High Levels of DNA Adducts in Human Colon Are Associated with Colorectal Cancer1

[CANCERRESEARCH55. 5611-5616. December 1, 1995] High Levels of DNA Adducts in Human Colon Are Associated with Colorectal Cancer1

Annie Pfohl-Leszkowicz, Yann Grosse, VÃ©roniqueCarriÃ¨re, Paul-Henri Cugnenc, Anne Berger, FranÃ§oiseCarnot, Philippe Beaune,2 and Isabelle de Waziers

Ecole Nationale SupÃ©rieureAgronomique de Toulouse, Laboratoire de Toxicologie et SÃ©curitÃ©Alimentaire, 145 avenue de Muret, 31076 Toulouse [A. P-L, Y. G.J; Institut National de la Sante et de la Recherche MÃ©dicaleU75, Centre Hospitalier Universitaire-Necker Enfants-Malades, UniversitÃ©RenÃ©Descartes, 156 rue de Vaugirard, 75730 Paris Cedex 15 [V. C., P. B., 1. d. W.]; Service de Chirurgie Digestive, HÃ´pital LaÃ«nnec, 42 rue de SÃ¨vres, 75007 Paris [P-H. C., A. B.]; and Service dAnatomo-pathologie, HÃ´pital LaÃ«nnec,42 rue de SÃ¨vres, 75007 Paris (F. C.], France

ABSTRACT been demonstrated in epithelium colon cells (18) and in colonic bacteria (19); thus, the production of reactive metabolites in situ in Colon cancer is one of the most frequent causes of cancer death in colonic mucosa is quite possible. western countries. Epidemiological studies suggest that colorectal cancer Therefore, we sought to determine DNA adduct levels in control can be attributed, at least in part, to carcinogens and mutagens present in the diet and/or the environment. The covalent binding of these xenobiotics colons from patients without colorectal adenocarcinoma and in non or their reactive metabolites to DNA is believed to initiate this chemical tumoral and tumoral tissues from patients with colorectal adenocar carcinogenesis. In the present study, using a 32P-postlabeling method, we cinoma. A potential difference between colonic mucosa from affected investigated DNA adduct levels in control colons from patients without (nontumoral tissue) and unaffected patients (sigmoiditis) could be in colorectal adenocarcinoma and in nontumoral and tumoral tissues from good agreement with the chemical origin of colorectal cancer. More patients with colorectal adenocarcinoma. Our results show that the DNA over, we compared nontumoral and tumoral tissues. adduct level is significantly higher (P < 0.001) in nontumoral than in We showed that both the quantity and variety of DNA adducts were control or tumoral colon samples. For the first time, we demonstrate in the highest in nontumoral tissues compared with tumoral tissues and, humans that the presence of numerous adducts in colonic mucosa is more interestingly, to mucosa from control patients. The relevance of associated with colorectal cancer, a finding in agreement with the impor these results to tumor biology, and to early detection of colorectal tance of chemical factors in causing this disease; therefore, after confir cancer, is discussed. mation of the link between DNA adducts and colorectal cancer, the measurement of DNA adduct levels in colon samples could constitute a useful approach to the early detection of colorectal cancer. MATERIALS AND METHODS Chemicals. Proteinase K, apyrase, and RNases A and Tl were purchased INTRODUCTION from Sigma Chemical Co. (St. Louis, MO); T4 polynucleotide kinase was purchased from PL Biochemicals (Milwaukee, WI); micrococcal nuclease and Colon cancer is one of the most frequent causes of cancer death in spleen phosphodiesterase were from Worthington Biochemicals (Freehold, developed countries (1). Both hereditary and, mainly, environmental NJ); nuclease P1 was from from Boehringer Mannheim (Mannheim, Germa factors (potential carcinogens and mutagens present in the diet and ny); [-y-32P]ATP (5000 Ci/mmol) was from Amersham (Buckinghamshire, United Kingdom); 5'-deoxynucleotide was from Sigma; phenol was from tobacco and alcohol consumption) contribute to the development of Appligene (Illkirch, France); polyethyleneimine was from Corcat (Portsmouth, colorectal cancer (2â€”5).The process of chemical carcinogenesis is VA); and cellulose sheet MN 301 was from Machery Nagel (Dllren,Germany). initiated by the covalent binding of carcinogens or their reactive Human Tissues. Human tissue samples were obtained, in accordance with metabolites to DNA, thus forming DNA adducts (6, 7). The presence French regulations and approval by the ethical committee, from the Depart of such adducts was demonstrated recently in human colonic mucosa ment of Surgery of HÃ´pitalLaÃ«nnec(patientsundergoing surgery for sigmoid (8). Theseadductscanbe removedby DNA repairprocessesorby cell itis and colorectal adenocarcinomas) and from the HÃ´pital Broussais (Paris, death, but they reach steady-state levels in carcinogen target tissues, France; control colons from organ donors). Clinical data from patients are reflecting the balance between exposure to carcinogens, adduct for given in Table 1. Eight control colon specimens were obtained from acciden mation, and adduct elimination. DNA adducts can result directly in tally deceased organ donors (6 males and 2 females) ages 23 Â±14 years. Twenty-one control colon specimens were obtained from individuals (1 1 males mutational events, leading potentially to cancer (9â€”11).These muta and 10 females), ages 63 Â±16 years, after resection of part of the organ tions in genes implicated in the control of cell growth lead to the following cured sigmoiditis. Control tissues were taken from these resected formation of small benign tumors (adenoma), which may grow and organs in regions without any macroscopic lesions. Tumoral and nontumoral may result finally in malignant tumors (carcinoma) (12). DNA car tissues were obtained from 18 patients (8 males and 10 females) ages 66 Â± 13 cinogen adducts in target tissues during chronic exposure seem to be years who underwent surgery for colorectal adenocarcinomas. Nontumoral dose related and could be predictive of human disease risk (13â€”15). specimens were taken from a macroscopically healthy region of the resected The liver is the most active organ in metabolizing xenobiotics into pieces at least 5 cm away from the tumor. Tumoral tisues were dissected out active metabolites. These metabolites then can be conjugated, ex from the middle of the tumor; the tumoral region was macroscopically visible. creted in bile, and liberated in the colon, and after activation by Part of each tissue sample was reserved for histopathological examination, and acetyltransferases, for instance, they may react with DNA (16, 17). the remainder was used for this investigation. 32pPostlabeling ofDNA Adducts. DNA from surgically removed control, Moreover, the presence of carcinogen-activating enzymes also has nontumoral, and tumoral colons was extracted and purified as described previously (20). Purified DNA (6 @g)was hydrolyzed enzymatically in 3'- Received 7/20/95; accepted 9/28/95. nucleotides before 5'-32P phosphorylation by [32P]ATP and T4 polynucleotide The costs of publication of this article were defrayed in part by the payment of page kinase according to the method described by Reddy and Randerath (21) and charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. modified by Pfohl-Leszkowicz et a!. (22). Adducts were separated by chro I This work was supported by l'[nstitut National de la Sante et de Ia Recherche matography on polyethyleneimine cellulose plates. Quantification was per MÃ©dicale,l'UniversitÃ© RenÃ©Descartes, la Ligue contre le Cancer Grant RS38, and formed by Cerenkov counting of individual spots, and the results were cx l'Association pour Ia Recherche contre le Cancer Grant 66â€”04. pressed after substraction of blank values. 2 To whom requests for reprints should be addressed, at INSERM U 75, CHU-Necker Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France. Phone: 33 1 4061 Statistical AnalySis. The level of significance was tested between control 56 33; Fax: 33 1 40 61 55 82; E-mail: [email protected]. and nontumoral tissues by the distribution-free Mann and Whitney unpaired 5611

investigatedAgePatient Table 1Clinical data of the patients

differentiationNormal Gender (yr)Diagnosis Dukes Astler-Coller Tumor colons 15 Fa 41 Traffic accident, kidney donor 16 F 11 Traffic accident, kidney donor 20 M 48 Traffic accident, kidney donor 21 M 16 Traffic accident, kidney donor 24 M 15 Traffic accident, kidney donor 32 M 15 Trafficaccident,kidneydonor 34 M 19 Traffic accident, kidney donor 41 M 22 Trafficaccident,kidneydonor Normal colons (sigmoiditis) 1285 F 54 Sigmoiditis 1305 M 47 Benign polyps 131 5 M 19 Sigmoiditis 132 5 M 63 Sigmoiditis 139 5 M 69 Sigmoiditis 141 5 M 48 Sigmoiditis 142 5 F 57 Sigmoiditis 1435 F 75 Sigmoiditis 144 5 M 47 Sigmoiditis 146 5 F 80 Sigmoiditis 147 5 F 81 Sigmoiditis 151 5 M 78 Sigmoiditis 153 5 M 76 Sigmoiditis 157S F 85 Sigmoiditis 159 5 F 73 Sigmoiditis 160 5 F 56 Sigmoiditis 165 5 M 63 Sigmoiditis 169 5 F 72 Sigmoiditis 171 5 M 54 Sigmoiditis 175 5 F 49 Sigmoiditis 176 5 M 73 Sigmoiditis Tumoral and nontumoral colons 124 T and NT M 57 Sigmoid adenocarcinoma A A Well differentiated 125 T and NT F 43 Colon adenocarcinoma B B2 Well differentiated 133 T and NT F 80 Sigmoid adenocarcinoma A BI Well differentiated 134 T and NT M 77 Sigmoid adenocarcinoma A BI Well differentiated 135 T and NT F 70 Right colon adenocarcinoma C Cl Moderately differentiated 138 T and NT M 75 Sigmoid adenocarcinoma C Cl Moderately differentiated 140 T and NT M 65 Sigmoid adenocarcinoma C Cl Well differentiated 145 NT F 48 Colorectaladenocarcinoma B B2 Welldifferentiated 148 T and NT F 66 Right colon adenocarcinoma B B2 Moderately differentiated 150 T and NT M 72 Left colon adenocarcinoma C Cl Well differentiated 155 T and NT F 54 Sigmoid adenocarcinoma D D Well differentiated 158 T and NT F 83 Rectum adenocarcinoma C C2 Well differentiated 162 T and NT M 81 Left colon adenocarcinoma B B2 Welldifferentiated 163 T and NT F 64 Right colon adenocarcinoma C Cl Well differentiated 166 T and NT M 77 Rectosigmoid adenocarcinoma C C 1 Well differentiated 170 T M 56 Sigmoid adenocarcinoma D D Well differentiated 172 T and NT F 44 Right colon adenocarcinoma C Cl Well differentiated 173 T F 80 Left colon adenocarcinoma C C2 Moderately differentiated a F, female; M, male; 5, control colons from patients with cured sigmoiditis; NT, nontumoral; T, tumoral colons from patients with colorectal adenocarcinoma. test and between nontumoral and tumoral tissues by the distribution-free adenocarcinoma (nontumoral and tumoral colons). Quantification of Wilcoxon paired t test (23). DNA adducts in control, nontumoral, and tumoral colon samples is presented in Table 2, and representative profiles of DNA adducts in RESULTS AND DISCUSSION each kind of tissue are shown in Fig. 1. No DNA adduct in control colons from organ donors (detection limit, 1 adduct/lO'Â° nucleotides) There is considerable evidence demonstrating the genotoxic activ was detected, whereas a few adducts were observed in control colons ity of numerous compounds present in the diet and in tobacco. These from patients operated on after cured sigmoiditis. Yet, the DNA compounds include mycotoxins, plant alkaloids, food additives, pes adduct level remained low; it was even undetectable in 4 of the 21 ticides, polycyclic aromatic hydrocarbons, and heterocyclic amines patients (sigmoiditis control colons) studied. Among the colon sam present in tobacco smoke and cooked fish and meat (3â€”5).The ples studied, 24 different adducts were detected; adducts 17â€”24were gastrointestinal tract is the first organ exposed, but these compounds seem to exert their deleterious effects exclusively in the colon. Indeed, so extremely rare and weak that they are not presented in either Table the incidence of cancer of the small intestine is 40â€”60times lower 2 or Fig. 1. The interindividual differences in the DNA adduct levels than that of colon (24), and this difference is in good agreement with were very great. The adduct levels ranged from 0.5 to 328.5 adducts/ the results of Hamada et a!. (25), who showed, in a Japanese popu l0@nucleotides. In most of the DNA samples that were positive in the lation with a mean age of 67 years, that the total level of DNA adducts 32P-postlabeling method, a dominant spot was detected, which ranged in the small intestine was 28-fold lower than in the colon. from 0.5 to 127 adducts/l09 nucleotides (adduct 1). Our result is in DNA adduct detection was carried out by the very sensitive 32P- accordance with a recent published study (8) showing the presence of postlabeling method on several types of colon samples from different a mucosa-specific DNA adduct in all adult colorectal samples studied. populations: organ donors (control colons), patients with cured sig On the other hand, some of these adducts were detectable only in moiditis (sigmoiditis control colons), and patients with colorectal samples from patients with colorectal cancer (adducts 6, 13, and 14). 5612

Table 2 Quant(fication of DNA adducts in humantissuesâ€•Adduct control, nontumoral, and tumoral colorectal

samplesÂ±1285â€•4.73.111.31302 160.810.945 67 8 9 10 11 12 13 14 15 0.8 50.50.50.41.4131 50.40.40.8132 51.751.150.853.75139 56.420.40.559.35141 51.61.614250143

53.21.62.12.31.811.0014451.10.9214650.91.1214750151

S5.31.71.10.959.051535015750159

51.61.71.31.71.91.22.912.3160 52.72.92.21.81.72.21.68.223.3165 S0.60.6169 S2.30.51.250.61.155.8171 50.40.4175 S2.32.317650.80.51.3124

NT46.16.2513.85.24.69.44.73.93.297.15125NT11.816.4311.91.151.846.05133

NT40.53.61.41.110.62214.22134NT0135

NT17.134.3138 I.9 15.3 NT0.60.20.31.1140 NT62.817.36.56.74.55.4144.51457.5 14.1 19.7 NT126.813.910.7645.516226.6148NT1.50.42.315011.7 19.5 12.5 0.4 NT0.50.5155 NT39.233.23318.145.459.814.714.3328.515815 55.8 NT15.23028.43.34.624.6[4.720140.8162 NT13.61917.82.1[2.519.796.8163NT11.615.827.416612.1

NT55918.10.8100.1172 17.2 NT81.212.914.216.59.37.77.8200.5124II.9 39 T8.51.52.41.52.21.523.21251.3 4.3 T2.51.551.411.557.01133 T0.61.40.51.22.31.17.1l34T0135

T0.70.60.651.95l38T0140

T4.60.44.41.32.80.80.614.9148 T0.50.40.9l5OT0155

T1.610.90.954.45l58T1.61.425l62T21.43.4[6311.21.2l66T1.61.13.717.4l7OT0.30.3l72T101.47.52.51.31.31.11.1127.2173

10.50.5

a Results are expressed as number of adducts/l09 nucleotides. @ b control colons from patients with cured sigmoiditis; NT, nontumoral; T, tumoral colons from patients with colorectal adenocarcinoma.

The average level of DNA adducts calculated for each group ported to occur in human lung tumor tissues compared with adjacent revealed that the highest amount of DNA adducts was observed in nonmalignant tissues (26). This could be due to lower adduct forma nontumoral samples (Fig. 14). Between control (sigmoiditis; tion. Indeed, in a previous study (27), we showed several changes in 4.7 Â±6.0 adducts/l09 nucleotides) and nontumoral (91.3 Â±95.6 drug-metabolizing enzymes in colorectal tumoral tissues compared adducts/109 nucleotides) colon samples, this difference was highly with nontumoral tissues. In tumoral tissues, a significant decrease in significant (P < 0.001 by the Mann and Whitney t test). In control cytochrome P450 3A, implicated in the metabolism of numerous colon samples, no more than 25 adducts/109 nucleotides were de carcinogens, such as aflatoxin (28), and an increase in glutathione tected, whereas in nontumoral colon samples, 11 of 16 patients (69%) transferase resulted in lower formation of electrophilic metabolites presented more than 25 adducts/109 nucleotides (Fig. 2B). These and, consequently, a lower formation of adducts. This also may have differences were even more striking when compared with the organ been due to faster adduct elimination. Adducts may be diluted by donor population. more rapid cell proliferation or aberrant DNA repair in the tumor. Finally, the mean level of DNA adducts from tumoral tissues Given the rapid cell turnover from 3 to 8 days in the human colon (6.15 Â± 8.2 adducts/109nucleotides) was significantly lower (29), the substancesformingDNA adductswould seemto originate (P < 0.002 by the Wilcoxon paired t test) compared with nontumoral mainly from recently consumed food, alcohol, and/or tobacco. Ob tissue from the same patient (Fig. 3). Differences in DNA adduct served differences from patient to patient could reflect: (a) variations levels between nontumoral and tumoral samples also have been re in food composition and, therefore, variations in genotoxic com 5613

A' B 300 0

C D Fig. I. Autoradiography of DNA adducts in human colon samples. Exposure time of 3 days for each sample. A, control colon from organ donor; B, control colon from patient with cured sigmoiditis; C. nontumoral colon; D, tumoral colon from the same patient with colorectal adenocarcinoma; E, corresponding mapping. Numbers correspond to adduct numbers inTable2. pounds to which these patients were exposed; (b) genetic or environ ratio. Finally, the population devoid of any known pathology, the mental variations in xenobiotic-metabolizing enzymes; or (c) the rate organ donors, is much younger and has no detectable adducts. This of elimination of these adducts. It is, therefore, interesting to observe absence of adducts could be due to age; however, two patients from that in the nontumoral tissues from patients with colon cancer, the this control population were older than than 40 years (41 and 48 adduct level is significantly higher than in every other tested category. years), and in colorectal cancer population, three patients were This would mean that these individuals either have had strong expo younger than 50 years (43, 44, and 48 years) and had high levels of sure to genotoxic compounds, a higher capacity to bioactive them, adducts (46.05, 200.5, and 226.6 adducts/l09 nucleotides, respective and/or a lower capacity to detoxicate them, and/or that they have a ly). Moreover, no correlation was found between the number of slower rate of elimination of these adducts. It also can be hypothesized adducts and the age in any population (data not shown). Therefore, we that the exposure remained similar between the time of initiation think that the high level of DNA adducts in nontumoral mucosa is (several years ago) and the time of the adduct measurement. quite relevant, because it was not found in either of the two control It was very difficult to find an ideal control population. Indeed, this populations. population should match for age and sex ratio and should be ideally Our results are, thus, the first to demonstrate, in humans, the devoid of any pathology that would render ethically impossible a presence of numerous adducts in nontumoral colon mucosa of patients colon sample resection. This is the reason why we chose the sigmoid with colorectal cancer. Moreover, they are in good agreement with the itis population as a control. In this population, the resection of the important role of dietary and environmental carcinogens in colorectal colon was performed when the inflammation was cured, the mucosa carcinogenesis (1, 5), in terms of exposition and of bioactivation. was macroscopically normal, and the regimen of these patients was However, although our results will need to be confirmed in a larger not strikingly or systematically different from that of the patients with population, they suggest that, at the time of colonoscopy or surgical colorectal carcinoma. Moreover, both populations (sigmoiditis and resection of part of the colon, some colonic cells could be taken, colorectal cancer) are perfectly comparable in terms of age and sex and DNA adduct levels could be determined. This is a simple 5614

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I@Ia @O@O@OLnOLnOLnOLnO @ in F.,- 0 C'sJ It) 0 C%J tfl N- 0 rsJ LI) CS S S I @- @- iâ€” ,- CS,j (@@J esj (%J C!) CY) @f) _@!f! 0 t) 0 S S I I S S S S S S S 0@-' 0 c%J LI) LI) 0 LI) 0 tn 0 LI) 0 @n 0 LI) 4-'@DL.@ r@-0 rsj LI) N.. 0 esi LI) N- 0 e%J @ C.â€” E ,- ,- Iâ€” I@ C@J rsJ e'J en en 00 Iâ€” L@ 0) 0 DNA-adducts/1 o@nucleotides @ z

@ Control (Sigmoiditis) U NonTumoral

Fig. 2. A, DNA adducts in control, nontumoral, and tumoral colorectal tissues; B, frequency distribution of DNA adducts from control (sigmoiditis) and nontumoral colorectal tissues.

procedure, because even paraffin-embedded tissues, if fixed less 350 U) than 48 h in formalin, are quite suitable for 32P-postlabeling 5@ analyses (30). The detection of high DNA adduct levels (>25 4-. 300' adducts/l09 nucleotides) could lead to close supervision of the 0 a, patient by means of regular colonoscopy, with the same time U 250 interval as recommended in Lynch syndrome. Indeed, Fig. 2 shows C that 69% of the colorectal population have more than 25 adducts/ 200 iO@nucleotides, whereas no patient with cured sigmoiditis has 0 such an adduct level. The detection of DNA adducts 6, 13, and 14, which are observed only in the nontumoral colon, would strengthen U) 4-' 150 U the significance of this measurement. The aim of future investigations will be to identify the adducts and 1 00 Cs the carcinogens that bind specifically to DNA in epithelial colonic 4k cells and, in particular, those leading to DNA adduct 1. Such a z 50 characterization could increase our knowledge of the carcinogens truly implicated in human colorectal carcinogenesis and the process of 0 their elimination.

L- Cs 0 ACKNOWLEDGMENTS E 0 @ E Iâ€” We thank Dr. A. Zweibaum for providing human colon samples from organ Iâ€” donors. C 0 z REFERENCES

Fig. 3. DNA adducts in human nontumoral and tumoral colorectal tissues from the I. Willet,W.Thesearchforthecausesofbreastandcoloncancer.Nature(Land.),338: same patients. 389â€”393,1989. 5615

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Annie Pfohl-Leszkowicz, Yann Grosse, Véronique Carrière, et al.

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