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Barrett’s oesophagus is an acquired precancerous condition that develops from mucosal injury incurred due to chronic gastro‐oesophageal reflux. The aim of this study was to determine if bile and/or acid components of the refluxate can induce DNA damage in vitro. The oesophageal cell lines FLO‐1 and HET1‐A were exposed to primary bile salts, individually or as a mixture, and the secondary bile salt sodium deoxycholate, in neutral or acidified media. Cells were then examined in the comet assay to measure DNA strand breaks. Cell viability was also monitored. Acidified media induced DNA damage in a pH‐ and time‐dependent manner. The primary bile compounds sodium glycocholate, glycocholic acid, sodium taurocholate and taurochenodeoxycholate, as an equimolar mixture (100 µM), caused a small but significant (P < 0.028) elevation in DNA damage, but only at neutral pH in FLO‐1 cells. Sodium deoxycholate (100 µM) caused a significant (P < 0.008) elevation in DNA damage in both cell lines, but again only at neutral pH. These data suggest that specific components of gastro‐oesophageal refluxate are capable of causing DNA damage and may participate in the genesis and progression of Barrett’s oesophagus via this mechanism. [ABSTRACT FROM AUTHOR]

Chromoendoscopy with methylene blue has been proposed to improve targeting of biopsies to specialised intestinal metaplasia and dysplasia in Barrett''s oesophagus. However, methylene blue can induce oxidative damage of DNA when photosensitised by white light. We show that damage to DNA is increased in Barrett''s mucosa after chromoendoscopy with methylene blue, an effect apparently dependent on presence of both methylene blue and endoscopic white light. Exposure of Barrett''s mucosa to DNA damage during endoscopy warrants caution since it could accelerate carcinogenesis. This risk needs to be carefully balanced against the possible benefit of improved early detection of preneoplastic lesions with methylene blue chromoendoscopy. [Copyright &y& Elsevier]

Deoxynivalenol (DON) is a ubiquitous contaminant of cereal crops in temperate regions of the world. It causes growth faltering and immune suppression in animals. Limited information is available on DON exposure in UK subpopulations. The objective of this study was to provide DON exposure assessment in a subset of pregnant women scheduled for an elective caesarean in a large multi-ethnic mother/infant birth cohort from Bradford, UK. Women aged 16-44 years (n = 85) provided a urine sample for DON analysis in the last trimester of pregnancy, and concurrently completed a food-frequency questionnaire (FFQ). The urinary DON biomarker was detected in all measured samples (geometric mean (GM) = 10.3 ng DON mg(-1) creatinine, range = 0.5-116.7 ng mg(-1)). Levels were higher in women classified as South Asian in origin (GM: 15.2 ng mg(-1); 95% CI = 10.7-21.5 ng mg(-1)) compared with non-South Asians (GM = 8.6 ng mg(-1); 95% CI = 6.6-11.8 ng mg(-1)), p = 0.02). Estimated DON intake from FFQ data and typical levels of DON contamination of food suggest that this was mainly due to higher levels of exposure from bread, particularly daily intake of DON from chapattis in South Asians (estimated mean = 2.4 µg day(-1); 95% CI = 1.2, 3.7 µg day(-1)) compared with non-South Asians (estimated mean = 0.2 µg day(-1); 95% CI = 0-0.4 µg day(-1)), p < 0.001. This is the first biomarker demonstration of DON exposure in pregnant women, and several urinary DON levels were the highest ever recorded in any study. A larger survey within this birth cohort is warranted to investigate any potential risk to mothers and their babies, from DON exposure during pregnancy.

Deoxynivalenol (DON) is a trichothecene mycotoxin found on wheat, maize and barley. In ecological surveys in China, DON and other trichothecenes have been implicated in acute poisoning episodes and linked with the incidence of esophageal cancer. In order to better understand exposure patterns, this pilot survey provided a combined measure of urinary un-metabolised or free DON (fD) and its glucuronide metabolite (DG) in a subset of 60 samples taken from the Shanghai Women's Health Study cohort, China. Samples were collected in 1997/1998 from women age 40-70 years. Urinary fD+DG combined was detected in 58/60 (96.7%) samples (mean 5.9 ng DON/mg creatinine; range nd-30.5); a similar frequency, and a mean level approximately half, of that previously observed for women in the UK. Wheat consumption was approximately 25% of that consumed by western diets; thus DON contamination of wheat may be higher in Shanghai than the UK. The de-epoxy metabolite of DON, a detoxification product observed in animals, was not detected, suggesting that humans may be particularly sensitive to DON due to a more restricted detoxification capacity., (© 2011 Taylor & Francis)

Evidence suggests that the majority of lung cancer patients have tumour-derived genetic alterations in circulating plasma DNA, and that this may be developed as a diagnostic tool. To this end, we have studied 60 individuals attending bronchoscopy clinic, with symptoms suspicious of lung cancer, for genetic alterations in bronchial mucosa biopsy (n = 47) and plasma (n = 40) DNA. Thirteen of 47 individuals from whom biopsies were taken displayed allelic loss of heterozygosity (LOH) in biopsy DNA for at least 1 of 4 markers. All 13 of these individuals had neoplastic tumour cells in their biopsies and were subsequently diagnosed with cancer. Thirteen of 40 individuals from whom plasma was taken displayed a plasma DNA LOH, and 12 of these 13 individuals were subsequently diagnosed with cancer. LOH in plasma was generally representative of LOH in the corresponding biopsy. In terms of sensitivity, using just 4 markers, biopsy LOH and plasma LOH were found in 13 of 44 (30%) and 12 of 29 (41%), respectively, of those patients subsequently diagnosed with cancer. Two patients were positive for LOH in plasma samples that pre-dated a diagnosis of cancer by several months. These data suggest that assay of genetic alterations in circulating plasma DNA may be developed as a useful addition to conventional techniques for the diagnosis of lung cancer.

Background: Esophageal adenocarcinoma commonly arises from a precancerous condition, Barrett's esophagus, in which the normal squamous epithelium is replaced by a columnar cell-lined epithelium. Genetic alterations occurring in this process could serve as biomarkers for the risk of malignant progression, improve surveillance, and contribute to early diagnosis. We examined two potential biomarkers, cyclin D1 and p53, in a prospective cohort of Barrett's esophagus patients., Methods: A total of 307 patients were enrolled in an endoscopic surveillance cohort, and esophageal biopsy specimens were collected at each endoscopy. Incident cases of adenocarcinoma were matched to control patients within the cohort by duration of follow-up, age, sex, and length of columnar cell-lined epithelium at recruitment. Biopsy specimens were analyzed for cyclin D1 and p53 protein levels by immunohistochemistry. Statistical tests were two-sided., Results: A total of 12 cases of adenocarcinoma occurred within the follow-up period, and tumor biopsy specimens from 11 cases stained positive for cyclin D1. Biopsy specimens from eight of these patients taken at recruitment also stained positive for cyclin D1. A case-control analysis of biopsy specimens obtained at recruitment revealed a statistically significantly increased risk of progression to adenocarcinoma in Barrett's esophagus patients whose biopsy specimens were cyclin D1 positive (odds ratio [OR] = 6. 85; 95% confidence interval [CI] = 1.57-29.91; P =.0106) but not in patients whose biopsy specimens were p53 positive (OR = 2.99; 95% CI = 0.57-15.76; P =.197)., Conclusions: Cyclin D1-positive staining could be a useful biomarker in identifying Barrett's esophagus patients at high risk of esophageal adenocarcinoma. Given the complexity of genetic alterations in the natural history of this cancer, additional biomarkers will be required to increase the sensitivity and specificity of molecular diagnosis.

Tobacco smoke is a major cause of both cancer and vascular disease. Although its carcinogenic role via induction of DNA damage and mutation is well established, the mechanisms involved in vascular disease remain unclear. One possibility is that DNA damage causes smooth muscle cell proliferation in the intima of arteries, thereby contributing to atherothrombotic processes. The binding of chemicals to DNA is modulated by detoxification enzymes, including glutathione S-transferases (GST) and microsomal epoxide hydrolase (EPXH). We therefore examined whether polymorphisms in these genes influence risk of cardiovascular disease. Blood was obtained from 398 patients admitted for angiographic investigation of chest pain and 196 age- and sex-matched controls. Patients were subdivided into those with and without previous acute myocardial infarction (AMI). DNA was analyzed for deletions in the GSTM1 and T1 genes and for substitutions in EPXH and GSTP1 genes. The GSTM1 null genotype occurred at a significantly lower frequency in the AMI patient group (48%) compared both to patients with no history of AMI (59%) and to the control group (57.2%). When subjects were stratified for smoking status, a significant association was observed only in smokers, suggesting the polymorphism is more important in the presence of tobacco smoke exposure. The association remained significant after adjusting for age, sex, and stenosis (presence or absence). No significant associations were observed between the other genotypes and cardiovascular disease (chi(2) test; P>0.1). The results of this study indicate that the GSTM1 null genotype is protective against AMI, an effect that is more marked in smokers. However, further study is required in order to elucidate the as yet unexplained, mechanisms underlying this association.

The nucleotide sequence of a rainbow trout transforming growth factor beta (TGF-beta) peptide is presented, which translates into a 382 amino acid precursor molecule containing a 20 amino acid leader and a mature peptide of 112 amino acids. The mature peptide has nine conserved cysteines and a conserved proline (position 36) and glycine (position 46), all characteristics of TGF-beta superfamily molecules. Within the precursor region are three glycosylation sites, two in common with known TGF-beta s, an integrin binding site (RGD) and the tetrabasic peptide cleavage site (RKKR). The full 3' untranslated region (UTR) consists of 542 nucleotides with a polyadenylation signal 16 nucleotides upstream of the poly(A) tail. The 5' UTR contains an open reading frame with the potential to encode an eleven amino acid peptide, which may have significance for regulation of TGF-beta translation. A wide tissue distribution of TGF-beta message was detected by RT-PCR; in blood leukocytes, kidney macrophages, brain, gill, and spleen tissue but not liver. A phylogenetic tree reveals the trout TGF-beta sequence is most related to xenopus TGF-beta 5, with these sequences and that of chicken TGF-beta 4 grouping with mammalian TGF-beta 1 s. The impact of the trout sequence on current theories of TGF-beta isotype evolution is discussed.

Expression of the ob gene and production of leptin have been examined on differentiation of rat fibroblastic preadipocytes to adipocytes in primary culture. Preadipocytes were obtained from the inguinal fat pad of suckling rats, and following differentiation the cells contained lipid droplets and the mRNAs for both lipoprotein lipase and adipsin were detected by Northern blotting. ob mRNA was not, however, detected on Northern blots, but analysis by RT-PCR indicated that the ob gene was expressed, particularly after differentiation. Measurement of leptin in the culture medium by ELISA showed that the ob gene product was secreted by adipocytes from approximately 4 days after the induction of differentiation. Leptin production was sustained over a 2-week period with a peak at 8-10 days post-induction. Dexamethasone stimulated leptin production, while an inhibition was observed with the beta-adrenoceptor agonist isoprenaline. These results demonstrate that following the differentiation of fibroblastic preadipocytes to adipocytes in primary culture, leptin is secreted with the cells responding to stimuli which regulate production of the hormone.

The acute effect of two selective beta 3-adrenoceptor agonists, BRL 35135A and ZD2079, on the expression of the ob gene and plasma leptin levels has been examined in mice. By 4-5 h after the administration of either beta 3-agonist to lean animals there was a major loss of ob mRNA from epididymal white adipose tissue. This was accompanied by a substantial fall in circulating leptin levels, as measured by an ELISA. Even 24 h after the first administration of beta 3-agonists, ob mRNA levels and circulating leptin levels remained low. In contrast to lean animals, treatment with BRL 35135A had only a minor effect on ob mRNA levels in obese (ob/ob) mice. Regulation of leptin production appears to involve a negative feedback loop to white adipose tissue through the sympathetic nervous system suppressing ob gene transcription via beta 3-adrenoceptors; an impairment in this loop is evident in the ob/ob mutant.

The development of a sensitive enzyme-linked immunosorbent assay (ELISA) is described for the quantitation of plasma leptin levels in both mice and rats. Approximately 1.5 ng leptin/ml plasma was detected in lean mice but significantly (p < 0.01) less was found in the plasma of ob/ob mice. In lean Zucker rats leptin circulated at approximately 4ng/ml plasma whereas levels were elevated more than 6-fold in fa/fa rats. Circulating leptin levels declined (p < 0.05) in lean rats which were fasted or exposed to 4 degrees C for 24h, but subsequently recovered to normal within 12h of refeeding or warming, respectively. Administration of insulin increased leptin levels in lean rats within 4h (p < 0.01). However, leptin levels were unchanged in fa/fa rats exposed to the same physiological stimuli.

A variety of supernatants were prepared by stimulating rainbow trout Oncorhynchus mykiss head kidney macrophages with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), or a leucocyte-derived macrophage-activating factor (I-MAF), individually and in combination. If generated using a 12-h stimulation period, such supernatants were found to elevate significantly the respiratory burst activity of target macrophages; that is, they contained a macrophage-derived MAF (m-MAF), but supernatants generated using a shorter incubation period showed no significant activity. Combinations of these treatments were particularly effective in generating m-MAF-containing supernatants. The elevation of respiratory burst activity by supernatants generated using combined treatments could be partially inhibited by prior treatment of the target macrophages with anti-TNF-alpha receptor 1 (TNFR1) monoclonal antibodies (mAbs). Similarly, treatment of macrophages with combinations of 1-MAF and m-MAF generated supernatants with potent m-MAF activity and this activity was partially inhibited by prior treatment of the target cells with anti-TNFR1 mAb. In addition, the presence of anti-transforming growth factor beta 1 (TGF-beta 1) serum while generating these latter supernatants resulted in significantly increased m-MAF activity. Such data suggest that fish leukocytes secrete a variety of potent macrophage-activating (TNF-alpha) and -deactivating (TGF-beta) factors.

Natural bovine transforming growth factor beta 1 (TGF-beta 1) was found to have both enhancing and suppressing effects on the respiratory burst activity of rainbow trout, Oncorhynchus mykiss, head kidney macrophages. Incubation with TGF-beta 1 alone increased respiratory burst activity using doses from 0.05 to 0.2 ng/mL. However, incubation of activated macrophages with TGF-beta 1 decreased their respiratory burst activity in a dose-dependent manner. Coincubation of macrophages with activating signals (macrophage activating factor containing supernatants and tumor necrosis factor alpha) and TGF-beta 1 inhibited the ability of such signals to increase respiratory burst activity. Similarly, pretreatment of macrophages with TGF-beta 1 prior to treatment with activating signals resulted in a time and dose-dependent decrease in activity relative to cells not treated with TGF-beta 1. These suppressive effects were largest using higher doses of TGF-beta 1 (1 ng/mL). The conservation of TGF-beta s and their possible mode of action are discussed.