation reached a plateau at about 35 PM 0,, while the rates of 0-demethylation and aromatic phydroxylation were still increasing at 223 PM 0, (air). 3. The rates of all three metabolic reactions of phenetole reached plateau levels at about 8 0 0,. ~ ~ 4. The rates of all three metabolic reactions of iso-propoxybenzene were still increasing ~ (air). ~ as 2 2 3 0,

5. The ratio of aromaticp-hydroxylation or 0-dealkylation to aromatic o-hydroxylation decreased in anisole metabolism, and showed no uniform change in phenetole and isopropoxybenzene metabolism with decreasing oxygen concentration. 6. T h e ratio of aromatic p-hydroxylation to 0-dealkylation was essentially constant over the range of oxygen concentration studied in anisole and phenetole metabolism, while in iso-propoxybenzene metabolism the ratio was different between higher and lower oxygen concentrations than 6 0 ~ ~ .

7. This series of compounds with increasing chain length did not show homologous changes in rates of product formation or 0, dependent of product formation.

Introduction Cytochromes P-450 in liver microsomes play an important role in the metabolism of a wide variety of compounds, including endogenous substrates such as steroids and fatty acids as well as exogenous substances (xenobiotics) (Lu and West 1980). For catalytic activity, cytochromes P-450 require reducing equivalents and molecular oxygen. Jones (198 1) mentioned that many drug-metabolizing reactions in vivo depend upon the ambient oxygen concentration, since molecular oxygen is necessary directly as a substrate or indirectly as a source for bioenergy formation. Oxygen concentration could also be important from the clinical point of view (Angus et al. 1990). Jones’ group (Jones 1981, 1984, Aw and Jones 1982) and some other groups (Von Kkrekjirta and Staudinger 1966, Hlavica and Kiese 1969, Fujii et al. 1981, Erickson et al. 1982, Tsuru et al. 1982, Pohl et al. 1984) have studied drugmetabolizing reactions at low oxygen concentrations using subcellular fractions or isolated hepatocytes. Generally the formation of oxidized metabolites has been

t Current address: Department of Biopharmaceutics, School of Pharmaceutical Sciences, Showa University, Hantanodai, Shinagawa-ku, Tokyo 142, Japan. t. T o whom all correspondence should be addressed. 0049-8254/92 $3.00 0 1992 Taylor and Francis Ltd

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H . Ohi et al.

reported to be decreased and to show dependence on oxygen concentrations only under hypoxic conditions. T h e actual oxygen concentrations in vivo is not the same as that in the atmosphere, and therefore the study of drug-metabolizing reactions should be carried out at an appropriate oxygen concentration. In the catalytic cycle of the cytochrome P-450, substrate binding alters the spin state of the haem iron. T h e equilibrium of spin state is affected by the lipophilicity of the substrate. This change in the spin state of the haem iron affects the susceptibilty of cytochrome P-450 to one-electron reduction, which consequently affects the binding of molecular oxygen to the haem iron. Thus, the substrate structure may affect the dependence of cytochrome P-450 on molecular oxygen. I n fact, the apparent Km(0,) values reported vary with different substrates (Von Kkrekjarto and Staudinger 1966, Boyd 1970, Bernhardt et al. 1973, Zachariah and Juchau. 1977, Jones and Mason 1978). However reported substrates are not structurally related. Previously, we reported that the metabolic pathways of anisole were altered by change in the oxygen concentration (Takahara et al. 1986). We chose anisole as the substrate, since the mixed-function oxidase system metabolizes anisole by two principal oxidation pathways, aromatic hydroxlation and 0-dealkylation, in spite of its very simple structure (Daly et al. 1968, Daly 1970). Therefore, the assessment of two different drug-metabolizing reactions is possible. In this study we investigated the alteration of the metabolic pathways of anisole homologues (anisole, phenetole and iso-propoxybenzene) under various oxygen concentrations to examine the relationship between the drug structures and the effect of oxygen concentration.

Materials and methods Materials All chemicals were of reagent grade unless otherwise indicated. NADP and glucose 6-phosphate were purchased from Boehringer Mannheim Biochemicals, Indianapolis, IN. Glucose 6-phosphate dehydrogenase was obtained from Sigma Chemical Co., St Louis, MO. Standard gases (1.0% and 10% 0, in N2) were purchased from Takachiho Chemical Industry, and gas mixtures of other desired concentrations (2.0 4 0 and 6.0% 0,in N,) were obtained with a gas divider apparatus (SGD-XC51, STEC Inc.). Oxygen concentration of the assay solution T h e oxygen concentrations of the assay solution equilibrated with the gases (1.0, 2.0,4.0, 6.0, 10% 0, in N,, and air) were determined to be 24,34,54,74, 113 and 223 p ~respectively, , by use of a Clark oxygen electrode. Preparation of rat h e r microsomes Male U'istar rat3 (ca. ISOg), obtained from Nippon Bio-Supp. Center, Tokyo, were used. T h e rats received intraperitoneal injections of sodium phenobarbital (60mg/kg) in saline once daily for three consecutive days, followed by 24 h starvation prior to killing by decapitation on the fourth day. Their livers, perfused via the portal vein with cold 1.19% KCI, were weighed, minced and subsequently homogenized in 4:l (v/w) cold sodium phosphate buffer (pH 7.4 in a Potter-Elvehjem type homogenizer. The homogenates were combined and centrifuged at 9000g for 20 min at 4°C. T h e supernatant fractions were centrifuged at 105000g for 1 h at 4"C, then the microsomal pellet obtained was resuspended in 1:l (v/w) cold sodium phosphate buffer (pH 7.4) and stored at -78°C until use. Protein was measured by the method of Lowry et a1.(1951) using bovine serum albumin as the standard. Microsomal incubation T h e formation rates of metabolites of anisole homologues were determined according to the method of Holtzman et al. (1983). with slight modifications. The system consisted of gassing towers with rubber seals at the top, connected with stainless-steel tubes. T h e gas was passed through the first gassing tower containing 0 . 1 sodium ~ phosphate buffer (pH 7.4). Then the humidified gas was passed successively through the towers containing the components (microsomes and glucose 6-phosphate dehydrogenase, NADP and glucose 6-phosphate) in the same buffer. T o avoid denaturation of the microsomes and glucose 6-phosphate dehydrogenase by gas bubbling, the gas was simply passed over them for 2h. The final reaction vessel was a 50ml Erlenmeyer flask. The mixture of microsomes and glucose 6-phosphate

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Oxygen concentration and drug metabolism

1331

dehydrogenase in the buffer was transferred to the reaction vessel with a gas-tight syringe, and then the substrate in 20p1 of acetone was added. After preincubation for 3 min the reaction was initiated by addition of the mixture of NADP and glucose 6-phosphate. The final volume of the mixture was 2.52m1, and it contained 4mg of microsomal protein, 0.4 mM NADP, 4 m glucose ~ 6-phosphate, 100mM KCI, 10 units of glucose 6-phosphate dehydrogenase, and 2 . 0 m ~substrate. The reaction was carried out for 5min at 37°C with continuous gassing (100-200ml/min) and shaking (100 strokes/min). T h e reaction was terminated by the addition of 15% ZnSO, solution (1 ml) through the seal. Then the seal was removed, and saturated Ba (OH), (1 ml) was added. T h e mixture was centrifuged, and the metabolites were extracted from the supernatant fraction (3 ml) with dichloromethane (7 ml x 3). The combined organic phase was carefully concentrated under reduced pressure at room temperature to prevent loss of the metabolites. Next, N,O-bis (trimethylsilyl) trifluoroacetamide and internal standard (2,4,6-trimethylphenol for anisole metabolites, o-hydroxyanisole for phenetole and iso-propoxybenzene metabolites) were added, and the mixture was heated at 70°C for 1 h. Aliquots of this solution were subjected to gas chromatography on a 2m column packed with 5% Silicon SE-52 on Chromosorb W AWDMCS 60/80 (GL Sciences). The column was held as 110°C and eluted with nitrogen (40ml/min). Peak areas were determined by electronic integration of the flame ionization detector output. In the inhibition experiment the reaction mixture contained the same components as above, but with the addition of S K F 525A or 7,sbenzoflavone to achieve a final concentration of 0.1 mM, and the reaction was carried out for 5 min under atmospheric conditions.

Results EfJects of oxygen concentration on anisole metabolism T h e metabolites of anisole by liver microsomes from phenobarbital-treated rats were the 0-demethylated product (phenol) and aromatic hydroxylated products (0-and p-hydroxyanisole); m-hydroxyanisole was not produced. Figure 1 shows the relationship between the rates of metabolite formation of anisole and oxygen concentration. With increase of oxygen concentration, the formation of o-hydroxyanisole became saturated at about 3 5 p O,, ~ while the formations of phenol and p-hydroxyanisole were still increasing at 223 ~ L 0, M (air). T h e formation of p-hydroxyanisole was greater than those of the two other and metabolites in the range of oxygen concentration examined. T h e apparent Km(02) V,,, for anisole o-hydroxylation were 80 p~ and 2.2 nmol/min per mg of protein, respectively. Effects of oxygen concentration on phenetole metabolism T h e metabolites of phenetole produced by liver microsomes from phenobarbitaltreated rats were the 0-deethylated product (phenol) and aromatic hydroxylated products (0-and p-hydroxylphenetole), analogously to the case of anisole. No side chain- or m-hydroxylated product was detected. T h e relationship between the rates of metabolite formation of phenetole and oxygen concentration is depicted in figure 2. With increase of oxygen concentration, the formation rates of the three metabolites similarly became saturated at about 8 0 p 02. ~ In this case, formations of p-hydroxyphenetole and phenol were almost the same, but that of o-hydroxyphenetole was small, in the range of oxygen concentration examined. T h e kinetic parameters calculated for these three reactions were as follows: K,(02)84, 143 and 83 p ~ V,,, , 9.45, 1.4 and 11.1 nmol/min per mg of protein, for 0-deethylation, o-hydroxylation and p-hydroxylation, respectively. Eflects of oxygen concentration on iso-propoxybenzene metabolism iso-Propoxybenzene was metabolized by liver microsomes from phenobarbitaltreated rats to form the 0-dealkylated product (phenol) and aromatic hydroxylated

H . Ohi et al.

1332 *.O

1

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100 is0 200 Oxygen concentration (wM)

250

Figure 1. Rate of formation of anisole metabolites by liver microsomes from phenobarbital-treated rats as a function of oxygen concentration. Each value is the m e a n f S D of three or four determinations. 0-dealkylation (-0-); o-hydroxylation (---0---); p-hydroxylation (- - - - 0 -- - -).

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Rate of formation of phenetole metabolites by liver microsomes from phenobarbital-treated rats as a function of oxygen concentration.

Each value is the mean? SD of three or four determinations. 0-dealkylation (-0-); o-hydroxylation (---&--); p-hydroxylation (- - - - 0 -- - -).

products (0-and p-hydroxy-iso-propoxybenzene). As in the case of phenetole, no mor side chain-hydroxylated product was detected. T h e formation rates of the three metabolites at each oxygen concentration are shown in figure 3. In all three cases the formation rates were still increasing at 223 PM O2(air). In addition, though more metabolites were formed at 223 PM 0 2there , was a 0 0,. ~ In ~the cases of anisole and small peak of formation rates at around 6 phenetole, such a peak was not observed.

Eflect of oxygen concentration on relative ratios of metabolic reactions Figure 4 shows the ratios of metabolic reactions at each oxygen concentration. In the case of anisole, as the oxygen concentration decreased, the relative ratios of 0-dealkylation and p-hydroxylation with respect to o-hydroxylation decreased gradually, while the ratio of p-hydroxylation to 0-dealkylation was not changed in the range of oxygen concentration studied (figure 4A). T h e ratio of p-hydroxylation to 0-dealkylation of phenetole was not changed in the range of oxygen concentration studied as in the case of anisole metabolism. However, the relative ratio of p-hydroxylation to o-hydroxylation increased at

Oxygen concentration and drug metabolism

50

0

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Figure 3.

100 IS0 200 Oxygen concentration (KMI

1333

250

Rate of formation of iso-propoxybenzene metabolites by liver microsomes from phenobarbital-treated rats as a function of oxygen concentration.

Each value is the mean+SD of three or four determinations. 0-dealkylation (-0-); o-hydroxylation (---U---); p-hydroxylation (- - - - 0 -- - -).

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Relative ratios of metabolites of anisole

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(C) formed by liver microsomes from phenobarbital-treated rats as a function of oxygen concentration. Each value is the mean+SD of three or four determinations. 0-dealkylation to o-hydroxylation (- 0 -); p-hydroxylation pto o-hydroxylation (--- 0---); hydroxylation to 0-dealkylation (---- 0

oxygen concentrations below about 6 0 p ~ contrary , to those of anisole, which decreased gradually with decreasing oxygen concentration (figure 4 A and 4 B). Thus, though the difference in structure between anisole and phenetole is small, those metabolic pathways were affected quite differently by the oxygen concentration. T h e change of the ratios in iso-propoxybenzene metabolism was not simple and altered at around 6 0 p~ 0,. T h e ratios of 0-dealkylation and p-hydroxylation to o-hydroxylation increased biphasically with decreasing oxygen concentration:

H . Ohi et al.

1334 Table 1 .

Effects of SKF 525A and 7,s-benzoflavone on the metabolism of anisole derivatives by liver microsomes from phenobarbital-treated rats.

SKF 5254 Anisole Phenetole iso-Propoxybenzene

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7,8- Benzojlavone Anisole Phenetole iso-Propox ybenzene

Metabolic reaction (percentage of control)

0-Dealkylation o-Hydroxylation p-Hydroxylation 44.6 f4.8' 3 5 0 f 1.3** 45,6+2.3* 37.1 f3.0'' 56.7 5 2.3' 54.7 f 1,9'* 56.6 k 6.7'* 36.5f3.3** 44.6 f6.2' 79.1 k 0 . 7 129.6 k 13.4f 1140f 13.8

133.0+ 5.0' 138.8 f9.5* 1 1 7.8f 17.3

98.8 f3.7 87.8f6.3* 91.7 k 5.3

Each value is the mean f SD of four determinations. * p

Effects of oxygen concentration on the metabolism of anisole homologues by rat liver microsomes.

1. The effects of oxygen concentration were studied on the metabolic pathways of anisole homologues (anisole, phenetole and isopropoxybenzene) catalys...
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