Regular Article Activities of Cytochrome P450 Enzymes in Liver and ...

Drug Metabol. Pharmacokin. 17 (1): 47–53 (2002).

Regular Article Activities of Cytochrome P450 Enzymes in Liver and Kidney Microsomes from Systemic Carnitine Deˆciency Mice with a Gene Mutation Organic Cation Transporter of Carnitine W Hiroshi YAMAZAKI, Hitomi IKETAKI, Ayaka SHIBATA,

Miki NAKAJIMA and Tsuyoshi YOKOI* Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan Summary: Juvenile visceral steatosis (jvs) mice, isolated from the C3H-H-29strain, exibit a systemic carnitine deˆciency (SCD) phenotype and develop fatty liver, hyperammonemia and hypoglycemia. This phenotype is caused by a missense mutation (Leu352Arg) of a sodium-dependent carnitine W organic cation transporter, Octn2 (Slc22a5). The jvs mouse could be a useful model for pharmacokinetics and drug metabolism studies concerning Octn2 substrate drugs. In the present study, the eŠects of the SCD phenotype on the cytochrome P450 (P450 or CYP) dependent activities of four endobiotic and seven xenobiotic oxidations catalyzed by liver and kidney microsomes from jvs mice were investigated. The jvs-type mutation was genotyped by PCR-RFLP. The contents of total P450 and NADPH-P450 reductase were similar in the the liver microsomes from male or female mice of the wild-type and those heterozygous or homozygous for the jvs-type mutation. The 6b-hydroxylation activities of testosterone and progesterone (marker for Cyp3a) based on the protein contents were 1.2- to 2.0-fold higher in liver microsomes from jvs W jvs-type mice compared to jvs W wt- or wt W wt-type mice. Coumarin 7-hydroxylation activijvs-type mice. The activities of lauric ties (marker for Cyp2a) were decreased to 0.7-fold in the male jvs W acid 12-hydroxylation (a marker for Cyp4a) and aniline p-hydroxylation (a marker for Cyp2e1) in liver jvs-type mice. Genotoxic activation of 2microsomes were increased 1.4- to 1.9-fold in female jvs W amino‰uorene (a marker for Cyp4b1) by male and female mouse kidney microsomes were not aŠected by the SCD phenotype. These results demonstrated that the SCD phenotype aŠected the P450-dependent catalytic activities in liver microsomes. The jvs mouse could provide valuable information in drug interaction and drug metabolism studies of OCTN2 substrate drugs and new compounds in development.

Key words: juvenile visceral steatosis (jvs) mouse; carnitine transporter Octn2; P450; lauric acid; testosterone; aniline 352 located within the sixth transmembrane domain of the organic cation transporter, Octn2 (Lu et al., 1998; Nezu et al., 1999). Mouse Octn2 has been shown to consist of 557 amino acids and has 85.5z identity with human OCTN2 (Nezu et al., 1999). Recently, human OCTN2, SLC22A5, has been cloned and analyzed the function has been analyzed (Nezu et al., 1999; Ohashi et al., 2001). OCTN2 has been shown to transport carnitine in a sodium-dependent manner, which is essential for fatty acid metabolism in mitochondria, and its functional defect leads to fatal systemic carnitine deˆciency in humans (Nezu et al., 1999). A defect of the carnitine transport system in the plasma membrane in systemic carnitine deˆciency patients who are autosomal recessive is characterized by

Introduction A C3H-H-29strain of mouse autosomal-recessively associated with microvesicular fatty inˆltration of viscera was ˆrst established in the Institute for Experimental Animals, Kanazawa University (Koizumi et al., 1988). The mice, named juvenile visceral steatosis (jvs) mice, revealed severe lipid accumulation in the liver within a few days after birth, and hypoglycemia, hyperammonemia, and growth retardation within 3 weeks after birth. The jvs mice exhibit a systemic carnitine ( b-hydroxyl-g-trimethylaminobutyric acid) deˆciency phenotype (Kuwajima et al., 1991). Recently, it has been reported that this phenotype was caused by a missense mutation from CTG (Leu) to CGG (Arg) at codon

Received: November 30, 2001; Accepted: January 15, 2002 * To whom correspondence should be addressed: Tsuyoshi YOKOI, Ph.D., Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan. TEL: +81-76-234-4407, FAX: +81-76-234-4407, E-mail: TYOKOI＠kenroku.kanazawa-u.ac.jp

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progressive cardiomyopathy, skeletal myopathy, hypoglycaemia and hyperammonaemia (Hashimoto et al., 1998, Nezu et al., 1999). Cytochrome P450 (P450, CYP) enzymes catalyze the oxidation of a broad spectrum of endobiotic and xenobiotic substrates. The resulting hydroxylated metabolites are more hydrophilic, facilitating renal and biliary excretion. It is well-known that the P450 enzyme levels are aŠected by many diseases (Howden et al., 1989; Paintaud et al., 1996). The jvs mice from the C3H-H-29strain have been reported to show markedly low levels of the hepatic urea cycle enzymes (Imamura et al., 1990). The marked decline in renal carnitine reabsorption has been thought to account for the systemic carnitine deˆciency in jvs mice. Carnitine administration is necessary to suppress the cardiac hypertrophy in jvs mice (Horiuchi et al., 1993). It has been suggested that the SCD phenotype may aŠect not only the regulation of the tissue-speciˆc expression of the urea cycle enzymes but also the regulation of their developmental induction (Tomomura et al., 1992). However, there are no reports on the eŠects of the SCD phenotype on drug metabolizing systems such as P450 enzymes in jvs mice. In the present study, the characteristics of P450 enzymes were investigated concerning the oxidation activities of endobiotics and xenobiotics in liver microsomes from jvs mice. Genotoxic activation of a procarcinogen by kidney microsomes was also investigated. Materials and Methods

Chemicals Carnitine was obtained from Sigma (St Louis, MO). The typical drug substrates, their metabolites, and reagents used in this study were obtained from sources described previously or were of the highest grade commercially available (Emoto et al., 2000a, b; Yamazaki and Shimada, 1999). Jvs Mice Male and female jvs mice from C3H W HeJ-jvs W jvs, jvs or -wt W wt strain were a generous gift from Drs. -wt W Hiroko Nikaido and Jun-Ichiro Hayakawa, Institute for Experimental Animals, Kanazawa University. Carnitine (1 mg W body W day) was subcutaneously administered to homo-mutated type mice from 10 to 21 days old. All mice were fed a diet containing 3.7z of total fat and 1z of carnitine. Individual liver microsomes and pooled kidney microsomes from mice (10–12 weeks old) were prepared in 10 mM Tris-HCl buŠer (pH 7.4) containing 0.10 mM EDTA and 20z (v W v) glycerol as described previously (Emoto et al., 2000a). Genotyping of Octn2 Gene in Mice The genotyping of the Octn2 ( Slc22a5) mutation causing an amino acid change T1114G was performed

Fig. 1. PCR-RFLP analysis for Octn2 gene in mouse genomic DNA. The PCR product of 204 bp was digested with a restriction enzyme of AvaII. Lanes 1 and 2, wt W wt-type male and female; Lanes 3 and 4, wt W jvs-type male and female; Lanes 5 and 6, jvs W jvs-type male and female, respectively.

according to the method described elsewhere (Nezu et al., 1999; Lu et al., 1998) with slight modiˆcations. Brie‰y, PCR ampliˆcation was performed in mixtures (ˆnal volume of 50 ml) containing 10×Taq buŠer, 0.2 mM dNTPs, 5 pmols of sense primer (5?gttgctgctccagctctctt-3?) and antisense primer (5?-aagggcagccgagatagaat-3?), genomic DNA (100 ng) isolated from mouse livers, and 1 U Taq polymerase (Greiner Japan, Tokyo, Japan). The ampliˆcation was perC for 1 min, annealing at formed by denaturation at 949 609 C for 2 min, and extension at 729 C for 30 sec for 30 cycles using a PCR Thermal Cycler Personal (Takara, Kyoto, Japan). The PCR products of 204 bp in intron 6 and exon 7 were digested with AvaII, electrophoresed using 15z polyacrylamide gels, and visualized with ethidium bromide. Homozygotes of jvs allele yielded 163 and 41 bp fragments, heterozygotes of the jvs W wild allele yielded 204, 163, and 41 bp fragments, and homozygotes of the wild allele yielded 204 bp fragments (Fig. 1).

Assays for Endobiotics and Xenobiotics Oxidation Activities Standard reaction mixtures in a ˆnal volume of 0.20 ml for the determination of the substrate oxidation by P450 enzymes included mouse liver microsomes (0.1–0.5 mg protein W ml) in 50–100 mM potassium phosphate buŠer (pH 7.4) containing an NADPH-generating system consisting of 0.5 mM NADP+, 5 mM glucose 6ml of glucose 6-phosphate dephosphate, and 0.5 unit W hydrogenase and 100 mM substrates, except for ethoxyresoruˆn (10 mM) and aniline (1 mM) (Emoto et al., 2000a, b). Incubation was carried out for 10 min at 379 C, except for the case of debrisoquine hydroxylation

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P450 activity in juvenile visceral steatosis mouse Table 1.

Total P450 and NADPH-P450 reductase contents in mouse liver microsomes. Genotype

Enzyme

P450 NPR

Sex

Male Female Male Female

wt W wt

0.712±0.077 0.564±0.055 0.042±0.004 0.061±0.011

wt W jvs

(100) (100) (100) (100)

nmol W mg protein 0.736±0.063 (103) 0.503±0.023 (89) 0.043±0.006 (102) 0.065±0.009 (107)

jvs W jvs

0.762±0.091 0.466±0.099 0.042±0.007 0.063±0.008

(107) (83) (100) (103)

wt ) group. Data are means±SD from 5 mice. Numbers in parentheses indicate the percent of control (wt W

which was 30 min (Kronbach et al., 1987). The formation of the metabolites was determined by HPLC analysis as described previously (Yamazaki and Shimada, 1997; Emoto et al., 2000a, b). Ethoxyresoruˆn Odeethylation and aniline p-hydroxylation activities were determined by ‰uorometric (Pohl and Fouts, 1980) and colorimetric (Imai et al., 1966) assays, respectively. Genotoxic activation of 2-amino‰uorene by mouse kidney microsomes was determined as described previously (Imaoka et al., 1995).

Other Assays The concentrations of P450 and protein were estimated spectrally by the methods of Omura and Sato (1964) and Lowry et al., (1951), respectively. The NADPHP450 reductase (NPR) contents were determined by the speciˆc NADPH-cytochrome c reduction activities of 3.0 mmol cytochrome c reduced W min W nmol NPR (Yamazaki et al., 1999). Statistical analysis was performed by Student's t test using InStat computer programs (Graphpad Software, San Diego, CA, USA). Results

Genotyping of Octn2 in jvs and Hepatic Microsomal P450 Systems The genotype of the Octn2 gene in jvs mice used in the present study was conˆrmed by PCR-RFLP (Fig. 1). The contents of total P450 and NPR in liver microsomes from male and female mice are shown in Table 1. The P450 contents in liver microsomes from male and female wild-type mice were 0.71 and 0.56 nmol W mg protein, respectively. The NPR contents in liver microsomes from male and female mice were ¿0.04 and ¿0.06 nmol W mg protein, respectively. Both the P450 and NPR levels in liver microsomes from male and female mice genotyped as wt W jvs and jvs W jvs were not wt-type mice. signiˆcantly diŠerent from those in wt W Catalytic Activities of Endobiotics in Liver Microsomes from jvs Mice The hydroxylation activities of testosterone, progesterone, estradiol, and lauric acid in liver microsomes from jvs mice were determined (Fig. 2). In male

jvs W jvs-type mice, the activities of estradiol 16a-hydroxylation (Fig. 2(A)), testosterone 6b-hydroxylation (Fig. 2(B)), and progesterone 6b-hydroxylation (Fig. 2 (C)) (markers for Cyp3a) were signiˆcantly ( pº0.01) increased 1.5- to 2.1-fold compared with the wild-type or heterozygous mice. The activities of testosterone 16ahydroxylation and progesterone 21-hydroxylation (markers for Cyp2c) in the homozygotes were also signiˆcantly ( pº0.05) higher than those in the wild-type or heterozygous mice. In female jvs W jvs-type mice, the lauric acid 12-hydroxylation activities (marker for Cyp4a) in liver microsomes were signiˆcantly ( pº0.01) increased to 1.9-fold compared with those in wild-type or heterozygous mice (Fig. 2(H)). In addition, testosterone 6b- and 16a-hydroxylation (Fig. 2(F)) and progesterone 6b- and 21-hydroxylation (Fig. 2(G)) were also signiˆcantly ( pº0.05) higher, as in the males. In contrast, the activities of estradiol 2-hydroxylation (Figs. 2(A) and 2(E), marker for Cyp1a) and testosterone 16b-hydroxylation (Figs. 2(B) and 2(F), marker for Cyp2b) in liver microsomes from male and female mice were similar among the three genotyped groups. Catalytic Activities of Xenobiotics in Liver Microsomes from jvs Mice The catalytic activities of the P450 forms in mouse liver microsomes were investigated using seven xenobiotics as substrates (Fig. 3). In male jvs W jvs-type mice, coumarin 7-hydroxylation activities (marker for Cyp2a) were signiˆcantly lower ( pº0.05, 0.7-fold) (Fig. 3(B)) and chlorzoxazone 6-hydroxylation activities were signiˆcantly higher ( pº0.05, 1.5-fold) (Fig. 3(F)) than in wild-type mice. In female jvs W jvs-type mice, the activities of bufuralol 1?-hydroxylation (Fig. 3(K), marker for Cyp2d) and the activities of chlorzoxazone 6hydroxylation (Fig. 3(M)) and aniline p-hydroxylation (Fig. 3(N)) (markers for Cyp2e1) were signiˆcantly increased ( pº0.01 or pº0.05) 1.2 - to 1.4-fold compared with the wild-type or heterozygous mice. In contrast, the activities of O-deethylations of ethoxyresoruˆn (Figs. 3(A) and 3(H), markers for Cyp1a) and ethoxycoumarin (Figs. 3(C) and 3(J), marker for Cyp1a and Cyp2b) and of debrisoquine 4-hydroxylation and M-1

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Fig. 2. Endobiotic oxidation activities in liver microsomes from male (A-D) and female (E-H) mice genotyped as wild-type ( ), heterozygous wt) group ( ), and homozygous ( ) for the mutation. Data are means±SD from 5 mice. Numbers in parentheses indicate percent of control (wt W or 15b-hydroxytestosterone (because we have no 15awith signiˆcant diŠerence (*pº0.05, **pº0.01). AD, androstenedione; 15-OH, 15a- and W wt) group. hydroxytestosterone standard at our hand). Numbers in parentheses indicate the percent of control (wt W

and M-2 metabolites formation (markers for Cyp2d), reported previously by Kronbach et al. (1987), were similar among the three genotyped groups of male and female mice (Figs. 3(E) and 3(L)).

Catalytic Activities in Kidney Microsomes from jvs Mice Genotoxic activation of 2-amino‰uorene by pooled mouse kidney microsomes was determined using S. typhimurium NM2009 (Table 2). Kidney microsomes from male mice had higher activities (¿6000 umu units W min W mg protein) than those from females, but the jvs W jvs-type mutation showed no eŠects in Octn2 in 2amino‰uorene activation. Discussion A limited number of cases (¿30) of systemic carnitine deˆciency patients have been reported in the world since 1973 (Pons and De Vivo, 1995). However, a conservative estimate of the overall prevalence of heterozygotes in Japan was 1.01z, with an estimated incidence of primary systemic carnitine deˆciency of 1 in 40,000 births (Koizumi et al., 1999). It has been suggested that many

cases are not recognized as systemic carnitine deˆciency. Secondary carnitine deˆciency may develop in patients with end-stage renal disease undergoing long-term hemodialysis (Evans et al., 2000). In these patients carnitine can be administered to restore the plasma and tissue levels. Administration of the anticonvulsant valproic acid may also induce hypocarnitinemia (Kendler, 1986). However, there is no clear information, to our knowledge concerning drug-metabolizing enzymes or drug interactions in primary systemic carnitine deˆciency patients. The jvs mouse is a model for fatal systemic carnitine deˆciency with a missense mutation (Leu352Arg) of Octn2 (Slc22a5) (Nezu et al., 1999). The eŠects of this mutation (T1114G) in the Octn2 ( Slc22a5) gene on cytochrome P450-dependent activities have not been clariˆed. Although much more information is available concerning the roles of individual P450 enzymes in drug metabolism in rats than in mice, it is generally accepted that the substrate speciˆcities of rat and mice P450 enzymes are roughly similar. In the present study, we investigated the levels of drug metabolizing P450 enzymes by determining the catalytic activities of endobiotics and

P450 activity in juvenile visceral steatosis mouse

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Fig. 3. Xenobiotics oxidation activities in liver microsomes from male (A-G) and female (H-N) mice genotyped as wild-type ( ), heterozygous wt ) group ( ), and homozygous ( ) for the mutation. Data are means±SD from 5 mice. Numbers in parentheses indicate percent of control (wt W with signiˆcant diŠerence (*pº0.05, **pº0.01). M-1 and M-2, unknown metabolites of debrisoquine (Kronbach et al., 1987). Numbers in parenwt) group. theses indicate the percent of control (wt W

xenobiotics by liver and kidney microsomes from mice wt, wt W jvs, or jvs W jvs. The contents of genotyped as wt W total P450 and NPR were not changed in the liver microsomes among these groups (Table 1). The activities of testosterone 6b-hydroxylation, progesterone 6bhydroxylation, and W or estradiol 16a-hydroxylation (Fig. 2), which have been reported to be catalyzed by mouse, rat, and human CYP3A enzymes (Kitada et al., 1988; Swinney, 1990) were higher in homozygous jvs W jvs-type mice than in wild-type or heterozygous mice. Although, those were activities of 2b-hydroxylations of testosterone and progesterone, other markers of Cyp3a enzymes, these activities in jvs W jvs mice were not higher than those in wild-type or heterozygous mice. This was probably because these metabolites would be minor products. Because of the higher rates of testosterone 16a-hydroxylation and progesterone 21-hydroxylation (Swinney, 1990; Yamazaki and Shimada, 1997), it is suggested that the levels of Cyp2c in liver microsomes from male and female jvs mice are also high. Coumarin 7-hydroxylation activities (Funae and Imaoka, 1993) in

liver microsomes from male jvs mice were decreased, suggesting lower Cyp2a5 levels in the male liver. The similarly low testosterone 15(a)-hydroxylation activities (Lindberg et al., 1992) among the three genotyped groups suggested that there were no diŠerences in Cyp2a4 expression. In addition, Cyp1a enzyme in liver microsomes were also not signiˆcantly diŠerent among the three genotyped groups. The Cyp4b1 levels in kidney microsomes determined by 2-amino‰uorene activation (Imaoka et al., 1995) were not aŠected by the jvstype mutation (Table 2). The induction of Cyp4a and Cyp2e1 enzymes in liver microsomes from female jvs mice were suggested by the higher activities of lauric acid 12-hydroxylation (Hardwick et al., 1987) and the activities of chlorzoxazone 6-hydroxylation and aniline p-hydroxylation (Park et al., 1986). On the other hand, the aniline p-hydroxylation activities were not higher in male jvs mice. It was not clear whether Cyp2e1 enzymes were induced in liver microsomes from male jvs mice, because higher chlorzoxazone 6-hydroxylation activities may be catalyzed by

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Table 2. Activation of 2-amino‰uorene by pooled kidney microsomes in S. typhimurium NM2009

1)

Genotype Sex

Male Female

wt W wt

wt W jvs

References

jvs W jvs

umu unit W min W mg protein 6020±482 (95) 5980±478 (95) 6320±506 (100) 1110±68 (100) 1150±80 (104) 1230±86 (111)

ml) and 2-Amino‰uorene (10 mM) was incubated with male (1 mg W ml) mouse liver microsomes for 2 h in the presence of S. female (5 mg W typhimurium NM2009. Data are means±SD of triplicate determinawt group. tions. Numbers in parentheses indicate z of wt W

Cyp3a enzymes as well as Cyp2e1 in rodents and humans (Jayyosi et al., 1995). The Cyp2d levels in female jvs liver may be slightly higher judging from the higher rates of bufuralol 1?-hydroxylation. Debrisoquine 4hydroxylation activities, a prototype marker for Cyp2d (Yamamoto et al., 1998, Masubuchi et al., 1997), appeared to be too low to detect the increased Cyp2d levels in liver microsomes from female jvs mice. Carnitine is required for the transport of long-chain fatty acids into the inner mitochondrial compartment for b-oxidation (Kendler, 1986). Jvs mice are known to show hepatic steatosis by low fatty acid metabolism in mitochondria that results from the decline in renal carnitine reabsorption. The hepatic steatosis was apparently more severe in female mice than in males in this study. This hepatic steatosis in female jvs mice may possibly lead to the induction of Cyp4a or Cyp2e1 enzymes in liver microsomes to eliminate fatty acids or triglycerides by oxidation. Therefore, physiological conditions of jvs mouse possibly aŠect the expression level or induction level of Cyp enzymes in jvs mice, which is an issue that should be clariˆed in the future. In conclusion, in the present study, the jvs mouse was shown to have signiˆcantly higher or lower levels of the marker activities for Cyp4a, Cyp3a, Cyp2e1, Cyp2c, Cyp2d or Cyp2a in liver microsomes compared to wildtype mice, in spite of the similarity in total P450 and NPR contents. The jvs mouse could be a useful for drug metabolism studies of OCTN2 deˆciency patients. In addition, the jvs mouse would give valuable information in drug interaction and drug metabolism studies of OCTN2 substrate drugs and new compounds in development. Acknowledgments: This work was supported in part by grant from the Ministry of Education, Science, Sports, and Culture of Japan. We thank Drs. Hiroko Nikaido and Jun-Ichiro Hayakawa for providing jvs mice and Mr. Brent Bell for critical reading of the manuscript.

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