O6-Alkyltransferase activity in normal human gastric mucosa

Cancer Letters, 54 (1990)

147

147-151

Elsevier Scientific Publishers Ireland Ltd.

06-Alkyltransferase

activity in normal human

gastric mucosa

G.W. Dyke”, J .L. Cravenb, R. Hallb, D.P. Cooper”, G. Soballad and R.C. Garnera Tamer Research Unit, Universiiy of York, Hestington, York YO1 5DD. bYork District Hospital, Wigginton Road, York Y03 7HE, Paterson Institute for Cancer Research, Christie Hospital and Ho/t Radium Institute, Manchester M20 9BX, and dLeeds General Infirmary, Great George Street, Leeds l-S1 3EX (U.K.) (Received 2 February 1990) (Revision received 14 August (Accepted 14 August 1990)

1990)

Summary The spectrum of actiuity of the DNA repair enzyme 06-alkyltransferase has been studied in a large series of normal stomachs in order to establish the baseline range of oalues for this enzyme. Sixty-eight patients with his~oIo~caIIy normal stomachs were biopsied during the course of upper gastrointestinal endoscopy and the biopsies assayed for 06-alkyl-transferase activity. A wide spectrum of actioity was found with ualues ranging from 38 fmor 06-guanine extracted/mg protein to ouer 400 ~moi/mg. This suggests that there may be wide interindiuidual dif’erences in susceptibility to alkylating actions in the human gastric mucosa. Keywords: alkyl~a~sferase; gastric mucosa; DNA repair. Introduction Although the incidence of gastric cancer is declining, it still accounts for approximately 10% of cancers [I]. These tumours probably arise as the result of an interaction between the gastric mucosa and mutagenic or carcinogenic compounds within the gastric juice. We have previously shown that a 0304-3835/90/$03,50

0

high percentage of individuals possess mutagenic gastric juice [Z] and have also demonstrated that there is no difference in gastric mutagenicity between groups of juice patients known to be at risk of developing gastric cancer - such as patients with pernicious anaemia or those who have previously undergone gastric surgery - and normal controls [2]. We hypothesise, therefore, that the risk of developing gastric cancer is primarily a function of the gastric mucosal response to carcinogens. In this respect, it is believed that the presence of N-nitroso compounds within the gastric juice is particularly important as these compounds have been strongly implicated in the aetiology of gastric cancer [3]. N-nitroso compounds have been shown both in vivo [4] and in vitro 151 to cause alky~ation of DNA which may be of particular biological significance if it occurs at the 06-position of guanine [6]. 06-alkylguanine damage can be repaired by the enzyme 06-alkyl-tranferase and a knowledge of the repair capability of gastric mucosa, with respect to this enzyme, may enable us to predict groups of patients at risk of developing gastric cancer. The 06-alkyltransferase enzyme acts by clearing the abnormal methyl groups from guanine and attaching them to a cysteine residue of its own protein [7]. This inacti-

1990 Elsevier Scientific Publishers lreland Ltd.

Published and Printed in Ireland

vates the enzyme and hence each cell must have a finite repair capability until a fresh enzyme is synthesised. Should the repair capability be exceeded and the cell enter a cycle of replication, the daughter strands may undergo point mutation through basepair mismatching [8]. Such damage has already been shown to lead to activation of the H-ras oncogene in MNU-induced rat mammary carcinomas [9] and it has been demonstrated in experimental animals that 06-alkyltransferase-deficient tissues are target tissues for alkylation-induced carcinogenesis. Previously published studies of 06-alkyltransferase activity in human tissues have suffered through having either small numbers [ 111 or heterogenous groups of diseased and normal subjects [12]. By using endoscopic biopsies as a source of tissue, we have investigated 06-alkyltransferase activity in a large number of subjects with normal gastric mucosa. We report here that the activity of this enzyme varies greatly between individuals. This suggests that certain patients may be at increased risk of suffering N-nitroso-induced DNA damage. Materials and methods Chemicals

Radiolabelled 3H-methylated DNA was a gift from Dr. G. Margison (Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester). AI] other chemicals were purchased from Sigma Chemical Chemical Company, Fancy Road, Poole, Dorset.

approximate weight 3-4 mg were taken from within 5 cm of the pylorus of patients with endoscopically normal stomachs, using conventional spring-loaded biopsy forceps. Biopsies were stored at - 20°C until assayed. At the same time, a biopsy was taken from an immediately adjacent site and sent, fixed in formalin, for routine histological examination. Preparation

of extracts

Biopsies were homogenised in 100 ~1 of TDEG buffer (50 mM Tris, 1 mM EDTA, 3 mM dithiothreitol, 5% glycerol) pH 7.8, using a miniature glass homogeniser. Remaining tissue fragments were sonicated briefly for 10 s in a further 100 ~1 buffer and the supernatants pooled. Debris was separated by centrifugation at 4 OC. Determination

of 06-alkyltransferase

activity

06-alkytransferase activity was estimated using a modification of the method of Pegg and Perry [13]. Assay mixtures comprised aliquots of the supernatant prepared above, TDEG buffer (pH 8.3), to a total volume of 150 ~1 and 100 ~1 of 3H-labelled methylated DNA of specific radioactivity 30 Ci/mmol. The pH of the final reaction mixture was 8.3. After 30 min incubation at 37OC, 50 ~1 of 10 mg/ml bovine serum albumin was added. All protein was precipitated with 100 ~1 4 M perchloric acid and residual DNA hydrolysed by heating at 70°C for 40 min. The protein was separated by centrifugation and was washed three times with 1 M perchloric acid. It was redissolved in 0.5 ml water, prior to scintillation counting.

Patients

Gastric biopsies were taken from 68 patients (41 male/27 female) with a mean age of 54 years (range 20-86 years) attending the Dyspepsia Clinic at Leeds General Infirmary.

Protein

estimation

The protein content of the biopsy homogenates was estimated by the method of Lowry [4]. Results

Endoscopy

All endoscopies were performed by an experienced endoscopist. Biopsies of

Plotting a graph of radioactivity of the protein precipitate after reaction with the

149

20

40

60

Volume

60

extract

100

(~11

Fig. 1. Typical curve showing uptake of radioactivity as a function of protein extract. 0-6-alkyltransferase activity is derived from linear portion of curve.

radioloabelled DNA against the volume of extract used, gives a curve of substratedependent enzyme kinetics (Fig. 1). Enzyme activity can then be calculated from the linear part of the graph, knowing the specific activity of the DNA. Enzyme activity was of 06-methylguanine expressed in fmol removed/mg protein (fmol/mg) . Table 1 shows the results of a small pilot study using multiple biopsies from the same patient. It demonstrates that this assay is

readily reproducible under the conditions described with a standard deviation between samples of less than 10%. Figure 2 demonstrates the range of activity of 06-alkyltransferase found in normal human gastric mucosa. There is a wide inter-individual variation of activity with a range from as little as 38 fmol/mg to over There is no correlation 400 fmol/mg. between enzyme activity and either age or sex. Scrutiny of Fig. 2 would also suggest that the range of activities does not follow the expected normal distribution, nor any of the recognisable patterns one might expect if, for example, the enzyme were under the control of a genetic polymorphism. It must be stressed, however, that the number of small and, patients studied is relatively hence, any genetic effect may be masked. Further studies with larger groups would be needed to clarify this point. Discussion This is the first study to look at 06-alkyltransferase levels in a large series of normal stomachs. It has demonstrated that enzyme activity can reliably be quantified even in small, endoscopic biopsies and, thus, is a technique which can readily be applied to large population surveys. It further demonstrates that, although enzyme activity was detectable in all samples

Table 1. Results of a preliminary study using multiple biopsies from the same patient. 06-alkyltransferase each biopsy was reproducible with standard deviations of less than 10% of the mean in all cases. Patient no.

06-alkyltransferase

activity in

S.D. as 46 of mean

activity

(fmol/mg)

Biopsy

1 2 3 4

1

2

3

4

mean

S.D.

370 234 480 198

457 256 463 176

446 241 453 163

446 205 492 189

434 234 472 181

43 21 17 15

9.9 8.9 3.6 8.2

FMOLES OS-METHYLGUANINE TRANSFERRED/MO PROTEIN Fig. 2. fmol/mg.

Distribution

of @-alkyltransferase

in normal

assayed, the range of activity encountered was wide. There was a > lo-fold difference between the most- and the least active samples. This cannot be accounted for by either variation in sampling site, as all biopsies were taken from the same area of stomach, or variation in storage conditions, as all biopsies were stored under the same conditions and for the same length of time. This variation has potentially important implications. As 06-alkyltransferase inactivates itself during the repair of DNA [7,15], it is theoretically possible for a cell to totally deplete itself of enzyme. Such a cell may then replicate with unrepaired DNA present, a situation which may lead to mutation. Gastric mucosal cells have a high turnover rate and are bathed in gastric juice containing Nnitroso compounds which have been shown to alkylate DNA [4,5]. Hence the stomach may be a particularly susceptible organ to this type of damage and certain individuals may be at greater risk of sustained Nnitroso-induced DNA damage than others. Although in humans no difference has yet been demonstrated between 06-alkyltrans-

human

gastric

Each bar represents

mucosa.

a spread

of 30

ferase levels in neoplastic and non-neoplastic stomachs, we speculate that the wide range of activity encountered may explain why certain individuals are at increased risk of developing gastric cancer. Acknowledgements This work was supported York Against Cancer.

by a grant from

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