Biochemical stress response in tetradifon exposed Daphnia magna and its relationship to individual growth and reproduction.

Environmental risk assessment of chemicals toxicity requires the use of costly and labor-intensive chronic data and short-term tests provide additional information. Energy budget is used by the animals for their growth, reproduction, and metabolism and it is reduced in case of toxic stress. Tetradifon acaricide is frequently used in the European Mediterranean region and it is implicated in aquatic environmental pollution. Previous studies showed that the EC(50)-24 h of tetradifon on Daphnia magna was 8.92 mg/L. Based on that, D. magna were exposed to sublethal tetradifon concentrations of 0.10, 0.18, 0.22 and 0.44 mg/L for five days in order to investigate their effect on intermediate metabolism. Caloric content was determined as biomarker of tetradifon toxicity. Results were analyzed using one-way analysis of variance (ANOVA) and Duncan's significant difference test was used to find differences between groups (alpha was set at p=0.05). Daphnids energy content decreased as tetradifon concentration increased. At 120-h caloric content was depleted >51% at pesticide concentrations of 0.18 mg/L and higher. In order to determine a possible link between the 5-d test and the 21-d chronic test, animals under short-term test were exposed to the same pesticide concentrations known to cause adverse effects on reproduction, growth and survival. Results from the present study indicated a good correlation between the proposed 5-day test and daphnid energy budget. Comparison between both, caloric content results and the chronic effect values obtained using life-table studies, suggested a good fit between them. These studies can be used as earlier, predictive and useful tests with comparable results to the classic chronic ones. Our results indicate that caution must be done about the use of tetradifon in the aquatic environment.

Laboratory investigation into the development of resistance of Daphnia magna to the herbicide molinate.

Daphnia magna (F0 generation) was exposed to different sublethal molinate concentrations (0, 3.77, 4.71, 6.28, 9.42, and 18.85 mg/L) during 21 days. Chronic toxicity tests, using the same herbicide concentrations, were also carried out during 21 days using neonates of F1 first brood (F1-1st) and F1 third brood (F1-3rd) offspring generations from the parentals (F0) preexposed to the herbicide. Finally, offspring (from F1-1st and F1-3rd broods) were transferred to herbicide-free medium during a 21-day recovery period. The alga Nannochloris oculata (5 x 10(5) cells/mL) was used as food in all the experiments. The effect of molinate on survival, reproduction, and growth was monitored for the selected daphnid generations. The parameters used to evaluate herbicide effect on reproduction were mean total young per female, mean brood size, time to first reproduction, mean number broods per female, and intrinsic rate of natural increase (r). Survival and growth (body length) were also determined after 21 days of exposure to molinate. Reproduction was significantly reduced when molinate concentration was increased in the medium. This effect was higher in the parental (F0) daphnids than in the F1-1st and F1-3rd offspring. In the recovery study, reproduction was still reduced in F1 generation daphnids (1st and 3rd), but only in those animals from parentals exposed to the highest molinate concentrations. The decreased with increasing concentrations of molinate in daphnids from the parental generation (F0). Significant differences were also found in daphnids from the F1 generation (exposure). The growth of the exposed organisms (F0 and F1) decreased, although the greatest decrease was found in the parental animals (F0) (25%) exposed to 9.42 mg/L molinate. F1 daphnids (1st and 3rd broods) from the recovery period did not show any significant difference in their growth after 21 days of study. Finally, survival was not affected after exposure to the selected molinate concentrations except in those daphnids from the F0 generation in which survival decreased 51% and 78% at the highest herbicide concentrations tested (9.42 and 18.85 mg/L). Our results suggest that the offspring daphnids seem to be adaptated to the herbicide molinate, showing more longevity and reproduction than their parentals.

Eel ATPase activity as biomarker of thiobencarb exposure.

European eels (Anguilla anguilla) were exposed to a sublethal thiobencarb concentration of 0.22 mg/L in a flow-through system for 96 h. Mg(2+) and Na(+)-K(+) adenosine triphosphatase (ATPase) activities were evaluated in gill and muscle tissues at 2, 12, 24, 48, 72, and 96 h of thiobencarb exposure. Gill ATPase activities were rapidly inhibited from 2h of contact onward. Highest inhibition was registered for Na(+), K(+)-ATPase (85%) from 2 to 12h. Both Mg(2+) and total ATPase were inhibited (>73%) during the first hours of toxicant exposure. At the end of the exposure period (96 h) ATPase activities were still different from those of the controls (>50%). Significant inhibition was detected in Na(+), K(+)-ATPase activity (80%) in muscle tissue after 2h and it was maintained over the entire exposure time. However, Mg(2+)-ATPase and total ATPase showed only perturbations after 2 h of exposure. Eels were exposed to 0.22 mg/L of thiobencarb for 96 h and then a recovery period in herbicide-free water was allowed for 192 h. Gill and muscle samples were removed at 8, 24, 72, 96, 144, and 192 h and ATPase activity was evaluated. Following 144 h of recovery, Mg(2+)- and Na(+), K(+)-ATPase activities, as well as total ATPase activity, in gills of those animals previously exposed to 0.22 mg/L of thiobencarb were still significantly different compared to controls. Thiobencarb seems to act to alter the ionic profiles. Since ion-dependent ATPases are known to regulate the influx and efflux of ions across the membrane to maintain the physiological requirements of the cells, the inhibition of Na(+), K(+)-ATPase probably induced osmoregulatory perturbations. On the other hand, thiobencarb exposure causes increases in the muscle water content of A. anguilla. The results indicated that water content increased significantly (>100% higher than the controls) during the first 24 h of exposure.

A new successful therapy for fetal chylothorax by intrapleural injection of maternal blood.

We present two cases of fetal chylothorax and hydrops diagnosed at 20 weeks' gestation, both of which underwent successful intrauterine treatment. In Case 1, a transient, near total resolution began 2 weeks after an iatrogenic hemothorax following a second thoracocentesis performed at 24 + 6 weeks. Because of pleural fluid reaccumulation, a Cesarean section was performed at 36 weeks. The 3805-g female neonate was admitted to neonatal intensive care but was discharged 50 days later in a healthy condition. In Case 2, resolution occurred after a third thoracocentesis and a second pleural injection of maternal blood, performed at 26 weeks. A 2660-g female neonate was delivered vaginally at 38 weeks. The infant remained asymptomatic and was discharged aged 4 days. Our experience suggests a possible useful role of intrapleural blood injection for the treatment of fetal chylothorax.

[Interest in the study of genetic variants of the promoter region of the UGT1A1 gene in neonatal jaundice]. / Interés del estudio de las variantes genéticas del promotor del gen UGT1A1 en la ictericia neonatal.

BACKGROUND: A relationship between polymorphism in the promoter region of the UGT1A1 gene (associated with Gilbert's syndrome) and the development of jaundice has recently been demonstrated. This polymorphism is due to (TA)7 instead of wild-type (TA)6. OBJECTIVE: To investigate the relationship between Gilbert's syndrome and neonatal jaundice by evaluating the distribution of (TA)7 in a population of newborns. METHODS: A total of 136 newborns were studied: 21 had neonatal jaundice, 69 were healthy and the remaining newborns had various diseases. DNA from each patient was used to amplify, by polymerase chain reaction, the promoter region of the UGT1A1 gene, which flanks the TATA box where the polymorphism is located. RESULTS: In the group without jaundice, 53 % of the newborns were normal (6/6 genotype), 40 % were 6/7 and 7 % were 7/7. In the group with jaundice, 33 % of the newborns were normal, 53 % were heterozygous (6/7) and 14 % were homozygous (7/7). Comparison of the groups revealed that the prevalence of UGTA1A polymorphism tended to be greater among jaundiced newborns (p 0.09). CONCLUSION: The results of this study suggest that there is a relationship between neonatal jaundice and Gilbert's syndrome among the Spanish population. These results, together with those of other authors, suggest that genetic screening for Gilbert's syndrome should be included in the investigation of neonatal jaundice in our population. Further studies with a greater number of subjects would determine the exact relationship between marked neonatal jaundice and IGTA1A polymorphism. Key words:

Interés del estudio de las variantes genéticas del promotor del gen en la ictericia neonatal / Interest in the study of genetic variants of the promoter region of the ugt1a1 gene in neonatal jaundice

Antecedentes: Recientemente se ha mostrado la asociación entre un polimorfismo en el promotor del gen UGT1A1 (asociado con el síndrome de Gilbert) y la presencia de ictericia. Este polimorfismo consiste en la existencia de siete repeticiones TA (TA)7, en lugar de seis (TA)6. Objetivo: Analizar la distribución del genotipo (TA)7 en una población de recién nacidos para determinar la posible relación entre el síndrome de Gilbert y la ictericia neonatal. Métodos: Se estudiaron 136 recién nacidos: 21 presentaron ictericia del resto de recién nacidos, 69 eran sanos y el resto mostró diferentes procesos, incluyendo 7 prematuros. El ADN de cada paciente fue utilizado para amplificar, mediante la reacción en cadena de la polimerasa, la región del promotor del gen de la UGT-1 que flanquea la caja TATA donde se encuentra el polimorfismo. Resultados: Grupo sin ictericia: 53% normales (genotipo 6/6); 40 % genotipo 6/7, y 7%, 7/7. Grupo con ictericia: 33% normales, 53% heterozigotos (6/7) y 14% homozigotos (7/7). Al comparar entre los grupos, los recién nacidos con ictericia tenían una tendencia a tener una mayor prevalencia del polimorfismo para el gen de la UGT-1 (p = 0,09). Conclusión: Este estudio sugiere una relación en la población española entre la ictericia neonatal y el síndrome de Gilbert. Estos datos y otros similares obtenidos por varios autores indican la idoneidad de incluir el escrutinio molecular para el síndrome de Gilbert en el protocolo diagnóstico de la ictericia neonatal. Evidentemente, estudios más amplios permitirán definir cuál es el grado exacto de relación entre la presencia de una ictericia neonatal marcada y la presencia de este polimorfismo (AU)

Structural features underlying selective inhibition of protein kinase CK2 by ATP site-directed tetrabromo-2-benzotriazole.

Two novel crystal structures of Zea mays protein kinase CK2alpha catalytic subunit, one in complex with the specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) and another in the apo-form, were solved at 2.2 A resolution. These structures were compared with those of the enzyme in presence of ATP and GTP (the natural cosubstrates) and the inhibitor emodin. Interaction of TBB with the active site of CK2alpha is mainly due to van der Waals contacts, with the ligand fitting almost perfectly the cavity. One nitrogen of the five-membered ring interacts with two charged residues, Glu 81 and Lys 68, in the depth of the cavity, through two water molecules. These are buried in the active site and are also generally found in the structures of CK2alpha enzyme analyzed so far, with the exception of the complex with emodin. In the N-terminal lobe, the position of helix alphaC is particularly well preserved in all the structures examined; the Gly-rich loop is displaced from the intermediate position it has in the apo-form and in the presence of the natural cosubstrates (ATP/GTP) to either an upper (with TBB) or a lower position (with emodin). The selectivity of TBB for CK2 appears to be mainly dictated by the reduced size of the active site which in most other protein kinases is too large for making stable interactions with this inhibitor.

Effects of thiobencarb herbicide to an alga (Nannochloris oculata) and the cladoceran (Daphnia magna).

Chronic toxicity studies were conducted with an algae (Nannochloris oculata) and the cladoceran (Daphnia magna) to determine their relative sensitivities to the thiocarbamate herbicide thiobencarb (S-4-chlorobenzyl diethylthiocarbamate). Most of the algal populations were initially affected by exposure to the herbicide. Thiobencarb concentrations higher than 0.5 mg/L significantly reduced algal densities after 24-h exposure. The 24-h static EC50 in D. magna was 3.01 mg/L. The sublethal effects of 0.3, 0.37, 0.5, 0.75, and 1.5 mg/L of thiobencarb concentrations on the survival, reproduction, and growth of D. magna were monitored for 21 days. The parameters used to determined the effect of the herbicide on D. magna were mean total young per female; mean brood size; days to first brood; intrinsic rate of natural increase (r); growth; and survival. Reproduction was significantly reduced at thiobencarb concentrations of 0.30 mg/L and higher while survival was affected after exposure to 0.75 and 1.5 mg/L of the pesticide. The r value decreased with increasing concentrations of thiobencarb. Growth, as measured by body length, was depressed significantly after exposure to all herbicide concentrations tested.

The crystal structure of the complex of Zea mays alpha subunit with a fragment of human beta subunit provides the clue to the architecture of protein kinase CK2 holoenzyme.

The crystal structure of a complex between the catalytic alpha subunit of Zea mays CK2 and a 23-mer peptide corresponding the C-terminal sequence 181-203 of the human CK2 regulatory beta subunit has been determined at 3.16-A resolution. The complex, composed of two alpha chains and two peptides, presents a molecular twofold axis, with each peptide interacting with both alpha chains. In the derived model of the holoenzyme, the regulatory subunits are positioned on the opposite side with respect to the opening of the catalytic sites, that remain accessible to substrates and cosubstrates. The beta subunit can influence the catalytic activity both directly and by promoting the formation of the alpha2 dimer, in which each alpha chain interacts with the active site of the other. Furthermore, the two active sites are so close in space that they can simultaneously bind and phosphorylate two phosphoacceptor residues of the same substrate.

The replacement of ATP by the competitive inhibitor emodin induces conformational modifications in the catalytic site of protein kinase CK2.

The structure of a complex between the catalytic subunit of Zea mays CK2 and the nucleotide binding site-directed inhibitor emodin (3-methyl-1,6,8-trihydroxyanthraquinone) was solved at 2.6-A resolution. Emodin enters the nucleotide binding site of the enzyme, filling a hydrophobic pocket between the N-terminal and the C-terminal lobes, in the proximity of the site occupied by the base rings of the natural co-substrates. The interactions between the inhibitor and CK2 alpha are mainly hydrophobic. Although the C-terminal domain of the enzyme is essentially identical to the ATP-bound form, the beta-sheet in the N-terminal domain is altered by the presence of emodin. The structural data presented here highlight the flexibility of the kinase domain structure and provide information for the design of selective ATP competitive inhibitors of protein kinase CK2.

Alterations on AChE activity of the fish Anguilla anguilla as response to herbicide-contaminated water.

The inhibition of both total and specific acetylcholinesterase activities was measured in the whole eyes of the yellow eel Anguilla anguilla after exposure to the carbamate thiobencarb. In vivo assays were conducted under a constant flow-through system of thiobencarb-contaminated water (1/60 LC(50) 96 h=0.22 ppm for 96 h) followed by a recovery period in clean water (192 h more). The results indicated a measurable level of AChE activity on eyes of control eels, which resulted in a sensitive indicator of the presence of thiobencarb in the water. The pesticide induced significant inhibitory effects on AChE activity ranging from 35% in total AChE activity to 75% in specific AChE activity. Following 1 week of recovery, AChE activity in eel eyes was still different from that of controls. Specific AChE activity remained significantly depressed (35%). On the other hand, protein content in whole eyes of pesticide-treated eels did not exhibit any significant difference from control animals. It has been accepted that a minimum of 20% reduction in brain AChE would be necessary to be indicative of exposure to anticholinergic agents; however, few data are reported about this effect on sense organs.

Thiobencarb toxicity and plasma AChE inhibition in the European eel.

The acute toxicity of the herbicide thiobencarb (S-4-chlorobenzyl diethylthiocarbamate) was determined for the European eel (Anguilla anguilla). The 24, 48, 72 and 96 hours median lethal concentrations (LC50) were 25.7, 21.7, 17.0 and 13.2 mg/L, respectively. Fish were also exposed to a sublethal thiobencarb concentration (1/60 LC50-96 hr = 0.22 mg/L) during 96 hours in a flow-through system and then an elimination period of 192 hours in clean water was allowed. Eels were removed and blood samples taken out at each exposure time and recovery period in order to evaluate AChE activity. Thiobencarb induced significant inhibitory effects on plasma AChE activity of A. anguilla from the first contact time with the toxicant. This inhibition (under 50% activity) was maintained during the entire exposure period (96 hours) and even those animals transferred to clean water showed plasma AChE activity different from the controls. Differences between total and specific AChE activity were detected during the exposure period. Total AChE activity in the plasma from animals transferred to a medium free of toxicant recovered its normal value while specific AChE activity remained depressed (< 50%) until five days later.

Pesticide toxicokinetics in fish: accumulation and elimination.

Bioaccumulation of fenitrothion in the European eel (Anguilla anguilla) was studied using two sublethal concentrations of this pesticide in a flow-through test system. The pesticide concentrations used were one-tenth (0.002 ppm) and one-fifth (0.04 ppm) the 96-h LC50 of fenitrothion in this species. Steady state was reached early (2 h) when the animals were exposed to 0.02 ppm of toxicant, and after 48 h when the animals were exposed to 0.04 ppm. Toxicokinetic parameters for fenitrothion in eel muscle (K1, K2, and T1/2) were calculated for both experiments. The highest bioconcentration factor was calculated for animals exposed to 0.04 ppm of toxicant, indicating the relationship between the bioconcentration in muscle tissue and the disposition of fenitrothion in the medium. After transfer to clean water (depuration phase), the animals rapidly eliminated the pesticide accumulated. Excretion rate constants (K2) were 0.06 and 0.04 h-1 in both experiments and the half-lives of fenitrothion in muscle tissue were less than 24 h. After 24 h of the recovery period, fenitrothion was not detected in eel muscle of those animals exposed to 0.02 ppm, and in eels exposed to 0.04 ppm of toxicant, a reduction of 91% was reached by the end of the period.

Uptake and elimination kinetics of a pesticide in the liver of the European eel.

This study was undertaken to determine the bioconcentration-elimination process of fenitrothion in the liver of the european eel (Anguilla anguilla). The animals were exposed to two sublethal fenitrothion concentrations corresponding to 1/5 and 1/10 of LC50 96-h in a flow-through test system. Uptake kinetics were determined from liver burdens measured at 2, 8, 24, 48, 56, 72 and 96 hours exposure. After pesticide exposure, animals were transferred to clean water for 72 hours. Fenitrothion elimination was determined after 12, 24, 48 and 72 hours of recovery period. The insecticide showed a high bioconcentration tendency. Steady-state was reached after 48 hours exposure when the animals where exposed to the lowest concentration. This was followed with a rapid elimination process when the animals were transferred to clean water. Animals exposed to 0.04 ppm showed an early accumulation of the toxicant into the liver, and a steady-state was reached after 8 hours. The elimination rate constant K2 of 0.0096 h-1 and 0.06 h-1 were estimated from the data. The biotransformation rate of fenitrothion in the European eel were low as indicated by a relatively short half-life (11.55 h) of the insecticide. An increase in the Hepatosomatic Index was observed after 96 hours of exposure, but no significant differences were found between control and exposed animals.

Bioconcentration and excretion of fenitrothion in the brain of the eel (Anguilla anguilla).

The bioconcentration of fenitrothion in the brain of the european eel (Anguilla anguilla) and its posterior elimination have been studied. Animals were exposed to a sublethal concentration of fenitrothion (0.04 mg/L) for 96 hours in a flow-through test system. After this pesticide exposure, animals were transferred to clean water for 72 hours more. Bioconcentration and elimination processes of fenitrothion were studied in blood and brain. This insecticide showed a strong tendency to bioconcentrate into selected tissues. A steady-state was observed in blood in few hours. Highest accumulation was detected in brain, where any steady-state could be observed. Elimination started rapidly from both tissues when a recovery period was allowed. Elimination kinetics were adjusted to one-compartment model. K2 of 0.015 and 0.044 hr-1 were calculated for fenitrothion in blood and brain. These K2 values were related with a relatively short half-live of fenitrothion in the analyzed tissues; probably due to the low biotransformation rate of this toxicant in the european eel. That fact would protect the animals against many biotransformation products even more toxic than the parent fenitrothion.

Effects of diazinon exposure on cholinesterase activity in different tissues of European eel (Anguilla anguilla).

Cholinesterase (ChE) activity was measured in brain, plasma, and whole eye of Anguilla anguilla experimentally exposed to a sublethal concentration of 0.042 mg/liter (0.50 of the 96-hr LC50) of the organophosphorous pesticide diazinon. Whole eye was the tissue which revealed higher values of ChE activity (8.17 micromol/min/g) in nonexposed animals. Brain, plasma, and whole eye ChE activity of A. anguilla was inhibited at 6, 24, 48, 72, and 96 hr of diazinon exposure. Pesticide induced significant inhibitory effects on the ChE activity of this species ranging from >70% inhibition in brain tissue to >90% in plasma samples. Brain and plasma presented technical difficulties in their collection. Due to its ease of collection, high ChE activity levels, and sensitivity to ChE pesticide inhibition, the use of whole eye is recommended for post-mortem ChE analysis in A. anguilla.

Chronic toxicity of fenitrothion to an algae (Nannochloris oculata), a rotifer (Brachionus calyciflorus), and the cladoceran (Daphnia magna).

Chronic toxicity studies were conducted with an algae (Nannochloris oculata), a rotifer (Brachionus calyciflorus), and a cladoceran (Daphnia magna) to determine their relative sensitivities to the organophosphorus insecticide fenitrothion. The cladoceran D. magna was the most sensitive of the three species. The no observed effect concentrations (NOECs) for the study with the algae (1.0 mg/liter) and for the rotifer (1.0 mg/liter) were higher than the NOEC (0.009 microgram/liter) and the LC50 of 24 hr (0.067 microgram/liter) for D. magna. Most of the algal populations were not initially affected by exposure to fenitrothion. Pesticide concentrations higher than 1.0 mg/liter significantly reduced algal densities after 72 hr exposure. The effects of chronic exposure of the rotifer B. calyciflorus to fenitrothion were evaluated using some demographic parameters: intrinsic rate of natural increase (r), generation time, net reproductive rate, and life expectancy. All the parameters studied decreased with increasing toxicant concentrations. The parameters used to determine the effect of the pesticide on D. magna reproduction were mean total young per female, mean brood size, mean time to first reproduction, and r. The r and the rest of the studied parameters were affected at 0.011-microgram/liter and higher fenitrothion concentrations. Growth, as measured by body length, was only depressed significantly at 0.011 microgram/liter pesticide.