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Paeonol promotion of DNA adduct formation and arylamines N-acetyltransferase activity in human colon tumour cells
Food and Chemical Toxicology 37 (1999) 327±334
Paeonol Promotion of DNA Adduct Formation and Arylamines NAcetyltransferase Activity in Human Colon Tumour Cells J. G. CHUNG Department of Medicine, China Medical College, 91 Hsueh-Shih Road, Taichung 400, Taiwan, Republic of China (Accepted 10 August 1998) AbstractÐPaeonol was used to determine any eects on the N-acetyltransferase (NAT) activity in human colon tumour cells as measured by HPLC exhibited for the amounts of N-acetyl-2-amino¯uorene (AAF) and N-acetyl-p-aminobenzoic acid (N-Ac-PABA) and remaining 2-amino¯uorene (AF) and p-aminobenzoic acid (PABA). The NAT activity in the human colon tumour intact cells and cytosols was promoted by paeonol in a dose-dependent manner, that is, the higher the concentrations of paeonol, the higher the promotion of NAT activity. The apparent Vmax values from NAT of human colon tumour cells were also promoted by paeonol in cytosols and in intact cells. The data also demonstrated that co-treatment with paeonol in the media an increase in AF±DNA adduct formation was seen in the human colon tumour cells. This report is the ®rst demonstration to show paeonol did promote human colon tumour cell NAT activity and AF±DNA adduct formation. # 1999 Elsevier Science Ltd. All rights reserved Keywords: 2-amino¯uorene; N-acetyl-2-amino¯uorene; p-aminobenzoic acid; N-acetyltransferase; paeonol; DNA adduct. Abbreviations: AAF = acetyl-2-amino¯uorene; AF = 2-amino¯uorene; BSA = bovine serum albumin; DMSO = dimethyl sulfoxide; DTT = dithiothreitol; EDTA = ethylenediaminetetraacetic acid; FBS = foetal bovine serum; N-Ac-PABA = N-acetyl-p-aminobenzoic acid; NAT = N-acetyltransferase; PABA = p-aminobenzoic acid.
INTRODUCTION
The arylamine carcinogens represent one of the documented classes of chemicals known to induce tumours in humans (Parker and Evans, 1984). The arylamine carcinogens require metabolic activation by host enzymes as a prerequisite to the initiation of carcinogenesis in target organs and tissues (Lower, 1982; Miller and Miller, 1981). NAcetylation, a major metabolic pathway of arylamines and hydrazines, is catalysed by cytosolic arylamine N-acetyltransferase (NAT) using acetyl coenzyme A as an acetyl donor (Weber and Hein, 1985). NAT also catalyses the formation of mutagenic products from heterocyclic dietary carcinogens that are derived largely from cooked meat and ®sh (Nagao and Sugimura, 1993). In humans, two distinct NAT genes (NAT1 and NAT2) have been identi®ed and sequenced, which encode for two
dierent human liver arylamine NATs (NAT1 and NAT2) (Blum et al., 1990; Ohsako and Deguchi, 1990). Humans exhibit genetic polymorphism in both NAT1 and NAT2 loci resulting in slow and rapid acetylator phenotypes; however, NAT1 activity is higher in bladder and colonic mucosa than NAT2, and the NAT1 enzyme also exhibits phenotypic variation among human tissues samples (Bell et al., 1995). Human epidemiological studies have suggested that there are associations between slow acetylator phenotype and bladder cancer (Cartwright et al., 1982), as well as between rapid acetylator phenotypes and colorectal cancer (Ilett et al., 1987; Lang et al., 1986). It has been reported that extrahepatic expression of acetylator-genotype dependent NAT activity occurs in human colon cells (Flammang et al., 1988; Kirlin et al., 1989a) and bladder (Kirlin et al., 1989b). The regulation of
0278-6915/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(99)00011-3
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the NAT loci in genetic variations of humans has been implicated as an important factor in determining susceptibility to arylamine carcinogens (Blum et al., 1990; Ohsako and Deguchi, 1990). Paeonol, 2-hydroxyl-4-methoxyacetophenone, is an antioxidant agent and has been reported to inhibit calcium in¯ux in cultured neonatal rat heart cells because paeonol may block the sodium calcium channel (Tang and Shi, 1991). Paeonol also has been demonstrated to show more potent radical scavenging and antioxidative eects than a-tocopherol (Yoshikawa et al., 1992). However, there is no available information that addresses the eects of paeonol on NAT activity. Thus, the present study was performed to determine whether paeonol could aect NAT activity and 2-amino¯uorene (AF)±DNA adduct formation in human colon tumour cells. The results of the present study demonstrated that using of (AF) and p-aminobenzoic acid (PABA) as substrates for NAT activity determination, paeonol did increase NAT activity and AF±DNA adduct formation in human colon tumour cells.
MATERIALS AND METHODS
Chemicals and reagents Paeonol, ethylenediaminetetraacetic acid (EDTA), PABA, N-Ac-PABA, acetylcarnitine, leupeptin, bovine serum albumin (BSA), phenylmethylsulfonyl¯uoride (PMSF), Tris, dimethyl sulfoxide (DMSO), dithiothreitol (DTT) and carnitine acetyltransferase were obtained from Sigma Chemical Co. (St Louis, MO, USA). Acetyl-coenzyme A was obtained from P-L Biochemicals Inc. (Milwaukee, WI, USA). AF and acetyl-2-amino¯uorene (AAF) were obtained from K and K Laboratories (Plainview, NY, USA). All of the chemicals used were reagent grade. Human colon tumour cell line Human colon tumour (adenocarcinoma) cell line (colo 205) was obtained from the Taipei Veterinary Hospital (Taipei, Taiwan) as described previously (Chen et al., 1998). The cells were placed into 75 cm2 tissue culture ¯asks and grown at 378C under a humidi®ed 5% CO2 atmosphere in RPMI 1640 medium (Sigma) supplemented with 10% foetal bovine serum (FBS) (Gibco BRL, Grand Island, NY, USA), 2% penicillin±streptomycin (10,000 U/ ml penicillin and 10 mg/ml streptomycin). Preparation of human colon tumour cell cytosols About 5 107 human colon tumour cells were placed in 2 ml of the lysis buer (20 mM Tris±HCl, pH 7.5 (at 48C), 1 mM DTT, 1 mM EDTA, 50 mM PMSF and 10 mM leupeptin) as previously described (Chung et al., 1993). The suspensions were centrifuged at 9000 g for 1 min in a model
3200 Eppendorf/Brinkman centrifuge, and the supernatant fraction was subsequently centrifuged at 10,000 g for 60 min. The supernatant was kept on ice for NAT activity and protein determinations. NAT activity determinations The determination of acetyl-CoA-dependent Nacetylation of PABA and AF were performed as described by Chung et al. (1993). NAT activity was expressed as nmol acetylated substrate per min per mg cytosolic protein. Protein concentrations in the human colon tumour cell cytosols were determined by the method of Bradford (1976) with BSA as the standard. All of the samples were assayed in triplicate. The data (mean 2 SD) were obtained from three individual experiments and each experiment contain three individual tests. Eects of various concentrations of paeonol on NAT activity of human colon tumour cell cytosols Paeonol was dissolved in DMSO at various concentrations, from 0.18 to 18,000 mM. The reaction mixtures consisted of 50 ml cytosols (colon tumour cells) diluted as required, 20 ml recycling mixture containing AF or PABA at selected concentrations for substrates, and 10 ml paeonol. The reactions were started by the addition of the AcCoA. The control reactions had 20 ml distilled water in place of AcCoA. Following these steps, the NAT activity was determined as described in NAT activity determination section in human colon tumour tissues (Chung et al., 1997). Eects of various concentrations of paeonol on NAT activity in intact human colon tumour cells Human colon tumour cells (in 1 ml RPMI 1640 media with glutamine and 10% FBS were incubated with arylamine substrate at 1 106 cells/ml in individual wells of 24-well cell culture plate with or without paeonol co-treatment for the incubation time (18 hr) at 378C in 95% air/5% CO2. At the conclusion of incubation, the cells and media were removed and centrifuged. For experiments with AF, the supernatant was immediately extracted with ethyl acetate/methanol (95:5), the solvent evaporated, and the residue redissolved in methanol and assayed for AAF as described above. For experiments with PABA, aliquots of the supernatant were assayed directly for N-Ac-PABA. Eects of paeonol on the kinetic constants of NAT from human colon tumour cells Cytosols of human colon tumour cells co-treated with and without 180 mM paeonol and selected concentrations of AF or PABA were determined for NAT activity as described above. All reactions were run in triplicate. For the intact cell studies, 1 106 human colon tumour cells were incubated with selected concentrations of AF or PABA with and without paeonol for 18 hr in a 378C incubator.
Paeonol promotion of DNA adduct formation and N-acetyltransferase activity
Following incubation, the cells and media suspensions were removed and centrifuged. In the experiment with AF, the supernatant was immediately extracted with ethyl acetate/methanol (95:5), the solvent evaporated, and the residue redissolved in methanol and assayed by HPLC. For the experiment with PABA, aliquots of the supernatant were assayed directly. All samples were run in triplicate. Detection and measurement of DNA adducts Detection and measurement of DNA adducts were performed as follows. Human colon tumour cells (colo 205) were incubated with AF and/or paeonol and recovered by centrifugation. The DNA was prepared using GNOME DNA isolation kit protocol. And dissolved in TE buer. The isolated nucleotides were labelled with 5 ml of a labelling mixture. The mixture contained 2.5 ml 10 buer (0.1 M Bicine, 0.1 M MgCl2, 0.1 M DTT, 0.01 M spermidine, pH 9), 8 U polynucleotide kinase and 20 mCi [g32P]ATP (5000±6000 Ci/mmol). The kinase reaction was run at 378C for 40 min. Then, 100 mU of apyrase (in 4 ml) was added and incubation continues 25 min (Levy and Weber, 1988). Postlabelled adducted nucleotides were separated by Beckman HPLC (pump 168 and detector 126) using Ultrasphere C18 reversed phase ion-pairing column 4.6 25 cm eluted at a ¯ow rate of 1.5 ml/min with 30 mM KPO4, pH 6.0, containing 10% CH3CN for 10 min followed by a linear gradient of 90% 30 mM KPO4, pH 6.0, 5 mM tetrabutylammonium phosphate and 50% CH3CN at 65 min. UV absorbance was followed at 254 nm. Samples (1 min = 1.5 ml) were collected and quantitated by scintillation spectrometry (Levy et al., 1994; Levy and Weber, 1988). Calculation of adduct formation was made by dividing the radioactivity in the adduct peak (after correction for recovery and eciency of counting) by the speci®c activity of the ATP used in labelling. Adduct levels are reported as the pmol adduct/mg DNA analysed (Levy et al., 1994; Levy and Weber, 1988). Statistical analysis Statistical analysis of the data was performed with an unpaired Student's t-test. The kinetic constants were calculated with the Cleland HYPER Program (Cleland, 1967) that performs linear regression using a least-squares method.
RESULTS
Eects of various concentrations of paeonol on NAT activity of human colon tumour cell cytosols and intact cells The means2 SD (standard deviation) of NAT activity co-treated with or without paeonol with both substrates are given in Tables 1 and 2. The data indicated that the NAT activity increased with
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Table 1. Eects of paeonol on human colon tumour cell NAT activity in cytosols Concn of paeonol Control 0.18 mM 1.8 mM 18 mM 180 mM 1800 mM 18,000 mM
2-AAF (nmol/min/mg protein)
N-Ac-PABA (nmol/min/mg protein)
1.28 2 0.23 1.262 0.26 1.302 0.30 1.402 0.33 a 1.642 0.20 b 1.94 20.42 c 2.32 20.46
1.06 20.22 1.07 20.26 1.10 20.27 1.23 20.20 a 1.39 20.29 b 1.8020.40 c 2.04 20.44
The cytosols of colo 205 cells were prepared as described in Materials and Methods. The AcCoA and paeonol concentrations were 0.1 mM and 0.18±18,000 mM, respectively. Values are means 2 SD of three individual experiments. a Diers between 180 mM paeonol and control; P < 0.05. b Diers between 1800 mM paeonol and control; P < 0.01. c Diers between 18000 mM paeonol and control; P < 0.001.
increased concentrations of paeonol both in cytosols and in intact cells. Paeonol induced a dose dependent eect on NAT activity in examined cytosols and intact cells. The NAT activity for the acetylation of AF (15, 30, 60 and 100 mM) measured from intact cells was 1.68 2 0.08, 3.10 2 0.34, 6.48 2 0.69 and 5.89 2 0.48 nmol/106 cells for acetylation of AF without co-treatment with paeonol. In the presence of 180 mM paeonol, the NAT activities were increased about 12±30% for AF (Fig. 1a). The NAT activity for the acetylation of PABA (15, 30, 60 and 100 mM) measured from intact cells was 1.18 2 0.07, 2.46 2 0.30, 5.122 0.43 and 5.06 2 0.46 nmol/106 cells, respectively, for acetylation of PABA without co-treatment with paeonol. In the presence of 180 mM paeonol, the NAT activities were increased about 15±28%, respectively (Fig. 1b). Eects of incubation time on AAF and N-Ac-PABA production by human colon tumour intact cells To determine the time course eect, 180 mM paeonol were incubated at 378C and harvested at 6, 12, 18 and 24 hr, respectively. Increased time of incubation led to increased AAF and N-Ac-PABA proTable 2. Eects of paeonol on human colon tumour cell NAT activity in intact cells Concn of paeonol
2-AAF (nmol/106 cells)
N-Ac-PABA (nmol/106 cells)
Control 0.18 mM 1.8 mM 18 mM 180 mM 1800 mM 18,000 mM
1.172 0.27 1.192 0.30 1.262 0.32 a 1.362 0.30 b 1.542 0.24 c 1.722 0.22 d 1.982 0.32
0.962 0.24 0.972 0.26 1.022 0.30 a 1.102 0.29 b 1.21 20.18 c 1.462 0.24 d 1.71 20.30
The suspensions of colo 205 cells were prepared as described in Materials and Methods. The AcCoA and paeonol concentrations were 0.1 mM and 0.18±18,000 mM, respectively. Values are means 2 SD of three individual experiments. a Diers between 18 mM paeonol and control; P < 0.05. b Diers between 180 mM paeonol and control; P < 0.01. c Diers between 1800 mM paeonol and control; P < 0.005. d Diers between 18,000 mM paeonol and control; P < 0.001.
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Fig. 1. Eects of paeonol on production of AAF and N-Ac-PABA by human colon tumour cells. Tumour cells were incubated as described for 18 hr at the concentrations of AF (panel A) and PABA (panel B) co-treatment with 180 mM paeonol. AAF and N-Ac-PABA were measured by HPLC assay. Each point represents the mean of triplicate assays of three incubations of cells. *Mean diers between paeonol and control; P < 0.05.
Paeonol promotion of DNA adduct formation and N-acetyltransferase activity
Fig. 2. Eects of incubation time on AAF and N-Ac-PABA production by human colon tumour cells. Tumour cells were incubated with AF (panel A) and PABA (panel B) at 60 mM with 180 mM paeonol co-treatment for the time shown. AAF and N-Ac-PABA were measured by HPLC assay. Each point represents the mean of triplicate assays of three incubations of cells. *Mean diers between paeonol and control; P < 0.05.
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J. G. Chung Table 3. Kinetic data for acetylation of 2-AF in human colon tumour cells In cytosols Km (mM)
Control Paeonol
In intact cells
Vmax (nmol/min/mg protein)
Km (mM)
28.572 5.14 38.572 6.18
6.9920.72 7.1220.94
7.04 2 1.22 7.49 2 0.48
a
Vmax (nmol/106 cells) b
40.00 26.09 48.20 27.16
Values are means 2SD n = 3. The acetyl CoA and paeonol concentrations were 0.1 mM and 180 mM, and the kinetic constants were calculated from the modi®ed HYPER Program of Cleland (1967). a Diers between 180 mM paeonol and control; P < 0.001. b Diers between 180 mM paeonol and control; P < 0.001.
Table 4. Kinetic data for acetylation of PABA in human colon tumour cells In cytosols Km (mM) Control Paeonol
In intact cells
Vmax (nmol/min/mg protein)
Km (mM)
Vmax (nmol/106 cells)
13.162 1.19 15.722 2.02
3.3720.46 3.4620.42
14.682 2.05 b18.872 1.82
2.21 2 0.17 2.24 2 0.26
a
Values are means2 SD n = 3. The acetyl CoA and paeonol concentrations were 0.1 mM and 180 mM and the kinetic constants were calculated from the modi®ed HYPER Program of Cleland (1967). a Diers between 180 mM paeonol and control; P < 0.05. b Diers between 180 mM paeonol and control; P < 0.05.
duction up to 24 hr (Fig. 2a,b). Figure 2 illustrates the course of AAF and N-Ac-PABA production from human colon tumour cells at 60 mM AF and PABA. Eects of paeonol on the kinetic constants of NAT from human colon tumour cells In the presence or absence of paeonol, speci®c concentrations of AF and PABA (0.373, 0.435, 0.543, 0.745, 1.102 and 2.205 mM, respectively) were added to the recycling mixtures for determining human colon tumour cell NAT kinetic constants. For the cytosol examinations, the apparent Km and Vmax values were 7.04 2 1.26 mM and 28.57 2 6.18 nmol/min/mg protein, respectively, for AF and 2.21 2 0.18 mM and 13.16 2 1.99 nmol/min/mg protein, respectively, for PABA (Tables 3 and 4). When 180 mM was added to the reaction mixtures, the apparent Vmax values were increased 28.4% and 17.2% for acetylation of AF and PABA, respectively. For the intact cell examinations, the apparent Vmax values were 6.99 2 0.72 mM and Table 5. DNA±AF adduct formation (pmol adduct/mg DNA) following 18 hr of incubation of colo 205 cells with or without 180 mM paeonol AF±DNA adducts (pmol/mg DNA) 60 mM
30 mM Colo 205 Colo 205 + paeonol a
0.48 20.16 0.69 20.20
a
b
0.702 0.12 0.982 0.23
Dier from colo 205 P<0.05 by two-tailed Student's t-test. Dier from colo 205 P<0.05 by two-tailed Student's t-test. Values are means 2SE of six separate preparations (colo 205 cells, incubation with AF, DNA preparation, postlabelling and HPLC). b
40.00 2 7.89 nmol/min/mg of protein, respectively, for AF, and 3.37 2 0.48 mM and 14.70 2 2.25 nmol/ min/mg protein, respectively, for PABA (Tables 3 and 4). When 180 mM paeonol was added to the reaction mixtures, the apparent Vmax values were increased 18.4% and 18.8% for acetylation of AF and PABA, respectively. Clearly, Vmax values for the human colon tumour cells NAT were increased in the presence of paeonol both in cytosols and intact cell examinations. Detection and measurement of DNA adducts Following an 18-hr incubation of colon tumour cells with AF in the presence of paeonol, cells were recovered and DNA was prepared, hydroylsed to nucleotides, adducted nucleotides were extracted into butanol (Gupta et al., 1988) and analysed by HPLC (Levy et al., 1994). The results indicate that colon tumour cells activate AF to a metabolite able to bind covalently with DNA, and also induced dose-dependent eect (Table 5).
DISCUSSION
Extrahepatic expression of acetylator-genotype dependent NAT activity also has been reported in tissues of human colon (Kirlin et al., 1989a). Earlier studies in our laboratory have already demonstrated that human colon tumour line (colo 205) contained NAT activity (Chen et al., 1998). Other investigators also reported that the acetyl-CoA-dependent arylamine NAT enzyme exists in many kinds of experimental animals, including humans, and the NAT has been shown to be involved in some chemical carcinogenesis (Bell et al., 1995; Minchin et al., 1992; Ohsako and Deguchi, 1990).
Paeonol promotion of DNA adduct formation and N-acetyltransferase activity
Acetylation may result in activation to DNA-reactive metabolites or, in some cases, detoxi®cation (Bell et al., 1995). The genetic variation in NAT activities among target organs or tissues may indicate dierent risks for arylamine-induced neoplasms. The present ®nding is important to oer information which showed that a plant compound did increase NAT activity, because paeonol has been identi®ed as an antioxidant agent. The substrate speci®city for NAT1 is dierent from that for NAT2 in humans (Weber and Hein, 1985). AF was chosen in the present study because it is the common substrate for both NAT1 and NAT2, while PABA is a substrate for NAT1 only, and our interest in comparing the metabolism of a carcinogen (AF) to a non-carcinogen (PABA). In order to simulate the conditions of the human large intestine, pH 7.5 was selected for all of the experiments because this pH is close to that of the normal large intestine. The data from the cytosol studies showed that there were signi®cant dierences of NAT activity between the control and paeonol treated groups. There were increases of 20±90% NAT activity after the cytosol were co-treated with paeonol in concentrations of 18, 180, 1800 and 18,000 mM. The data from the intact cell studies showed that there were signi®cant dierences of NAT activity between the controls and the paeonol treatment groups which occurred at 18 hr. Collectively, the present studies demonstrated that paeonol can promote the NAT activity of human colon tumour cells in cytosols and in intact cells and the promotion has a dosedependent eect, that is, the higher the concentration of paeonol, the higher the promotion of NAT activity. The data indicated that paeonol increased apparent values of Vmax, but it is did not change Km values both in cytosols and intact cell examinations. Thus, paeonol might act as a competitive promoter in the reaction. It has been reported that colorectal adenoma and cancer risks were related to meat intake and acetylator status (Robert-Thomson et al., 1996). Arylamine NAT activity towards AF and PABA has been detected in human colon tumour cell lines (Gupta et al., 1988). Our earlier studies already demonstrated that human colon tumour cell lines produced acetylated metabolites from AF and PABA. The present results also demonstrated that the produced acetylated metabolites increased gradually as the incubation time increased up to 18 hr for AF for PABA. The decrease in AAF production at 100 mM AF was possibly due to toxicity and loss of viable cells (data not shown). The results are in agreement with an earlier study on mice leucocytes (Chung et al., 1993; Levy and Weber, 1994). It was also demonstrated that an increase in the concentration of AF and PABA, the production of AAF and N-Ac-PABA was increased.
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The present data also demonstrated that co-treatment with paeonol in the media of intact colon tumour cells did increase AF±DNA adduct formation. It also showed that increased AF in the media led to increased AF±DNA adduct formation in the control and with paeonol co-treated studies. The values from present studies were considerably higher than the values of 0.01±0.6 pmol adduct/mg DNA reported for human mononuclear leucocytes incubated under the same conditions (unpublished data). But it was clearly indicated that paeonol did increase AF±DNA adduct formation in examined human colon tumour cells. Collectively, the increasing of AF acetylation led to the increase AF±DNA adduct formation. Based on the result from paeonol cotreatment demonstrated that paeonol promote AF acetylation and AF±DNA adduct formation in human colon tumour cells. Carcinogen±DNA adduct formation is an important step in chemical carcinogenesis. The exact mechanism by which paeonol promotes AF±DNA adduct formation needs further investigation. Many studies have attempted to relate acetylation phenotype to cancer susceptibility. Except for bladder cancer, in other tumours including colon tumours, the relationship between acetylator phenotypes and susceptibility is not clear. Recently, other investigators have demonstrated that the combination of fast-acetylator phenotypes with a high consumption of meat increased the risk of colorectal cancer (Robert-Thomson et al., 1996). The relationship between meat intake, acetylator status and colorectal carcinogenesis may involve exposure to heterocyclic amines that are generated by cooking meat for long periods at high temperatures. The present study oers information about how paeonol promotes NAT activity, because NATs are expressed in human ileum and colon (Ilett et al., 1987) and are able to metabolize arylamine compounds. However, it is not known whether the increase in NAT activity would increase tumour production or whether paeonol could promote the development of colon cancer. It is dicult at this point to extrapolate the quantity of paeonol that would need to be consumed by humans to potentially promote the NAT activity of human colon tumour. Further investigations are needed. One important point is that other investigators have already pointed out that elevated levels of NAT activity are associated with increased sensitivity to the mutagenic eects of many arylamines (Einisto et al., 1991). Furthermore, it was reported that the attenuation of liver NAT activity is associated with several disease processes such as bladder and breast cancer (Weber and Hein, 1985). REFERENCES
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Paeonol Promotion of DNA Adduct Formation and Arylamines NAcetyltransferase Activity in Human Colon Tumour Cells J. G. CHUNG Department of Medicine, China Medical College, 91 Hsueh-Shih Road, Taichung 400, Taiwan, Republic of China (Accepted 10 August 1998) AbstractÐPaeonol was used to determine any eects on the N-acetyltransferase (NAT) activity in human colon tumour cells as measured by HPLC exhibited for the amounts of N-acetyl-2-amino¯uorene (AAF) and N-acetyl-p-aminobenzoic acid (N-Ac-PABA) and remaining 2-amino¯uorene (AF) and p-aminobenzoic acid (PABA). The NAT activity in the human colon tumour intact cells and cytosols was promoted by paeonol in a dose-dependent manner, that is, the higher the concentrations of paeonol, the higher the promotion of NAT activity. The apparent Vmax values from NAT of human colon tumour cells were also promoted by paeonol in cytosols and in intact cells. The data also demonstrated that co-treatment with paeonol in the media an increase in AF±DNA adduct formation was seen in the human colon tumour cells. This report is the ®rst demonstration to show paeonol did promote human colon tumour cell NAT activity and AF±DNA adduct formation. # 1999 Elsevier Science Ltd. All rights reserved Keywords: 2-amino¯uorene; N-acetyl-2-amino¯uorene; p-aminobenzoic acid; N-acetyltransferase; paeonol; DNA adduct. Abbreviations: AAF = acetyl-2-amino¯uorene; AF = 2-amino¯uorene; BSA = bovine serum albumin; DMSO = dimethyl sulfoxide; DTT = dithiothreitol; EDTA = ethylenediaminetetraacetic acid; FBS = foetal bovine serum; N-Ac-PABA = N-acetyl-p-aminobenzoic acid; NAT = N-acetyltransferase; PABA = p-aminobenzoic acid.
INTRODUCTION
The arylamine carcinogens represent one of the documented classes of chemicals known to induce tumours in humans (Parker and Evans, 1984). The arylamine carcinogens require metabolic activation by host enzymes as a prerequisite to the initiation of carcinogenesis in target organs and tissues (Lower, 1982; Miller and Miller, 1981). NAcetylation, a major metabolic pathway of arylamines and hydrazines, is catalysed by cytosolic arylamine N-acetyltransferase (NAT) using acetyl coenzyme A as an acetyl donor (Weber and Hein, 1985). NAT also catalyses the formation of mutagenic products from heterocyclic dietary carcinogens that are derived largely from cooked meat and ®sh (Nagao and Sugimura, 1993). In humans, two distinct NAT genes (NAT1 and NAT2) have been identi®ed and sequenced, which encode for two
dierent human liver arylamine NATs (NAT1 and NAT2) (Blum et al., 1990; Ohsako and Deguchi, 1990). Humans exhibit genetic polymorphism in both NAT1 and NAT2 loci resulting in slow and rapid acetylator phenotypes; however, NAT1 activity is higher in bladder and colonic mucosa than NAT2, and the NAT1 enzyme also exhibits phenotypic variation among human tissues samples (Bell et al., 1995). Human epidemiological studies have suggested that there are associations between slow acetylator phenotype and bladder cancer (Cartwright et al., 1982), as well as between rapid acetylator phenotypes and colorectal cancer (Ilett et al., 1987; Lang et al., 1986). It has been reported that extrahepatic expression of acetylator-genotype dependent NAT activity occurs in human colon cells (Flammang et al., 1988; Kirlin et al., 1989a) and bladder (Kirlin et al., 1989b). The regulation of
0278-6915/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(99)00011-3
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the NAT loci in genetic variations of humans has been implicated as an important factor in determining susceptibility to arylamine carcinogens (Blum et al., 1990; Ohsako and Deguchi, 1990). Paeonol, 2-hydroxyl-4-methoxyacetophenone, is an antioxidant agent and has been reported to inhibit calcium in¯ux in cultured neonatal rat heart cells because paeonol may block the sodium calcium channel (Tang and Shi, 1991). Paeonol also has been demonstrated to show more potent radical scavenging and antioxidative eects than a-tocopherol (Yoshikawa et al., 1992). However, there is no available information that addresses the eects of paeonol on NAT activity. Thus, the present study was performed to determine whether paeonol could aect NAT activity and 2-amino¯uorene (AF)±DNA adduct formation in human colon tumour cells. The results of the present study demonstrated that using of (AF) and p-aminobenzoic acid (PABA) as substrates for NAT activity determination, paeonol did increase NAT activity and AF±DNA adduct formation in human colon tumour cells.
MATERIALS AND METHODS
Chemicals and reagents Paeonol, ethylenediaminetetraacetic acid (EDTA), PABA, N-Ac-PABA, acetylcarnitine, leupeptin, bovine serum albumin (BSA), phenylmethylsulfonyl¯uoride (PMSF), Tris, dimethyl sulfoxide (DMSO), dithiothreitol (DTT) and carnitine acetyltransferase were obtained from Sigma Chemical Co. (St Louis, MO, USA). Acetyl-coenzyme A was obtained from P-L Biochemicals Inc. (Milwaukee, WI, USA). AF and acetyl-2-amino¯uorene (AAF) were obtained from K and K Laboratories (Plainview, NY, USA). All of the chemicals used were reagent grade. Human colon tumour cell line Human colon tumour (adenocarcinoma) cell line (colo 205) was obtained from the Taipei Veterinary Hospital (Taipei, Taiwan) as described previously (Chen et al., 1998). The cells were placed into 75 cm2 tissue culture ¯asks and grown at 378C under a humidi®ed 5% CO2 atmosphere in RPMI 1640 medium (Sigma) supplemented with 10% foetal bovine serum (FBS) (Gibco BRL, Grand Island, NY, USA), 2% penicillin±streptomycin (10,000 U/ ml penicillin and 10 mg/ml streptomycin). Preparation of human colon tumour cell cytosols About 5 107 human colon tumour cells were placed in 2 ml of the lysis buer (20 mM Tris±HCl, pH 7.5 (at 48C), 1 mM DTT, 1 mM EDTA, 50 mM PMSF and 10 mM leupeptin) as previously described (Chung et al., 1993). The suspensions were centrifuged at 9000 g for 1 min in a model
3200 Eppendorf/Brinkman centrifuge, and the supernatant fraction was subsequently centrifuged at 10,000 g for 60 min. The supernatant was kept on ice for NAT activity and protein determinations. NAT activity determinations The determination of acetyl-CoA-dependent Nacetylation of PABA and AF were performed as described by Chung et al. (1993). NAT activity was expressed as nmol acetylated substrate per min per mg cytosolic protein. Protein concentrations in the human colon tumour cell cytosols were determined by the method of Bradford (1976) with BSA as the standard. All of the samples were assayed in triplicate. The data (mean 2 SD) were obtained from three individual experiments and each experiment contain three individual tests. Eects of various concentrations of paeonol on NAT activity of human colon tumour cell cytosols Paeonol was dissolved in DMSO at various concentrations, from 0.18 to 18,000 mM. The reaction mixtures consisted of 50 ml cytosols (colon tumour cells) diluted as required, 20 ml recycling mixture containing AF or PABA at selected concentrations for substrates, and 10 ml paeonol. The reactions were started by the addition of the AcCoA. The control reactions had 20 ml distilled water in place of AcCoA. Following these steps, the NAT activity was determined as described in NAT activity determination section in human colon tumour tissues (Chung et al., 1997). Eects of various concentrations of paeonol on NAT activity in intact human colon tumour cells Human colon tumour cells (in 1 ml RPMI 1640 media with glutamine and 10% FBS were incubated with arylamine substrate at 1 106 cells/ml in individual wells of 24-well cell culture plate with or without paeonol co-treatment for the incubation time (18 hr) at 378C in 95% air/5% CO2. At the conclusion of incubation, the cells and media were removed and centrifuged. For experiments with AF, the supernatant was immediately extracted with ethyl acetate/methanol (95:5), the solvent evaporated, and the residue redissolved in methanol and assayed for AAF as described above. For experiments with PABA, aliquots of the supernatant were assayed directly for N-Ac-PABA. Eects of paeonol on the kinetic constants of NAT from human colon tumour cells Cytosols of human colon tumour cells co-treated with and without 180 mM paeonol and selected concentrations of AF or PABA were determined for NAT activity as described above. All reactions were run in triplicate. For the intact cell studies, 1 106 human colon tumour cells were incubated with selected concentrations of AF or PABA with and without paeonol for 18 hr in a 378C incubator.
Paeonol promotion of DNA adduct formation and N-acetyltransferase activity
Following incubation, the cells and media suspensions were removed and centrifuged. In the experiment with AF, the supernatant was immediately extracted with ethyl acetate/methanol (95:5), the solvent evaporated, and the residue redissolved in methanol and assayed by HPLC. For the experiment with PABA, aliquots of the supernatant were assayed directly. All samples were run in triplicate. Detection and measurement of DNA adducts Detection and measurement of DNA adducts were performed as follows. Human colon tumour cells (colo 205) were incubated with AF and/or paeonol and recovered by centrifugation. The DNA was prepared using GNOME DNA isolation kit protocol. And dissolved in TE buer. The isolated nucleotides were labelled with 5 ml of a labelling mixture. The mixture contained 2.5 ml 10 buer (0.1 M Bicine, 0.1 M MgCl2, 0.1 M DTT, 0.01 M spermidine, pH 9), 8 U polynucleotide kinase and 20 mCi [g32P]ATP (5000±6000 Ci/mmol). The kinase reaction was run at 378C for 40 min. Then, 100 mU of apyrase (in 4 ml) was added and incubation continues 25 min (Levy and Weber, 1988). Postlabelled adducted nucleotides were separated by Beckman HPLC (pump 168 and detector 126) using Ultrasphere C18 reversed phase ion-pairing column 4.6 25 cm eluted at a ¯ow rate of 1.5 ml/min with 30 mM KPO4, pH 6.0, containing 10% CH3CN for 10 min followed by a linear gradient of 90% 30 mM KPO4, pH 6.0, 5 mM tetrabutylammonium phosphate and 50% CH3CN at 65 min. UV absorbance was followed at 254 nm. Samples (1 min = 1.5 ml) were collected and quantitated by scintillation spectrometry (Levy et al., 1994; Levy and Weber, 1988). Calculation of adduct formation was made by dividing the radioactivity in the adduct peak (after correction for recovery and eciency of counting) by the speci®c activity of the ATP used in labelling. Adduct levels are reported as the pmol adduct/mg DNA analysed (Levy et al., 1994; Levy and Weber, 1988). Statistical analysis Statistical analysis of the data was performed with an unpaired Student's t-test. The kinetic constants were calculated with the Cleland HYPER Program (Cleland, 1967) that performs linear regression using a least-squares method.
RESULTS
Eects of various concentrations of paeonol on NAT activity of human colon tumour cell cytosols and intact cells The means2 SD (standard deviation) of NAT activity co-treated with or without paeonol with both substrates are given in Tables 1 and 2. The data indicated that the NAT activity increased with
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Table 1. Eects of paeonol on human colon tumour cell NAT activity in cytosols Concn of paeonol Control 0.18 mM 1.8 mM 18 mM 180 mM 1800 mM 18,000 mM
2-AAF (nmol/min/mg protein)
N-Ac-PABA (nmol/min/mg protein)
1.28 2 0.23 1.262 0.26 1.302 0.30 1.402 0.33 a 1.642 0.20 b 1.94 20.42 c 2.32 20.46
1.06 20.22 1.07 20.26 1.10 20.27 1.23 20.20 a 1.39 20.29 b 1.8020.40 c 2.04 20.44
The cytosols of colo 205 cells were prepared as described in Materials and Methods. The AcCoA and paeonol concentrations were 0.1 mM and 0.18±18,000 mM, respectively. Values are means 2 SD of three individual experiments. a Diers between 180 mM paeonol and control; P < 0.05. b Diers between 1800 mM paeonol and control; P < 0.01. c Diers between 18000 mM paeonol and control; P < 0.001.
increased concentrations of paeonol both in cytosols and in intact cells. Paeonol induced a dose dependent eect on NAT activity in examined cytosols and intact cells. The NAT activity for the acetylation of AF (15, 30, 60 and 100 mM) measured from intact cells was 1.68 2 0.08, 3.10 2 0.34, 6.48 2 0.69 and 5.89 2 0.48 nmol/106 cells for acetylation of AF without co-treatment with paeonol. In the presence of 180 mM paeonol, the NAT activities were increased about 12±30% for AF (Fig. 1a). The NAT activity for the acetylation of PABA (15, 30, 60 and 100 mM) measured from intact cells was 1.18 2 0.07, 2.46 2 0.30, 5.122 0.43 and 5.06 2 0.46 nmol/106 cells, respectively, for acetylation of PABA without co-treatment with paeonol. In the presence of 180 mM paeonol, the NAT activities were increased about 15±28%, respectively (Fig. 1b). Eects of incubation time on AAF and N-Ac-PABA production by human colon tumour intact cells To determine the time course eect, 180 mM paeonol were incubated at 378C and harvested at 6, 12, 18 and 24 hr, respectively. Increased time of incubation led to increased AAF and N-Ac-PABA proTable 2. Eects of paeonol on human colon tumour cell NAT activity in intact cells Concn of paeonol
2-AAF (nmol/106 cells)
N-Ac-PABA (nmol/106 cells)
Control 0.18 mM 1.8 mM 18 mM 180 mM 1800 mM 18,000 mM
1.172 0.27 1.192 0.30 1.262 0.32 a 1.362 0.30 b 1.542 0.24 c 1.722 0.22 d 1.982 0.32
0.962 0.24 0.972 0.26 1.022 0.30 a 1.102 0.29 b 1.21 20.18 c 1.462 0.24 d 1.71 20.30
The suspensions of colo 205 cells were prepared as described in Materials and Methods. The AcCoA and paeonol concentrations were 0.1 mM and 0.18±18,000 mM, respectively. Values are means 2 SD of three individual experiments. a Diers between 18 mM paeonol and control; P < 0.05. b Diers between 180 mM paeonol and control; P < 0.01. c Diers between 1800 mM paeonol and control; P < 0.005. d Diers between 18,000 mM paeonol and control; P < 0.001.
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Fig. 1. Eects of paeonol on production of AAF and N-Ac-PABA by human colon tumour cells. Tumour cells were incubated as described for 18 hr at the concentrations of AF (panel A) and PABA (panel B) co-treatment with 180 mM paeonol. AAF and N-Ac-PABA were measured by HPLC assay. Each point represents the mean of triplicate assays of three incubations of cells. *Mean diers between paeonol and control; P < 0.05.
Paeonol promotion of DNA adduct formation and N-acetyltransferase activity
Fig. 2. Eects of incubation time on AAF and N-Ac-PABA production by human colon tumour cells. Tumour cells were incubated with AF (panel A) and PABA (panel B) at 60 mM with 180 mM paeonol co-treatment for the time shown. AAF and N-Ac-PABA were measured by HPLC assay. Each point represents the mean of triplicate assays of three incubations of cells. *Mean diers between paeonol and control; P < 0.05.
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J. G. Chung Table 3. Kinetic data for acetylation of 2-AF in human colon tumour cells In cytosols Km (mM)
Control Paeonol
In intact cells
Vmax (nmol/min/mg protein)
Km (mM)
28.572 5.14 38.572 6.18
6.9920.72 7.1220.94
7.04 2 1.22 7.49 2 0.48
a
Vmax (nmol/106 cells) b
40.00 26.09 48.20 27.16
Values are means 2SD n = 3. The acetyl CoA and paeonol concentrations were 0.1 mM and 180 mM, and the kinetic constants were calculated from the modi®ed HYPER Program of Cleland (1967). a Diers between 180 mM paeonol and control; P < 0.001. b Diers between 180 mM paeonol and control; P < 0.001.
Table 4. Kinetic data for acetylation of PABA in human colon tumour cells In cytosols Km (mM) Control Paeonol
In intact cells
Vmax (nmol/min/mg protein)
Km (mM)
Vmax (nmol/106 cells)
13.162 1.19 15.722 2.02
3.3720.46 3.4620.42
14.682 2.05 b18.872 1.82
2.21 2 0.17 2.24 2 0.26
a
Values are means2 SD n = 3. The acetyl CoA and paeonol concentrations were 0.1 mM and 180 mM and the kinetic constants were calculated from the modi®ed HYPER Program of Cleland (1967). a Diers between 180 mM paeonol and control; P < 0.05. b Diers between 180 mM paeonol and control; P < 0.05.
duction up to 24 hr (Fig. 2a,b). Figure 2 illustrates the course of AAF and N-Ac-PABA production from human colon tumour cells at 60 mM AF and PABA. Eects of paeonol on the kinetic constants of NAT from human colon tumour cells In the presence or absence of paeonol, speci®c concentrations of AF and PABA (0.373, 0.435, 0.543, 0.745, 1.102 and 2.205 mM, respectively) were added to the recycling mixtures for determining human colon tumour cell NAT kinetic constants. For the cytosol examinations, the apparent Km and Vmax values were 7.04 2 1.26 mM and 28.57 2 6.18 nmol/min/mg protein, respectively, for AF and 2.21 2 0.18 mM and 13.16 2 1.99 nmol/min/mg protein, respectively, for PABA (Tables 3 and 4). When 180 mM was added to the reaction mixtures, the apparent Vmax values were increased 28.4% and 17.2% for acetylation of AF and PABA, respectively. For the intact cell examinations, the apparent Vmax values were 6.99 2 0.72 mM and Table 5. DNA±AF adduct formation (pmol adduct/mg DNA) following 18 hr of incubation of colo 205 cells with or without 180 mM paeonol AF±DNA adducts (pmol/mg DNA) 60 mM
30 mM Colo 205 Colo 205 + paeonol a
0.48 20.16 0.69 20.20
a
b
0.702 0.12 0.982 0.23
Dier from colo 205 P<0.05 by two-tailed Student's t-test. Dier from colo 205 P<0.05 by two-tailed Student's t-test. Values are means 2SE of six separate preparations (colo 205 cells, incubation with AF, DNA preparation, postlabelling and HPLC). b
40.00 2 7.89 nmol/min/mg of protein, respectively, for AF, and 3.37 2 0.48 mM and 14.70 2 2.25 nmol/ min/mg protein, respectively, for PABA (Tables 3 and 4). When 180 mM paeonol was added to the reaction mixtures, the apparent Vmax values were increased 18.4% and 18.8% for acetylation of AF and PABA, respectively. Clearly, Vmax values for the human colon tumour cells NAT were increased in the presence of paeonol both in cytosols and intact cell examinations. Detection and measurement of DNA adducts Following an 18-hr incubation of colon tumour cells with AF in the presence of paeonol, cells were recovered and DNA was prepared, hydroylsed to nucleotides, adducted nucleotides were extracted into butanol (Gupta et al., 1988) and analysed by HPLC (Levy et al., 1994). The results indicate that colon tumour cells activate AF to a metabolite able to bind covalently with DNA, and also induced dose-dependent eect (Table 5).
DISCUSSION
Extrahepatic expression of acetylator-genotype dependent NAT activity also has been reported in tissues of human colon (Kirlin et al., 1989a). Earlier studies in our laboratory have already demonstrated that human colon tumour line (colo 205) contained NAT activity (Chen et al., 1998). Other investigators also reported that the acetyl-CoA-dependent arylamine NAT enzyme exists in many kinds of experimental animals, including humans, and the NAT has been shown to be involved in some chemical carcinogenesis (Bell et al., 1995; Minchin et al., 1992; Ohsako and Deguchi, 1990).
Paeonol promotion of DNA adduct formation and N-acetyltransferase activity
Acetylation may result in activation to DNA-reactive metabolites or, in some cases, detoxi®cation (Bell et al., 1995). The genetic variation in NAT activities among target organs or tissues may indicate dierent risks for arylamine-induced neoplasms. The present ®nding is important to oer information which showed that a plant compound did increase NAT activity, because paeonol has been identi®ed as an antioxidant agent. The substrate speci®city for NAT1 is dierent from that for NAT2 in humans (Weber and Hein, 1985). AF was chosen in the present study because it is the common substrate for both NAT1 and NAT2, while PABA is a substrate for NAT1 only, and our interest in comparing the metabolism of a carcinogen (AF) to a non-carcinogen (PABA). In order to simulate the conditions of the human large intestine, pH 7.5 was selected for all of the experiments because this pH is close to that of the normal large intestine. The data from the cytosol studies showed that there were signi®cant dierences of NAT activity between the control and paeonol treated groups. There were increases of 20±90% NAT activity after the cytosol were co-treated with paeonol in concentrations of 18, 180, 1800 and 18,000 mM. The data from the intact cell studies showed that there were signi®cant dierences of NAT activity between the controls and the paeonol treatment groups which occurred at 18 hr. Collectively, the present studies demonstrated that paeonol can promote the NAT activity of human colon tumour cells in cytosols and in intact cells and the promotion has a dosedependent eect, that is, the higher the concentration of paeonol, the higher the promotion of NAT activity. The data indicated that paeonol increased apparent values of Vmax, but it is did not change Km values both in cytosols and intact cell examinations. Thus, paeonol might act as a competitive promoter in the reaction. It has been reported that colorectal adenoma and cancer risks were related to meat intake and acetylator status (Robert-Thomson et al., 1996). Arylamine NAT activity towards AF and PABA has been detected in human colon tumour cell lines (Gupta et al., 1988). Our earlier studies already demonstrated that human colon tumour cell lines produced acetylated metabolites from AF and PABA. The present results also demonstrated that the produced acetylated metabolites increased gradually as the incubation time increased up to 18 hr for AF for PABA. The decrease in AAF production at 100 mM AF was possibly due to toxicity and loss of viable cells (data not shown). The results are in agreement with an earlier study on mice leucocytes (Chung et al., 1993; Levy and Weber, 1994). It was also demonstrated that an increase in the concentration of AF and PABA, the production of AAF and N-Ac-PABA was increased.
333
The present data also demonstrated that co-treatment with paeonol in the media of intact colon tumour cells did increase AF±DNA adduct formation. It also showed that increased AF in the media led to increased AF±DNA adduct formation in the control and with paeonol co-treated studies. The values from present studies were considerably higher than the values of 0.01±0.6 pmol adduct/mg DNA reported for human mononuclear leucocytes incubated under the same conditions (unpublished data). But it was clearly indicated that paeonol did increase AF±DNA adduct formation in examined human colon tumour cells. Collectively, the increasing of AF acetylation led to the increase AF±DNA adduct formation. Based on the result from paeonol cotreatment demonstrated that paeonol promote AF acetylation and AF±DNA adduct formation in human colon tumour cells. Carcinogen±DNA adduct formation is an important step in chemical carcinogenesis. The exact mechanism by which paeonol promotes AF±DNA adduct formation needs further investigation. Many studies have attempted to relate acetylation phenotype to cancer susceptibility. Except for bladder cancer, in other tumours including colon tumours, the relationship between acetylator phenotypes and susceptibility is not clear. Recently, other investigators have demonstrated that the combination of fast-acetylator phenotypes with a high consumption of meat increased the risk of colorectal cancer (Robert-Thomson et al., 1996). The relationship between meat intake, acetylator status and colorectal carcinogenesis may involve exposure to heterocyclic amines that are generated by cooking meat for long periods at high temperatures. The present study oers information about how paeonol promotes NAT activity, because NATs are expressed in human ileum and colon (Ilett et al., 1987) and are able to metabolize arylamine compounds. However, it is not known whether the increase in NAT activity would increase tumour production or whether paeonol could promote the development of colon cancer. It is dicult at this point to extrapolate the quantity of paeonol that would need to be consumed by humans to potentially promote the NAT activity of human colon tumour. Further investigations are needed. One important point is that other investigators have already pointed out that elevated levels of NAT activity are associated with increased sensitivity to the mutagenic eects of many arylamines (Einisto et al., 1991). Furthermore, it was reported that the attenuation of liver NAT activity is associated with several disease processes such as bladder and breast cancer (Weber and Hein, 1985). REFERENCES
Bell D. A., Badawi A. F., Lang N. P., Ilett F. F., Kadlubar F. F. and Hirvonen A. (1995) Polymorphism
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