Rat oesophageal cytochrome P450 (CYP) - Oxford Academic

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Carcinogenesis vol.22 no.11 pp.1877–1883, 2001

Rat oesophageal cytochrome P450 (CYP) monooxygenase system: comparison to the liver and relevance in N-nitrosodiethylamine carcinogenesis

Luis F.Ribeiro Pinto2, Emanuela Moraes, Rodolpho M.Albano, Maria C.Silva, Wagner Godoy, Tina Glisovic1 and Matti A.Lang1

extent, be due to the tissue specific expression of the activating enzymes.

Departamento de Bioquı´mica, IBRAG, Universidade do Estado do Rio de Janeiro, Av. 28 de Setembro, 87, fundos, 4° andar, Vila Isabel, Rio de Janeiro, RJ, Brasil and 1Department of Pharmaceutical Biosciences, Division of Biochemistry, Uppsala University, Uppsala, Sweden

Introduction

2To

whom correspondence should be addressed Email: [email protected]

N-nitrosodiethylamine (NDEA) is able to induce tumours in the rat oesophagus. It has been suggested that this could be due to tissue specific expression of NDEA activating cytochrome P450 enzymes. We investigated this by characterizing the oesophageal monooxygenase complex of male Wistar rats and comparing it with that of the liver. Total amount of cytochrome P450, NADPH P450 reductase, cytochrome b5 and cytochrome b5 reductase of the oesophageal mucosa was ~7% of what was found in the liver. In addition, major differences were found in the cytochrome P450 isoenzyme composition between these organs: CYP 2B1/2B2 and CYP3A were found only in the liver, whereas CYP1A1 was constitutively expressed only in the oesophagus. Of the two well-known nitrosamine metabolizing enzymes, CYP2A3 was found only in the oesophagus whereas CYP2E1 was exclusively expressed in the liver. Catalytic studies, western blotting and RT– PCR analyses confirmed the expression of CYP2A3 in the oesophagus. CYP2A enzymes are known to be good catalysts of NDEA metabolism. Oesophageal microsomes had a Km for NDEA metabolism, which was about onethird of that of hepatic microsomes, but they showed similar activities when compared per nmol of total P450. NDEA activity in the oesophagus was significantly increased by coumarin (CO), which also induced oesophageal CYP2A3. Immunoinhibition of the microsomal NDEA activity showed that up to 70% of this reaction is catalysed by CYP2A3 in the oesophagus, whereas no inhibition of the hepatic NDEA activity could be achieved by the antiCYP2A5 antibody. NDEA, but not N-nitrosodimethylamine (NDMA) inhibited the oesophageal metabolism of CO. The results of the present investigation show major differences in the enzyme composition of the oesophageal and hepatic monooxygenase complexes, and are in accordance with the hypothesis that the NDEA organotropism could, to a large Abbreviations: CO, coumarin; COH, coumarin-7-hydroxylase; CYP, cytochrome P450; ECOD, 7-ethoxycoumarin-o-deethylase; EROD, 7-ethoxyresorufin-o-deethylase; 3-MC, 3-methylcholanthrene; NDEA, N-nitrosodiethylamine; NDMA, N-nitrosodimethylamine; NMAA, N-nitrosomethylamylamine; NMBA, N-nitrosomethylbutylamine; NMBzA, N-nitrosomethylbenzylamine; NNN, N-nitrosonornicotine; PB, phenobarbital; PROD, 7pentoxyresorufin o-depenthylase; PY, pyrazole; SDS–PAGE; sodium dodecylsulphate polyacrylamide gel electrophoresis; RT–PCR, reverse transcription– polymerase chain reaction. © Oxford University Press

Nitrosamines are versatile carcinogens, being able to induce tumours in the oesophagus of experimental animals (1). In the rat, the oesophagus is the main target for tumour induction by nitrosamines (1). As these carcinogens need to be metabolically activated by cytochrome P450 (CYP) enzymes to exert their carcinogenic effects, this organotropism suggests that the oesophagus express CYP enzymes that efficiently metabolize nitrosamines. In Wistar rats, a commonly used experimental model for oesophageal carcinogenesis, N-nitrosodiethylamine (NDEA) induces tumours at low doses. When higher doses are given for a shorter period, tumours originate mainly in the liver (1). Reasons for such interorgan differences are currently unknown but it has been hypothesized that this could be due to different enzymes being responsible for the metabolic activation of NDEA in the two organs (2). Different kinetics of hepatic and oesophageal NDEA metabolism have, indeed, been demonstrated (2,3), suggesting that the enzymes responsible for its metabolism in the two organs could be different. However, only the hepatic NDEA metabolising enzyme, CYP2E1, has been identified (4). In order to confirm this hypothesis and to learn about the possible reasons for NDEA organotropism, it would be important to identify the enzyme(s) that metabolize NDEA in the oesophagus. Ethanol is a substrate of CYP2E1 (5) and a major aetiological factor of oesophageal carcinogenesis (6). When coadministered with NDEA it changes the distribution and metabolism of this nitrosamine in vivo (2,3) and promotes oesophageal but not hepatic cancer (7). This result is in accordance with the inhibition of hepatic but not oesophageal metabolism of NDEA by ethanol, and further suggests that the enzymes responsible for NDEA metabolism in the two organs are different. Additionally, rat liver presents a very low coumarin 7hydroxylase (COH), a reaction catalysed exclusively by some CYP2A enzymes, such as mouse CYP2A5 and human CYP2A6 (8). These enzymes are also known to be efficient catalysts of NDEA metabolism (9). The purpose of this work was to compare the isoenzyme composition of the microsomal monooxygenase complexes of rat liver and oesophagus and to identify the key enzymes responsible for oesophageal NDEA metabolism. The results show major differences in the isoenzyme composition between the two organs and give evidence that CYP2A3, the rat orthologue of mouse CYP2A5 and human CYP2A6, rather than CYP2E1, is playing a central role in oesophageal NDEA metabolism. Our results confirm the previous hypothesis that different enzymes are responsible for NDEA metabolism in liver and oesophagus, and shed light on the biochemical 1877

L.F.Ribeiro Pinto et al.

mechanisms underlying the organotropism of NDEA carcinogenesis. Materials and methods Chemicals Unless stated otherwise all chemicals and enzymes were obtained from Sigma. [14C]N-nitrosodimethylamine (NDMA) (54 mCi/mmol, 98% purity) was synthesized and purified as described previously (10). Treatments and microsomal preparation Male Wistar rats (200 ⫾ 30 g) from FIOCRUZ Central Animal House breeding stock, were kept in 12 h light and dark cycle. Thirty rats were treated with phenobarbital (PB) according to Phillips et al. (11). Another thirty rats were treated with ethanol (ET) as described by Yang et al. (5), in order to induce the levels of CYP2E1 in the liver. A third group of thirty rats was treated with 3-methylcholanthrene (3-MC) as described by Rodrigues and Prough (12), in order to achieve maximum induction of CYP1A1 in the liver. A fourth and fifth group of thirty rats were treated with coumarin (CO) and pyrazole (PY), respectively, as described previously (13). After treatment, the rats were starved overnight, then killed by CO2 asphyxiation, and the liver and oesophagus were removed to ice-cold homogenizing buffer (50 mM Tris pH 7.4/0.15 M KCl), with the liver being immediately frozen at –70°C. The oesophageal mucosa was quickly stripped off and immediately frozen at –70°C (14). The preparation of hepatic and oesophageal microsomes was done based on a method described previously (14), with some modifications. The oesophageal mucosa was opened longitudinally, cut into small pieces at 0°C and homogenized in 5 vol. of homogenizing buffer with 3⫻ 10 passes using a motor driven Duall (Kontes, USA) glass homogenizer. After the initial centrifugation at 9000 g for 20 min, the supernatant was saved and the pellet was resuspended in 3 vol. of homogenizing buffer and recentrifuged at 9000 g for 20 min. The combined supernatants were pooled and centrifuged at 105 000 g for 60 min. The final microsomal pellet was then resuspended in 1 vol. of storage buffer (50 mM Tris/20% glycerol pH 7.4), divided into small aliquots and stored at –70°C. For spectrophotometric studies, the microsomal pellet was resuspended in 2 vol. of homogenizing buffer, resedimented by centrifugation to reduce haemoglobin contamination and the final pellet was resuspended in 1 vol. of storage buffer. Measurement of enzymatic activities NADPH-cytochrome P450 reductase activity was measured using the artificial electron acceptor cytochrome C (15). NADH-dependent cytochrome b5 reductase was measured as described by Mihara and Sato (16) and ethoxyresorufin (EROD)- and pentoxyresorufin (PROD)-dealkylase activities were measured according to Burke et al. (17). NDMA demethylase activity was measured as described by Yang et al. (5), except that 1.0 mg of liver microsomal protein from untreated or 0.5 mg from ethanol treated rats was incubated with 0.2 mM NDMA for 20 min. Alternatively, 0.05 mg of liver or 0.5 mg of oesophageal microsomes were incubated with 40 µM [14C]NDMA (54 mCi/mmol, 98% purity) for up to 30 min, as described by Levin et al. (18). Under these conditions, the rate of production of formaldehyde was linear with respect to both protein content and time. NDEA metabolism was measured based on the method described by Yoo et al. (4), with some modifications: the protein content of oesophageal microsomes was 100 µg, and of liver microsomes was 50 µg, and NDEA concentration was 100 µM, in a final incubation volume of 100 µl. The reaction was carried out for 5 min with liver microsomes or 30 min with oesophageal microsomes, making the rate of formation of the acetaldehyde derivative linear under these conditions. For kinetic constant determinations of NDEA metabolism by oesophageal and hepatic microsomes, the reaction had NDEA concentrations varying from 20 to 160 µM for oesophageal microsomes and from 15 to 1500 µM for hepatic microsomes. 7-Ethoxycoumarin-o-deethylase (ECOD) activity of liver microsomes was measured according to Aitio (19) by incubating 0.4 mg of liver microsomal protein with 300 µM ethoxycoumarin for 5 min. ECOD activity of oesophageal microsomes was determined as for the liver, except that the microsomal protein content was 0.5 mg, and the reaction was carried out for 15 min. Coumarin-7-hydroxylation (COH) catalysed by liver and oesophageal microsomes was carried out as for ECOD, except that the substrate was CO, and the incubation time was 30 min. An NADPH-generating system consisting of 0.5 mM NADP, 10 mM glucose-6-phosphate and 1 U/ml of glucose-6-phosphate dehydrogenase was used in all CYP-dependent reactions. To investigate whether a polyclonal antibody against CYP2A5 (20), CO or different nitrosamines could inhibit the microsomal enzymatic activities, variable amounts were incubated with microsomes for 5 min at room temperature, then at 37°C for 2 min, after which the reaction was started by adding the substrate.

1878

Spectrophotometric measurements The total CYP content of liver and oesophageal microsomes was determined as described by Omura and Sato (21), and Jackobson and Cinti (22), cytochrome b5 content was determined according to Omura and Sato (21) and Matsubara et al. (23) in microsomes of liver and oesophagus, respectively, and protein concentration by the method of Lowry et al. (24). Gel electrophoresis of proteins and western blotting Microsomal proteins were electrophoresed (25) in an 8% resolving gel, except for western blot of cytochrome b5 reductase (10% resolving gel) and of cytochrome b5 (13% resolving gel). After electrophoresis, the gels were electroblotted to a nitrocellulose membrane (Hybond-ECL, Amersham, NJ, USA). The membrane was then blocked, incubated with antibodies, and developed by enhanced chemiluminescence according to the manufacturer’s instructions (ECL Western-Blot Kit, Amersham), or using an alkaline phosphatase kit (for CYP2A), according to the manufacturer’s instructions (Bio-Rad, CA, USA). Antibodies raised in rabbits against rat NADPH-P450 reductase, cytochrome b5, cytochrome b5 reductase and CYP2B1/2B2 (11) were a generous gift of Dr E.A.Shephard, from University College London. Rabbit antibodies against rat CYP1A1, 2E1 and 3A were obtained from Amersham and the rabbit antibody against mouse CYP2A5 has been characterized previously (20). RT–PCR and DNA sequencing Total RNA was extracted from oesophagus and liver using Trizol, following the manufacturer’s instructions (Gibco BRL, CA, USA). The RNA was quantified by spectrophotometer and 400 ng of RNA from each tissue was used for reverse-transcription in a 20 µl reaction using the Superscript II Reverse Transcriptase (RT) (Gibco BRL) following the manufacturer’s instructions. The PCR reaction was done using 0.5 µl of the RT reaction mixture and using primers specific for CYP2A3 in the conditions described by Hellmold et al. (26), or for cyclofiline (27). Five microlitres of the PCR reaction from oesophagus and liver were separated on a 6% polyacrylamide gel and silver stained (28). For DNA sequencing, the PCR products were separated on a 2% agarose gel with TAE buffer [40 mM Tris–acetate pH 7.6/1 mM Na2 EDTA], the specific bands were excised with a clean scalpel blade, and the product was purified with Geneclean (BIO 101, CA, USA). The products were directly sequenced using 100 ng of the gel-purified PCR product following the procedures outlined by Lee (29) using [α-33P]dATP. Sequencing reactions were run on a 6% denaturing polyacrylamide gel at 35 W for 2 h. The gel was then fixed, dried and exposed to Kodak Biomax MR film. Statistical analysis and densitometry Statistical analysis was made by Student’s or Welch t-test. Statistical evaluation and kinetic analysis were done using an Instat 2.01 and GraphPad Prism programs (GraphPad Software, CA, USA). Densitometric analysis was done using GS-670 imaging densitometer (Bio-Rad).

Results The amount of CYP and the other major components of the monooxygenase complex in oesophageal and liver microsomes is shown in Table I, as analysed spectrophotometrically and confirmed by immunoblotting and densitometric analysis whenever possible (blots not shown). The total CYP content of oesophageal microsomes is 6.9% of that in the liver microsomes, and roughly the same ratio was found for the other components. The composition of the major carcinogen metabolizing CYP enzymes of liver and oesophageal microsomes was compared by western blot analysis using antibodies against CYP1A1, CYP2A5, CYP2B1/2B2, CYP2E1 and CYP3A1, and confirmed by catalytic studies when necessary. Microsomes were isolated from both untreated rats and after treatment with appropriate inducers. In accordance with previous studies (5), the ethanol inducible CYP2E1 was found in liver microsomes of both untreated and ethanol treated rats (Figure 1a). In contrast, no immunoreactive protein could be detected in oesophageal microsomes. Based on band intensities in the western blot, the induction of the hepatic CYP2E1 by ethanol is not obvious. However, this was confirmed by measuring the

Rat oesophageal cytochrome P450 monooxygenase system

Table I. Comparison between the oesophageal and liver monooxygenase system Microsomal component

Oesophagus

Total CYP (nmol/mg protein) P450 reductase activitya Cytochrome b5 (nmol/mg protein) Cytochrome b5 reductase activitya Protein (mg/g tissue)

0.07 45.9 0.04 3.2 3.9

⫾ ⫾ ⫾ ⫾ ⫾

0.003 2.9 0.014 0.11 0.38

Liver

Ratio (oesophagus/liver)

0.99 ⫾ 0.043 558 ⫾ 155 0.55 ⫾ 0.062 71 ⫾ 0.6 9.4 ⫾ 2.3

0.069 0.08 (0.07)b 0.07 (0.028)b 0.045 (0.18)b 0.41

are given as µmol cytochrome C reduced/min/mg protein for P450 reductase; and mmol ferricyanide reduced/min/mg protein for cytochrome b5 reductase. bLevels, determined by western blot and densitometry, refer to the protein (taken as mean ⫾ SD of six determinations). aActivities

microsomal NDMA demethylation activity, a reaction catalysed by CYP2E1 (5), which showed an ~4-fold increase after ethanol treatment (2.5 ⫾ 0.31 and 0.63 ⫾ 0.05 nmol/min⫻mg protein for ethanol treated and control livers, respectively). No NDMA metabolism could be detected with oesophageal microsomes prepared from control or ethanol treated rats, even when the extremely sensitive radiometric assay (5,18), and 10 times more oesophageal than hepatic microsomes were used. This supports the results observed in the western blot showing that the enzyme playing a major role in the hepatic nitrosamine metabolism, CYP2E1, is not expressed in the oesophagus of rats. No CYP2B1/2B2 protein could be detected in oesophageal microsomes, even after induction by PB and if excessive amounts of microsomal protein were used in the western blot (Figure 1b). The induction of the hepatic CYP2B proteins by PB, is shown by the increased intensity of the specific band as compared with that of the untreated animals (Figure 1b). The double band recognized in microsomes from untreated rats probably represents CYP2B1 and CYP2B2 because, due to their high homology, the antibody reacts with both of them (11,30). PROD was also not detected with uninduced or PB treated oesophageal microsomes. These results seem to be in contrast with the results of Chen et al. (31), who found traces of CYP2B1/2B2 in the oesophagus of untreated rats. No traces of CYP3A enzymes were found in oesophageal microsomes, even if a 10-fold increase in oesophageal microsomal protein was used for western blot in relation to the hepatic microsomes (Figure 1c). As reported previously, CYP1A1 was not found to be expressed in liver microsomes of untreated rats, but was strongly expressed after the 3-MC treatment (Figure 1d) (30). In the oesophagus, CYP1A1 is expressed at low levels in untreated rats and, similarly to the liver, it was induced by the 3-MC treatment (Figure 1d). Densitometric analysis revealed that in 3-MC treated rats, the amount of CYP1A1 present in oesophageal microsomes corresponds to ~4% of that present in liver microsomes. Western blot analysis was carried out with oesophageal and hepatic microsomes using an antibody against mouse CYP2A5 (20). Mouse liver microsomes were used as a positive control. Figure 1e shows that oesophageal, but not liver microsomes, from untreated rats express an immunologically similar protein with the same molecular weight as mouse CYP2A5. Pretreatment of the rats with CO or PY, inducers of mouse CYP2A5 (13), did not seem to significantly increase the level of this protein in the rat oesophagus. The possibility that the rat oesophagus expresses a gene similar to mouse CYP2A5 was further investigated by RT– PCR using primers specific for rat CYP2A3 mRNA, an

orthologous form of CYP2A5 (26). Figure 2 shows that a PCR product of the expected size, 398 bp, is amplified with the primers used (26). In contrast, the RT–PCR product obtained with liver mRNA was of a different size, which suggested that it did not represent CYP 2A3 mRNA. The identity of the amplified fragments was confirmed by sequencing, which showed that the one amplified in the oesophagus corresponds to CYP2A3, whereas the one from liver was identical to rat liver CYP2A1/2A2 (data not shown). Next, we investigated the microsomal metabolism of NDEA, CO and ethoxycoumarin of the liver and the oesophagus. Whereas NDEA is known to be a substrate of both CYP2E1 (4) and CYP2A5 (9), and, to a minor extent, of some other CYP enzymes, CO is a specific substrate of CYP2A5 and CYP2A3 (9,13,32). Although CYP2A5 presents a high catalytic activity in the metabolism of ethoxycoumarin, this compound is a substrate for many different CYP enzymes. Oesophageal microsomes presented a lower Km and Vmax for NDEA metabolism than did liver microsomes (13.18 ⫾ 5.74 µM and 0.174 ⫹ 0.014 nmol/min/mg microsomal protein for oesophageal microsomes, against 39.58 ⫾ 4.45 µM and 1.684 ⫹ 0.041 nmol/min/mg microsomal protein for liver microsomes). However, Table II shows that when NDEA activity of the two organs are compared per nmol of total CYP, NDEA metabolism in the oesophagus is higher than that found in the liver. Conversely, COH activity is roughly the same in both organs when compared per mg of microsomal protein, but oesophageal activity is much higher than that found in the liver when the values are expressed per nmol of total CYP. Contrary to this, oesophageal ECOD activity is much lower than that found in liver, either when comparing per mg of microsomal protein or per nmol of total CYP. The anti-CYP2A5 antibody was used in immunoinhibition analysis of the microsomal COH and NDEA activities of the rat oesophagus. Table III shows the maximal inhibition that could be achieved by using excessive amounts of the antibody in the reaction mixtures. A near complete inhibition of the oesophageal COH activity was achieved by the antibody (Table III), indicating that the protein recognized by the antibody was the major, if not the only, catalyst of this reaction in oesophageal microsomes. In comparison, about two-thirds of the NDEA metabolism was inhibited by the antibody. This suggests that whereas CYP2A3 plays a major role in the metabolism of NDEA in the oesophagus, other enzymes may contribute as well. In contrast to the oesophagus, the hepatic microsomal COH and NDEA activities were not inhibited at all by the anti-CYP2A5 antibody (Table III). Table III also shows that CO inhibits NDEA metabolism significantly in oesophageal, but not in hepatic microsomes, thus confirming the involvement of CYP2A3 in the metabolism of NDEA in oesophagus. 1879

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Fig. 2. RT–PCR analysis of CYP2A3 in oesophagus and liver from untreated rats. Lane 1, Bluescript SK⫹ digested with HinfI (456, 396 and 356 bp); lane 2, oesophagus; lane 3, liver; lane 4, control 1 (without reverse transcriptase); lane 5, control 2 (without cDNA); lane 6, PCR negative control.

Table II. NDEAd, COH and ECOD activities of oesophageal and hepatic microsomesa Activity per mg microsomal protein per nmol total CYPb Oesophageal NDEAd COH ECOD

Hepatic

Oesophageal

Hepatic

0.121 (⫾0.02) 1.11 (⫾0.004) 1.73 (⫾0.23) 1.12 (⫾0.004) 0.72 (⫾0.03) 0.96 (⫾0.02) 10.29 (⫾0.002) 0.97 (⫾0.02) 7.1 (⫾2.4) 430 (⫾21) 101.4 (⫾34.3) 434.3 (⫾22.2)

are expressed as mean ⫾ SD of six determinations, and given as nmol acetaldehyde formed/min. bTaken as reference data from Table I. aResults

Fig. 1. (a–e). Western blot analysis of oesophageal microsomes (OM) and liver microsomes (LM) from untreated rats (UT), and after treatment with ethanol (ET) PB, 3-MC, PY or CO by five antibodies against CYP isoenzymes. Molecular weight markers [except for (e) CYP2A blotting]: catalase (58.1 kDa), alcohol dehydrogenase (39.8 kDa), carbonic anhydrase (29.0 kDa). For further details, see Materials and methods. (a) CYP2E1; in OM and LM from UT and ET treated rats. Lane 1, molecular weight markers; lanes 3–5, LM/UT (0.66 µg); lanes 2, 6 and 7, LM/ET (0.66 µg); lane 8, OM/UT (28 µg); lane 9, OM/ET (28 µg). (b) CYP3A; in OM and LM from UT rats. Lane 1, OM (17 µg); lane 2, OM (8.5 µg); lane 3, molecular weight markers; lane 4, LM (7.5 µg); lane 5, LM (1.8 µg). (c) CYP2B1/2B2; in OM and LM from UT and PB treated rats. Lane 1, OM/UT (28 µg); lane 2, OM/PB (28 µg); lane 3, molecular weight markers; lanes 4 and 5, LM/PB (2 µg); lanes 6 and 7, LM/UT (2 µg). (d) CYP1A1; in OM and LM from UT and 3-MC treated rats. Lane 1, OM/UT (20 µg); lanes 2 and 3, OM/3-MC (20 µg); lane 4, molecular weight markers; lane 5, LM/UT (3.7 µg); lanes 6 and 7, LM/3-MC (1.0 µg). (e) CYP2A5; in OM, LM and LMM (microsomes from mouse liver-positive control) from UT, CO and PY treated rats. Lane 1, molecular weight markers (phosphorylase B 97.4 kDa; bovine serum albumin 66.2 kDa; ovalbumin 45 kDa; carbonic anhydrase 31 kDa); lane 2, LMM/UT (6 µg); lane 3, OM/UT (20 µg); lane 4, OM/CO (20 µg); lane 5, OM/PY (20 µg); lane 6, LM/UT (20 µg); lane 7, LM/CO (20 µg); lane 8, LM/PY (20 µg).

Table IV shows the effect of CO and PY pretreatment of rats on NDEA metabolism and COH activity of oesophageal and hepatic microsomes. CO produced a statistically significant induction of oesophageal NDEA metabolism (2.5-fold), and of COH activity (2.3-fold). This is in accordance with previous studies where CO has been shown to elevate nasal epithelial CYP2A3 expression and COH activity (13). The western blot analysis did not show any clear increment of the oesophageal CYP2A3 protein (Figure 1e), but this may be due to the relative insensitivity of the western blot as compared with 1880

the enzymatic assay. PY produced a modest, non-significant increment of NDEA and COH activity, suggesting that it may not be able to induce CYP2A3 in the oesophagus. Conversely, the modest increase in hepatic COH activity by PY and CO could be due to induction of the hepatic CYP enzymes, which are able to metabolize CO at low rate with the concentrations used in the assays (Table IV). PY was also able to increase the hepatic NDEA activity, probably as a consequence of the induction of the hepatic CYP2E1 by this compound (5). Finally, Figure 3 shows that NDEA, but not NDMA, is able to inhibit oesophageal COH in a concentration-dependent fashion. This in accordance with previous results, which showed that NDEA is a good substrate for CYP2A enzymes (9), and is able to inhibit oesophageal CO metabolism (32). Discussion In investigations on oesophageal xenobiotic metabolism, a microsomal fraction is often used, which is obtained by a method optimized for the liver (31,33–36). The characteristics of such microsomes have, however, not been described in detail. Therefore, we used the NADPH-cytochrome P450 reductase as a marker enzyme for the endoplasmic reticulum for preparation of microsomes from extra-hepatic tissues (37). We found that the yield of microsomes from the liver was about twice as much as from the oesophagus, but that the enrichment factor of endoplasmic reticulum of oesophageal and hepatic microsomes is comparable. This suggests that the method used for isolation of hepatic microsomes can be applied for the oesophagus with minor modifications. Although the total CYP content of the oesophageal microsomes is only 7% of that of liver microsomes, NDEA and other nitrosamines are known to be metabolized in the oesophagus at a relatively high rate, often leading to high levels of alkylation of oesophageal DNA (2,38,39). This seems somewhat surprising, as the enzymes that contribute to hepatic nitrosamine metabolism, CYP 2B1/2B2, CYP2E1 and CYP3A (30), are

Rat oesophageal cytochrome P450 monooxygenase system

Table III. Inhibition of rat hepatic and oesophageal COH activity by anti-CYP2A5 antibody and NDEAd activity by anti-CYP2A5 antibody or by COa Microsomes

COH activity

Hepatic Oesophageal

COc

NDEAd activity

Control

Antibodyb

Control

Antibodyb

0.89 ⫾ 0.089 0.78 ⫾ 0.07

0.82 ⫾ 0.057 (92%)d 0.082 ⫾ 0.003(11%)e

1.02 ⫾ 0.06 0.136 ⫾ 0.03

1.07 ⫾ 0.07 (105%) 0.057 ⫾ 0.002 (42%)e

0.78 ⫾ 0.14 (76%) 0.087 ⫾ 0.005 (64%)e

are expressed as mean ⫾ SD of six determinations, and given as nmol acetaldehyde formed/min⫻mg microsomal protein or pmol umbelliferone formed/min⫻mg microsomal protein. bTwo milligrams of anti-CYP2A5 antibody/mg microsomal protein. cFifty micromolars of CO. dPercentage of control. eSignificantly (P ⬍ 0.05) different from control activity. aResults

Table IV. Microsomal NDEAd and COH activity of the oesophagus and the liver of untreated rats (C) and after treatment with PY and COa Activity

NDEAd COH

Treatment

Hepatic microsomes

C

PY

CO

C

PY

CO

0.121 (⫾0.004) 0.72 (⫾0.03)

0.258 (⫾0.11) 0.94(⫾0.16)

0.301b (⫾0.09) 1.62b (⫾0.38)

1.11 (⫾0.02) 0.96(⫾0.02)

1.74 (⫾0.49) 3.36b (⫾0.23)

1.14 (⫾0.03) 1.32b (⫾0.06)

are expressed as mean ⫾ SD of six determinations, and given as nmol acetaldehyde formed/min⫻mg microsomal protein for NDEAd, or pmol umbelliferone formed/min⫻mg microsomal protein for COH. (P ⬍ 0.05) different from control values.

aResults

bSignificantly

Fig. 3. Inhibition of rat oesophageal COH (500 µM) activity by NDMA and NDEA. (a) Results are expressed as mean ⫾ SD of six determinations, and given as pmol umbelliferone formed/min⫻mg microsomal protein. (b) Significantly (P ⬍ 0.05) different from control values.

not expressed in the oesophagus. This suggests that some other oesophageal CYP enzymes are efficient catalysts of nitrosamine metabolism. The presence and induction of CYP1A1 in the oesophagus agrees with previous data showing that the oesophageal CYP1A1 is inducible by polycyclic aromatic hydrocarbons (36,40) and suggest that the oesophagus has a capacity to metabolize these carcinogens. However, CYP1A1 does not seem to play a significant role in the metabolism of NDEA, or other nitrosamines (36,41; our unpublished data). An enzyme highly similar to mouse CYP2A5 was found to be constitutively expressed in the oesophagus of untreated rats. In previous investigations such an enzyme has been found in the rat nasal epithelium (13,32) and Kimura et al. (42) have isolated an orthologous enzyme from rat lung, assigned as CYP2A3, with a 98% sequence similarity to CYP2A5. We showed here that the oesophagus expresses CYP2A3, whereas

it is absent from the rat liver, suggesting that in the rat, enzyme(s) similar to CYP2A5 are extra-hepatic. CYP2A3 has recently been detected in oesophageal microsomes of untreated rats as two immunoreactive bands, one relative to CYP2A3, and another of a lower molecular weight (43). Contrary to that we found using the same antibody against CYP2A5, only one immunoreactive band in oesophageal microsomes of untreated rats. We do not know the reasons for such discrepancy, but direct sequencing of our RT–PCR product did not suggest that CYP2A enzymes, other than CYP2A3, are expressed in the oesophagus of untreated rats. The amplification of CYP2A1/2A2 with liver RNA indicates that the primers used were not specific to CYP2A3, and that if other similar CYP2A enzymes were present in the oesophagus, they would probably result in different products being amplified and sequenced. Until now, there was very scarce data available concerning NDEA metabolism in the oesophagus. This is in part because CYP enzymes expressed in the oesophagus have been poorly characterized and the kinetics of oesophageal nitrosamine metabolism have proven difficult to determine, being often obtained with high concentrations of nitrosamines, which may not reflect the situation in vivo. We had measured NDEA metabolism in the oesophagus previously, but the data were obtained using oesophageal epithelium (3). However, in this work we were able to measure kinetic constants of NDEA metabolism catalysed by oesophageal microsomes, using low concentrations of NDEA. The Km for NDEA metabolism in oesophageal microsomes is about one-third of that of hepatic microsomes, in agreement to those obtained previously by Swann et al. (2), and by us (3) using tissue slices. This difference in Km may help to explain why lower doses of NDEA induce predominantly oesophageal tumours, whereas higher doses of this carcinogen cause mostly hepatic tumours (1). Furthermore, when the microsomal metabolism of NDEA in both tissues is compared per nmol of total CYP, the 1881

L.F.Ribeiro Pinto et al.

oesophagus possesses higher activity than what is found in the liver. Several studies (9,13,31,32) have shown that nitrosamines can be good substrates of CYP2A enzymes, although the Km and Vmax may vary significantly, depending on the structure of the molecule. We have shown, for example, that whereas both NDMA and NDEA can be metabolized by mouse CYP2A5 and human CYP2A6, NDEA is a much better substrate (lower Km, higher Vmax) than NDMA for these enzymes (NDMA is a better substrate for CYP2E1) (9). CYP2A3 appears to be an efficient catalyst of NDEA activation in the rat nose (13). Other good substrates of CYP2A3 are the oesophageal carcinogens N-nitrosomethylamylamine (NMAA) (31), N-nitrosomethylbenzylamine (NMBzA) (32) and N-nitrosonornicotine (NNN) (13,32). Our data suggest that CYP2A3 is responsible for most of the NDEA metabolism in the oesophagus of untreated rats. However, based on the differences in NDEA inhibition achieved in our previous work with rat nasal microsomes (13), and in this work with oesophageal microsomes, it seems that CYP2A3 is not the only enzyme to be involved in NDEA metabolism in the oesophagus. In this regard, a novel cytochrome P450, designated CYP2B21 has been recently cloned from the rat oesophagus, but its catalytic properties are still not known (44). Although CYP2E1 is involved in the metabolism of NDEA in rat liver (4), our results show that this CYP is not expressed in the oesophagus. This agrees with the fact that NDMA, which is exclusively metabolized by this enzyme in the rat at low concentration (5), is not able to induce oesophageal tumours (1), and does not methylate oesophageal DNA (2,39). Furthermore, ethanol, which is an inhibitor of CYP2E1 metabolism in the rat liver (2,3,5), is not able to inhibit oesophageal NDEA metabolism (2). In accordance, Ahn et al. (36) did not detect CYP2E1, CYP2B1/2B2 or CYP3A in the oesophagus of untreated rats. CYP2E1 has been shown to efficiently catalyse the demethylation, but not the dealkylation of unsymmetric nitrosamines, such as NMAA (31), nitrosomethylbutylamine (NMBA) and NMBzA (45), all potent oesophageal carcinogens (1). Therefore, the absence of CYP2E1 in the oesophagus can explain why this tissue catalyses the dealkylation of these nitrosamines at a much higher rate than catalyses the demethylation, when compared with the liver (14,39). It must be pointed out that NDMA can be metabolized by cultured human oesophageal explants (46) and microsomes (47,48), and that CYP2E1 is expressed in human oesophagus (48, our unpublished data). CYP3A is also expressed in the oesophagus of some patients (48). Therefore, the expression of some CYP enzymes in human oesophagus seems to be different from that of rat oesophagus, the most susceptible species in oesophageal experimental carcinogenesis (2). We conclude that major differences were found in the CYP enzyme composition between the oesophagus and liver of rats. CYP2B1/2B2, CYP2E1 and CYP3A were expressed only in the liver, CYP1A1, in both organs, and CYP2A3 only in the oesophagus. Furthermore, CYP2A3 seems to be responsible for a great part, but not for the entire metabolism of NDEA in the oesophagus. Work is being carried out at the moment in our laboratory to study the mode of regulation of CYP2A3 in the oesophagus and other extra-hepatic tissues of the rat, as well as to try to characterize the other enzyme(s) responsible for NDEA metabolism in the rat oesophagus. 1882

Acknowledgements We thank Dr E.A.Shephard, University College, London, UK, for donating some of the antibodies. This work was generously supported by the Cancer Research Campaign, Third World Academy of Sciences, grant no. 97-208, and by ‘Fundac¸ a˜ o de Amparo a Pesquisa do Estado do Rio de Janeiro-FAPERJ’.

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