The synthetic estrogen 17α-ethinyl estradiol (EE2) disturbs reproduction and causes gonadal malformation in fish. Effects on the transcription of genes involved in gonad development and function that could serve as sensitive biomarkers of reproductive effects in the field is, however, not well known. We have studied mRNA expression in testes and liver of adult zebrafish (Danio rerio) males treated with 0, 5 or 25ng/L EE2for 14days. qPCR analysis showed that the mRNA expression of four genes linked to zebrafish male sex determination and differentiation, Anti-Mullerian Hormone, Double sex and mab-related protein, Sry-related HMG box-9a and Nuclear receptor subfamily 5 group number 1b were significantly decreased by 25ng/L, but not 5ng/L EE2 compared with the levels in untreated fish. The decreased transcription was correlated with a previously shown spawning failure in these males (Reyhanian et al., 2011. Aquat Toxicol 105, 41–48), suggesting that decreased mRNA expression of genes regulating male sexual function could be involved in the functional sterility. The mRNA level of Cytochrome P-45019a, involved in female reproductive development, was unaffected by hormone treatment. The transcription of the female-specific Vitellogenin was significantly induced in testes. While testicular Androgen Receptor and the Estrogen Receptor-alpha mRNA levels were unchanged, Estrogen receptor-beta was significantly decreased by 25ng/L EE2. Hepatic Estrogen Receptor-alpha mRNA was significantly increased by both exposure concentrations, while Estrogen Receptor-beta transcription was unaltered. The decreased transcription of male-predominant genes supports a demasculinization of testes by EE2 and might reflect reproductive disturbances in the environment.
Little research on the epigenetic phenomenon of DNA methylation has been performed with invertebrates. However, a few studies have shown that effects of transient chemical exposure can be transferred to non-exposed generations through epigenetic inheritance. If this is a wide-spread phenomenon it may have major consequences for current ecological risk assessments. The presence of DNA methylation in Daphnia magna has only very recently been demonstrated, although not quantified. In the present study, the overall content of 5-methyl-2'-deoxycytidine (5 mdC) in the DNA of D. magna was determined. Additionally, the effect of transgenerational Zn exposure on the 5 mdC content was assessed. An optimized LC-MS-MS method proved to be very suitable for measuring overall levels of 5 mdC in relatively small samples of D. magna DNA. The average [5 mdC]/[dG] ratio ranged from 0.13% to 0.81%. A slight but significant decrease in DNA methylation was found in the offspring (F1) of Zn exposed daphnids in the parental generation. However, this effect did not propagate into the next generation (F2). The presence of DNA methylation in D. magna and the finding that different exposure histories can entail different levels of methylation, open the way for further investigations on the implications of epigenetics in aquatic toxicology.
This study was performed to evaluate the effects, if any, of aromatic nitroxides, namely, indolinic nitroxides, on membrane fluidity of rat epithelial cells using steady-state fluorescence. These nitroxides are being increasingly considered as new and versatile compounds to reduce oxidative stress in biological systems. Hence, the results obtained in this study will give more insights on the interaction of these compounds with biological structures which at present is lacking, especially in view of their possible application as antioxidant therapeutic agents. The probes DPH and Laurdan which give information on the hydrophobic and hydrophilic–hydrophobic regions of the membrane bilayer, respectively, showed that nitroxide 1 (1,2-dihydro-2-methyl-3H-indole-3-one-1-oxyl) significantly increases membrane fluidity, whereas the corresponding phenylimino nitroxide derivative 2 (1,2-dihydro-2-methyl-3H-indole-3-phenylimino-1-oxyl) leads to membrane rigidification. The aliphatic nitroxide TEMPO included in this study for comparison produced no modifications. Consequently, it appears that the structure of the heterocyclic rings (aromatic or aliphatic) and the substituents may affect membrane fluidity differently.
It may be hypothesised that as the bioavailable background concentration of an essential metal increases (within natural limits), the natural tolerance (to the metal) of the acclimated/adapted organisms and communities will increase. In this study the influence of acclimation to different copper concentrations on the sensitivity of the freshwater cladoceran Daphnia magna Straus was investigated. D. magna was acclimated over three generations to environmentally relevant copper concentrations ranging from 0.5 to 100 μg Cu/l (copper activity: 7.18×10−15 to 3700×10−12 M Cu2+). A modified standard test medium was used as culture and test medium. Medium modifications were: reduced hardness (lowered to 180 mg CaCO3/l) and addition of Aldrich humic acid at a concentration of 5 mg DOC/l (instead of EDTA). The effects of acclimation on these organisms were monitored using acute mortality assays and long-term assays in which life table parameters, copper body concentrations and energy reserves were used as test endpoints. Our results showed a two-fold increase in acute copper tolerance with increasing acclimation concentration for second and third generation organisms. Copper acclimation concentrations up to 35 μg Cu/l (80 pM Cu2+) did not affect the net reproduction and the intrinsic growth rate. The energy reserves of the acclimated daphnids revealed an Optimal Concentration range (OCEE) and concentrations between 5 and 12 μg Cu/l (0.5–4.1 pM Cu2+) and 1 and 35 μg Cu/l (0.023–80 pM Cu2+) seemed to be optimal for first and third generation daphnids, respectively. Lower and higher copper concentrations resulted in deficiency and toxicity responses. It was also demonstrated that up to 35 μg Cu/l, third generation daphnids were able to regulate their total copper body concentration. These results clearly indicate that bioavailable background copper concentrations present in culture media have to be considered in the evaluation of toxicity test results, especially when the toxicity data are used for water quality guideline derivation and/or ecological risk assessment for metals.
Juvenile rainbow trout, Oncorhynchus mykiss, were exposed to the synthetic estrogen 17α-ethinylestradiol (EE2) through injection (1, 10, 25 and 50 μg EE2/g fish/week) and via water exposure (1, 10 and 100 ng EE2/l). After seven (injection and water exposure) and 14 days (only for water exposure), blood and plasma vitellogenin concentrations were quantified using indirect endpoints, i.e. plasma alkaline-labile phosphorus (ALP), plasma protein and plasma calcium. In addition, the relative gonad (GSI) and liver weight (HSI) were recorded. Actual plasma vitellogenin concentrations were measured with an enzyme immunoassay. Only fish injected with 50 μg EE2/g fish had a significantly higher gonad weight. No concentration-dependent changes in the HSI were detected in fish exposed via the water, but a significant dose-dependent increase of the HSI was observed in fish injected with EE2. Exposure of rainbow trout to EE2 had a significant effect on all tested plasma parameters. Plasma protein, phosphoprotein and calcium concentrations were significantly higher after two weeks exposure to 100 ng EE2/l. Fish injected with 10, 25 and 50 μg EE2/g fish exhibited increased plasma protein concentrations after 1 week. Compared to the controls, plasma ALP and calcium levels were significantly higher in all injected fish. A significant and positive correlation was observed between all three plasma parameters and between these indirect parameters and the actual plasma vitellogenin concentrations. These findings indicate that both the plasma ALP and the plasma calcium assay have a similar sensitivity as that of available antibody-based assays (EIA), at least in EE2 exposure studies, and thus these assays can provide a rapid, simple and cost-effective alternative to available immunoassays.
A previously developed biotic ligand model (BLM) was validated for its capacity to predict acute 48-h EC50 values of copper to Daphnia magna in 25 reconstituted media with different pH values and concentrations of artificial dissolved organic carbon, Ca, Mg and Na. Before the BLM validation, fitting of measured (with a copper ion-selective electrode) and calculated (with the BLM) Cu2+-activity was performed by adjusting the wham model V (i.e. the metal–organic speciation part of the BLM) copper–proton exchange constant to pKMHA=1.9. Using this value, the 48-h EC50 values observed agreed very well with BLM-predicted EC50 values for tests performed at pH<8, but not at all for tests performed at pH>8. Additional experiments demonstrated that this was due to toxicity of the CuCO3 complex, which is the most abundant inorganic copper species at pH>8. This was incorporated into the initial BLM by allowing the binding of CuCO3 (next to Cu2+ and CuOH+) to the biotic ligand of D. magna. The affinity of CuOH+ and CuCO3 for the biotic ligand was approximately five- and 10-fold lower than that of Cu2+, respectively. With the refined BLM, 48-h EC50 values could be accurately predicted within a factor of two not only in all 25 reconstituted media, but also in 19 natural waters. This validated and refined BLM could support efforts to improve the ecological relevance of risk assessment procedures applied at present.
1. The roles of the inhibitory neurotransmitter GABA and the excitatory neurotransmitter glutamate in anoxic survival and anoxic death are discussed, with particular reference to the brain. 2. It is pointed out that the metabolic relationship between GABA and glutamate causes the neural levels of GABA to increase and glutamate to decrease during anoxia. 3. It is suggested that increased levels of GABA could mediate metabolic depression, and, thus, anoxic survival in ectothermic as well as endothermic vertebrates. Furthermore, evidence for a role of GABA in hibernation is discussed. 4. A hypothesis is presented suggesting that hypoxia has been a selective pressure in conserving GABA and glutamate as major inhibitory and excitatory neurotransmitters in vertebrates as well as invertebrates.
1. Single sucrose gap recordings showed that spontaneous action potentials of rat ileal smooth muscle consisted of slow waves and superimposed spikes which generated rhythmic contractions. As external potassium was raised, the resting potential progressively depolarized. 2. Calcium-free salines inhibited spontaneous mechanical activity and inhibited the plateau phase of the action potential, but spontaneous spike depolarizations persisted. 3. Verapamil, nifedipine and diltiazem all inhibited spontaneous mechanical activity and the plateau phase of the action potential, while in addition diltiazem augmented spike amplitude. 4. Mn ions also inhibited mechanical activity and the action potential plateau, without affecting spike activity while the calcium ionophore A23187 enhanced both mechanical and electrical activity with a pronounced effect on spike amplitude. 5. These results are consistent with the view that the plateau phase of the ileal smooth muscle action potential is dependent upon an influx of extracellular calcium possibly through voltage dependent slow calcium channels.
1. Changes in the erythrocyte system of frogs poisoned with tetrachlorwinfos depend on the sex of the animals and the dose of pesticide applied. They are a result of the pathomorphological changes due to translocation of fluids from the tissues to the circulation and swelling of the blood cells. 2. Changes in the leucocyte system of frogs are caused by several mechanisms: lytic action of the pesticide on the blood cell membrane, the stressogenic effect of the agent and enhanced activity of the reticuloendothelial system. 3. The appearance of typical changes due to stress, after even the lowest dose of tetrachlorwinfos, and low LD50 values indicate that this pesticide is highly toxic for frogs. 4. The relatively high susceptibility of frogs to intoxication with tetrachlorwinfos is probably the result of a high affinity of cholinesterase to this pesticide, because of the presence of the P = O bond in its molecule.
1. The toxic effect of deltamethrin, asymmethrin, alphamethrin, cypermethrin and cypermethrin High cis in nymphs III of Triatoma infestans was lower at 28 than 16-degrees-C.2. Prostration in treated nymphs III occurred more rapidly at 28 than 16-degrees-C.3. Symptoms of poisoning rapidly disappeared when nymphs III intoxicated were moved from 16 to 28-degrees-C.4. Nymphs III exposed to low doses of pyrethroids at 28-degrees-C, which did not show symptoms of poisoning, were rapidly affected when they were moved to 16-degrees-C.
1. The high-K (60 mM)-induced ileal tonic tensions were not affected after 5 min with (0.01-1 mM) Cr6+. However, they were inhibited dose-dependently by Cr6+ after 30 min application. 2. Cr6+ inhibited the tonic response to K more strongly than the phasic response. 3. Cr6+ did not displace Ca(2+)-concentration-response curves to the right, although it reduced the Ca2+ response size. 4. The low affinity binding sites of Ca2+ and the tissue ATP concentration during K contraction was not entirely inhibited after 5 min with 0.5 mM Cr6+. However, they were fairly inhibited after 30 min with the Cr6+. 5. We suggest that Cr6+ probably reduced tension by the interference of Ca2+ influx at the cell membrane resulting from the inhibitory action on mitochondria.
1. The study has been carried out on Wistar rats. The aim of the present study was to trace the effect of aluminum on enzyme activities and hematological parameters on erythrocytes. 2. Aluminum decreased activities of acetylcholinesterase, glutathione reductase, glucose-6-phosphate dehydrogenase, and lactate dehydrogenase in the erythrocytes of the animals tested. 3. In the peripheral blood, a significant decrease in the erythrocyte count, hemoglobin level and hematocrit index and increased percentage of reticulocytes and polychromatophilic erythrocytes were observed. 4. The increase in the neutrophilic granulocyte and lymphocyte count was significant. 5. An inhibitory effect of aluminum on the phagocytic activity of granulocytes was also observed.
1. Novel antitumor and antimicrobial glycoproteins were found in the sea hares. These glycoproteins were purified to apparent homogeneity from Aplysia kurodai, Aplysia juliana and Dolabella auricularia, and designated as aplysianins, julianins and dolabellanins, respectively. 2. The nine isolated glycoproteins lysed all the tumor cells tested but did not lyse normal white and red blood cells. 3. The glycoproteins completely inhibited the synthesis of DNA and RNA by tumor cells within 2 hr and caused tumor lysis within 15 hr. 4. Tumor lysis was inhibited by the presence of N-acetylneuraminic acid, suggesting that the recognition of the sugar moiety is a key step in the cytolysis by antitumor glycoproteins from sea hares. 5. These antitumor glycoproteins, except dolabellanin P, also showed antimicrobial activities. 6. The factors were active for Gram-positive and -negative bacteria and some fungi, and their action was not cytocidal but cytostatic. 7. They exerted the antibacterial action by inhibiting nucleic acid synthesis, as does a DNA-inhibiting chemotherapeutic drug. 8. The sequence of the N-terminal part of dolabellanin A was similar to other antibacterial peptides from arthropoda, amphibia and mammals, suggesting that dolabellanin-like antibacterial peptides are common throughout the animal kingdom.
1. Trimethadione (TMO) has the properties required of probe drugs for the evaluation of hepatic oxidizing capacity in vivo. 2. TMO is demethylated to dimethadione (DMO), its only metabolite, in the liver after oral administration. 3. In rats with various types of hepatic intoxicated-, induced- and partially hepatectomized-rats, the serum DMO/TMO ratios, which were measured on blood samples obtained by a single collection 2 hr after oral administration of TMO, correlated well with the degree of hepatic damage or induction. 4. This finding suggests that TMO may be used as a probe drug in the rapid determination of the functional reserve mass of the liver as well as the hepatic oxidizing capacity.
1. Most of the components of the mixed function oxidase (MFO) in hepatic microsomes were reduced by corticosterone implants, and the degree of the reduction in females and at an older age was greater than those in males and at a younger age. 2. Ascorbic acid (AA) prevented the reduction in the MFO caused by corticosterone implants. 3. The activities of aniline hydroxylase and aminopyrine N-demethylase were enhanced by corticosterone implants regardless of AA supplementation. 4. The activity of NADPH-cytochrome c reductase in male broiler was greater than that in females under normal conditions. 5. Corticosterone implants and dietary AA had less influence on the antibody production, especially to T-cell dependent antigen.
1. Mammary gland of mouse (Mus musculus), rat (Rattus rattus), guinea pig (Cavia porcellus), cow (Bos taurus) and pig (Sus scrofa) contains different but always high concentrations of histamine.2. Generally, the tissue histamine is localized in mast cells, although non-mast cell histamine immuno-reactivity is also present in mammary glands of the mouse, cow and pig. No histamine immunoreactive nerves could be detected.3. Mammary glands are able to synthesize and inactivate histamine; the activity of specific histidine decarboxylase and at least one of the catabolizing enzyme-could be demonstrated.4. Histamine fulfils basic criteria for being involved in physiological function of mammary glands.
1. We have studied the binding characteristics of the hepatic VPRs expressed by rat, cow, pig, sheep and human and have demonstrated species heterogeneity. 2. These species differences are manifested as variation in the VPR capacity (rat > cow > pig > human > sheep), and in the affinity of these receptors for their natural ligand AVP (rat = human > pig = cow). 3. A single class of VPRs, with high affinity for AVP, is present in rat, cow, human and pig liver. In contrast, ovine hepatocytes do not express a VPR. 4. The affinity of the V1a-selective antagonist d(CH2)5Tyr(Me)2AVP is highly dependent on the species utilised, such that rat > human > cow.
1. The effects of temperature on the topical toxicity of cypermethrin and fenvalerate to fifth-instar larvae of the fall armyworm, Spodoptera frugiperda, were investigated, as well as their effects on ataxia and prostration. Activities of carboxylesterase and glutathione S-transferase were also assessed.2. Pyrethroid toxicities were characterized by a negative temperature coefficient (TC) between 15 and 27-degrees-C, and a positive TC between 27 and 38-degrees-C. Fenvalerate was more toxic at all test temperatures.3. The median time for the onset of ataxia and prostration decreased as temperature increased.4. Activity (mu mol/min/mg protein) of carboxylesterase and glutathione S-transferase was significantly lower for fenvalerate-treated insects (217.4 and 17.2, respectively) than for cypermethrin (344.7, 39.7).
1. We compared the effects of four anesthetics on heart rate, dorsal and ventral aortic blood pressure, and electrocardiograms of rainbow trout (Oncorhynchus mykiss).2. Exposure to the local anesthetics tricaine methanesulfonate (MS-222) and benzocaine hydrochloride (BZH) produced minimal cardiovascular alterations. Mean dorsal aortic pressure (DAP) decreased during exposure to MS-222, and mean DAP and mean ventral aortic pressure (VAP) increased 15% during recovery from BZH.3. Exposure to the general anesthetic 2-phenoxyethanol (2-PE) or the hypnotic agent etomidate (ET) dramatically decreased heart rate and blood pressures and altered EKG patterns.4. During recovery, VAP and DAP increased above baseline for an extended period. Heart rate and EKG patterns rapidly returned to normal.
1. In rainbow trout liver, dexamethasone caused a reduction in cytochrome P450 1A1 proteins but no change in P450 3A proteins as measured by Western blots using specific anti P450 1A1 and P450 3A sera.2. After dexamethasone administration, the level of P450 1A1 was down to 14% and 8% of the control values at 24 and 48 hr, respectively.3. There was no change in P450 1A1 mRNA abundance at 24 hr but a decrease to 45% of the control value was observed at 48 hr after dexamethasone.4. There is, therefore, a differential response between P450 1A1 and P450 3A to dexamethasone in the trout liver.5. The initial decrease in P450 1A1 protein probably occurs at the translational level while both translational and transcriptional levels are involved 48 hr after dexamethasone administration.
