Deoxynivalenol exposure assessment in a cohort of pregnant women from Bradford, UK.

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.

A biomarker survey of urinary deoxynivalenol in China: the Shanghai Women's Health Study.

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.

Removal of red light minimizes methylene blue-stimulated DNA damage in oesophageal cells: implications for chromoendoscopy.

Barrett's oesophagus (BO) carries an increased risk of progression to oesophageal adenocarcinoma. Chromoendoscopy with methylene blue (MB) can be used to facilitate identification of BO and target areas for biopsy. If photoexcited, MB can generate reactive oxygen species and genotoxic photodegradation products leading to DNA damage. We have previously demonstrated that levels of DNA damage are increased in BO following MB chromoendoscopy. The aim of this study was to investigate whether DNA damage, as measured by the comet assay, can be minimized during chromoendoscopy by varying MB concentration and light wavelength using an in vitro model. OE33 cells were treated with MB (0.015-15 mM) and exposed to white light (WL). Cells were also illuminated with WL fractions (580-700, 480-580, 350-480, <575, <610 and <688 nm) in the presence of MB. At clinically relevant concentrations, WL illumination of MB (15 mM) caused significant DNA damage in vitro (P < 0.001). Illumination of MB with red light (580-700 nm) also stimulated high levels of DNA damage in OE33 cells (P < 0.001). This effect was not observed with green or blue light. Filtering WL to remove red light wavelengths (>575 nm) reduced DNA damage and apoptosis to control levels in MB-treated cells. In addition, reducing the concentration of MB 10-fold markedly reduced the DNA-damaging effect of MB in vitro. The results show that photoactivation of MB by red light is responsible for the majority of DNA damage. Simple modifications to MB chromoendoscopy, such as filtering out red light from endoscopic WL or reducing MB concentration, are likely to limit DNA damage induced by the procedure.

IGFBP-3 and IGFBP-10 (CYR61) up-regulation during the development of Barrett's oesophagus and associated oesophageal adenocarcinoma: potential biomarkers of disease risk.

Dys-regulation of the insulin-like growth factor (IGF) system increases the risk of a number of malignancies. The aim of this study was to investigate the role of members of the IGF binding protein (IGFBP) superfamily in the development of oesophageal adenocarcinoma (EAC) and their possible use as markers of disease risk. Expression of IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-10/CYR61 was assessed using Real-Time-polymerase chain reaction (PCR) and immunohistochemistry in oesophageal tissues from Barrett's oesophagus (BE) patients with and without associated EAC, and in control subjects. IGFBP-3, IGFBP-4, and IGFBP-10/CYR61 mRNA levels were up-regulated in Barrett's (n=17) and tumour tissue of EAC patients (n=18) compared with normal tissue of control subjects without BE or EAC (n=18) (p<0.001). Over-expression of IGFBP-3 and IGFBP-10/CYR61 proteins was observed in Barrett's, dysplastic and tumour tissue of EAC cases (n=47 for IGFBP-10; n=39 for IGFBP-3) compared with adjacent normal epithelium (p<0.050). Notably, IGFBP-3, IGFBP-4, and IGFBP-10/CYR61 expression in Barrett's tissue of EAC cases (n=17) was significantly (p<0.001) higher than in Barrett's tissue of BE patients with no sign of progression to cancer (n=15). Overall, the results suggest that members of the IGFBP superfamily are up-regulated during oesophageal carcinogenesis and merit further investigation as markers of EAC risk.

TP53 and progression from Barrett's metaplasia to oesophageal adenocarcinoma in a UK population cohort.

BACKGROUND AND AIMS: Oesophageal adenocarcinoma frequently develops on a background of metaplastic Barrett's epithelium. The development of malignancy is accompanied by genetic alterations, which may be promising biomarkers of disease progression. METHODS: A case control study was conducted nested within a large unselected population based cohort of Barrett's patients. Incident oesophageal malignancies and high grade dysplasias were identified. For each case up to five controls were matched on age, sex, and year of diagnosis. Biopsies from the time of diagnosis of Barrett's epithelium were stained immunohistochemically for TP53, cyclin D1, cyclooxygenase 2 (COX-2), and beta-catenin proteins. RESULTS: Twenty nine incident oesophageal malignancies and six cases of high grade dysplasia were identified. The odds of diffuse or intense TP53 staining were substantially elevated in biopsies from patients who developed oesophageal adenocarcinoma compared with controls (odds ratio (OR) 11.7 (95% confidence interval (CI) 1.93, 71.4)). This difference was also present when all cases were considered (OR 8.42 (95% CI 2.37, 30.0). Despite the association with TP53 staining, only 32.4% of cases had an initial biopsy showing diffuse/intense TP53 staining. There were no significant associations between cyclin D1, COX-2, or beta-catenin staining and case control status. The OR for positive staining for both TP53 and COX-2 was markedly increased in cases compared with controls (OR 27.3 (95% CI 2.89, 257.0)) although only 15% of cases had positive staining for both markers. CONCLUSIONS: Immunohistochemical detection of TP53 expression is a biomarker of malignant progression in Barrett's oesophagus but sensitivity is too low to act as a criterion to inform endoscopic surveillance strategies. Additional biomarkers are required which when combined with TP53 will identify, with adequate sensitivity and specificity, Barrett's patients who are at risk of developing cancer.

Acid and bile salts induce DNA damage in human oesophageal cell lines.

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 microM), caused a small but significant (P < 0.028) elevation in DNA damage, but only at neutral pH in FLO-1 cells. Sodium deoxycholate (100 microM) 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.

Chromoendoscopy with methylene blue and associated DNA damage in Barrett's oesophagus.

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.

DNA damage levels are raised in Barrett's oesophageal mucosa relative to the squamous epithelium of the oesophagus.

Barrett's oesophagus (BE) is a pre-malignant metaplastic tissue predisposing to oesophageal adenocarcinoma (EC), and gastro-oesophageal reflux is a risk factor for both conditions. Reflux of acid and bile can cause mucosal injury and initiate chronic inflammation. These processes can induce DNA damage, possibly via an oxidative stress mechanism, thus increasing the likelihood of progression from Barrett's metaplasia to dysplasia and finally carcinoma. The comet assay was optimized for the detection of DNA damage (strand breaks and alkali-labile sites) in oesophageal biopsies, including incorporation of the DNA repair enzyme Fapy-DNA glycosylase (Fpg). Fpg allows the detection of 8-hydroxy-2-deoxyguanosine (8-OHdG) sites, a known pro-mutagenic DNA lesion. BE patients were recruited from BE surveillance clinics and oesophageal biopsies collected at endoscopy. Comet analysis revealed significantly increased (p < 0.001) DNA damage in Barrett's epithelium compared with matched squamous epithelium, with median % tail DNA values of 25.1% (first to third quartile 21.7-29.6%) and 18.6% (first to third quartile 16.9-21.4%), respectively. The median % tail DNA was up to 70% higher in the matched BE tissue compared with squamous epithelium from the same patient. Fpg sensitive sites were demonstrated in both tissue types at similar levels. The raised level of DNA damage in the premalignant BE may contribute to the accumulation of genetic alterations occurring during progression to EC. Understanding these underlying mechanisms provides a basis for cancer prevention strategies in BE patients.

Deoxynivalenol-induced immunomodulation of human lymphocyte proliferation and cytokine production.

Mycotoxins are a structurally diverse group of secondary metabolites produced by different genera of fungi, and include deoxynivalenol (DON), T-2 toxin, aflatoxin B1 (AFB1) and fumonisin B1 (FB1). Despite widespread human exposure and potent immunomodulation in animals, their effects on the human immune system remain to be defined. In this study, the effect of these toxins on human lymphocyte proliferation was evaluated using the MTT assay. Additionally, the effect of DON on cytokine profiles was measured. A 50% inhibition in cell proliferation was observed with a DON concentration of 216 ng/ml. T-2 toxin was more potent with 50% inhibition between 1 and 5 ng/ml. Negligible effects were observed with AFB1 and FB1, and a mixture of DON with either FB1 or AFB1 did not show any synergistic effects in this assay. Short-term treatment of PHA-stimulated lymphocytes with DON (100, 200 and 400 ng/ml) modulated the kinetics of IL-2, IL-4 and IL-6 production. IL-2 levels were up to 12-fold higher (P<0.05) in comparison to control levels at toxin concentrations of 200 and 400 ng/ml 72 h after treatment. IL-4 levels were only slightly elevated and IL-6 levels were slightly inhibited by these DON concentrations. The kinetics of cytokine production was followed for an extended period of 8-9 days at DON concentrations of 200 and 400 ng/ml. At the lower DON concentration (200 ng/ml), IL-2 levels were elevated 17-25-fold with a concomitant mild elevation in IFN-gamma. Consistent with earlier experiments, IL-6 levels were slightly suppressed by DON at this concentration. At 400 ng/ml, IL-2 levels were again significantly (P<0.05) elevated until 6 days post-treatment, while the effects on IL-4 and IL-6 were less marked. These data suggest DON has potent effects on human lymphocyte cytokine production which merit investigation in exposed human populations.

Genetic alterations in bronchial mucosa and plasma DNA from individuals at high risk of lung cancer.

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.

Prospective study of cyclin D1 overexpression in Barrett's esophagus: association with increased risk of adenocarcinoma.

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.

Glutathione S-transferase M1 null genotype is associated with a decreased risk of myocardial infarction.

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 effect of hOGG1 and glutathione peroxidase I genotypes and 3p chromosomal loss on 8-hydroxydeoxyguanosine levels in lung cancer.

Polymorphic genes for the peroxide scavenger glutathione peroxidase I (GPX1) and 8-hydroxydeoxyguanosine (8-OHdG) DNA glycosylase/apurinic (AP) lyase (hOGG1) map to loci on chromosome 3p which are subject to frequent loss of heterozygosity (LOH) in lung tumours. Levels of the pro-mutagenic, oxidative DNA lesion 8-OHdG, were measured in 37 paired normal and tumorous lung specimens using HPLC with electrochemical detection. Lung tumours were also analysed for 3p LOH by fluorescent PCR with Genescan analysis. No significant difference was observed between 8-OHdG levels in tumour [7.7 +/- 6.7 (mean +/- SE) 8-OHdG/10(6) 2'-deoxyguanosine (dG)] and normal (8.1 +/- 8.8 8-OHdG/10(6) dG) lung tissue. Adduct levels in normal lung tissue DNA were not associated with constitutive hOGG1 genotype although there was a trend towards lower 8-OHdG levels in individuals possessing the ALA6 GPX1 polymorphism. Lung tumours exhibiting 3p LOH (40%) contained higher levels of 8-OHdG adducts (10.9 +/- 2.6 8-OHdG/10(6) dG) (P = 0.05) and lower GPX1 enzyme activity [45.5 nmol glutathione (GSH)/min/mg] (P = 0.09) when compared with tumours without LOH at these sites (5.55 +/- 0.87 8-OHdG/10(6) dG and 63.6 nmol GSH/min/mg, respectively). In conclusion, tumours with 3p LOH at loci associated with hOGG1 and GPX1 appear to have compromised oxidative defence mechanisms as measured by reduced GPX1 enzyme activity and elevated 8-OHdG levels and this may affect the prognosis of lung cancer patients.

Expression of an inducible nitric oxide synthase gene in rainbow trout Oncorhynchus mykiss.

Using oligonucleotide primers based on mammalian nitric oxide synthases (NOS), expression of an inducible NOS (iNOS) gene was detected in head kidney and gill tissue of bacterially-challenged rainbow trout. Three overlapping fragments were amplified by RT-PCR and used to construct a contiguous sequence of 1410bp, with high nucleotide homology to iNOS in birds (61%) and mammals (57-59%). The nucleotide sequence translated in one reading frame to produce a partial peptide containing 470 amino acids, with 69-71% amino acid homology with mammalian iNOS, 81% homology with chicken iNOS and 85% homology with a partial (492bp) goldfish iNOS sequence. In vitro stimulation of head kidney macrophages with LPS also induced expression of the trout iNOS RNA, with optimal expression seen using 20-50 microg/ml LPS at 2h to 6h post-stimulation. The evolutionary and functional significance of the trout iNOS sequence are discussed.

Characterization of cytochrome P450 expression in human oesophageal mucosa.

The expression of cytochrome (CYP) P450 enzymes in human oesophageal mucosa was investigated in a total of 25 histologically non-neoplastic surgical tissue specimens by using specific antibodies in immunoblots and by RT-PCR mRNA analysis. The presence of CYP1A, 2E1, 3A and 4A enzymes was demonstrated by both techniques; CYP2A reactive protein was also detected by immunoblot. The presence of CYP4B1 mRNA was established but no specific antibody was available for detection of the corresponding protein by immunoblot. CYP2B6/7 mRNA was not detected in any sample. The mRNA transcripts for CYP1A1, 2E1, 4A11 and 4B1 were consistently detected in the majority of samples (>84%), whereas CYP1A2 mRNA was only detected in 11 of 19 specimens examined. An RT-PCR method to differentiate CYP3A4 and 3A5 mRNA was developed. This demonstrated CYP3A5 mRNA expression in all samples tested, whereas CYP3A4 mRNA was not detectable, suggesting that CYP3A5 is the major CYP3A protein in human oesophagus. There were significant interindividual variations in the amount of proteins, ranging from 8-fold for CYP4A to 43-fold for CYP2E1. For each patient, data on exposure to risk factors for oesophageal cancer were available, including tobacco smoke, alcohol, gastro-oesophageal reflux and hot beverage consumption. None of these risk factors or other patient characteristics (age, sex, tumour location and tumour stage) were correlated with the protein level of the individual CYP enzymes as determined by quantitation of immunoblot staining. However, the small series of samples precludes any strong conclusion concerning the lack of such correlations. There were no differences between squamous cell carcinomas and adenocarcinomas in either the qualitative or quantitative expression of the CYP enzymes. These data demonstrate that a range of CYP enzymes are expressed in human oesophageal mucosa and indicate that this tissue has the capacity to activate chemical carcinogens to reactive DNA binding metabolites.

Parasite-induced anorexia: leptin, insulin and corticosterone responses to infection with the nematode, Nippostrongylus brasiliensis.

The nematode parasite, Nippostrongylus brasiliensis, induces a biphasic anorexia in its rat host. The mechanisms, underlying this anorexia and its possible advantages to the host or parasite are unknown. We have investigated the effect of acute (12-24 h) and chronic (2-17 days) infections on plasma concentrations of leptin, insulin and corticosterone, and on hypothalamic expression of neuropeptide Y, galanin and corticotrophin-releasing factor genes. Plasma leptin was elevated in infected rats relative to uninfected ad libitum-fed controls and pair-fed controls in 12 h infections initiated at dark onset and in infections of 2 days' duration. At other times prior to parasite expulsion, plasma leptin in infected and pair-fed rats was lower than that of uninfected ad libitum-fed controls, reflecting the existing state of negative energy balance. Elevated plasma leptin concentrations in infected rats at day 2 post-infection were accompanied by reduced neuropeptide Y gene expression in the hypothalamic arcuate nucleus compared with both ad libitum control and pair-fed animals, and by lowered corticotrophin-releasing factor gene expression in the paraventricular nucleus relative to pair-feds. Twelve hour infections were characterized by a substantial increase in plasma corticosterone that was independent of reduced food intake, and in 12 h infections initiated at dark onset, where plasma leptin was elevated, there was also increased plasma insulin concentration in infected rats. In longer infections, differences between the groups in plasma insulin and corticosterone concentration were only observed at day 4 post-infection. In summary, perturbations to leptin, insulin and corticosterone signals early in infection may have a causative role and might feed back onto hypothalamic gene expression, whereas subsequent changes in these parameters are more likely to be secondary to negative energy balance.

Isolation of the first piscine transforming growth factor beta gene: analysis reveals tissue specific expression and a potential regulatory sequence in rainbow trout (Oncorhynchus mykiss).

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.