R.C. Garner

Mutations in APC, Kirsten-ras, and p53—alternative genetic pathways to colorectal cancer

Colorectal cancer is one of the most significant causes of cancer death. A genetic model for colorectal cancer has been proposed in which the sequential accumulation of mutations in specific genes, including adenomatous polyposis coli (APC), Kirsten-ras (K-ras), and p53, drives the transition from healthy colonic epithelia through increasingly dysplastic adenoma to colorectal cancer. We have characterized tumor mutation spectra in a large cohort of colorectal cancer patients. In marked contrast to the predictions of the sequential model of mutation accumulation, only 6.6% of tumors were found to contain mutations in APC, K-ras, and p53, with 38.7% of tumors containing mutations in only one of these genes. The most common combination of mutations was p53 and APC (27.1%), whereas mutations in both p53 and K-ras were extremely rare. Statistical analysis (two-sided Fishers exact test) confirmed that mutations in K-ras and p53 co-occurred less frequently than expected by chance (P < 0.01, Fishers exact test). This finding suggests that these mutations lie on alternate pathways of colorectal tumor development. The heterogeneous pattern of tumor mutations in our patient cohort suggests that multiple alternative genetic pathways to colorectal cancer exist and that the widely accepted genetic model of cancer development is not representative of the majority of colorectal tumors.

Analysis of DNA adducts by accelerator mass spectrometry in human breast tissue after administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and benzo[a]pyrene

Epidemiological evidence has suggested an association between meat consumption and the risk of breast cancer. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine found in cooked meat, has been implicated in the aetiology of breast cancer and has been shown to induce tumour formation in rodent mammary glands. In addition, polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P) which has also been shown to induce tumour formation at a number of sites in rodents including the breast, are produced during the cooking of meat through the pyrolysis of fats. The aim of this study was to examine the bioavailability of these compounds to human breast tissue and their ability to bind to DNA to form DNA adducts. Patients undergoing breast surgery at York District Hospital were orally administered prior to surgery a capsule containing 20microg of 14C PhIP (182kBq, specific activity 2.05GBq/mmol) or 5microg of 14C B[a]P (36kBq, specific activity 1.81GBq/mmol). At surgery, normal and tumour breast tissue was resected and tissue concentrations of carcinogen measured by liquid scintillation counting and DNA adduct levels by accelerator mass spectrometry (AMS) were subsequently determined. It was found that both 14C PhIP and 14C B[a]P were able to reach the target organ where they had the ability to form DNA adducts. The level of adducts ranged from 26.22-477.35 and 6.61-208. 38 adducts/10(12) nucleotides following administration of 14C PhIP and 14C B[a]P, respectively, with no significant difference observed between levels in normal or tumour tissue. In addition, the data obtained in this study were comparable to adduct levels previously found in colon samples following administration of the same compounds to individuals undergoing colorectal surgery. This is the first report that these two carcinogens bind to human breast DNA after administration of a defined low dose.

Comparative biotransformation studies of MeIQx and PhIP in animal models and humans

MeIQx and PhIP are putative carcinogenic heterocyclic amines formed during the cooking of meat and fish. Using accelerator mass spectrometry, we have investigated the metabolism and macromolecule binding of 14C-labelled MeIQx and PhIP in human cancer patients compared to the rat. Following oral administration of MeIQx and PhIP, more DNA adducts were formed in human colon tissue compared with rats. Differences were also observed between rats and humans in the metabolite profile and urine excretion for these compounds. These results suggest humans metabolise heterocyclic amines differently to laboratory rodents and question their use as models of human risk.

Measurement of ‘unscheduled’ DNA synthesis in HeLa cells by liquid scintillation counting after carcinogen treatment

HeLa cells, conditioned in an arginine-deficient medium to reduce DNA S-phase synthesis, were treated with one of four ultimate carcinogens (MNNG, BrMBA, N-acetoxy AAF and EMS) and one precarcinogen, AFB1. All treated cells preferentially incorporated [3H] thymidine as a result of DNA repair monitored by liquid scintillation counting of the extracted DNA. The cells showed some capacity to activate AFB1, but repair synthesis was much increased if a rat liver mixed function oxidase preparation was also present. At equimolar concentrations the various carcinogens stimulated different amounts of DNA repair; this variation was not proportional to the carcinogenic potency of the chemicals tested. Reasons for this are discussed as is the use of this technique as a screen for chemical carcinogens.

DNA damage as measured by 32P-postlabelling in normal and pre-malignant gastric mucosa.

DNA was extracted from the gastric mucosa of 69 patients and analysed for the presence of DNA adducts by 32P-postlabelling. Adduct levels found in patients with histologically normal gastric mucosa were compared with levels found in patients displaying evidence of chronic atrophic gastritis or intestinal metaplasia, both of which may be considered pre-malignant conditions. Adduct patterns were the same for all patients, but the highest adduct levels were found in the latter two groups. Mean adduct levels were also higher in patients with abnormal gastric mucosa, but there was no statistically significant difference in adduct levels between the normal and pre-malignant groups (P > 0.05, Mann-Whitney U test). Thus 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.

Effect of vitamin C upon gastric mucosal O6-alkyltransferase activity and on gastric vitamin C levels

The repair enzyme O6-alkyltransferase will repair O6-methylguanine adducts in human DNA. In gastric mucosal DNA these adducts may be formed as a result of exposure to nitrosamines within the gastric lumen. The formation of these nitrosamines may be inhibited by vitamin C. We have examined the effect of oral vitamin C supplementation upon intragastric vitamin C levels and gastric mucosal O6-alkyltransferase levels in 48 patients. Intragastric vitamin C levels were significantly elevated in those patients with normal gastric mucosal histology after treatment, although a variable response in intragastric vitamin C to supplementation was seen in the presence of chronic atrophic gastritis. Gastric mucosal O6-alkyltransferase activities ranged from 100 to 950 fmol/mg protein before vitamin C administration. The range of enzyme activity was similar after the course of vitamin C (62-1137 fmol/mg) but O6-alkyltransferase activities were found to be higher in 33 of the 48 patients following treatment (P < 0.01). Once again this effect was more pronounced in patients with normal gastric mucosa than those displaying evidence of chronic atrophic gastritis. We speculate that inhibition of intragastric nitrosation by vitamin C results in decreased formation of O6-methylguanine-DNA. In consequence, less O6-alkyltransferase is consumed in repairing these adducts resulting in higher tissue levels of this enzyme.

O6-Alkyltransferase activity in normal human gastric mucosa

The spectrum of activity of the DNA repair enzyme O6-alkyltransferase has been studied in a large series of normal stomachs in order to establish the baseline range of values for this enzyme. Sixty-eight patients with histologically normal stomachs were biopsied during the course of upper gastrointestinal endoscopy and the biopsies assayed for O6-alkyl-transferase activity. A wide spectrum of activity was found with values ranging from 38 fmol O6-guanine extracted/mg protein to over 400 fmol/mg. This suggests that there may be wide inter-individual differences in susceptibility to alkylating actions in the human gastric mucosa.

O6-Alkyltransferase activity in normal and abnormal gastric mucosa

The activity of the DNA repair enzyme O6-alkyltransferase has been studied in a series of stomachs with abnormal gastric mucosa and the activities found compared with those in normal stomachs. Enzyme activities found in stomachs with the macroscopic abnormalities of gastric ulcer, duodenal ulcer or gastric cancer were not significantly different from normal. In those stomachs where there was histological evidence of chronic atrophic gastritis or intestinal metaplasia however enzyme activities (mean 398 fmole/mg) were significantly higher than normal (mean activity 228 fmole/mg activity P < 0.001). We speculate that the conditions which stimulate these histological changes also give rise to induction of O6-alkyltransferase.