Mutagenesis, Vol. 16, No. 5, 377-383,
September 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Lack of change in the levels of liver and kidney cytochrome P-450 isozymes in p53(+/–) knockout mice treated with N-butyl-N-(4-hydroxybutyl)nitrosamine
Laboratory of Radiochemistry, Gifu Pharmaceutical University, 6-1 Mitahora-higashi 5-chome, Gifu 502-8585, Japan and 1 First Department of Pathology, Osaka City University Medical School, 4-54 Asahi-machi 1-chome, Abeno-ku, Osaka 545-8585, Japan
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We have previously shown that p53(+/–) knockout mice are highly sensitive to urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in spite of a lack of effects of p53 heterozygosity on N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN) excretion in urine. To determine the influence of p53 deficiency on in vitro formation of BCPN, mutagenicity of BBN and BCPN and levels of several cytochrome P450 (CYP) isozymes, groups of five p53(+/–) knockout and wild-type mice (littermates), as well as animals of the C57BL/6 parental strain, were administered 0.025% BBN in their drinking water for 4 weeks. The livers and kidneys were then used for analyses of BBN metabolism, western immunoblotting and Ames liquid incubation. BBN treatment caused a slight decrease in BCPN formation in the livers of C57BL/6 mice, but there was no significant difference between p53 knockout, wild-type and C57BL/6 mice. In kidney BCPN formation in p53 knockout mice was 33–46% less than that in their wild-type counterparts. Using anti-rat CYP antibodies, CYP1A2, 2B9/10, 2E1 and 3A11/13 were constitutively detected in liver microsomes and CYP2E1 and 3A11/13 in the kidney. Densitometric determination of these CYP proteins revealed no significant variation in levels detected in both tissues among the four groups of mice. BBN and BCPN were not mutagenic for Salmonella typhimurium TA100 in either the absence or presence of liver S9 from untreated mice and rats and from p53 knockout mice treated with BBN. In conclusion, p53 deficiency and BBN had no enhancing effects on metabolism of BBN to BCPN and expression of the CYP isozymes typically responsible for activation of environmental carcinogens, including both of the N-nitrosamines tested, and their mutagenicity, indicating that the high susceptibility of p53(+/–) knockout mice is not attributable to metabolic activation in liver and kidney by CYP isozymes or urinary excretion of BCPN.
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p53 gene mutations have been observed in 37–62% of human bladder cancers (Sidransky et al., 1991
; Fujimoto et al., 1992; Sarkis et al., 1993; Kusser et al., 1994; Uchida et al., 1995; Koga et al., 2000). N-Butyl-N-(4-hydroxybutyl)nitrosamine (BBN) is a potent urinary bladder carcinogen in mice (Bertram et al., 1972; Hirose et al., 1976), causing lesions that are histologically similar to neoplasms in man, such as non-papillary, invasive transitional cell carcinomas (TCC) and squamous cell carcinomas (Hirose et al., 1976; Tamano et al., 1991). In contrast, most urinary bladder tumors in rats are papillary and superficial TCCs (Ito et al., 1969; Fukushima et al., 1976). Furthermore, frequent p53 mutations in invasive urinary bladder carcinomas in NON/Shi mice are detected at similar levels to those observed for human high-grade invasive carcinomas (Yamamoto et al., 1995).
The oxidative metabolite of BBN, N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), is known to play a decisive role in tumor induction in the urinary bladder by BBN (Hashimoto et al., 1972,1974; Okada et al., 1975). BBN and BCPN are metabolized through the 
-hydroxylation pathway, which is thought to lead to formation of reactive species that bind to DNA within the target organ (Airoldi et al., 1990,1992a); O6-(4-hydroxybutyl)guanine [O6-(4-OH-Bu)G] and O6-butylguanine (O6-BuG) have been detected in urotherial DNA of rats given BBN (Airoldi et al., 1994). In the rat BBN is efficiently metabolized by phenobarbital (PB)-inducible cytochrome P-450 (CYP) in liver and acetone-inducible CYP in bladder to active species (Airoldi et al., 1992b). It has been reported that rat liver and urinary bladder metabolize BBN (Irving and Daniel, 1987; Airoldi et al., 1987) and N-nitrosodibutylamine (DBN) (Suzuki et al., 1983) to BCPN and BBN, respectively. However, to date no studies have compared the in vitro metabolic potencies of any organs, including liver in mice and kidney in any animal species.
The -oxidation of carcinogenic N-nitrosamines is also reported to be catalyzed by CYP isozymes, such as CYP2B1/2 and rat CYP2E1 and CYP2A6. CYP2B1 and 2B2 activate N-nitrosodialkylamines with alkyl chains longer than propyl groups (Mori et al., 1986; Kawanishi et al., 1992; Shu and Hollenberg, 1996) and also N-nitrosodimethylamine (DMN) at high substrate concentration, whereas rat CYP2E1 activates DMN and N-nitrosodiethylamine (DEN) at lower substrate concentrations (Yang et al., 1987). In addition, hepatic CYP2A6 in humans is involved in the metabolic activation of DEN and the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (Yamazaki et al., 1992). Carcinogens such as heterocyclic amines (HCA), aminoazo compounds (Degawa et al., 1987,1990,1992; Kleman et al., 1990) and N-nitrosobis(2-oxopropyl)amine (BOP) (Nishikawa et al., 1997) are known to induce hepatic CYP isozymes responsible for their activation. Masuko et al. (1987) reported that BBN induces rat CYP1A2 in the bladder, but not in the liver, kidney, esophagus or intestine, whereas BBN has no effect on CYP2B1/2 in any organs.
Previously we reported that p53(+/–) knockout mice are very much more sensitive to urinary bladder carcinogenesis by BBN than the parent strain, C57BL/6 mice (Ozaki et al., 1998). Although considerable attention has been focused on the susceptibility to chemical carcinogenesis in p53 knockout mice (Kemp et al., 1993; Tennant et al., 1995; Finch et al., 1998), little is known about the effect of p53 deficiency on metabolic activation of carcinogens and CYP levels. With regard to associations of metabolic enzymes with tumor suppressor gene mutations, Romkes et al. (1996) reported that low CYP3A4 and high CYP2D6 activity in vivo are selectively observed with p53 mutations and Rb mutations, respectively, in aggressive bladder cancers. CYP3A activity is also reported to be ~33% lower in primary hepatocytes from p53(–/–) knockout mice than in those from wild-type mice (Shimoji et al., 1996). In the present study we report results for in vitro transformation of BBN to BCPN and mutagenic activation by liver and kidney S9 from p53(+/–) knockout, wild-type and C57BL/6 mice treated with BBN. Western blot analyses were also performed for several CYP isozymes known to activate typical environmental carcinogens in order to clarify the effects of p53 deficiency on metabolic activation of BBN.
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Chemicals
BBN (CAS no. 3817-11-6) was obtained from Tokyo Kasei Kogyo Co. (Tokyo, Japan) and BCPN (CAS no. 38252-74-3) was synthesized in our laboratory according to the method of Okada et al. (1978). Prior to assay for mutagenicity, its purity was confirmed to be >99% by high performance liquid chromatography (HPLC). 2-Amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1)·HCl (CAS no. 67730-11-4), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) (CAS no. 76180-96-6), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) acetate (CAS no. 62450-07-1) and DEN (CAS no. 55-18-5) were purchased from Wako Pure Chemicals (Osaka, Japan). N-Benzylimidazole (BI) (CAS no. 4238-71-5) was obtained from Aldrich Chemical Co. (Milwaukee, WI) and glucose 6-phosphate dehydrogenase (G6PDH), glucose 6-phosphate (G6P), NADP+, NADPH, NADH and ATP were from Oriental Yeast Co. (Tokyo, Japan). All other commercial products were of the purest grade available.
Animal treatment and tissue preparation
Heterozygous male p53 knockout [p53(+/–)] and wild-type [p53(+/+)] mice were purchased from GenPharm International (Palo Alto, CA) and mice of the C57BL/6 parent strain were from Charles River Japan (Atsugi, Japan). The animals were housed in plastic cages (five mice per cage) with woodchip bedding in a room maintained at 24 ± 2°C and 40–70% humidity with a 12 h light/dark cycle and received Oriental MF diet (Oriental Yeast Co.) and tap water ad libitum. At 7–8 weeks of age groups of five C57BL/6 mice, knockout mice and their wild-type littermates were administered 0.025% BBN in the drinking water. After 4 weeks treatment livers and kidneys were perfused in situ with ice-cold sterile 1.15% KCl and 25% homogenates in 1.15% KCl were prepared. Male 6-week-old Wistar rats (Japan SLC, Hamamatsu, Japan) were orally administered, via a stomach tube, 75 mg/kg BI suspended in 500 µl of 0.5% Tween 20 once daily for 3 days and then livers were similarly perfused and homogenized. Tissue S9 and microsomal fractions from the 25% homogenates were prepared using established procedures (Mori et al., 1985) and total CYP content was spectroscopically determined by the method of Omura and Sato (1964).
Metabolism of BBN by S9 mix
The incubation mixture was prepared according to the method of Suzuki et al. (1983) except for a final volume of 250 µl and a substrate concentration of 0.4 mM instead of 5 ml and 8 mM, respectively. The S9 mix consisted of 75 µl of liver or kidney S9, 0.1 M sodium phosphate buffer, pH 7.4, 8 mM MgCl2, 33 mM KCl, 4 mM NADP+ and 5 mM G6P. BBN in dimethyl sulfoxide (50 µl) and the S9 mix were incubated for 1–5 h with shaking (100 strokes/min) at 37°C. Then the incubation mixture was added to an equal volume of acetonitrile and centrifuged for 10 min at 8000 g. After clarification of the supernatant by passage through a Sartorius Minisart RC4 filter (0.2 µm pore size; Sartorius Co., Göttingen, Germany), a 10 µl aliquot of the acetonitrile solution was directly injected into the HPLC instrument, a Shimazu LC9A (Shimazu Co., Kyoto, Japan). BBN and BCPN were separated using a Jasco Finepak SIL C18 column (4.6 mm i.d.x25 cm) with acetonitrile/20 mM sodium acetate buffer, pH 4.5 (3:7 v/v) as the solvent (flow rate 1.0 ml/min) and measured at 239 nm (0.16 absorbance units full scale). Under the chromatographic conditions described above BBN and BCPN had retention times of 10.9 and 7.8 min, respectively.
Mutation assay
DEN, Glu-P-1·HCl and Trp-P-2 acetate (all 100 µl) were dissolved in water and IQ (100 µl), BBN and BCPN (50 µl) in dimethyl sulfoxide. The mutagenicity of these carcinogens was checked in the presence of liver or kidney S9, according to the methods described by Nagao et al. (1977) and Koide et al. (1999). The amount of tissue S9 was 150 µl/plate, except for liver S9 for HCAs (10 µl/plate). The S9 mix contained the cofactors 4 mM NADPH, 4 mM NADH, 0.5 U G6PDH, 5 mM G6P and 5 mM ATP for HCAs, 4 mM NADP+ and 5 mM G6P for DEN (Koide et al., 1999) and 8 mM NADH instead of 4 mM NADP+ for BBN and BCPN (Nagao et al., 1977). Salmonella typhimurium TA100 and TA98 tester strains were employed for the N-nitrosamines and HCAs, respectively.
Western blotting
Goat anti-rat polyclonal antibodies for CYP1A1/2, CYP2B1/2, CYP2E1, CYP3A2 and NADPH-CYP reductase (Daiichi Pure Chemicals, Tokyo, Japan) were used as primary antibodies. Gel electrophoresis and blot analysis were performed as described in detail previously (Koide et al., 1999), according to the established methods of Laemmli (1970) and Towbin et al. (1979), respectively.
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Total BBN intake in p53 knockout mice was essentially the same as for wild-type and C57BL/6 mice. There were no statistically significant differences in the mean body and tissue weights among p53 knockout, wild-type and C57BL/6 mice treated with BBN and untreated C57BL/6 mice. Total CYP contents in their liver microsomes were 0.78 ± 0.08 (mean ± SD), 0.85 ± 0.10, 0.83 ± 0.10 and 0.80 ± 0.11 nmol/mg, respectively.
Figure 1 shows data for the abilities of mouse liver and kidney to metabolize BBN to BCPN. BCPN formation increased linearly with incubation time in both liver and kidney; after 5 h 101.0 (84% of the substrate) and 48.6 nmol (40%) BCPN were produced by liver and kidney, respectively. Treatment of C57BL/6 mice with BBN caused an 18–34% decrease (P < 0.05) in hepatic BCPN formation at 1–4 h incubation times, but there was no significant difference between p53 knockout, wild-type and C57BL/6 mice. In contrast, BCPN formation by kidney S9 was 33–46% less (P < 0.05) in p53 knockout than in wild-type mice and the renal formation was also lower (P < 0.01) compared with C57BL/6 controls, with or without BBN treatment.
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) mice and from five C57BL/6 (
), wild-type (
) and p53 knockout (•) mice given BBN. Results are means of three to four determinations. *P < 0.05, compared with the untreated group (A) and with wild-type mice (B); Student's t-test.Western blots for CYP isozymes and NADPH-CYP reductase in liver microsomes from the four groups of mice are shown in Figure 2
. Proteins were positively detected with antibodies against rat CYP1A1/2, 2B1/2, 2E1 and 3A2 and NADPH-CYP reductase. Mouse CYP1A2 was constitutively expressed (Figure 2A, lane 1) and essentially co-migrated with rat CYP1A2 (56 kDa, Figure 2A, lane 5). With the anti-CYP2B1/2 antibody one faint band and two distinct bands were found in liver microsomes from C57BL/6 mice (Figure 2B, lane 1), with molecular weights of 55.5, 53 and 51 kDa. The 55.5 kDa band completely co-migrated with CYP2B2 and the levels of the 53 and 51 kDa proteins were almost equivalent. With the anti-CYP3A2 antibody a major band (54.5 kDa) and three minor bands (53.5, 52 and 51.5 kDa) were found (Figure 2C, lane 1), but the molecular weight of major band was slightly different from standard CYP3A2 (54 kDa). The anti-rat CYP2E1 antibody recognized a major band (54 kDa) and faint bands, including a 52 kDa protein (Figure 2D, lane 1). The 52 kDa proteins detected with antibodies for CYP3A2 and rat CYP2E1 can be assigned to the CYP2C isozyme, since these antibodies are known to cross-react with CYP2C11, as shown in Figure 2C and D, lanes 5. Mouse NADPH-CYP reductase was detected as a single band co-migrating with the rat standard (Figure 2E, lane 1). Densitometric analyses of tissues in three to six experiments for lanes 1–4 showed no significant differences in the levels (pmol/mg protein) of CYP isozymes and NADPH-CYP reductase among the four groups.
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In kidney microsomes two proteins (54 and 52 kDa) were detected with the anti-rat CYP2E1 antibody (Figure 3A
, lane 1). The level of kidney CYP2E1 (8 pmol/mg protein) was only ~10% of that in liver. With the anti-CYP3A2 antibody (Figure 3B, lane 1) two distinct bands were detected and the levels of both were almost the same (0.79 and 0.73 pmol/mg protein), but were about 150 times less than that of the 54.5 kDa protein in liver. In contrast, no immunoreactive bands were observed with anti-CYP2B1/2 and CYP1A1/2 antibodies in kidney, as shown in Figure 3C and D. Anti-rat liver NADPH-CYP reductase recognized one band (Figure 3E, lane 1) and the level in kidney (18.5 pmol/mg protein) was ~50% of that in liver. Comparison of lanes 2–4 with lane 1 (three to four experiments) indicated that neither p53 deficiency nor BBN treatment affected the levels of these CYP proteins and NADPH-CYP reductase.
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To confirm the potential of liver and kidney S9 from the four groups of mice to mediate mutagenic activation of carcinogens, the mutagenicities of Glu-P-1, IQ, Trp-P-2 and DEN were tested in S.typhimurium strain TA98 or TA100 (Table I
). Glu-P-1, IQ and Trp-P-2 showed clear mutagenicity in the TA98 strain in the presence of liver S9 from untreated mice and BBN treatment slightly decreased the mutagenic activity. In the presence of kidney S9 the numbers of revertant TA100 colonies with DEN and HCAs were less than twice and over two or three times the spontaneous rate, respectively, indicating slight activation, but there was no decrease with BBN treatment. These findings with liver and kidney are in accordance with their metabolic potential for BCPN formation from BBN. On the other hand, 5–50 µmol BBN and BCPN showed negative mutagenicity in the presence of liver S9 from all four groups of mice and 50 µmol BCPN was slightly toxic to Salmonella, as shown in Figure 4. In order to confirm the mutagenicity of BBN and BCPN, this was also checked in the presence of liver S9 from rats treated with BI. As shown in Figure 5, BBN showed dose-dependent mutagenicity under these conditions, but not with S9 from untreated rats, whereas 5–30 µmol BCPN lacked mutagenicity, independent of rat liver S9.
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) or presence of liver S9 pooled from each three untreated ( |
Discussion |
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The mobilities of the mouse liver or kidney CYP1A2 and 2E1 proteins detected in the present experiment were the same as standards from rats, in agreement with earlier findings of Raunio et al. (1990) and Nakajima et al. (1993). However, the molecular weights of the mouse CYP2B and CYP3A proteins were slightly different from those of CYP2B1 and CYP3A2, except for that of CYP2B2 (55.5 kDa). It is reported that the molecular weights of CYP2B10 and 2B9 are 56 and 53 kDa (Honkakoski et al., 1992
; Nemoto et al., 1995), respectively, and that three bands can be detected in mouse liver using rabbit polyclonal antibodies for CYP2B1, of which the highest and middle bands are assigned to CYP2B10 and 2B9, respectively (Sakuma et al., 1999). Therefore, the 55.5 and 53 kDa proteins constitutively detected in mouse liver (Figure 2
) can be identified as CYP2B10 and 2B9, respectively. For further confirmation, the induction effect of PB must be examined. The mouse CYP3A subfamily is comprised of CYP3A11, CYP3A13 and CYP3A16 (Itoh et al., 1994). It has been reported that two CYP3A isozymes are detected in livers from male BALB/c (Riley et al., 1993) and NMRI (Villard et al., 1994) mice and that the level of the upper band is much higher than that of the lower band. The molecular weights of CYP3A11 and CYP3A13, calculated from their amino acid sequences, are reported to be 57.853 and 57.491 kDa, respectively (Yanagimoto et al., 1992,1994). In this study of hepatic CYP3A isozymes the level of the 54.5 kDa protein was ~10 times higher than that of the 53.5 kDa protein. Together with the earlier findings that CYP3A11 mRNA is predominantly expressed in mouse liver (Yanagimoto et al., 1992) and that CYP3A16 mRNA is specifically present in the liver of fetal and neonatal mice (Itoh et al., 1994), it is reasonable that the 54.5 and 53.5 kDa proteins can be assigned to CYP3A11 and CYP3A13, respectively. Also, the 52 kDa protein detected together with the CYP3A and 2E isozymes may be CYP2C29, since this isozyme is known to be present at the highest level of the five CYP2C isozymes in mouse liver (Luo et al., 1998).
Expression of mouse CYP2E1 in the kidney was higher than that of CYP3A11 (Figure 3), and vice versa in the liver. This is in agreement with previous observations for the kidney of male C57BL/6 mice (Henderson et al., 1990). Accordingly, it is clear that expression of CYP2E1, CYP3A11 and CYP3A13 differ greatly between the liver and kidney in this strain of mouse. The fact that CYP1A1/2 and CYP2B9/10 were not detected in kidney microsomes at all is in line with previous data indicating that CYP1A isoforms are not expressed in the kidneys of either NIH/Swiss mice or Wistar and F344/NCr rats (Sesardic et al., 1990; Nerurkar et al., 1993). In contrast, there are conflicting reports on expression of kidney CYP2B isozymes in mice and rats; kidney CYP2B9 and CYP2B10 are present at higher levels than kidney CYP2E1 in male C57BL/6 mice but not in females (Henderson et al., 1990), while CYP2B proteins are not found in the kidney from either male NMRI mice (Seree et al., 1996) or male F344/Crl rats (Wardlaw et al., 1998). For these detections, however, different assay kits were employed; Henderson et al. and Seree et al. or Wardlaw et al. using 125I-labeled protein A and an enhanced chemiluminescence system (ECL Western Blotting Detection Reagents; Amersham Pharmacia Biotech, Little Chalfont, UK), respectively, while we applied a streptavidin/biotin/peroxidase complex approach (ABC; Wako Pure Chemicals, Osaka, Japan). It is known that the detection limit with ECL kits (1 pg protein) is lower than with 125I-labeled protein A (300 pg protein) (Gillespie and Hudspeth, 1991) and that in the case of ABC it is 30 pg (Koide et al., 1999). Accordingly, the situation regarding CYP2B isozymes in kidney remains to be clarified.
Although mice were exposed to ~50 mg BBN/kg/day for 4 weeks, treatment exerted no obvious effects on the levels of any CYP isozymes determined in liver or kidney. This is in accordance with the findings of Masuko et al. (1987) for rat CYP1A1/2 and 2B1/2 in these organs. However, since slight induction of hamster CYP2B isozymes by 50 mg BOP/kg was observed in the liver 6 h after treatment (Nishikawa et al., 1997), it may be necessary to determine the effect of BBN on expression of CYP2B9/10 in mouse liver immediately after a single injection. On the other hand, enzymatic -oxidation is known to contribute to metabolic activation of several N-nitrosodialkylamines (Druckrey et al., 1967; Magee and Barnes, 1967) and the hepatic CYP isozymes involved, such as CYP2B1/2, rat CYP2E1 and CYP2A6, differ in their actions, depending on the length of the alkyl chain and the substrate concentration (Yang et al., 1987; Yamazaki et al., 1992; Shu and Hollenberg, 1996). Moreover, rabbit CYP4B1 in the bladder activates DBN (Schulze et al., 1990) and mouse CYP4B1 is known to be a major form in kidney (Imaoka et al., 1997). BBN induces CYP1A2 in rat bladder epithelium (Masuko et al., 1987) and O6-BuG and O6-(4-OH-Bu)G can be detected in urothelial cells of rats given BBN at levels ~10-fold higher than those in the liver (Airoldi et al., 1994). Consequently, for urinary bladder carcinogenesis by BBN in mice induction of CYP1A2 and 4B1, as well as the CYP2E and 2B isozymes (Airoldi et al., 1992b), in the bladder might be more critical than that in the liver and kidney. Bladder CYP4B1 has been detected using ECL kits (Imaoka et al., 1997), and by gas chromatography/thermal energy analysis with very high sensitivity for various N-nitrosamines, it has been demonstrated that BBN is transformed to BCPN by rat urinary bladder (Airoldi et al., 1987). Accordingly, it is necessary to determine the metabolic capacity and levels of CYP isozymes in mouse bladder by these highly sensitive methods. Nevertheless, from the present result that four carcinogens were not or only slightly mutagenic in the presence of kidney S9 (Table I) and the previous findings that BP hydroxylation and its DNA binding in rat and human bladder epithelial cells (Autrup et al., 1981) and mutagenic activation of 3,3'-dichlorobenzidine in the umu test by mouse bladder microsomes is much less than with either kidney or liver (Imaoka et al., 1997), it is reasonable to assume that in the Ames test various procarcinogens may show no clear mutagenicity in the presence of bladder S9 with CYP expression similar or less pronounced than in the kidney (Vanderslice et al., 1985).
It has been reported that levels of CYP2E1 and CYP3A11/13 in the liver and kidney from CYP1A2(–/–) knockout mice (Genter et al., 1998) and hepatic activities of CYP1A2, CYP2B9/10 and CYP2E1 in metallothionein-I transgenic mice (Iszard et al., 1995) are not different from those in the respective wild-type mice. On the other hand, CYP3A activities are low in bladder cancers of patients with p53 mutations (Romkes et al., 1996) and in hepatocytes from p53(–/–) knockout mice (Shimoji et al., 1996). In the present study expression of CYP3A11 and 3A13, as well as other CYP isozymes detected in the liver and kidney, were similar in p53(+/–) knockout mice and their wild-type littermates, as well as the original parent strain (Figures 2 and 3). One reason for the apparent discrepancy might be that actual CYP3A activity is lower in p53(+/–) knockout mice than wild-type mice, in spite of the same level of expression. Another is that there may be differences in nullizygous and heterozygous p53 knockout mice. It has been verified by Southern and northern hybridization, RNA PCR, protein immunoprecipitation and immunoblot experiments that intact p53 is synthesized in p53(+/–) knockout mice, but expression from the wild-type p53 allele and p53 protein levels in heterozygous mice are clearly less than in wild-type mice (Donehower et al., 1992). Heterozygous p53-deficient mice are highly susceptible to development of spontaneous embryonal carcinomas and malignant lymphomas (Donehower et al., 1992) and also to experimental induction of skin or urinary bladder carcinomas (Kemp et al., 1993; Tennant et al., 1995), in addition to those induced by BBN (Ozaki et al., 1998).
Nagao et al. (1977) reported the mutagenic activity of BBN to be ~2.6-fold higher in the presence of rat liver S9 with NADH than with NADPH. This is the reason why NADH was used as the cofactor in the present study. BBN requires liver S9, for example from Aroclor-treated rats, to exhibit mutagenicity (Nagao et al., 1977; Rumruen and Pool, 1984), in agreement with our results obtained with liver S9 from BI-treated rats, with which potent induction of enzymes responsible for activation of various carcinogens, including N-nitrosamines, is observed (Mori et al., 1993). BCPN is known to be mutagenic without metabolic activation but not in the presence of liver S9 from polychlorinated biphenyl-treated rats in strain TA1535 (Nagao et al., 1977). Furthermore, Pool et al. (1988) described BCPN as not being mutagenic in either strain TA1535 or TA100, with or without liver S9 from Aroclor-treated rats. This is supported by the present finding that BCPN did not show mutagenicity, independent of the presence of liver S9 from control or BBN-treated mice and also BI-treated rats. Moreover, the negative mutagenicity of BCPN in the presence of mouse and rat liver S9 is in line with that of N-nitrosomethyl-3-carboxypropylamine with rat or hamster liver activation (Lijinsky and Andrews, 1983), indicating that in bladder carcinogenesis by BBN, water-soluble BCPN might be excreted into the urine, incorporated into the bladder cells and further metabolized to 4-(N-butylnitrosamino)-4-hydroxybutyric acid lactone, which shows potent effects in mutation and repair tests in four strains tested (Mochizuki et al., 1980). Whatever, neither p53 deficiency nor BBN treatment resulted in elevated mutagenicity of BBN and BCPN, despite the potential of mouse liver S9 to activate HCAs and DEN, reflecting no changes in levels of microsomal CYP isozymes in either liver or kidney.
Irving and Daniel (1987) reported that 0.5 mM BBN is rapidly metabolized by rat hepatocytes, ~90% being transformed into BCPN within 4 h. Pastorelli et al. (1988) also described 
-oxidation to BCPN as being predominant in rat hepatocytes. The present findings with liver and kidney similarly suggest that -oxidation of BBN is the major pathway in the mouse. BBN and p53 deficiency caused a slight decrease in BCPN formation by liver and kidney, respectively. However, it is reported that the oxidation of BBN to BCPN can be attributed to alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) (Irving 1988; Irving and Daniel, 1988). Whether hepatic or renal ADH and ALDH might be inhibited by BBN or p53 deficiency now requires investigation, given the fact that levels of CYP isozymes did not change.
In conclusion, the present study has demonstrated that p53 deficiency and BBN treatment exert no enhancing effects on metabolism of BBN to BCPN, mutagenic activation or levels of CYP isozymes and NADPH-CYP reductase in the mouse. Consequently, the data indicate that high susceptibility to BBN carcinogenesis in p53(+/–) knockout mice is not attributable to alterations in the levels of CYP isozymes and metabolic activation in liver or kidney, in support of a previous finding that a high level of cell proliferation is important, rather than mutations of the p53 gene (Ozaki et al., 1998).
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2 To whom correspondence should be addressed. Tel: +81 58 237 3931; Fax: +81 58 237 5979; Email: ymori{at}gifu-pu.ac.jp
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References |
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Received on July 6, 2000; accepted on April 11, 2001.
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