Respiratory impairment in crustaceans and molluscs due to exposure to heavy metals.

1. We have assessed, using current literature, the respiratory consequences of water-borne heavy metal exposure in crustaceans and molluscs. 2. We suggest that in lethal and sub-lethal concentrations the essential metals Cu and Zn act on the respiratory system primarily by disrupting gill function which results in the development of internal hypoxia, although reparation can be accomplished even at "high" sub-lethal concentrations. 3. The more toxic xenobiotes such as Hg (and perhaps to a lesser extent Cd) may interfere with the respiratory system at every level of organisation including cellular respiration itself.     

Profenofos induced DNA damage in freshwater fish, Channa punctatus (Bloch) using alkaline single cell gel electrophoresis

The aim of the present study was to evaluate the induced genotoxicity (DNA damage) due to organophosphate pesticide profenofos (PFF) in gill cells of freshwater fish Channa punctatus using single cell gel electrophoresis (SCGE)/Comet assay. The 96h LC(50) value of PFF (50% EC) was estimated for the fish species in a semistatic system and then three sub-lethal of LC(50) concentrations viz the sub-lethal 1, sub-lethal 2 and sub-lethal 3 concentrations were determined as 0.58ppb, 1.16ppb and 1.74ppb, respectively. The fish specimens were exposed to these concentrations of the pesticide and the gill tissue samplings were done on 24h, 48h, 72h and 96h post exposure for assessment of DNA damage in terms of percentage of DNA in comet tails. In general, a concentration dependent response was observed in the gill cells with induction of maximum DNA damage at the highest concentration of PFF. The results of the present investigation indicated that PFF could potentially induce genotoxic effect in fish, even in sub-lethal concentrations and SCGE as a sensitive and reliable tool for in vivo assessment of DNA damage caused by the genotoxic agents.     

Toxicity of cypermethrin in Labeo rohita fingerlings: biochemical,enzymatic and haematological consequences

The effect of exposure to sub-lethal concentrations of cypermethrin, a synthetic pyrethroid pesticide, on biochemical parameters of muscle, blood and enzyme activities in brain, liver and kidney of the Indian major carp, Labeo rohita was studied. The sub-lethal exposure studies were done for up to 45 days at 1/10 and 1/50 of 96 h LC(50) of cypermethrin. The 96 h LC(50) was found to be 0.139 ppm. RNA levels decreased while DNA levels were elevated. Acid phosphatase was unchanged while alkaline phosphatase was depleted. Brain acetylcholinesterase activity was decreased significantly (P<0.05) over a period of 45 days at both cypermethrin concentrations. Lactate dehydrogenase activity in brain and liver was elevated, but inhibited in kidney. Succinate dehydrogenase and ATPase activities were depleted in brain, kidney and liver. There was a decrease in serum protein level over control at both concentrations of the pyrethroid. Blood glucose level and total leucocytes were elevated compared with controls at either concentration from day 15 to day 45. Haemoglobin percentage and total erythrocytes decreased in both sub-lethal concentrations. Extracts of the herb Datura stramonium were effective in countering the toxicity of this pesticide. Our data suggest that sub-lethal exposure of cypermethrin alters the biochemical, haematological parameters and enzymes of organs tissue and exert stress on the fish. Plant extracts may be useful in counteracting some of these effects.     

Adaptation of Campylobacter jejuni to biocides used in the food industry affects biofilm structure,adhesion strength,and cross-resistance to clinical antimicrobial compounds

The emergence of biocide-adapted Campylobacter jejuni strains that developed into biofilms and their potential to develop clinical resistance to antimicrobial compounds was studied. C. jejuni was grown in sub-lethal concentrations of five biocides used in the food industry. C. jejuni exhibited adaptation to these biocides with increased minimum inhibitory concentrations. The 3-D structures of the biofilms produced by the biocide-adapted cells were investigated by atomic force microscopy (AFM). The results revealed marked variability in biofilm architecture, including ice-crystal-like structures. Adaptation to the biocides enhanced biofilm formation, with significant increases in biovolume, surface coverage, roughness, and the surface adhesion force of the biofilms. Adaptation to commercial biocides induced resistance to kanamycin and streptomycin. This study suggests that the inappropriate use of biocides may lead to cells being exposed to them at sub-lethal concentrations, which can result in adaptation of the pathogens to the biocides and a subsequent risk to public health.     

Acclimatable cardiac and ventilatory responses to copper in the freshwater crayfish Procambarus clarkii

Mortality and physiological tests following exposure to waterborne copper were performed in the red swamp crayfish Procambarus clarkii from a central Italian population. Mortality tests gave an estimated 96 h LC50 value (with 95% confidence limits) of 162 (132-211) mg L(-1) waterborne copper II. Variations in cardiac and ventilatory rates were simultaneously monitored using a non-invasive plethysmographic technique. In experiments with different sub-lethal copper concentrations (control, 0.5, 1 and 10 mg L(-1)) performed at different times (3, 6, 96 h), copper exposure elicited a reduction in both heart and scaphognatite rates. Following exposure to 10 mg L(-1) copper for 96 h, the heart and scaphognatite rates decreased to about 35% of the initial values. The reduction was fully reversible, since crayfish exposed to 0.5, 1 and 10 mg Cu L(-1) for 96 h resumed control rates after a 3-h residence in clean water. In crayfish pre-exposed (96 h) to sub-lethal copper concentrations (0.1 and 1 mg L(-1)) and then held in control water (3 h), the reduction of heart and scaphognatite rates after exposure to 10 mg Cu L(-1) were significantly lower than in specimens pre-exposed to control water. Therefore, copper induces a concentration and time dependent reduction of both cardiac and ventilatory activity in P. clarkii; these responses can be reduced or fully abolished by pre-exposure to sub-lethal levels of the metal.     

Complex physiological traits as biomarkers of the sub-lethal toxicological effects of pollutant exposure in fishes

Complex physiological traits, such as routine aerobic metabolic rate or exercise performance, are indicators of the functional integrity of fish that can reveal sub-lethal toxicological effects of aquatic pollutants. These traits have proved valuable in laboratory investigations of the sub-lethal effects of heavy metals, ammonia and various xenobiotics. It is not known, however, whether they can also function as biomarkers of the complex potential range of effects upon overall functional integrity caused by exposure to mixtures of chemicals in polluted natural environments. The current study used portable swimming respirometers to compare exercise performance and respiratory metabolism of fish exposed in cages for three weeks to either clean or polluted sites on three urban European river systems: the river Lambro, Milan, Italy; the rivers Blythe, Cole and Tame, Birmingham, UK; and the river Amstel, Amsterdam, The Netherlands. The UK and Italian rivers were variously polluted with high levels of both bioavailable heavy metals and organics, and the Amstel by mixtures of bioavailable organics at high concentrations. In both the UK and Italy, indigenous chub (Leuciscus cephalus) exposed to clean or polluted sites swam equally well in an initial performance test, but the chub from polluted sites could not repeat this performance after a brief recovery interval. These animals were unable to raise the metabolic rate and allocate oxygen towards exercise in the second trial, an effect confirmed in successive campaigns in Italy. Swimming performance was therefore a biomarker indicator of pollutant exposure in chub exposed at these sites. Exposure to polluted sites on the river Amstel did not affect the repeat swimming performance of cultured cloned carp (Cyprinus carpio), indicating either a species-specific tolerance or relative absence of heavy metals. However, measurements of oxygen uptake during swimming revealed increased rates of routine aerobic metabolism in both chub and carp at polluted sites in all of the rivers studied, indicating a sub-lethal metabolic loading effect. Therefore, the physiological traits of exercise performance and metabolic rate have potential as biomarkers of the overall sub-lethal toxic effects of exposure to complex mixtures of pollutants in rivers, and may also provide insight into why fish do not colonize some polluted environments.     

Chromosomally-determined induced tolerance to copper in Escherichia coli

Pre-exposure of Echerichia coli to sub-lethal concentrations of cupric sulphate induced copper tolerance: pre-exposed (habituated) organisms were essentially unaffected by concentrations of Cu²⁺ which completely prevented colony formation by non-habituated ones. The observed copper tolerance was not dependent on the selection of copper-resistant mutants but resulted from a phenotypic change in the organisms during the pre-exposure period.     

Diverse modulation of spa transcription by cell wall active antibiotics in Staphylococcus aureus

ABSTRACT: BACKGROUND: The aim of this study was to investigate the effect of various classes of clinically relevant antibiotics at sub-lethal concentrations on virulence gene expression and biofilm formation in Staphylococcus aureus. FINDINGS: LacZ promoter fusions of genes related to staphylococcal virulence were used to monitor the effects of antibiotics on gene expression in a disc diffusion assay. The selected genes were hla and spa encoding alpha-hemolysin and Protein A, respectively and RNAIII, the effector molecule of the agr quorum sensing system. The results were confirmed by quantitative real-time PCR. Additionally, we monitored the effect of subinhibitory concentrations of antibiotics on the ability of S. aureus to form biofilm in a microtiter plate assay. The results show that sub-lethal antibiotic concentrations diversely modulate expression of RNAIII, hla and spa. Consistently, expression of all three genes were repressed by aminoglycosides and induced by fluoroquinolones and penicillins. In contrast, the beta-lactam sub-group cephalosporins enhanced expression of RNAIII and hla but diversely affected expression of spa. The compounds cefalotin, cefamandole, cefoxitin, ceftazidime and cefixine were found to up-regulate spa, while down-regulation was observed for cefuroxime, cefotaxime and cefepime. Interestingly, biofilm assays demonstrated that the spa-inducing cefalotin resulted in less biofilm formation compared to the spa-repressing cefotaxime. CONCLUSIONS: We find that independently of the cephalosporin generation, cephalosporins oppositely regulate spa expression and biofilm formation. Repression of spa expression correlates with the presence of a distinct methyloxime group while induction correlates with an acidic substituted oxime group. As cephalosporines target the cell wall penicillin binding proteins we speculate that subtle differences in this interaction fine-tunes spa expression independently of agr.     

A histological study of the response of the interrenal cells of the goldfish (Carassius auratus) to treatment with sodium lauryl sulphate

Treatment of goldfish ( Carassius auratus ) with sub-lethal concentrations of an anionic detergent sodium lauryl sulphate for 1, 2 or 4 weeks produced histological changes in the interrenal (steroidogenic) cells of the head kidney which are indicative of a cellular activation. Significant increases in both the nuclear diameter of these cells and the number of nucleoli contained in the nuclei occurred after 1 week's treatment, although these effects were more pronounced after 2 and 4 weeks. There were little associated alterations in chromaffin cell activity. Preliminary experiments with another pollutant, zinc sulphate, suggest that it exerts a similar action to sodium lauryl sulphate on the interrenal cells. It is suggested the increases in corticosteroid production which occur after exposure of fish to sub-lethal concentrations of pollutants may be important in the development of a generalized disease syndrome and to the long-term success of fish populations.     

Modification of bacterial cell membrane dynamics and morphology upon exposure to sub inhibitory concentrations of ciprofloxacin

Ciprofloxacin (CPX), a second generation fluoroquinolone antibiotic, is used as a primary antibiotic for treatment against gastroenteritis, drug-resistant tuberculosis, and malignant otitis externa. CPX is a broad spectrum antibiotic that targets the DNA gyrase of both Gram-positive and Gram-negative bacteria. Irrational and improper usage of CPX results in emergence of CPX resistant organisms emphasizing the importance of using lethal doses of CPX. Here, we have systematically analysed the effect of CPX at sub lethal concentrations on live E. coli membrane and growth dynamics. As a result of CPX interaction at sub-lethal concentrations, we detected filamentation of the bacterial cells during cell division. Although CPX is a DNA targeting antibiotic and did not result in considerable increase of live E. coli cell surface roughness, we observed significant enhancement in the lipid diffusion coefficients possibly due to disrupted lipid packing or altered lipid composition. Interestingly, we seem to observe slightly higher extent of lipid diffusion alteration when bacterial inner membrane specific label FM4-64 was used in comparison to the non-specific membrane dye. Both these results are contrary to that observed in bacterial cells for colistin, a membrane targeting antibiotics. Our work highlights the need for using multiple, complementary surface and depth sensitive techniques to obtain information on the realistic nature of bacterial cell membrane remodelling due to non-membrane targeting antibiotics. Our work could have implications for identification of potential biomembrane markers at sub-lethal concentrations even for antibiotics which are non-membrane targeting that could help in unravelling pathways for emergence of antimicrobial resistance.     

Susceptibility and fitness cost of Aedes albopictus on their survivability after the exposure to the insecticide

The present study was undertaken to investigate the lethal and sub-lethal effects of insecticides on the life history of Aedes albopictus mosquitoes from urban and sub-urban areas in Penang, Malaysia. The female mosquitoes aged two to five days old were exposed to; (1) diagnostic dose (lethal dose) at 5% malathion and 0.75% permethrin; and (2) sub-lethal concentration of 1.5% malathion, and 0.2% permethrin, respectively and adult female survivors from sub-lethal concentrations were evaluated for fitness parameters. Fecundity, fertility, adult longevity, development time and survival of mosquitoes were the crucial point in their life history had studied. Aedes albopictus species from urban Sg. Dua strain has developed high resistance towards 0.75% permethrin and was confirmed on resistance after 24 h of reading. While sub-urban Batu Maung strain is still susceptible to both 5% malathion and 0.75% permethrin. At the sub-lethal dose, we discovered 0.2% permethrin insecticide have significantly more effects on the fitness cost of Ae. albopictus as compared to 1.5% malathion; with decreasing on fecundity, lesser time was needed to reach each development stages, and more male adult was emerged compared to female mosquitoes for both urban and sub-urban strains. Whereas, malathion insecticide only affected the number of eggs laid by the parent mosquitoes and the development time to reach adult stages. Even though, Ae. albopictus mosquitoes had developed resistance towards permethrin resulted in decreasing mortality, but subsequent effects on their fitness cost still continued on the first generation. Thus, it will benefit in reducing the transmission of mosquito-borne diseases.     

The effects of organic and inorganic phosphates on fertilization and early development in the sea urchin Lytechinus variegatus (Echinodermata: Echinoidea)

The embryos of the sea urchin Lytechinus variegatus are capable of surviving chronic exposure to inorganic sodium phosphate and organic triethyl phosphate concentrations as high as 6 and 1000 mg l(-1) seawater, respectively. However, chronic exposure to sub-lethal concentrations of these phosphates may cause arrested or abnormal embryonic development. We measured fertilization success and percentages of normal, arrested and abnormal embryos exposed to low, medium and high sub-lethal concentrations of inorganic and organic phosphate. Fertilization success was significantly reduced in all phosphate treatments. After attaining the 4-cell stage, embryos exposed to the highest phosphate concentrations displayed arrested development. Percentages of abnormally developing embryos showed a strong concentration dose-response with a significant increase in abnormal embryonic development with increasing phosphate concentration. Overall, these results indicate that the gametes and embryos of L. variegatus may provide a rapid and sensitive model bioassay for the evaluation of phosphate pollutants in marine systems. Our findings also indicate that shallow-water populations of L. variegatus spawning in areas subjected to inorganic and organic phosphate pollutants may suffer detrimental effects on fertilization and embryonic development.     

N uptake and growth responses to sub-lethal freezing in the grass Poa pratensis L.

Background and aims

Climate warming has the potential to increase both the exposure and vulnerability of grass roots to frost in temperate regions by reducing snow cover and altering the timing of cold acclimation. Despite a strong research focus on the direct effects of freezing on grass mortality, the direct sub-lethal effects of freezing on grass performance have not been well-characterized. We examined sub-lethal responses of the grass Poa pratensis to variation in the timing, severity, rate and length of freezing.

Methods

We assessed short term root functional responses (¹⁵N uptake) and longer term plant growth responses to freezing administered both under controlled conditions in a refrigerated incubator, and in the field by manipulating snow and litter cover.

Results

In fall and spring, ¹⁵N uptake declined in response to 1?day of freezing down to ?10?°C or to 3?days of freezing at ?5?°C, whereas in winter, ¹⁵N uptake was insensitive to freezing. Long term growth responses were similar, with reduced growth only occurring for grasses frozen for 3?days at ?5?°C in spring, but not for grasses frozen in fall or winter. Snow and litter removal intensified soil freezing over winter, but did not significantly affect plant growth.

Conclusions

Our results demonstrate that while P. pratensis is relatively tolerant to frost damage over winter, it may be vulnerable to sub-lethal frost effects in fall, and particularly in spring. These sub-lethal effects occur at temperatures approximately 15–20?°C warmer than the published LT₅₀ values for this species.     

The acute phase protein haptoglobin and its relation to oxidative status in piglets undergoing weaning-induced stress

Haptoglobin (Hp) prevents the hemoglobin driven generation of hydroxyl radicals and lipid peroxides. Hp can reduce the neutrophil respiratory burst and is an antioxidative molecule in its own right. We aimed to evaluate Hp concentrations, oxidative stress and antioxidative capacity in blood during weaning and to characterise potential relationships between these parameters. Two batches of 10 piglets each (2 trials) weaned at the age of 27-30 days were fed a starter feed mix ad libitum. Blood samples were taken 1 week before weaning and at weekly intervals thereafter. Oxidative stress was monitored via the D-ROM system, antioxidative capacity was measured with the TEAC assay and Hp concentrations were measured by ELISA. Neutrophil phagocytic activity and oxidative burst were examined via flow-cytometry. Body weights were recorded weekly. Hp concentrations were increased in both trials post-weaning (P < 0.01); oxidative stress and oxidative burst were elevated in trial I (P < 0.005). In trial I, Hp and ROM values returned to baseline levels at 6 weeks post-weaning. The piglets in trial II showed respiratory symptoms and maintained elevated Hp concentrations. ROM values and Hp were related (r = 0.58; P < 0.01). Hp and body weight gain were inversely related post-weaning.     

Sub-lethal concentrations of activated complement increase rat lymphocyte glutamine utilization and oxidation while lethal concentrations cause death by a mechanism involving ATP depletion

Nucleated cells are more resistant to complement-mediated cell death than anucleated cells such as erythrocytes. There are few reports concerning the metabolic response of nucleated cells subjected to sub-lethal complement attack. It is possible that the rate of utilization of specific metabolic fuels by the cell is increased to enhance cell defence. We have measured the maximum activity of hexokinase, citrate synthase, glucose 6-phosphate dehydrogenase and glutaminase in rat mesenteric lymphocytes exposed to sub-lethal concentrations of activated complement (present in zymosan-activated serum, ZAS). These enzymes were carefully selected as they indicate changes of flux in glycolysis, TCA cycle, pentose phosphate pathway and glutaminolysis, respectively. The only enzyme activity to change on exposure of lymphocytes to ZAS was glutaminase, which was enhanced approximately by two-fold. Although rates of both glutamine and glucose utilization were enhanced by exposure to ZAS, only the rate of oxidation of glutamine was increased. Complement kills anucleated cells by simple osmotic lysis. However, it is likely that some nucleated cells will display characteristics of an ordered death mechanism and we have demonstrated that the concentration of lymphocyte ATP is dramatically decreased by activated complement. Nevertheless, the extent of cell death could be significantly reduced by the addition of inhibitors of the nuclear enzyme poly (ADP-ribose) polymerase (PARP). We conclude that glutamine metabolism is not only important for lymphocyte proliferative responses but is also important for cell defence from sub-lethal concentrations of activated complement. The rapid rate of complement-induced lymphocyte death reported here is suggested to be a consequence of over-activation of the nuclear enzyme PARP and ATP depletion.     

Effect of Ischemic Preconditioning on Mitochondrial Dysfunction and Mitochondrial P53 Translocation after Transient Global Cerebral Ischemia in Rats

Transient global brain ischemia induces dysfunctions of mitochondria including disturbance in mitochondrial protein synthesis and inhibition of respiratory chain complexes. Due to capacity of mitochondria to release apoptogenic proteins, ischemia-induced mitochondrial dysfunction is considered to be a key event coupling cerebral blood flow arrest to neuronal cell death. Ischemic preconditioning (IPC) represents an important phenomenon of adaptation of central nervous system (CNS) to sub-lethal short-term ischemia, which results in increased tolerance of CNS to the lethal ischemia. In this study we have determined the effect of ischemic preconditioning on ischemia/reperfusion-associated inhibition of mitochondrial protein synthesis and activity of mitochondrial respiratory chain complexes I and IV in the hippocampus of rats. Global brain ischemia was induced by 4-vessel occlusion in duration of 15 min. Rats were preconditioned by 5 min of sub-lethal ischemia and 2 days later, 15 min of lethal ischemia was induced. Our results showed that IPC affects ischemia-induced dysfunction of hippocampal mitochondria in two different ways. Repression of mitochondrial translation induced during reperfusion of the ischemic brain is significantly attenuated by IPC. Slight protective effect of IPC was documented for complex IV, but not for complex I. Despite this, protective effect of IPC on ischemia/reperfusion-associated changes in integrity of mitochondrial membrane and membrane proteins were observed. Since IPC exhibited also inhibitory effect on translocation of p53 to mitochondria, our results indicate that IPC affects downstream processes connecting mitochondrial dysfunction to neuronal cell death.     

Oxidative stress,DNA damage,and histological alterations in Bombyx mori exposed orally to pesticide dimethoate

Pesticides are an essential part of agricultural practices that ward off pathogens and diseases from the agricultural crop. However, apart from target organisms, these chemicals also have adverse effects on non-target organisms. Dimethoate is an insecticide used extensively in agriculture and horticulture practices worldwide. We used the silkworm Bombyx mori as a model organism to study the effect of commercial formulation of dimethoate (Dimethoate-30% EC) on the gut, silk gland, and fat body tissues. LD₅₀ of dimethoate-30% EC on silkworm (B. mori) was 997 ppm, as reported in a previous study. We used concentrations of 25, 50, and 100 ppm in our experiments. Our results showed that sub-lethal doses of dimethoate caused weight loss and induced damage at the histological level to the mid-gut, silk gland, and fat body of B. mori. It also caused a decrease in the level of antioxidants like CAT, SOD, GPx, GSH, and GST, indicating that dimethoate has produced a shift of ROS balance towards free radical generation and therefore resulted in overall damage to this organism. Sub-lethal doses of this pesticide also caused lipid peroxidation in the silk gland, gut, and fat body of B. mori, damaging these tissues. The disruption was also seen in the mid-gut and middle silk gland at the DNA level, where it caused single-strand breaks, as was revealed by single cell gel electrophoresis studies. Damage at histological, biochemical, and molecular levels was most extreme at a concentration of 100 ppm, the highest sub-lethal concentration given to B. mori.     

Sub-lethal plasma ammonia accumulation and the exercise performance of salmonids

The proposal that plasma ammonia accumulation might impair the swimming performance of fish was first made over a decade ago, and has now proven to be the case for a number of salmonid species. The first experimental evidence was indirect, when a negative linear relationship between plasma ammonia concentrations and maximum sustainable swimming speed (U(crit)) was found following the exposure of brown trout (Salmo trutta) to sub-lethal concentrations of copper in soft acidic water. Since then, negative linear relationships between plasma ammonia concentration and U(crit) have been demonstrated following exposure of brown trout, rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) to elevated water ammonia. For brown trout, the relationships between plasma ammonia and U(crit) were remarkably similar following either exposure to elevated water ammonia or to sub-lethal copper. This indicates that the impairment of swimming performance resulting from exposure to sub-lethal concentrations of heavy metals may be attributable in large part to an accumulation of endogenous ammonia. The negative relationship between plasma ammonia concentration and U(crit) was similar in size-matched rainbow and brown trout but, under similar regimes of ammonia exposure, rainbow trout were able to maintain a significantly lower plasma ammonia concentration, revealing inter-specific differences in ammonia permeability and/or transport. One primary mechanism by which ammonia accumulation may impair exercise performance is a partial depolarisation of membrane potential in tissues such as the brain and white muscle. This may prejudice the co-ordination of swimming movements and reduce or abolish the development of muscle tension, thus, compromising swimming efficiency and performance at the top end of the range.