Safrole-like DNA adducts in oral tissue from oral cancer patients with a betel quid chewing history
Chiu-Lan Chen1,
Chin-Wen Chi1,3,
Kuo-Wei Chang2 and
Tsung-Yun Liu1,3,4
1 Institute of Pharmacology and
2 Institute of Oral Biology, National Yang-Ming University and
3 Department of Medical Research and Education, Veterans General HospitalTaipei, Taipei, Taiwan, Republic of China
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Abstract
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Betel quid (BQ) chewing has been associated with an increased risk of oral squamous cell carcinoma (OSCC) and oral submucous fibrosis (OSF). Piper betle inflorescence, which contains 15 mg/g safrole, is a unique ingredient of BQ in Taiwan. Chewing such prepared BQ may contribute to safrole exposure in human beings (420 µM safrole in saliva). Safrole is a known rodent hepatocarcinogen, yet its carcinogenicity in human beings is largely undetermined. In this study, using a 32P-post-labeling method, we have found a high frequency of safrole-like DNA adducts in BQ-associated OSCC (77%, 23/30) and non-cancerous matched tissue (NCMT) (97%, 29/30). This was in contrast to the absence (< 1/109 nucleotides) of such adducts in all of non-BQ-associated OSCC and their paired NCMT (P < 0.001). Six of seven OSF also exhibited the same safrole-like DNA adduct. The DNA adduct levels in OSF and NCMT were significantly higher than in OSCC (P < 0.05). Using co-chromatography and rechromatography techniques, we further demonstrated that these adducts were identical to synthetic safroledGMP adducts as well as DNA adducts from 1'-hydroxysafrole-treated HepG2 cells. These results suggest that safrole forms stable safroleDNA adducts in human oral tissue following BQ chewing, which may contribute to oral carcinogenesis.
Abbreviations: BQ, betel quid; dAMP, 2'-deoxyadenosine 3'-monophosphate; dGMP, 2'-deoxyguanosine 3'-monophosphate; NCMT, non-cancerous matched tissue; OSCC, oral squamous cell carcinoma; OSF, oral submucous fibrosis; PAPS, 3'-phosphoadenosine 5'-phosphosulfate.
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Introduction
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Oral squamous cell carcinoma (OSCC) is one of the most common malignant neoplasms in Asian countries. It is the fifth leading cause of male cancer mortality in Taiwan (1). The International Agency for Research on Cancer concluded that OSCC is associated with betel quid (BQ) chewing together with tobacco or cigarette smoking (2). Oral submucous fibrosis (OSF) is also a frequent precancerous condition in BQ chewers (3). In Taiwan, tobacco is not included in the preparation used for BQ chewing. Nevertheless, epidemiological studies showed that BQ chewing is still the main cause of OSCC and OSF in Taiwan (4,5). The most popular way to chew BQ in Taiwan is a combination of the areca nut, Piper betle inflorescence (sometimes substituted by betel leaf) and lime paste. Piper betle inflorescence contains a high concentration (15 mg/g) of safrole (6). Consequently, chewing BQ containing Piper betle inflorescence may contribute to safrole exposure (420 µM in saliva during chewing) (7).
Safrole is classified as a weak hepatocarcinogen in mice and rats (8). The carcinogenicity of safrole is mediated through 1'-hydroxysafrole formation, followed by sulfonation to an unstable sulfuric acid ester that reacts to form stable safroleDNA adducts (9). 1'-Hydroxysafrole, the proximate carcinogen of safrole, was detected in the liver, urine and bile of animals treated with safrole (8). However, 1'-hydroxysafrole was not detected in human subjects who received 1.66 mg [14C]safrole (10). The reason for this discrepancy remains unknown. It is plausible that the administrated dose or the assay sensitivity is insufficient for the detection of this metabolite. Using the 32P-post-labeling technique, the most sensitive method for detection of DNA adducts (11), stable safroleDNA adducts can be detected in both rodent liver and other tissues treated with safrole (1214). However, the effect of safrole in human beings has not been documented. In this study, using the 32P-post-labeling technique, we determined the presence of safrole-like DNA adducts in oral tissues of BQ users.
Histologically confirmed OSCC (n = 36) and OSF (n = 7) tissues were obtained from the Department of Dentistry, according to a protocol approved by the committee for the conduct of human research at the Veterans General HospitalTaipei. Specimens were kept at 70°C immediately after excision until DNA extraction. History of BQ chewing, cigarette smoking, alcohol consumption and other clinical parameters were carefully recorded. Normal gingival tissues (n = 14) were obtained from healthy volunteers undergoing tooth extraction. DNA was extracted by a conventional phenol/chloroform procedure (15).
1'-Hydroxysafrole was synthesized from vinyl bromide and piperonal (16). 1'-Hydroxysafrole was then reacted with 2'-deoxyguanosine 3'-monophosphate (dGMP) or 2'-deoxyadenosine 3'-monophosphate (dAMP) in the presence of sulfotransferase and 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The incubation mixture contained 3 mM MgCl2, 0.1 mM PAPS, 5 mM dGMP or dAMP, hepatic cytosol from ICR mice (2 mg protein) and 4 mM 1'-hydroxysafrole in a final volume of 2 ml of 50 mM TrisHCl buffer (pH 7.4). After incubation at 37°C for 3 h, 5-fold cold acetone was added and the mixture was centrifuged at 10 000 g at 4°C for 20 min. The supernatant was analyzed by the 32P-post-labeling assay. HepG2 cells were first cultured in DMEM medium containing 10% fetal bovine serum, then changed to serum-free medium and incubated with 400 µM 1'-hydroxysafrole for 24 h prior to adduct analysis.
Four micrograms of coded DNA samples were assayed by the nuclease P1-enhanced 32P-post-labeling procedure as described by Reddy and Randerath (13). The [
-32P]ATP (6000 Ci/mmol; NEN Life Science, Boston, MA)-labeled safroleDNA adducts were resolved on polyethyleneiminecellulose TLC plates (Machery-Nagel, Düren, Germany) with two different buffer systems. Development condition A consisted of 2.3 M sodium phosphate, pH 6.0 (D1) and 1.8 M lithium formate, 4.25 M urea, pH 3.5 (D2) (bottom to top); 0.36 M lithium chloride, 0.22 M TrisHCl, 3.8 M urea, pH 8.0 (D3) and 1.7 M sodium phosphate, pH 6.0 (D4) (left to right). SafroleDNA adducts were detected by autoradiography and quantified by scintillation counting. Adduct levels are expressed as relative adduct labeling (RAL)x108 values (11). The sensitivity of this assay was limited to 1 adduct/109 nucleotides. To compare the adducts detected in OSCC with safroleDNA adducts in 1'-hydroxysafrole-treated HepG2 cells, the two DNA samples were mixed before enzymatic digestion and, using the above-mentioned condition A, the resulting chromatogram was compared with maps derived from individual DNA samples. These adducts were also compared by a different development condition designated condition B: 1.7 M sodium phosphate, pH 6.0 (D1); 1.9 M lithium formate, 3.8 M urea, pH 3.5 (D2); 0.36 M sodium phosphate, 0.23 M TrisHCl, pH 8.0 (D3); 1.7 M sodium phosphate, pH 6.0 (D4) (17). To further characterize these adducts, excised adducts from individual chromatograms were extracted with 4 N ammonium hydroxide:isopropanol (1:1 v/v) and rechromatographed for further separation (18,19).
Thirty-six patients with OSCC were enrolled in this study. The sex difference (34 males, two females) closely resembles the distribution of BQ chewers (21:1) in the Taiwanese population (20). The most common primary site of OSCC was the buccal mucosa (14 cases) followed by the tongue (seven cases) and gingiva (five cases). The majority of BQ chewers (30 cases) were also cigarette smokers (27 cases). Seven male patients with OSF were also included in this study, all of whom were BQ chewers and cigarette smokers.
We detected two major and two minor DNA adducts on autoradiograms from 1'-hydroxysafrole-treated HepG2 cells under condition A (Figure 1A
). The levels of spot 1 and spot 2 were 105.0 and 22.3 adducts/108 nucleotides, respectively. This map was qualitatively similar to DNA adducts found in safrole-treated mice as described by Reddy and Randerath (13). The two major safroleDNA adducts have been identified as N2-(trans-isosafrol-3'-yl) 2'-deoxyguanosine (spot 1) and N2-(safrol-1'-yl) 2'-deoxyguanosine (spot 2) (21). The nature of the two minor safroleDNA adducts (spots 3 and 4) remains unclear. In this study, one major spot (spot 1) in the fingerprints of OSCC and non-cancerous matched tissue (NCMT) (Figure 1B and C
) as well as in OSF samples was detected. In addition, one minor spot (spot 2) was occasionally noticed (Figures 1C and 2B and E
). The chromatographic location of this major DNA adduct is qualitatively similar to spot 1 as shown by others (1214). On the other hand, no adduct was detectable on the autoradiogram of the control gingival sample (Figure 1D
). The location of the major DNA adducts in oral tissues was qualitatively similar to that of the safroleDNA adduct (spot 1) from 1'-hydroxysafrole-treated HepG2 cells (Figure 1A
). In this development system, interference by benzo[a] pyrene and other macromolecular DNA adducts which result from cigarette smoking was unlikely since these adducts are much more hydrophobic than safrole adducts (data not shown).

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Fig. 1. Autoradiograms of polyethyleneiminecellulose TLC maps of 32P-labeled digests of DNA. (A) DNA from HepG2 cells treated with 400 µM 1'-hydroxysafrole for 24 h; (B) DNA from an OSCC (patient no. 47); (C) DNA from NCMT (no. 48); (D) DNA from normal gingival tissue. Adducts were visualized by autoradiography employing Kodak Biomas MR imaging film with an intensifying screen for 24 h at 70°C, except for (A), which was exposed for 6 h.
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Fig. 2. Co-chromatography of 32P-labeled safroleDNA adducts. (A) dGMP reacted with 1'-hydroxysafrole; (B) DNA from an OSCC (no. 48); (C) A (1-fold) mixed with B (3-fold); (D) dAMP reacted with 1'-hydroxysafrole; (E) DNA from NCMT (no. 48); (F) D (1-fold) mixed with E (3-fold). Chromatographs were developed under condition B. Autoradiographs were obtained after 24 h at 70°C, except for (A), which was exposed for 10 h.
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In order to identify the nature of the DNA adducts, DNA from OSCC was mixed with 1'-hydroxysafrole-treated HepG2 DNA, then digested and developed in TLC plates. Under condition A, the major and minor spots co-migrated with safroleDNA adducts 1 and 2 (spots 1 and 2). Co-migration of such adducts with safroleDNA adducts 1 and 2 was demonstrable under development condition B (data not shown). To characterize these adducts further, we reacted dGMP and dAMP with 1'-hydroxysafrole and separated the products in a TLC plate using development condition B (Figure 2A and D
). As shown in Figure 2C and F
, the adduct product co-migrated with adduct 1 obtained from safroledGMP, but not with dAMP derivatives (spot 5). The nature of spots 1 and 2 in OSCC were further demonstrated to be identical to the 1'-hydroxysafrole-treated DNA adducts by two different solvent systems in 1-dimensional TLC (Figure 3
). The major (spot 1) and minor (spot 2) adducts were confirmed as chromatographically indistinguishable from safroledGMP adducts using two distinct development conditions, co-chromatography and rechromatography.

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Fig. 3. Rechromatography analysis of 32P-labeled safroleDNA adducts. Solvents used for rechromatography were (A) 4 N ammonium hydroxide:isopropanol (1:1 v/v); (B) 0.65 M sodium chloride, 0.25 M boric acid, 0.005 M EDTA, 4 M urea, pH 8.0. OSCC tissue DNA (400 µg) was digested, nuclease P1 enriched and concentrated, then one fiftieth was labeled with [ -32P]ATP for 2-dimensional chromatography. Spot 1 was eluted from 2-dimensional TLC plates of DNA samples from six different OSCC tissues (lanes 16) and from 1'-hydroxysafrole-treated HepG2 cells (4 µg) (lane 7). Spot 2 was similarly excised and eluted from HepG2 cells (lane 8) and OSCC tissues (lanes 9 and 10). Lane 11 was sample 1 and 1'-hydroxysafrole-treated HepG2 DNA co-migrated in 2-dimensional TLC and then eluted for rechromatography. Lanes 1 and 9 and 5 and 10 are from the same individual. Adducts were visualized by autoradiography employing Kodak Biomas MR imaging film with an intensifying screen for 12 h at 70°C.
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The safrole-like DNA adduct was present in 77% (23/30) of OSCC, 97% (29/30) of NCMT from OSCC patients and 86% (6/7) of OSF patients with BQ use. In contrast, this safrole-like DNA adduct was not detectable in gingival tissues from 14 persons without BQ chewing. The presence of this adduct in oral samples is highly correlated with BQ chewing (Fisher's exact test, P < 0.001). The safrole-like DNA adduct levels in OSCC, NCMT and OSF were 4.0 ± 0.9 (range 019.4), 9.7 ± 2.7 (range 065.3) and 7.8 ± 1.5 (range 011.7) per 108 nucleotides, respectively (Table I
). The DNA adduct levels in OSF and NCMT were significantly higher than that in OSCC (MannWhitney rank sum test, P < 0.05).
The formation of covalent adducts of DNA is generally regarded as a critical event in the initiation of chemical carcinogenesis. Consequently, measurement of DNA adducts may be a powerful tool to demonstrate a possible association between carcinogen exposure and cancer risk. The 32P-post-labeling assay combined with the nuclease P1 enhancement technique provides a highly sensitive method for detecting adducts in human tissue (11). Using this analysis, we demonstrated the presence of a safrole-like DNA adduct in oral DNA samples from OSCC and OSF patients and the presence of such an adduct is highly correlated with BQ chewing.
By giving safrole to mice, Randerath et al. reported that the loss of safrole adducts from hepatic DNA was biphasic: a rapid loss during the first week followed by a much slower decline for up to 20 weeks. The early rapid adduct loss was probably related to repair processes, while the latter may be due to cell turnover in carcinogen-exposed tissues rather than DNA repair (12). In this study, we detected one major and/or one minor adduct in oral tissues instead of four spots as seen in 1'-hydroxysafrole-treated HepG2 cells and mice hepatic tissues. There may be a number of reasons for this difference. First, adduct 1 forms more easily than other adducts. Second, adduct 1 is resistant to repair processes. Last, safrole requires a two-step metabolic activation to form DNA adducts and the metabolic capacity of oral tissue is lower than that of liver tissue. Consequently, the major adduct levels are lower and the minor adducts are not detectable in oral tissues.
Adduct levels in a cell are dependent on pharmacodynamic and pharmacokinetic factors, adduct stability and the rates of cell turnover and DNA repair. Substantial amounts of safrole-like DNA adducts were detected in most of the BQ-associated subjects and the levels of safrole-like DNA adducts were significantly higher in NCMT than in OSCC. Observations of a higher carcinogenDNA adduct level in NCMT rather than tumors has been reported in lung, colorectal and breast cancer (18,22,23). A tentative explanation for this phenomenon is that the level of DNA adducts may have been diluted during rapid DNA replication and tumor cell turnover. Another possibility is that the OSCC is less capable of activating the carcinogen; such a phenomenon has been observed in animal models (24).
Although the generation of safroleDNA adducts in rodents is well defined (1214), the role of these adducts in cancer development is not clear since there is no in vivo evidence highlighting the susceptibility to mutation of safroleDNA adducts. Recently, Daimon et al. demonstrated that the genotoxic effects of safrole might result from covalent DNA modification in rat liver (14). However, the role of safroleDNA adducts in non-target tissues and oral mucosa is not known at present. DNA adduction is known to be associated with the activation of proto-oncogenes. Activation of the c-Ha-ras proto-oncogene by point mutations in codon 61 has been found in hepatocellular carcinomas of safrole-treated B6C3F1 mice (25), implicating the involvement of safrole-related DNA adducts in mutagenesis. The involvement of safroleDNA adducts during oral carcinogenesis needs further study.
In conclusion, using the 32P-post-labeling technique, we demonstrated the presence of safrole-like DNA adducts in oral tissues associated with BQ exposure. The results suggest that stable safroleDNA adducts derived from Piper betle inflorescence, an ingredient of Taiwanese BQ, may be associated with oral carcinogenesis.
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Acknowledgments
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This study was supported in part by grants from the National Science Council (NSC 87-2314-B-075-029-M14) and Department of Health (DOH 88-HR-802), Republic of China.
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Notes
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4 To whom correspondence should be addressed at: Department of Medical Research and Education, Veterans General Hospital-Taipei, Taipei, Taiwan 11217, Republic of China Email: tyliu{at}vghtpe.gov.tw 
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Received April 1, 1999;
revised August 31, 1999;
accepted September 3, 1999.