The role of nitric oxide in the cardiovascular response to chronic and acute hypoxia in White Leghorn chicken (Gallus domesticus).

AIM: Prenatal hypoxia due to placental insufficiency results in deleterious phenotypes and compensatory mechanisms including increased sympathetic tone. Utilizing the embryonic chicken model, we investigated (i) changes in nitric oxide (NO)-mediated tone in response to chronic hypoxic development and (ii) the in vivo role of NO-mediated tone during acute hypoxic exposure, which has not been previously studied. We hypothesized that NO tone on the cardiovascular system would be unaffected by chronic hypoxic incubation in White Leghorn chicken (Gallus domesticus) embryos. METHODS: We measured arterial pressure, heart rate and femoral blood flow (via a Doppler flow probe) in response to acute hypoxia (10% O2 ) and pharmacological manipulations in normoxic- and hypoxic (15% O2 )-incubated embryos. This was performed at 70 and 90% of total incubation time (21 days). At 70% of incubation (day 15), blood volume and chorioallantoic membrane development are maximal; 90% of incubation (day 19) is 1 day prior to lung ventilation. RESULTS: Acute hypoxic exposure decreased femoral flow in both 90% groups, but increased femoral artery resistance in the hypoxic group. NO tone increased during development, but was not affected by hypoxic incubation. Inhibition of NO production by L-NAME (100 mg kg(-1) ) revealed that NO plays a significant role in the flow response to hypoxia. CONCLUSION: Chronic hypoxic incubation has no effect on cardiovascular NO tone during White Leghorn chicken development. In the intact animal, NO function during acute hypoxic stress is suppressed by hypoxic incubation, indicating that chronic hypoxic stress dampens the NO contribution.

Intravascular infusion of PEGylated Au nanoparticles affects cardiovascular function in healthy mice.

Recent advances of nanotechnology in clinical settings have spurred the development of various complex engineered nanoparticles (NPs). NPs share characteristics with ultrafine particles (UFPs; <1 µm) that can cross the pulmonary epithelium and disturb cardiovascular functions. Since these particles are injected directly into the blood stream, it is imperative to clarify whether NPs disrupt cardiovascular functions similar to UFPs. Therefore, we investigated whether engineered polyethylene glycol (PEG)-coated aluminum NPs for biomedical uses disturb cardiovascular functions in healthy mice. Mean arterial blood pressure (MAP) was measured in mice chronically instrumented with telemetric blood pressure transducers, and NPs were administered intravenously (10 mg kg(-1)). The NPs caused a prolonged lowering of MAP 7 days after injection (119.3 ± 3.3 vs. 97.4 ± 7.5 min(-1)), with no effect on the endothelial function as revealed by normal endothelial function of small vessels mounted in a myograph.

Fecundity, 17beta-estradiol concentrations and expression of vitellogenin and estrogen receptor genes throughout the ovarian cycle in female Eastern mosquitofish from three lakes in Florida.

Previous studies of Eastern mosquitofish in contaminated Lake Apopka, Florida, have documented reduced sperm count and sexual behaviour in males but increased fecundity and liver weight in females, compared to nearby reference lakes. Liver weight can be an indicator of vitellogenin (Vtg) synthesis in fish, such as the mosquitofish. It was therefore hypothesized that estrogenic organochlorine pesticides, present at elevated concentrations in animals from Lake Apopka, could cause the reproductive disorders in males, as well as increase female fecundity. We initiated a test of this hypothesis by examining the relationship between 17beta-estradiol (E2) tissue concentrations, hepatic estrogen receptor alpha (ERalpha) and Vtg A, B and C gene expression and fecundity in sexually mature female Eastern mosquitofish from Lake Apopka and two reference lakes, Lake Woodruff and Lake Orange. We observed that female Eastern mosquitofish from one site in contaminated Lake Apopka produced fewer but bigger embryos than females from the other Lake Apopka site and two reference sites. However, female E2 concentrations and hepatic ERalpha and Vtg A, B and C gene expression showed no overall differences among the four sites, and it is therefore unlikely that the differences in fecundity were caused by estrogenic EDCs. In addition, we observed no induction of any of the three Vtg genes in male Eastern mosquitofish at the two Lake Apopka sites. Based on the well-documented high sensitivity of Vtg induction as a biomarker of xenoestrogen exposure, the evidence from the present study does not support the hypothesis that estrogenic EDCs are affecting reproduction in Eastern mosquitofish living in Lake Apopka. Our experimental design tested specifically for effects mediated via the ER, and e.g. antiandrogenic DDT metabolites might still be of importance for mosquitofish reproduction in Lake Apopka.

Antiandrogenic pesticides disrupt sexual characteristics in the adult male guppy Poecilia reticulata.

Environmental contaminants have been identified as endocrine disruptors through their antiandrogenic activity. Thus, as androgen receptor antagonists, the fungicide vinclozolin and the principal DDT metabolite p,p'-DDE have been demonstrated to induce demasculinization in rats. Whether this is also the case in fish remains to be demonstrated. For a period of 30 days, groups of adult male guppies were exposed to vinclozolin, p,p'-DDE, or the therapeutic antiandrogen flutamide (used as positive control) applied to the fodder at concentrations between 0.1 and 100 microg/g fodder. Subsequently, sexual characteristics of relevance to the male reproductive capacity were measured and compared with untreated control fish. All three chemicals caused profound alterations at increasing levels of biological organization, even in these fully matured males. At the cellular level, the three compounds induced a significant reduction in the number of ejaculated sperm cells. At the organ level, the sexually attractive orange-yellow coloration was reduced in area and discolored, and treated fish also had smaller testes. Further, at the organismal level, computer-aided behavior analyses demonstrated a severe disruption in male courtship behavior. We conclude that this demasculinization is consistent with an antiandrogenic action of vinclozolin and p,p'-DDE and is likely to compromise reproductive capability in this fish.

Sexual characteristics are altered by 4-tert-octylphenol and 17beta-estradiol in the adult male guppy (Poecilia reticulata).

Endocrine-disrupting chemicals (EDCs) are believed to interfere with animal reproduction, but only few biomarkers above the cellular level have been developed to assess the adverse effects of these chemicals. Using the guppy (Poecilia reticulata) as a model organism, studies have been undertaken with the object of comparing biomarker responses to EDCs at increasing levels of biological complexity. Short-term exposure to the xenoestrogen 4-tert-octylphenol and the natural estrogen 17beta-estradiol (E2) changed important sexual characteristics in the adult male guppy. Both compounds increased the number of sperm cells in the ejaculates, reduced the area and color intensity of the sexually attractive orange spots, and inhibited testis growth. In the case of E2, the effects on sperm count and coloration were still evident after 3 months of recovery in clean water. Preliminary results on male reproductive capability demonstrated that treated males produced fewer offspring than untreated fish, indicating an impairment of reproduction itself.

Guppy sexual behavior as an effect biomarker of estrogen mimics.

There is widespread concern that some environmental chemicals can reduce the reproductive capability of humans and wildlife by mimicking natural estrogens and disrupting endocrine function. This potential threat to animal populations posed by xenoestrogens has, hardly surprisingly, been met by an intensive global effort to identify and develop biomarkers suitable for screening chemicals for estrogen mimicking capacity. Despite this effort, there are few biomarkers capable of linking exposure to xenoestrogens to impaired reproductive capability. The reproductive success of most animals depends strongly on the ability to perform the appropriate sexual behavior. The sexual display of the male guppy is strongly linked to reproductive success and is readily quantified under laboratory conditions. This preliminary study demonstrates that exposure of adult male guppies to water weakly contaminated with either natural estrogen (17beta-estradiol) or the xenoestrogen (4-tert-octylphenol) causes a dramatic decrease in the rate and intensity of sexual display. It is concluded that quantitative analysis of the sexual display of male guppies holds great promise as a biomarker at the organismal level for the effects of estrogen mimicking xenobiotics.

Pesticide uptake and locomotor behaviour in the woodlouse: an experimental study employing video tracking and (14)C-labelling.

: The toxicity of soil pesticide residues to target and non-target organisms depends on the amount of chemical absorbed by the organism. One of the principle factors governing chemical uptake is the amount of chemical encountered by the animal and, hence, the area of soil contacted. This in turn, depends on the locomotor behaviour of the animal. In the present study, the relationship between the uptake of soil residues of an organophosphate insecticide and locomotor behaviour was examined, employing (14)C-labelled dimethoate and computer-aided video tracking. Groups of male woodlice, Porcellio scaber (Isopoda), walked freely for 22 h on a soil substrate treated with three application rates of the pesticide. A strong correlation was found between pesticide uptake and path length, mean velocity and time spent in locomotor acivity, which is consistent with previously reported modelling studies. Our data suggest a linear relationship for all locomotor parameters except for path length at the highest application rate, where uptake was best described by an inverse exponential relationship. All doses induced hyperactivity in terms of time spent in locomotor activity. However, when compared with a untreated control group, the most pronounced effects were displayed at the lowest dose where path length, mean velocity and turning rate were also significantly different. The number of shifts between locomotor active and inactive periods in the experimental period increased with increasing application rate.

Elevated copper levels during larval development cause altered locomotor behavior in the adult carabid beetle Pterostichus cupreus L. (Coleoptera: Carbidae).

It is generally believed that copper causes changes in carabid communities indirectly by reducing food availability, because these animals are frequently found to have only slightly elevated metal contents even close to pollution sources. Using computer-centered video tracking, the locomotor behavior of adult Pterostichus cupreus carabid beetles was quantified after being raised on copper-contaminated food and soil during larval development. Copper was found to have an acute toxic effect measured in larval mortality, to cause a slight increase in the developmental period of males, but not to effect the emergence weights of adults of either sex. This toxic effect on the larvae was preserved through pupation to the surviving adults, which were normal in size and appearance, but displayed a dramatically depressed locomotor behavior. Copper analysis of these adults revealed that copper levels were either the same as or only slightly elevated in comparison with controls. The findings suggest that the altered locomotor behavior is associated with copper-induced internal structural damage during larval development and therefore expresses a prolonged or permanent effect. Such changes in locomotor behavior are likely to reduce the fitness of the animal under field conditions.

Effects of the pyrethroid insecticide Cypermethrin on the locomotor activity of the wolf spider Pardosa amentata: quantitative analysis employing computer-automated video tracking.

Wildlife in areas surrounding arable land is almost inevitably exposed to pesticide spray. Even at doses far below the lethal level, this presents a threat to vulnerable species. The widely used pyrethroid insecticides, including Cypermethrin, are known for their direct effect on the locomotor apparatus of animals, inducing varying degrees of paresis. Quantitative measurements of the voluntary locomotion of animals express an integrated response to changes in biochemical and physiological processes. In the present study, the effect of Cypermethrin on the voluntary locomotion of the wolf spider Pardosa amentata was quantified in an open field setup, using computer-automated video tracking. Each spider was recorded for 24 hr prior to pesticide exposure. After topical application of 4.6 ng of Cypermethrin, the animal was recorded for a further 48 hr. Finally, after 9 days of recovery, the spider was tracked for 24 hr. Initially, Cypermethrin induced an almost instant paralysis of the hind legs and a lack of coordination in movement seen in the jagged and circular track appearance. This phase culminated in total quiescence, lasting approximately 12 hr in males and 24-48 hr in females. Following paresis, the effects of Cypermethrin were evident in reduced path length, average velocity, and maximum velocity and an increase in the time spent in quiescence. Also, the pyrethroid disrupted the consistent distributions of walking velocity and periods of quiescence seen prior to pesticide application. Our results suggest that normal locomotion had returned 9 days after Cypermethrin application, but that recovery of high velocities was still incomplete.

Quantitative analysis of spider locomotion employing computer-automated video tracking.

The locomotor activity of adult specimens of the wolf spider Pardosa amentata was measured in an open-field setup, using computer-automated colour object video tracking. The x,y coordinates of the animal in the digitized image of the test arena were recorded three times per second during four consecutive 12-h periods, alternating between white and red (lambda > 600 nm) illumination. Male spiders were significantly more locomotor active than female spiders under both lighting conditions. They walked, on average, twice the distance of females, employed higher velocities, and spent less time in quiescence. Both male and female P. amentata were significantly less active in red light (simulated dark environment) than in white light. The results also revealed that P. amentata administers its walking velocity and periods of quiescence according to consistent distributions, which can be approximated by simple mathematical expressions. It was found that this species spends exponentially decreasing time at increasing velocities. The number of quiescent periods, however, follow a power decay distribution at increasing quiescent period duration.

The effect of Cu (II) on the electro-olfactogram (EOG) of the Atlantic salmon (Salmo salar L) in artificial freshwater of varying inorganic carbon concentrations.

The effect of inorganic copper species was studied by recording the receptor potential, electro-olfactogram (EOG), from the olfactory epithelium of Atlantic salmon (Salmo salar L). In a series of experiments, the olfactory organ was irrigated with aqueous copper solutions with concentrations of the free cupric ion (Cu2+) ranging from 0.2 to 9.7 microM. The diverse copper species were created by varying the amount of bicarbonate (NaHCO3) in artificial freshwater solutions of equal total copper concentrations. In general, these copper solutions induced a slow depolarization of the baseline followed by a hyperpolarization. The amplitudes of these variations in baseline potentials increased with increasing concentrations of Cu2+ ion, i.e., decreasing concentrations of NaHCO3. Stimulating the olfactory epithelium with L-alanine during the copper exposure evoked atypical EOG responses. The amplitudes and form of the EOGs changed drastically with increasing Cu2+ concentrations, with significant correlation between the reduction in EOG amplitudes and the Cu2+ concentration. The results indicate that among the copper species tested the toxic effect is caused mainly by the dissolved Cu2+ ion. The results also suggest that the Cu2+ ion exerts its toxic effects on the transduction mechanisms of the olfactory receptor cells. The different EOG profiles obtained in response to varying Cu2+ concentrations indicate that this ion affects the transduction mechanisms at different stages.

Histochemical distribution of zinc in the brain of the rainbow trout, Oncorhynchos myciss. II. The diencephalon.

The distribution of zinc in the diencephalon of the rainbow trout, Oncorhynchos myciss, is described in the present paper, which is the second in a series of three reporting for the first time the distribution of a heavy metal in the fish brain. The Neo-Timm method was used for the histochemical demonstration of zinc. The staining was essentially confined to the neuropil, in all probability representing stained axon terminals, but stained nerve cell bodies were observed in the nucleus lateralis geniculatus and the nucleus cerebellosus hypothalami. Stained fibers were never seen. The staining gave rise to a consistent, specific distribution pattern, which accorded well with the diencephalic nuclei defined on the basis of cytoarchitectural criteria. The diencephalon was in general stained with much higher intensity than the telencephalon, in surprising contrast to the state of affairs in the mammalian, reptilian, and avian brain. In species of these classes, high staining intensities are observed almost exclusively in the telencephalon. The Neo-Timm staining was predominantly distributed in the nuclei of the periventricular zone, but some internal (migrated) nuclei did show a positive staining reaction, namely the nucleus lateralis geniculatus, the anterior thalamic nucleus, the nucleus diffusus tori lateralis, and the nucleus cerebellosus hypothalami. The zinc distribution pattern has been compared with the terminal fields of afferent projections, known from experimental studies, and with the distribution of substance P. The possible function of zinc in synaptic vesicles is considered.

Histochemical distribution of zinc in the brain of the rainbow trout, Oncorhynchos myciss. I. The telencephalon.

The present paper which describes the distribution of zinc in the telencephalon of the rainbow trout, Oncorhynchos myciss, is the first report on the distribution of a heavy metal in the fish brain. Zinc was demonstrated histochemically by silver enhancement using the Neo-Timm method. The staining was mainly confined to the neuropil, but both moderately and intensely stained nerve cell bodies were of common occurrence. Stained fibers were never observed. The staining revealed a specific distribution pattern which could easily be correlated with the telencephalic nuclei defined on the basis of cytoarchitectural features. However, the telencephalon stained much more weakly than the rest of the brain, in striking contrast to the situation in the reptilian, mammalian, and avian brain. In these classes, high staining intensities are observed almost exclusively in the telencephalon. The staining was essentially restricted to the nuclei of the ventral telencephalic area. In the dorsal telencephalic area, only the medial and central zones and medial part of the posterior zone showed comparable staining intensities. The Neo-Timm staining pattern lends support to the view that the pallio-subpallial boundary is between the medial and dorsal zones of the dorsal telencephalic area. The distribution of zinc has been compared with the terminal field of afferent projections, known from experimental mapping, and also with the distribution of substance P and vasoactive intestinal peptide. Finally, the possible functional implications of zinc in synaptic vesicles are considered.

Ultrastructural localization of silver in rat testis and organ distribution of radioactive silver in the rat.

The deposition of silver after a single intravenous injection (2 micrograms Ag g-1 body weight) was studied in the testes of Wistar rats 24 h and 1 and 2 weeks after dosing with radiolabelled 110AgNo3 (2 micrograms Ag and 1.2 kBq g-1 body weight). Also, the temporal accumulation of silver during the experimental period was monitored in the blood, testes, epididymides, kidney, liver and brain. The subcellular distribution of silver within the testes was demonstrated by using the histochemical technique of autometallography. Silver was cleared rapidly from the blood. After an initial rise, concentrations in organs remained almost stable throughout the experimental period. Silver was especially abundant in interstitial macrophages and in the basement membrane. Deposits of silver were found in all cell types of spermatogenesis and in the lysosomes of the Sertoli cells.

Structural and functional effects of heavy metals on the nervous system, including sense organs, of fish.

1. Today, fish in the environment are inevitably exposed to chemical pollution. Although most hazardous substances are present at concentrations far below the lethal level, they may still cause serious damage to the life processes of these animals. 2. Fish depend on an intact nervous system, including their sense organs, for mediating relevant behaviour such as food search, predator recognition, communication and orientation. 3. Unfortunately, the nervous system is most vulnerable and injuries to its elements may dramatically change the behaviour and consequently the survival of fish. 4. Heavy metals are well known pollutants in the aquatic environment. Their interaction with relevant chemical stimuli may interfere with the communication between fish and environment. 5. The affinity for a number of ligands and macromolecules makes heavy metals most potent neurotoxins. 6. The present Mini-Review highlights some aspects of how trace concentrations of mercury, copper and lead affect the integrity of the fish nervous system; structurally, physiologically and biochemically.

Differential effects of mercurial compounds on the electroolfactogram (EOG) of salmon (Salmo salar L.).

The effects on the salmon (Salmo salar L.) electroolfactogram (EOG) of the two mercurials, mercuric chloride (HgCl2) and methylmercuric chloride (CH3HgCl), were studied. The EOG responses were evoked by stimulating the olfactory epithelium with 340 microM L-alanine for 10 sec every second minute during a 1-hr period. Each EOG response consisted of an initial peak component followed by a sustained component with an amplitude about 40% below the peak value. Three experimental series, each comprising six fish, were carried out. In the first series, the rosette was irrigated solely with artificial "fresh water." In the two other series, a 5-min exposure to mercury (HgCl2 or CH3HgCl, at 10(-5) M) was included after 10 min and a 15-min exposure after 45 min. The mercuric ion (Hg2+) eliminated the peak response within 2 min and suppressed the sustained response to about 35%. During the subsequent irrigation with mercury-free fresh water, both EOG components regained about 50% of their initial amplitudes. In contrast, methylmercury induced a steady and parallel decline of both the peak and the sustained responses, which were not reversed by rinsing the epithelium with fresh water. The results of this study demonstrate the vulnerability of the olfactory receptor function in fish to mercury exposure. Also, they show the very different effects of inorganic and organic mercurials upon the EOG.

Histochemical demonstration of mercury in the olfactory system of salmon (Salmo salar L.) following treatments with dietary methylmercuric chloride and dissolved mercuric chloride.

The deposition of organic and inorganic mercury compounds was studied histochemically in the salmon (Salmo salar L.) olfactory system. One group of salmon was given fodder pellets containing methylmercuric chloride (CH3HgCl, 99 micrograms Hg/g) for 4 weeks. Other groups of fish were exposed to dissolved mercuric chloride (HgCl2, 270 micrograms Hg/liter) for 2, 6, and 12 hr, respectively. In both series of experiments, the radioisotope 203Hg was included in order to determine the accumulation of mercury in the olfactory system. Gamma-spectrometry showed that both mercury compounds accumulated in the olfactory rosettes and their nerves. Tissue sections from the rosettes and olfactory nerves were subjected to autometallographic silver enhancement, thereby rendering mercury deposits visible for light and electron microscopy. Microscopic analysis demonstrated an intense and comprehensive Hg deposition in the axons and Schwann cells of both methylmercury- and inorganic mercury-exposed fish. On the other hand, the two mercury compounds showed different staining patterns in the sensory epithelium. The silver grains evoked by methylmercury were localized predominantly in lysosome-like inclusions within the receptor cells, while those produced by HgCl2 exposure were situated mainly along the borders of neighboring cells. The present findings that organic and inorganic mercury compounds were deposited in the olfactory system along its whole length, from the receptor cell apices to the brain, support the electrophysiological results presented elsewhere (Baatrup et al., 1990, Ecotoxicol. Environ. Safety 20, 269-276).

Darkfield illumination improves microscopic detection of metals in Timm's stained tissue.

Deposits of trace or toxic metals can be quickly identified by light microscopical surveys of tissue sections stained for metals by variants of Timm's silver enhancement method. The present work shows that the small, isolated silver grains that label isolated deposits of metal in tissue are undetectable in brightfield light microscopy but are easily detected in darkfield microscopy. Darkfield illumination is therefore recommended for improving the detection of trace or toxic metals in tissue.

Mercury-selenium interactions in relation to histochemical staining of mercury in the rat liver.

Selenium has been suggested to enhance the histochemical staining of mercury when sections of tissue are subjected to the silver-enhancement method. In the present study, histochemical staining patterns of mercury in tissue sections of rat livers were compared with the actual content of organic and inorganic Hg in the livers, in both the presence and the absence of Se. Rats were injected intravenously with 5 micrograms of Hg g-1 body weight as methyl [203Hg] mercury chloride (MeHg) or as [203Hg]mercuric chloride (Hg2+). After 2 h, half the rats received an additional intraperitoneal injection of 2 micrograms of Se g-1 body weight as sodium [75Se]selenite. All the rats were killed 1 h later. Homogenized liver samples were prepared for mercury analysis by two different methods: alkaline digestion and ultrasonic disintegration. Quantitative chemical analysis based on benzene extraction of the radioactively labelled Hg compounds showed that the chemical form of mercury, either organic or inorganic, was preserved from its administration to its deposition in the liver. Light and electron microscopy demonstrated that no silver enhancement of Hg occurred when MeHg alone was present in the sections of tissue, whereas MeHg accompanied by Se induced a moderate deposition of silver grains. In contrast, sections containing Hg2+ alone yielded some staining, and the addition of Se increased the staining dramatically. The results of the present study show that acute selenite pretreatment is a prerequisite for the histochemical demonstration of methyl mercury, and greatly increases the staining of inorganic mercury when applying the silver-enhancement method.