Mutagenesis, Vol. 16, No. 6, 523-528,
November 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
S9 induction by the combined treatment with cyclohexanol and albendazole
1 Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Apartado Postal 70228, 04510 México, D.F., 2 Torre de Investigación `Joaquín Cravioto', Instituto Nacional de Pediatría, Insurgentes Sur, 3700-C, 04530 México, D.F. and 3 Hospital Juárez de México, Av. Instituto Politécnico Nacional 5160, 07760 México D.F., Mexico
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Abstract |
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Cyclohexanol (CH) is an industrial solvent capable of inducing cytochrome P450 (CYP) enzymes including the CYP2E and CYP2B subfamilies. S9 from CH treated rats is able to activate several N-nitrosamines that are poorly activated by Aroclor 1254, phenobarbital/ß-naphthoflavone (PB/NF) or 3-methylcholanthrene S9 fractions into mutagens detected by the Salmonella typhimurium Ames test. Additionally, albendazole (ABZ) is a widely used anthelmintic drug and a potent inducer of the CYP1A subfamily. Since CYP1A, -2B and -2E subfamilies are implicated in the activation of several environmental mutagens/carcinogens, we studied CYP induction in the rat liver by the combined effect of these two compounds, and used S9 derived from it in the Salmonella/microsome assay to compare with S9 obtained from Aroclor or PB/NF treated rats. Total CYP content in hepatic microsomes was induced by Aroclor, but not by any of the other chemical combinations. Western blot and enzymatic activity analysis revealed quantitative but not qualitative differences in the CYP subfamilies present in the different microsomal fractions; all of the chemicals used increased the levels of CYP1A1/2, CYP2B1/2 and CYP2E1 with respect to control microsomes. CYP3A was not modified by the different treatments. When tested in the Ames test, Aroclor S9 and PB/NF S9 were the most effective in the activation of benzo[a]pyrene and 3-methylcholanthrene which are metabolized mainly by CYP1A1; additionally, the highest mutagenic potency of 2-aminofluorene and N-nitrosodipropylamine, which are activated by CYP1A2 and CYP2B, respectively, were obtained with PB/NF S9. All these compounds were also activated when CH/ABZ S9 was used as the exogenous source of metabolism. Mutagens like N-nitrosopyrrolidine and N-nitrosodimethylamine, activated by CYP2E1, were detected only when CH/ABZ S9 was used, and the effectiveness of the different S9 fractions in activating cyclophosphamide decreased in the following order: Aroclor = PB/NF > CH/ABZ > control. From these experiments we can conclude that the individual CYP- inducing properties of ABZ and CH complement each other when the two compounds are administered in conjunction and that the resulting S9 fraction is able to activate several known mutagens in the Ames test.
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Introduction |
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Preparation of hepatic S9 fraction, required as an integral part of standard in vitro genotoxicity assays, involves pretreatment of the animals with an inducing agent. A mixture of polychlorinated biphenyls, designated Aroclor 1254, has been used for a long time for this purpose, but its detrimental environmental impact (Clare, 1989
) has prompted a re-evaluation of the continuing use of Aroclor 1254, and alternatives have been proposed. Matsushima et al. (1976) showed that a combination of phenobarbital/ß-naphthoflavone (PB/NF) administered intraperitoneally (i.p.) to rats is a good inducer of hepatic microsomal enzymes; different routes of administration have also been successfully used (Elliot et al., 1992; García Franco et al., 1999). Since the original proposal by Matsushima to replace Aroclor 1254 with the combination of PB/NF, mutagens activated by CYP1A, and to a minor extent CYP2B, as well as enzymatic activities for these two subfamilies, have been used in order to validate the use of S9 induced by different chemicals in the in vitro metabolic systems for genotoxicity assays (Guengerich et al., 1982; Callander et al., 1995; García Franco et al., 1999). Nevertheless, the CYP2E subfamily participates in the activation of environmental nitrosamines (Yang et al., 1991) and is poorly induced, or even suppressed, by Aroclor 1254 or PB/NF (Guengerich et al., 1982; Thomas et al., 1987). Cyclohexanol (CH) is an industrial solvent that induces the CYP2E1 and CYP2B1/2 isozymes (Espinosa-Aguirre et al., 1996, 1997) and albendazole (ABZ) is an anthelmintic drug with a high potential to induce CYP1A1/2 (Souhaili-El Amri et al., 1988; Asteinza et al., 2000); both compounds display lower toxicity than other P450 inducers and they are not under regulatory control. Based on the latter advantages, along with the CYP induction properties of these two substances, we compared the mutagenicity induced in the Ames test by reference chemicals activated either by S9 induced with the ABZ/CH combination, or by S9 induced with Aroclor or with PB/NF. Biochemical characterization of the three different S9 fractions, including enzymatic activities and western blot analysis, were included. |
Materials and methods |
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Chemicals
Nicotinamide adenine dinucleotide phosphate (ß-NADP), glucose-6- phophate (G6P), N-nitrosodipropylamine (NDPA), N-nitrosopyrrolidine (NPYR), N-nitrosodimethylamine (NDMA), benzo[a]pyrene (B[a]P), 3-methylcholanthrene (3-MC), 2-aminofluorene (2-AF), cyclophosphamide (CP), anti-mouse immunoglobulin IgG peroxidase conjugate, methoxyresorufin, 7-pentoxyresorufin, 7-benzyloxyresorufin, resorufin, ethoxyresorufin, albendazole, ß-naphthoflavone (NF), 4-nitrophenol and 4-nitrocatechol were purchased from Sigma (St Louis, MO). Cyclohexanol was obtained from J.T.Baker (Phillipsburg, NJ). Goat polyclonal anti-rat CYP1A1/2, CYP2B1/2 and CYP2E1, along with microsome standards for each CYP, were manufactured by Daiichi Pure Chemicals (Tokyo, Japan) and purchased from Gentest (Woburn, MA). Chemicals for electrophoresis and nitrocellulose membranes were purchased from Bio-Rad (Richmond, CA).
Animal treatment
Male Wistar rats (weighing 200–250 g) were housed in polypropylene cages and kept in a 12 h light/dark cycle in an animal care facility. Animals were allowed unrestricted access to laboratory rodent chow and distilled water.
Four groups of animals were used (four animals/group). One group was treated with 2.5% (v/v) CH in drinking water for 4 days (Espinosa Aguirre et al., 1997). On the third day of CH treatment, animals were administered a single i.p. dose of ABZ (50 mg/kg) in corn oil and were killed 24 h later (Asteinza et al., 2000). In a second group, rats were injected intraperitoneally with a single dose of Aroclor 1254 (500 mg/kg) and killed on day 5 of induction (Ames et al., 1975). PB/NF induction was done following the scheme proposed by Matsushima et al. (1979) with some modifications: animals received three daily i.p. doses of PB (60 mg/kg) and an additional i.p. injection of 30 mg/kg on day 4; NF was injected i.p. to the same rats at a dose of 80 mg/kg on day 3 and the animals were killed on day 5. The last group, used as a control, received four daily doses of corn oil and were killed on day 5.
Preparation of S9 and microsomal fraction
Liver S9 fractions were prepared according to Maron and Ames (1983). Briefly, livers from each group were pooled, minced into small pieces and homogenized in 150 mM KCl (3 ml/g liver wet weight). After centrifugation at 9000 g for 10 min, supernatants were decanted, distributed in 2 ml aliquots and stored at –70°C until use. A portion of each S9 fraction obtained as described was centrifuged at 100 000 g for 60 min; the pellets were resuspended in an equal volume of 100 mM potassium phosphate buffer (pH 7.4) and centrifuged. Microsomal fractions were finally resuspended in 100 mM potassium phosphate (pH 7.4), 1.0 mM EDTA, 0.1 mM DTT and 20% glycerol and stored at –70°C. All solutions and glassware were kept at 4°C. Protein concentrations in microsomal fractions were determined according to Bradford (1976).
Mutagenesis assay
Salmonella typhimurium strains TA1535, TA98 and TA100 donated by Dr B.N.Ames (University of California, Berkeley) were used for mutagenesis assays. TA1535 was used to detect the mutagenicity of CP; TA98 for B[a]P and 2-AF and TA100 for 3-MC, NDPA, NPYR and NDMA. Pre-incubation was performed according to Yahagi et al. (1977) with some modifications. A 0.1 ml aliquot of overnight bacterial culture in nutrient broth, 0.5 ml S9 mixture (30 or 10% S9 fraction, 8 mM MgCl2, 33 mM KCl, 4 mM NADP, 5 mM G6P, 100 mM sodium phosphate buffer, pH 6.5) and 0.1 ml of appropriately diluted chemical solution were added to sterile tubes. This mixture was incubated at 37°C for 60 min, mixed with 2 ml top agar at 45°C and poured onto minimal agar plates. Revertant colonies were counted after a 48 h incubation period at 37°C.
Gel electrophoresis and immunoblot analysis
Polyacrylamide slab gel electrophoresis (PAGE) was carried out at room temperature in the presence of SDS using the discontinuous buffer system described by Laemmli (1970). Each microsomal protein sample was loaded at a concentration of 1 µg/well. The slab separating gel (7x8x0.075 cm) contained 7.5% acrylamide with 0.2% bis-acrylamide. After electrophoresis, proteins were transferred to a 8x6 cm nitrocellulose membrane following the method described by Towbin et al. (1979). For the detection of CYP1A1/A2, -2B1/B2 and -2E1, nitrocellulose sheets were treated with 5% skimmed milk in phosphate-buffered saline (PBS, blocking solution) during 4–24 h at 4°C, followed by incubation with individual anti-CYP antibodies diluted 1:750 in the blocking solution for an additional 1 h, washed with PBS containing 0.03% Tween 20 and incubated with rabbit anti-mouse IgG peroxidase conjugated secondary antibody (1:2000 dilution) for 1 h at room temperature. The nitrocellulose sheets were washed as before and the immunocomplexes were detected using 3',3-diaminobenzidine and hydrogen peroxide.
Biochemical determinations
Alkoxyresorufin O-dealkylases (AROD)
Total cytochome P450 were measured by the method of Omura and Sato (1964). Microsomal AROD activities were measured spectrofluorometrically by monitoring the formation of resorufin according to Burke's method (Burke et al., 1985, 1994) with some modifications: excitation and emission wavelengths were set at 520 and 585 nm, respectively. Substrate (10–40 µl), 1.91–1.94 ml buffer A (50 mM Tris–HCl, 25 mM MgCl2, pH 7.6) and 10–40 µl microsomal sample were placed in a fluorimeter cuvette and incubated at 37°C for 3 min. Reactions were started by the addition of 500 µM NADPH (20 µl from a 50 mM solution in buffer A). With a total reaction volume of 2 ml, the cuvette was then placed in the fluorimeter and the reactions followed for 3 min, recording the fluorescence reading every 15 s. Substrates were dissolved in dimethylsulfoxide as follows: 50 µM ethoxyresorufin, 0.5 mM methoxyresorufin, 1.0 mM pentoxyresorufin and 1.0 mM benzyloxyresorufin. Catalytic activities were calculated from a standard curve of resorufin (0–50 pmol/ml).
4-Nitrophenol hydroxylase (4-NPH)
Hydroxylation of 4-nitrophenol to 4-nitrocatechol was determined by a modification of the method described by Koop (1986). 4-Nitrophenol (0.2 mM) was dissolved in 50 mM Tris–HCl, 25 mM MgCl2, pH 7.4. 930 µl of this solution and 50 µl microsomal samples were incubated at 37°C for 5 min. Reactions were started by adding 20 µl 50 mM NADPH and incubation continued for 10 min more. Reaction mixtures were stopped by the addition of 0.5 ml 0.6 N perchloric acid followed by centrifugation. 4-Nitrocatechol formation was then spectrophotometrically determined in 1 ml supernatant plus 0.1 ml 10 N NaOH at 510 nm. A standard curve with 4-nitrocatechol (5–50 nmol/ml) was used to calculate microsomal activity.
Erythromycin N-demethylase (END)
Erythromycin N-demethylation was assessed spectrophotometrically by measuring the production of formaldehyde according to Alexidis et al. (1996). The incubation mixture (1 ml final volume) contained 10 mM erythromycin, 1 mg microsomal protein and buffer (50 mM Tris–HCl, 10 mM MgCl2, 150 mM KCl). The mixture was pre-incubated 3 min at 37°C, the reactions started by the addition of 10 mM NADPH and incubation was continued for 10 min more. The reaction was stopped by the addition of 0.5 ml 12.5% trichloroacetic acid followed by centrifugation at 5600 r.p.m. Clear supernatant (1 ml) was mixed with 1 ml Nash reagent (Nash, 1953), heated at 50°C for 30 min and measured spectrophotometrically at 412 nm. A standard formaldehyde solution (100 nmol/ml) was used for the calibration curve.
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Results |
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Enzyme activity determinations in hepatic microsomes from rats induced by the different treatments are shown in Table I
. Total CYP was highly induced by Aroclor (up to 468%) but not by PB/NF (up to 122%) or by CH/ABZ, in which a 13% reduction from controls was noted. EROD activity was significantly enhanced in hepatic microsomes from Aroclor- and PB/NF-treated rats (183- and 162-fold over controls, respectively). CH/ABZ pretreatment induced control EROD activity by only 22-fold.
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The PB/NF combination induced the PROD and BROD activities seen in control microsomes 2.5-fold more than Aroclor. Conversely, Aroclor was 3-fold more effective than PB/NF to induce MROD activity (Table I
). On the other hand, although CH/ABZ induced EROD activity by 22-fold over control and MROD, PROD and BROD activities reached a 10-fold induction, this regimen was the less effective. The highest induction of 4-nitrophenolhydroxylase activity was obtained with the CH/ABZ regimen, reaching 408% over the control, followed by Aroclor and PB/NF with 233 and 150%, respectively. CYP3A associated erythromycin-N-demethylase activity was not induced by any of the three regimens tested.
The pattern of modulation of the CYP1A1/2, -2B1/2 and -2E1 immunoreactive proteins stimulated by the different inducers tested paralleled that of the related enzyme activities in that both parameters showed an induction over controls (Figure 1).
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The biochemical data described above predict that S9 fractions, obtained from animals treated with either one of the three different inducers, are able to `activate' standard pro-mutagens in the Salmonella/microsome test. Different S9-dependent mutagens were chosen for this purpose: B[a]P and 3-MC are activated by CYP1A1; 2-AF by CYP1A2; NDPA by CYP2B1; NPYR and NDMA by CYP2E1 and CP by CYP2B and CYP3A. Aroclor and PB/NF S9 activated B[a]P to a similar extent, producing 17 and 12 revertants per microgram (rev/µg), respectively, contrasting with control S9 that produced 3 rev/µg (Table II
). Additionally, PB/NF and Aroclor S9 elevated 13- and 8-fold the mutagenic potency of 3-MC with respect to S9 from control rats (Table II). Although not with the same efficiency, CH/ABZ S9 also activated the two polycyclic compounds inducing a 2-fold (B[a]P) and 5-fold (3-MC) increase in their mutagenic potency over controls. 2-Aminofluorene was the only mutagen activated by control S9 and when tested in the presence of S9 mixtures prepared from PB/NF, Aroclor and CH/ABZ treated rats, the activation was enhanced approximately 2-fold over control. Control S9 was unable to activate NDPA but the three inducing mixtures used allowed its detection as a mutagen in the Ames test (Table II). The mutagenic potencies obtained were 0.58, 0.83 and 1.48 rev/µg from CH/ABZ, Aroclor and PB/NF S9, respectively.
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Mutagenicity by NPYR and NDMA was achieved only with CH/ABZ S9. In the presence of Aroclor or PB/NF S9, these promutagens were unable to duplicate the spontaneous reversion of TA100 tester strain contrasting with an 8-fold (for NPYR) and 323-fold (for NDMA) enhancement over the control when CH/ABZ S9 was used (Table II
). Finally, the mutagenic potency of CP was 0.5 rev/µg when activated with either Aroclor or PB/NF S9, 0.16 rev/µg with CH/ABZ S9 and 0.06 rev/µg with control S9 (Table II). |
Discussion |
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Previous studies in our laboratory have shown that the industrial solvent CH acts as an antimutagenic agent when assayed in vitro and is an in vivo inducer of cytochrome P450 in the rat (Espinosa-Aguirre et al., 1996
, 1997). Exposure of rats to this agent in drinking water gives rise to increased hepatic concentrations of the CYP2B and CYP2E subfamilies. On the other hand, the widely used anthelmintic drug ABZ is a potent inducer of the CYP1A subfamily (Asteinza et al., 2000). CYP1A, -2B and -2E subfamilies constitute the main groups of enzymes involved in the activation of environmental mutagens/carcinogens and, therefore, their level of activity in in vitro metabolic systems (hepatic S9 fractions from rodents) used for the detection of environmental genotoxicants is of great importance.
The potential of inducers to be used in the production of S9 for in vitro genotoxicity assays has usually been evaluated using the Ames assay (Matsushima et al., 1976), but recently, enzymatic assays associated with the presence of CYP enzymes have gained importance in addition to microbial assays (Callander et al., 1995; García Franco et al., 1999). However, these methods have been mainly focused at exploring the activity of CYP1A and CYP2B subfamilies, even though the CYP2E1 enzyme is also involved in the metabolic activation of important environmental mutagens belonging to the nitrosamine family of chemicals, such as nitrosodimethylamine, nitrosodiethylamine, nitrosodipropylamine, nitrosodibutylamine, nitrosopyrrolidine and nitrosonornicotine among others. Since Aroclor 1254 and the combination of PB/NF are poor inducers of CYP2E1, hepatic S9 fractions obtained from animals treated with these inducing mixtures fail to activate NDMA at low concentrations (Matsushima et al., 1976; Yoshikawa et al., 1982; Callander et al., 1995).
The present work was designed to explore the possibility of a complementary effect between ABZ and CH to induce the three CYP subfamilies involved in mutagen activation. Our results showed that Aroclor was the only compound that produced a 5-fold induction of total CYP, confirming the data reported by others (Callander et al., 1995), in contrast to a 13% reduction observed in CH/ABZ microsomes (Table I). PB/NF treatment did not modify the total CYP content seen in control microsomes (Table I). Taking into account that CYP1A, -2B and -2E subfamilies represent just 11% of the total CYP in rat liver (Lewis, 1996), an inducer of total CYP does not necessary constitute a good S9 inducer for mutagenicity tests. On the other hand, a reduction in total CYP could be due to a reduced expression of the CYP2C subfamily that accounts for 65% of total CYP in rat liver, and there is no evidence of its implication in mutagen activation.
Results obtained from the Ames test, western blots and enzymatic activities showed that treatment of rats with CH/ABZ was less efficient than Aroclor and PB/NF in inducing the hepatic CYP1A and CYP2B subfamilies (Tables I and II; Figure 1).
Microsomes from Aroclor- and PB/NF-treated rats displayed a high EROD activity (associated with CYP1A1) and the highest protein concentration in western blot analysis (Table I; Figure 1). These results agreed with the Ames test in which S9 fractions obtained with these chemicals were more effective in activating B[a]P and 3-MC to mutagens, as detected with the strains TA98 and TA100, respectively (Table II).
Although Aroclor was the best inducer of CYP1A2 (Table I; Figure 1), S9 from rats treated with PB/NF was the most effective in activating 2-AF in the Ames test (Table II). Modulation by Aroclor of phase II detoxifying enzymes, or other CYPs not measured in this work, could be responsible for this effect.
The protocol for CH/ABZ induction also produced an increment in CYP1A1/2 proteins and activities (Table I; Figure 1) and S9 from rats treated with this mixture was able to mediate the mutagenicity of B[a]P, 3-MC and 2-AF (Table II). Moreover, Aroclor and CH/ABZ S9 have equally activated the last two mutagens.
Induction of CYP2B1 and CYP2E1 mediated the enhanced genotoxicity elicited by NDPA (Shu and Hollenberg, 1990) and by using inhibitory monoclonal antibodies against these enzymes, the same authors (Shu and Hollenberg, 1996) had demonstrated a 65% contribution of CYP2B1 in the depropylation of NDPA. Our data fit well these results in that S9 from PB/NF- and Aroclor-treated rats were the most effective in activating NDPA (Table II). On the other hand, CH/ABZ S9 increased 10-fold the mutagenic potency of NDPA as seen with control S9 (Table II).
The ethanol-inducible enzyme CYP2E1 activated NDMA into their mutagenic metabolites in the Ames test (Haag and Sipes, 1980; Garro et al., 1981). CYP 2E1 is also involved in the 
-hydroxylation of NPYR, enhancing its mutagenic potency when CYP2E1 inducers were used in the production of the S9 fraction (Burke et al., 1994). Of all the CYP-inducing mixtures used in this work, the CH/ABZ mixture was the most effective in elevating the hepatic microsomal 4-NPH activity associated with CYP2E1 (Table I) and, hence, the mutagenicity of NDMA and NPYR were clearly detected only when CH/ABZ S9 was used (Table II). Aroclor and PB/NF S9 were reported to activate NDMA in the Ames test at doses up to 500–5000 µg/plate (Callander et al., 1995).
CYP2B, and to a minor extent CYP3A, subfamilies are involved in cyclophosphamide transformation to its 4-hydroxylated genotoxic derivative (Chang et al., 1993; Roy et al., 1999); therefore, the higher mutagenic potency of this anticancer prodrug was obtained with S9s from Aroclor- and PB/NF-treated rats (Table II). Since CYP3A was not modified by the different treatments (Figure 1, Table I), our data suggest that CYP2B is the major enzyme involved in the mutagenic activation of CP according to previous reports by Ellard et al. (1991) and Roy et al. (1999).
It is now well established that a wide variety of foods, particularly cured meat products, smoked fish, dried malt and alcoholic beverages, contain trace levels of carcinogenic nitrosamines (Scalan, 1983; Havery and Fazio, 1985; Österdahl, 1991). The detection of these chemicals in environmental samples using microbial mutagenicity tests depends on an effective metabolic system `enriched' with CYP2E1. For this purpose, the combination of CH/ABZ to induce hepatic CYP in rats provides a better option than Aroclor and PB/NF.
In conclusion, the CH/ABZ regime used in this work results in the induction of CYP1A, -2B and -2E subfamilies. Hepatic S9 from CH/ABZ-treated rats efficiently activates promutagens metabolized by these CYP subfamilies and, consequently, represents a good alternative for the in vitro detection of genotoxic agents activated mainly by CYP2E1.
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Acknowledgments |
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The authors would like to express their appreciation to Dr Regina Montero Montoya for critically reviewing the manuscript and to M.S.Clementina Castro Hernández for her excellent technical assistance.
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Notes |
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4 To whom correspondence should be addressed at: Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Apartado Postal 70228, Ciudad Universitaria, 04510 México D.F., México. Email: jjea{at}servidor.unam.mx
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References |
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Ames,B.N., McAnn,J. and Yamasaki,E. (1975) Method for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutat. Res., 31, 347–364.[Web of Science][Medline]
Alexidis,A.N., Commandeur,J.N. and Rekka,E.A. (1996) Novel piperidine derivatives: inhibitory properties towards cytochrome P450 isoforms, and cytoprotective and cytotoxic characteristics. Environ. Toxicol. Pharmacol., 1, 81–88.
Asteinza,J., Camacho-Carranza,R., Reyes-Reyes,R.E., Dorado-González,V. and Espinosa-Aguirre,J.J. (2000) Induction of cytochrome P450 enzymes by albendazole treatment in the rat. Environ. Toxicol. Pharmacol., 9, 31–37.[Medline]
Bradford,M.M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248–254.[Web of Science][Medline]
Burke,M.D., Thompson,S., Elcombe,C.R., Halpert,J., Haaparanta,T. and Mayer,R.T. (1985) Ethoxy-, pentoxy-, and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450. Biochem. Pharmacol., 34, 3337–3345.[Web of Science][Medline]
Burke,M.D., Thompson,S., Weaver,R.J., Wolf,C.R. and Mayer,R.T. (1994) Cytochrome P450 specificities of alkoxyresorufin O-dealkylation in human and rat liver. Biochem. Pharmacol., 48, 923–936.[Web of Science][Medline]
Callander,R.D., MacKay,J.M., Clay,P., Elcombe,C.R. and Elliott,B.M. (1995) Evaluation of phenobarbital/ß-naphthoflavone as an alternative S9-induction regime to Aroclor 1254 in the rat for use in in vitro genotoxicity assays. Mutagenesis, 10, 517–522.
Clare,C. (1989) The state of play for PCBs. BIBRA Bull., 28, 111–114.
Chang,T.K.H., Weber,G.F., Crespi,C.L. and Waxman,D.J. (1993) Differential activation of cyclophosphamide and ifosphamide by cytochrome P450 2B and 3A in human liver microsomes. Cancer Res., 53, 5629–5637.
Ellard,S., Mohammed,Y., Dogra,S., Wolfel,C., Doehmer,J. and Parry,J.M. (1991) The use of genetically engineered V79 chinese hamster cultures expressing rat liver CYP1A1, 1A2 and 2B1 cDNAs in micronucleus assays. Mutagenesis, 6, 461–470.
Elliot,B.M., Combes,R.D., Elcombe,C.R., Gatehouse,D.G., Gibson,G.G., MacKay,J.M. and Wolf,R.C. (1992) Report of the UK Environmental Mutagen Society working party. Alternatives to Aroclor 1254-induced S9 in in vitro genotoxicity assays. Mutagenesis, 7, 175–177.
Espinosa-Aguirre,J.J., Rubio,J., Cassani,M., Nosti,R., Caballero,S., González,I. and Martínez,G. (1996) Induction of microsomal enzymes in liver of rats treated with cyclohexanol. Mutat. Res., 368, 103–107.[Web of Science][Medline]
Espinosa-Aguirre,J.J., Rubio,J., López,I., Nosti,R. and Asteinza,J. (1997) Characterization of the CYP isozyme profile induced by cyclohexanol. Mutagenesis, 12, 159–162.
García Franco,S., Domínguez,G. and Pico,J.C. (1999) Alternatives in the induction and preparation of phenobarbital/naphthoflavone-induced S9 and their activation profiles. Mutagenesis, 14, 323–326.
Garro,A.J., Seitz,H.K. and Lieber,C.S. (1981) Enhancement of dimethylnitrosamine metabolism and activation to a mutagen following chronic ethanol consumption. Cancer Res., 41, 120–124.
Guengerich,F.P., Dannan,G.A., Wright,S.T., Martin,M.V. and Kaminsky,S. (1982) Purification and characterization of liver microsomal cytochromes P-450: Electrophoretic, spectral, catalytic, and immunochemical properties and inducibility of eight isozymes isolated from rats treated with phenobarbital or ß-naphthoflavone. Biochemistry, 21, 6019–6030.[Medline]
Haag,S. and Sipes,G. (1980) Differential effects of acetone or Aroclor 1254 pretreatment on the microsomal activation of dimethylnitrosamine to a mutagen. Mutat. Res., 74, 431–438.[Web of Science][Medline]
Havery,D.C. and Fazio,T. (1985) Human exposure to nitrosamines from foods. Food Technol., 39, 80–83.
Koop,D.R. (1986) Hydroxylation of p-nitrophenol by rabbit ethanol-inducible cytochrome P-450 isozyme 3a. Mol. Pharmacol., 29, 399–404.[Abstract]
Laemmli,U.K. (1970) Maturation of the head of bacteriophage T4. I. DNA packaging events. Nature, 227, 680–685.[Medline]
Lewis,D.F.V. (1996) Introduction. In Rubinstein,M.H. and Wilson,C.G. (eds) Cytochrome P450, Structure, Function and Mechanism. Taylor and Francis, London, pp. 1–54.
Maron,D.M. and Ames,B.N. (1983) Revised methods for the Salmonella mutagenicity test. Mutat. Res., 113, 173–215.[Web of Science][Medline]
Matsushima,T., Sawamura,M., Hara,K. and Sugimura,T. (1976) A safe substitute for polychlorinated biphenyls as an inducer of metabolic activation system. In DeSerres,F.J., Fouts,J.R., Bend,J.R. and Philpot,R.M. (eds) In Vitro Metabolic Activation in Mutagenesis Testing. Elsevier/North-Holland, Amsterdam, pp. 85–88.
Nash,T. (1953) The colormetric estimation of formaldehyde by means of the Hantzen reactions. Biochem. J., 55, 416–421.[Web of Science][Medline]
Omura,T. and Sato,R. (1964). The carbon monoxide-binding pigment of liver microsomes. I. Evidence of its hemoprotein nature. J. Biol. Chem., 239, 2370–2378.
Österdahl,B.G. (1991) Occurrence of and exposure to N-nitrosamines in Sweden: A review. IARC Scientific Publications No. 105, IARC, Lyon, pp. 235–237.
Roy,P., Yu,L.J., Crespi,C.L. and Waxman,D.J. (1999) Development of a substrate-activity based approach to identify the major human liver P-450 catalysts of cyclophosphamide and ifosfamide activation based on cDNA-expressed activities and liver microsomal P-450 profiles. Drug Metab. Dispos., 27, 655–666.
Scalan,R.A. (1983) Formation and occurrence of nitrosamines in food. Cancer Res., 43, 2435–2440.[Web of Science]
Shu,L. and Hollenberg,P. (1990) Role of cytochrome P450 in DNA damage induced by N-nitrosodialkylamines in cultured rat hepatocytes. Carcinogenesis, 3, 569–576.
Shu,L. and Hollenberg,P. (1996) Identification of the cytochrome P450 isozymes involved in the metabolism of N-nitrosodipropyl-, N-nitrosodibutyl- and N-nitroso-n-butyl-n-propylamine. Carcinogenesis, 17, 839–848.
Souhaili-El Amri,H., Fargetton,X., Benoit,E., Totis,M. and Batt,A.M. (1988) Inducing effects of albendazole on rat liver drug-metabolizing enzymes and metabolite pharmacokinetics. Toxicol. Appl. Pharmacol., 92, 141–149.[Web of Science][Medline]
Thomas,P.E., Bandiera,F., Maines,S.L., Ryan,D.E. and Levin,W. (1987) Regulation of cytochrome P450j, a high affinity N-nitrosodimethylamine demethylase, in rat hepatic microsomes. Biochemistry, 26, 2280–2289.[Medline]
Towbin,H., Staehelin,T. and Gordon,J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl Acad. Sci. USA, 76, 4350–4354.
Yahagi,Y., Nagao,M., Seino,Y., Matsushima,T., Sugimura,T. and Okada,M. (1977) Mutagenicities of N-nitrosamines on Salmonella. Mutat. Res., 48, 121–130.[Web of Science][Medline]
Yang,C.S., Smith,T., Ishizaki,H. and Hong,J.Y. (1991) Enzyme mechanism in the metabolism of nitrosamines. In O'Neill,I.K., Chen,J. and Bartsh,H. (eds) Relevance to Human Cancer of N-Nitroso Compounds, Tobacco Smoke and Mycotoxins. IARC Scientific Publications No. 105, IARC, Lyon, pp. 265–274.
Yoshikawa,K., Nohmi,T., Miyata,R. and Ishidate,M. (1982) Differences in liver homogenates from Donryu, Fischer, Sprague–Dawley and Wistar strains of rat in the drug-metabolizing enzyme assay and the Salmonella/hepatic S9 activation test. Mutat. Res., 96, 167–186.[Web of Science][Medline]
Received on July 3, 2001; accepted on July 10, 2001.
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