Mutagenicity of 7H-dibenzo[c,g]carbazole and its tissue specific derivatives in genetically engineered Chinese hamster V79 cell lines stably expressing cytochrome P450

Mutation Research 517 (2002) 135–145

Mutagenicity of 7H-dibenzo[c,g]carbazole and its tissue specific derivatives in genetically engineered Chinese hamster V79 cell lines stably expressing cytochrome P450 Alena Gábelová∗ , Timea Farkašová, Gabriela Baˇcová, Soˇna Robichová Cancer research Institute, Department of Mutagenesis and Carcinogenesis, Vlárska, 7, 833 91 Bratislava, Slovak Republic Received 29 October 2001; received in revised form 8 March 2002; accepted 8 March 2002

Abstract Genetically engineered Chinese hamster V79 cell lines with stable expression of human cytochrome P4501A1 and 1A2 were used to characterize the particular form of P450 enzymes capable of activating 7H-dibenzo[c,g]carbazole (DBC) and its tissueand organ-specific derivatives, N-methylDBC (N-MeDBC) and 5,9-dimethylDBC (diMeDBC). In addition, a V79 cell line with co-expression of CYP1A2 together with a phase II enzyme, N-acetyltransferase was utilized to study the role of an entire metabolic activation system in biotransformation of these carbazoles. The rise of 6-thioguanine resistant (6-TGr ) mutations was followed as a marker of biological activity of these agents. None of the carbazoles elevated significantly the frequency of mutations in the parental V79MZ cell line lacking any cytochrome P450 (CYP) activity or in the V79NH cells expressing N-acetyltransferase activity. A variable, however, increase of mutations was found in the cell lines expressing CYP activity. Both DBC, a potent liver and skin carcinogen, and N-MeDBC, a specific sarcomagen, increased significantly (P < 0.001) the frequency of 6-TGr mutations in V79MZh1A1 cells, expressing the human CYP1A1; in contrast, a strict hepatocarcinogen diMeDBC was devoid of any activity. All carbazoles elevated significantly the level of mutations in the V79MZh1A2 cell line expressing the human CYP1A2, N-MeDBC was most efficient. Co-expression of CYP1A2 together with NAT activity significantly reduced or totally eliminated the mutagenicity of all carbazoles. These data confirm that CYP1A1 is explicitly involved in the activation of sarcomagenic DBC derivatives, whereas CYP1A2 is included in biotransformation of all DBC derivatives. Reactive intermediates formed due to CYP1A2 activation are substrate for conjugation reactions mediated by N-acetyltransferase. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Genetically engineered V79 cell lines; 6-Thioguanine resistant mutations; 7H-dibenzo[c,g]carbazole; N-methyldibenzo[c,g]carbazole; 5,9-dimetyldibenzo[c,g]carbazole

1. Introduction A characteristic hallmark of many chemical carcinogens is their strict organ- or tissue-specificity. While the target organs of the aromatic amines are liver and ∗ Corresponding author. Tel.: +421-2-59327-202; fax: +421-2-59327-506. E-mail address: [email protected] (A. G´abelov´a).

urinary bladder, these agents are inactive against epidermis. In contrast, polycyclic aromatic hydrocarbons (PAHs) have strong local activity against lung and skin but have no effect on liver. A number of approaches have been undertaken to identify the factors responsible for this phenomenon. One reason of such strict specificity could be the chemical structure of the molecule that determines the mode of biotransformation. Most of the chemical carcinogens require

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metabolic activation to reactive electrophilic intermediates before they can bind covalently to DNA and other macromolecules and exert their mutagenic and carcinogenic effects [1]. A majority of activation reactions involves oxidations that are primarily mediated by cytochrome P450 (CYP) enzymes. Cytochrome P450s represent a superfamily of the most important activation enzymes catalyzing oxidative metabolism of various endogenous substances as well as xenobiotics [2]. It is supposed that differences in the expression of drug-metabolizing enzymes in the target tissues could also greatly contribute to the variation in the cellular response to chemical carcinogens. Although, the phase II enzymes catalyzing the conjugation reactions play a key role mainly in the detoxification processes, they could be also involved in the activation pathways of some procarcinogens [3,4]. 7H-dibenzo[c,g]carbazole (DBC), a ubiquitous environmental pollutant, is formed during the incomplete combustion of organic materials. DBC is a component of a variety of complex mixtures as tobacco smoke condensate, synthetic coal fuel and coal tar. Carcinogenicity of DBC was demonstrated in a number of different species including mouse, rat, hamster and dog [5]. IARC [6,7] listed DBC as a potential human carcinogen, though the extent of human exposure is unknown [8]. DBC is unusual in its carcinogenic re-

sponse, it induces tumors at the site of its application as well as at distant sites particularly in the liver. The chemical structure of DBC joins the features of PAHs via the aromatic skeleton with a typical bay region, while the pyrrolic NH group has a chemical behavior similar to that of the arylamides (Fig. 1). To study the mechanisms of DBC biotransformation, various methylated derivatives were synthesized by substitution of a methyl group at different position of the molecule. Among these derivatives, diMeDBC and N-MeDBC manifested specific tropism for the liver and skin, respectively. Metabolic studies in vivo [9] and in vitro [10,11] suggested that the sarcomagenic activity of DBC probably depends on PAH-type activation, whereas the heterocyclic nitrogen of DBC could play a key role in liver carcinogenicity. Hydroxylation at the aromatic ring, leading to phenols, is the major metabolic pathway in which the positions 3 and 5 of DBC are involved [10]. It is assumed that the position 3 plays an important role in the sarcomagenic activity. Monomethylation of DBC at this position resulted in a suppression of sarcomagenic activity and in an increase in hepatocarcinogenicity [12]. A severe hepatic toxicity and carcinogenicity is related to a specific metabolic activation of the NH group [10,13]. This judgement is supported by the fact that blocking the activation of the nitrogen by N-methylation leads

Fig. 1. Formulas of DBC and its tissue specific derivatives, N-MeDBC and diMeDBC.

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to drop of any hepatocarcinogenic potential [14]. The need for a free access to the NH group for activating enzymes supports also the disappearance of hepatic activity by steric hindrance due to the presence of two methyl groups at the positions 6 and 8 of DBC [12,15]. Moreover, the structurally related compounds O- and S-isoesters of DBC, dinaphtho(2,1,1 ,2 ) furan and dinaphtho(2,1,1 ,2 ) thiophene, respectively, lack carcinogenic activity altogether [16]. All attempts to identify N-hydroxylated or any other NH-associated derivative among DBC metabolites, as is the case for the liver active aromatic amines, however, have failed probably because of their instability [10]. The role of cytochrome P4501A-subfamily in biotransformation of DBC derivatives was suggested in in vitro experiments on primary mouse embryo fibroblasts [17]. DBC derivatives with sarcomagenic activity apart from diMeDBC induced DNA-adduct formation in these cells. Moreover, pretreatment of these cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a specific inducer of CYP1A-subfamily, [18], furthermore stimulated DNA-adduct formation. The in vivo evaluation of CYP1A-family induction by methylated derivatives of DBC as a measure of susceptibility to carcinogenesis in liver and skin yielded ambiguous results. While a clear correlation was found between the tissue-specificity of DNA binding and the capacity of particular DBC derivative to induce skin or liver tumors, no direct relationship was observed in their capacity to induce CYP1A1/1A2 [12]. Pretreatment of mouse strains with various inducers of CYP1A-family led even to the total or at least partial inhibition of diMeDBC-DNA-adduct formation in the liver [19]. Farkašová et al. [20] have recently demonstrated that CYP1A1 is probably involved in biotransformation of sarcomagenic DBC derivatives. While DBC and N- MeDBC increased significantlly the level of micronuclei in the genetically engineered V79 cells with stable expression of human CYP1A1, the organspecific hepatocarcinogen diMeDBC was inefficient. The aim of this study was to confirm the unique role of CYP1A1 in biotransformation of sarcomagenic derivatives of DBC and evaluate the function of CYP1A2 and conjugating enzyme, NAT, in biotransformation pathway of DBC and its tissue specific derivatives, N-methylDBC and 5,9-dimethylDBC. Mutagenicity of these carbazoles was measured in Chinese hamster V79 cell lines with stable expres-

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sion of human CYP1A1 and CYP1A2 with or without co-expression of NAT as well as in the parental V79MZ cells lacking any CYP activity and V79NH cells expressing acetyltransferase and nitroreductase activities. Benzo[a]pyrene (BaP), a well-known PAH, and aflatoxin B1 (AFB1), a proven hepatocarcinogen, were used as positive controls in this study. 2. Materials and methods 2.1. Cell lines V79 Chinese hamster cell lines: parental V79MZ lacking any CYP activity, V79MZh1A1 stably expressing human cytochrome P4501A1, V79MZh1A2 expressing human cytochrome P4501A2 [21], V79NH is devoid of CYP activity but expresses nitroreductase and N-acetyl-o-transferase (NAT2) activities and V79NH1A2 co-expressing of human CYP1A2 with NAT2 and nitroreductase activities [22,23]. Genetically engineered V79 cell lines were generously provided by Dr J. Doehmer (Munich, Germany). V79 cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 4.5 g/l glucose and 10% fetal calf serum and antibiotics (penicillin 200 U/ml; streptomycin and kanamycin 100 ␮g/ml). Genetically engineered V79 cells were cultured in the presence of 400 ␮g/ml geneticin (G418), which was not present during incubation with tested agents. 2.2. Chemicals DBC (CAS No. 194-59-2), N-MeDBC, diMeDBC were kindly provided by Dr. F. Périn, Institute Curie, France. The methyl derivatives were synthesized and tested for purity as described elsewhere [10,11,13]. BaP (CAS No. 50-32-8) and AFB1 (CAS No. 1162-65-8) were purchased from Sigma. All other chemicals used for cell cultivation (MEM, FCS, antibiotics) were purchased from GIBCO. The stock solutions of DBC, N-MeDBC, diMeDBC, AFB1, and BaP in DMSO (2 mM) were kept at −20 ◦ C and diluted immediately before use in medium. 2.3. Treatment of cells V79 cells (3 × 105 ) were plated on several petri dishes (∅ = 100 mm) and incubated at 37 ◦ C in a 5%

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CO2 atmosphere for 24 h. Next day 24 h cell exposure was performed. The stock solutions (2 mM) were diluted to the adequate concentrations in DMSO, from which 10 ␮l were added to the medium to reach the final concentrations 0.5, 1.0 and 2.5 ␮M in the case of BaP and carbazoles or 0.01, 0.05 and 0.1 ␮M in the case of AFB1. The treatment was finished by removal of the medium and two times wash of cells with fresh one.

petri dishes (∅ = 100 mm) for the analysis of 6-TGr mutations at the next expression time. Colonies of mutations were stained by methylene blue (1% solution) and counted 10 days after adding of 6-tg. The frequency of 6-TGr mutations per 1 × 105 viable cells was calculated at 6th and 9th day of sampling. The results were statistically evaluated by the Student’s t-test.

2.4. Colony forming ability of V79 cells (CFA)

3. Results

After finishing the treatment the cells were trypsinized and 3 × 102 V79 cells were plated on several petri dishes (∅ = 60 mm, in triplicate per sample). On the 7th day after treatment, they were stained with methylene blue (1% solution) and the number of colonies was counted. From the ratio of the number of colonies/cells plated the colony forming ability (CFA) in percentage was calculated.

3.1. Mutagenicity in the V79MZ and V79NH cells lacking any CYP activity

2.5. HPRT mutations A respreading mutation assay proposed by Chasin [24] and modified by Slameˇnová and Gábelová [25] was used for detection of 6-thioguanine resistant (6-TGr ) mutations. In brief, after finishing the treatment the cells were trypsinized and diluted as follows: (i) 3 × 102 V79 cells were plated on petri dishes (∅ = 60 mm, in triplicate per sample) to determine the cytotoxic effect of the agents as described above, (ii) 3.5 × 105 V79 cells per dish were plated on three petri dishes (∅ = 100 mm) for further cultivation. The V79 cells were kept by regular subculture at a certain cell density per surface unit in order to avoid overcrowding. At the 7th and 9th day of expression, the yield of 6-TGr mutations was measured. At that time the V79 cells from each sample were plated (a) on five petri dishes (∅ = 100 mm) at density of 2 × 105 per dish for detection of 6-TGr mutations; 1 h later, after attaching the cells, the selective agent 6-thioguanine (6-tg) was added to these dishes in the final concentration 5 ␮g/ml, (b) on three petri dishes (∅ = 60 mm) at density 3 × 102 cells per dish for estimation of viability of the cells (necessary for calculation of the frequency of 6-TGr mutations at appropriate time expression), (c) at the 7th day only, 3.5 × 105 V79 cells per plate from each sample were plated on three

The parental V79MZ cells lacking any drug-metabolizing activity and V79NH cells without any CYP activity but expressing nitroreductase and NAT2 activities were treated with equimolar concentration (1 ␮M) of DBC, N-MeDBC, diMeDBC, BaP and AFB1 for 24 h. Induction of 6-TGr mutations, an endpoint of biological activity of carbazoles, was followed at the 7th and the 9th day of expression. Under these conditions of treatment none of the agents increased significantly the frequency of 6-TGr mutations over the control (Tables 1 and 2). In contrast, the mutation frequencies were even slightly reduced. This decrease of mutations in the treated cells, however, was not due to the cytotoxicity of particular agent because the viability did not decrease below 65%. 3.2. Mutagenicity in genetically engineered V79 cell lines with stable expression of human cytochrome P4501A1 The ability of DBC, N-MeDBC, and diMeDBC to induce mutations in the V79 cell line with stable expression of human cytochrome P4501A1 was measured in the concentration range from 0.5 to 2.5 ␮M after 24 h of exposure (Table 3). Under these conditions of treatment DBC and its two tissue specific derivatives manifested a variable cytotoxic effect. The viability of the V79MZh1A1 treated cells spans mainly from 59 to 96%, only at the concentration 2.5 ␮M of N-MeDBC the viability was reduced to 51%. The mutagenicity of BaP was followed at the same concentrations as carbazoles,

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Table 1 The level of 6-TGr mutations per 1 × 105 viable V79MZ cells treated for 24 h with DBC, N-MeDBC, diMeDBC, BaP or AFB1 Agent

Control DBC N-MeDBC diMeDBC BaP AFB1

Concentration (␮M)

0.0 1.0 1.0 1.0 1.0 1.0

Cloning efficiency (%)a

Expression timea

0 min

R

7 Days

100.0 76.7 83.1 105.3 100.0 87.9

2.68 1.95 0.52 0.77 <0.20 2.52

88.00 67.56 73.11 92.67 88.00 77.33

± ± ± ± ± ±

1.45 3.66 3.91 3.86 1.41 4.49

± ± ± ±

0.79 0.96 0.70 0.44

± 0.64

MF

9 Days

1.00 0.73 0.19 0.29 <0.07 0.94

3.08 2.99 1.17 1.09 0.53 2.78

± ± ± ± ± ±

MF 1.25 1.49 0.96 0.91 0.72 1.44

1.00 0.97 0.39 0.35 0.17 0.90

R: ratio of cloning efficiency in treated to untreated cells, MF: ratio of induced to spontaneous mutations. a Values represent mean ± S.D.

however, the cytotoxicity of BaP ranged from 40 to 15%. Both DBC and N-MeDBC induced a statistically significant (P < 0.01) increase of mutations in these cells. The tissue specific sarcomagen N-MeDBC was even more efficient in mutation induction than the positive control BaP. Neither dose nor time-dependent yield of 6-TGr mutations was found. A comparison of the relative mutation frequency (MF) among these three compounds led to following ranking: N-MeDBC > BaP > DBC. The organ-specific hepatocarcinogen diMeDBC did not elevate the level of mutations in the V79MZh1A1 cells. A slight but significant increase of mutations was found only at the concentration 0.5 ␮M at the 7th day of expression (P < 0.05), however, this rise was abolished at the 9th day of expression (Table 3). These data confirmed that human cytochrome CYP1A1 is explicitly involved in the biotransformation of DBC derivatives with sarcomagenic activity.

3.3. Mutagenicity in genetically engineered V79 cell lines with stable expression of human cytochrome P4501A2 and CYP1A2 with co-expression with NAT No strong cytotoxic effect of carbazoles in the concentration range from 0.5 to 2.5 ␮M was found in both the V79MZh1A2 and V79NHh1A2 cells. The viability of treated V79MZh1A2 cells was comparable to untreated cells except for diMeDBC at concentration 2.5 ␮M (Table 4). In contrast, a strong cytotoxicity was estimated at these concentrations for AFB1 due to activation via CYP1A2 (data not shown), therefore in the mutation experiments the genotoxicity of AFB1 was tested in the concentrations ranging from 0.01 to 0.1 ␮M (Table 4). Co-expression of N-acetyltransferase together with CYP1A2, however, led to a marked loss of cytotoxicity primarily in the case of AFB1 (Table 5). All carbazoles increased the level of 6-TGr mutations in the V79MZh1A2 cells (Table 4). DBC and

Table 2 The level of 6-TGr mutations per 1 × 105 viable V79NH cells treated for 24 h with DBC, N-MeDBC, diMeDBC, BaP or AFB1 Agent

Control DBC N-MeDBC diMeDBC BaP AFB1

Concentration (␮M)

0.0 1.0 1.0 1.0 1.0 1.0

Cloning efficiency (%)a

Expression timea

0 min

R

7 Days

100.0 106.5 92.8 103.1 104.8 111.1

1.53 0.77 0.42 0.66 1.04 0.29

91.45 97.42 84.87 94.26 95.89 101.67

± ± ± ± ± ±

5.34 12.16 3.37 6.39 10.31 17.56

± ± ± ± ± ±

1.12 0.63 0.38 0.79 0.64 0.44

MF

9 Days

1.00 0.50 0.27 0.43 0.68 0.19

1.71 1.33 >0.11 1.69 1.36 1.51

R: ratio of cloning efficiency in treated to untreated cells, MF: ratio of induced to spontaneous mutations. a Values represent mean ± S.D.

± 1.09 ± 0.46 ± 0.91 ± 0.51 ± 0.64

MF 1.00 0.78 0.06 0.99 0.79 0.88

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Table 3 The level of 6-TGr mutations per 1 × 105 viable V79MZh1A1 cells treated for 24 h with DBC, N-MeDBC, diMeDBC or BaP Agents

Concentration (␮M)

Cloning efficiency (%)a

Expression timea

0 min

R

7 Days

MF

9 Days

MF

DBC

0.0 0.5 1.0 2.5

114.89 68.10 67.90 66.90

± 5.23 ± 3.40 ± 7.80 ±1.35

100.0 59.3 59.1 58.2

5.55 18.19 17.14 23.52

± ± ± ±

1.37 5.21∗∗∗ 4.87∗∗∗ 3.48∗∗∗

1.00 3.28 3.09 4.24

2.41 15.08 9.15 15.50

± ± ± ±

1.29 2.22∗∗∗ 2.28∗∗∗ 3.95∗∗∗

1.00 6.26 3.79 6.43

N-MeDBC

0.0 0.5 1.0 2.5

110.78 73.76 96.00 51.67

± ± ± ±

4.73 3.24 1.53 3.06

100.0 66.6 86.7 46.6

1.04 27.94 34.41 25.51

± ± ± ±

0.64 3.14∗∗∗ 5.35∗∗∗ 2.27∗∗∗

1.00 26.86 33.09 24.53

0.832 19.92 24.55 30.94

± ± ± ±

0.69 2.47∗∗∗ 1.01∗∗∗ 3.36∗∗∗

1.00 24.00 29.58 37.28

diMeDBC

0.0 0.5 1.0 2.5

115.22 67.67 70.11 90.00

± ± ± ±

9.98 9.20 14.42 6.64

100.0 58.7 60.1 78.1

3.87 5.61 3.30 4.59

± ± ± ±

1.08 1.74∗ 1.28 3.04

1.00 1.45 0.85 1.19

4.37 3.86 1.61 3.52

± ± ± ±

1.44 1.48 0.63 1.48

BaP

0.0 0.5 1.0 2.5

110.58 75.33 59.00 85.67

± ± ± ±

4.75 3.76 4.71 11.29

100.0 68.1 53.3 77.5

2.54 31.34 29.04 34.06

± ± ± ±

1.96 4.27∗∗∗ 3.24∗∗∗ 5.59∗∗∗

1.00 12.34 11.43 13.41

3.14 24.45 32.46 35.34

± ± ± ±

1.43 3.14∗∗∗ 5.11∗∗∗ 3.72∗∗∗

1.00 0.88 0.37 0.81 1.00 7.78 10.34 11.25

R: ratio of cloning efficiency in treated to untreated cells, MF: ratio of induced to spontaneous mutations. a Values represent mean ± S.D. ∗ Significantly different P < 0.05. ∗∗∗ Significantly different P < 0.001. Table 4 The level of 6-TGr mutations per 1 × 105 viable V79MZh1A2 cells treated for 24 h with DBC, N-MeDBC, diMeDBC or AFB1 Agents

Concentration (␮M)

Cloning efficiency (%)a

Expression timea

0 min

7 Days

R

MF

9 Days

MF

DBC

0.0 0.5 1.0 2.5

92.86 87.22 74.86 84.67

± ± ± ±

3.72 1.56 12.41 4.84

100.0 93.9 80.1

0.44 3.97 4.46 5.96

± ± ± ±

0.41 0.93∗∗∗ 0.89∗∗∗ 0.92 ∗∗∗

1.00 9.02 10.14 13.55

0.98 5.78 4.41 3.80

± ± ± ±

0.58 0.82∗∗∗ 1.29∗∗∗ 1.14∗∗∗

1.00 5.89 4.50 3.88

N-MeDBC

0.0 0.5 1.0 2.5

93.22 88.33 92.00 88.16

± ± ± ±

1.82 2.08 0.88 5.89

100.0 94.5 98.7 94.6

0.44 5.26 19.02 18.86

± ± ± ±

0.41 1.98∗∗∗ 4.87∗∗∗ 3.71∗∗∗

1.00 11.95 43.22 42.86

0.98 6.44 15.33 15.46

± ± ± ±

0.58 1.18∗∗∗ 3.21∗∗∗ 2.91∗∗∗

1.00 6.57 15.64 15.78

diMeDBC

0.0 0.5 1.0 2.5

111.67 92.76 85.33 59.66

± ± ± ±

3.22 3.56 9.89 1.15

100.0 83.3 76.4 53.4

0.55 2.25 1.71 3.73

± ± ± ±

0.44 1.16∗ 0.58∗ 0.57∗∗

1.00 4.10 3.11 6.78

0.44 1.56 2.95 1.99

± ± ± ±

0.41 0.58∗ 2.04∗ 0.24∗

1.00 3.54 6.70 4.52

AFB1

0.0 0.01 0.05 0.1

128.90 107.0 44.89 7.54

± ± ± ±

16.97 8.14 5.36 2.56

100.0 83.0 34.8 5.8

5.68 13.91 14.68 1.58

± ± ± ±

1.27 1.53∗∗∗ 2.12∗∗∗ 0.47

1.00 2.44 2.58 0.28

4.78 12.14 19.44 1.66

± ± ± ±

1.06 1.72∗∗∗ 4.77∗∗∗ 0.93

1.00 2.54 4.07 0.35

R: ratio of cloning efficiency in treated to untreated cells, MF: ratio of induced to spontaneous mutations. a Values represent mean ± S.D. ∗ Significantly different P < 0.05. ∗∗ Significantly different P < 0.01. ∗∗∗ Significantly different P < 0.001.

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Table 5 The level of 6-TGr mutations per 1 × 105 viable V79NHh1A2 cells treated for 24 h with DBC, N-MeDBC, diMeDBC or AFB1 Agents

Concentration (␮M)

Cloning efficiency (%)a

Expression timea

0 min.

R

7 Days

MF

9 Days

MF

DBC

0.0 0.5 1.0 2.5

89.11 82.67 82.53 83.72

± ± ± ±

3.19 1.52 4.52 4.61

100.0 92.8 92.6 93.9

3.48 2.87 3.48 2.39

± ± ± ±

0.88 0.55 2.52 1.59

1.00 0.82 1.00 0.69

2.84 2.47 2.58 2.00

± ± ± ±

1.59 1.31 1.73 1.56

1.00 0.87 0.91 0.70

N-MeDBC

0.0 0.5 1.0 2.5

89.67 77.56 88.53 79.59

± ± ± ±

5.16 5.21 3.02 7.07

100.0 86.5 98.7 88.7

2.45 4.71 3.06 3.89

± ± ± ±

1.58 1.91∗∗ 1.74 2.16

1.00 1.92 1.25 1.59

2.41 4.96 2.97 3.27

± ± ± ±

1.22 3.30∗ 1.92 1.52

1.00 2.06 1.23 1.36

diMeDBC

0.0 0.5 1.0 2.5

97.44 84.77 90.99 74.99

± ± ± ±

6.15 4.65 9.42 7.31

100.0 87.0 93.3 76.9

2.29 1.42 2.20 1.28

± ± ± ±

1.58 1.09 1.18 0.64

1.00 0.62 0.96 0.56

2.68 1.37 2.99 1.26

± ± ± ±

1.71 0.64 1.41 1.02

1.00 0.51 1.12 0.47

AFB1

0.0 0.01 0.05 0.1

104.00 97.22 87.98 91.88

± ± ± ±

5.54 6.51 9.31 8.94

100.0 93.5 84.6 88.3

2.82 2.16 1.31 1.10

± ± ± ±

1.52 0.89 0.68 0.82

1.00 0.76 0.46 0.39

1.89 2.55 1.49 1.06

± ± ± ±

0.94 1.33 0.73 0.72

1.00 1.35 0.79 0.56

R: ratio of cloning efficiency in treated to untreated cells, MF: ratio of induced to spontaneous mutations. a Values represent mean ± S.D. ∗ Significantly differentP < 0.5. ∗∗ Significantly different P < 0.01.

diMeDBC induced approximately the same relative MF in these cells, while N-MeDBC was twice more efficient. The mutagenicity of these compounds decreased in the order N-MeDBC > DBC = diMeDBC > AFB1. The mutation frequency induced by N-MeDBC in the V79MZh1A2 cells rose linearly

with increasing concentrations (0.5 and 1 ␮M) at both the 7th and the 9th day of expression (r = 0.91 and 0.98, respectively) but followed a saturation at concentration 2.5 ␮M. DBC gave rise to a dose-dependent growth of mutations (r = 0.84) only at the 7th day of expression, however, no dose or time dependent

Fig. 2. Mean relative mutation frequencies induced by DBC, N-MeDBC, diMeDBC, BaP and AFB1 in the genetically engineered V79 cells.

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increase in mutation frequencies was induced by diMeDBC (Table 4). The co-expression of NAT together with human CYP1A2 led either to a total elimination or at least to a significant reduction of any mutagenicity of all carbazoles (Table 5). The relative MF induced solely by the lowest concentration of N-MeDBC (0.5 ␮M) at both the 7th and the 9th day of expression was significantly reduced in comparison to the relative MF found in the V79MZh1A2 cells (Tables 4 and 5, respectively). To compare the mutagenicity of particular compound in the individual V79 cell lines expressing different form of cytochrome P450, the data from both the 7th and the 9th day of expression for particular agent were pooled together and the mean relative MF was calculated (Fig. 2).

4. Discussion It is supposed that selective expression of CYP forms could contribute to tissue-specificity of many chemical carcinogens. Several approaches have been used to identify the role of particular CYP on carcinogen metabolism in the target organ. One valuable tool for studying P450-mediated metabolism of xenobiotics might be the genetically engineered cells stably expressing different cytochromes P450 [26]. These cells can be expected to show higher sensitivity to short-lived active intermediates, since the metabolites can directly react with target macromolecules in the same cells. Chinese hamster V79 cells lack any cytochrome P450 activity but, nevertheless, contain NADPH-cytochrome P450 reductase and are therefore suitable for transfection with the cDNA encoded CYP [21]. V79-derived cell lines are best suited for mutagenicity studies; their doubling time is of 12 h, cloning efficiency is more than 80%, and the hprt gene located on X chromosome is in hemizygote state. Moreover, the results are of high reproducibility. Using the genetically engineered Chinese hamster V79 cell lines we studied the role of two cytochromes P450, CYP1A1 and CYP1A2, in biotransformation of tissue and organ-specific derivatives of DBC. CYP1A1 and CYP1A2 belong to a cytochrome P4501A family that is coded by two genes, Cyp1A1 and Cyp1A2. Although, these CYP1A1/1A2 bear 72% amino acid

sequence homology, they differ in their distribution in the cells of various organs and tissues and exhibit distinct but broad substrate specificity [27,28]. Cytochrome P4501A1 is expressed in the skin and lung, and only after induction in the liver, whereas cytochrome P4501A2 has been detected nearly entirely in the liver. Moreover, substrates for cytochrome P4501A1 are PAHs, while cytochrome P4501A2 is involved mainly in biotransformation of aromatic amines [29,30]. These phenomena suggest that differences of CYP1A1/1A2 expression in the skin and liver could greatly contribute to the variation in cellular response to carbazoles, i.e. might explain their tissueand organ-specificity. A previous in vitro study on mouse embryo fibroblasts suggested the role of cytochrome P4501A in metabolism of DBC derivatives with sarcomagenic activity [17]. Afterwards experiments on the genetically engineered V79 cells with stable expression of human CYP1A1 supported the role of CYP1A1 in metabolism of sarcomagenic derivatives of DBC [31]. Taras-Valéro et al. [12], however, did not find any correlation between the levels of DNA-adducts and CYP1A1 expression either in the liver or in the skin after parenteral routes of DBC derivative administration, but differences in the induction pattern between genes Cyp1A1 and Cyp1A2 might contribute to explain these discrepancies [27]. In our mutation experiments both DBC and N-MeDBC caused significant increase of 6-TGr mutations in the V79MZh1A1cell line (Table 3). The specific sarcomagen N-MeDBC induced even higher level of mutations than the positive control, BaP, while the parental molecule DBC was less efficient. On the other hand, diMeDBC did not increase the mutation frequency in the V79MZh1A1 cells. Our results were in a very good correlation with the finding of Farkašová et al. [20] who did not detect any micronucleus formation in the V79MZh1A1 cells after treatment with diMeDBC. The lack of any genotoxicity of diMeDBC in the V79MZh1A1 cells suggests that diMeDBC is probably not a substrate for CYP1A1 enzyme or the intermediates formed via CYP1A1 activation are not genotoxic altogether. Using the transgenic MutaTM Mouse model it was shown that diMeDBC did not induce any DNA-adducts or gene mutations in the skin that is not the target organ for this derivative [32].

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Both DBC and diMeDBC possess hepatocarcinogenic activity, they induce tumors and DNA-adducts in the liver DNA [14,15,32]. In contrast, the specific sarcomagen N-MeDBC even when administered at very high doses initiates only trace level of DNA binding in this organ [12,14]. Hepatocarcinogenicity of DBC and diMeDBC is followed with severe cytotoxicity manifested by mitochondrial vacuolisation and destruction [33]. At low (<10 ␮M) DBC concentrations the dead cells are removed by apoptosis [34]. It is supposed that the heterocyclic nitrogen strongly affects the biological activity of DBC and plays an important role in liver carcinogenicity. The chemical properties of this pyrrolic NH group resemble those of the arylamides [9–11]. Activation of many liver carcinogens including arylamines, arylamides, nitrosoamines and aflatoxines is mediated by CYP1A2 [27,35]. For that reason the role of CYP1A2 in biotransformation of tissue specific DBC derivatives was evaluated. All three carbazoles elevated significantly the level of mutations in the V79MZh1A2 with stable expression of human CYP1A2 (Table 4). The strict sarcomagen, N-MeDBC, induced approximately the same level of gene mutations in the V79MZh1A2 cells as in the V79MZh1A1 cells (Tables 3 and 4, respectively). These results clearly demonstrated that N-MeDBC is a substrate for CYP1A2 as well. Therefore, the lack of any biological activity of N-MeDBC in the liver cannot be explained by the absence of its biotransformation in this organ. On the basis of these results the failure of N-MeDBC to induce tumors in the liver might be explained by two mechanisms. First, reactive intermediates formed in liver due to CYP1A2 activation are rapidly trapped by other drug metabolising enzymes taking part in detoxification process, e.g. NAT, glutathione-S-transferases, etc.; second, due to a strong hydrophobicity, N-MeDBC is able to induce tumors only in situ, i.e. at the site of its application. This phenomenon is a characteristic feature of PAHs. Co-expression of NAT2 together with CYP1A2 in the V79NHh1A2 cells led to a clear-cut reduction of mutations induced by all agents including N-MeDBC (Table 5). This finding suggests that a rapid removal of the reactive intermediates by conjugation reaction may explain the inactivity of N-MeDBC in the liver, albeit the hydrophobicity cannot be excluded as well. Previously Périn et al. [11] showed in the Ames ex-

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periments that N-MeDBC was highly mutagenic by activation with a low concentration of S9 fraction or pure microsomes, however, the presence of cytosolic fraction had an inhibitory effect on its activity. Mutation experiments confirmed that CYP1A1 is involved only in the metabolic activation of sarcomagenic DBC derivatives while CYP1A2 mediates the biotransformation of all DBC derivatives, including the tissue specific sarcomagen, N-MeDBC. Reactive intermediates formed due to CYP1A2 activation were substrate for NAT2, a phase II enzyme. Subsequent acetylation of proximate intermediates led to the elimination of any mutagenicity of DBC derivatives. The impact of NAT on CYP1A1-generated intermediates cannot be excluded. These data indeed indicate that CYP1A2 is not the ultimate cytochrome P450 involved in hepatocarcinogenicity of DBC derivatives. Further experiments are required to elucidate the role of other drug-metabolising and conjugating enzymes in the biological activity of DBC derivatives. A promising tool in this study might be the genetically engineered V79 cell lines with stable expression of different forms of cytochrome P450.

Acknowledgements The authors wish to thank Dr. F. Périn, Department of Genotoxicity and Carcinogenicity, Institute Curie, France, who provided the DBC derivatives and Mrs. A. Vokáliková for excellent technical assistance. This study was supported by VEGA Grant 2/6032/99. References [1] E.C.M.J.A. Miller, The metabolism of chemical carcinogens to reactive electrophiles and their possible mechanisms of action in carcinogenesis, in: C.E. Searle (Ed.), Chemical Carcinogens, AC Monograph 173, American Chemical Society, Washington (DC) 1973, pp. 737–762. [2] D.R. Nelson, L. Koymans, T. Kamataki, J.J. Stegeman, R. Feyereisen, D.J. Waxman, M.R. Waterman, O. Gotoh, M.J. Coon, R.W. Estabrook, I.C. Gunsalus, D.W. Nebert, P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature, Pharmacogenetics 6 (1996) 1–42. [3] J.A. Miller, Y.J. Surh, Historical perspectives on conjugation-dependent bioactivation of foreign compounds, in: M.V. Anders, W. Dekant (Eds.), Conjugation-Dependent Carcinogenicity and Toxicity of Foreign Compounds, Advances

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