Mutagenesis vol. 19 no. 3 pp. 245-250,
May 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
Comparative study in the Ames test of benzo[a]pyrene and 2-aminoanthracene metabolic activation using rat hepatic S9 and hepatocytes following in vivo or in vitro induction
1Department of Biochemical Pharmacology, Chemical Research Center of the Hungarian Academy of Sciences, H1525 Budapest, Hungary and 2Environmental Mutagenesis Laboratory, Fodor Jozsef National Center for Public Health, National Institute of Environmental Health, H1097 Budapest, Hungary
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Abstract |
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We studied the replacement of hepatic S9 with in vivo and in vitro induced hepatocytes as a metabolic activation system with the aim of broadening the possibilities of mutagenic assays. Rats were pretreated with ß-naphthoflavone (BNF), phenobarbital (PB), 3-methylcholanthrene (MC) and a combination of BNF and PB (BNF + PB). Mutagenic activation of benzo[a]pyrene (BP) and 2-aminoanthracene (2AA) by hepatic S9 and hepatocytes was determined in the Ames test. Primary rat hepatocytes were used for in vitro induction and were used as the activating system in the Ames test. In vivo BNF treatment greatly increased the metabolic activation capacity of hepatic S9 and hepatocytes towards BP. With regard to 2AA activation, S9 and hepatocytes showed different BNF induction profiles. PB treatment reduced the mutagenicity of both compounds. Although ethoxyresorufin O-dealkylase (EROD) activity of S9 from BNF + PB-treated animals was almost 30-fold greater than the control, its effectiveness in activation of 2AA was below the control level. A large part of the EROD activity of control cells was lost during culture, together with the ability to activate 2AA, however, 72 h of MC induction increased EROD activity to 200-fold of the control, which corresponds to 28% of that of in vivo induced hepatocytes. The mutagenic potential of BP activated by in vitro induced hepatocytes was 10-fold above the control, which is 47% of the mutagenicity detected following in vivo induction. In vitro induced hepatocytes increased 2AA mutagenicity to 14.6-fold over the control, which corresponds to 68% of in vivo induction. Our results suggest that primary culture of hepatocytes provides a useful model for the study of the role of metabolic activation processes concerning enzyme activity of cytochromes P450 and other metabolic enzymes and induction profiles of different inducers.
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Introduction |
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Many carcinogens and mutagens have to be metabolized before their mutagenic activity can be detected. With few exceptions, success in detecting carcinogens as mutagens by mutagenicity assays is to a large extent due to the incorporation of metabolic systems, usually rat liver S9 fraction, to mimic mammalian xenobiotic metabolism. There is an increasing need for metabolically competent systems to study the metabolic activation of promutagens with different molecular structures and to predict their relevant carcinogenic potency. Although the metabolic capacity of rat liver S9 fraction is generally enhanced by in vivo induction of rat metabolic enzymes, this system suffers from a number of limitations. These are primarily related to the complexity of the metabolic pathways responsible for the generation or, on the contrary, the detoxification of ultimate mutagenic metabolites involving different phase I and phase II enzymes, which are present at either the baseline level or increased levels following induction (Kranendonk et al., 2000
; Zheng et al., 2002).
The use of liver S9 fraction completed with NADPH as the only cofactor in the metabolic system restricts the study of metabolic activation to phase I enzymes, mainly cytochromes P450. The role of phase II enzymes in either activation or detoxification is beyond the possibilities of this method. A chance to study the role of the complete metabolic system is the employment of isolated hepatocytes, in which all of the metabolic pathways function, including the transport processes (Vienneau et al., 1995). Another remarkable advantage of the use of hepatocytes as a metabolic activator system is that as they can be induced in vitro after preparation and the effect of inducers can be measured separately or in combination. In addition, the number of animals can be greatly reduced, as hepatocytes obtained from one rat can be used for the study of numerous inducers. Also, this model is suitable to determine the mutagenic potency of compounds to which humans may be exposed, using human hepatocytes following in vitro induction. The effect of exposure of humans to various environmental xenobiotics on the activation of mutagens can be modelled by the in vitro induction of human hepatocytes by these compounds.
Bacterial mutagenicity assays (the Ames test) have been in use for more than two decades (Ames et al., 1975). They are used to both elucidate the biochemical mechanism of mutagenesis and to screen for the genotoxicity of pharmaceuticals and environmental pollutants. At first a mixture of polychlorinated biphenyls, like Aroclor 1254, was used as inducing agent, which was replaced by a combined treatment with phenobarbital (PB) and ß-naphthoflavone (BNF) (Callander et al., 1995; Garcia-Franco et al., 1999). Growing knowledge concerning signal transduction events triggered by xenobiotics either specifically or non-specifically suggests that most of the examined inducers regulate several drug metabolism genes, hence, it is difficult to predict the phenotypic outcomes from gene expression profiles concerning metabolic activation of an actual mutagen (Nebert, 2002). There are a number of papers describing the role of cytochromes P450, especially CYP1A1, CYP1A2 and CYP1B1, in the activation of different mutagens (benzo[a]pyrene and arylamines) (Oda et al., 2001; Sparfel et al., 2002). These cytochrome P450 isoforms are highly induced by BNF. Other mutagens, like 2-aminofluorene and 6-aminochrysene, are activated by PB inducible isoenzymes (Lubet et al., 1989). It is very rare that the effect of these two inducers is additive (like in the case of 2-acetylaminofluorene), moreover, several lines of evidence suggest that these compounds inhibit the inductive effect of each other. Contradictory effects of inducers on the activation of mutagens with similar chemical structures have been described. It has been reported that the activation of 2-aminoanthracene (2AA) is decreased by PB treatment (Carriere et al., 1992) while the opposite effect has been measured for 6-aminochrysene and 2-acetylaminofluorene (Lubet et al., 1989). These findings query the usefulness of combined PB + BNF treatment, which may result in misleading risk assessment.
The aim of this work was to study the effect of PB, MC and BNF induction on the metabolic activation of chemically different mutagens, 2AA and benzo[a]pyrene (BP). The effect of replacement of rat S9 fraction with hepatocytes prepared from treated or non-treated animals was also investigated. We also compared the sensitivity of the assay employing isolated rat hepatocytes induced in vivo and in vitro, respectively.
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Materials and methods |
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Chemicals
BNF, PB, MC, BP, 2AA, Williams’ medium E, collagenase, 7-ethoxyresorufin and 7-pentoxyresorufin were purchased from Sigma Chemical Co. (St Louis, MO). NADPH and NADP were from Reanal (Hungary). Glucose 6-phosphate and glucose 6-phosphate dehydrogenase were purchased from Calbiochem (La Jolla, CA).
Animal treatment
Male Wistar rats (Charles-River, Hungary) weighing 200–250 g were housed in polypropylene cages and kept on a 12 h light/dark cycle in an animal care facility. The animals were allowed free access to laboratory rodent chow and tap water. The animals were treated daily for 3 days p.o. with 80 mg/kg PB dissolved in distilled water, 20 mg/kg MC or 80 mg/kg BNF dissolved in corn oil, respectively. PB + BNF-treated animals were treated with 80 mg/kg PB + 80 mg/kg BNF in corn oil p.o. daily for 3 days. The control animals were untreated.
Preparation of S9 fraction
S9 fractions were prepared from pooled livers of each group of three rats for all treatments. The livers were minced and homogenized in 0.01 M phosphate buffer (pH 7.4) containing 1.15% KCl (3 ml/g liver wet wt). The homogenates were centrifuged at 9000 g for 30 min at 4°C. The supernatants were stored at –70°C until use. The protein content was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard.
Preparation of hepatocytes
Hepatocytes were prepared from treated and untreated male Wistar rats (200–250 g) by in situ liver collagenase perfusion according to the method of Seglen (1976). Cell viability (>90%) was determined by trypan blue exclusion. The freshly isolated hepatocytes were suspended in Williams’ medium E containing 20 mM HEPES and used for mutagenesis assay within 2 h after preparation.
In vitro induction of hepatocytes
Cells prepared from untreated rats were seeded on collagen-coated dishes at a density of 10 x 106 and 1.33 x 106 cells/dish (100 and 30 mm diameter), respectively, in Williams’ medium E containing 5% foetal calf serum, 100 nM insulin, 100 nM dexamethasone, 2.5 µg/ml amphotericin B, 0.1 mg/ml gentamicin and 30 nM Na2SeO3. Calf serum and amphotericin B were present for the first 24 h, then omitted. Cells were maintained at 37°C in a humidified atmosphere of 95% air, 5% CO2. Four hours after plating and every day thereafter the medium was changed to Williams’ medium E supplemented with 3.7 µM MC in DMSO. The control medium contained vehicle (0.1% DMSO). The induction period lasted for 72 h. The cells, washed with phosphate-buffered saline (PBS), were released from the collagen layer by collagenase treatment for 10 min in Hanks balanced salt solution and washed twice with PBS. The freshly prepared hepatocytes were suspended in Williams’ medium E containing 20 mM HEPES and used for mutagenesis assay within 2 h after preparation. The number of cells was determined on the bases of protein content measured by the method of Lowry et al. (1951). The total cell protein isolated from 106 cells was 1.84 ± 0.17 mg.
7-Ethoxyresorufin O-dealkylase (EROD) and 7-pentoxyresorufin O-dealkylase (PROD) activity measurements
7-Ethoxy- and 7-pentoxyresorufin O-dealkylation were determined according to Burke et al. (1985) with some modifications. S9 fractions prepared from the liver of treated or untreated animals were used instead of microsomes, when S9 fractions were the metabolic activating system in the mutagenesis assays. S9 fractions were also prepared from hepatocytes when hepatocytes were used in the Ames test. The incubation test consisted of 0.1 M Tris–HCl (pH 7.4), 10 µM substrate, 10 µM dicumarole, 0.5 mM NADPH, 10 mM glucose 6-phosphate, 2 mM MgCl2, 3 U/ml glucose 6-phosphate dehydrogenase. 0.5 mg/ml S9 for EROD and 0.3 mg/ml S9 for PROD activity measurements, respectively, in a final volume of 1 ml. The reaction lasted for 5, 10 and 15 min and was terminated by the addition of 2 ml of ice-cold methanol. The enzyme activities were determined spectrofluorometrically (550/589 nm) based on a standard curve of resorufin (0–500 pmol/ml).
Mutagenesis assay
Bacterial strains. Salmonella typhimurium strains TA98 and TA100 were obtained from Bruce N.Ames (University of California, Berkeley, CA) and stored as frozen master copies at –70°C (Maron and Ames, 1983). Cultures were grown at 37°C for 10 h with shaking in Oxoid nutrient broth no. 2 (CM67) (Oxoid Ltd, Basingstoke, UK).
Preincubation procedure. All experiments were carried out using a preincubation modification (Yahagi et al., 1975) of the standard plate incorporation assay (Ames et al., 1975). Briefly, 0.5 ml of S9 mix (or 0.1 M phosphate buffer, pH 7.4), 0.1 ml of bacterial culture and 0.05 ml of test solution (or solvent) were added to each tube. When hepatocytes were used for metabolic activation instead of S9 mix the preincubation mixture consisted of 0.5 ml of hepatocyte suspension in Williams’ medium E containing 20 mM HEPES, 0.1 ml of bacterial culture and 0.05 ml of test solution (or solvent). The 0.5 ml of hepatocyte suspension contained 0.5 x 106 cells for 2AA activation and 2 x 106 cells for BP activation. In pilot studies these cell densities were found to ensure optimal conditions for linear cell number versus revertant number response. Triplicate tubes were prepared for each dose of mutagen. The mixture was vortexed, then allowed to incubate at 37°C with shaking for 30 min. Following this preincubation period, 2.0 ml of molten top agar (45°C) supplemented with histidine and biotin was dispensed into the tubes, which were immediately vortexed and the contents poured onto the surface of bottom minimal glucose agar (Vogel and Bonner, 1956) in 90 x 15 mm disposable Petri dishes. When the agar overlay had solidified, the plates were inverted and placed in a 37°C incubator. After incubation for approximately 60 h the revertant colonies were counted.
Statistics
Mutagenic potency was calculated from the linear portion of the dose–response curve.
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Results |
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The extent of induction following in vivo PB, BNF and BNF + PB treatment was checked by EROD and PROD activity measurements (Figure 1). EROD activity was significantly enhanced in the hepatic S9 fraction from BNF- and BNF + PB-treated rats (29- and 22-fold over controls, respectively). PB treatment induced EROD activity by 2.6-fold. PB and BNF + PB treatment resulted in similar increases in the PROD activity (42- and 40-fold, respectively). BNF increased PROD activity by 3-fold over the control.
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We analysed the mutagenic activity of BP and 2AA as model compounds in the Ames test using hepatic S9 from BNF-, PB- and BNF + PB-treated rats and S9 from untreated rats as a control. BP showed low mutagenic activity with control S9, but using S9 prepared from BNF-treated rats mutagenicity was greatly increased (12.4-fold over control) (Figure 2). PB treatment doubled the mutagenic activity of BP, however, in combination with BNF PB reduced the inductive effect of BNF by 50%. In the case of 2AA, the highest mutagenic potency in the lower, linear range of mutagen concentrations was observed with S9 from untreated rats. Up to 0.333 µg/plate 2AA, mutagenic potency decreased to 43, 9 and 18% of the control with S9 from BNF-, PB- and BNF + PB-treated rats, respectively. At higher 2AA concentrations BNF treatment slightly enhanced the metabolic activation of 2AA compared with the control.
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The mutagenic potencies of BP and 2AA were measured using isolated hepatocytes from BNF- and MC-treated and untreated rats (Figure 3). BP was weakly mutagenic when control hepatocytes were used for metabolic activation. The mutagenic activity of BP was increased similarly to 10- and 12-fold of the control with hepatocytes from MC- and BNF-treated rats, respectively. 2AA showed marked mutagenicity even with the control hepatocytes. In contrast to the results obtained with S9 fraction (Figure 2B), BNF treatment markedly induced 2AA activation by hepatocytes (5.4-fold of control). A significant difference was observed in the mutagenic activity of 2AA when comparing metabolic activation by hepatocytes from MC- and BNF-treated rats (177 and 544%, respectively).
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The extent of in vitro induction was determined as EROD activity of S9 prepared from hepatocytes. The control cells lost 85% of EROD activity during culturing compared with that in freshly isolated hepatocytes (13.4 ± 3.5 and 2.1 ± 0.6 pmol/mg protein/min, respectively). MC treatment resulted in a 200-fold increase in EROD activity after 72 h induction, which corresponds to 28% of the EROD activity of hepatocytes prepared from in vivo induced rats (407 ± 39 and 1437 ± 196 pmol/mg protein/min, respectively). BP activated by in vitro MC-induced hepatocytes was 10-fold more mutagenic than the control, which was 47% of the mutagenic potential achieved by in vivo induced hepatocytes (Figure 4A). The mutagenicity of 2AA activated by control hepatocytes following 72 h culture decreased to 8% compared with the effect of freshly isolated cells. Seventy-two hours induction increased the mutagenic effect of 2AA to 14.6-fold of the control, which corresponds to 68% of the mutagenicity measured with in vivo induced hepatocytes (Figure 4B).
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Discussion |
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The present study was designed to compare the metabolic activation of 2AA and BP, as model procarcinogen compounds, by different metabolic systems, rat hepatic S9 and hepatocytes induced in vivo or in vitro, respectively. The potential of inducers to be used in the production of S9 for genotoxicity assays has usually been determined by the Ames test for model procarcinogens. For about two decades polychlorinated biphenyls, like Aroclor 1254, were used, which induce a wide range of cytochrome P450 enzymes (Borlak and Thum, 2001
). Due to its high toxicity and harmful environmental impact the replacement of Aroclor 1254 by combined BNF + PB induction was proposed based on enzymatic assays concerning CYP enzyme activities (Guengerich et al., 1982; Callander et al., 1995). However, these inducers mainly explore the activity of the CYP1A1/1A2, CYP1B1 and CYP2B subfamilies, even though other enzymes, such as CYP2E, are also involved in the metabolic activation of some mutagens (Yamazaki et al., 1992; Escobar-Garcia et al., 2001). In this study hepatic S9 fractions prepared from control and BNF-, PB- or BNF + PB-treated rats were applied to determine which inducer provides the highest rate of metabolic activation. Our results demonstrate that BNF treatment greatly increased the metabolic activation capacity of hepatic S9 towards BP but not towards 2AA. The involvement of CYP1A1 and CYP1B1 in the metabolic activation of BP and the role of CYP1A2 in the activation of 2AA have been described by many authors, and recently proved by applying mutagenicity tester bacteria expressing mammalian biotransformation enzymes (Kranendonk et al., 1999, 2000). In agreement with the findings mentioned, our data show that BNF treatment resulted in a sharp increase in CYP1A1/1A2 activity, measured as EROD activity, in accordance with the elevation of BP and 2AA mutagenicity in the presence of hepatocytes. After PB treatment 2AA mutagenicity markedly decreased compared with the control, in agreement with the report of Carriere et al. (1992), and using PB in combination with BNF, PB prevented the inductive effect of BNF. Some authors have described that CYP1A2 enzyme activity is decreased by PB treatment (Wortelboer et al., 1991; Rushmore and Kong, 2002), however, the EROD activity of S9 fraction derived from PB-treated rats increased. The elevated EROD activity is probably due to CYP1A1. However, 2AA is activated by CYP1A2, which explains the discrepancy between elevated EROD activity and decreased 2AA activation observed with PB-treated S9. At the same time, using PB in combination with BNF, EROD activity decreased by 25% of the activity of S9 from BNF-treated rats. This loss of enzyme activity could be the reason for the decreased mutagenicity of BP, which is known to be activated by CYP1A1 (Bauer et al., 1995). The low mutagenicity of 2AA cannot be explained by decreased CYP1A1/1A2 activity, as the EROD activity measured in S9 of BNF + PB-treated rats was almost 30-fold higher than that of the control. Similar paradoxical results were found with Aroclor 1254, which induces a number of CYP enzymes, including CYP1A1/1A2, whereas liver S9 prepared from Aroclor-treated animals was less effective in activating 2AA than the control (Zeiger et al., 1979). Our results are in good agreement with these findings; BNF + PB treatment induced both EROD and PROD activity, however, the mutagenicity of 2AA decreased. Little information is available concerning 2AA metabolism and the role played by different cytochromes P450 other than CYP1A1/1A2 in its metabolism. The involvement of multiple CYP isoenzymes in both the activation and inactivation of 2AA is feasible. Overall, our results demonstrate that in the case of both BP and 2AA the combination of BNF with PB diminishes the metabolic activation potency of S9 compared with the effect of BNF alone.
Considering the results obtained with hepatic S9 as the activating system, another polycyclic aromatic hydrocarbon MC was chosen for in vivo hepatocyte induction. As for the experiments using hepatic S9, the untreated hepatocytes poorly activated BP (as we presumed), since the CYP1A1 level was negligible in the control hepatocytes. Hepatocytes from BNF-treated rats increased BP mutagenicity 12-fold compared with the control and MC treatment resulted in almost the same effect. 2AA activated by BNF-induced hepatocytes was almost as mutagenic as it was with BNF-induced S9. However, huge differences were observed when comparing the metabolic activation effect of control hepatocytes and control S9. Probably due to detoxification processes, the mutagenic activity of 2AA activated by control hepatocytes was 15-fold lower than that activated by control S9. A reason for the elevated mutagenic potential of 2AA activated by BNF-induced hepatocytes compared with the control might be that the detoxifying mechanisms are not induced by BNF to the same extent. It is worth mentioning that MC treatment was much less effective in the case of 2AA than in that of BP, which indicates differences between the induction spectra of BNF and MC. Our results demonstrate that substitution of hepatic S9 by hepatocytes provides a useful system for metabolic activation. The use of hepatocytes offers the possibility of studying the effect of a broad spectrum of enzyme inducers on the same cell preparation with regard to metabolic activation of promutagens. Several investigators have reported that during cell preparation and culture much of the enzyme activity of some cytochromes P450 vanishes (LeCluyse et al., 1996; Richert et al., 2002). In our study, over 3 days of culture 85% of EROD activity was lost. Other P450 activities were not measured in this study, but similar loss of enzyme activity has been reported for CYP2B, CYP3A and CYP4A (Richert et al., 2002). The decrease in EROD activity did not alter the mutagenic potential of BP significantly, since CYP1A1 activity was originally very low in control cells. Although the 200-fold increase in EROD activity on in vitro MC treatment corresponds to only 28% of the EROD activity of hepatocytes induced in vivo, this elevated enzyme activity resulted in a high mutagenic potential of BP which was 47% of that observed when using in vivo induced hepatocytes. LeCluyse et al. (1999) reported similar differences between in vitro and in vivo induction concerning CYP1A, CYP2B, CYP3A and CYP4A activities. The significant loss of mutagenic activity of 2AA after 72 h culture supports the importance of a constitutive enzyme like CYP1A2 in the activation process.
In conclusion, our results suggest that the combined BNF + PB induction protocol is inadequate to ensure optimal conditions for all promutagens in mutagenicity assays. The use of hepatocytes for metabolic activation provides a useful system to find the best inducer, and the effect of other enzymes besides cytochromes P450 can also be taken into consideration. Extrapolation of the mutagenic potential of a compound from rat to human is often misleading due to differences in enzyme activities, in substrate specificity and in regulation between species (Hakura et al., 1999; Bogaards et al., 2000). Another advantage of this model is that using human hepatocytes induced in vitro for metabolic activation enables direct human risk assessment concerning the mutagenic potential of compounds. Further studies with this system should be carried out in order to obtain more information on cytochromes P450 other than CYP1A1 and CYP1A2 taking part in 2AA activation and to gain new insights into the use of human hepatocytes for risk assessment.
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Acknowledgement |
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This work was supported by a grant from the National Science and Research Fund (OTKA, T037635).
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Notes |
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3To whom correspondence should be addressed. Tel: +36 1 4384141; Fax: +36 1 3257554; Email: jemnitz{at}chemres.hu
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