Neurovascular coupling and oxygenation are decreased in hippocampus compared to neocortex because of microvascular differences.

The hippocampus is essential for spatial and episodic memory but is damaged early in Alzheimer's disease and is very sensitive to hypoxia. Understanding how it regulates its oxygen supply is therefore key for designing interventions to preserve its function. However, studies of neurovascular function in the hippocampus in vivo have been limited by its relative inaccessibility. Here we compared hippocampal and visual cortical neurovascular function in awake mice, using two photon imaging of individual neurons and vessels and measures of regional blood flow and haemoglobin oxygenation. We show that blood flow, blood oxygenation and neurovascular coupling were decreased in the hippocampus compared to neocortex, because of differences in both the vascular network and pericyte and endothelial cell function. Modelling oxygen diffusion indicates that these features of the hippocampal vasculature may restrict oxygen availability and could explain its sensitivity to damage during neurological conditions, including Alzheimer's disease, where the brain's energy supply is decreased.

Dietary variables associated with DNA N7-methylguanine levels and O6-alkylguanine DNA-alkyltransferase activity in human colorectal mucosa.

Components of human diets may influence the incidence of colorectal adenomas, by modifying exposure or susceptibility to DNA-damaging alkylating agents. To examine this hypothesis, a food frequency questionnaire was used to assess the diet of patients recruited for a case-referent study where biopsies of normal colorectal mucosa were collected during colonoscopy and subsequently analysed for DNA N7-methylguanine (N7-MeG) levels, as an indicator of exposure, and activity of the DNA repair protein O6-alkylguanine DNA-alkyltransferase (MGMT), as an indicator of potential susceptibility. Cases with histologically proven colorectal adenomas (n = 38) were compared with referents (n = 35) free of gastrointestinal neoplasia. The case group consumed significantly more red meat (4.5 versus 3.4 servings/week, P < 0.05), processed meats, (4.7 versus 3.2 servings/week, P < 0.05) and % food energy as fat (34.9 versus 30.7%, P < 0.001). N7-MeG [mean: 95% confidence interval (CI)] levels were significantly lower in the group that consumed the highest proportion of dietary fibre/1000 kcal in comparison with the group with the lowest intake (0.61; 0.35-0.86 versus 1.88; 0.88-2.64 micromol/mol dG, P < 0.05). N7-MeG levels were also inversely associated with folate consumption (P < 0.05). MGMT activity (mean; 95% CI) was significantly higher in the group with the lowest consumption of vegetables than in the group with the greatest vegetable consumption (7.02; 5.70-8.33 versus 4.93; 3.95-5.91 fmol/microg DNA, P < 0.05). Our results are consistent with the hypothesis that dietary factors may modify exposure or susceptibility, respectively, to DNA damage by alkylating agents.

Human colorectal mucosal O6-alkylguanine DNA-alkyltransferase activity and DNA-N7-methylguanine levels in colorectal adenoma cases and matched referents.

BACKGROUND AND AIMS: O(6)-alkylguanine DNA-alkyltransferase (MGMT) provides protection against alkylating agent-induced GC-->AT transition mutations. Such mutations are frequently seen in the KRAS oncogene of large colorectal adenomas, but whether adenoma or mutational risk in humans is influenced by MGMT activity and alkylating agent exposure is unclear. Hence, MGMT activity and, as an indicator of alkylating agent exposure, DNA-N7-methylguanine (N7-MeG) levels were determined in the normal tissue of patients with and without adenomas. METHODS: Biopsy specimens of normal colorectal mucosa were collected during colonoscopy from 85 patients with histologically proved colorectal adenomas (cases) and from 85 patients free of gastrointestinal neoplasia (referents) matched by age, sex and biopsy location. MGMT activity and N7-MeG levels were measured in colorectal tissue extracts and DNA, respectively. RESULTS: MGMT activity was higher in the normal mucosa of cases than in referents (6.65+/-3.03 vs 5.61+/-2.74 fmol/micro g DNA, p = 0.01). On stratification of cases, MGMT activity was found to be considerably greater in the normal mucosa of cases with large adenomas (p = 0.003) and slightly higher in cases with a GC-->AT transition mutation in the K-ras gene (p = 0.03). Elevated MGMT levels were associated with an increased risk of adenoma (OR 1.17, 95% CI 1.03 to 1.33 per unit increase in activity). Detectable levels of N7-MeG were found in DNA from 89% of cases and 93% of referents, with levels ranging from <0.1 to 7.7 micro mol/mol dG. Cases and referents had similar DNA-N7-MeG levels. CONCLUSIONS: Human exposure to methylating agents is widespread. MGMT activity is increased in the normal mucosa of patients with adenomas.

Inactivation of nitric oxide by rat cerebellar slices.

Nitric oxide (NO) functions as an intercellular messenger throughout the brain. For this role to be performed efficiently, there must be a mechanism for neutralizing NO, but whether an active biological process exists, or whether NO is lost mainly through diffusion is unclear. To investigate this issue, rat cerebellar slices were exposed to constant levels of NO and the cGMP generated within the slice used as an indicator of NO concentrations therein. NO was about 1000-fold less potent in slices (EC50, 1 microM) than in separated cells from the same tissue (EC50, 1.6 nM), consistent with access of NO to the slice interior being greatly hindered by inactivation. Supporting this interpretation, immunohistochemical analysis indicated a marked concentration gradient of cGMP across the thickness of slices exposed to subsaturating NO concentrations, signifying a marked NO gradient. Several known NO-degrading processes, including reaction with lipid peroxyl radicals, erythrocytes and superoxide ions, were eliminated as contributing factors, indicating a novel mechanism. A diffusion-inactivation model was used to estimate the kinetics of NO consumption by the slices. The best fits to experimental data indicated a Michaelis-Menten-type reaction having a Vmax of 1-2 microM s-1 and a Km of around 10 nM. The rates predict that inactivation would impose a very short half-life (<10 ms) on NO in physiological concentrations (up to 10 nM) and that it would play an important role in shaping the NO concentration profiles when it is synthesized by multiple nearby sites.

Longitudinal variation in O(6)-alkylguanine DNA-alkyltransferase activity in the human colon and rectum.

In a systematic study of O(6)-alkylguanine DNA-alkyltransferase activity in the human colon and rectum, tumours were found to occur in regions of low activity. These results are consistent with the hypothesis that O(6)-alkylguanine DNA-alkyltransferase levels and alkylating agent exposure may be important determinants of large bowel tumorigenesis.

Host determinants of DNA alkylation and DNA repair activity in human colorectal tissue: O(6)-methylguanine levels are associated with GSTT1 genotype and O(6)-alkylguanine-DNA alkyltransferase activity with CYP2D6 genotype.

There is increasing evidence that alkylating agent exposure may increase large bowel cancer risk and factors which either alter such exposure or its effects may modify risk. Hence, in a cross-sectional study of 78 patients with colorectal disease, we have examined whether (i) metabolic genotypes (GSTT1, GSTM1, CYP2D6, CYP2E1) are associated with O(6)-methyldeoxyguanosine (O(6)-MedG) levels, O(6)-alkylguanine-DNA alkyltransferase (ATase) activity or K-ras mutations, and (ii) there was an association between ATase activity and O(6)-MedG levels. Patients with colon tumours and who were homozygous GSTT1(*)2 genotype carriers were more likely than patients who expressed GSTT1 to have their DNA alkylated (83 versus 32%, P=0.03) and to have higher O(6)-MedG levels (0.178+/-0.374 versus 0.016+/-0.023 micromol O(6)-MedG/mol dG, P=0.04) in normal, but not tumour, DNA. No such association was observed between the GSTT1 genotype and the frequency of DNA alkylation or O(6)-MedG levels in patients with benign colon disease or rectal tumours. Patients with colon tumours or benign colon disease who were CYP2D6-poor metabolisers had higher ATase activity in normal tissue than patients who were CYP2D6 extensive metabolisers or CYP2D6 heterozygotes. Patients with the CYP2E1 Dra cd genotype were less likely to have a K-ras mutation: of 55 patients with the wild-type CYP2E1 genotype (dd), 23 had K-ras mutations, whereas none of the 7 individuals with cd genotype had a K-ras mutation (P=0.04). No other associations were observed between GSTT1, GSTM1, CYP2D6 and CYP2E1 Pst genotypes and adduct levels, ATase activity or mutational status. O(6)-MedG levels were not associated with ATase activity in either normal or tumour tissue. However, in 15 patients for whom both normal and tumour DNA contained detectable O(6)-MedG levels, there was a strong positive association between the normal DNA/tumour DNA adduct ratio and the normal tissue/tumour tissue ATase ratio (r(2)=0.66, P=0.001). These results indicate that host factors can affect levels both of the biologically effective dose arising from methylating agent exposure and of a susceptibility factor, the DNA repair phenotype.

Development and application of a sensitive and rapid immunoassay for the quantitation of N7-methyldeoxyguanosine in DNA samples.

N7-Methyldeoxyguanosine (N7-MedG) in DNA is a biomarker of exposure to environmental and endogenous methylating agents and may be of use in epidemiological studies. To quantitate N7-MedG in human samples, a sensitive assay system that uses only small quantities of DNA (<10 microg) is required. To this end, polyclonal antibodies against the imidazole ring-opened form of N7-MedG have been used to develop a highly sensitive immunoslot blot (ISB) assay. The limit of detection of the assay is 0.10 micromol of N7-MedG/mol of deoxyguanosine (dG) using 1 microg of DNA per analysis. The method was optimized using in vitro-methylated calf thymus DNA and then applied to a study of DNA methylation in liver and brain tissues of mice following a single iv dose of the antitumor agent Temozolomide. The amount of N7-MedG in both tissues was strictly proportional to dose over a range of 10-200 mg of Temozolomide/kg of body weight. The ISB assay was then validated using pyloric DNA of rats treated with N-methyl-N'-nitro-N-nitrosoguanidine and DNA samples from human bladder tumors, for both of which N7-MedG levels had already been quantitated by an HPLC/(32)P-postlabeling method previously described. The results showed a high degree of correlation (r = 0.98) between the two assays. The ISB assay was then applied to a range of human samples. A series of peripheral blood mononuclear cell DNA samples from cancer patients following treatment with Temozolomide had levels of N7-MedG ranging from 0.22 to 320 micromol/mol of dG. DNA samples from colon carcinoma and normal colorectal mucosa from individuals not known to be exposed to methylating agents contained levels of 0.11-1.34 micromol of N7-MedG/mol of dG. The ISB assay offers the potential for the rapid and high-throughput analysis of DNA obtained from routine biopsies and blood samples, thus enabling the determination of the extent of human exposure to environmental and endogenous sources of methylating agents in large-scale biomonitoring studies.

Elevated levels of the pro-carcinogenic adduct, O(6)-methylguanine, in normal DNA from the cancer prone regions of the large bowel.

BACKGROUND: The pro-mutagenic lesion O(6)-methyldeoxyguanosine (O(6)-MedG), a marker of exposure to many N-nitroso compounds (NOC), can be detected in normal and tumour DNA isolated from colorectal tissue. The biological significance of this exposure is, as yet, unknown but in situ NOC formation is bacterially catalysed suggesting that NOC formation and potentially DNA alkylation will vary throughout the large bowel. AIMS: To determine if O(6)-MedG levels in colorectal DNA vary within the large bowel. PATIENTS: We studied 62 men and women undergoing surgery for colorectal tumours in the north west of England. METHODS: O(6)-MedG levels were measured in paired normal and tumour DNA samples. DNA was digested to nucleosides, fractionated by HPLC, and purified O(6)-MedG quantified by a radioimmunoassay. RESULTS: O(6)-MedG was detected in 27 out of a total of 62 (43%) normal DNA samples and in 30 of 58 (52%) tumour DNA samples: it was present at concentrations of <0. 01-0.94 and <0.01-0.151 micromol O(6)-MedG/mol deoxyguanosine for normal and tumour DNA, respectively. Levels of O(6)-MedG in normal, but not tumour, DNA from the proximal colon were lower than those found in DNA from either the sigmoid colon (p=0.03) or rectum (p=0. 05). When the analysis was restricted to samples that contained O(6)-MedG, similar results were obtained in that O(6)-MedG levels in normal DNA were lower in the proximal colon than in the sigmoid colon (p=0.04) or rectum (p=0.03). CONCLUSIONS: DNA alkylation varied within the large bowel possibly due to in situ NOC formation and was highest in areas of the colon and rectum where the highest incidence of large bowel tumours occurs, suggesting that DNA alkylation may play a role in the aetiology of colorectal cancer.

Determinants of O(6)-alkylguanine-DNA alkyltransferase activity in normal and tumour tissue from human colon and rectum.

O(6)-Alkylguanine-DNA-alkyltransferase (ATase) is an important modulator of alkylating agent-induced toxicity and carcinogenicity, but those factors which influence the expression of this repair protein in human tissues are poorly characterised. In this study, we have determined ATase levels in macroscopically normal and tumour tissues from 76 individuals with benign or malignant colorectal disease. All tissue samples had detectable ATase activity, with values ranging from 35 to 451 fmol/mg protein. ATase activity in normal rectal tissue was significantly higher than that in normal tissue from the sigmoid colon (148 +/- 76 vs. 100 +/- 40 fmol/mg protein, p = 0.01), whereas ATase levels within different regions of the colon (proximal vs. sigmoid colon) were similar. In normal tissue, inter-individual variation in ATase activity was 4-fold in the colon and 6-fold in the rectum, whereas in tumour tissue the corresponding figures were approx. 13.0- and 7-fold, respectively. There was no detectable difference in normal tissue ATase activity between individuals with benign or malignant disease of the colon. Normal and tumour tissue ATase activities were strongly correlated in the sigmoid colon (r = 0.80) and rectum (r = 0.59) but not the caecum (r = -0.03). In a multivariate analysis, ATase activity in normal colon tissue increased with age (p = 0.01) and current smoking (p = 0.06), whereas tumour ATase activity increased only with use of anti-histamines (p = 0.05). In rectal tumour tissue, activity decreased with age (p = 0.05) and use of anti-muscarinic medications (p = 0.01): in normal rectal tissue, no modulating factors were identified.

Synchronous and metachronous gastric adenocarcinoma: case report and literature review.

Whilst synchronous adenocarcinoma of the stomach is well documented, metachronous primary disease is exceedingly rare. We report a man with a family history of colonic and gastric cancer, who underwent a resection of a Duke's C adenocarcinoma of the rectum, aged 56 years, and a proximal partial gastrectomy for synchronous stage 1 gastric adenocarcinomas of the lesser curve, aged 61 years. Nine years later, a metachronous gastric primary was discovered in the gastric remnant, necessitating total gastrectomy. Total gastrectomy is the operation of choice for synchronous gastric primaries as it ensures clearance and prevents metachronous growth. However, it may not be appropriate for all gastric cancer as operative morbidity and mortality are increased, and because synchronicity and metachronicity of gastric cancer are uncommon. Moreover, there are no consistent data to demonstrate a survival advantage for total compared with partial gastrectomy for operable gastric cancer. If, after partial gastrectomy, synchronous disease is detected in the resected specimen (as in this reported case), endoscopic surveillance for metachronous disease is advised, since this may be amenable to surgical cure.

Low O6-alkylguanine DNA-alkyltransferase activity in normal colorectal tissue is associated with colorectal tumours containing a GC-->AT transition in the K-ras oncogene.

O6-alkylguanine DNA-alkyltransferase (ATase) provides protection against the toxic, mutagenic and carcinogenic effects of alkylating agents, principally by removing the promutagenic lesion O6-alkylguanine from DNA. Differences in ATase activity in human tissue may thus determine mutational susceptibility. As GC-->AT transitions, which can be induced by O6-alkylguanine in DNA, are commonly observed in the K-ras oncogene of alkylating agent induced animal tumours and in human colorectal tumours, we have examined whether differences in ATase activity may affect the risk of K-ras mutations in humans with colorectal tumours. NTase activity in normal tissue from individuals with a K-ras mutation in colorectal tissue and more specifically a GC-->AT transition (but not a transversion mutation) was significantly lower than that in individuals without a mutation (P < 0.01). Thus, individuals with low ATase activity in normal tissue (i.e. below the median) were at increased risk of having a transition (OR 10.1; 95% CI 1.9-99.0), but not a transversion mutation (OR 1.7; 95% CI 0.3-12.2). There were no significant differences in tumour ATase activity in individuals with or without a mutation. These results suggest that ATase can protect colorectal tissue against the mutagenic effects of alkylating agents and furthermore, that alkylating agent exposure plays a role in the aetiology of colorectal tumours containing a GC-->AT transition in the K-ras oncogene.

Frequency of Ki-ras mutations and DNA alkylation in colorectal tissue from individuals living in Manchester.

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.

Biliary-cutaneous fistula: an uncommon complication of retained gallstones following laparoscopic cholecystectomy.

A biliary-cutaneous fistula occurring as a late complication of laparoscopic cholecystectomy is described. The fistula was associated with retained gallstone fragments following intra-operative spillage and attempted retrieval. The fate of intra-peritoneal gallstones is reviewed and preventative measures discussed.

The detection of alkylation damage in the DNA of human gastrointestinal tissues.

Damage arising from putative environmental sources has been found in the DNA of the gastric and colorectal mucosae of patients presenting with gastrointestinal disorders from the South Manchester area. O6-Methylguanine (O6-MeG) in the range 0.010- greater than 0.300 mu moles mole-1 adenine was heterogeneously distributed both between and within individuals. The pattern of alkylation of tissue DNA appears to differ when comparison is made between gastric and colorectal samples. Most of the gastric tumour DNA samples were alkylated (5/6; 0.087 +/- 0.097), whereas the DNA of the associated mucosa was alkylated less frequently (2/7) and to a lesser extent; (0.017 +/- 0.030; P = 0.07). Conversely, colorectal tumour DNA was alkylated infrequently (1/7) and to a lower extent (0.003 +/- 0.007) than the DNA of the adjacent mucosa (8/10 samples alkylated with a mean of 0.083 +/- 0.106; P = less than 0.01), or indeed of any other tissue. Although increased levels of DNA damage in tissue associated with malignant disease have been indicated by independent studies of DNA damage at other cancer sites, significant differences were not observed in the present report, neither was there any suggestion of a relationship with smoking or alcohol consumption. The data provided by this report indicate that exposure to putative environmental alkylating agents occurs in the UK at levels comparable to those previously detected in areas of higher cancer risk. Although we cannot determine the extent to which this DNA damage is attributable to normal background exposures, it is evident that the alkylation of tissue DNA occurs and is not uniform. In conjunction with other reports, therefore these differences may begin to provide indications of mechanisms that could be of relevance in the aetiology of gastrointestinal cancers.

Detection of O6-methylguanine in human DNA.

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.

Increased risk of cancer at multiple sites after gastric surgery for peptic ulcer.

Of 5018 patients who had undergone gastric surgery at St James Hospital, Balham, at least 25 years ago, death certificates have been received for 2768, whilst 1746 patients are still alive and are flagged (Office of Populations, Censuses and Surveys (OPCS) will notify us of their death and its cause) and only 504 could not be traced. Mortality from cancers of various organs has been determined using a 'years at risk' calculation in five year bands. There was no increase in mortality risk from any cancers during the first 15 postoperative years, but from 20 years after operation there was a significant excess risk not only of cancer of the stomach (4.5-fold), but also of the large bowel (1.6-fold), bronchus (3.9-fold), pancreas (4.0-fold), biliary tract (9.1-fold), oesophagus (2.3-fold), bladder (2.4-fold), breast (4.0-fold), and cancer of all sites (3.3-fold). These findings are consistent with the production in the operated-upon stomach of circulating carcinogens with a 20 year latency period.

Urinary N-nitrosoproline excretion: a further evaluation of the nitrosamine hypothesis of gastric carcinogenesis in precancerous conditions.

Measurement of N-nitroso compounds in gastric juice by different methods has given conflicting results. In order to resolve this controversy, we have assessed endogenous nitrosation by the independent N-nitrosoproline excretion test in subjects who had previously undergone gastric juice analysis by one of these methods. Ten Polya gastrectomy, 10 pernicious anaemia and nine matched control subjects were fed 380 mg of nitrate in beetroot juice and 500 mg proline. N-nitrosoproline (N-Pro) synthesised intragastrically from these precursors, and quantitatively excreted by the kidneys, was measured in 24 hour urine samples (collection checked by creatinine clearance). N-Pro excretion (mean +/- SEM) was reduced (p less than 0.01) in pernicious anaemia (1.1 +/- 0.8 ng/day) compared with matched control (18.0 +/- 7.2 ng/day), and also tended to be lower (NS) in polya gastrectomy (3.2 +/- 2.3 ng/day). Twenty four hour intragastric pH was monitored on a separate occasion in 23 of the 29 subjects; 13 were hypoacidic (pH greater than 4 greater than 50% of 24 hours) and 10 were acidic. N-Pro yields were reduced (p less than 0.01) in the hypoacidic group (0.9 +/- 0.6 ng/day) compared with the acidic group (17.9 +/- 6.6 ng/day), and N-Pro was negatively associated with mean intragastric pH (tau = -0.53, p = 0.001). We conclude that endogenous synthesis of this specific N-nitroso compound is favoured by low rather than high pH. These results are concordant with those previously reported in gastric juice from the same subjects and suggest that nitrosation is chemically rather than bacterially mediated, contrary to the nitrosamine hypothesis of gastric carcinogenesis.