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DNA adducts detected in human gastric mucosa
Cancer Detection and Prevention 27 (2003) 209–215
DNA adducts detected in human gastric mucosa Mohammed Abdul Momen, MD, Yasumasa Monden, MD, PhD, Kunimi Hamada, MD, PhD, Kansei Komaki, MD, PhD, Kazuya Kondo, MD, PhD, Atsushi Umemoto, MD, PhD∗ Second Department of Surgery, School of Medicine, University of Tokushima, 3-18-15, Kuramoto-cho, Tokushima 770-8503, Japan Accepted 25 February 2003
Abstract Human gastrointestinal neoplasms are mostly developed from the mucosa, not from the adjacent muscle layer. DNA adducts in the mucosa and adjacent muscle layer of the non-tumoral part of stomach from 19 patients with gastric neoplasms and from six newborns were analyzed by 32 P-postlabeling, and then compared them with those of representative colon or small intestine sample. Five kinds of mucosa-specific DNA adducts (G1–5) were found in all of the adult stomach samples, but were entirely absent from the adjacent muscle layers and from the newborn stomachs. In addition, several common background adducts were also present in both the mucosa and muscle layer. G2 was the same DNA adduct as Si2 in the small intestine and C1 in the colon, and G3 was the same as Si1 in the small intestine. Thus, it was demonstrated that the mucosa of the stomach was exposed to DNA-reactive substances. © 2003 International Society for Preventive Oncology. Published by Elsevier Science Ltd. All rights reserved. Keywords: DNA adduct; 32 P-postlabeling; Carcinogenesis; Stomach; Digestive organ
1. Introduction Gastric cancer is the most common malignancy in many countries, although it is declining in frequency in almost all populations [1]. The highest incidence of gastric cancer worldwide was previously found in Japan, but it has also decreased comparatively, and colorectal cancer has increased greatly over the last three decades and it may cross the incidence of gastric cancer in near future [2]. Epidemiological studies suggest that the Westernization of Japanese dietary habits is the main cause for this change [3]. Studies of dietary factors indicated that the risk for stomach cancer is associated with high intake of smoked, salted, pickled, and preserved foods (rich in salt, nitrite, nitrosoable compounds and preformed N-nitroso compounds), high intake of carbohydrates and low intake of fruits, vegetables and milk [2,4,5], although studies of factors other than dietary factors suggest that concurrent or previous infection with Helicobacter pylori or Epstein–Barr (EB) virus is also a risk factor [6]. Molecular biological studies have revealed that genetic alterations in genes such as c-Ki-ras, c-erbB-2, K-sam, hst/int-2, c-met, c-myc, p53, APC, DCC and RB1, as well as LOH in several chromosomes play a role in ∗ Corresponding author. Tel.: +81-88-633-7143; fax: +81-88-633-7144. E-mail address: [email protected] (A. Umemoto).
gastric carcinogenesis [7]. It has been suggested that DNA adduct formation by dietary carcinogens may be an important cause of such gene mutations. Although the level of human exposure to a given dietary carcinogen should be quite low, covalent DNA adducts have been found in the human digestive tract by means of 32 P-postlabeling [8–11]. A notable clinical observation is that most cancers of the digestive tract develop from cells of the mucosa which are constantly exposed to intraluminal contents, not from cells of the muscle layer adjacent to the mucosa. This leads to the simple idea that DNA modifications by carcinogens leading to critical mutations that cause cancer may be more frequent in the mucosa than in the muscle layer. In our previous study, DNA adduct formation in the normal mucosa and muscle layer of the colon and small intestine of patients suffering from neoplastic diseases was examined by means of 32 P-postlabeling [9,10]. Covalent DNA adducts were found in both the mucosa and the muscle layer of the human intestine and were classified into two types; i.e. common background DNA adducts that are present in both the mucosa and muscle layer and mucosa-specific DNA adducts. Only one spot was identified as a mucosa-specific DNA adduct of the colon (C1) while two were identified as those of the small intestine (Si1 and 2) by means of thin layer chromatography. Since the mucosa-specific DNA adducts are associated only with mucosa and not with the
0361-090X/03/$30.00 © 2003 International Society for Preventive Oncology. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0361-090X(03)00065-5
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muscle layer and are not present in corresponding neonatal tissues, it is postulated that these DNA adducts play an initiating role in cancer of the colon and small intestine. In the present study, we attempted to detect mucosa-specific DNA adducts in the human stomach, and to compare them with the mucosa-specific DNA adducts of the colon and small intestine.
2. Materials and methods
further incubated at 37 ◦ C for 30 min. The mixture was successively extracted with 1 vol. each of phenol (saturated with 0.1 M Tris–HCl (pH 8.0)), a 1:1 mixture of phenol/sevag (chloroform/isoamyl alcohol, 24:1) and sevag. DNA was precipitated by adding 0.1 vol. of 5 M NaCl and 1 vol. of cold absolute ethanol. The DNA precipitate was collected, washed twice with 70% cold ethanol and dissolved in 1 ml of 0.01 × SSC/1 mM EDTA (1 × SSC = 0.15 M NaCl/0.015 M sodium citrate). The DNA was further purified by digestion with RNases followed by proteinase K, and the above extraction procedures were repeated.
2.1. Chemicals 2.4. RNase A, RNase T1, micrococcal nuclease and spleen phosphodiesterase were purchased from Worthington Biochemical Co. (Freehold, NJ). Nuclease P1 and T4 polynucleotide kinase were obtained from Yamasa Shoyu Co. (Choshi, Japan) and Pharmacia Fine Chemicals (Uppsala, Sweden), respectively. Proteinase K was purchased from Sigma (St. Louis, MO, USA). [␥-32 P]adenosine-5 -triphosphate (>7000 Ci/mmol) was obtained from ICN Radiochemicals (Irvine, CA). Polyethyleneimine-cellulose sheets (Polygram Cell 300 PEI) were purchased from Machery– Nagel (Düren, Germany). 2.2. Human tissues Non-cancerous parts of the stomach wall were collected from 19 adult patients (14 males and 5 females; 48–87 years old; mean 66 years old) who had undergone surgery for neoplasms of the stomach. The wall of the adult stomach was separated into the mucosal and muscle layers immediately after resection. Stomach samples (combined layers) from six newborns who died without having ingested breast milk were collected at autopsy. The informed consent of their families was taken prior to investigation. The ethical standards of the committee on human experimentation of the institution in which the experiments were done were followed. For the comparative study, DNA samples of normal tissues of the colon from an 83-year-old female patient with colon cancer and the small intestine from a 71-year-old male patient with ileus which showed representative mucosa-specific DNA adduct profiles in our previous study were used. The specimens were stored at −80 ◦ C until analysis. 2.3. DNA purification DNA was isolated as described with minor modifications [12]. Briefly, frozen tissue (0.25 g) was thawed in 3 ml of 1.0% SDS/10 mM EDTA/20 mM Tris–HCl (pH 7.4) and treated with a Polytron homogenizer for 30 s. The homogenate was incubated at 37 ◦ C for 30 min with a mixture of RNase A (200 g/ml) and RNase T1 (34 units/ml). After addition of proteinase K (500 g/ml), the homogenate was
32 P-postlabeling
DNA adducts were analyzed by means of the nucleoside bisphosphate procedure with adduct enrichment by nuclease P1 as follows [13]. Ten micrograms of DNA (2 mg/ml) was digested to deoxynucleoside 3 -monophosphates by micrococcal nuclease and spleen phosphodiesterase at 37 ◦ C for 3.5 h [14]. Thereafter, the mixture was digested with nuclease P1 for 1 h. The digest was then converted to 5 -32 P-labeled deoxynucleoside 3 ,5 -bisphosphates using [␥-32 P]ATP and T4 polynucleotide kinase at 37 ◦ C for 1 h. The 32 P-labeled adducts were purified by chromatography on PEI-cellulose sheets with 2.3 M sodium phosphate (pH 6.0) at room temperature for 15 h. The part of the sheet around the origin was cut out and the adducts remaining at the origin were contact-transferred to new PEI-cellulose sheets (8.5 cm × 8.5 cm) for conventional two-dimensional chromatography. The adducts were separated with 4.05 M lithium formate/7.65 M urea (pH 3.5) from the bottom to the top (D1) of the sheets and with 1.2 M lithium chloride/0.5 M Tris–HCl/8 M urea (pH 8.0) from left to right (D2) followed by 1.7 M sodium phosphate buffer (pH 6.0) with a 3.5 cm paper wick in the same direction as D2. The adducts were visualized by autoradiography using Kodak XAR-5 film with an intensifying screen (Dupont Quanta III) at −80 ◦ C. To quantify the DNA adduct level, the number of total nucleotides was measured by one-dimensional PEI-cellulose TLC after appropriate dilution of the labeled digest [13]. The radioactivity level of each excised spot was measured, and the relative adduct labeling (RAL) value was calculated. 2.5. High-resolution comparative study To determine more exactly the mucosa-specific DNA adducts of the stomach, high-resolution two-dimensional chromatography was performed; i.e. the adducts at the TLC origin as well as those separated by the conventional method were contact-transferred to larger PEI-cellulose sheets (14 cm × 14 cm), and developed with 2.5 M lithium formate/6.0 M urea (pH 3.5) from the bottom to the top (D1) and with 1.2 M lithium chloride/0.4 M Tris–HCl/6.4 M urea (pH 8.0) from left to right (D2) followed by 1.7 M sodium phosphate buffer (pH 6.0). DNA samples from the paired
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mucosa and muscle layer of the stomach of adult patient #1, the whole gastric wall of newborn patient #4, the small intestinal mucosa of the male patient with ileus (71 years old), and the colonic mucosa of the female patient with colon cancer (83 years old) were analyzed by this method. For the co-chromatographic study, 32 P-labeled digests of the gastric mucosa DNA and small intestine or colonic mucosa DNA were spotted at the same origin of TLC sheets and developed.
3. Results In the paired stomach mucosa and muscle layer of all adult patients, covalent DNA adducts were detected in the human stomach (Fig. 1). The DNA adducts could be classified into two types, namely, common background DNA adducts and mucosa-specific DNA adducts, as seen previously in the case of the colon or small intestine. Several common background DNA adducts were detected in both the mucosa and muscle layer of all adult patients, with similar patterns of prevalence. Inter-individual differences in the levels of common background DNA adducts were small. These common background DNA adducts were also found in the stomachs of the six newborns. In addition to the common background DNA adducts, five mucosa-specific DNA adducts (G1–5) were found in the gastric mucosa at mean levels of 0.11–0.35 adducts/108 nucleotides, but these were entirely absent in the mucosaadjacent muscle layer of the adult stomachs and in the combined layers of the newborn stomachs (Fig. 1 and Table 1). There was no muscle layer-specific DNA adduct which was
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not present in the mucosa. All five mucosa-specific DNA adducts were not always present in every person, but the G3 adduct was present in all cases. The total mucosa-specific DNA adduct level of patients who smoked (0.87 ± 0.99 adducts/108 nucleotides) was slightly but not significantly higher than that of the non-smoking patients (0.72 ± 0.29). In the patients with ‘mucosa-origin’ carcinoma (patients #1–17), there was no relationship between the histological type of carcinoma and the total level of mucosa-specific DNA adducts. However, among 7 undifferentiated carcinoma cases (diffuse type; poorly differentiated adenocarcinoma and signet ring cell carcinoma), 6 cases had the G1 adduct, while among 10 differentiated carcinoma cases (intestinal type; well and moderately differentiated adenocarcinoma), only 1 case had the G1 adduct (Table 1). The presence of G2–5 adducts was not related to the histological type. The average age of the cases positive for G1 (59.0 ± 9.1 years old) was slightly younger than that of cases positive for G2 (65.3 ± 7.9 years old), G3 (64.5 ± 8.2 years old), G4 (65.6 ± 6.6 years old) and G5 (64.6 ± 10.3 years old). In addition, among seven G1-positive cases, only one case was G4 positive, while among 10 G1-negative cases, 6 cases were G4 positive. Thus, it appeared that there was a complementary relationship between G1 and 4. In our previous study, a high-resolution co-chromatographic study using large TLC sheets showed that mucosaspecific DNA adducts of the small intestine (Si2) were identical to mucosa-specific DNA adducts of the colon (C1) (10). In the present high-resolution study, two more mucosa-specific DNA adducts in addition to those in the conventional study were detected in the gastric mucosa of
Fig. 1. 32 P-postlabeling profiles of DNA adducts of the stomach of adult patients. The normal part of the stomach wall of the adult patients was separated into the mucosa and adjacent muscle layer, and pairs of these DNA samples were analyzed by 32 P-postlabeling with nuclease P1 enrichment. A–a, B–b, C–c and D–d are paired mucosa (capital letters) and muscle layer (small letters) samples from patients #12, 8, 1 and 2, respectively. Closed arrows ( ) indicate the mucosa-specific adducts (G1–5). Open arrows ( ) indicate the common background adducts.
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Table 1 Mucosa-specific DNA adducts in the human stomach Patients
Age (years)
Sex
Smoking
DNA adduct level (RAL × 108 )
Histology
G1
G2
G3
G4
G5
Total
Adult 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
68 73 72 63 72 48 67 74 55 74 67 57 50 67 58 64 68
M M M M M M F M M M M M F F M M F
–a + + + + + – + + – + + – – + + –
– 0.09 – 0.12 – 0.33 0.36 – 0.05 – – 0.08 0.04 – – – –
0.12 0.12 – 0.11 0.10 0.07 0.06 0.14 0.10 0.13 0.11 0.10 – 0.11 – 0.11 –
1.18 0.64 0.42 0.66 0.39 0.38 0.31 0.59 0.23 0.35 0.40 0.41 0.07 0.18 0.09 0.14 0.12
0.82 – 0.62 – 0.37 – – – 0.32 – 0.05 – – 0.14 0.13 – –
0.73 0.37 – – – – – – – 0.08 – – 0.40 – 0.11 – –
2.85 1.22 1.04 0.89 0.86 0.78 0.73 0.73 0.70 0.56 0.56 0.51 0.51 0.43 0.33 0.25 0.12
Wb Pc W Md P Se S W P M M P P W M W W
18 19
87 68
M F
– –
– –
– –
0.07 0.05
– –
0.04 –
0.11 0.05
BLf MLg
7/19
13/19
19/19
7/19
6/19
– – – – – –
– – – – – –
– – – – – –
– – – – – –
– – – – – –
0/6
0/6
0/6
0/6
0/6
Adduct positive ratio Newbornh 1 2 3 4 5 6
1 2 1 1 7 1
M M F M F M Adduct positive ratio
0 0 0 0 0 0
a
Not detected. W: well differentiated adenocarcinoma. c P: poorly differentiated adenocarcinoma. d M: moderately differentiated adenocarcinoma. e S: signet ring cell carcinoma. f BL: benign leiomyoma. g ML: malignant lymphoma. h Age is in days. b
patient #1 (the arrows marked with “?”, Fig. 2A and B). The pattern of the mucosa-specific adducts on the present TLC was slightly different from that on conventional TLC. The G2/Si2 spot only showed tailing from a left to right direction, although this tailing was not observed in the conventional TLC. The co-chromatographic study clarified that among the stomach mucosa-specific DNA adducts, G2 was identical to Si2 or C1 (Fig. 2F and G), and G3 was identical to Si1 (Fig. 2F). Thus, the number of mucosa-specific DNA adducts decreased in the order stomach-small intestinecolon. G2 and 3, which were commonly observed in intestinal DNA, were the most common mucosa-specific DNA adducts in the stomach. The high-resolution TLC study also showed that the pattern of the common background DNA adducts in the newborn stomach was very similar to that in the adult stomach (Fig. 2A–C).
4. Discussion The 32 P-postlabeling assay is highly sensitive and suitable for detecting a very low level of DNA adducts in human samples. Although it lacks adduct specificity, a diverse spectrum of DNA adducts containing one aromatic ring or a bulky non-aromatic moiety can be detected by this method [15]. It is not known that such bulky DNA modifications in human cells exert normal physiological functions. On the other hand, many of the carcinogens so far tested bind DNA in vivo and in vitro [16], and there are reports that DNA adduct-forming capacity and carcinogenic potency of many polycyclic aromatic hydrocarbons or alkylating agents are well correlated in animals [17,18]. Of course, it should be noted from animal experiments that DNA adducts do form in non-target tissues and these is no evidence that the formation
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Fig. 2. High-resolution comparative study of DNA adducts in the stomach, small intestine and colon using large TLC sheets. (A) Gastric mucosa of adult patient #1. (B) Gastric muscle layer of adult patient #1. (C) Whole gastric wall of newborn patient #4. (D) Small intestinal mucosa of the male patient with ileus (71 years old). (E) Colonic mucosa of the female patient with colon cancer (83 years old). (F) Co-chromatography of A and D. (G) Co-chromatography of A and E. Closed arrows ( ) indicate the mucosa-specific DNA adducts. The numbers of mucosa-specific DNA adducts correspond to those of Fig. 1. Open arrows ( ) indicate the common background DNA adducts.
of DNA adducts can be directly linked to gene mutation or cancer development in animals and humans. Although DNA adducts is a reliable marker for carcinogen exposure, only DNA adducts is not sufficient event in the multiple stage of carcinogenesis. However, it is supposed that the present results give some implicative information about the association between mucosa-specific DNA adducts and gastrointestinal carcinogenesis. Since gastric cancer is mostly developed from the mucosa, not from the adjacent muscle layer, DNA adducts which exist only in the mucosa but not in the muscle layer are possibly responsible for “mucosa-origin” gastric carcinomas. Although many DNA adducts were detected in the mucosa and the muscle layer of the stomach, comparison of the mucosa with the adjacent muscle layer reveals two types of adducts, mucosa-specific DNA adducts and common background DNA adducts. The level of the mucosa-specific
DNA adducts is likely to be correlated with the incidence of ‘mucosa-origin’ carcinoma, as shown by the following findings. The mucosa-specific DNA adducts were entirely absent in the adjacent muscle layer, in which cancer is rare. The mucosa-specific DNA adducts were also absent in newborns who had had no chance to develop gastric cancer. Furthermore, epidemiological studies show that the annual crude rates of incidence of stomach and colorectal cancers per 100,000 in Japan are 105.8, 57.5 (male, female) and 42.5, 33.8 (male, female), respectively, while the rate is quite low (0.4) for cancers in the small intestine [19]. In agreement with these findings, our previous studies and the present study showed that the total mucosa-specific DNA adduct level in the small intestine (0.13 ± 0.07 adducts/108 nucleotides) [10], was about 28-fold lower than that in the colon (3.64 ± 7.92 adducts/108 nucleotides) [9], and five-fold lower than that in the stomach (0.70 ± 0.61
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adducts/108 nucleotides). Of course the idea that DNA adduct level at a single point in time in patients may be associated with the feasibility of cancer is simplistic. DNA adduct level is determined by many factors such as enzyme activities of activation/inactivation of carcinogens and rate of DNA repair. The present findings may suggest that the mucosa-specific DNA adducts play some role in carcinogenesis in the human stomach and/or intestine, but there is another possibility that the mucosa-specific DNA adduct may simply be related to the process of aging and nothing to do with the induction of gastric carcinomas. To clarify the association between the mucosa-specific DNA adducts and carcinogenesis of the stomach, it is necessary to identify the substances that form these DNA adducts. This might be learned by comparing individuals with and without gastric carcinoma, although it is very rare to obtain the stomach samples from non-cancerous patients. Histologically, gastric carcinoma is classified into two types, undifferentiated carcinoma (diffuse type) with high malignant potential which arises from the ordinary mucosa, and differentiated one (intestinal type) with low malignant potential, which arises from the intestinal metaplastic epithelium [20]. In the present study the, G1 adduct was rather specifically detected in somewhat younger patients with undifferentiated carcinoma. This may suggest that the G1 adduct induces specific gene mutation(s) leading to undifferentiated carcinoma. With respect to histology, Dyke et al. previously compared the gastric mucosa DNA adducts from normal subjects with those from patients with chronic atrophic gastritis and intestinal metaplasia, which may be considered pre-malignant conditions [21]. They could not find any difference in adduct patterns or adduct levels for any patients, and concluded that DNA adduct levels do not correlate with the presence of histological abnormalities in the stomach and are not useful as a marker of malignant potential. But, according to our findings, there is a possibility of reaching a different conclusion, if the mucosa-specific DNA adducts are selectively analyzed in these samples. The origin of the DNA adducts detected in the stomach may be dietary carcinogens, because the incidence of gastric cancer is closely associated with dietary habits. Among the possible dietary carcinogens for gastric cancer, DNA adducts of heterocyclic amines or alkylation by N-nitroso compounds cannot be detected by the nuclease P1-enrichment method of 32 P-postlabeling. Although progressive decrease of mucosa-specific DNA adduct types was shown from the upper stomach to the lower colon, this does not necessarily indicate that these mucosal adducts were formed through direct exposure from the intraluminal direction of carcinogens which were present in the gastrointestinal content. For example, when human dietary carcinogen, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), was administered into the rat stomach, adduct formation was induced mainly through the circulatory exposure, but partly through the intraluminal exposure [22,23]. However, in the case of 2-amino1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the DNA
adduct formation through intraluminal exposure was negligible (unpublished data). In addition to dietary factors, cigarette smoking is also an important risk factor for gastric cancer. Dyke et al. previously demonstrated that in male subjects, adduct levels were significantly greater in the DNA of smokers than in that of non-smokers by comparing DNA adducts in the tumor tissue of smokers and non-smokers with gastric cancer by the 32 P-postlabeling method [24]. The present results also showed the same tendency, although there was no significant difference. In contrast, the common background DNA adducts observed in both the mucosa and muscle layer and also in different organs (stomach, small intestine and colon) showed no relationship between the adduct level and the incidence of cancer, indicating that they are not associated with carcinogenesis in the digestive tract. Some common background DNA adducts may be artifacts produced during assays, but others may be formed by endogenous factors, since (1) some of them were also present in tissues from newborns, who were presumably less exposed to environmental carcinogens than adults and (2) the levels of these adducts were rather similar inter-individually, and in the mucosa and muscle layer, or even among the stomach, small intestine and colon. Our search for endogenous substances forming common background DNA adducts revealed that some hormonal steroids in humans can form covalent DNA adducts in vitro [25]. The common background DNA adducts found in the human digestive organs may correspond with the ‘I-compounds’ reported by Randerath et al. [26]. Finally, it should be emphasized that the separate preparation of the mucosa and muscle layer and comparison of their DNA adducts were essential for distinguishing cancer-responsible adducts from unimportant adducts.
Acknowledgements This work was supported by Grants-in-Aids from the Ministry of Education, Science, Sports and Culture of Japan. We thank Akio Kubota and Masahiro Nakayama, Osaka Medical Center and Research Institute for Maternal and Child Health, Japan for providing samples from newborns. The doctoral fellowship of Mohammed Abdul Momen from the Ministry of Education, Science, Sports and Culture of Japan is gratefully acknowledged.
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DNA adducts detected in human gastric mucosa Mohammed Abdul Momen, MD, Yasumasa Monden, MD, PhD, Kunimi Hamada, MD, PhD, Kansei Komaki, MD, PhD, Kazuya Kondo, MD, PhD, Atsushi Umemoto, MD, PhD∗ Second Department of Surgery, School of Medicine, University of Tokushima, 3-18-15, Kuramoto-cho, Tokushima 770-8503, Japan Accepted 25 February 2003
Abstract Human gastrointestinal neoplasms are mostly developed from the mucosa, not from the adjacent muscle layer. DNA adducts in the mucosa and adjacent muscle layer of the non-tumoral part of stomach from 19 patients with gastric neoplasms and from six newborns were analyzed by 32 P-postlabeling, and then compared them with those of representative colon or small intestine sample. Five kinds of mucosa-specific DNA adducts (G1–5) were found in all of the adult stomach samples, but were entirely absent from the adjacent muscle layers and from the newborn stomachs. In addition, several common background adducts were also present in both the mucosa and muscle layer. G2 was the same DNA adduct as Si2 in the small intestine and C1 in the colon, and G3 was the same as Si1 in the small intestine. Thus, it was demonstrated that the mucosa of the stomach was exposed to DNA-reactive substances. © 2003 International Society for Preventive Oncology. Published by Elsevier Science Ltd. All rights reserved. Keywords: DNA adduct; 32 P-postlabeling; Carcinogenesis; Stomach; Digestive organ
1. Introduction Gastric cancer is the most common malignancy in many countries, although it is declining in frequency in almost all populations [1]. The highest incidence of gastric cancer worldwide was previously found in Japan, but it has also decreased comparatively, and colorectal cancer has increased greatly over the last three decades and it may cross the incidence of gastric cancer in near future [2]. Epidemiological studies suggest that the Westernization of Japanese dietary habits is the main cause for this change [3]. Studies of dietary factors indicated that the risk for stomach cancer is associated with high intake of smoked, salted, pickled, and preserved foods (rich in salt, nitrite, nitrosoable compounds and preformed N-nitroso compounds), high intake of carbohydrates and low intake of fruits, vegetables and milk [2,4,5], although studies of factors other than dietary factors suggest that concurrent or previous infection with Helicobacter pylori or Epstein–Barr (EB) virus is also a risk factor [6]. Molecular biological studies have revealed that genetic alterations in genes such as c-Ki-ras, c-erbB-2, K-sam, hst/int-2, c-met, c-myc, p53, APC, DCC and RB1, as well as LOH in several chromosomes play a role in ∗ Corresponding author. Tel.: +81-88-633-7143; fax: +81-88-633-7144. E-mail address: [email protected] (A. Umemoto).
gastric carcinogenesis [7]. It has been suggested that DNA adduct formation by dietary carcinogens may be an important cause of such gene mutations. Although the level of human exposure to a given dietary carcinogen should be quite low, covalent DNA adducts have been found in the human digestive tract by means of 32 P-postlabeling [8–11]. A notable clinical observation is that most cancers of the digestive tract develop from cells of the mucosa which are constantly exposed to intraluminal contents, not from cells of the muscle layer adjacent to the mucosa. This leads to the simple idea that DNA modifications by carcinogens leading to critical mutations that cause cancer may be more frequent in the mucosa than in the muscle layer. In our previous study, DNA adduct formation in the normal mucosa and muscle layer of the colon and small intestine of patients suffering from neoplastic diseases was examined by means of 32 P-postlabeling [9,10]. Covalent DNA adducts were found in both the mucosa and the muscle layer of the human intestine and were classified into two types; i.e. common background DNA adducts that are present in both the mucosa and muscle layer and mucosa-specific DNA adducts. Only one spot was identified as a mucosa-specific DNA adduct of the colon (C1) while two were identified as those of the small intestine (Si1 and 2) by means of thin layer chromatography. Since the mucosa-specific DNA adducts are associated only with mucosa and not with the
0361-090X/03/$30.00 © 2003 International Society for Preventive Oncology. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0361-090X(03)00065-5
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muscle layer and are not present in corresponding neonatal tissues, it is postulated that these DNA adducts play an initiating role in cancer of the colon and small intestine. In the present study, we attempted to detect mucosa-specific DNA adducts in the human stomach, and to compare them with the mucosa-specific DNA adducts of the colon and small intestine.
2. Materials and methods
further incubated at 37 ◦ C for 30 min. The mixture was successively extracted with 1 vol. each of phenol (saturated with 0.1 M Tris–HCl (pH 8.0)), a 1:1 mixture of phenol/sevag (chloroform/isoamyl alcohol, 24:1) and sevag. DNA was precipitated by adding 0.1 vol. of 5 M NaCl and 1 vol. of cold absolute ethanol. The DNA precipitate was collected, washed twice with 70% cold ethanol and dissolved in 1 ml of 0.01 × SSC/1 mM EDTA (1 × SSC = 0.15 M NaCl/0.015 M sodium citrate). The DNA was further purified by digestion with RNases followed by proteinase K, and the above extraction procedures were repeated.
2.1. Chemicals 2.4. RNase A, RNase T1, micrococcal nuclease and spleen phosphodiesterase were purchased from Worthington Biochemical Co. (Freehold, NJ). Nuclease P1 and T4 polynucleotide kinase were obtained from Yamasa Shoyu Co. (Choshi, Japan) and Pharmacia Fine Chemicals (Uppsala, Sweden), respectively. Proteinase K was purchased from Sigma (St. Louis, MO, USA). [␥-32 P]adenosine-5 -triphosphate (>7000 Ci/mmol) was obtained from ICN Radiochemicals (Irvine, CA). Polyethyleneimine-cellulose sheets (Polygram Cell 300 PEI) were purchased from Machery– Nagel (Düren, Germany). 2.2. Human tissues Non-cancerous parts of the stomach wall were collected from 19 adult patients (14 males and 5 females; 48–87 years old; mean 66 years old) who had undergone surgery for neoplasms of the stomach. The wall of the adult stomach was separated into the mucosal and muscle layers immediately after resection. Stomach samples (combined layers) from six newborns who died without having ingested breast milk were collected at autopsy. The informed consent of their families was taken prior to investigation. The ethical standards of the committee on human experimentation of the institution in which the experiments were done were followed. For the comparative study, DNA samples of normal tissues of the colon from an 83-year-old female patient with colon cancer and the small intestine from a 71-year-old male patient with ileus which showed representative mucosa-specific DNA adduct profiles in our previous study were used. The specimens were stored at −80 ◦ C until analysis. 2.3. DNA purification DNA was isolated as described with minor modifications [12]. Briefly, frozen tissue (0.25 g) was thawed in 3 ml of 1.0% SDS/10 mM EDTA/20 mM Tris–HCl (pH 7.4) and treated with a Polytron homogenizer for 30 s. The homogenate was incubated at 37 ◦ C for 30 min with a mixture of RNase A (200 g/ml) and RNase T1 (34 units/ml). After addition of proteinase K (500 g/ml), the homogenate was
32 P-postlabeling
DNA adducts were analyzed by means of the nucleoside bisphosphate procedure with adduct enrichment by nuclease P1 as follows [13]. Ten micrograms of DNA (2 mg/ml) was digested to deoxynucleoside 3 -monophosphates by micrococcal nuclease and spleen phosphodiesterase at 37 ◦ C for 3.5 h [14]. Thereafter, the mixture was digested with nuclease P1 for 1 h. The digest was then converted to 5 -32 P-labeled deoxynucleoside 3 ,5 -bisphosphates using [␥-32 P]ATP and T4 polynucleotide kinase at 37 ◦ C for 1 h. The 32 P-labeled adducts were purified by chromatography on PEI-cellulose sheets with 2.3 M sodium phosphate (pH 6.0) at room temperature for 15 h. The part of the sheet around the origin was cut out and the adducts remaining at the origin were contact-transferred to new PEI-cellulose sheets (8.5 cm × 8.5 cm) for conventional two-dimensional chromatography. The adducts were separated with 4.05 M lithium formate/7.65 M urea (pH 3.5) from the bottom to the top (D1) of the sheets and with 1.2 M lithium chloride/0.5 M Tris–HCl/8 M urea (pH 8.0) from left to right (D2) followed by 1.7 M sodium phosphate buffer (pH 6.0) with a 3.5 cm paper wick in the same direction as D2. The adducts were visualized by autoradiography using Kodak XAR-5 film with an intensifying screen (Dupont Quanta III) at −80 ◦ C. To quantify the DNA adduct level, the number of total nucleotides was measured by one-dimensional PEI-cellulose TLC after appropriate dilution of the labeled digest [13]. The radioactivity level of each excised spot was measured, and the relative adduct labeling (RAL) value was calculated. 2.5. High-resolution comparative study To determine more exactly the mucosa-specific DNA adducts of the stomach, high-resolution two-dimensional chromatography was performed; i.e. the adducts at the TLC origin as well as those separated by the conventional method were contact-transferred to larger PEI-cellulose sheets (14 cm × 14 cm), and developed with 2.5 M lithium formate/6.0 M urea (pH 3.5) from the bottom to the top (D1) and with 1.2 M lithium chloride/0.4 M Tris–HCl/6.4 M urea (pH 8.0) from left to right (D2) followed by 1.7 M sodium phosphate buffer (pH 6.0). DNA samples from the paired
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mucosa and muscle layer of the stomach of adult patient #1, the whole gastric wall of newborn patient #4, the small intestinal mucosa of the male patient with ileus (71 years old), and the colonic mucosa of the female patient with colon cancer (83 years old) were analyzed by this method. For the co-chromatographic study, 32 P-labeled digests of the gastric mucosa DNA and small intestine or colonic mucosa DNA were spotted at the same origin of TLC sheets and developed.
3. Results In the paired stomach mucosa and muscle layer of all adult patients, covalent DNA adducts were detected in the human stomach (Fig. 1). The DNA adducts could be classified into two types, namely, common background DNA adducts and mucosa-specific DNA adducts, as seen previously in the case of the colon or small intestine. Several common background DNA adducts were detected in both the mucosa and muscle layer of all adult patients, with similar patterns of prevalence. Inter-individual differences in the levels of common background DNA adducts were small. These common background DNA adducts were also found in the stomachs of the six newborns. In addition to the common background DNA adducts, five mucosa-specific DNA adducts (G1–5) were found in the gastric mucosa at mean levels of 0.11–0.35 adducts/108 nucleotides, but these were entirely absent in the mucosaadjacent muscle layer of the adult stomachs and in the combined layers of the newborn stomachs (Fig. 1 and Table 1). There was no muscle layer-specific DNA adduct which was
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not present in the mucosa. All five mucosa-specific DNA adducts were not always present in every person, but the G3 adduct was present in all cases. The total mucosa-specific DNA adduct level of patients who smoked (0.87 ± 0.99 adducts/108 nucleotides) was slightly but not significantly higher than that of the non-smoking patients (0.72 ± 0.29). In the patients with ‘mucosa-origin’ carcinoma (patients #1–17), there was no relationship between the histological type of carcinoma and the total level of mucosa-specific DNA adducts. However, among 7 undifferentiated carcinoma cases (diffuse type; poorly differentiated adenocarcinoma and signet ring cell carcinoma), 6 cases had the G1 adduct, while among 10 differentiated carcinoma cases (intestinal type; well and moderately differentiated adenocarcinoma), only 1 case had the G1 adduct (Table 1). The presence of G2–5 adducts was not related to the histological type. The average age of the cases positive for G1 (59.0 ± 9.1 years old) was slightly younger than that of cases positive for G2 (65.3 ± 7.9 years old), G3 (64.5 ± 8.2 years old), G4 (65.6 ± 6.6 years old) and G5 (64.6 ± 10.3 years old). In addition, among seven G1-positive cases, only one case was G4 positive, while among 10 G1-negative cases, 6 cases were G4 positive. Thus, it appeared that there was a complementary relationship between G1 and 4. In our previous study, a high-resolution co-chromatographic study using large TLC sheets showed that mucosaspecific DNA adducts of the small intestine (Si2) were identical to mucosa-specific DNA adducts of the colon (C1) (10). In the present high-resolution study, two more mucosa-specific DNA adducts in addition to those in the conventional study were detected in the gastric mucosa of
Fig. 1. 32 P-postlabeling profiles of DNA adducts of the stomach of adult patients. The normal part of the stomach wall of the adult patients was separated into the mucosa and adjacent muscle layer, and pairs of these DNA samples were analyzed by 32 P-postlabeling with nuclease P1 enrichment. A–a, B–b, C–c and D–d are paired mucosa (capital letters) and muscle layer (small letters) samples from patients #12, 8, 1 and 2, respectively. Closed arrows ( ) indicate the mucosa-specific adducts (G1–5). Open arrows ( ) indicate the common background adducts.
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Table 1 Mucosa-specific DNA adducts in the human stomach Patients
Age (years)
Sex
Smoking
DNA adduct level (RAL × 108 )
Histology
G1
G2
G3
G4
G5
Total
Adult 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
68 73 72 63 72 48 67 74 55 74 67 57 50 67 58 64 68
M M M M M M F M M M M M F F M M F
–a + + + + + – + + – + + – – + + –
– 0.09 – 0.12 – 0.33 0.36 – 0.05 – – 0.08 0.04 – – – –
0.12 0.12 – 0.11 0.10 0.07 0.06 0.14 0.10 0.13 0.11 0.10 – 0.11 – 0.11 –
1.18 0.64 0.42 0.66 0.39 0.38 0.31 0.59 0.23 0.35 0.40 0.41 0.07 0.18 0.09 0.14 0.12
0.82 – 0.62 – 0.37 – – – 0.32 – 0.05 – – 0.14 0.13 – –
0.73 0.37 – – – – – – – 0.08 – – 0.40 – 0.11 – –
2.85 1.22 1.04 0.89 0.86 0.78 0.73 0.73 0.70 0.56 0.56 0.51 0.51 0.43 0.33 0.25 0.12
Wb Pc W Md P Se S W P M M P P W M W W
18 19
87 68
M F
– –
– –
– –
0.07 0.05
– –
0.04 –
0.11 0.05
BLf MLg
7/19
13/19
19/19
7/19
6/19
– – – – – –
– – – – – –
– – – – – –
– – – – – –
– – – – – –
0/6
0/6
0/6
0/6
0/6
Adduct positive ratio Newbornh 1 2 3 4 5 6
1 2 1 1 7 1
M M F M F M Adduct positive ratio
0 0 0 0 0 0
a
Not detected. W: well differentiated adenocarcinoma. c P: poorly differentiated adenocarcinoma. d M: moderately differentiated adenocarcinoma. e S: signet ring cell carcinoma. f BL: benign leiomyoma. g ML: malignant lymphoma. h Age is in days. b
patient #1 (the arrows marked with “?”, Fig. 2A and B). The pattern of the mucosa-specific adducts on the present TLC was slightly different from that on conventional TLC. The G2/Si2 spot only showed tailing from a left to right direction, although this tailing was not observed in the conventional TLC. The co-chromatographic study clarified that among the stomach mucosa-specific DNA adducts, G2 was identical to Si2 or C1 (Fig. 2F and G), and G3 was identical to Si1 (Fig. 2F). Thus, the number of mucosa-specific DNA adducts decreased in the order stomach-small intestinecolon. G2 and 3, which were commonly observed in intestinal DNA, were the most common mucosa-specific DNA adducts in the stomach. The high-resolution TLC study also showed that the pattern of the common background DNA adducts in the newborn stomach was very similar to that in the adult stomach (Fig. 2A–C).
4. Discussion The 32 P-postlabeling assay is highly sensitive and suitable for detecting a very low level of DNA adducts in human samples. Although it lacks adduct specificity, a diverse spectrum of DNA adducts containing one aromatic ring or a bulky non-aromatic moiety can be detected by this method [15]. It is not known that such bulky DNA modifications in human cells exert normal physiological functions. On the other hand, many of the carcinogens so far tested bind DNA in vivo and in vitro [16], and there are reports that DNA adduct-forming capacity and carcinogenic potency of many polycyclic aromatic hydrocarbons or alkylating agents are well correlated in animals [17,18]. Of course, it should be noted from animal experiments that DNA adducts do form in non-target tissues and these is no evidence that the formation
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Fig. 2. High-resolution comparative study of DNA adducts in the stomach, small intestine and colon using large TLC sheets. (A) Gastric mucosa of adult patient #1. (B) Gastric muscle layer of adult patient #1. (C) Whole gastric wall of newborn patient #4. (D) Small intestinal mucosa of the male patient with ileus (71 years old). (E) Colonic mucosa of the female patient with colon cancer (83 years old). (F) Co-chromatography of A and D. (G) Co-chromatography of A and E. Closed arrows ( ) indicate the mucosa-specific DNA adducts. The numbers of mucosa-specific DNA adducts correspond to those of Fig. 1. Open arrows ( ) indicate the common background DNA adducts.
of DNA adducts can be directly linked to gene mutation or cancer development in animals and humans. Although DNA adducts is a reliable marker for carcinogen exposure, only DNA adducts is not sufficient event in the multiple stage of carcinogenesis. However, it is supposed that the present results give some implicative information about the association between mucosa-specific DNA adducts and gastrointestinal carcinogenesis. Since gastric cancer is mostly developed from the mucosa, not from the adjacent muscle layer, DNA adducts which exist only in the mucosa but not in the muscle layer are possibly responsible for “mucosa-origin” gastric carcinomas. Although many DNA adducts were detected in the mucosa and the muscle layer of the stomach, comparison of the mucosa with the adjacent muscle layer reveals two types of adducts, mucosa-specific DNA adducts and common background DNA adducts. The level of the mucosa-specific
DNA adducts is likely to be correlated with the incidence of ‘mucosa-origin’ carcinoma, as shown by the following findings. The mucosa-specific DNA adducts were entirely absent in the adjacent muscle layer, in which cancer is rare. The mucosa-specific DNA adducts were also absent in newborns who had had no chance to develop gastric cancer. Furthermore, epidemiological studies show that the annual crude rates of incidence of stomach and colorectal cancers per 100,000 in Japan are 105.8, 57.5 (male, female) and 42.5, 33.8 (male, female), respectively, while the rate is quite low (0.4) for cancers in the small intestine [19]. In agreement with these findings, our previous studies and the present study showed that the total mucosa-specific DNA adduct level in the small intestine (0.13 ± 0.07 adducts/108 nucleotides) [10], was about 28-fold lower than that in the colon (3.64 ± 7.92 adducts/108 nucleotides) [9], and five-fold lower than that in the stomach (0.70 ± 0.61
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adducts/108 nucleotides). Of course the idea that DNA adduct level at a single point in time in patients may be associated with the feasibility of cancer is simplistic. DNA adduct level is determined by many factors such as enzyme activities of activation/inactivation of carcinogens and rate of DNA repair. The present findings may suggest that the mucosa-specific DNA adducts play some role in carcinogenesis in the human stomach and/or intestine, but there is another possibility that the mucosa-specific DNA adduct may simply be related to the process of aging and nothing to do with the induction of gastric carcinomas. To clarify the association between the mucosa-specific DNA adducts and carcinogenesis of the stomach, it is necessary to identify the substances that form these DNA adducts. This might be learned by comparing individuals with and without gastric carcinoma, although it is very rare to obtain the stomach samples from non-cancerous patients. Histologically, gastric carcinoma is classified into two types, undifferentiated carcinoma (diffuse type) with high malignant potential which arises from the ordinary mucosa, and differentiated one (intestinal type) with low malignant potential, which arises from the intestinal metaplastic epithelium [20]. In the present study the, G1 adduct was rather specifically detected in somewhat younger patients with undifferentiated carcinoma. This may suggest that the G1 adduct induces specific gene mutation(s) leading to undifferentiated carcinoma. With respect to histology, Dyke et al. previously compared the gastric mucosa DNA adducts from normal subjects with those from patients with chronic atrophic gastritis and intestinal metaplasia, which may be considered pre-malignant conditions [21]. They could not find any difference in adduct patterns or adduct levels for any patients, and concluded that DNA adduct levels do not correlate with the presence of histological abnormalities in the stomach and are not useful as a marker of malignant potential. But, according to our findings, there is a possibility of reaching a different conclusion, if the mucosa-specific DNA adducts are selectively analyzed in these samples. The origin of the DNA adducts detected in the stomach may be dietary carcinogens, because the incidence of gastric cancer is closely associated with dietary habits. Among the possible dietary carcinogens for gastric cancer, DNA adducts of heterocyclic amines or alkylation by N-nitroso compounds cannot be detected by the nuclease P1-enrichment method of 32 P-postlabeling. Although progressive decrease of mucosa-specific DNA adduct types was shown from the upper stomach to the lower colon, this does not necessarily indicate that these mucosal adducts were formed through direct exposure from the intraluminal direction of carcinogens which were present in the gastrointestinal content. For example, when human dietary carcinogen, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), was administered into the rat stomach, adduct formation was induced mainly through the circulatory exposure, but partly through the intraluminal exposure [22,23]. However, in the case of 2-amino1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the DNA
adduct formation through intraluminal exposure was negligible (unpublished data). In addition to dietary factors, cigarette smoking is also an important risk factor for gastric cancer. Dyke et al. previously demonstrated that in male subjects, adduct levels were significantly greater in the DNA of smokers than in that of non-smokers by comparing DNA adducts in the tumor tissue of smokers and non-smokers with gastric cancer by the 32 P-postlabeling method [24]. The present results also showed the same tendency, although there was no significant difference. In contrast, the common background DNA adducts observed in both the mucosa and muscle layer and also in different organs (stomach, small intestine and colon) showed no relationship between the adduct level and the incidence of cancer, indicating that they are not associated with carcinogenesis in the digestive tract. Some common background DNA adducts may be artifacts produced during assays, but others may be formed by endogenous factors, since (1) some of them were also present in tissues from newborns, who were presumably less exposed to environmental carcinogens than adults and (2) the levels of these adducts were rather similar inter-individually, and in the mucosa and muscle layer, or even among the stomach, small intestine and colon. Our search for endogenous substances forming common background DNA adducts revealed that some hormonal steroids in humans can form covalent DNA adducts in vitro [25]. The common background DNA adducts found in the human digestive organs may correspond with the ‘I-compounds’ reported by Randerath et al. [26]. Finally, it should be emphasized that the separate preparation of the mucosa and muscle layer and comparison of their DNA adducts were essential for distinguishing cancer-responsible adducts from unimportant adducts.
Acknowledgements This work was supported by Grants-in-Aids from the Ministry of Education, Science, Sports and Culture of Japan. We thank Akio Kubota and Masahiro Nakayama, Osaka Medical Center and Research Institute for Maternal and Child Health, Japan for providing samples from newborns. The doctoral fellowship of Mohammed Abdul Momen from the Ministry of Education, Science, Sports and Culture of Japan is gratefully acknowledged.
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