Identification of newly recognized serotype 1c as the most prevalent Shigella flexneri serotype in northern rural Vietnam.

We investigated the identity of 37 Shigella flexneri strains that had previously been isolated from northern rural Vietnam (Son Tay Province) and described as untypable. Twenty-four isolates reacted with MASF 1c, a monoclonal antibody specific for S. flexneri serotype 1c. A further ten untypable isolates were found to be rough mutants (no longer expressing O-antigen) that were derived from serotype 1c strains. Pulsed-field gel electrophoresis demonstrated that these strains consisted of many different clones, indicating serotype 1c was well established in this region in the late 1990s. Serotype 1c was the most prevalent S. flexneri serotype isolated in the Son Tay Province, accounting for about 40% of S. flexneri isolates. Subsequent isolation of S. flexneri serotype 1c in this region and elsewhere in Vietnam confirmed that serotype 1c is of genuine importance in Vietnam.

Measurement of cytochrome P4501A induction in dab (Limanda limanda) and other teleosts with species-specific cDNA probes: isolation and characterisation of dab cDNA and its use in expression studies with beta-naphthoflavone-treated fish.

Induction of cytochrome P4501A (CYP1A) in fish is an important biomarker in marine monitoring programmes but a number of factors complicate interpretation of data based on catalytic activity. To provide additional analytical tools, we have cloned and sequenced entire (dab) and partial cDNAs (flounder, turbot, sand eel) from several fish species. A detailed analysis comparing the new sequences to those on the database (13 sequences) is presented and identifies an invariant, teleost-specific sequence (195-IVVSVANVICGMCFGRRYDH-214) which might be the basis for production of a species cross-reactive antibody. Northern and slot blots of fish RNA (sand eel, plaice, turbot, flounder and dab) showed extensive cross-species hybridisation with each of the cDNAs (sand eel, plaice, turbot, flounder and dab). The exception was turbot RNA, which only gave adequate hybridisation when the turbot probe was used. Attempts to normalise the hybridisation data to GAPDH mRNA were not satisfactory since there were significant species differences in expression of this gene and expression was suppressed (20-40%) by beta-naphthoflavone treatment. The CYP1A probes indicated induction levels relative to untreated dab of: plaice (five-fold); turbot (12-fold); flounder (12-fold); and dab (10-fold). The study demonstrates the relative ease with which species-specific molecular probes can be generated and used.

Paralytic shellfish poisoning toxins induce xenobiotic metabolising enzymes in Atlantic salmon (Salmo salar).

Paralytic shellfish poisoning (PSP) toxins have been implicated as the causative agent of a number of fish kills. Exposure experiments indicate that fish are susceptible to PSPs by intraperitoneal (i.p.) and oral administration, while sampling of fish affected by toxic blooms reveals that these toxins can be accumulated. In spite of the potential impact to marine fisheries, little research has been conducted on the potential metabolism and detoxification of PSPs in marine fishes. Previous work by this group has shown that the xenobiotic metabolising enzyme (XME) cytochrome P-450 (CYP1A) is induced in Atlantic salmon (Salmo salar) following i.p. exposure to saxitoxin (STX). Salmon injected i.p. with sub-lethal doses of STX show a four- to eight-fold induction of hepatic CYP1A (as shown by ethoxyresorufin-O-deethylase activity) over controls after 96 h. Results presented here show that the phase II XME glutathione S-transferase (GST) is also induced in salmon following PSP exposure. Post smolts were exposed to three injections of PSPs (2 micrograms STXeq/kg) over 21 days. Injection of both STX and PSPs extracted from a toxic strain of dinoflagellate (Alexandrium fundyense, CCMP 1719) resulted in induction of hepatic GST, as measured by activity for 1-chloro 2,4-dinitrobenzene. Such inductions indicate a potential role for XMEs in PSP metabolism. Possible roles for other enzymes are also discussed.

The induction of metallothionein in tissues of the Norway lobster Nephrops norvegicus following exposure to cadmium, copper and zinc: the relationships between metallothionein and the metals.

Nephrops norvegicus were exposed simultaneously to cadmium, copper and zinc over an 18-day period. Exposure concentrations were control, 1, 5 and 25 microg litre(-1) for cadmium and copper and 8, 40 and 200 microg litre(-1) for zinc. Concentrations of cadmium, copper, zinc and metallothionein were measured in homogenates of both the gill and the hepatopancreas. Quantification of metallothionein was carried out by differential pulse polarography. Cadmium concentrations increased significantly in the gill and hepatopancreas of both male and female animals in response to increases in exposure concentration. In contrast, the concentration of copper and zinc increased significantly in the gills of males, but not in females. In the hepatopancreas, neither copper nor zinc resulted in significant changes in concentrations of these metals. Metallothionein concentrations in the gill and hepatopancreas were increased significantly in relation to metal exposure in both males and females. Concentrations of cadmium and metallothionein in both the gill and hepatopancreas of males and females were positively correlated. Copper in the hepatopancreas also showed positive relationships with MT concentrations in males, but not in females. This study suggested that cadmium MTs in the gill and hepatopancreas of Nephrops norvegicus could be used as a sensitive tool to detect cadmium contamination in the lobsters, although this was not true for copper and zinc.

The effects of in vivo exposure to cadmium, copper and zinc on the activities of gill ATPases in the Norway lobster, Nephrops norvegicus.

Norway lobsters, Nephrops norvegicus were exposed for 18 days to combinations of cadmium, copper and zinc at three sublethal concentrations (1, 5, 25 micrograms/L for Cd and Cu, and 8, 40 and 200 micrograms/L for Zn). Male animals were exposed to all three concentrations, while female animals were studied only in one (highest) concentration of the metals. Activities of Na,K-ATPase and both oligomycin-sensitive and insensitive Mg-ATPase were investigated in relation to metal exposure. A group of male Nephrops of different size groups from a single location in the Clyde Sea was also sampled to investigate the relationship between size and ATPase activity. Cadmium concentrations increased significantly with increases in exposure concentrations in both male and female animals, whereas copper and zinc increased significantly only in the highest treatment in males. There was no significant difference in concentrations of the metals in control male and female animals, whereas males accumulated significantly higher levels of copper and zinc in the highest concentrations of the metals. Control males showed higher activity of Na,K-ATPase than control females. Na,K-ATPase activity was significantly inhibited in male animals exposed to metals whereas in female animals there was only a significant inhibition in total Mg-ATPase activity. Na,K-ATPase activity had a negative relationship with copper in males, while in treated females this ATPase had positive relationship with zinc and copper. There were negative relationships between the activities of Na,K-ATPase and oligomycin sensitive Mg-ATPase with carapace length of the animals.

The impact of sewage sludge exposure on the reproduction of the sand goby, Pomatoschistus minutus.

Adult sand gobies were exposed to 0.1% sewage sludge for 19 weeks prior to the end of spawning. Exposure to sewage sludge had a significant effect on male mortality rates but no significant effects on the gonadosomatic index of males or females. There were no major effects of sludge exposure on testes androgen content or on testes release of androgens after in vitro gonadotrophin stimulation. Fecundity and the number of larvae produced were not significantly affected by the sludge exposure. There was a tendency for eggs and larvae from sludge-exposed females to have a higher mortality rate. At a population level, however, there was a major reduction in the number of eggs and larvae produced in the sludge-exposed population which reflected a failure of some females to spawn. Of the larvae produced, 60-70% from sludge-exposed parents were lighter and had a larger yolk-sac volume compared to larvae from non-exposed parents which may have indicated impaired yolk utilisation.

Characterization of the microsomal epoxide hydrolase of hepatic microsomes of the common dab,Limanda limanda.

Epoxide hydrolase of microsomal membranes of the common dab (Limanda limanda) has been characterized using p-nitrostyrene oxide as substrate. Under the conditions of assay used, the turnover number with this substrate was higher than found for the more frequently used styrene oxide and steady state kinetics were observed. The enzyme had a KM of 0.12 mM and optima for pH and temperature between pH 8-10.2 and 50-60°C respectively. Enzyme activity was unaffected by low concentrations of ionic and non-ionic detergents but was inhibited by higher concentrations of Lubrol and Brij. The enzyme protein did not react with monospecific antibodies to rat or human microsomal epoxide hydrolase during Western blotting. Large inter-individual variation in enzyme activity was found but the enzyme does not appear to be expressed in a gender-specific way. Fish were administered a wide range of hydrocarbons which are known to alter the expression of cytochrome P450 1A but these had no effect other than benzothiophene which caused a small increase in enzyme activity.

Renal, respiratory and ionic regulation in Atlantic salmon (Salmo salar L.) kelts following transfer from fresh water to seawater.

Atlantic salmon may return to the sea after spawning in fresh water. These fish, known as kelts, reportedly show a limited ability to hypoosmoregulate. However, this study shows that fresh-water-adapted kelts exposed to seawater demonstrate rapid adaptation (within 48 h) in osmoregulatory parameters to values characteristics of seawater-adapted salmonids. The urine flow rate falls from 1.2 to 0.2 ml.kg-1.h-1 within 24 h. Over the same period, urine osmolality increases from 48 mosmol.kg-1 to become isosmotic with the plasma, and Mg2+ secretion by the kidney tubules elevates the urine concentration from 0.5 to 100 mmol.l-1. As is characteristic for marine teleosts, kelts drink seawater and process the ingested water in the gut to replace body water lost by osmosis to the hyperosmotic medium. Seawater exposure causes a marked hypoxia, arterial oxygen tension falling by 43% within minutes and persisting for at least 4 days at this low level. This is associated with large changes in blood pH and acid-base balance. The physiological mechanisms involved in adaptation to a hyperosmotic external medium are discussed, and the osmoregulatory capacity of kelts is compared with that of salmon at other stages of the life cycle.

Sites of interactions of surfactants with beta-adrenergic responses in trout (Salmo gairdneri) gills.

The aims of this study were to investigate the effects of surfactants on the beta-adrenergic responses of the arterio-arterial (lamellar) vascular pathway of isolated gills from the trout Salmo gairdneri. The resistance of the arterio-arterial vasculature was monitored by measuring the input pressure of isolated gills perfused at a constant flow and at constant efferent pressure. Specific beta-adrenergic stimulation with isoprenaline produced a dose-dependent reduction in vascular resistance which was mimicked by the presence of cyclic 8-(4-chlorophenylthio) adenosine-3':5'-monophosphate (8-cpt cyclic AMP). These results suggest that cyclic AMP is involved as an intracellular second messenger of beta-adrenergic regulatory responses of branchial haemodynamics. Surfactants in the perfusate at nominal concentrations of 1.0 mumol 1(-1) (0.3-0.5 mg 1(-1)) inhibited the response of the branchial tissue to isoprenaline but did not affect the response to 8-cpt cyclic AMP. This suggests that the surfactant effects are the result of events occurring within the cell membrane. Furthermore because the surfactants used affected the efficacy, rather than the affinity, of the isoprenaline response then the mechanism of action is unlikely to be an effect on agonist/receptor interaction but may be further along the stimulus response chain, perhaps affecting receptor/regulatory nucleotide/adenylate cyclase interaction or via direct inhibition of the cyclase itself.

Hemodynamics and potentials in isolated flounder gills: effects of catecholamines.

Branchial hemodynamics and transepithelial potentials were investigated in an isolated perfused gill preparation from the flounder (Platichthys flesus L.). An analysis of the pressure-flow profiles suggests that the gill is compliant but that the physical relationships of pressure and flow are complex. Epinephrine has a bimodal effect with an initial vasoconstriction followed by a vasodilation, the latter response being much smaller than that usually seen in teleosts. Pharmacological analysis shows 1) an alpha-mediated vasoconstriction of the arterial and venous pathway, associated with a redistribution of flow away from the venous pathway, and 2) a small beta-mediated vasodilation of the arterial pathway only. An electrical potential (inside positive) was measured across the isolated gill, bathed and perfused with an identical saline. This potential was recorded, despite the venous system being open to the external bath, and was independent of both arterial and venous flows. Epinephrine caused a significant decline in this potential, which could be resolved into a large inhibitory alpha-mediated response and a small beta-mediated stimulation.