• Detection of O6-methylguanine in human DNA.

      Saffhill, Roy; Badawi, Alaa F; Hall, C Nicholas; Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, UK. (1988)
      Using very specific antibodies in sensitive radioimmunoassays for O6-methyldeoxyguanosine (O6-medGuo), we have been able to detect the presence of this modification in human DNA. Since O6-medGuo is not likely to be a normal component of DNA, its presence must be due to exposure to environmental alkylating agents. We have studied two groups of samples, one of which appears to have received exposure to an alkylating agent (so that most members of the group show a detectable level of O6-medGuo). Although some individuals in the other group showed detectable O6-medGuo (sometimes at levels exceeding those observed in the first group), the majority showed undetectable levels. This may indicate that they had much less exposure to environmental alkylating agents than the first group and that some individuals may have received additional exposures due to other factors such as life style or drugs that they may have been given.
    • Frequency of Ki-ras mutations and DNA alkylation in colorectal tissue from individuals living in Manchester.

      Jackson, Peta E; Hall, C N; Badawi, Alaa F; O'Connor, Peter J; Cooper, Donald P; Povey, Andrew C; Cancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, United Kingdom. (1996-05)
      Most human colorectal cancers arise through the accumulation of a series of genetic alterations such as point mutations within the Ki-ras and p53 genes, but the chemical carcinogens that may be implicated in these events are still unidentified. In a previous study, we showed that DNA from human colorectal tissue contained O6-methyldeoxyguanosine (O6-MedG), a promutagenic lesion arising from exposure to as yet unidentified methylating agents. To address whether such exposure may result in oncogene activation in human colorectal tumors, we examined another series of paired normal and tumor DNA samples from the lower intestinal tract for the presence of O6-MedG in DNA (as a marker of exposure) and for mutations within the Ki-ras gene. After isolation by high pressure liquid chromatography, O6-MedG was quantified by a radioimmunoassay with a limit of detection of 0.01 mumol O6-MedG/mol dG. The frequencies of methylation were 33%, 52%, and 48% for normal DNA and 58%, 32%, and 63% for tumor DNA isolated from the cecum, sigmoid colon, and rectum, respectively. Overall, 35% of the individuals had no detectable O6-MedG in the DNA from both their tumor and normal tissue. Ki-ras mutations were initially identified by a restriction site mutation assay and then sequenced to ascertain the mutations thus detected. The frequencies of mutations in tumor DNA isolated from the cecum, sigmoid colon, and rectum were 28%, 29%, and 42%, respectively. DNA isolated from macroscopically normal tissue was found to contain Ki-ras mutations in 14% of sigmoid colon samples and 12% of rectal samples. Most base mutations were in codon 12 (72%), and 64% were GC-->AT transitions: 28% and 8% were GC-->TA and CG-->CG transversions, respectively. All mutations were at the second base of either codon 12 or codon 13 except for a single GC-->TA transversion at the first base of codon 13 in a rectal tumor sample. There was no association between the presence of O6-MedG in DNA from either normal or tumor tissue or both normal and tumor tissue and the incidence of Ki-ras mutations or GC-->AT transitions in mutated Ki-ras genes. It remains to be determined, however, whether there is a relationship between methylating-agent exposure and Ki-ras mutations, as (i) the presence of O6-MedG in colorectal DNA in these samples may not represent the exposure when Ki-ras mutational activation was occurring (i.e., at some unknown time in the past), (ii) interindividual differences in repair-enzyme activity may alter susceptibility to a mutational event after exposure, (iii) the predominant mutagen in the colon and rectum may not be a methylating agent (e.g., nitric oxide), and (iv) exposure to methylating agents need not result in oncogene activation in human tissues but may perhaps promote the emergence of the mutator phenotype.
    • Involvement of alkylating agents in schistosome-associated bladder cancer: the possible basic mechanisms of induction.

      Badawi, Alaa F; Mostafa, M H; O'Connor, Peter J; Department of Environmental Studies, Alexandria University, Egypt. (1992-04-30)
      Carcinoma of the urinary bladder is a common malignancy in many tropical and subtropical countries. There is a well documented sequela of chronic urinary schistosomal infection and bladder cancer associated with schistosomiasis is a major cause of morbidity and mortality in the endemic areas. Experimental bladder cancer can be induced in schistosome-infected animals. Multiple factors have been suggested as causative agents in schistosome-associated bladder carcinogenesis and the N-nitroso compounds appear to be of particular importance. These agents have long been suspected to play a major role in the aetiology of a variety of human cancers. A model for the induction of bladder cancer associated with schistosomiasis is proposed which takes into account the interrelationships between different factors resulting from the infection, especially the role of alkylating agents that can contribute to the induction of this neoplasm.
    • Promutagenic methylation damage in bladder DNA from patients with bladder cancer associated with schistosomiasis and from normal individuals.

      Badawi, Alaa F; Mostafa, M H; Aboul-Azm, T; Haboubi, N Y; O'Connor, Peter J; Cooper, Donald P; Institute for Graduate Studies and Research, University of Alexandria, Egypt. (1992-05)
      Radioimmunoassays (RIAs) have been used to detect the promutagenic lesion O6-methyldeoxyguanosine (O6-MedG) in DNA isolated from the bladder tissues of Egyptian patients presenting with bladder carcinoma and concomitant schistosomiasis (bilharziasis), a parasitic disease known to be associated with the presence of N-nitrosamines in the urine. Alkylation damage was present in the DNA of the majority of the samples (44/46, 96%); 38 samples were of tumour tissue and 8 from uninvolved bladder mucosa. Levels of O6-MedG ranged from undetectable (ND; i.e. less than 0.01 mumol O6-MedG/mol dG) to 0.485 mumol/mol dG with an overall mean of 0.134 +/- 0.10 mumol/mol dG, including the two samples that were below the limit of detection. In contrast, methylation damage was detected in only 4/12 (33%) of the DNA samples from normal bladder tissue of European origin. In these samples levels of O6-MedG ranged from ND to 0.225 mumol/mol dG with an overall mean of 0.046 +/- 0.082 mumol O6-MedG/mol dG. These results confirm that alkylation events can be detected in the DNA of schistosome-infected human bladder tissue. The methylation of uninvolved and tumour tissue DNA to similar extents suggests that the alkylating intermediate may have been present in the urine. These results indicate the need for further investigation to determine whether relationships exist between levels of DNA damage and the prevalence of schistosome infection and/or the extent and type of bacterial infection that frequently accompanies this disease.