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D P Cooper

Publications and source records attributed to D P Cooper.

At least 37 records · Page 2Linked to original sources
PubMed1994

Optimization of 32P-postlabelling assays for the quantitation of O6-methyl and N7-methyldeoxyguanosine-3'-monophosphates in human DNA.

The 3' and 5'-monophosphates of O6-methyldeoxyguanosine and N7-methyldeoxyguanosine were chemically synthesized. Using these standards with deoxyguanosine-3'-monophosphate (dGp) as an internal standard, conditions were optimized to quantify O6-methyldeoxyguanosine-3'-monophosphate (O6-MedGp) and N7-methyldeoxyguanosine-3'-monophosphate (N7-MedGp) by 32P-postlabelling. Under optimal conditions, the labelling efficiencies of O6-MedGp and N7-MedGp were respectively approximately 100 and approximately 15%, with detection limits of approximately 1.1 and approximately 6.0 fmol respectively using 10 pmol dGp or 0.8 fmol of O6-MedGp if 2 pmol of dGp was used. The assay developed for O6-MedGp was then applied to the quantitation of [3H]-O6-MedGp and O6-MedGp isolated from DNA digests by immunoaffinity separation. The standard curve generated from the use of [3H]-O6-MedGp, thus isolated, was identical to that generated previously using the chemically synthesized O6-MedGp, indicating that no inhibitory factors co-eluted with the O6-MedGp. After passage through two immunocolumns, recovery of 4 and 40 fmol of O6-MedGp was approximately 30%. Four human stomach samples were analysed by combining this immunoaffinity purification with 32P-post-labelling: levels ranged from 0.21 to 0.86 mumol O6-MedGp/mol dG. Further DNA samples, isolated from the human colon, were fractionated by anion-exchange HPLC and the N7-MedGp and O6-MedGp containing fractions were purified by reverse-phase HPLC and immunoaffinity chromatography respectively. Adduct-containing fractions were dried and 32P-postlabelled. Whereas O6-MedGp was detected at levels between 0.3 and 3.4 mumol O6-MedGp/mol dG, no N7-MedGp was detected in these samples, probably due to depurination of N7-MedGp to N7-methylguanine or reduced assay sensitivity resulting from contaminating nucleotides and/or unidentified radioactivity eluting close to the N7-methyldeoxyguanosine-5'-monophosphate.

Chromatography, Affinity↗
PubMed1993

Tissue and cell specific methylation, repair and synthesis of DNA in the upper gastrointestinal tract of Wistar rats treated with single doses of N-methyl-N'-nitro-N-nitrosoguanidine.

Several potential cancer risk factors have been monitored concurrently in the upper gastrointestinal tract of young adult male Wistar rats given single (i.g.) doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) which readily induces forestomach tumours under these conditions. Radioimmunoassay was used to determine the formation of O6-methyl-2'-deoxyguanosine (O6-MedG) in DNA after MNNG doses of 1, 5, 25 or 50 mg/kg and was found to be highest in the pylorus, with progressively lower levels in the corpus, forestomach, duodenum, oesophagus and jejunum. Immunohistochemical procedures showed that cells with nuclei containing O6-MedG were heterogeneously distributed in these tissues. O6-Alkylguanine-DNA alkyltransferase activity in untreated animals was highest in the mucosae of the corpus, lower and relatively similar in that of the pylorus, duodenum and jejunum and lowest in the tissues of oesophagus and forestomach. Estimates of DNA synthesis and cell proliferation indicated a 5-fold increase in the DNA labelling index in the forestomach whereas perturbations of DNA synthetic activity in the other tissues of the upper gastrointestinal tract were much less marked. As a result of these changes, cells with nuclei that contained O6-MedG and were also undergoing DNA synthesis (determined by sequential immunohistochemical analysis and autoradiography) were found most commonly in the forestomach and to a lesser extent in the pylorus. This distribution of replicating damaged cells corresponds with the relative tumour yields in these upper gastrointestinal tract tissues and such cells are the probable targets in this single dose carcinogenesis regime. Thus, whilst the highest concentration of O6-MedG did not correlate tumour incidence, the overall risk for tumour induction did correlate with a significant level of DNA damage, a lower capacity for DNA repair and a marked increase in DNA synthesis over the constitutive level in the target cells. Carcinogenic risk in this system is therefore more readily determined by studying several risk factors simultaneously.

Animals↗
PubMed1993

Tissue and cell specific methylation, repair and synthesis of DNA in the upper gastrointestinal tract of Wistar rats treated with N-methyl-N'-nitro-N-nitrosoguanidine via the drinking water.

Several potential cancer risk factors have been monitored concurrently in the upper gastrointestinal tract of young male Wistar rats given N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) via the drinking water, a regimen that induces a high yield of tumours in the pylorus and to a lesser extent in the duodenum. Radioimmunoassay was used to determine the amounts of O6-methyl-2'-deoxyguanosine (O6-MedG) formed in the tissue DNA of rats given MNNG at doses of 40 or 80 micrograms/ml for periods of 3, 6 and 12 weeks. The highest adduct concentration was found in the pylorus with progressively lower concentrations in the corpus and duodenum, jejunum, forestomach and oesophagus. Between 3 and 12 weeks these adduct levels decreased in all tissues and there was no evidence of a dose dependent accumulation of O6-MedG. When analysed by immunohistochemistry the distribution of cells with nuclei containing O6-MedG was seen to be heterogeneous in the various tissues. O6-Alkylguanine-DNA alkyltransferase activity increased during the 12 weeks of MNNG treatment in oesophagus and forestomach, but decreased to approximately 50% of the initial value in the corpus, pylorus, duodenum and jejunum. The major changes in DNA synthesis and cell proliferation were the marked upward expansion (i.e. towards the lumen) of the zone of replicating cells in the glands of the pylorus and the greatly increased numbers of replicating damaged cells (i.e. cells that contained O6-MedG whilst undergoing DNA synthesis) as determined by sequential immunohistochemical analysis and autoradiography. Such cells are the probable target cells in this chronic dose carcinogenesis regime. Although similar changes also occurred in the glands of the corpus these were of lesser extent and the changes of labelling index in the oesophagus and forestomach were relatively minor. In the duodenum, MNNG treatment led to erosion of the upper part of the glands so that the zone of cells containing O6-MedG overlapped with the zone of proliferating cells resulting in the formation of many replicating damaged cells. Thus, as in the single dose study (see preceding paper) the distribution of replicating damaged cells coincides with the tumour yield in the tissues of the upper gastrointestinal tract. As in the case of single doses of MNNG the risk factors for carcinogenesis are, a significant level of DNA damage, a lower capacity for DNA repair and an increased DNA synthetic activity, again suggesting that carcinogenic risk cannot readily be determined by studying risk factors individually.

Animals↗
PubMed1993

Promutagenic methylation damage in liver DNA of mice infected with Schistosoma mansoni.

Male and female BK-TO mice were infected with different numbers of Schistosoma mansoni cercariae under standard environmental conditions. Promutagenic methylation damage (O6-methyldeoxyguanosine; O6-MedG) was detected in liver DNA, but not in kidney, spleen or bladder DNA of infected animals. It was shown that levels of hepatic O6-MedG increased with increasing intensities of schistosomal infection. Possible mechanisms of action are discussed. These include the activating effects of schistosomes and their products on murine macrophages and subsequent endogenous formation of N-nitroso compounds by the activated macrophages.

Animals↗
PubMed1993

Combined micropreparative techniques with synchronous fluorescence spectroscopy or 32P-postlabelling assay for carcinogen-DNA adduct determination.

Methods for the detection of carcinogen-DNA adducts require both sensitivity and specificity. The 32P-postlabelling assay is highly sensitive but lacks adduct specificity. The strategy reported herein combines micropreparative techniques including HPLC and immunoaffinity chromatography (IAC) to enhance chemical specificity. The resultant assays have retained sensitivity for human DNA analysis. Combined HPLC and 32P-postlabelling has detected 7-methyldeoxyguanosine in human lung samples, while combined IAC and 32P-postlabelling has detected O6-methyldeoxyguanosine adducts in stomach tissue. The limits of detection are one adduct in 10(7) and 10(8) unmodified deoxyguanosine (dG), respectively. IAC was combined with 32P-postlabelling to detect polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human lung. The detection limit was one adduct in 10(7) dG. Our laboratory has also employed synchronous fluorescence spectroscopy (SFS) for the detection of adducts formed from benzo[a]pyrene in human lung. Complex fluorescence matrices suggest the presence of other PAH-DNA adducts. Both the SFS assay and the 32P-postlabelling assay were applied to a series of human samples and a high correlation was found for adduct levels. The development of such assays using synthetic standards, internal standards, determination of calibration curves and validation with corroborating methods is required for chemically specific and sensitive adduct detection.

Benzo(a)pyrene↗
PubMed1992

Normal human breast xenografts activate N-nitrosodimethylamine: identification of potential target cells for an environmental nitrosamine.

Normal human breast tissue maintained as xenografts in female Balb/c (nu/nu) athymic mice is capable of metabolising N-nitrosodimethylamine (NDMA) to active intermediates that will react with DNA. Administration of NDMA to mice with slow-release implants of 17 beta-oestradiol which provide human physiological (luteal phase) circulating oestrogen levels and increase cell proliferation in the xenograft (Laidlaw et al., 1992), leads to an apparent increase in the extent of reaction with DNA compared to controls without oestrogen implants. In mice with oestrogen implants, measurements of the amounts of the promutagenic lesion, O6-methyl-2'-deoxyguanosine formed in DNA clearly indicated a dose related increase in the extent of reaction. Detection of O6-methyl-2'-deoxyguanosine using immunohistochemical procedures revealed that the nuclei of cells of the glandular epithelium, supportive tissue and adipose tissue, in decreasing order of prevalence, were positively stained for the presence of this DNA lesion. Epithelial cells, which are the putative target cells for carcinogenesis in the breast, are therefore prone to promutagenic damage as a result of exposure to an environmental nitrosamine.

Animals↗
PubMed1992

Immunoaffinity purification combined with 32P-postlabelling for the detection of O6-methylguanine in DNA from human tissues.

Three different activated supports were used to immobilize alpha-O6-methyldeoxyguanosine (O6-MedG) monoclonal antibody. Affinity gels based on CNBr Sepharose, Affigel Hz and periodate-activated Sepharose (PIAS) were able to bind 1.4, 1.5 and 6.2 nmol [3H]O6-MedG/mg immobilized IgG respectively, and recovery of bound material exceeded 95% in all cases. Significant non-specific binding of normal nucleosides occurred only when using the CNBr gel. PIAS alpha-O6-MedG affinity gel was able to purify O6-MedG-3'-monophosphate from digested synthetic oligonucleotides and in vitro-methylated calf thymus DNA to allow subsequent detection by 32P-postlabelling and two-dimensional TLC. The combined method was applied to three human samples and O6-MedG levels of 0.39, 0.38 and 0.45 mumol/mol 2'-deoxyguanosine were found. The minimum detection limit of the combined method is expected to be approximately 1 O6-MedG in 10(8) normal 2'-deoxyguanosines (i.e. approximately 30 lesions per human cell) when 100 micrograms of DNA is used.

Chromatography, Thin Layer↗
PubMed1992

Promutagenic methylation damage in bladder DNA from patients with bladder cancer associated with schistosomiasis and from normal individuals.

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.

Age Factors↗
PubMed1991

32P-postlabelling of alkylated thymidines using Epstein-Barr virus encoded thymidine kinase.

Alkylated nucleotides have been detected by 32P-postlabelling using the enzyme T4 polynucleotide kinase which phosphorylates the 3'-mononucleotides to give the 3',[5'-32P]bisphosphates. These may then be separated by two-dimensional TLC as the bisphosphates or the [5'-32P]monophosphates. We describe here an alternative approach using the Epstein-Barr virus (EBV) encoded thymidine kinase (TK) to directly phosphorylate adducted nucleosides to give the [5'-32P]monophosphates. Using a series of methyl, ethyl and butyl thymidines EBV-encoded TK was shown to phosphorylate a wide range of adducted thymidines with varying degrees of labelling efficiency; N3-methyl thymidine was labelled with the highest efficiency and O4-ethyl thymidine the lowest. Whereas O4-methyl thymidine was labelled at a higher efficiency than O2-methyl thymidine, O4-ethyl and O4-butyl thymidines were labelled at a much lower efficiency than the corresponding O2-alkyl thymidines. Labelling efficiency increased with pH in the range pH 7 to pH 9, but the relative labelling efficiency was ATP independent. This direct phosphorylation of adducted nucleosides offers an alternative approach to the detection of alkylated residues in DNA which may complement current postlabelling procedures.

Adenosine Triphosphate↗
PubMed1991

Isolation and cDNA cloning of a rat O6-alkylguanine-DNA-alkyltransferase gene, molecular analysis of expression in rat liver.

A rat O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA has been isolated from a rat liver cDNA library by hybridization with the human homologue. The candidate 806 bp cDNA was sequenced and shown to contain a 630 bp open reading frame that could encode a protein of 22.2 kd. Fluorography of labelled ATase indicates a 24 kd protein which is consistent with several previous reports. The derived amino acid sequence demonstrated 81% similarity with the human ATase and 94% identity over a 67 residue region encompassing the putative alkyl acceptor site. Peptide sequences derived from cleaved homogeneous rat ATase have been located in the predicted protein providing additional confirmation of the identity of the cDNA. A 1.05 kb mRNA has been detected in rat liver by Northern analysis; treatment of adult rats with 2-acetylaminofluorene causes an approximately 10-fold induction of this message in liver. Following site directed mutagenesis of the 806 bp cDNA, the 630 bp protein coding sequence has been ligated into an Escherichia coli expression vector to achieve ATase levels of greater than 3% of total protein in bacterial extracts.

2-Acetylaminofluorene↗
PubMed1991

DNA methylation in specific cells of rat liver by N-nitrosodimethylamine and N-nitrosomethylbenzylamine.

Dose-response curves for the O6-methylation of guanine in the hepatic DNA of Wistar and Sprague-Dawley rats were determined after administration of N-nitrosomethylbenzylamine (NMBzA) or N-nitrosodimethylamine (NDMA). Similar results were obtained for both rat strains but methylation of hepatic DNA by NDMA was approximately 9-fold more efficient than with NMBzA when doses were compared on a molar basis. Comparison by immunohistochemical analysis of the distribution of nuclei containing O6-methylguanine within the liver lobules showed that both agents tended to alkylate cells close to the central veins at the lower doses. With increasing doses, the band width of alkylated cells around the central vein increased, spreading in the case of NDMA virtually into the portal zones, whereas with NMBzA the zone of alkylated nuclei reached little more than halfway from the central vein to the portal zone. These differences in the distribution of alkylated cells may explain the differing hepatic responses to these two nitrosamines.

Animals↗
PubMed1990

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.

Adult↗
PubMed1990

Purification to apparent homogeneity and partial amino acid sequence of rat liver O6-alkylguanine-DNA-alkyltransferase.

O6-alkylguanine-DNA-alkyltransferase (ATase) activity was increased in rat liver from 80 to 320 fmoles/mg total protein 48 h after administration of 2-acetylaminofluorene at 60 mg/kg body weight. This tissue was used as a source of ATase which was purified by ammonium sulphate precipitation and DNA-cellulose, molecular exclusion and ion exchange chromatography (IEC). IEC purified material showed a major 24 kDa band after polyacrylamide gel electrophoresis (PAGE) with silver staining. Fluorography of purified ATase following incubation with [3H]-methylated substrate DNA and PAGE showed a single band at 24 kDa suggesting that, as with bacterial ATases, the protein itself accepts the alkyl group from O6-alkylguanine in substrate DNA during the repair reaction. Further purification of the protein using reverse phase HPLC resulted in a single peak representing approximately 125,000 fold purification. This was subjected to amino-terminal sequencing and it was found that the protein was blocked at the amino-terminal end: it was cleaved using trypsin or cyanogen bromide and the amino acid sequence of several reverse phase HPLC purified fragments was determined.

Amino Acid Sequence↗
PubMed1988

Longitudinal chromatic aberration of the human eye and wavelength in focus.

The wavelength in focus on the retina was determined for 14 observers from measurements of the longitudinal chromatic aberration of the eye. Measurements were made at two viewing distances: 3 m and 40 cm. The results show a large amount of individual variability for the wavelength in focus at each distance. The range of values at 3 m was 457 to 593 nm with a mean of 518 nm, and 375 to 548 nm with a mean of 468 nm at 40 cm. The chromatic aberration measured at 3 m is the same as previously reported data by other investigators for distant viewing. The average results of this study indicate very little difference for the chromatic aberration measured at near compared to distant viewing but, again, considerable individual variability exists.

Accommodation, Ocular↗
PubMed1987

Characterisation and nucleotide sequence of ogt, the O6-alkylguanine-DNA-alkyltransferase gene of E. coli.

The plasmid pO61 that was isolated from an E. coli genomic DNA library and codes for O6-alkylguanine (O6AG) DNA alkyltransferase (ATase) activity (1) has been further characterised. Subclones of the 9 Kb insert of pO61 showed that the ATase activity was encoded in a 2Kb Pst1 fragment but a partial restriction endonuclease map of this was different to that of the E. coli ada gene that codes for O6-AG and alkylphosphotriester dual ATase protein. Fluorographic analyses confirmed that the molecular weight of the pO61-encoded ATase was 19KDa i.e. similar to that of the O6AG ATase function that is cleaved from the 39KDa ada protein but rabbit polyclonal antibodies to the latter reacted only very weakly with the pO61-encoded protein. A different set of hybridisation signals was produced when E. coli DNA, which had been digested with a variety of restriction endonucleases was probed with 2Kb Pst 1 fragment or the ada gene. These results provided evidence for the existence of a second ATase gene in E. coli. The 2Kb Pst-1 fragment of pO61 was therefore sequenced and an open reading frame (ORF) that would give rise to a 19KDa protein was identified. The derived amino acid sequence of this showed a 93 residue region with 49% homology with the O6AG ATase region of the ada protein and had a pentamer and a heptamer of identical sequence separated by 34 amino acids in both proteins. The pentamer included the alkyl accepting cysteine residue of the ada O6AG ATase. The hydrophobic domains were similarly distributed in both proteins. Shine-Dalgarno, -10 and -35 sequences were identified and the origin of transcription was located by primer extension and S1 nuclease mapping. The amino-terminal amino acid sequence of the protein was as predicted from the ORF.

Amino Acid Sequence↗
PubMed1985

Cloning of the E. coli O6-methylguanine and methylphosphotriester methyltransferase gene using a functional DNA repair assay.

Alkylating agents react with various nitrogen and oxygen atoms in DNA and many of the products are substrates for repair processes. Oxygen atom derivatives such as O6-methylguanine (O6-meG) O4-methylthymine and methylphosphotriesters (MP) have been shown to undergo repair by methyl group removal. The proteins involved in the latter reaction can be considered to be methyltransferases (MT) because their action results in the transfer of the methyl group to a cysteine residue within a polypeptide. A rapid and sensitive assay for MT activity has been developed and used to screen extracts of bacteria harbouring an E. coli genomic DNA library carried in a plasmid vector. We report here the cloning of an E. coli gene coding for O6-meG and MP MT repair functions. These two activities reside on a 37Kd protein that can undergo a host-dependent cleavage to produce an 18Kd protein which contains only O6-meG MT and a 13Kd protein which contains only MP MT.

Cloning, Molecular↗
PubMed1985

Methylated purines formed in DNA by dimethylnitrosamine in rats previously exposed to hepatotoxic and hepatocarcinogenic regimes: effects on the repair of O6-methylguanine.

Studies of mammalian systems for the repair of O6-methylguanine in DNA have revealed large differences in the capacities of tissues and cells to perform this function and in the case of rat liver it has been shown that the O6-methylguanine repair system can be stimulated by exposure to hepatotoxic and hepatocarcinogenic regimes. In this report an assessment is made of possible relationships between toxic liver injury, DNA synthesis, cell proliferation and DNA repair by treating Wistar rats with agents selected to provide differing degrees of liver involvement. The effects of long-term (20 week) treatments with acetylaminofluorene (15 mg/kg/day), quinoxaline 1,4-dioxide (10 mg/kg/day), 4-aminobiphenyl-HCl (15 mg/kg/day) and pronethalol (20 mg/kg/day) were assessed, using the same strain of animals in which the original toxicity and carcinogenicity data were obtained. Repair of O6-methylguanine produced in liver DNA by a low, non-toxic dose (2 mg/kg) of [14C]dimethylnitrosamine was increased 3-4-fold throughout the period of treatment with acetylaminofluorene, to a lesser extent by quinoxaline 1,4-dioxide and 4-aminophenyl-HCl and not at all in the case of pronethalol. No evidence was obtained to indicate a direct relationship between O6-methylguanine repair and either the induced hepatotoxicity or the ensuing increased rates of DNA synthesis which occur following exposure to these agents.

2-Acetylaminofluorene↗