Laboratory of Radiochemistry, Gifu Pharmaceutical University, 6-1, Mitahora-higashi 5-chome, Gifu 502-8585, Japan, 1Department of Pathology, Osaka City University Medical School, 1-4-3, Asahi-machi, Abeno-ku, Osaka 545-8585, Japan, 2Japan Bioassay Research Center, Japan Industrial Safety and Health Association, 2445 Hirasawa, Hadano, Kanagawa 257-0015, Japan
*To whom correspondence should be addressed. Tel: +81 58 237 3931; Fax: +81 58 237 5979; E-mail: [email protected]
†These authors contributed equally to this work.
Received on February 29, 2012; revised on September 18, 2012; accepted on October 1, 2012
Alcohol consumption is frequently associated with various cancers and the enhancement of the metabolic activation of carcinogens has been proposed as a mechanism underly-ing this relationship. The ethanol-induced enhancement of N-nitrosodiethylamine (DEN)-mediated carcinogenesis can be attributed to an increase in hepatic activity. However, the mechanism of elevation of N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis remains unclear. To elucidate the mechanism underlying the role of ethanol in the enhance-ment of NMBA-induced oesophageal carcinogenesis, we eval-uated the hepatic and extrahepatic levels of the cytochrome P450 (CYP) and mutagenic activation of environmental carcinogens by immunoblot analyses and Ames preincuba-tion test, respectively, in F344 rats treated with ethanol. Five weeks of treatment with 10% ethanol added to the drink-ing water or two intragastric treatments with 50% ethanol, both resulted in elevated levels of CYP2E1 (1.5- to 2.3-fold) and mutagenic activities of DEN, N-nitrosodimethylamine and N-nitrosopyrrolidine in the presence of rat liver S9 (1.5- to 2.4-fold). This was not the case with CYP1A1/2, CYP2A1/2, CYP2B1/2 or CYP3A2, nor with the activities of 2-amino-3-methylimidazo[4,5-f]quinoline, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, aflatoxin B1 or other N-nitroso com-pounds (NOCs), including NMBA. Ethanol-induced elevations of CYP2A and CYP2E1 were observed in the oesophagus (up to 1.7- and 2.3-fold) and kidney (up to 1.5- and 1.8-fold), but not in the lung or colon. In oesophagus and kidney, the mutagenic activities of NMBA and four NOCs were markedly increased (1.3- to 2.4-fold) in treated rats. The application of several CYP inhibitors revealed that CYP2A were likely to contribute to the enhancing effect of ethanol on NMBA activa-tion in the rat oesophagus and kidney, but that CYP2E1 failed to do so. These results showed that the enhancing effect of eth-anol on NMBA-induced oesophageal carcinogenesis could be attributed to an increase in the metabolic activation of NMBA by oesophageal CYP2A during the initiation phase, and that this occurred independently of CYP2E1.
Introduction
Epidemiological studies have shown that alcohol consumption is associated with cancers of the upper gastrointestinal tract, liver,
large bowel and breast. It is suspected that there is also an asso-ciation with cancers of the lung and pancreas. Approximately 3.6% of cancers worldwide are associated with the chronic con-sumption of alcohol (1). Ethanol-induced cancers are caused by various molecular mechanisms including the genotoxic effects of acetaldehyde, the production of reactive oxygen species and the inhibition of S-adenosyl-l-methionine synthesis (2). The meta-bolic activation of exogenous and endogenous carcinogens by cytochrome P450 (CYP) 2E1 is also one of the routes by which ethanol exerts its carcinogenic action. Ethanol is known to be a hepatic CYP2E1 inducer in rodents, and there are a few reports on the induction of other CYP isozymes by ethanol in rats (3). CYP2A, CYP2B and CYP2E are specifically involved in the metabolic activation of environmental N-nitroso compounds (NOCs) to produce carcinogenic end products. Rat CYP2E1 predominantly activates N-nitrosodimethylamine (DMN), N-nitrosodiethylamine (DEN) and N-nitrosopyrrolidine (NPYR), whereas CYP2A and CYP2B1/2 activate N-nitrosodialkylamines that possess relatively long alkyl chains (4,5). Administration of ethanol in the drinking water of F344 rats showed an enhancing effect on DEN-induced (6) and N-nitrosomethylbenzylamine (NMBA)-induced (7) oesophageal carcinogenesis during the ini-tiation phase. It has been reported that chronic ethanol treatment reduces UDP-glucuronyltransferase (UGT) 2B mRNA and pro-tein levels and UGT activity in the rat liver (8,9). It has also been suggested that UGT2B1 is likely to be the enzyme responsible for the glucuronidation of some NOCs (10). Therefore, we pos-tulated that ethanol increases NOC-induced carcinogenesis by enhancing its metabolic activation or by suppressing its inacti-vation. A previous report showed that supplementing drinking water with 10% ethanol for 2 weeks increased the mutagenic activation of DEN, DMN and NPYR by CYP2E1 in rats, but did not affect the mutagenic activation of NMBA by CYP2B1/2 or the activities of the three types of UGT (11). However, the fail-ure to observe any enhancement of NMBA activation might have been attributable to insufficient duration of ethanol treatment.
NMBA is known to be the most potent carcinogen in the rat oesophagus. This is irrespective of its mode of administration and requires metabolic activation to have a mutagenic or carcinogenic effect (12). The metabolism of NMBA is inhibited to >95% by CO and 70% by SKF-525A, which are both typical CYP inhibitors, indicating that there is a relationship between NMBA activation and CYP activity (13). In our previous report we showed that, in a phenobarbital-induced rat liver, NMBA is mutagenically activated by CYP2B1 and CYP2B2, but not by CYP2E1 (11). In contrast to this, other researchers have proposed that CYP2A3 (14) or CYP2E1 (15) contribute to the metabolic activation of NMBA. The activation of NMBA is observed not only in the liver but also in the oesophagus. The total CYP content in oesophageal microsomes is only 7% of that in liver microsomes (16). Oesophageal mucosal microsomes from male Sprague–Dawley rats can produce benzaldehyde and formaldehyde from NMBA at rates of 1/5 and 1/60 of their respective hepatic levels (13). The O6-methylated guanine level in rats treated
161
Mutagenesis vol. 28 no. 2 pp. 161–169, 2013Advance Access publication 15 January 2013
with NMBA was six times higher in oesophageal DNA than in hepatic DNA (17). In addition, ethanol is known to increase the total CYP content (18) and CYP2E1 enzyme protein in the rat oesophagus (15,19). This suggests that metabolic activation in target organs may play an important role in ethanol-induced carcinogenesis. Our previous report showed that suppression of NMBA-induced oesophageal carcinogenesis by curcumin could be attributed to a decrease in the metabolic activation of NMBA by oesophageal CYP2B1 during the initiation phase (20). However, only limited data exist about the effect of ethanol on metabolic activation by the CYP protein in extrahepatic tissues such as rat oesophagus.
To elucidate the mechanism(s) underlying the enhancement of NMBA-induced oesophageal tumorigenesis by ethanol, we assayed the hepatic and extrahepatic levels of microsomal CYP enzymes that are known to activate typical environmental carcinogens in F344 rats treated with ethanol and/or NMBA. Furthermore, the mutagenic activation of these carcinogens by tissue S9 and the three types of hepatic UGT was assayed. We also report on the CYP forms that are responsible for the meta-bolic activation of NMBA in the liver, oesophagus, kidney, lung and colon of ethanol-treated rats.
Materials and methods
ChemicalsMetyrapone, NPYR and orphenadrine were obtained from Aldrich Chemical (Milwaukee, WI, USA) and aflatoxin B1 (AFB1) was obtained from Makor Chemicals (Jerusalem, Israel). Bilirubin, coumarin, DEN, DMN, ethanol, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), methoxsalen, 4-methyl-pyrazole, 4-nitrophenol, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), SKF-525A, testosterone, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) acetate and UDP-glucuronic acid were purchased from Wako Pure Chemicals (Osaka, Japan) and NMBA was purchased from Sakai Laboratory (Fukui, Japan). UDP-[14C(U)]glucuronic acid was obtained from American Radiolabeled Chemicals (St Louis, MO, USA) and ATP, glucose-6-phosphate (G6P), G6P dehydrogenase (G6PDH), NADH, NADP+ and NADPH were purchased from Oriental Yeast (Tokyo, Japan). All other commercial products were of the purest grade available. N-nitrosobis(2-hydroxypropyl)amine (BHP) and N-nitroso-2,6-dimethylmorpholine (NDMM) were synthesised in our labo-ratory as described previously (5).
Animal treatment and tissue preparationAnimal experiments were executed following the guidelines for the care and use of experimental animals set out by Gifu Pharmaceutical University and Osaka City University (Osaka, Japan).
Six-week-old male F344/DuCrj and F344/N Slc rats were purchased from Charles River Japan (Hino, Shiga, Japan) and Japan SLC, Inc. (Hamamatsu, Japan), respectively. The rats were housed in wire cages (2–3 rats/cage) and maintained under standard laboratory conditions. Twenty-eight male F344/DuCrj rats were divided into four groups of seven animals. Rats in groups 1 and 2 were given access to tap water ad libitum; group 1 rats were subcu-taneously treated with 20% dimethyl sulphoxide (DMSO) three times per week for 5 weeks and served as the controls; group 2 rats received injections of 0.5 mg/kg NMBA dissolved in 20% DMSO three times per week for 5 weeks. Rats in groups 3 and 4 were treated for 5 weeks with 10% etha-nol added to their drinking water and simultaneously treated with vehicle or NMBA, respectively. Rats were decapitated 24 h after the last dose of vehicle or NMBA. In addition, 32 male F344/N Slc rats were divided into two groups. They were twice treated orally with 50% ethanol (0.5 ml/kg) or unsupplemented drinking water (the second dose was given 12 h after the first). Rats were decapitated 24 h after the last administration. Harvested liv-ers and lungs were perfused in situ with ice-cold sterile 1.15% KCl solution and 25% homogenates were prepared in 1.15% KCl. Oesophagi, kidneys and colons were rinsed in ice-cold 1.15% KCl after harvesting. Oesophageal and colonic mucosae were stripped off the submucosae and muscular tissues. S9 and microsomal fractions from these tissues were prepared using established procedures (5).
Western blotsGoat anti-rat polyclonal antibodies against CYP1A1/2, CYP2B1/2 and CYP2E1, rabbit anti-rat antibodies against CYP3A2 (Nosan Co., Yokohama, Japan) and goat anti-human polyclonal antibodies against CYP2A (Santa Cruz Biotechnology, Santa Cruz, CA) were used as the primary antibodies. Gel elec-trophoresis and blot analyses were performed as described previously (20).
Mutation assayAll tests were performed using the Ames preincubation assay (21). Seven NOCs, BHP, DEN, DMN, NDMM, NMBA, NNK and NPYR were dissolved in 100 μl of water and all other carcinogens in 50 μl of DMSO. The muta-genicities of Trp-P-2 and IQ (0.03 μg/plate), AFB1 (1 μg), NPYR (0.25 mg), DMN, DEN, NMBA and NNK (1 mg), BHP and NDMM (10 mg) were assayed in the presence of liver, oesophagus, kidney, lung and colon S9 using estab-lished procedures (5,11,20). The tissue S9 fraction volume was 10 μl/plate for heterocyclic amines and 150 μl for NOCs and AFB1. Salmonella typhimurium tester strains TA100 and TA98 were used for the seven NOCs and the other carcinogens, respectively. The S9 mix contained the following cofactors: 4 mM NADPH, 4 mM NADH, 0.5 U G6PDH, 5 mM G6P and 5 mM ATP (except for the NOCs, where 4 mM NADP+ and 5 mM G6P were used). In experiments on CYP inhibition, 25 μM methoxsalen, 1.0 mM coumarin and metyrapone, 0.25 mM SKF-525A, 0.2 mM orphenadrine and 0.1 mM 4-methylpyrazole were preincubated with the carcinogen substrate and S9 mix (20,22,23).
Assay of UGT activityUGT activity on bilirubin and 4-nitrophenol in liver microsomes was assayed according to the method described by Heirwegh et al. (24) and Isselbacher et al. (25), respectively. UGT activity on testosterone was determined using UDP-[14C(U)]glucuronic acid as described by Matern et al. (26).
Statistical analysesStatistical analyses of the two groups were performed using a Student’s t-test. One-way analysis of variance (ANOVA) followed by a Tukey–Kramer test were used for comparing the four groups with the help of the computer pack-age KyPlot version 2.0 (Kyence Inc., Tokyo, Japan). P < 0.05 was considered significant.
Results
Figure 1 shows the immunoblots and levels of microsomal CYP proteins in male F344 rats treated with NMBA and ethanol for 5 weeks. Oesophageal CYP2E1, CYP2B1, CYP1A1, CYP1A2 and CYP3A2 were constitutively detected with an antibody against hepatic CYP in the vehicle group (group 1, lane 1). The hepatic CYP2A1/2 and oesophageal CYP2A proteins were constitutively detected with antibody against human CYP2A in the vehicle group. CYP2A proteins migrated slightly slower than the standard CYP2A6. The hepatic (left) and oesophageal (right) levels of CYP2E1 in rats treated with 10% ethanol in drinking water (groups 3 and 4, lanes 3 and 4) were 2.1- to 2.3-fold and 1.8- to 2.3-fold higher, respectively, than those observed in group 1 rats. The CYP2E1 level was unchanged in rats subcutaneously treated with 0.5 mg/kg NMBA (group 2, lane 2). The level of oesophageal CYP2A was slightly increased in ethanol-treated group, but no significant differences were observed. There were no significant differences in hepatic levels of constitutively detected CYP2A1/2, CYP2B2, CYP1A2 and CYP3A2 and oesophageal levels of CYP2B1, CYP1A1, CYP1A2 and CYP3A2 among the four groups. Neither hepatic CYP2B1 and CYP1A1 nor oesophageal CYP2B2 were expressed in any group of rats. Figure 2 shows the immunoblots of CYP proteins in the lung, kidney and colon of rats in group 1 (V) and 3 (E). CYP2A, CYP2E1, CYP2B1 and CYP1A1 in the lung; CYP2A, CYP2E1 and CYP2B1 in the kidney; and CYP2A, CYP2E1, CYP2B1 and CYP3A2 in the colon were constitutively detected with antibodies against hepatic CYP in the vehicle group. Treatment with ethanol significantly increased renal CYP2E1 1.8-fold compared with that for
group 1 rats (P < 0.01). In contrast, there were no significant alterations in the levels of CYP2E1 in the lung and colon and other detected CYP isoforms in the three tissues.
To confirm the evidence for CYP induction, the potency of ethanol was further checked in hepatic and extrahepatic micro-somes from F344/N Slc rats, which is genetically similar to F344/DuCrj, orally treated with 50% ethanol twice (correspond-ing to the daily intake in drinking water containing 10% ethanol without acute toxicities). Figure 3 shows the immunoblots of CYP proteins in the liver, oesophagus, kidney, lung and colon. In addition to the results shown in Figures 1 and 2, CYP2E1 levels in the liver, oesophagus and kidney in ethanol-treated rats were 1.5-, 1.7- and 1.4-fold higher, respectively, than those observed in control rats. Oesophageal and renal CYP2A levels in the treated group were significantly higher than those in the vehicle group (1.7- and 1.5-fold, respectively) although there were no significant alterations in the levels of pulmonary and colonic CYP2E1 and CYP2A, hepatic CYP2A1/2 and other detected CYP isoforms in the five tissues.
To clarify the potential of liver S9 to mediate the mutagenic activation of carcinogens, the mutagenicities of NMBA and eight other carcinogens known to be metabolically activated by specific CYP enzymes were tested in Salmonella strains TA100 and TA98. Table I shows the mutagenic activities of six NOCs including NMBA, two heterocyclic amines and AFB1 in the presence of liver S9 mix from rats treated with NMBA and ethanol for 5 weeks. Liver S9 from ethanol-treated rats (groups
3 and 4) increased the mutagenic activities of NPYR, DMN and DEN 1.8-, 2.8- and 1.5-fold, respectively, in TA100 strain compared with that from group 1 rats. No significant altera-tions in the mutagenicity were observed with NMBA, BHP and NDMM, and there were no significant differences between groups 1 and 2 in the mutagenic activities of any NOCs. A sig-nificant effect on the mutagenic activities of IQ, Trp-P-2 and AFB1 in TA98 strain was not observed.
Figure 4 shows the mutagenic activities of NPYR, DMN, DEN, NMBA and NNK in the presence of liver, oesopha-gus, kidney, lung and colon S9 from rats orally treated with the vehicle or 50% ethanol two times. Mutagenic activities of NPYR, DMN and DEN with liver S9 increased up to 1.5-fold compared with those in the vehicle group by ethanol treatment, although those of NMBA and NNK did not (A). The mutagenic activities of the five NOCs, including NMBA, in the presence of oesophagus and kidney S9 in ethanol-treated rats increased 1.3- to 1.4-fold or 1.4- to 1.9-fold, respectively, compared with those in control rats (B and C). However, no significant altera-tions in the mutagenicity of the five NOCs in the presence of lung or colon S9 were produced (D and E).
In an attempt to obtain more information about CYP species responsible for NMBA activation, typical selective and non-selective CYP inhibitors were used in mutagenic activation assays. Figure 5 shows the effects of specific and non-specific inhibitors on the mutagenic activity of NMBA in the presence of liver, oesophagus, kidney, lung and colon S9 from 50% ethanol-treated
Fig. 1. Immunoblots and densitometric determination of the expression of CYP protein in liver (left) and oesophageal (right) microsomes from rats treated with NMBA, ethanol or both. Microsomes were pooled from seven rats each treated with vehicle (group 1, lane 1) or 0.5 mg/kg NMBA three times per week for 5 weeks (group 2, lane 2), with 10% ethanol in the drinking water for 5 weeks (group 3, lane 3), or NMBA + ethanol (group 4, lane 4). Lane 5 contains CYP standards from male Sprague–Dawley rats treated with acetone (B), phenobarbital (PB) (C and E) or 3-methylcholanthrene (MC) (D), and the CYP2A6 standard obtained from a human B lymphoblastoid cell line (A). Liver microsomes contained 8.0 μg (A), 0.4 μg (B–D) and 1.0 μg (E) microsomal protein, and the values represent the means ± SD of microsomal protein (pmol/mg) obtained from 4 to 8 experiments. Oesophageal microsomes contained 30 μg microsomal protein, and the values represent the means ± SD of the ratio to the vehicle group (comprising 3–4 experiments) expressed in arbitrary units. *P < 0.05, compared with the vehicle group (one-way ANOVA followed by Tukey–Kramer test). n.d. = not detected.
rat. Coumarin and methoxsalen are used for specific inhibition of CYP2A activity. Metyrapone, orphenadrine and 4-methylpyrazole are specific inhibitors for CYP2B1/2, CYP2B1 and CYP2E1, respectively, whereas SKF-525A is a non-specific inhibitor. SKF-525A inhibited the mutagenic activation of NMBA in the presence of liver, oesophagus, kidney, lung and colon S9 by 56–94%. Metyrapone inhibited mutagenic activation in the presence of liver S9 by 32%, whereas orphenadrine, methoxsalen and 4-methylpyrazole showed no significant inhibition (A). Alternatively, coumarin and methoxsalen decreased the mutagenic activity of NMBA in the presence of oesophagus (37–53%) and kidney S9 (33–47%) (B and C). Metyrapone and orphenadrin also inhibited the mutagenic activity of NMBA in the oesophagus (21–23%) and kidney (47–50%), but 4-methylpyrazole did not show significant inhibition in these tissues. Five selective inhibitors showed no significant alterations in the activity of NMBA with lung or colon S9 (D and E).
Table II summarises the effects of NMBA and ethanol treat-ment on hepatic UGT activities towards 4-nitrophenol, bilirubin and testosterone in liver microsomes (groups 1–4). Treatment with NMBA, ethanol or both for 5 weeks did not produce sig-nificant differences in the three types of UGT activities among the four groups. Similarly, no significant alteration in these activities was observed when treated with 50% ethanol.
Discussion
Alterations to hepatic xenobiotic-metabolising enzymes reflect the activation of environmental carcinogens and can predict their carcinogenic potential. NMBA treatment produced no obvious effects on the levels of any CYP subfamilies, mutagenic activation by liver S9 or UGT activities. It has been confirmed by our previous findings that no significant effects are observed in these expressions and activities by NMBA, N-nitrosobis(2-oxopropyl)nitrosamine or N-butyl-N-(4-hydroxybutyl)nitrosamine (11,20,27,28). A 5-week treatment with 10% ethanol administered via drinking water enhanced the mutagenic activation of DEN, DMN and NPYR, reflecting an increase in hepatic CYP2E1. These results are in accordance with our previous reports, which demonstrated the mutagenic activation of DMN and DEN by CYP2E1 after a 2-week treatment with ethanol (11). Similar observations were obtained after intragastric treatments twice with 50% ethanol, suggesting that specific CYP induction does not depend on the route of ethanol administration. Interestingly, the levels of other hepatic CYPs and the activation of NMBA were not induced even by a 5-week treatment. The mutagenic activity of NMBA in the presence of liver S9 from ethanol-treated rats was significantly inhibited by metyrapone, but
Fig. 2. Immunoblots and densitometric determination of the expression of the CYP protein in lung, kidney and colon microsomes from rats treated with ethanol. Microsomes were pooled from seven rats each treated with vehicle (lane 1, V) or 10% ethanol (lane 2, E) for 5 weeks. Lanes acetone, PB and MC contain CYP standards, and the CYP2A6 standard was obtained from a human B lymphoblastoid cell line. The values represent means ± SD of the ratio to the vehicle group (comprising 4–6 experiments) expressed in arbitrary units. The electrophoresis assays were carried out with 10 μg microsomal protein, except for the case of colon microsomes, of which 60 μg microsomal protein was loaded. *P < 0.01, compared with the vehicle group (Student’s t-test). n.d. = not detected.
not by 4-methylpyrazole. This supports our previous finding that NMBA was mutagenically activated by hepatic CYP2B2, but not by CYP2E1 in phenobarbital (PB)-induced rats (11). Therefore, it is reasonable to suppose that the mutagenic activation of NMBA was not enhanced by ethanol in the rat liver. Conflicting results have been reported for the inductive potentials of UGT activities by ethanol and we have discussed this matter in a previous report (11). NMBA is known to be a substrate for UGT (29), but no alterations to the activities of the three forms of hepatic UGT were observed in either group of ethanol-treated rats. Consequently, ethanol does not affect the hepatic activation or detoxification of NMBA by these enzymes in rats.
It has been reported that ethanol treatment induces not only oesophageal cancer but also pulmonary, renal and colorectal cancers in rodents (30,31). Ethanol caused an increase in the
levels of CYP2E1 in oesophageal and renal tissue, but not in the lung or colon. These results are consistent with other findings in which the CYP2E1 level is induced in the oesophagus (15,19) and kidney (32) by feeding rats with liquid diets containing ethanol. In contrast, it has also been reported that pulmonary and colonic CYP2E1 is induced by ethanol treatment in rats (33,34). The induction of CYP2E1 by ethanol can be attributed to protein stabilisation by protection from cytosolic degradation (35). Furthermore, the ethanol induction of renal and hepatic CYP2E1 involves an additional transcriptional component when blood ethanol concentrations exceed a threshold value of 200–300 mg/dl (32). Therefore, this discrepancy may be due to a lower induction of CYP2E1 by ethanol in the lung (1.2- to 3.0-fold) compared with that of the kidney (5.0- to 9.0-fold) (36). Together with reports that ethanol does not produce an increase in colonic CYP2E1 (19), induction in the lung
Fig. 3. Immunoblots and densitometric determination of the expression of the CYP protein in liver, oesophagus, kidney, lung and colon microsomes from rats treated orally with 50% ethanol. These microsomes were pooled from 10 rats that were each treated twice with vehicle (V) or ethanol (E). Lanes acetone, PB and MC contain CYP standards, and the CYP2A6 standard was obtained from a human B lymphoblastoid cell line. The values represent the means ± SD as described in the legends for Figure 1 (liver and oesophagus) and Figure 2 (kidney, lung and colon). *P < 0.05, compared with the vehicle group (Student’s t-test). n.d. = not detected.
Table I. Mutagenic activities of NOCs, HCAs and AFB1 in the S. typhimurium tester strains TA100 and TA98 in the presence of liver S9 from rats treated with NMBA, ethanol or both for 5 weeks
Group Treatment TA100 revertants/plate TA98 revertants/plate
Each test was carried out in duplicate (4–8 plates) with liver S9 pooled from seven rats. Data are given as means ± SD after subtraction of spontaneous revertants (TA100, 110-135; TA98, 13-19). The values in parentheses show the ratio to the activity obtained with the vehicle group.*P < 0.01, compared with the vehicle group (one-way ANOVA followed by Tukey–Kramer test).
and colon may be undetectable in these experiments. In rats, CYP2A1 and CYP2A2 are predominantly expressed in the liver, whereas CYP2A3 is expressed in extrahepatic tissues. Oesophageal and renal CYP2A were significantly increased by intragastric treatment with 50% ethanol, supporting previous findings that renal CYP2A was increased 1.9-fold, albeit not significantly, by a combination of ethanol and a low-fat diet (32). However, clear enhancement of CYP2A was not observed in these tissues from rats treated with 10% ethanol in the drinking water. The reason for this discrepancy is entirely unknown, but activation of NMBA in rat oesophagus by ethanol performed on the initiation phase. Although other researchers have reported that CYP2A3 mRNA was not detected in the kidneys of untreated rats (37). We tested CYP2A activity in the rat kidney by using the inhibitory effect of coumarin and methoxsalen on the mutagenic activity of NMBA in the presence of kidney S9. Additionally, this is the first report in which ethanol has been shown to induce oesophageal CYP2A protein in rodents. Oesophageal CYP2A3 plays a role in the metabolic activation of methyl-n-amylnitrosamine, DEN and NMBA (14,16,38,39), suggesting that ethanol may enhance the oesophageal carcinogenesis that is initiated by these carcinogens. Expression of the CYP2A3 gene is subject to a complex regulatory mechanism that varies from tissue to tissue (37). In conjunction with the finding that ethanol did not induce hepatic CYP2A1/2, further investigation of the mechanism of tissue-specific ethanol induction of CYP2A is required.
Enhancement of the mutagenic activities of DEN, DMN and NPYR with tissue S9 by ethanol was observed in the
oesophagus, kidney and liver. In addition, ethanol increased the mutagenicity of NMBA and NNK in the presence of oesophageal or renal S9. These results are consistent with the finding that an addition of 5% ethanol to drinking water increases the mutagenicity of NMBA in the rat oesophagus (40). They are also in agreement with reports that NMBA-induced oesophageal carcinogenesis is promoted by ethanol in drinking water (7) or liquid diets containing ethanol (19). This enhancement would be associated with increases not in CYP2E1 but in CYP2A because this activation was suppressed by coumarin and methoxsalen but not 4-methylpyrazole. This is in accordance with findings that the CYP2A3 enzyme, expressed in baculovirus, catalyses the conversion of NMBA to benzaldehyde (14) and that the oesophageal CYP2A3 level in the rat can be a determinant of its ability to metabolise NMBA in vivo (39). However, the inhibition by coumarin and methoxsalen was lower than that observed with SKF-525A in the oesophagus or kidney. This supports the finding that a CYP subfamily other than CYP2A3 is responsible for catalysing the methylene hydroxylation of NMBA (39,41). Metyrapone and orphenadrine produced a small but significant inhibition of the mutagenicities in these tissues. Suppression of NMBA-induced oesophageal carcinogenesis by curcumin can be attributed to a reduction in the metabolic activation of NMBA by oesophageal CYP2B1 (20). The inhibition by SKF-525A was also observed in lung and colon, indicating that other unconfirmed CYP species were associated with activation in these tissues. In conjunction with these findings, multiple CYP species might be relevant
NPYR DMN DEN NMBA NNK0
50
100
150
200TA
100
Reve
rtan
ts/p
late
NPYR DMN DEN NMBA NNK0
50
100
150
200
250
TA10
0 Re
vert
ants
/pla
te
NPYR DMN DEN NMBA NNK0
30
60
90
120
150
TA10
0 Re
vert
ants
/pla
te
NPYR DMN DEN NMBA NNK0
200
400
600
800
1000TA
100
Reve
rtan
ts/p
late
NPYR DMN DEN NMBA NNK0
50
100
150
200
TA10
0 Re
vert
ants
/pla
te
*
**
**
*
B
D E
**
**
A
*
*
*
C
Fig. 4. Effect of two treatments with 50% ethanol on the mutagenic activities of NMBA, DEN, DMN and NPYR in the S. typhimurium strain TA100 in the presence of liver (A), oesophagus (B), kidney (C), lung (D) or colon (E) S9. Each test was carried out in duplicate (4–8 plates) with tissue S9 pooled from 16 rats each treated with vehicle (open bars) or 50% ethanol (solid bars). Each bar represents the means ± SD after subtraction of spontaneous revertants (89–105). *P < 0.05, compared with the vehicle group (Student’s t-test).
to activation of NMBA and responsible enzymes depend on the class of the tissue. Anyway, this is the first demonstration that ethanol exerts an enhancing effect on the mutagenic activation of NMBA by an action on oesophageal CYP2A in rats. In addition, activation of NMBA is predominantly associated with multiple CYP proteins including CYP2A and CYP2B, but not in the case for CYP2E.
In conclusion, it has been demonstrated that ethanol administered either to drinking water or intragastrically results in an enhancing effect on the levels of oesophageal and renal CYP2A and CYP2E1 and hepatic CYP2E1, and the mutagenic
activation of five NOCs, including NMBA, in the oesophagus and kidney. Consequently, this suggests that this ethanol-mediated enhancement of NMBA-induced oesophageal carcinogenesis in F344 rats can be attributed to an increase in the metabolic activation of NMBA by oesophageal CYP2A. This occurs during the initiation phase without the contribution of metabolic activation or inactivation by glucuronidation in the liver. Furthermore, activation of NMBA is predominantly associated with CYP2A and CYP2B, but not CYP2E. This difference suggests that N-nitrosodialkylamines with bulky groups are activated by CYP2A and CYP2B, whereas those with relatively short chain(s) are activated by CYP2E1 (42). The present data also suggest that treatment with ethanol may enhance the carcinogenesis initiated by DMN (43) and NPYR (4) or the oesophageal and renal carcinogenesis initiated by other carcinogens activated by CYP2A or CYP2E1 (44). Tissue-specific activation of N-nitrosopiperidine and NPYR contributes to organ-specific carcinogenicities (45), suggesting that CYP expression in the relevant organ may play an important role in tumorigenesis by inducing NOCs during the initiation phase.
Funding
This work was supported (in part) by a grant from the Smoking Research Foundation.
Metyrapone
Orphenadrine
4-Methylpyrazole
Coumarin
Methoxalen
SKF-525A0
20
40
60
80
100
% o
f con
trol
A
*
*
Metyrapone
Orphenadrine
4-Methylpyrazole
Coumarin
Methoxalen
SKF-525A0
20
40
60
80
100
% o
f con
trol
D
*
Metyrapone
Orphenadrine
4-Methylpyrazole
Coumarin
Methoxalen
SKF-525A0
20
40
60
80
100
% o
f con
trol
E
*
Metyrapone
Orphenadrine
4-Methylpyrazole
Coumarin
Methoxalen
SKF-525A0
20
40
60
80
100
% o
f con
trol
C
*
*
***
Metyrapone
Orphenadrine
4-Methylpyrazole
Coumarin
Methoxalen
SKF-525A0
20
40
60
80
100
% o
f con
trol
B
*
**
* *
Fig. 5. Effect of CYP inhibitors on the mutagenic activity of NMBA on liver (A), oesophagus (B), kidney (C) lung (D) or colon (E) S9 from rats treated with 50% ethanol. Each bar represents the mean ± SD (4–6 plates) expressed as a percentage of the control. Activities of control samples are referred to the data of tissue S9 from rats treated with 50% ethanol shown in Figure 4. *P < 0.01, compared with the vehicle group (one-way ANOVA followed by Tukey–Kramer test).
Table II. UDPGT activities in liver microsomes from rats treated with NMBA, ethanol or both
The authors very thanks to Prof. Shin-ichi Kondo for proofing this article and giving many crucial advices.
Conflict of interest statement: None declared.
References
1. Seitz, H. K. and Stickel, F. (2007) Molecular mechanisms of alcohol-medi-ated carcinogenesis. Nat. Rev. Cancer, 7, 599–612.
2. Phillips, B. J. and Jenkinson, P. (2001) Is ethanol genotoxic? A review of the published data. Mutagenesis, 16, 91–101.
3. Johansson, I., Ekström, G., Scholte, B., Puzycki, D., Jörnvall, H. and Ingelman-Sundberg, M. (1988) Ethanol-, fasting-, and acetone-inducible cytochromes P-450 in rat liver: regulation and characteristics of enzymes belonging to the IIB and IIE gene subfamilies. Biochemistry, 27, 1925–1934.
4. Yang, C. S., Patten, C., Lee, M. J., Li, M., Yoo, J. S., Pan, J. and Hong, J. (1987) Enzymatic Mechanisms in the Metabolic Activation of N-nitrosodialkylamines. IARC Scientific Publication, Lyon.
5. Mori, Y., Yamazaki, H., Toyoshi, K., Makino, T., Obara, T., Yokose, Y. and Konishi, Y. (1985) Mutagenic activation of carcinogenic N-nitrosopropylamines by rat liver: evidence for a cytochrome P-450 dependent reaction. Carcinogenesis, 6, 415–420.
6. Aze, Y., Toyoda, K., Furukawa, F., Mitsumori, K. and Takahashi, M. (1993) Enhancing effect of ethanol on esophageal tumor development in rats by initiation of diethylnitrosamine. Carcinogenesis, 14, 37–40.
7. Kaneko, M., Morimura, K., Nishikawa, T., Wanibuchi, H., Osugi, H., Kinoshita, H., Koide, A., Mori, Y. and Fukishima, S. (2002) Weak enhanc-ing effects of simultaneous ethanol administration on chemically induced rat esophageal tumorigenesis. Oncol. Rep., 9, 1069–1073.
8. Kardon, T., Coffey, M. J., Bánhegyi, G., Conley, A. A., Burchell, B., Mandl, J. and Braun, L. (2000) Transcriptional induction of bilirubin UDP-glucuronosyltransrase by ethanol in rat liver. Alcohol, 21, 251–257.
9. Kundu, R., Dasgupta, S., Biswas, A., Bhattacharya, A., Pal, B. C., Bandyopadhyay, D., Bhattacharya, S. and Bhattacharya, S. (2008) Cajanus cajan Linn. (Leguminosae) prevents alcohol-induced rat liver damage and augments cytoprotective function. J. Ethnopharmacol., 118, 440–447.
10. Wiench, K., Frei, E., Schroth, P. and Wiessler, M. (1992) 1-C-glucuronidation of N-nitrosodiethylamine and N-nitrosomethyl-n-pentylamine in vivo and in primary hepatocytes from rats pretreated with inducers. Carcinogenesis, 13, 867–872.
11. Mori, Y., Koide, A., Kobayashi, Y., Morimura, K., Kaneko, M. and Fukushima, S. (2002) Effect of ethanol treatment on metabolic activation and detoxification of esophagus carcinogenic N-nitrosamines in rat liver. Mutagenesis, 17, 251–256.
12. Lijinsky, W., Saavedra, J. E., Reuber, M. D. and Singer, S. S. (1982) Esophageal carcinogenesis in F344 rats by nitrosomethylethylamines sub-stituted in the ethyl group. J. Natl Cancer Inst., 68, 681–684.
13. Labuc, G. E. and Archer, M. C. (1982) Esophageal and hepatic microsomal metabolism of N-nitrosomethylbenzylamine and N-nitrosodimethylamine in the rat. Cancer Res., 42, 3181–3186.
14. Gopalakrishnan, R., Morse, M. A., Lu, J., Weghorst, C. M., Sabourin, C. L., Stoner, G. D. and Murphy, S. E. (1999) Expression of cytochrome P450 2A3 in rat esophagus: relevance to N-nitrosobenzylmethylamine. Carcinogenesis, 20, 885–891.
15. Tsutsumi, M., George, J., Ishizawa, K., Fukumura, A. and Takase, S. (2006) Effect of chronic dietary ethanol in the promotion of N-nitrosomethylbenzylamine-induced esophageal carcinogenesis in rats. J. Gastroenterol. Hepatol., 21, 805–813.
16. Pinto, L. F., Moraes, E., Albano, R. M., Silva, M. C., Godoy, W., Glisovic, T. and Lang, M. A. (2001) Rat oesophageal cytochrome P450 (CYP) monooxygenase system: comparison to the liver and relevance in N-nitrosodiethylamine carcinogenesis. Carcinogenesis, 22, 1877–1883.
17. Kouros, M., Mönch, W., Reiffer, F. J. and Dehnen, W. (1983) The influ-ence of various factors on the methylation of DNA by the oesophageal carcinogen N-nitrosomethylbenzylamine. I. The importance of alcohol. Carcinogenesis, 4, 1081–1084.
18. Farinati, F., Lieber, C. S. and Garro, A. J. (1989) Effects of chronic ethanol consumption on carcinogen activating and detoxifying systems in rat upper alimentary tract tissue. Alcohol. Clin. Exp. Res., 13, 357–360.
19. Shimizu, M., Lasker, J. M., Tsutsumi, M. and Lieber, C. S. (1990) Immunohistochemical localization of ethanol-inducible P450IIE1 in the rat alimentary tract. Gastroenterology, 99, 1044–1053.
20. Mori, Y., Tatematsu, K., Koide, A., Sugie, S., Tanaka, T. and Mori, H. (2006) Modification by curcumin of mutagenic activation of carcinogenic
N-nitrosamines by extrahepatic cytochromes P-450 2B1 and 2E1 in rats. Cancer Sci., 97, 896–904.
21. Yahagi, T., Nagao, M., Seino, Y., Matsushima, T. and Sugimura, T. (1977) Mutagenicities of N-nitrosamines on Salmonella. Mutat. Res., 48, 121–129.
22. Yamazaki, H., Mimura, M., Sugahara, C. and Shimada, T. (1994) Catalytic roles of rat and human cytochrome P450 2A enzymes in testosterone 7 alpha- and coumarin 7-hydroxylations. Biochem. Pharmacol., 48, 1524–1527.
23. Koide, A., Fuwa, K., Furukawa, F., Hirose, M., Nishikawa, A. and Mori, Y. (1999) Effect of cigarette smoke on the mutagenic activation of environ-mental carcinogens by rodent liver. Mutat. Res., 428, 165–176.
24. Heirwegh, K. P., Van de Vijver, M. and Fevery, J. (1972) Assay and prop-erties of dititonin-activated bilirubin uridine diphosphate glucuronyltrans-ferase from rat liver. Biochem. J., 129, 605–618.
25. Isselbacher, K. J., Chrabas, M. F. and Quinn, R. C. (1962) The solubiliza-tion and partial purification of a glucuronyl transferase from rabbit liver microsomes. J. Biol. Chem., 237, 3033–3036.
26. Matern, H., Heinemann, H. and Matern, S. (1994) Radioassay of UDP-glucuronosyltransferase activities toward endogenous substrates using labeled UDP-glucuronic acid and an organic solvent extraction procedure. Anal. Biochem., 219, 182–188.
27. Murai, T., Mori, Y., Tatematsu, K., Koide, A., Hagiwara, A., Makino, S., Mori, S., Wanibuchi, H. and Fukushima, S. (2005) Differences in suscep-tibility to N-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder carcinogenesis between SD/gShi rats with spontaneous hypospermatogen-esis and SD/cShi rats with spontaneous hydronephrosis. Cancer Sci., 96, 637–644.
28. Tatematsu, K., Koide, A., Hirose, M., Nishikawa, A. and Mori, Y. (2011) Effect of cigarette smoke on mutagenic activation of environmental car-cinogens by cytochrome P450 2A8 and inactivation by glucuronidation in hamster liver. Mutagenesis, 26, 323–330.
29. Wiessler, M. and Rossnagel, G. (1987) Alpha-glucuronides of N-nitrosomethylbenzylamine. IARC. Sci. Publ., 84, 170–172.
30. Anderson, L. M., Chhabra, S. K., Nerurkar, P. V., Souliotis, V. L. and Kyrtopoulos, S. A. (1995) Alcohol-related cancer risk: a toxicokinetic hypothesis. Alcohol, 12, 97–104.
31. Seitz, H. K., Czygan, P., Simanowski, U., Waldherr, R., Veith, S., Raedsch, R. and Kommerell, B. (1985) Stimulation of chemically induced rectal carcinogenesis by chronic ethanol ingestion. Alcohol Alcohol, 20, 427–433.
32. Ronis, M. J., Huang, J., Longo, V., Tindberg, N., Ingelman-Sundberg, M. and Badger, T. M. (1998) Expression and distribution of cytochrome P450 enzymes in male rat kidney: effects of ethanol, acetone and dietary condi-tions. Biochem. Pharmacol., 55, 123–129.
33. Hakkak, R., Korourian, S., Ronis, M. J., Ingelman-Sundberg, M. and Badger, T. M. (1996) Effects of diet and ethanol on the expression and localization of cytochromes P450 2E1 and P450 2C7 in the colon of male rats. Biochem. Pharmacol., 51, 61–69.
34. Carlson, G. P. (1990) Induction of N-nitrosodimethylamine metabolism in rat liver and lung by ethanol. Cancer Lett., 54, 153–156.
35. Roberts, B. J., Song, B. J., Soh, Y., Park, S. S. and Shoaf, S. E. (1995) Ethanol induces CYP2E1 by protein stabilization. Role of ubiquitin conjugation in the rapid degradation of CYP2E1. J. Biol. Chem., 270, 29632–29635.
36. Zerilli, A., Lucas, D., Amet, Y., Beauge, F., Volant, A., Floch, H. H., Berthou, F. and Menez, J. F. (1995) Cytochrome P-450 2E1 in rat liver, kidney and lung microsomes after chronic administration of ethanol either orally or by inhalation. Alcohol Alcohol., 30, 357–365.
37. Robottom-Ferreira, A. B., Aquino, S. R., Queiroga, R., Albano, R. M. and Ribeiro Pinto, L. F. (2003) Expression of CYP2A3 mRNA and its regu-lation by 3-methylcholanthrene, pyrazole, and beta-ionone in rat tissues. Braz. J. Med. Biol. Res., 36, 839–844.
38. Chen, S. C., Wang, X., Xu, G., Zhou, L., Vennerstrom, J. L., Gonzalez, F., Gelboin, H. V. and Mirvish, S. S. (1999) Depentylation of [3H-pentyl]methyl-n-amylnitrosamine by rat esophageal and liver microsomes and by rat and human cytochrome P450 isoforms. Cancer Res., 59, 91–98.
39. Gopalakrishnan, R., Gupta, A., Carlton, P. S., Morse, M. A. and Stoner, G. D. (2002) Functional role of cytochrome p-450 2a3 in N-nitrosomethylbenzylamine metabolism in rat esophagus. J. Toxicol. Environ. Health Part A, 65, 1077–1091.
40. de Boer, J. G., Yang, H., Holcroft, J. and Skov, K. (2004) Chemoprotection against N-nitrosomethylbenzylamine-induced mutation in the rat esophagus. Nutr. Cancer, 50, 168–173.
41. von Weymarn, L. B., Felicia, N. D., Ding, X. and Murphy, S. E. (1999) N-Nitrosobenzylmethylamine hydroxylation and coumarin 7-hydroxyla-tion: catalysis by rat esophageal microsomes and cytochrome P450 2A3 and 2A6 enzymes. Chem. Res. Toxicol., 12, 1254–1261.
42. Kushida, H., Fujita, K., Suzuki, A., Yamada, M., Endo, T., Nohmi, T. and Kamataki, T. (2000) Metabolic activation of N-alkylnitrosamines in geneti-cally engineered Salmonella typhimurium expressing CYP2E1 or CYP2A6 together with human NADPH-cytochrome P450 reductase. Carcinogenesis, 21, 1227–1232.
43. Schwarz, M., Wiesbeck, G., Hummel, J. and Kunz, W. (1982) Effect of ethanol on dimethylnitrosamine activation and DNA synthesis in rat liver. Carcinogenesis, 3, 1071–1075.
44. Navasumrit, P., Ward, T. H., O’Connor, P. J., Nair, J., Frank, N. and Bartsch, H. (2001) Ethanol enhances the formation of endogenously and exogenously derived adducts in rat hepatic DNA. Mutat. Res., 479, 81–94.
45. Wong, H. L., Murphy, S. E., Wang, M. and Hecht, S. S. (2003) Comparative metabolism of N-nitrosopiperidine and N-nitrosopyrrolidine by rat liver and esophageal microsomes and cytochrome P450 2A3. Carcinogenesis, 24, 291–300.
