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Metabolism of nicotine and induction of CYP1A forms in precision-cut rat liver and lung slices
Toxicology in Vitro 18 (2004) 179–185 www.elsevier.com/locate/toxinvit
Metabolism of nicotine and induction of CYP1A forms in precision-cut rat liver and lung slices Roger J. Price, Anthony B. Renwick, David G. Walters, Philip J. Young, Brian G. Lake* BIBRA International Ltd., Woodmansterne Road, Carshalton, Surrey SM5 4DS, UK and Centre for Toxicology, School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK Received 30 September 2002
Abstract The aim of this study was to investigate xenobiotic metabolism and induction of cytochrome P450 (CYP) forms in precision-cut rat liver and lung slices, employing nicotine as a model compound. Freshly cut rat liver and lung slices metabolised nicotine to the major metabolite cotinine. Observed Km values for cotinine formation in liver and lung slices were 323 and 41.7 mM, respectively, with corresponding Vmax values of 47.2 and 3.21 pmol/min/mg protein, respectively. Rat liver and lung slices were cultured for 48 h with Aroclor 1254, benzo(a)pyrene, nicotine and cotinine. Both Aroclor 1254 and benzo(a)pyrene produced a marked induction of CYP1A-dependent 7-ethoxyresorufin O-deethylase activity in both liver and lung slices. However, while nicotine induced 7-ethoxyresorufin O-deethylase activity in lung slices, but not in liver slices, cotinine did not induce enzyme activity in either liver or lung slices. Overall, while higher rates of nicotine metabolism were observed in rat liver slices, nicotine-induced CYP1A form induction was observed in lung slices. These results demonstrate the usefulness of precision-cut tissue slices for studying tissue differences in xenobiotic metabolism and CYP form induction. # 2003 Elsevier Ltd. All rights reserved. Keywords: Precision-cut tissue slices; Nicotine metabolism; Cytochrome P450; CYP1A form induction
1. Introduction In recent years much effort has been devoted to the development of in vitro systems to study the metabolism and toxicity of xenobiotics. One potentially very valuable in vitro model system is the precision-cut tissue slice technique, which was originally developed at the University of Arizona (Krumdieck et al., 1980; Parrish et al., 1995). This technique has been applied to various tissues including the liver, kidney lung, heart and spleen (Parrish et al., 1995; Bach et al., 1996; de Kanter et al., 1999, 2002). For the lung, an agarose embedding procedure Abbreviations: CLint, intrinsic clearance; CYP, cytochrome P450; DMSO, dimethyl sulphoxide; EBSS, Earle’s balanced salt solution; Km, the concentration of substrate giving half maximal velocity; LC-MS-MS, liquid chromatography-mass spectrometry-mass spectrometry; MEMa, Minimal Essential Medium Alpha; MRM, multiple reaction monitoring; Vmax, the maximal velocity. * Corresponding author: Tel.: +44-20-8652-1006; fax: +44-208661-7029. E-mail address: [email protected] (B.G. Lake). 0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2003.08.012
has been developed which provides a solid support for this tissue, thus permitting the preparation and culture of precision-cut lung slices (Stefaniak et al., 1992; Parrish et al., 1995). Major advantages of the tissue slice technique include the maintenance of tissue architecture, so that all cell types are present and the ready application of the technique to different tissues and species (Parrish et al., 1995; Bach et al., 1996; de Kanter et al., 1999; Lerche-Langrand and Toutain, 2000). Precision-cut liver slices have been extensively used for studies of xenobiotic metabolism and toxicity and to investigate the induction of cytochrome P450 (CYP) forms (Lake et al., 1993, 1998; Parrish et al., 1995; Bach et al., 1996; Lerche-Langrand and Toutain, 2000). Compared to liver slices, fewer studies on the metabolism and toxicity of xenobiotics have been performed with lung slices (Parrish et al., 1995; Bach et al., 1996). Recent studies in our laboratory have demonstrated that cultured rat lung slices can be employed to study the induction of CYP1A1 forms (Lake et al., 2001, 2003).
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The aim of this study was to evaluate the usefulness of the tissue slice technique for assessing organ differences in xenobiotic metabolism and CYP form induction. Nicotine was selected as a model compound for these studies as other investigations have demonstrated tissue differences in both nicotine metabolism and effects on xenobiotic metabolising enzymes. Apart from tobacco, nicotine is present in certain foodstuffs including tomatoes, potatoes, cauliflower and certain teas (Davis et al., 1991). Nicotine is metabolised by various pathways, of which cotinine is the primary product of the C-oxidation pathway of nicotine biotransformation (Kyerematen and Vesell, 1991). While the liver is considered to be the major site of nicotine biotransformation, metabolism also occurs in the lung and kidney (Shigenaga et al., 1988; Kyerematen and Vesell, 1991; Foth, 1995). Nicotine is 0 0 metabolised by CYP forms to a nicotine 1 (5 )-iminium ion which is converted to cotinine by a cytosolic aldehyde oxidase enzyme (McCoy et al., 1989; Hammond et al., 1991; Kyerematen and Vesell, 1991; Nakayama et al., 1993; Foth, 1995). In rat liver nicotine is metabolised by CYP1A2, CYP2B1, CYP2C11 and other CYP forms; CYP2B1 also being constitutively expressed in rat lung (Hammond et al., 1991; Guengerich, 1993; Nakayama et al., 1993; Foth, 1995; Nims and Lubet, 1996). Nicotine metabolism by the lung has been demonstrated in studies with both perfused organs and subcellular fractions (Mattammal et al., 1987; Shigenaga et al., 1988; Kyerematen and Vesell, 1991; Foth, 1995). In other studies the effect of nicotine on rat hepatic, renal and pulmonary CYP1A forms has been investigated. Following a single subcutaneous dose nicotine was shown to induce pulmonary 7-ethoxyresorufin O-deethylase activity and levels of CYP1A1 apoprotein (Iba et al., 1998). In contrast to nicotine, administration of either subcutaneous or intraperitoneal doses of cotinine did not induce enzyme activity or CYP1A1 apoprotein levels in either the liver, kidney or lung (Iba et al., 1998). The effect of nicotine administration for 30 days in a nutritionally balanced liquid diet has also been investigated (Iba et al., 1999). Nicotine treatment induced 7-ethoxyresorufin O-deethylase activity in all three tissues, the effect being most marked in the lung. Treatment with nicotine also resulted in a dose-dependent increase in CYP1A1 apoprotein in the lung, whereas in the liver CYP1A1 was induced only at the highest dose level examined (Iba et al., 1999). In the present study, the metabolism of nicotine to cotinine has been studied in freshly prepared precisioncut rat liver and lung slices. Cultured rat liver and lung slices were used to study the effects of nicotine and cotinine on hepatic and pulmonary CYP1A forms. To serve as positive controls for induction of CYP forms, liver and lung slices were also treated with two known inducers of xenobiotic metabolism (Conney, 1986;
Okey, 1990), namely the polychlorinated biphenyl mixture Aroclor 1254 and benzo(a)pyrene.
2. Materials and methods 2.1. Materials All tissue culture media were obtained from Gibco BRL (Life Technologies Ltd., Paisley, Scotland). The sources of the other tissue culture materials were as described previously (Beamand et al., 1993). Nicotine, cotinine, cotinine-d3,7-ethoxyresorufin, resorufin, benzo(a)pyrene, enzyme cofactors and buffers were obtained from Sigma-Aldrich Company Ltd (Poole, Dorset, UK). Aroclor 1254 was the generous gift of the Monsanto Chemical Company (St Louis, MO, USA). 2.2. Animals Male Sprague–Dawley rats were obtained from Harlan Olac (Bicester, Oxon, UK) and were allowed free access to R and M No.1 laboratory animal diet (Special Diets Services, Witham, Essex, UK) and water. The animals were housed in mesh-floored cages in accommodation maintained at 22 3 C with a relative humidity of 40–70% and allowed to acclimatise to these conditions for at least 7 days before use. Rats (10–14 weeks old) were killed by exsanguination under sodium pentobarbitone anaesthesia (60 mg/kg, ip). The livers were perfused in situ, prior to excision, with 20 ml of icecold oxygenated (95% O2/5% CO2) Earle’s balanced salt solution (EBSS) containing 25 mm d-glucose, 50 mg/ml gentamicin and 2.5 mg/ml fungizone (Price et al., 1998). After excision the livers and lungs were placed into icecold oxygenated EBSS containing the above additions. 2.3. Preparation of precision-cut liver and lung slices Tissue cylinders were prepared from liver samples with a 10 mm diameter motor-driven tissue coring tool. The trachea was cannulated and the lungs instilled at 37 C with a 0.75% (w/v) agarose solution under a constant hydrostatic pressure of 20 cm as described previously (Placke and Fisher, 1987; Price et al., 1995a,b; Stefaniak et al., 1992). After allowing the agarose to gel at 4 C, tissue cylinders were prepared with an 8 mm motor-driven tissue coring tool. From the tissue cylinders liver slices (around 250 mm thickness) and lung slices (around 500 mm thickness) were prepared in cold oxygenated EBSS containing 25 mm d-glucose, 50 mg/ml gentamicin and 2.5 mg/ml fungizone using a Krumdieck tissue slicer (Alabama Research and Development Corporation, Munford, AL, USA). Tissue slice thickness was determined as described previously (Price et al., 1998).
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2.4. Culture of liver and lung slices Tissue slices were floated onto Vitron (Vitron Inc., Tucson, AZ, USA) type C titanium roller inserts (two slices per insert) and cultured in glass vials at 37 C containing 1.7 ml culture medium employing a Vitron dynamic organ culture incubator. Liver slices were cultured in Williams’ medium E containing 2 mm l-glutamine, 0.1 mm insulin, 0.1 mm dexamethasone, 50 mg/ml gentamicin and 2.5 mg/ml fungizone under an atmosphere of 95% O2/5% CO2, whereas lung slices were cultured in MEMa medium, containing all the above additions except l-glutamine, under an atmosphere of 95% air/5% CO2. After 1 h, the medium was replaced with fresh medium and subsequently the medium was changed every 24 h. The test compounds were dissolved in dimethyl sulphoxide (DMSO) and added to the culture medium so that the final DMSO concentration was 0.4% (v/v) in all vials including the control (no test compound) cultures. 2.5. Biochemical investigations After incubation, liver and lung slices were washed in 0.154 M KCl containing 50 mM Tris–HCl pH 7.4 and then homogenised (2 slices in 2 ml) in this medium by sonication (Beamand et al., 1993). Liver and lung slice homogenates were stored at 80 C prior to analysis. 7-Ethoxyresorufin O-deethylase activity was determined at 37 C in spectrofluorimeter cuvettes containing 2 mm substrate, 8 mm dicumarol, 250 mm NADPH, tissue slice homogenate (liver 0.2 ml, lung 1.2 ml) and 50 mm TrisHCl buffer pH 8.4 in a final volume of 2 ml. Resorufin formation was monitored with time at wavelengths of 535 nm excitation and 582 nm emission. Protein content was determined by the method of Lowry et al. (1951) employing bovine serum albumin as standard.
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Hitchin, Herts, UK) attached to an HP1100 series HPLC (Hewlett-Packard Ltd., Bracknell, Berkshire, UK) were used to introduce samples into a Micromass Quattro LC Triple Quadrupole Mass Spectrometer (Micromass UK Ltd., Altrincham, Cheshire, UK) using an Electrospray Z-spray1 source. Chromatography of 50 ml aliquots of the extracts was performed with a 1003 mm Hypersil-3 BDS C18 column (ThermoHypersil-Keystone, Runcorn, Cheshire, UK) with a 102 mm guard cartridge of the same material and isocratic elution with a mobile phase of acetonitrile, methanol and 10 mm ammonium acetate (50:30:20 v/v/ v) at a flow rate of 0.75 ml/min and column temperature of 35 C. The eluate was passed directly into the triple quadrupole mass spectrometer operating in positive atmospheric pressure chemical ionisation mode with probe and source block temperatures of 400 C and 100 C, respectively, using nitrogen for desolvation and nebulisation and a corona voltage of 3.8 kV to initiate ionisation. Cotinine and cotinine-d3 (retention times 0.76 min) were selectively detected by multiple reaction monitoring (MRM) using argon and quantitation was carried out at five levels against triplicate aqueous authentic standards of cotinine using cotinine-d3 as internal standard. MRM function (parent and daughter ions) were 177.08!79.91 and 180.10!79.91 for cotinine and cotinine-d3, respectively. Cone voltage and collision energy were 32 and 20 V, respectively, for cotinine and 35 and 23 V, respectively, for cotinine-d3. 2.7. Statistical analysis Statistical analysis of data was performed by one-way analysis of variance. Comparisons between means were made use the least significant difference test.
3. Results 2.6. Metabolism of nicotine 3.1. Metabolism of nicotine in rat liver and lung slices Freshly cut rat liver and lung slices were incubated in phenol red free RPMI 1640 medium containing 0–1000 mm nicotine for periods of 30 and 180 min, respectively. At the end of the incubations the tissue slices were homogenised (Beamand et al., 1993) by sonication in the culture medium and the tissue slice/medium homogenates stored at 80 C prior to analysis by liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS). Tissue slice homogenates (1.0 ml) were treated with 0.025 ml of 0.880 g/ml ammonia and 0.025 ml of 600 ng/ml cotinine-d3 (internal standard) and extracted with 8 ml of dichloromethane for 30 min. After centrifugation at 1700 g for 10 min at 4 C the organic phase was evaporated to dryness and reconstituted in 300 ml liquid chromatography mobile phase (see below). An HTS PAL autosampler (Presearch Ltd.,
Freshly cut rat liver and lung slices were cultured in medium containing 0–1000 mm nicotine for periods of 30 and 180 min, respectively. Preliminary experiments established that cotinine formation was linear with respect to time in both liver and lung slices over these incubation periods (data not shown). At the end of the incubations, the liver and lung slices were harvested and homogenised by sonication in the culture medium. Levels of cotinine were determined by LC-MS-MS assay of the tissue slice/medium homogenates. Nicotine was metabolised to cotinine by both rat liver (Fig. 1A) and lung (Fig. 1B) slices. Kinetic analysis of rates of cotinine formation in rat liver and lung slices was performed by both Michaelis-Menten (Fig. 1) and Eadie–Hofstee plots. Calculated Km and Vmax values for
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Fig. 1. Freshly cut rat liver (A) and lung (B) slices were incubated with 0–1000 mM nicotine for 30 and 180 min, respectively. Levels of cotinine formation in tissue slice/medium homogenates were determined by LC-MS-MS. Results are presented as meanS.E.M. of 3 experiments.
cotinine formation were lower in rat lung than in rat liver slices (Table 1). Intrinsic clearance (CLint, i.e. Vmax/Km) values for cotinine formation were also calculated. When expressed per unit of tissue slice homogenate protein (i.e. in units of ml/min/mg protein), CLint values were similar in rat liver and lung slices (Table 1). However, when scaled for total organ protein content and for relative organ weight, CLint values were eleven times greater in rat liver than in lung slices (Table 1). 3.2. Induction of CYP1A forms in cultured rat liver and lung slices CYP1A form induction was monitored by measurement of 7-ethoxyresorufin O-deethylase activity in rat liver and lung slice whole homogenates. To serve as positive controls for induction of CYP1A forms, rat liver and lung slices were cultured for 48 h with 0 and 10 mg/ml Aroclor 1254 and 0, 2 and 20 mM benzo(a)pyrene. The treatment of rat liver (Fig. 2A) and lung (Fig. 2B) slices with 10 mg/ml ARO for 48 h markedly induced tissue slice whole homogenate 7-ethoxyresorufin Odeethylase activity. Enzyme activity was also markedly induced in liver (Fig. 2C) and lung (Fig. 2D) slices by treatment with 2 and 20 mm benzo(a)pyrene. While the
induction of 7-ethoxyresorufin O-deethylase activity was more marked in rat liver slices treated with 2 mm than with 20 mm benzo(a)pyrene, enzyme induction in lung slices was somewhat more marked at the higher concentration of benzo(a)pyrene examined. No studies were undertaken to ascertain if high concentrations of benzo(a)pyrene were cytotoxic to cultured rat liver slices. The treatment of rat liver slices for 48 h with 5–200 mm nicotine resulted in a significant reduction in whole homogenate 7-ethoxyresorufin O-deethylase activity (Fig. 3A). In contrast, enzyme activity in rat lung slices was significantly induced to 370% of control by treatment with 50 mm nicotine (Fig. 3B). The treatment of liver (Fig. 3C) and lung (Fig. 3D) slices with 2–200 mm cotinine for 48 h had no statistically significant effect on 7-ethoxyresorufin O-deethylase activity.
4. Discussion Many studies have utilised precision-cut tissue slices to evaluate species differences in xenobiotic metabolism and xenobiotic-induced toxicity (Parrish et al., 1995; Bach et al., 1996; Lerche-Langrand and Toutain, 2000). Another potential application of precision-cut tissue
Table 1 Kinetics of nicotine metabolism to cotinine by precision-cut rat liver and lung slices Tissue
Liver Lung a b c d
Nicotine metabolism kineticsa Km (mM)b
Vmax (pmol/min/mg protein)b
CLint (ml/min/mg protein)c
CLint (ml/min/organ weight per kg body weight)d
323 14 41.7 15.9
47.23.2 3.210.46
0.1460.007 0.1180.054
1.030.05 0.0930.043
Results are presented as mean S.E.M. of three experiments. Km and Vmax values calculated from Michaelis–Menten and Eadie–Hofstee plots. Intrinsic clearance (CLint) values calculated as Vmax/Km. Scaled CLint values allowing for liver and lung total protein content and relative organ weight.
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Fig. 2. Rat liver (A,C) and lung (B,D) slices were cultured for 48 h in medium containing either 0 (control, DMSO only) and 10 mg/ml Aroclor 1254 (A,B) or 0 (control), 2 and 20 mm benzo(a)pyrene (C,D). 7-Ethoxyresorufin O-deethylase activity was determined in tissue slice whole homogenates. Results are presented as meanS.E.M. of 3–4 experiments. Values significantly different from control (DMSO only treated tissue slices) are: **P<0.01; ***P<0.001.
slices is their use to evaluate organ differences in the metabolism and toxic and other effects of xenobiotics. For example, rodent and human liver, kidney, lung and intestine slices have used for comparative studies on the metabolism of cyclosporin A and derivatives, 7-ethoxycoumarin, 7-hydroxycoumarin, lidocaine and testosterone (Vickers et al., 1992; 1997; Price et al., 1995a; de Kanter et al., 1999, 2002). The aim of this study was to evaluate the usefulness of precision-cut tissue slices to evaluate organ differences in xenobiotic metabolism and CYP form induction. For these investigations rat liver and lung slices were used and nicotine was selected as the model compound. While the liver is considered to be the major site of nicotine biotransformation, metabolism also occurs in the lung (Mattammal et al., 1987; Shigenaga et al., 1988; Kyerematen and Vesell, 1991; Foth, 1995). Nicotine can be metabolised by various pathways, of which cotinine is the primary product of the C-oxidation pathway of nicotine biotransformation (Kyerematen and Vesell, 1991). A number of CYP forms can metabolise nicotine 0 0 to a nicotine 1 (5 )-iminium ion which is subsequently
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Fig. 3. Rat liver (A,C) and lung (B,D) slices were cultured for 48 h in medium containing either 0 (control, DMSO only) and 2–200 mm nicotine (A,B) or medium containing 0 (control) and 2–200 mm cotinine (C,D). 7-Ethoxyresorufin O-deethylase activity was determined in tissue slice whole homogenates. Results are presented as meanS.E.M. of 3 experiments. Values significantly different from control (DMSO only treated slices) are: *P< 0.05; **P <0.01.
converted to cotinine by cytosolic aldehyde oxidase (McCoy et al., 1989; Hammond et al., 1991; Kyerematen and Vesell, 1991; Nakayama et al., 1993; Foth, 1995). One hepatic CYP form involved in this pathway is CYP2B1, which is also constitutively expressed in rat lung (Hammond et al., 1991; Guengerich, 1993; Nakayama et al., 1993; Foth, 1995; Nims and Lubet, 1996). Nicotine, but not cotinine, has also been shown to induce CYP1A forms in rat liver and lung, the effect being more marked in the lung than in the liver (Iba et al., 1998, 1999). In agreement with previous studies using perfused organs and subcellular fractions, nicotine was metabolised to cotinine by freshly cut rat liver and lung slices. The observed Km values for cotinine formation were lower in rat lung than in liver slices. This may suggest that different enzymes or different ratios of enzymes (e.g. CYP forms) are responsible for nicotine metabolism in these two tissues. When scaled for total organ protein content and for relative organ weight, CLint values were greater in rat liver than in lung slices. Although liver slices are a valuable liver intact cell model system for studying pathways of xenobiotic
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metabolism, they have been reported to be less useful than hepatocytes for obtaining estimates of CLint due to problems of substrate uptake into all cells of a liver slice (Worboys et al., 1995, 1997; Houston and Carlile, 1997). However, recent studies with tolbutamide, a low clearance compound, demonstrated that rat liver slice data could accurately predict the in vivo intrinsic clearance of this compound (Haenen et al., 2002). In addition, the sum of rates of 7-ethoxycoumarin metabolism by rat liver, kidney, lung and small intestine slices, was similar to the observed rate of in vivo metabolism of this compound (de Kanter et al., 2002). Overall, there appears to be scope for further studies to evaluate the usefulness of precision-cut tissue slices to provide data on tissue differences in rates of xenobiotic metabolism. Studies conducted in a number of laboratories have demonstrated that cultured precision-cut rat liver slices may be utilised for CYP form induction studies (Lake et al., 1993, 1998; Lerche-Langrand and Toutain, 2000). In addition, studies conducted in our laboratory have demonstrated the induction of CYP1A1 by Aroclor 1254 and other inducing agents in cultured precision-cut rat lung slices, as determined by increases in 7-ethoxyresorufin O-deethylase activity and CYP1A1 mRNA and apoprotein levels (Lake et al., 2001, 2003). In the present study, the treatment of rat lung slices with nicotine was found to result in the induction of CYP1Adependent 7-ethoxyresorufin O-deethylase activity. In contrast, nicotine did not induce enzyme activity in cultured liver slices and cotinine did not induce enzyme activity in either liver or lung slices. The functional viability of the rat liver and lung slice preparations used for these studies was demonstrated by the marked induction in both tissues of 7-ethoxyresorufin O-deethylase activity by Aroclor 1254 and benzo(a)pyrene. Both Aroclor 1254 and benzo(a)pyrene are known to induce CYP1A forms in rat liver and lung (Conney, 1986; Okey, 1990). Overall, these results are in agreement with previous in vivo studies where nicotine has been shown to produce a greater induction of CYP1A-dependent activity in rat lung than in liver (Iba et al., 1998, 1999). In summary, nicotine was metabolised to cotinine by rat liver and lung slices and also induced CYP1Adependent enzyme activity in culture lung slices. These results show the usefulness of the precision-cut tissue slice technique for studying tissue differences in xenobiotic metabolism and CYP form induction.
Acknowledgements We are grateful to the UK Food Standards Agency (contract number T01011) and to the European Union (EU BIOTECH Contract Number BI04-CT97-2145) for financial support of these studies.
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Price, R.J., Renwick, A.B., Barton, P.T., Houston, J.B., Lake, B.G., 1998. Influence of slice thickness and culture conditions on the metabolism of 7-ethoxycoumarin in precision-cut rat liver slices. ATLA 26, 541–548. Price, R.J., Renwick, A.B., Beamand, J.A., Esclangon, F., Wield, P.T., Walters, D.G., Lake, B.G., 1995a. Comparison of the metabolism of 7-ethoxycoumarin and coumarin in precision-cut rat liver and lung slices. Food and Chemical Toxicology 33, 233–237. Price, R.J., Renwick, A.B., Wield, P.T., Beamand, J.A., Lake, B.G., 1995b. Toxicity of 3-methylindole, 1-nitronaphthalene and paraquat in precision-cut rat lung slices. Archives of Toxicology 69, 405–409. Shigenaga, M.K., Trevor, A.J., Castagnoli Jr., N., 1988. Metabolismdependent covalent binding of (S)-[5-3H]nicotine to liver and lung microsomal macromolecules. Drug Metabolism and Disposition 16, 397–402. Stefaniak, M.S., Krumdieck, C.L., Spall, R.D., Gandolfi, A.J., Brendel, K., 1992. Biochemical and histological characterization of agar-filled precision-cut rat lung slices in dynamic organ culture as an in vitro tool. In vitro Toxicology 5, 7–19. Vickers, A.E.M., Fischer, V., Connors, S., Fisher, R.L., Baldeck, J.P., Maurer, G., Brendel, K., 1992. Cyclosporin A metabolism in human liver, kidney, and intestine slices. Comparison to rat and dog slices and human cell lines. Drug Metabolism and Disposition 20, 802–809. Vickers, A.E.M., Jimenez, R.M., Spaans, M.C., Pflimlin, V., Fisher, R.L., Brendel, K., 1997. Human and rat lung biotransformation of cyclosporin A and its derivatives using slices and bronchial epithelial cells. Drug Metabolism and Disposition 25, 873–880. Worboys, P.D., Bradbury, A., Houston, J.B., 1995. Kinetics of drug metabolism in rat liver slices. Rates of oxidation of ethoxycoumarin and tolbutamide, examples of high- and low-clearance compounds. Drug Metabolism and Disposition 23, 393–397. Worboys, P.D., Bradbury, A., Houston, J.B., 1997. Kinetics of drug metabolism in rat liver slices. III. Relationship between metabolic clearance and slice uptake rate. Drug Metabolism and Disposition 25, 460–467.
Metabolism of nicotine and induction of CYP1A forms in precision-cut rat liver and lung slices Roger J. Price, Anthony B. Renwick, David G. Walters, Philip J. Young, Brian G. Lake* BIBRA International Ltd., Woodmansterne Road, Carshalton, Surrey SM5 4DS, UK and Centre for Toxicology, School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK Received 30 September 2002
Abstract The aim of this study was to investigate xenobiotic metabolism and induction of cytochrome P450 (CYP) forms in precision-cut rat liver and lung slices, employing nicotine as a model compound. Freshly cut rat liver and lung slices metabolised nicotine to the major metabolite cotinine. Observed Km values for cotinine formation in liver and lung slices were 323 and 41.7 mM, respectively, with corresponding Vmax values of 47.2 and 3.21 pmol/min/mg protein, respectively. Rat liver and lung slices were cultured for 48 h with Aroclor 1254, benzo(a)pyrene, nicotine and cotinine. Both Aroclor 1254 and benzo(a)pyrene produced a marked induction of CYP1A-dependent 7-ethoxyresorufin O-deethylase activity in both liver and lung slices. However, while nicotine induced 7-ethoxyresorufin O-deethylase activity in lung slices, but not in liver slices, cotinine did not induce enzyme activity in either liver or lung slices. Overall, while higher rates of nicotine metabolism were observed in rat liver slices, nicotine-induced CYP1A form induction was observed in lung slices. These results demonstrate the usefulness of precision-cut tissue slices for studying tissue differences in xenobiotic metabolism and CYP form induction. # 2003 Elsevier Ltd. All rights reserved. Keywords: Precision-cut tissue slices; Nicotine metabolism; Cytochrome P450; CYP1A form induction
1. Introduction In recent years much effort has been devoted to the development of in vitro systems to study the metabolism and toxicity of xenobiotics. One potentially very valuable in vitro model system is the precision-cut tissue slice technique, which was originally developed at the University of Arizona (Krumdieck et al., 1980; Parrish et al., 1995). This technique has been applied to various tissues including the liver, kidney lung, heart and spleen (Parrish et al., 1995; Bach et al., 1996; de Kanter et al., 1999, 2002). For the lung, an agarose embedding procedure Abbreviations: CLint, intrinsic clearance; CYP, cytochrome P450; DMSO, dimethyl sulphoxide; EBSS, Earle’s balanced salt solution; Km, the concentration of substrate giving half maximal velocity; LC-MS-MS, liquid chromatography-mass spectrometry-mass spectrometry; MEMa, Minimal Essential Medium Alpha; MRM, multiple reaction monitoring; Vmax, the maximal velocity. * Corresponding author: Tel.: +44-20-8652-1006; fax: +44-208661-7029. E-mail address: [email protected] (B.G. Lake). 0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2003.08.012
has been developed which provides a solid support for this tissue, thus permitting the preparation and culture of precision-cut lung slices (Stefaniak et al., 1992; Parrish et al., 1995). Major advantages of the tissue slice technique include the maintenance of tissue architecture, so that all cell types are present and the ready application of the technique to different tissues and species (Parrish et al., 1995; Bach et al., 1996; de Kanter et al., 1999; Lerche-Langrand and Toutain, 2000). Precision-cut liver slices have been extensively used for studies of xenobiotic metabolism and toxicity and to investigate the induction of cytochrome P450 (CYP) forms (Lake et al., 1993, 1998; Parrish et al., 1995; Bach et al., 1996; Lerche-Langrand and Toutain, 2000). Compared to liver slices, fewer studies on the metabolism and toxicity of xenobiotics have been performed with lung slices (Parrish et al., 1995; Bach et al., 1996). Recent studies in our laboratory have demonstrated that cultured rat lung slices can be employed to study the induction of CYP1A1 forms (Lake et al., 2001, 2003).
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The aim of this study was to evaluate the usefulness of the tissue slice technique for assessing organ differences in xenobiotic metabolism and CYP form induction. Nicotine was selected as a model compound for these studies as other investigations have demonstrated tissue differences in both nicotine metabolism and effects on xenobiotic metabolising enzymes. Apart from tobacco, nicotine is present in certain foodstuffs including tomatoes, potatoes, cauliflower and certain teas (Davis et al., 1991). Nicotine is metabolised by various pathways, of which cotinine is the primary product of the C-oxidation pathway of nicotine biotransformation (Kyerematen and Vesell, 1991). While the liver is considered to be the major site of nicotine biotransformation, metabolism also occurs in the lung and kidney (Shigenaga et al., 1988; Kyerematen and Vesell, 1991; Foth, 1995). Nicotine is 0 0 metabolised by CYP forms to a nicotine 1 (5 )-iminium ion which is converted to cotinine by a cytosolic aldehyde oxidase enzyme (McCoy et al., 1989; Hammond et al., 1991; Kyerematen and Vesell, 1991; Nakayama et al., 1993; Foth, 1995). In rat liver nicotine is metabolised by CYP1A2, CYP2B1, CYP2C11 and other CYP forms; CYP2B1 also being constitutively expressed in rat lung (Hammond et al., 1991; Guengerich, 1993; Nakayama et al., 1993; Foth, 1995; Nims and Lubet, 1996). Nicotine metabolism by the lung has been demonstrated in studies with both perfused organs and subcellular fractions (Mattammal et al., 1987; Shigenaga et al., 1988; Kyerematen and Vesell, 1991; Foth, 1995). In other studies the effect of nicotine on rat hepatic, renal and pulmonary CYP1A forms has been investigated. Following a single subcutaneous dose nicotine was shown to induce pulmonary 7-ethoxyresorufin O-deethylase activity and levels of CYP1A1 apoprotein (Iba et al., 1998). In contrast to nicotine, administration of either subcutaneous or intraperitoneal doses of cotinine did not induce enzyme activity or CYP1A1 apoprotein levels in either the liver, kidney or lung (Iba et al., 1998). The effect of nicotine administration for 30 days in a nutritionally balanced liquid diet has also been investigated (Iba et al., 1999). Nicotine treatment induced 7-ethoxyresorufin O-deethylase activity in all three tissues, the effect being most marked in the lung. Treatment with nicotine also resulted in a dose-dependent increase in CYP1A1 apoprotein in the lung, whereas in the liver CYP1A1 was induced only at the highest dose level examined (Iba et al., 1999). In the present study, the metabolism of nicotine to cotinine has been studied in freshly prepared precisioncut rat liver and lung slices. Cultured rat liver and lung slices were used to study the effects of nicotine and cotinine on hepatic and pulmonary CYP1A forms. To serve as positive controls for induction of CYP forms, liver and lung slices were also treated with two known inducers of xenobiotic metabolism (Conney, 1986;
Okey, 1990), namely the polychlorinated biphenyl mixture Aroclor 1254 and benzo(a)pyrene.
2. Materials and methods 2.1. Materials All tissue culture media were obtained from Gibco BRL (Life Technologies Ltd., Paisley, Scotland). The sources of the other tissue culture materials were as described previously (Beamand et al., 1993). Nicotine, cotinine, cotinine-d3,7-ethoxyresorufin, resorufin, benzo(a)pyrene, enzyme cofactors and buffers were obtained from Sigma-Aldrich Company Ltd (Poole, Dorset, UK). Aroclor 1254 was the generous gift of the Monsanto Chemical Company (St Louis, MO, USA). 2.2. Animals Male Sprague–Dawley rats were obtained from Harlan Olac (Bicester, Oxon, UK) and were allowed free access to R and M No.1 laboratory animal diet (Special Diets Services, Witham, Essex, UK) and water. The animals were housed in mesh-floored cages in accommodation maintained at 22 3 C with a relative humidity of 40–70% and allowed to acclimatise to these conditions for at least 7 days before use. Rats (10–14 weeks old) were killed by exsanguination under sodium pentobarbitone anaesthesia (60 mg/kg, ip). The livers were perfused in situ, prior to excision, with 20 ml of icecold oxygenated (95% O2/5% CO2) Earle’s balanced salt solution (EBSS) containing 25 mm d-glucose, 50 mg/ml gentamicin and 2.5 mg/ml fungizone (Price et al., 1998). After excision the livers and lungs were placed into icecold oxygenated EBSS containing the above additions. 2.3. Preparation of precision-cut liver and lung slices Tissue cylinders were prepared from liver samples with a 10 mm diameter motor-driven tissue coring tool. The trachea was cannulated and the lungs instilled at 37 C with a 0.75% (w/v) agarose solution under a constant hydrostatic pressure of 20 cm as described previously (Placke and Fisher, 1987; Price et al., 1995a,b; Stefaniak et al., 1992). After allowing the agarose to gel at 4 C, tissue cylinders were prepared with an 8 mm motor-driven tissue coring tool. From the tissue cylinders liver slices (around 250 mm thickness) and lung slices (around 500 mm thickness) were prepared in cold oxygenated EBSS containing 25 mm d-glucose, 50 mg/ml gentamicin and 2.5 mg/ml fungizone using a Krumdieck tissue slicer (Alabama Research and Development Corporation, Munford, AL, USA). Tissue slice thickness was determined as described previously (Price et al., 1998).
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2.4. Culture of liver and lung slices Tissue slices were floated onto Vitron (Vitron Inc., Tucson, AZ, USA) type C titanium roller inserts (two slices per insert) and cultured in glass vials at 37 C containing 1.7 ml culture medium employing a Vitron dynamic organ culture incubator. Liver slices were cultured in Williams’ medium E containing 2 mm l-glutamine, 0.1 mm insulin, 0.1 mm dexamethasone, 50 mg/ml gentamicin and 2.5 mg/ml fungizone under an atmosphere of 95% O2/5% CO2, whereas lung slices were cultured in MEMa medium, containing all the above additions except l-glutamine, under an atmosphere of 95% air/5% CO2. After 1 h, the medium was replaced with fresh medium and subsequently the medium was changed every 24 h. The test compounds were dissolved in dimethyl sulphoxide (DMSO) and added to the culture medium so that the final DMSO concentration was 0.4% (v/v) in all vials including the control (no test compound) cultures. 2.5. Biochemical investigations After incubation, liver and lung slices were washed in 0.154 M KCl containing 50 mM Tris–HCl pH 7.4 and then homogenised (2 slices in 2 ml) in this medium by sonication (Beamand et al., 1993). Liver and lung slice homogenates were stored at 80 C prior to analysis. 7-Ethoxyresorufin O-deethylase activity was determined at 37 C in spectrofluorimeter cuvettes containing 2 mm substrate, 8 mm dicumarol, 250 mm NADPH, tissue slice homogenate (liver 0.2 ml, lung 1.2 ml) and 50 mm TrisHCl buffer pH 8.4 in a final volume of 2 ml. Resorufin formation was monitored with time at wavelengths of 535 nm excitation and 582 nm emission. Protein content was determined by the method of Lowry et al. (1951) employing bovine serum albumin as standard.
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Hitchin, Herts, UK) attached to an HP1100 series HPLC (Hewlett-Packard Ltd., Bracknell, Berkshire, UK) were used to introduce samples into a Micromass Quattro LC Triple Quadrupole Mass Spectrometer (Micromass UK Ltd., Altrincham, Cheshire, UK) using an Electrospray Z-spray1 source. Chromatography of 50 ml aliquots of the extracts was performed with a 1003 mm Hypersil-3 BDS C18 column (ThermoHypersil-Keystone, Runcorn, Cheshire, UK) with a 102 mm guard cartridge of the same material and isocratic elution with a mobile phase of acetonitrile, methanol and 10 mm ammonium acetate (50:30:20 v/v/ v) at a flow rate of 0.75 ml/min and column temperature of 35 C. The eluate was passed directly into the triple quadrupole mass spectrometer operating in positive atmospheric pressure chemical ionisation mode with probe and source block temperatures of 400 C and 100 C, respectively, using nitrogen for desolvation and nebulisation and a corona voltage of 3.8 kV to initiate ionisation. Cotinine and cotinine-d3 (retention times 0.76 min) were selectively detected by multiple reaction monitoring (MRM) using argon and quantitation was carried out at five levels against triplicate aqueous authentic standards of cotinine using cotinine-d3 as internal standard. MRM function (parent and daughter ions) were 177.08!79.91 and 180.10!79.91 for cotinine and cotinine-d3, respectively. Cone voltage and collision energy were 32 and 20 V, respectively, for cotinine and 35 and 23 V, respectively, for cotinine-d3. 2.7. Statistical analysis Statistical analysis of data was performed by one-way analysis of variance. Comparisons between means were made use the least significant difference test.
3. Results 2.6. Metabolism of nicotine 3.1. Metabolism of nicotine in rat liver and lung slices Freshly cut rat liver and lung slices were incubated in phenol red free RPMI 1640 medium containing 0–1000 mm nicotine for periods of 30 and 180 min, respectively. At the end of the incubations the tissue slices were homogenised (Beamand et al., 1993) by sonication in the culture medium and the tissue slice/medium homogenates stored at 80 C prior to analysis by liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS). Tissue slice homogenates (1.0 ml) were treated with 0.025 ml of 0.880 g/ml ammonia and 0.025 ml of 600 ng/ml cotinine-d3 (internal standard) and extracted with 8 ml of dichloromethane for 30 min. After centrifugation at 1700 g for 10 min at 4 C the organic phase was evaporated to dryness and reconstituted in 300 ml liquid chromatography mobile phase (see below). An HTS PAL autosampler (Presearch Ltd.,
Freshly cut rat liver and lung slices were cultured in medium containing 0–1000 mm nicotine for periods of 30 and 180 min, respectively. Preliminary experiments established that cotinine formation was linear with respect to time in both liver and lung slices over these incubation periods (data not shown). At the end of the incubations, the liver and lung slices were harvested and homogenised by sonication in the culture medium. Levels of cotinine were determined by LC-MS-MS assay of the tissue slice/medium homogenates. Nicotine was metabolised to cotinine by both rat liver (Fig. 1A) and lung (Fig. 1B) slices. Kinetic analysis of rates of cotinine formation in rat liver and lung slices was performed by both Michaelis-Menten (Fig. 1) and Eadie–Hofstee plots. Calculated Km and Vmax values for
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Fig. 1. Freshly cut rat liver (A) and lung (B) slices were incubated with 0–1000 mM nicotine for 30 and 180 min, respectively. Levels of cotinine formation in tissue slice/medium homogenates were determined by LC-MS-MS. Results are presented as meanS.E.M. of 3 experiments.
cotinine formation were lower in rat lung than in rat liver slices (Table 1). Intrinsic clearance (CLint, i.e. Vmax/Km) values for cotinine formation were also calculated. When expressed per unit of tissue slice homogenate protein (i.e. in units of ml/min/mg protein), CLint values were similar in rat liver and lung slices (Table 1). However, when scaled for total organ protein content and for relative organ weight, CLint values were eleven times greater in rat liver than in lung slices (Table 1). 3.2. Induction of CYP1A forms in cultured rat liver and lung slices CYP1A form induction was monitored by measurement of 7-ethoxyresorufin O-deethylase activity in rat liver and lung slice whole homogenates. To serve as positive controls for induction of CYP1A forms, rat liver and lung slices were cultured for 48 h with 0 and 10 mg/ml Aroclor 1254 and 0, 2 and 20 mM benzo(a)pyrene. The treatment of rat liver (Fig. 2A) and lung (Fig. 2B) slices with 10 mg/ml ARO for 48 h markedly induced tissue slice whole homogenate 7-ethoxyresorufin Odeethylase activity. Enzyme activity was also markedly induced in liver (Fig. 2C) and lung (Fig. 2D) slices by treatment with 2 and 20 mm benzo(a)pyrene. While the
induction of 7-ethoxyresorufin O-deethylase activity was more marked in rat liver slices treated with 2 mm than with 20 mm benzo(a)pyrene, enzyme induction in lung slices was somewhat more marked at the higher concentration of benzo(a)pyrene examined. No studies were undertaken to ascertain if high concentrations of benzo(a)pyrene were cytotoxic to cultured rat liver slices. The treatment of rat liver slices for 48 h with 5–200 mm nicotine resulted in a significant reduction in whole homogenate 7-ethoxyresorufin O-deethylase activity (Fig. 3A). In contrast, enzyme activity in rat lung slices was significantly induced to 370% of control by treatment with 50 mm nicotine (Fig. 3B). The treatment of liver (Fig. 3C) and lung (Fig. 3D) slices with 2–200 mm cotinine for 48 h had no statistically significant effect on 7-ethoxyresorufin O-deethylase activity.
4. Discussion Many studies have utilised precision-cut tissue slices to evaluate species differences in xenobiotic metabolism and xenobiotic-induced toxicity (Parrish et al., 1995; Bach et al., 1996; Lerche-Langrand and Toutain, 2000). Another potential application of precision-cut tissue
Table 1 Kinetics of nicotine metabolism to cotinine by precision-cut rat liver and lung slices Tissue
Liver Lung a b c d
Nicotine metabolism kineticsa Km (mM)b
Vmax (pmol/min/mg protein)b
CLint (ml/min/mg protein)c
CLint (ml/min/organ weight per kg body weight)d
323 14 41.7 15.9
47.23.2 3.210.46
0.1460.007 0.1180.054
1.030.05 0.0930.043
Results are presented as mean S.E.M. of three experiments. Km and Vmax values calculated from Michaelis–Menten and Eadie–Hofstee plots. Intrinsic clearance (CLint) values calculated as Vmax/Km. Scaled CLint values allowing for liver and lung total protein content and relative organ weight.
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Fig. 2. Rat liver (A,C) and lung (B,D) slices were cultured for 48 h in medium containing either 0 (control, DMSO only) and 10 mg/ml Aroclor 1254 (A,B) or 0 (control), 2 and 20 mm benzo(a)pyrene (C,D). 7-Ethoxyresorufin O-deethylase activity was determined in tissue slice whole homogenates. Results are presented as meanS.E.M. of 3–4 experiments. Values significantly different from control (DMSO only treated tissue slices) are: **P<0.01; ***P<0.001.
slices is their use to evaluate organ differences in the metabolism and toxic and other effects of xenobiotics. For example, rodent and human liver, kidney, lung and intestine slices have used for comparative studies on the metabolism of cyclosporin A and derivatives, 7-ethoxycoumarin, 7-hydroxycoumarin, lidocaine and testosterone (Vickers et al., 1992; 1997; Price et al., 1995a; de Kanter et al., 1999, 2002). The aim of this study was to evaluate the usefulness of precision-cut tissue slices to evaluate organ differences in xenobiotic metabolism and CYP form induction. For these investigations rat liver and lung slices were used and nicotine was selected as the model compound. While the liver is considered to be the major site of nicotine biotransformation, metabolism also occurs in the lung (Mattammal et al., 1987; Shigenaga et al., 1988; Kyerematen and Vesell, 1991; Foth, 1995). Nicotine can be metabolised by various pathways, of which cotinine is the primary product of the C-oxidation pathway of nicotine biotransformation (Kyerematen and Vesell, 1991). A number of CYP forms can metabolise nicotine 0 0 to a nicotine 1 (5 )-iminium ion which is subsequently
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Fig. 3. Rat liver (A,C) and lung (B,D) slices were cultured for 48 h in medium containing either 0 (control, DMSO only) and 2–200 mm nicotine (A,B) or medium containing 0 (control) and 2–200 mm cotinine (C,D). 7-Ethoxyresorufin O-deethylase activity was determined in tissue slice whole homogenates. Results are presented as meanS.E.M. of 3 experiments. Values significantly different from control (DMSO only treated slices) are: *P< 0.05; **P <0.01.
converted to cotinine by cytosolic aldehyde oxidase (McCoy et al., 1989; Hammond et al., 1991; Kyerematen and Vesell, 1991; Nakayama et al., 1993; Foth, 1995). One hepatic CYP form involved in this pathway is CYP2B1, which is also constitutively expressed in rat lung (Hammond et al., 1991; Guengerich, 1993; Nakayama et al., 1993; Foth, 1995; Nims and Lubet, 1996). Nicotine, but not cotinine, has also been shown to induce CYP1A forms in rat liver and lung, the effect being more marked in the lung than in the liver (Iba et al., 1998, 1999). In agreement with previous studies using perfused organs and subcellular fractions, nicotine was metabolised to cotinine by freshly cut rat liver and lung slices. The observed Km values for cotinine formation were lower in rat lung than in liver slices. This may suggest that different enzymes or different ratios of enzymes (e.g. CYP forms) are responsible for nicotine metabolism in these two tissues. When scaled for total organ protein content and for relative organ weight, CLint values were greater in rat liver than in lung slices. Although liver slices are a valuable liver intact cell model system for studying pathways of xenobiotic
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metabolism, they have been reported to be less useful than hepatocytes for obtaining estimates of CLint due to problems of substrate uptake into all cells of a liver slice (Worboys et al., 1995, 1997; Houston and Carlile, 1997). However, recent studies with tolbutamide, a low clearance compound, demonstrated that rat liver slice data could accurately predict the in vivo intrinsic clearance of this compound (Haenen et al., 2002). In addition, the sum of rates of 7-ethoxycoumarin metabolism by rat liver, kidney, lung and small intestine slices, was similar to the observed rate of in vivo metabolism of this compound (de Kanter et al., 2002). Overall, there appears to be scope for further studies to evaluate the usefulness of precision-cut tissue slices to provide data on tissue differences in rates of xenobiotic metabolism. Studies conducted in a number of laboratories have demonstrated that cultured precision-cut rat liver slices may be utilised for CYP form induction studies (Lake et al., 1993, 1998; Lerche-Langrand and Toutain, 2000). In addition, studies conducted in our laboratory have demonstrated the induction of CYP1A1 by Aroclor 1254 and other inducing agents in cultured precision-cut rat lung slices, as determined by increases in 7-ethoxyresorufin O-deethylase activity and CYP1A1 mRNA and apoprotein levels (Lake et al., 2001, 2003). In the present study, the treatment of rat lung slices with nicotine was found to result in the induction of CYP1Adependent 7-ethoxyresorufin O-deethylase activity. In contrast, nicotine did not induce enzyme activity in cultured liver slices and cotinine did not induce enzyme activity in either liver or lung slices. The functional viability of the rat liver and lung slice preparations used for these studies was demonstrated by the marked induction in both tissues of 7-ethoxyresorufin O-deethylase activity by Aroclor 1254 and benzo(a)pyrene. Both Aroclor 1254 and benzo(a)pyrene are known to induce CYP1A forms in rat liver and lung (Conney, 1986; Okey, 1990). Overall, these results are in agreement with previous in vivo studies where nicotine has been shown to produce a greater induction of CYP1A-dependent activity in rat lung than in liver (Iba et al., 1998, 1999). In summary, nicotine was metabolised to cotinine by rat liver and lung slices and also induced CYP1Adependent enzyme activity in culture lung slices. These results show the usefulness of the precision-cut tissue slice technique for studying tissue differences in xenobiotic metabolism and CYP form induction.
Acknowledgements We are grateful to the UK Food Standards Agency (contract number T01011) and to the European Union (EU BIOTECH Contract Number BI04-CT97-2145) for financial support of these studies.
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