Matrilysin-1 mediates bronchiolization of alveoli, a potential premalignant change in lung cancer

Matrilysin-1 (also called matrix metalloproteinase-7) is expressed in injured lung and in cancer but not in normal epithelia. Bronchiolization of the alveoli (BOA), a potential precursor of lung cancer, is a histologically distinct type of metaplasia that is composed of cells resembling airway epithelium in the alveolar compartment. We demonstrate that there is increased expression of matrilysin-1 in human lesions and BOA in the CC10-human achaete-scute homolog-1 transgenic mouse model. Forced expression of the matrilysin-1 gene in immortalized human normal airway epithelial BEAS-2B and HPLD1 cells, which do not normally express matrilysin-1, promoted cellular migration, suggesting a functional link for BOA formation via bronchiolar cell migration. In addition, matrilysin-1 stimulated proliferation and inhibited Fas-induced apoptosis, while a knockdown by RNA interference decreased cell growth, migration, and increased sensitivity to apoptosis. Western blotting demonstrated increased levels of phospho-p38 and phospho-Erk1/2 kinases after matrilysin-1 expression. Gene expression analysis uncovered several genes that were related to cell growth, migration/movement, and death, which could potentially facilitate bronchiolization. In vivo, the formation of BOA lesions was reduced when CC10-human achaete-scute homolog-1 mice were crossed with matrilysin-1 null mice and was correlated with reduced matrilysin-1 expression in BOA. We conclude that matrilysin-1 may play an important role in the bronchiolization of alveoli by promoting proliferation, migration, and attenuation of apoptosis involving multiple genes in the MAP kinase pathway.

Constitutive expression of human keratin 14 gene in mouse lung induces premalignant lesions and squamous differentiation

Squamous cell carcinoma accounts for 20% of all human lung cancers and is strongly linked to cigarette smoking. It develops through premalignant changes that are characterized by high levels of keratin 14 (K14) expression in the airway epithelium and evolve through basal cell hyperplasia, squamous metaplasia and dysplasia to carcinoma in situ and invasive carcinoma. In order to explore the impact of K14 in the pulmonary epithelium that normally lacks both squamous differentiation and K14 expression, human keratin 14 gene hK14 was constitutively expressed in mouse airway progenitor cells using a mouse Clara cell specific 10 kDa protein (CC10) promoter. While the lungs of CC10-hK14 transgenic mice developed normally, we detected increased expression of K14 and the molecular markers of squamous differentiation program such as involucrin, loricrin, small proline-rich protein 1A, transglutaminase 1 and cholesterol sulfotransferase 2B1. In contrast, wild-type lungs were negative. Aging CC10-hK14 mice revealed multifocal airway cell hyperplasia, occasional squamous metaplasia and their lung tumors displayed evidence for multidirectional differentiation. We conclude that constitutive expression of hK14 initiates squamous differentiation program in the mouse lung, but fails to promote squamous maturation. Our study provides a novel model for assessing the mechanisms of premalignant lesions in vivo by modifying differentiation and proliferation of airway progenitor cells. © The Author 2008. Published by Oxford University Press. All rights reserved.

Whole Slide Image Cytometry: a Novel Method for the Detection and Quantification of Abnormal DNA Contents in Barrett’s Oesophagus

Background: Barrett's oesophagus (BO) is a well recognized precursor of the majority of cases of oesophageal adenocarcinoma (OAC). Endoscopic surveillance of BO patients is frequently undertaken in an attempt to detect early OAC, high grade dysplasia (HGD) or low grade dysplasia (LGD). However histological interpretation and grading of dysplasia is subjective and poorly reproducible. The alternative flow cytometry and cytology-preparation image cytometry techniques require large amounts of tissue and specialist expertise which are not widely available for frontline health care.
Methods: This study has combined whole slide imaging with DNA image cytometry, to provide a novel method for the detection and quantification of abnormal DNA contents. 20 cases were evaluated, including 8 Barrett's specialised intestinal metaplasia (SIM), 6 LGD and 6 HGD. Feulgen stained oesophageal sections (1µm thickness) were digitally scanned in their entirety and evaluated to select regions of interests and abnormalities. Barrett’s mucosa was then interactively chosen for automatic nuclei segmentation where irrelevant cell types are ignored. The combined DNA content histogram for all selected image regions was then obtained. In addition, histogram measurements, including 5c exceeding ratio (xER-5C), 2c deviation index (2cDI) and DNA grade of malignancy (DNA-MG), were computed.
Results: The histogram measurements, xER-5C, 2cDI and DNA-MG, were shown to be effective in differentiating SIM from HGD, SIM from LGD, and LGD from HGD. All three measurements discriminated SIM from HGD cases successfully with statistical significance (pxER-5C=0.0041, p2cDI=0.0151 and pDNA-MG=0.0057). Statistical significance is also achieved differentiating SIM from LGD samples with pxER-5C=0.0019, p2cDI=0.0023 and pDNA-MG=0.0030. Furthermore the differences between LGD and HGD cases are statistical significant (pxER-5C=0.0289, p2cDI=0.0486 and pDNA-MG=0.0384).
Conclusion: Whole slide image cytometry is a novel and effective method for the detection and quantification of abnormal DNA content in BO. Compared to manual histological review, this proposed method is more objective and reproducible. Compared to flow cytometry and cytology-preparation image cytometry, the current method is low cost, simple to use and only requires a single 1µm tissue section. Whole slide image cytometry could assist the routine clinical diagnosis of dysplasia in BO, which is relevant for future progression risk to OAC.

Incidence of colorectal cancer in a population-based cohort of patients with Barrett's oesophagus

Objective. Previous studies have shown a positive association between colorectal cancer and Barrett's oesophagus, but this association is disputed. No population-based studies have examined the incidence of this cancer in patients with Barrett's oesophagus. Material and methods. The present study comprised a population-based cohort of patients with Barrett's oesophagus (constructed using pathology reports of all oesophageal biopsies in Northern Ireland 1993-99; cohort subclassified according to whether specialized intestinal metaplasia (SIM) was present, absent, or not commented on in biopsies). Cases of colorectal cancer were identified by linking with the Northern Ireland Cancer Registry. The comparison group used was the general population in Northern Ireland. Results. A total of 2969 patients with Barrett's oesophagus were followed for a total of 14,014 person-years (mean 4.7 years). SIM was present in 1670 patients (56.2%), absent in 545 (18.4%) and not commented on in 754 (25.4%). Colorectal cancer was diagnosed in 39 patients; 22 patients had cancer diagnosed at least 6 months after diagnosis of Barrett's oesophagus. There was no increased risk of colorectal cancer: the standardized incidence ratio (SIR) for cancer diagnosed at least 6 months after entry into the cohort was 0.82 (95% CI, 0.48-1.17); this risk did not alter with SIM status or gender. To assess a possible effect of diagnostic bias, we calculated SIRs for cancers occurring after at least 3 months, after at least 1 month and at any time after diagnosis of Barrett's oesophagus. These were 0.94 (0.57-1.30), 1.09 (0.69-1.48) and 1.46 (1.00-1.92), respectively. Conclusions. The incidence of colorectal cancer was not elevated in patients with Barrett's oesophagus. Diagnostic bias may explain why previous studies have found an association. © 2005 Taylor & Francis.

Symptoms and Endoscopic Features at Barrett's Esophagus Diagnosis: Implications for Neoplastic Progression Risk

OBJECTIVES: Risk stratification of Barrett's esophagus (BE) patients based on clinical and endoscopic features may help to optimize surveillance practice for esophageal adenocarcinoma (EAC) development. The aim of this study was to investigate patient symptoms and endoscopic features at index endoscopy and risk of neoplastic progression in a large population-based cohort of BE patients.

METHODS: A retrospective review of hospital records relating to incident BE diagnosis was conducted in a subset of patients with specialized intestinal metaplasia from the Northern Ireland BE register. Patients were matched to the Northern Ireland Cancer Registry to identify progressors to EAC or esophageal high-grade dysplasia (HGD). Cox proportional hazards models were applied to evaluate the association between endoscopic features, symptoms, and neoplastic progression risk.

RESULTS: During 27,997 person-years of follow-up, 128 of 3,148 BE patients progressed to develop HGD/EAC. Ulceration within the Barrett's segment, but not elsewhere in the esophagus, was associated with an increased risk of progression (hazard ratio (HR) 1.72; 95% confidence interval (CI): 1.08–2.76). Long-segment BE carried a significant sevenfold increased risk of progression compared with short-segment BE; none of the latter group developed EAC during the study period. Conversely, the absence of reflux symptoms was associated with an increased risk of cancer progression (HR 1.61; 95% CI: 1.05–2.46).

CONCLUSIONS: BE patients presenting with a long-segment BE or Barrett's ulcer have an increased risk of progressing to HGD/EAC and should be considered for more intense surveillance. The absence of reflux symptoms at BE diagnosis is not associated with a reduced risk of malignant progression, and may carry an increased risk of progression.

Intakes of dietary folate and other B vitamins are associated with risks of esophageal adenocarcinoma, Barrett's esophagus, and reflux esophagitis

Folate is implicated in carcinogenesis via effects on DNA synthesis, repair, and methylation. Efficient folate metabolism requires other B vitamins and is adversely affected by smoking and alcohol. Esophageal adenocarcinoma (EAC) may develop through a process involving inflammation [reflux esophagitis (RE)] leading to metaplasia [Barrett’s esophagus (BE)] and carcinoma. Within a population-based, case-control study, we investigated associations between dietary folate and related factors and risks of EAC, BE, and RE. EAC and BE cases had histologically confirmed disease; RE cases had endoscopically visible inflammation. Controls, age-sex frequency matched to EAC cases, were selected through population and general practice registers. Participants underwent structured interviews and completed food-frequency questionnaires. Multivariate ORs and 95% CIs were computed using logistic regression. A total of 256 controls and 223 EAC, 220 BE, and 219 RE cases participated. EAC risk decreased with increasing folate intake (OR highest vs. lowest = 0.56; 95% CI: 0.31, 1.00; P-trend < 0.01). Similar trends were found for BE (P-trend < 0.01) and RE (P-trend = 0.01). Vitamin B-6 intake was significantly inversely related to risks of all 3 lesions. Riboflavin intake was inversely associated with RE. Vitamin B-12 intake was positively associated with EAC. For EAC, there was a borderline significant interaction between folate intake and smoking (P-interaction = 0.053); compared with nonsmokers with high (≥median) folate intake, current smokers with low intakes (<median) had an 8-fold increased risk (OR: 8.15; 95% CI: 3.61, 18.40). The same group had increased BE risk (OR: 2.93; 95% CI: 1.24, 6.92; P-interaction = 0.12). Folate and other dietary methyl-group factors are implicated in the etiology of EAC and its precursors.

Effect of isomalt consumption on faecal microflora and colonic metabolism in healthy volunteers

Due to its low digestibility in the small intestine, a major fraction of the polyol isomalt reaches the colon. However, little is known about effects on the intestinal microflora. During two 4-week periods in a double-blind, placebo-controlled, cross-over design, nineteen healthy volunteers consumed a controlled basal diet enriched with either 30 g isomalt or 30 g sucrose daily. Stools were collected at the end of each test phase and various microbiological and luminal markers were analysed. Fermentation characteristics of isomalt were also investigated in vitro. Microbiological analyses of faecal samples indicated a shift of the gut flora towards an increase of bifidobacteria following consumption of the isomalt diet compared with the sucrose diet (P

Role of capsular modified heptose in the virulence of Campylobacter jejuni

The Campylobacter jejuni capsular polysaccharide is important for virulence and often contains a modified heptose. In strain ATCC 700819 (a.k.a. NCTC 11168), the modified heptose branches off from the capsular backbone and is directly exposed to the environment. We reported previously that the enzymes encoded by wcaG, mlghB and mlghC are involved in heptose modification. Here, we show that inactivation of any of these genes leads to production of capsule lacking modified heptose and alters the transcription of other capsule modification genes differentially. Inactivation of mlghB or mlghC, but not of wcaG, decreased susceptibility to bile salts and abrogated invasion of intestinal cells. All mutants showed increased sensitivity to serum killing, especially wcaG::cat, and had defects in colonization and persistence in chicken intestine, but did not show significant differences in adhesion, phagocytosis and intracellular survival in murine macrophages. Together, our findings suggest that the capsular heptose modification pathway contributes to bacterial resistance against gastrointestinal host defenses and supports bacterial persistence via its role in serum resistance and invasion of intestinal cells. Our data further suggest a dynamic regulation of expression of this pathway in the gastrointestinal tract.

Neuromusculature of Gyrodactylus rysavyi, a monogenean gill and skin parasite of the catfish Clarias gariepinus

Phalloidin fluorescence technique, enzyme cytochemistry and immunocytochemistry in conjunction with confocal scanning laser microscopy were used for the first time to describe the nervous and muscle systems of the viviparous monogenean parasite, Gyrodactylus rysavyi inhabiting the gills and skin of the Nile catfish Clarias gariepinus. The body wall muscles are composed of an outer layer of circular fibres, an intermediate layer of paired longitudinal fibres and an inner layer of well-spaced bands of diagonal fibres arranged in two crossed directions. The musculature of the pharynx, intestine, reproductive tract and the most prominent muscles of the haptor were also described. Two characteristic muscular pads were found lying in the anterior region of the haptor in close contact with the hamuli. To each one of these pads, a group of ventral extrinsic muscles was connected. The role of this ventral extrinsic muscle in the body movement was discussed. The mechanism operating the marginal hooklets was also discussed. The central nervous system (CNS) consists of paired cerebral ganglia from which three pairs of longitudinal ventral, lateral and dorsal nerve cords arise. The nerve cords are connected at intervals by many transverse connectives. The CNS is better developed ventrally than dorsally or laterally and it has the highest reactivity for all neuroactive substances examined. Both the central and the peripheral nervous system (PNS) are bilaterally symmetrical. Structural and functional correlates of the neuromusculature of the pharynx, haptor and reproductive tracts were explained. The results implicated acetylcholine, FMRFamide-related peptides (FaRPs) and serotonin in sensory and motor function. The results were compared with those of the monogeneans Macrogyrodactylus clarii and M. congolensis inhabiting the gills and skin respectively of the same host fish C. gariepinus.

Identification of lactic acid bacteria strains modulating incretin hormone secretion and gene expression in enteroendocrine cells

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones released from intestinal enteroendocrine (EE) cells and have well-established glucose-lowering actions. Lactic acid bacteria (LAB) colonise the human intestine, but it is unknown whether LAB and EE cells interact. Acute co-culture of LAB with EE cells showed that certain LAB strains elicit GLP-1 and GIP secretion (13-194-fold) and upregulate their gene expression. LAB-induced incretin hormone secretion did not appear to involve nutrient mechanisms, nor was there any evidence of cytolysis. Instead PCR array studies implicated signalling agents of the toll-like receptor system, e.g. adaptor protein MyD88 was decreased 23-fold and cell surface antigen CD14 was increased 17-fold. Mechanistic studies found that blockade of MyD88 triggered significant GLP-1 secretion. Furthermore, blocking of CD14 completely attenuated LAB-induced secretion. A recent clinical trial clearly shows that LAB have potential for alleviating type 2 diabetes, and further characterisation of this bioactivity is warranted.

Localization of polymorphic N-acetyltransferase (NAT2) in tissues of inbred mice

Like humans, mice exhibit polymorphism in the N-acetylation of aromatic amines, many of which are toxic and/or carcinogenic. Mice have three N-acetyltransferase (Nat) genes, Nat1, Nat2 and Nat3, and Nat2 is known to be polymorphic. There is a dramatic difference in the acetylation of NAT2 substrates by blood from fast (C57BL/6J) compared with slow acetylator (A/J) mice. However, the acetylation of these substrates by liver cytosols from the two strains is very similar. In order to determine whether the expression of the NAT2 protein corresponded with the activities measured, a polyclonal antipeptide antisera was raised against the C-terminal decapeptide of NAT2 and characterized using recombinant murine NAT2 antigen. Enzyme-linked immunosorbent assays (ELISAs) demonstrated that the anti-NAT2 antiserum bound in a concentration-dependent fashion to recombinant NAT2. Immunochemical analysis of mouse liver cytosols from C57BL/6J or A/J livers indicated that the level of NAT2 protein expressed in the two strains was similar. Immunohistochemical staining of C57BL/6J liver with anti-NAT2 antiserum showed that NAT2 was expressed in hepatocytes throughout the liver although the intensity of staining in the perivenous (centrilobular) region was higher than that in the periportal region. NAT2 was also detected in epithelial cells in the lung, kidney, bladder, small intestine and skin as well as in erythrocytes and lymphocytes in the spleen and hair follicles and sebaceous glands in the skin. Characterization of the distribution of NAT2 will be of value in elucidating the role of polymorphic N-acetylation in protecting the organism from environmental insults as well as in endogenous metabolism.

Autonomic modification of intestinal smooth muscle contractility

Intestinal smooth muscle contracts rhythmically in the absence of nerve and hormonal stimulation because of the activity of pacemaker cells between and within the muscle layers. This means that the autonomic nervous system modifies rather than initiates intestinal contractions. The practical described here gives students an opportunity to observe this spontaneous activity and its modification by agents associated with parasympathetic and sympathetic nerve activity. A section of the rabbit small intestine is suspended in an organ bath, and the use of a pressure transducer and data-acquisition software allows the measurement of tension generated by the smooth muscle of intestinal walls. The application of the parasympathetic neurotransmitter ACh at varying concentrations allows students to observe an increase in intestinal smooth muscle tone with increasing concentrations of this muscarinic receptor agonist. Construction of a concentration-effect curve allows students to calculate an EC50 value for ACh and consider some basic concepts surrounding receptor occupancy and activation. Application of the hormone epinephrine to the precontracted intestine allows students to observe the inhibitory effects associated with sympathetic nerve activation. Introduction of the drug atropine to the preparation before a maximal concentration of ACh is applied allows students to observe the inhibitory effect of a competitive antagonist on the physiological response to a receptor agonist. The final experiment involves the observation of the depolarizing effect of K+ on smooth muscle. Students are also invited to consider why the drugs atropine, codeine, loperamide, and botulinum toxin have medicinal uses in the management of gastrointestinal problems.

Physiologic indicators of vitamin A status.

Physiologic indicators reflect the functional consequences of vitamin A deficiency and may be particularly useful for detecting early perturbations in vitamin A status. In conjunctival impression cytology (CIC), epithelial morphology and the presence or absence of mucin spots and goblet cells allow samples, obtained by applying filter paper to the temporal conjunctiva, to be characterized as normal or typical of vitamin A-deficient keratinizing metaplasia. The validity of CIC has been established with reference to other indicators of vitamin A status, and a prevalence of > or =20% abnormal results has been suggested as indicative of a public health problem. However, interpretation of specimens requires considerable training, and nonresponsiveness to supplementation is a frequent problem, which limits the utility of CIC as a method for evaluating the impact of intervention programs. Several simplified field protocols for dark adaptation have been developed, including one in which dark adaptation is assessed by the responsiveness of the pupil to light. Night blind subjects have consistently shown abnormal results on this test, and a significant response to placebo-controlled dosing has been demonstrated for children and pregnant women. Scores have correlated significantly with serum retinol and relative dose response. Pupillary dark adaptation testing is acceptable to most children as young as 2 y old. Limitations of this technique include a time course for recovery after dosing as long as 4-6 wk, a testing time of 20 min, and the need for 1-3 d of training. Given its low cost, noninvasive nature, and lack of the need to transport samples, pupillary dark adaptation offers advantages over other techniques for assessing a population's vitamin A status.

Magnitude differences in bioactive compounds, chemical functional groups, fatty acid profiles, nutrient degradation and digestion, molecular structure and metabolic characteristics of protein in newly developed yellow-seeded and black-seeded canola lines.

Recently, new lines of yellow-seeded (CS-Y) and black-seeded canola (CS-B) have been developed with chemical and structural alteration through modern breeding technology. However, no systematic study was found on the bioactive compounds, chemical functional groups, fatty acid profiles, inherent structure, nutrient degradation and absorption, or metabolic characteristics between the newly developed yellow- and black-seeded canola lines. This study aimed to systematically characterize chemical, structural, and nutritional features in these canola lines. The parameters accessed include bioactive compounds and antinutrition factors, chemical functional groups, detailed chemical and nutrient profiles, energy value, nutrient fractions, protein structure, degradation kinetics, intestinal digestion, true intestinal protein supply, and feed milk value. The results showed that the CS-Y line was lower (P ≤ 0.05) in neutral detergent fiber (122 vs 154 g/kg DM), acid detergent fiber (61 vs 99 g/kg DM), lignin (58 vs 77 g/kg DM), nonprotein nitrogen (56 vs 68 g/kg DM), and acid detergent insoluble protein (11 vs 35 g/kg DM) than the CS-B line. There was no difference in fatty acid profiles except C20:1 eicosenoic acid content (omega-9) which was in lower in the CS-Y line (P < 0.05) compared to the CS-B line. The glucosinolate compounds differed (P < 0.05) in terms of 4-pentenyl, phenylethyl, 3-CH3-indolyl, and 3-butenyl glucosinolates (2.9 vs 1.0 μmol/g) between the CS-Y and CS-B lines. For bioactive compounds, total polyphenols tended to be different (6.3 vs 7.2 g/kg DM), but there were no differences in erucic acid and condensed tannins with averages of 0.3 and 3.1 g/kg DM, respectively. When protein was portioned into five subfractions, significant differences were found in PA, PB1 (65 vs 79 g/kg CP), PB2, and PC fractions (10 vs 33 g/kg CP), indicating protein degradation and supply to small intestine differed between two new lines. In terms of protein structure spectral profile, there were no significant differences in functional groups of amides I and II, α helix, and β-sheet structure as well as their ratio between the two new lines, indicating no difference in protein structure makeup and conformation between the two lines. In terms of energy values, there were significant differences in total digestible nutrient (TDN; 149 vs 133 g/kg DM), metabolizable energy (ME; 58 vs 52 MJ/kg DM), and net energy for lactation (NEL; 42 vs 37 MJ/kg DM) between CS-Y and CS-B lines. For in situ rumen degradation kinetics, the two lines differed in soluble fraction (S; 284 vs 341 g/kg CP), potential degradation fraction (D; 672 vs 590 g/kg CP), and effective degraded organic matter (EDOM; 710 vs 684 g/kg OM), but no difference in degradation rate. CS-Y had higher digestibility of rumen bypass protein in the intestine than CS-B (566 vs 446 g/kg of RUP, P < 0.05). Modeling nutrient supply results showed that microbial protein synthesis (MCP; 148 vs 171 g/kg DM) and rumen protein degraded balance (DPB; 108 vs 127 g/kg DM) were lower in the CS-Y line, but there were no differences in total truly digested protein in small intestine (DVE) and feed milk value (FMV) between the two lines. In conclusion, the new yellow line had different nutritional, chemical, and structural features compared to the black line. CS-Y provided better nutrient utilization and availability.

Mercury in feral fish: distribution, accumulation and toxicity

O principal objectivo desta dissertação foi estudar a acumulação de mercúrio em vários tecidos de peixes marinhos, a sua relação com factores biológicos e as respectivas respostas bioquímicas. O trabalho realizado permitiu obter novos conhecimentos sobre a acumulação de mercúrio em peixes, possibilitando avaliar a influência da biodisponibilidade do elemento e as suas possíveis implicações no ambiente. O trabalho foi desenvolvido na Ria de Aveiro (Portugal), uma zona costeira onde existe um gradiente ambiental de mercúrio, o que oferece a oportunidade de estudar a sua acumulação e os seus efeitos tóxicos em condições realísticas. As amostragens foram efectuadas em dois locais considerados críticos em termos de contaminação por mercúrio – Largo do Laranjo (L1 e L2) e num local afastado da principal fonte de poluição, usado como termo de comparação (Referência; R); L1 e L2 corresponderam a locais moderadamente e altamente contaminados, respectivamente. Foram escolhidos juvenis de duas espécies ecologicamente diferentes e representativas da comunidade piscícola local, a tainha garrento (Liza aurata) e o robalo (Dicentrarchus labrax). Em cada local foram recolhidas amostras de água e de sedimento para determinação de mercúrio. Foram quantificadas as concentrações de mercúrio total (T-Hg) e orgânico (O-Hg) em vários tecidos dos peixes, escolhidos tendo em conta a sua função relativamente à toxicocinética e toxicodinâmica de metais. As respostas antioxidantes (Catalase- CAT, glutationa peroxidase- GPx, glutationa reductase- GR, glutationa –S-transferase- GST e conteúdo em glutationa total- GSHt), o dano peroxidativo (LPO) e o conteúdo em metalotioninas (MTs) foram também avaliados. A acumulação de T-Hg foi semelhante para as duas espécies de peixes estudadas, embora D. labrax tenha apresentado concentrações tendencialmente maiores. Ambas as espécies demonstraram capacidade de reflectir o grau de contaminação ambiental existente, indicando claramente que a acumulação depende da concentração ambiental. A acumulação revelou-se específica de cada tecido. O padrão da acumulação em L. aurata foi rim > fígado > músculo > cérebro > guelras > sangue e em D. labrax foi fígado > rim > músculo > cérebro ≈ guelras > sangue. Relativamente à acumulação de OHg, verificou-se que D. labrax exibiu concentrações mais elevadas que L. aurata. Todos os tecidos foram capazes de reflectir diferenças entre R e L2. Os níveis de O-Hg no fígado, músculo e nos conteúdos intestinais foram diferentes entre espécies, sendo mais elevados para D. labrax. As guelras e o intestino foram os tecidos onde se obtiveram os valores mais baixos de O-Hg e observaram-se valores idênticos para as duas espécies. Com excepção das guelras, as concentrações de O-Hg variaram em função do valor observado nos conteúdos intestinais, indicando que a alimentação é a via dominante da acumulação. As concentrações de O-Hg nos conteúdos intestinais revelaram ser uma informação relevante para prever a acumulação de O-Hg nos tecidos, pois verificou-se uma razão praticamente constante entre o teor de mercúrio no fígado, no músculo e nos conteúdos intestinais. A percentagem de O-Hg no músculo e no fígado variou de acordo com o grau de contaminação ambiental e com o tipo de assimilação preferencial do elemento (alimentação vs. água), sugerindo que o fígado exerce um papel protector em relação à acumulação de mercúrio nos outros órgãos. Ambas as espécies de peixes demonstraram ser boas sentinelas da contaminação ambiental com mercúrio (T-Hg e O-Hg), sendo o cérebro e o músculo os tecidos que melhor reflectiram o grau de acumulação com o elemento. A análise conjunta dos dados de bioacumulação e de respostas ao stress oxidativo permitiram estabelecer uma relação entre as concentrações de mercúrio nas guelras, fígado, rim e cérebro e a sua toxicidade. As respostas do cérebro aos efeitos tóxicos do mercúrio revelaram ser específicas de cada espécie. Enquanto que para o cérebro de L. aurata se verificou um decréscimo de todos os parâmetros antioxidantes estudados nos locais contaminados, sem haver evidência de qualquer mecanismo compensatório, no D. labrax observaram-se respostas ambivalentes, que indicam por um lado a activação de mecanismos adaptativos e, por outro, o decréscimo das respostas antioxidantes, ou seja, sinais de toxicidade. Embora em ambas as espécies de peixe fosse evidente uma condição pró-oxidante, o cérebro parece possuir mecanismos compensatórios eficientes, uma vez que não se verificou peroxidação lipídica. As respostas antioxidantes do cérebro de D. labrax foram comparadas em diferentes períodos do ano - quente vs. frio. O período quente mostrou ser mais crítico, uma vez que no período frio não se verificaram diferenças nas respostas entre locais, ou seja, a capacidade antioxidante do cérebro parece ser influenciada pelos factores ambientais. As guelras revelaram susceptibilidade à contaminação por mercúrio, uma vez que se verificou uma tendência para o decréscimo da actividade de CAT em L2 e ausência de indução em L1. O fígado e o rim demonstraram mecanismos adaptativos face ao grau de contaminação moderada (L1), evidenciados pelo aumento de CAT. O rim também demonstrou adaptabilidade face ao grau elevado de contaminação (L2), uma vez que se verificou um aumento GST. Embora o grau de susceptibilidade tenha sido diferente entre os órgãos, não se verificou peroxidação lipídica em nenhum. A determinação do conteúdo em MTs em D. labrax e em L. aurata revelou que este parâmetro depende não só da espécie, mas também do tecido em causa. Assim, em D. labrax foi observado um decréscimo de MTs no cérebro, bem como a incapacidade de síntese de MTs no sangue, guelras, fígado, rim e músculo. Em L. aurata observou-se um aumento do conteúdo em MTs no fígado e no músculo. Estes resultados indicam que a aplicabilidade das MTs como biomarcador de exposição ao mercúrio parece ser incerta, revelando limitações na capacidade de reflectir os níveis de exposição ao metal e por consequência o grau de acumulação. Este trabalho comprova a necessidade de se integrarem estudos de bioacumulação com biomarcadores de efeitos, de modo a reduzir os riscos de interpretações erróneas, uma vez que as respostas nem sempre ocorrem para os níveis mais altos de contaminação ambiental com mercúrio.