Insights into growth kinetics and roles of enzymes of Krebs' cycle and sulfur oxidation during exochemolithoheterotrophic growth of Achromobacter aegrifaciens NCCB 38021 on succinate with thiosulfate as the auxiliary electron donor.

Achromobacter aegrifaciens NCCB 38021 was grown heterotrophically on succinate versus exochemolithoheterotrophically on succinate with thiosulfate as auxiliary electron donor. In batch culture, no significant differences in specific molar growth yield or specific growth rate were found for the two growth conditions, but in continuous culture in the succinate-limited chemostat, the maximum specific growth yield coefficient increased by 23.3% with thiosulfate present, consistent with previous studies of endo- and exochemolithoheterotrophs and thermodynamic predictions. Thiosulfate oxidation was coupled to respiration at cytochrome c551, and thiosulfate-dependent ATP biosynthesis occurred. Specific activities of cytochrome c-linked thiosulfate dehydrogenase (E.C. 1.8.2.2) and two other enzymes of sulfur metabolism were significantly higher in exochemolithoheterotrophically grown cell extracts, while those of succinyl-transferring 2-oxoglutarate dehydrogenase (E.C. 1.2.4.2), fumarate hydratase (E.C. 4.2.1.2) and malate dehydrogenase (NAD+, E.C. 1.1.1.37) were significantly lower-presumably owing to less need to generate reducing equivalents during Krebs' cycle, since they could be produced from thiosulfate oxidation.

Pharmaceutical Metabolism in Fish: Using a 3-D Hepatic In Vitro Model to Assess Clearance.

At high internal doses, pharmaceuticals have the potential for inducing biological/pharmacological effects in fish. One particular concern for the environment is their potential to bioaccumulate and reach pharmacological levels; the study of these implications for environmental risk assessment has therefore gained increasing attention. To avoid unnecessary testing on animals, in vitro methods for assessment of xenobiotic metabolism could aid in the ecotoxicological evaluation. Here we report the use of a 3-D in vitro liver organoid culture system (spheroids) derived from rainbow trout to measure the metabolism of seven pharmaceuticals using a substrate depletion assay. Of the pharmaceuticals tested, propranolol, diclofenac and phenylbutazone were metabolised by trout liver spheroids; atenolol, metoprolol, diazepam and carbamazepine were not. Substrate depletion kinetics data was used to estimate intrinsic hepatic clearance by this spheroid model, which was similar for diclofenac and approximately 5 fold higher for propranolol when compared to trout liver microsomal fraction (S9) data. These results suggest that liver spheroids could be used as a relevant and metabolically competent in vitro model with which to measure the biotransformation of pharmaceuticals in fish; and propranolol acts as a reproducible positive control.

DNA double-strand breaks in incubating female common eiders (Somateria mollissima): Comparison between a low and a high polluted area.

Alterations in the genetic material may have severe consequences for individuals and populations. Hence, genotoxic effects of environmental exposure to pollutants are of great concern. We assessed the impact of blood concentrations of persistent organic pollutants (POPs) and mercury (Hg) on DNA double-strand break (DSB) frequency, in blood cells of a high-exposed Baltic, and lower exposed Arctic population of common eiders (Somateria mollissima). Furthermore, we examined whether the genotoxic response was influenced by antioxidant concentration (plasma total glutathione (tGSH) and total antioxidant capacity) and female body mass. The DNA DSB frequency did not differ between the two populations. We found significant positive relationships between Hg and DNA DSB frequency in Baltic, but not in Arctic eiders. Although both p,p'-DDE and PCB 118 had a lesser effect than Hg, they exhibited a positive association with DNA DSB frequency in Baltic eiders. Antioxidant levels were not important for the genotoxic effect, suggesting alternative mechanisms other than GSH depletion for the relationship between Hg and DNA DSBs. Hence, the Baltic population, which is considered to be endangered and is under the influence of several environmental stressors, may be more susceptible to genotoxic effects of environmental exposure to Hg than the Arctic population.

Physiological niche and geographical range in European diving beetles (Coleoptera: Dytiscidae).

Geographical ranges vary greatly in size and position, even within recent clades, but the factors driving this remain poorly understood. In aquatic beetles, thermal niche has been shown to be related to both the relative range size and position of congeners but whether other physiological parameters play a role is unknown. Metabolic plasticity may be critical for species occupying more variable thermal environments and maintaining this plasticity may trade-off against other physiological processes such as immunocompetence. Here we combine data on thermal physiology with measures of metabolic plasticity and immunocompetence to explore these relationships in Deronectes (Dytiscidae). While variation in latitudinal range extent and position was explained in part by thermal physiology, aspects of metabolic plasticity and immunocompetence also appeared important. Northerly distributed, wide-ranging species apparently used different energy reserves under thermal stress from southern endemic congeners and differed in their antibacterial defences. This is the first indication that these processes may be related to geographical range, and suggests parameters that may be worthy of exploration in other taxa.

Antioxidant Responses in Relation to Persistent Organic Pollutants and Metals in a Low- and a High-Exposure Population of Seabirds.

Oxidative stress occurs when there is an imbalance between the production of reactive oxygen species (ROS) and antioxidant defense. Exposure to pollutants may increase ROS and affect antioxidant levels, and the resulting oxidative stress may negatively affect both reproduction and survival. We measured concentrations of 18 persistent organic pollutants (POPs) and 9 toxic elements in blood, as well as total antioxidant capacity (TAC), total glutathione (tGSH), and carotenoids in plasma of Baltic and Arctic female common eiders (Somateria mollissima) (N = 54) at the end of their incubation-related fasting. The more polluted Baltic population had higher TAC and tGSH concentrations compared to the Arctic population. Carotenoid levels did not differ between populations. The effect of mixtures of pollutants on the antioxidants was assessed, and the summed molar blood concentrations of 14 POPs were positively related to TAC. There was no significant relationship between the analyzed pollutants and tGSH concentrations. The adaptive improvement of the antioxidant defense system in the Baltic population may be a consequence of increased oxidative stress. However, both increased oxidative stress and energy allocation toward antioxidant defense may have adverse consequences for Baltic eiders at the incubation stage, when energy resources reach an annual minimum due to incubation-related fasting.

Hyperbaric oxygen enhances neutrophil apoptosis and their clearance by monocyte-derived macrophages.

Neutrophil apoptosis and clearance by macrophages are essential for wound healing. Evidence suggests that hyperbaric oxygen (HBO) exposure may enhance neutrophil apoptosis, but HBO effects leading to neutrophil clearance by macrophages are still unclear. In the current study, bovine neutrophils and monocyte-derived macrophages (MDMΦ) were co-cultured under HBO (97.9% O2, 2.1% CO2 at 2.4 atm absolute (ATA)) (1 atm = 101.325 kPa), hyperbaric normoxia (8.8% O2 at 2.4 ATA), normobaric hyperoxia (95% O2, 5% CO2), normoxia (air), and normobaric hypoxia (5% O2, 5% CO2). Phagocytosis of fresh and 22 h aged neutrophils by MDMΦ was increased after HBO pre-treatment, assessed using flow cytometry and light microscopy. Enhanced clearance of neutrophils was accompanied by an increase in H2O2 levels following HBO pre-treatment with upregulation of IL-10 (anti-inflammatory cytokine) mRNA expression in LPS-stimulated MDMΦ that had ingested aged neutrophils. TNF-α (pro-inflammatory cytokine) gene expression did not change in LPS-stimulated MDMΦ that had ingested fresh or aged neutrophils after HBO, pressure, and hyperoxia. These findings suggest that HBO-activated MDMΦ participate in the clearance of apoptotic cells. Uptake of neutrophils by MDMΦ exposed to HBO may contribute to resolution of inflammation, because HBO induced up-regulation of IL-10 mRNA expression.

Non-transferrin-bound iron is associated with biomarkers of oxidative stress, inflammation and endothelial dysfunction in type 2 diabetes.

AIMS: To investigate the association between circulating non-transferrin-bound iron [NTBI], and markers of oxidative stress, endothelial function and inflammation in subjects with type 2 diabetes and non-diabetic subjects with varying degrees of obesity. METHODS: Plasma NTBI was measured by HPLC, together with total iron, iron-binding capacity, transferrin saturation and soluble transferrin receptor, together with total and reduced ascorbate, malondialdehyde [MDA], E-selectin and high-sensitivity c-reactive protein [hs-CRP] in groups of 28 subjects with type 2 diabetes, 28 non-obese controls and 17 obese non-diabetic subjects. RESULTS: Levels of NTBI were higher than controls in the diabetes group, but the total serum iron levels were lower. MDA levels were higher than controls in both the diabetes and obese groups, and this was associated with higher levels of oxidised ascorbate. hs-CRP levels were higher in both the diabetes and obese groups, and E-selectin was significantly higher in the diabetes group. There were strong positive correlations between HbA1c levels and NTBI [P<0.01], HbA1c and E-selectin [P<0.001] and NTBI and E-selectin [P<0.02] in the diabetes group. CONCLUSION: These results support the hypothesis that iron-mediated oxidative stress may be a mechanism linking poor glycaemic control with vascular dysfunction in type 2 diabetes.

A novel field transplantation technique reveals intra-specific metal-induced oxidative responses in strains of Ectocarpus siliculosus with different pollution histories.

A novel field transplantation technique, in which seaweed material is incorporated into dialysis tubing, was used to investigate intra-specific responses to metals in the model brown alga Ectocarpus siliculosus. Metal accumulation in the two strains was similar, with higher concentrations in material deployed to the metal-contaminated site (Ventanas, Chile) than the pristine site (Quintay, Chile). However, the oxidative responses differed. At Ventanas, strain Es147 (from low-polluted site) underwent oxidative damage whereas Es524 (from highly polluted site) was not affected. Concentrations of reduced ascorbate (ASC) and reduced glutathione (GSH) were significantly higher in Es524. Activities of the antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR) all increased in Es524, whereas only SOD increased in Es147. For the first time, employing a field transplantation technique, we provide unambiguous evidence of inter-population variation of metal-tolerance in brown algae and establish that antioxidant defences are, in part, responsible.

Copper-induced intra-specific oxidative damage and antioxidant responses in strains of the brown alga Ectocarpus siliculosus with different pollution histories.

Inter- and intra-specific variation in metal resistance has been observed in the ecologically and economically important marine brown macroalgae (Phaeophyceae), but the mechanisms of cellular tolerance are not well elucidated. To investigate inter-population responses of brown seaweeds to copper (Cu) pollution, the extent of oxidative damage and antioxidant responses were compared in three strains of the filamentous brown seaweed Ectocarpus siliculosus, the model organism for the algal class Phaeophyceae that diverged from other major eukaryotic groups over a billion year ago. Strains isolated from locations with different pollution histories (i.e. LIA, from a pristine site in Scotland; REP and Es524 from Cu-contaminated sites in England and Chile, respectively) were exposed to total dissolved Cu concentrations (CuT) of up to 2.4 µM (equivalent to 128 nM Cu(2+)) for 10 d. LIA exhibited oxidative stress, with increases in hydrogen peroxide (H2O2) and lipid peroxidation (measured as TBARS levels), and decreased concentrations of photosynthetic pigments. Es524 presented no apparent oxidative damage whereas in REP, TBARS increased, revealing some level of oxidative damage. Adjustments to activities of enzymes and antioxidant compounds concentrations in Es524 and REP were strain and treatment dependent. Mitigation of oxidative stress in Es524 was by increased activities of superoxide dismutases (SOD) at low CuT, and catalase (CAT) and ascorbate peroxidase (APX) at all CuT, accompanied by higher levels of antioxidants (ascorbate, glutathione, phenolics) at higher CuT. In REP, only APX activity increased, as did the antioxidants. For the first time evidence is presented for distinctive oxidative stress defences under excess Cu in two populations of a species of brown seaweed from environments contaminated by Cu.

Comparison of intermittent and continuous exposures to inorganic mercury in the mussel, Mytilus edulis: accumulation and sub-lethal physiological effects.

Aquatic organisms are often subject to intermittent exposure to pollutants in real ecosystems. This study aimed to compare mercury accumulation and the physiological responses of mussels, Mytilus edulis during continuous and intermittent exposure to the metal. Mussels were treated in a semi-static, triplicated design to either a control (no added Hg) or 50 µg l(-1) Hg as HgCl2 in continuous (daily) or intermittent (2 day exposure, 2 days in clean seawater alternately) exposure for 14 days. A time-dependent increase in Hg accumulation was observed in the continuous exposure, while the intermittent treatment showed step-wise changes in Hg concentrations with the exposure profile, especially in the gills. At the end of the experiment, tissue Hg concentrations were significantly increased in the continuous compared to the intermittent exposure for digestive gland (4 fold), gonad and remaining soft tissue (>2 fold), but not for the gill and adductor muscle. There was no observed oxidative damage at the end of the experiment as measured by the thiobarbituric acid reactive substances (TBARS) concentrations in tissues from all treatments. However, total glutathione was significantly decreased in the gill and digestive gland of both the continuous and intermittent exposure by the end of the experiment. The neutral red retention ability of the haemocytes was not affected, but total haemocyte counts were significantly decreased (<2 fold) in the intermittent compared to the continuous exposure. Histopathological examinations showed less pathology in the gill, but more inflammation in the digestive gland of mussels for the intermittent compared to the continuous exposure. Overall, the results showed that Hg accumulation from intermittent exposure was less than that of the continuous exposure regime, but the sub-lethal responses are sometimes more severe than expected in the former.

Large changes in NAD levels associated with CD38 expression during HL-60 cell differentiation.

NAD is an important cofactor involved in multiple metabolic reactions and as a substrate for several NAD-dependent signalling enzymes. One such enzyme is CD38 which, alongside synthesising Ca(2+)-releasing second messengers and acting as a cell surface receptor, has also been suggested to play a key role in NAD(+) homeostasis. CD38 is well known as a negative prognostic marker in B-CLL but the role of its enzymatic activity has not been studied in depth to date. We have exploited the HL-60 cell line as a model of inducible CD38 expression, to investigate CD38-mediated regulation intracellular NAD(+) levels and the consequences of changes in NAD(+) levels on cell physiology. Intracellular NAD(+) levels fell with increasing CD38 expression and this was reversed with the CD38 inhibitor, kuromanin confirming the key role of CD38 in NAD(+) homeostasis. We also measured the consequences of CD38 expression during the differentiation on a number of functions linked to NAD(+) and we show that some but not all NAD(+)-dependent processes are significantly affected by the lowered NAD(+) levels. These data suggest that both functional roles of CD38 might be important in the pathogenesis of B-CLL.

Effects of hyperoxia on the permeability of 16HBE14o- cell monolayers--the protective role of antioxidant vitamins E and C.

The use of hyperoxia for critically ill patients is associated with adverse impacts resulting in lung injury accompanied by inflammation. The aim of this study was to evaluate aspects of mechanisms that contribute to hyperoxia-induced disruption of the epithelial permeability barrier, and also the protective effects of the antioxidants α-tocopherol and ascorbate. 16HBE14o- cells were cultured as monolayers at an air-liquid interface for 6 days, after which transepithelial electrical resistance reached 251.2 ± 4.1 Ω.cm(2) (mean ± standard error of the mean). They were then exposed for 24 h to normoxia (21% O2, 5% CO2), hyperoxia (95% O2, 5% CO2), hyperoxia with 10(-7) M α-tocopherol, hyperoxia with 10(-7) M ascorbate, hyperoxia with 10(-6) M ascorbate, and hyperoxia with a combination of α-tocopherol and ascorbate (10(-7) M and 10(-6) M, respectively). Significant reductions (P < 0.05) in transepithelial electrical resistance seen after hyperoxia (with or without antioxidants) were associated with reductions in the levels of zona occludens-1 (ZO-1) observed by immunohistochemistry, and downregulation of ZO-1 expression (P < 0.01) as compared with normoxia. In contrast, the expression levels of interleukin (IL)-8, IL-6 and tumour necrosis factor-α (TNF-α) were increased after hyperoxia (P < 0.01), and marked increases in the levels of these cytokines (ELISA) were seen in the medium (P < 0.001) as compared with normoxia. The antioxidant vitamins E and C had a partial protective effect against the hyperoxia-induced reduction in ZO-1 levels and the increase in levels of the proinflammatory cytokines IL-8, IL-6, and TNF-α. In conclusion, hyperoxia-induced epithelial disruption is associated with tight junction weakening, and induction of a proinflammatory environment.

Effects of hyperbaric oxygen treatment on antimicrobial function and apoptosis of differentiated HL-60 (neutrophil-like) cells.

AIMS: Neutrophil apoptosis is important in the resolution of inflammation in chronic wounds. Hyperbaric oxygen (HBO) therapy, an intermittent inhalation of 100% oxygen at greater than atmospheric pressure, appears to be an effective treatment for chronic wounds. The aim was to use HL-60 cells differentiated using all-trans retinoic acid (ATRA) (neutrophil-like cells) to test the hypothesis that an HBO-induced increase in antimicrobial activity might lead to an increase in apoptosis, thereby contributing to neutrophil clearance from chronic wounds. MAIN METHODS: ATRA differentiated HL-60 cells, an in vitro neutrophil model, were used to test the effects of normoxia, hypoxia (5% O2), hyperoxia (95% O2), hyperbaric normoxia (pressure) (8.8% O2 at 2.4 ATA) and HBO (97.9% O2 at 2.4 ATA) on antimicrobial function [NBT staining, superoxide and H2O2 production, and phagocytosis activity] and apoptosis (caspase 3/7 activity and morphological changes observed using SYBR Safe staining). KEY FINDINGS: A single 90min HBO exposure caused an increase in the respiratory burst activity of neutrophil-like cells post exposure. Phagocytosis of Staphylococcus aureus was also increased. HBO pre-treatment had a pro-apoptotic effect, increasing caspase 3/7 activity and causing morphological changes associated with apoptosis. SIGNIFICANCE: The potential detrimental effect of enhanced antimicrobial activity induced by HBO may be offset by enhanced apoptosis. Both hyperoxia and pressure alone seemed to contribute to the HBO-induced increases in antimicrobial activity and apoptosis, although there was no consistent pattern. These data contribute to explaining the effectiveness of HBO in the treatment of chronic wounds.

Comparison of intermittent and continuous exposures to cadmium in the blue mussel, Mytilus edulis: accumulation and sub-lethal physiological effects.

Little is known about the bioaccumulation responses of shellfish to metals during intermittent compared to continuous exposure. There is also the concern that the toxicity of intermittent events may not be the same as that from the steady-state continuous exposures. The aim of the present study was to determine whether there was any difference between cadmium (Cd) accumulation, or Cd-dependent biological responses, in tissues of blue mussels (Mytilus edulis) during intermittent compared to continuous Cd exposure. Tissues and hemolymph were collected from M. edulis exposed for 14 days to either control (no added Cd, only seawater), or 50 µg/l Cd as CdCl2 in continuous or intermittent profile (2 day exposure, 2 days in clean seawater alternately); and sub-lethal responses examined using a suite of assays including total glutathione, thiobarbituric acid reactive substances (TBARS), neutral red retention, total hemocyte counts, hemolymph Na(+) and K(+), plasma glucose and histopathology. A time-dependent accumulation of the Cd was observed in tissues of mussels after continuous exposure, while the intermittent exposure showed step-wise changes in the hemolymph and gonad. Tissue Cd concentration in the continuous exposure was significantly increased (≥2 fold) for most tissues compared to the intermittent exposure. No clear differences were seen between the continuous and intermittent exposure for most end points measured apart from a 2 fold significant increase in hemocyte infiltration in the digestive gland of the continuous exposure compared to the intermittent exposure. Overall, the data showed that the Cd accumulation was generally greater in the continuous exposure regime, but despite this, most of the biological responses being similar in both regimes.

Growth performance and starch utilization in common carp (Cyprinus carpio L.) in response to dietary chromium chloride supplementation.

A nutrition trial was conducted on juvenile common carp (Cyprinus carpio), initial mean body weight 15 ± 0.4 g within a controlled facility at 25 ± 0.5°C. Six diets containing various levels of supplementary Cr (0, 0.2, 0.5, 1.0, 1.5, and 2.0) mg Cr/kg of diet as Cr chloride hexahydrate were fed to carp for a period of 10 weeks. Lower growth performance was observed in fish fed on the control diet and the diet supplemented with the highest level of Cr (2.0mg Cr/kg). Although fish fed 0.5mg Cr/kg showed the best growth performance, this was not significantly different (P>0.05) from fish fed 1.0mg Cr/kg. The regression of plasma glucose concentration was linear (R(2)=0.97 and P value=0.001) as the Cr content of the diet increased (up to 1.5mg Cr/kg). Cr carcass content was elevated with an increasing level of dietary Cr supplementation up to 1.5mg Cr/kg; but fish fed on the diet supplemented with the highest level of Cr (2.0mg Cr/kg) showed a decrease in Cr carcass content. Histological examination to evaluate the impact of different Cr supplementation on liver and gut tissues showed notable changes. The higher level of Cr (2.0mg Cr/kg) in the diet gave rise to elevated hepatocyte vacuolization and changes in gut tissue morphology. It appeared that Cr chloride significantly improved growth within a defined range (0.2-1.5) mg Cr/kg without any negative impact, while 2.0mg Cr/kg in carp diet seems to be the threshold for the initiation of toxicity.

Hyperoxia-induced ciliary loss and oxidative damage in an in vitro bovine model: the protective role of antioxidant vitamins E and C.

Although elevated oxygen fraction is used in intensive care units around the world, pathological changes in pulmonary tissue have been shown to occur with prolonged exposure to hyperoxia. In this work a bovine bronchus culture model has been successfully used to evaluate the effects of hyperoxia on ciliated epithelium in vitro. Samples were cultured using an air interface method and exposed to normoxia, 21% O(2) or hyperoxia, 95% O(2). Cilial coverage was assessed using scanning electron microscopy (SEM). Tissue damage (lactate dehydrogenase, LDH, in the medium), lipid peroxidation (thiobarbituric acid reactive substances, TBARS), DNA damage (comet assay), protein oxidation (OxyBlot kit) and antioxidant status (total glutathione) were used to assess whether the hyperoxia caused significant oxidative stress. Hyperoxia caused a time-dependent decline (t(½)=3.4d compared to 37.1d under normoxia) in cilial coverage (P<0.0001). This was associated with a significant increase in the number of cells (2.80 ± 0.27 × 10(6) compared to 1.97 ± 0.23 × 10(6)ml(-1) after 6d), many apparently intact, in the medium (P<0.05); LDH release (1.06 ± 0.29 compared to 0.83 ± 0.36 µmol min(-1)g(-1) after 6d; P<0.001); lipid peroxidation (352 ± 16 versus 247 ± 11 µmol MDA g(-1) for hyperoxia and normoxia, respectively); % tail DNA (18.7 ± 2.2 versus 11.1 ± 1.5); protein carbonyls (P<0.05); and total glutathione (229 ± 20 µmol g(-1) versus 189 ± 15 µmol g(-1)). Vitamins E (10(-7)M) and C (10(-6) or 10(-7)M) alone or in combination (10(-7)M and 10(-6)M, respectively) had a significant protective effect on the hyperoxia-induced reduction in percentage cilial coverage (P<0.05). In conclusion, hyperoxia caused damage to cultured bovine bronchial epithelium and denudation of cilia. The antioxidant vitamins E and C significantly protected against hyperoxia-induced cilia loss.

Merging nano-genotoxicology with eco-genotoxicology: an integrated approach to determine interactive genotoxic and sub-lethal toxic effects of C(60) fullerenes and fluoranthene in marine mussels, Mytilus sp.

Whilst there is growing concern over the potential detrimental impact of engineered nanoparticles (ENPs) on the natural environment, little is known about their interactions with other contaminants. In the present study, marine mussels (Mytilus sp.) were exposed for 3 days to C(60) fullerenes (C(60); 0.10-1 mg l(-1)) and a model polycyclic aromatic hydrocarbon (PAH), fluoranthene (32-100 µg l(-1)), either alone or in combination. The first two experiments were conducted by exposing the organisms to different concentrations of C(60) and fluoranthene alone, in order to determine the effects on total glutathione levels (as a measure of generic oxidative stress), genotoxicity (DNA strand breaks using Comet assay in haemocytes), DNA adduct analyses (using (32)P-postlabelling method) in different organs, histopathological changes in different tissues (i.e. adductor muscle, digestive gland and gills) and physiological effects (feeding or clearance rate). Subsequently, in the third experiment, a combined exposure of C(60) plus fluoranthene (0.10 mg l(-1) and 32 µg l(-1), respectively) was carried out to evaluate all endpoints mentioned above. Both fluoranthene and C(60) on their own caused concentration-dependent increases in DNA strand breaks as determined by the Comet assay. Formation of DNA adducts however could not be detected for any exposure conditions. Combined exposure to C(60) and fluoranthene additively enhanced the levels of DNA strand breaks along with a 2-fold increase in the total glutathione content. In addition, significant accumulation of C(60) was observed in all organs, with highest levels in digestive gland (24.90 ± 4.91µg C(60) g(-1) ww). Interestingly, clear signs of abnormalities in adductor muscle, digestive gland and gills were observed by histopathology. Clearance rates indicated significant differences compared to the control with exposure to C(60), and C(60)/fluoranthene combined treatments, but not after fluoranthene exposure alone. This study demonstrated that at the selected concentrations, both C(60) and fluoranthene evoke toxic responses and genetic damage. The combined exposure produced enhanced damage with additive rather than synergistic effects.

Screening microorganisms for insulin binding reveals binding by Burkholderia multivorans and Burkholderia cenocepacia and novel attachment of insulin to Aeromonas salmonicida via the A-layer.

Exposure to microorganisms is considered an environmental factor that can contribute to Type 1 diabetes. Insulin-binding proteins (IBPs) on microorganisms may induce production of antibodies that can react with the human insulin receptor (HIR) with possible consequences in developing a diabetic autoimmune response against HIR and insulin. The interaction of insulin with microorganisms was studied by screening 45 microbial species for their ability to bind insulin. Binding assays were performed using labelled insulin to identify insulin-binding components on the microorganisms. Burkholderia multivorans and Burkholderia cenocepacia isolated from patients with cystic fibrosis (CF) and the fish pathogen Aeromonas salmonicida were the only strains of those tested, which showed insulin-binding components on their cell surfaces. Further work with A. salmonicida suggested that the insulin-binding activity of A. salmonicida is due to the A-layer. A mutant of A. salmonicida lacking the A-layer showed binding, but at a much reduced rate suggesting another insulin-binding component in addition to the high affinity of the A-protein. Soluble protein lysates were subjected to Western ligand blotting using peroxidase-labelled insulin to detect IBPs. Two positive IBPs were apparent at approximately 30 and 20 kDa in lysates from Burkholderia strains, but no IBP was detected in A. salmonicida lysates.

Effects of the microbial secondary metabolites pyrrolnitrin, phenazine and patulin on INS-1 rat pancreatic ß-cells.

The effects on pancreatic ß-cell viability and function of three microbial secondary metabolites pyrrolnitrin, phenazine and patulin were investigated, using the rat clonal pancreatic ß-cell line, INS-1. Cells were exposed to 10-fold serial dilutions (range 0-10 µg mL(-1)) of the purified compounds for 2, 24 and 72 h. After 2 h exposure, only patulin (10 µg mL(-1)) was cytotoxic. All compounds showed significant cytotoxicity after 24 h. None of the compounds altered insulin secretion with 2 and 20 mM glucose after 2 h. However, after 24 h treatment, phenazine and pyrrolnitrin (10 and 100 ng mL(-1)) potentiated insulin production and glucose-stimulated insulin secretion, whereas patulin had no effect. Exposure (24 h) to either phenazine (100 ng mL(-1)) or pyrrolnitrin (10 ng mL(-1)) caused similar increases in the Ca(2+) content of INS-1 cells. The outward membrane current was inhibited after 24 h exposure to either phenazine (100 ng mL(-1)) or pyrrolnitrin (10 or 100 ng mL(-1)). This study presents novel data suggesting that high concentrations of pyrrolnitrin and phenazine are cytotoxic to pancreatic ß-cells and thus possibly diabetogenic, whereas at lower concentrations these agents are nontoxic and may be insulinotropic. The possible role of such agents in the development of cystic fibrosis-related diabetes is discussed.

The effect of material choice on biofilm formation in a model warm water distribution system.

Water distribution systems (WDS) are composed of a variety of materials and may harbour potential pathogens within surface-attached microbial biofilms. Biofilm formation on four plumbing materials, viz. copper, stainless steel 316 (SS316), ethylene propylene diene monomer (EPDM) and cross-linked polyethylene (PEX), was investigated using scanning electron microscope (SEM)/confocal microscopy, ATP-/culture-based analysis, and molecular analysis. Material 'inserts' were incorporated into a mains water fed, model WDS. All materials supported biofilm growth to various degrees. After 84 days, copper and SS316 showed no significant overall differences in terms of the level of biofilm formation observed, whilst PEX supported a significantly higher level of biofilm. EPDM exhibited gross contamination by a complex, multispecies biofilm, at a level significantly higher than was observed on the other materials, regardless of the analytical method used. PCR-DGGE analysis showed clear differences in the composition of the biofilm community on all materials after 84 days. The primary conclusion of this study has been to identify EPDM as a potentially unsuitable material for use as a major component in WDS.