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PubMed2004

Toxicity, histopathological alterations and immunohistochemical CYP1A induction in the early life stages of the seabream, Sparus aurata, following waterborne exposure to B(a)P and TCDD.

In this paper, the toxicity (percentage of hatching and LC50) and histopathological alterations induced by benzo(a)pyrene (B(a)P; 0.032, 0.056, 0.1, 0.32, 0.56 and 0.1 microg l(-1)) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 0.025, 0.05, 0.1, 1.5 and 5 pg l(-1)) have been studied in the early life stages of the seabream, Sparus aurata, from 0 to 15 days post-hatching (dph). Toxicity assays showed that the percentage of hatching decreased with increasing the contaminant concentration. Moreover, the number of hatched larvae was lower for TCDD-exposed eggs in comparison with the B(a)P exposed ones. The sensitivity of the larvae, in terms of LC50, towards B(a)P and TCDD increased with age of the larvae. The LC50 were 0.81 microg l(-1) for B(a)P and 4.37 pg l(-1) for TCDD in neonate larvae and 0.11 microg l(-1) for B(a)P and 1.45 pg l(-1) for TCDD in 5 dph larvae. For histopathological examination, samples from LC50 experiments were taken at different concentrations of B(a)P (between 0.032 and 0.1 microg l(-1)) and TCDD (between 0.025 and 5 pg l(-1)). In both, B(a)P- and TCDD-exposed larvae, a concentration-dependency of the histopathological alterations was detected. In contrast, an age-dependency was not clearly detected, possibly due to the lack of development of mostly the organs in the early life stages. Cytoplasmic vacuolization of hepatocytes, as well as subcutaneous edema and necrosis of the trunk musculature, were the most common histopathological disorders detected in both B(a)P- and TCDD-exposed larvae. On the other hand, there were differences in histopathology on exposure to B(a)P and TCDD. Epithelial desquamation in gills, lack of inflation of the swim bladder, as well as lesions in the nervous system were specific for TCDD, while hepatic, vascular and muscular alterations were common for both toxicants. In parallel to the histopathological examinations, immunohistochemical analyses on cytochrome P450-1A isoenzyme (CYP1A) expression were performed on the same samples. The basal/constitutive distribution of CYP1A and its induction was also analysed in similar stages of larval development of the seabream under control conditions and after sublethal exposure to B(a)P (between 0.032 and 0.1 microg l(-1)) and TCDD (between 0.025 and 5 pg l(-1)). During the endogenous nutrition period (from hatching until 4 dph), constitutive CYP1A immunoreactivity was observed in the syncytium and in the matrix of the yolk sac. On the other hand, during exogenous feeding (between 4 and 10 dph), basal CYP1A immunoreactivity was detected in vascular hepatic system, whereas exocrine pancreas showed no reactivity. In gut, basal CYP1A immunoreactivity was restricted to the intestinal brush border and the apical cytoplasm of some enterocytes. Induced CYP1A immunoreactivity in B(a)P-exposed larvae was detected within cytoplasm of hepatocytes, intestinal enterocytes and endothelial cells of the heart. Finally, in TCDD-exposed larvae, CYP1A induction was also detected in pancreatic acinar cells, as well as in renal epithelial cells. The results of this study provided preliminary evidence that constitutive and inducible CYP1A organ distribution in S. aurata larvae was similar to that existing in adult fish. Moreover, exposure to TCDD was more toxic for the larvae and induced more CYP1A that exposure to B(a)P.

Animals↗
PubMed1982

Gastro-duodenal motor and transit disturbances associated with Haemonchus contortus infection in sheep.

Abomasal and duodenal motility was investigated by electromyography in 4 lambs fitted with permanent abomasal and duodenal cannulae and infected twice with 25,000 Haemonchus contortus larvae at an interval of 39 days. In addition, abomasal and duodenal pH, flow rate of digesta of the duodenum, and faecal water content were determined. The 80-90 min cycles of antral contractions were shortened for at least 3 weeks after infection. This was accompanied by a 26% increase in the number of duodenal migrating myoelectric complexes/day during the intraparietal development of the larvae (10-12 days). Concomitantly, the duodenal flow of digesta increased from 215 +/- 22 ml/h to 318 +/- 15 ml/h 4 days after infection and was associated with a higher water content of the faeces. The frequency of the migrating myoelectric complexes, but not the duration of antral motor cycles (or the duodenal flow rate), was correlated with both gastric (P less than 0.01) and duodenal (P less than 0.05) pH after the first and second infections. The duodenal flow rate was correlated with the Cl- concentrations of the abomasal contents (P less than 0.01) and with the duodenal pH (P less than 0.05). It is concluded that, (i) the duodenal motor disturbances associated with experimental H. contortus infection are related to the alterations in abomasal gastric acid secretion which occurs during the histotrophic phase of larval development and (ii) the increased duodenal flow is a consequence of ionic permeability changes of the gastro-intestinal mucosae.

Abomasum↗
PubMed1999

Immunocytochemical distribution of cytochrome P4501A (CYP1A) in developing gilthead seabream, Sparus aurata.

CYP1A is a major inducible enzyme in the metabolism of xenobiotic substrates. In this paper we investigate by means of immunohistochemistry, the tissue distribution of constitutive cytochrome P4501A (CYP1A) during the period of endogenous nutrition (from hatching until day 4) in developing gilthead seabream, Sparus aurata larvae. For this purpose, a polyclonal antiserum (BN-1, Biosense Laboratories) directed against conserved piscine CYP1A sequences was used on paraffin-embedded sections from seabream larvae. From hatching onward, CYP1A immunoreactivity was observed in the following tissues and cells: syncytial, oil-globule envelopes and matrix of the yolk-sac, kidney (epithelia of renal tubules), cardiac muscle cells, skin epidermal cells, troncal musculature, enterocytes of different intestinal regions, goblet cells of the bucco-pharyngeal region, gill epithelial cells and the endothelia of the vascular system of various tissues (especially from liver and brain). Moreover, eye (retina), olfactory epithelium and some positive nerve fibers located in the proximity of the olfactory bulbs and running ventrally toward the posterior brain were strongly CYP1A immunoreactive. In general, the intensity of immunostaining increased with larval development.

Animals↗
PubMed2004

Comparing genomic expression patterns across species identifies shared transcriptional profile in aging.

We developed a method for systematically comparing gene expression patterns across organisms using genome-wide comparative analysis of DNA microarray experiments. We identified analogous gene expression programs comprising shared patterns of regulation across orthologous genes. Biological features of these patterns could be identified as highly conserved subpatterns that correspond to Gene Ontology categories. Here, we demonstrate these methods by analyzing a specific biological process, aging, and show that similar analysis can be applied to a range of biological processes. We found that two highly diverged animals, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, implement a shared adult-onset expression program of genes involved in mitochondrial metabolism, DNA repair, catabolism, peptidolysis and cellular transport. Most of these changes were implemented early in adulthood. Using this approach to search databases of gene expression data, we found conserved transcriptional signatures in larval development, embryogenesis, gametogenesis and mRNA degradation.

Age Factors↗
PubMed2005

Survival strategy of Echinococcus multilocularis in the human host.

As exemplified by "aborted" calcified liver lesions commonly found in patients from endemic areas, Echinococcus multilocularis metacestodes develop only in a minority of individuals exposed to infection with the papasite. Clinical research has disclosed some aspects of the survival strategy of E. multilocularis in human hosts. Clinical observations in liver transplantation and AIDS suggest that suppression of cellular/Th1-related immunity increases disease severity. Most of the studies have stressed a role for CD8+ T cells and for Interleukin-10 in the development of tolerance. A spontaneous secretion of IL-10 by the PBMC seems to be the immunological hallmark of patients with progressive forms of alveolar echinococcosis (AE). IL-10-induced inhibition of effector macrophages, but also of antigen-presenting dendritic cells, may be operating and allowing parasite growth and survival. The genetic correlates of susceptibility to infection with E. multilocularis are clearer in humans than in the mouse model. A significant link between MHC polymorphism and clinical presentation of AE has been shown, and the spontaneous secretion of IL-10 in patients with a progressive AE is higher in patients with the HLA DR3+, DQ2+ haplotype. Clustering of cases in certain families, in communities otherwise exposed to similar risk factors, also points to immuno-genetic predisposition factors that may allow the larva to escape host immunity more easily. The first stage of larval development may be crucial in producing "danger signals" stimulating the initial production of cytokines. Therapeutic use of Interferon alpha is an attempt to foil the survival strategy of E. multilocularis.

Animals↗
PubMed2002

Molecular cloning and developmental expression of zinc finger transcription factor MTF-1 gene in zebrafish, Danio rerio.

Metal-responsive transcription factor, MTF-1 is a zinc finger protein, shown to be essential for embryonic development. Homozygous knockout mouse embryos for MTF-1 die in utero at day 14 of gestation, due to liver decay. In the present study, we report the complete nucleotide sequence of cDNA encoding zebrafish MTF-1 and the amino acid sequence similarity with that of mouse, human, fish and Drosophila. The size of the zebrafish MTF-1 cDNA is 3,379 bp and the coding region (1,779 bp) encodes a polypeptide of 593 amino acids. The putative zinc finger and transactivation domains comprised by zebrafish MTF-1 were also determined. The zebrafish MTF-1 shows high identity of 97, 93, 93 and 67% in the DNA binding zinc finger domain and 51, 44, 48 and 20% overall identity with fugu, human, mouse and Drosophila, respectively. RT-PCR results show the maternal expression of MTF-1 transcripts. The pattern of MTF-1 gene expression during embryonic and early larval development was studied by whole-mount in situ hybridization using DIG-labeled anti-sense RNA probe. Stronger and ubiquitous expression was observed during the embryonic stages whereas, specific expression, especially in the neural parts, was observed throughout the stages studied after hatching.

Amino Acid Sequence↗
PubMed1990

Temperature, thyroxine, and induced metamorphosis in tadpoles of a primitive frog, Ascaphus truei.

To understand the environmental factors and endocrine mechanisms which regulate the extended period (2 to 4 years) of larval development in Ascaphus, the effect of temperature and thyroxine (T4 by immersion) on metamorphic changes of the tail, limb buds, operculum, anal fold, oral disc, and teeth were studied in premetamorphic and prometamorphic tadpoles. (1) Control tadpoles did not undergo metamorphic change at any of the temperatures tested. (2) When tadpoles were kept in thyroxine solutions at 5 degrees, there were no changes in developmental morphology, and tadpoles kept at 18 and 20 degrees showed significant metamorphic changes in 5 to 6 days, but there was a high mortality at the higher T4 concentrations. The optimum temperature is about 10 degrees, and significant changes were completed in 3 weeks. (3) The premetamorphic tadpoles were less sensitive to T4 than prometamorphic tadpoles. The most responsive tissues are teeth and operculum, and the tail tissue shows the lowest sensitivity. (4) Duration of exposure to hormone and concentration also affect the growth or regression of larval tissue.

Animals↗
PubMed1984

Two isoenzymes of glyceraldehyde-3-phosphate dehydrogenase in Caenorhabditis elegans. Isolation, properties, and immunochemical characterization.

Two glyceraldehyde-3-phosphate dehydrogenases have been separated and purified from the nematode Caenorhabditis elegans. As defined by starch gel electrophoresis, the faster-migrating isoenzyme, glyceraldehyde-3-phosphate dehydrogenase-2, increases its activity during postembryonic development. In contrast, the slower-migrating isoenzyme, glyceraldehyde-3-phosphate dehydrogenase-1, is enriched in isolated embryos. Both isoenzymes were initially purified by ammonium sulfate fractionation, gel filtration, and NAD+-agarose affinity chromatography. The separation of both isoenzymes as well as their purification to homogeneity was obtained by preparative chromatofocusing. The subunit molecular weight of each isoenzyme is 38,500 +/- 500. A tetrameric native molecular weight of 157,000 +/- 2000 was determined for glyceraldehyde-3-phosphate dehydrogenase-2. Monospecific rabbit polyclonal antibodies were initially raised against the major isoenzyme and subsequently used to characterize both isoenzymes. Staphylococcus aureas V8 protease digests of each isoenzyme were separated electrophoretically and stained immunochemically, providing evidence that the two isoenzymes differed in their amino acid sequences. Developmental immunocytochemical studies suggest that the embryonic-enriched isoenzyme, glyceraldehyde-3-phosphate dehydrogenase-1, is present in all cells. The second isoenzyme, exhibiting the major activity during postembryonic larval development, may define a body-wall-muscle specific activity which is located within the actin-containing I and A zones of the nematode's sarcomeres.

Aging↗
PubMed1998

Effects of cold stress on survival and reproduction of Haematobia irritans (Diptera: Muscidae).

Adult horn fly, Haematobia irritans (L.), survival, mating success, and ovarian development were measured following exposure of pupae to 4 degrees C for 2, 3, and 4 wk, and were compared with an untreated control group held at 29 degrees C. Survival varied inversely with duration of low-temperature exposure, and ranged from 92.1% in the control to 7.0% following a 4-wk exposure. Mating success also varied inversely with duration of low-temperature exposure, and ovarian development was delayed, especially in the week 4 group. Reproductive and population parameters were measured in a 2nd cohort of horn flies following exposure of pupae to 1-4 wk at 4 degrees C, and in a control. Adult longevity varied inversely with duration of exposure. Mean longevity was greatest in the week 1 group at 17.6 d, followed by the control at 14.7 d. Week 4 flies lived an average of 2.2 d and produced no eggs. The preoviposition period was longest in the week 3 group at 7.3 d. In cohort 2, control and week 1 flies produced the highest number of eggs, eggs per female, and eggs per day compared with other groups. Eggs were produced over a mean of 6.8 d by week 3 flies compared with 25.4 d by week 1 flies. Reproductive rates varied inversely with low-temperature exposure duration, as did F1 generation egg hatch, larval development, and adult emergence. Survivorship and fecundity schedules are discussed with respect to the effects of low-temperature exposure duration. The effects of low-temperature exposure duration on horn fly reproduction and life-history patterns are discussed with respect to rearing, storage, and ecology.

Animals↗
PubMed2003

Bioactive aldehydes from diatoms block the fertilization current in ascidian oocytes.

The effects of bioactive aldehydes from diatoms, unicellular algae at the base of the marine food web, were studied on fertilization and early development processes of the ascidian Ciona intestinalis. Using whole-cell voltage clamp techniques, we show that 2-trans-4-trans-decadienal (DD) and 2-trans-4-cis-7-cis-decatrienal (DT) inhibited the fertilization current which is generated in oocytes upon interaction with the spermatozoon. This inhibition was dose-dependent and was accompanied by inhibition of the voltage-gated calcium current activity of the plasma membrane. DD and DT did not inhibit the subsequent contraction of the cortex. Moreover, DD specifically acted as a fertilization channel inhibitor since it did not affect the steady state conductance of the plasma membrane or gap junctional (GJ) communication within blastomeres of the embryo. On the other hand, DD did affect actin reorganization even though the mechanism of action on actin filaments differed from that of other actin blockers. Possibly this effect on actin reorganization was responsible for the subsequent teratogenic action on larval development. The effect of DD was reversible if oocytes were washed soon after fertilization indicating that DD may specifically target certain fertilization mechanisms. Thus, diatom reactive aldehydes such as DD may have a dual effect on reproductive processes, influencing primary fertilization events such as gating of fertilization channels and secondary processes such as actin reorganization which is responsible for the segregation of cell lineages. These findings add to a growing body of evidence on the antiproliferative effects of diatom-derived aldehydes. Our results also report, for the first time, on the action of a fertilization channel blocker in marine invertebrates.

Aldehydes↗
PubMed2000

Identification and validation of a gene involved in anchorage-independent cell growth control using a library of randomized hairpin ribozymes.

We have developed a library of hairpin ribozyme genes that can be delivered and expressed in mammalian cells with the purpose of identifying genes involved in a specific phenotype. By applying the appropriate phenotypic selection criteria in tissue culture, we can enrich for ribozymes that knock down expression of an unknown gene or genes in a particular pathway. Once specific ribozymes are selected, their target binding sequence is used to identify and clone the target gene. We have applied this technology to identify a putative tumor suppressor gene that has been activated in HF cells, a nontransformed revertant of HeLa cells. Using soft agar growth as the selection criteria for gain of transformation, we have isolated ribozymes capable of triggering anchorage-independent growth. Isolation of one of these ribozymes, Rz 568, led to the identification and cloning of the human homologue of the Drosophila gene ppan, a gene involved in DNA replication, cell proliferation, and larval development. This novel human gene, PPAN, was verified as the biologically relevant target of Rz 568 by creating five additional "target validation" ribozymes directed against additional sites in the PPAN mRNA. Rz 568 and all of the target validation ribozymes reduced the level of PPAN mRNA in cells and promoted anchorage-independent growth. Exogenous expression of PPAN in HeLa and A549 tumor cells reduced their ability to grow in soft agar, underscoring its role in regulating anchorage-dependent growth. This study describes a novel method for gene discovery where the intracellular application of hairpin ribozyme libraries was used to identify a novel gene based solely on a phenotype.

Agar↗
PubMed2004

Transcriptional regulation in cowpea bruchid guts during adaptation to a plant defence protease inhibitor.

Cowpea bruchid, when fed on a diet containing the soybean cysteine protease inhibitor soyacystatin N (scN), activates an array of counter-defence genes to adapt to the negative effects of the inhibitor and regain its normal rate of feeding and development. A collection of 1920 cDNAs was obtained by differential subtraction with cDNAs prepared from guts of the 4th instar larvae of scN-adapted (reared on scN-containing diet) and scN-unadapted (reared on regular scN-free diet) cowpea bruchids. Subsequent expression profiling using DNA microarray and Northern blot analyses identified ninety-four transcript species from this collection that are responsive to dietary scN. scN-adapted insects induced genes encoding protein and carbohydrate digestive enzymes, probably to help meet their carbon and nitrogen requirements. Up-regulation of antimicrobial and detoxification protein genes may represent a generalized defence response. Genes down-regulated by scN reflected physiological adjustments of the cowpea bruchids to scN challenge. A large portion of the responsive genes, presumably involved in carrying out the counter-defence response, were of unknown function. The full-length cDNA of an scN-inducible cathepsin B-like cysteine protease was obtained. Its transcriptional response to scN during larval development contrasts with the pattern of the cathepsin L family, the major digestive enzymes. These results suggest cathepsin B-like cysteine proteases may play a crucial role in cowpea bruchid adaptation to dietary scN.

Adaptation, Physiological↗
PubMed2006

Effects of temperature stress and pesticide exposure on fluctuating asymmetry and mortality of Copera annulata (Selys) (Odonata: Zygoptera) larvae.

Although there have been some investigations into the effects of insecticide on the level of fluctuating asymmetry (FA) of adult damselflies, the cooperative effects of environmental factors on FA of larval damselflies were known little. This paper explored effects of exposure to temperature and pesticide on larval development of the damselfly Copera annulata (Selys). A conventional life history trait (mortality) and developmental instability (estimated by calculating fluctuating asymmetry of bilaterally symmetrical structures) were used to measure stresses in this paper. The results showed that temperature and different concentrations of pesticide produced significant effects only on developmental stability of some characters. The FA values of three traits decreased at lower concentrations, then increased slowly with increased insecticide doses. The FA values of four traits decreased slowly with increased temperatures. However, the interaction between different concentrations of insecticide and temperature was complicated and only produced significant effects on five traits. Insecticide treatment did not significantly affect mortality of the larvae of damselfly. However, mortality was significantly positively associated with temperature. There were significantly negative associations between mortality and the FA values of three traits. These results may be caused by higher mortality and short rearing time although we did not find the significant effects of concentrations on mortality. Therefore, we speculate FA may be induced if larval damselflies were treated during longer term and FA has potential as a more specific bioindicator of stresses if we guarantee enough longer rearing time without higher mortality under stressful environment.

Animals↗
PubMed1993

Brain creatine kinase activity during ontogeny of the cichlid fish oreochromis mossambicus and the clawed toad Xenopus laevis, influence of gravity?

The development of creatine kinase (CK) activity was studied in the brain of cichlid fish and clawed toads. The activity of CK in the whole brain of the fish decreases immediately after hatching (stage 6) from values of about 135 nmol substrate cleaved/mg protein/min to a value of about 105 at stage 8 (5 days post hatch at 20 degrees C). With the exception of a significant peak (125 nmol) between stages 9 and 10 (7 and 9 days respectively, post hatch at 20 degrees C) and a small intermediate peak at stages 12 and 13 (about 10 days post hatch at 20 degrees C) a constant level of about 100 nmol cleaved substrate is maintained until maturity. In contrast, CK activity was determined to be 3-fold higher in the whole brain of the clawed toad. With the exception of two significant peaks at stages 47 and 49 (5 and 12 days respectively, post fertilization at 23 degrees C) a value of about 360 nmol was found during larval development and metamorphosis, as well as in the adult brain. In investigating the possible influence of gravity on CK activity during early ontogeny of the brain both animal species were exposed to hyper-gravity (3 +/- 1 g) for 7 days. A significant decrease of total CK activity of 20% was found in the fish brain and of about 5% in the toad.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed2005

Influence of saccharose on the development of cercariae from Schistosoma mansoni strains BH and SJ.

The development of cercariae from Schistosoma mansoni strains BH and SJ in Biomphalaria glabrata and Biomphalaria tenagophila treated with saccharose was studied. The molluscs were maintained in dechlorinated tap water containing 0.01% saccharose. After one week of treatment with saccharose, B. glabrata and B. tenagophila were exposed to ten S. mansoni miracidia, from BH and SJ strains respectively. Control snails of both species were maintained in dechlorinated tap water without saccharose and exposed to the same number of miracidia. There was no significant difference between the infection rates of snails treated or not with saccharose. However, the two groups of B. glabrata had significantly greater infection rates than the corresponding B. tenagophila groups. Molluscs treated with saccharose had a lower survival rate, with the greatest mortality occurring immediately before and at the beginning of cercariae release. Treatment with saccharose did not result in the release of more cercariae, but larvae from molluscs so treated showed a greater capacity to penetrate mouse skin, which was attributed to the greater energy supply during larval development in the mollusc.

Animals↗
PubMed2006

Biological control of nematode parasites in sheep.

In a world in which sheep producers are facing increasing problems due to the rapid spread of anthelmintic resistance, the battle against gastrointestinal parasitic nematodes is a difficult one. One of the potential new tools for integrated control strategies is biological control by means of the nematode-destroying microfungus Duddingtonia flagrans. This fungus forms sticky traps that catch developing larval stages of parasitic nematodes in the fecal environment. When resting spores (chlamydospores) of this fungus are fed daily to grazing animals for a period of time, the pasture infectivity and thus, the worm burden of grazing animals are lowered, especially in young lambs. Research has been conducted throughout the world covering many different climates and management systems. An Australian parasite model showed that if the fungus performs efficiently (> or =90% reduction in worm burden) for 2 or 3 mo, it should contribute significantly to a reduction in the number of dead lambs otherwise occurring when managed only by anthelmintic treatment and grazing management. Feeding or field trials have clearly demonstrated that dosing with a few hundred thousand spores per kilogram of live BW not only reduced the number of infective larvae but also increased the BW of the lambs compared with controls not given fungus. Initial Australian work with feeding spores by means of a block formulation or a slow-release device has shown some promise, but further work is needed to fully develop these delivery systems. In tropical Malaysia, small paddock trials and field studies resulted in significant improvements, in terms of lower worm burdens and increased live BW, when feeding half a million spores daily to grazing lambs. Additional benefits have been observed when the fungus is employed in combination with a fast rotational grazing system. Research has also demonstrated that spores can be delivered in slightly moist feed block material, but only if such blocks are consumed rapidly, because of their very short shelf life. In the northern, temperate Danish climate it has been demonstrated that daily feeding of half a million spores per kilogram of live BW can lead to significant production benefits, with increased live BW gain in fungus-exposed animals. Biological control of parasitic nematodes in sheep seems to hold promise for the future, but to be able to assist producers, the optimal delivery system needs to be refined and further developed. In addition, more work will be needed to define the best use of this technology in different geographic regions.

Animals↗
PubMed1999

Amphibians as a model to study endocrine disruptors: II. Estrogenic activity of environmental chemicals in vitro and in vivo.

Several environmental chemicals are known to have estrogenic activity by interacting with development and functions of endocrine systems in nearly all classes of vertebrates. In order to get a better insight of potential estrogenic effects on amphibians caused by environmental pollution this study aims to develop a model for investigating endocrine disruptors using the amphibian Xenopus laevis. In that model the potential estrogenic activity of endocrine disruptors is determined at several levels of investigation: (I) binding to liver estrogen receptor; (II) estrogenicity in vitro by inducing vitellogenin synthesis in primary cultured hepatocytes; and (III) in vivo effects on sexual development. Here we deal with establishing methods to assay estrogenic activity of environmental chemicals in vitro and in vivo. In vitro we used a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) technique to determine mRNA-induction of the estrogenic biomarker vitellogenin in primary cultured hepatocytes of male Xenopus laevis. Time courses of vitellogenin-mRNA in the presence and absence of 10(-6) M 17 beta-estradiol (E2) resulted in a marked loss of mRNA from controls after 2 days while E2 treatment kept vitellogenin-mRNA at a relatively stable level. After 36 h of incubation estrogenic activities of E2, 4-nonylphenol (NP), and 2,2-bis-(4-hydroxyphenyl)-propan (bisphenol A) at concentrations ranging from 10(-10) to 10(-5) M were assayed by RT-PCR of vitellogenin-mRNA and showed the following ranking of dose-dependent potency: E2 > NP > bisphenol A. These in vitro results were confirmed further by in vivo experiments determining sexual differentiation of Xenopus laevis after exposure to E2 and environmental chemicals during larval development. Concentrations of 10(-7) and 10(-8) M E2 as well as 10(-7) M of NP or bisphenol A caused a significant higher number of female phenotypes compared to controls indicating a similar ranking of estrogenic potencies in vivo as in vitro. In addition, butylhydroxyanisol and octylphenol, both showed feminization at 10(-7) M while octylphenol was also effective at 10(-8) M. In summary these results demonstrate for the first time the use of a semiquantitative RT-PCR technique for screening estrogenicity by assaying mRNA induction of the estrogenic biomarker vitellogenin in vitro. The combination of this newly developed method with classical exposure experiments is necessary for determination of the biological significance of estrogenic chemicals.

Animals↗
PubMed2002

Ochlerotatus albifasciatus in rain pools of Buenos Aires: seasonal dynamics and relation to environmental variables.

The immature stages of Ochlerotatus albifasciatus develop in temporary pools. The present study aims at evaluating the seasonal dynamics of the aquatic stages of this mosquito, also analyzing the relationship among their presence and breeding success to some relevant climatic and environmental variables in the ephemeral rain pools of an urban park. Nineteen cohorts of O. albifasciatus that developed synchronously after rain events were recorded in all seasons. The proportions of mosquito-positive pools were significantly higher during the fall-winter period than in the spring-summer months (p < 0.001). The presence of this mosquito species was positively related to the amount of rain (p < 0.001), whereas negatively correlated to air temperature (p < 0.05) within a 5.2 to 29.7 degrees C range. The distribution of the number of cohorts per pool throughout the year was grouped (variance/mean: 3.96), indicating that these habitats were not equally suitable as breeding sites. The immature stages of O. albifasciatus were detected in pools belonging to all of the categories of surface area, depth, duration, vegetation cover, and insolation. However, the proportion of pools where immature mosquitoes were detected was positively and significantly related to surface, depth, duration, and vegetation cover. On the other hand, the proportion of mosquito-positive pools was higher at an intermediate insolation degree. Our results suggest that although preimaginal stages were present in all seasons, high temperatures may be unfavorable to larval development, and substrate vegetation may regulate water temperature. The positive relationship between the proportion of mosquito-positive pools and pool size and duration might reflect a strategy of O. albifasciatus to accomplish immature development.

Animals↗