Meeting report: international workshop on endocrine disruptors: exposure and potential impact on consumers health.

The French Agency for Food, Environmental and Occupational Health and Safety (Anses) hosted a two-day workshop on Endocrine Disruptors: Exposure and Potential Impact on Consumers Health, bringing together participants from international organizations, academia, research institutes and from German, Swedish, Danish and French governmental agencies. The main objective of the workshop was to share knowledge and experiences on endocrine disruptors (ED) exposure and potential impact on consumers' health, to identify current risk assessment practices and knowledge gaps and issue recommendations on research needs and future collaboration. The following topics were reviewed: (1) Definition of ED, (2) endpoints to be considered for Risk assessment (RA) of ED, (3) non-monotonic dose response curves, (4) studies to be considered for RA (regulatory versus academic studies), (5) point of departure and uncertainty factors, (6) exposure assessment, (7) regulatory issues related to ED. The opinions expressed during this workshop reflect day-to-day experiences from scientists, regulators, researchers, and others from many different countries in the fields of risk assessment, and were regarded by the attendees as an important basis for further discussions. Accordingly, the participants underlined the need for more exchange in the future to share experiences and improve the methodology related to risk assessment for endocrine disrupters.

The Mouse Genome Database (MGD): from genes to mice--a community resource for mouse biology.

The Mouse Genome Database (MGD) forms the core of the Mouse Genome Informatics (MGI) system (http://www.informatics.jax.org), a model organism database resource for the laboratory mouse. MGD provides essential integration of experimental knowledge for the mouse system with information annotated from both literature and online sources. MGD curates and presents consensus and experimental data representations of genotype (sequence) through phenotype information, including highly detailed reports about genes and gene products. Primary foci of integration are through representations of relationships among genes, sequences and phenotypes. MGD collaborates with other bioinformatics groups to curate a definitive set of information about the laboratory mouse and to build and implement the data and semantic standards that are essential for comparative genome analysis. Recent improvements in MGD discussed here include the enhancement of phenotype resources, the re-development of the International Mouse Strain Resource, IMSR, the update of mammalian orthology datasets and the electronic publication of classic books in mouse genetics.

The Mouse Genome Database (MGD): integrating biology with the genome.

The Mouse Genome Database (MGD) is one component of the Mouse Genome Informatics (MGI) system (http://www.informatics.jax.org), a community database resource for the laboratory mouse. MGD strives to provide a comprehensive knowledgebase about the mouse with experiments and data annotated from both literature and online sources. MGD curates and presents consensus and experimental data representations of genetic, genotype (sequence) and phenotype information including highly detailed reports about genes and gene products. Primary foci of integration are through representations of relationships between genes, sequences and phenotypes. MGD collaborates with other bioinformatics groups to curate a definitive set of information about the laboratory mouse and to build and implement the data and semantic standards that are essential for comparative genome analysis. Recent developments in MGD discussed here include an extensive integration of the mouse sequence data and substantial revisions in the presentation, query and visualization of sequence data.

Synthesis of heparin partial structures and their binding activities to platelets.

A synthetic pentasaccharide corresponding to the antithrombin III-binding region in heparin was also found to bind to human platelets. To identify the platelet-binding site in the pentasaccharide which is expected to be a novel sequence in heparin responsible for its platelet-binding, five partial structures of this particular pentasaccharide were synthesized. In a competitive assay using [3H]-heparin, a trisaccharide, O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-glucopyranosyl)-1--> 4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1-->4)-2-deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranose, was concluded to be a high-affinity site for heparin's binding to platelets.

A novel crosslinking reagent and its application for the detection and isolation of heparin-binding protein(s) on the platelet surface.

A new hetero-bifunctional photo crosslinking reagent, 2-(4-azidoanilyl)-4-(4-azabicyclo-[2,2, 2]hexylammonio)-6-morpholino-1,3,5-triazine chloride, was designed to detect and isolate heparin-binding protein(s) that may act as heparin-receptor(s) on the platelet surface. In a preliminary study using ethanol as a model substrate, the reagent was shown to react with the alcoholic hydroxy group under mild conditions and its crosslinking photoreactivity was high. The reagent effectively formed similar covalent bonds with heparin, while preserving its anticoagulant anti-Xa activity. [3H]Heparin labeled with this reagent crosslinked to antithrombin III very specifically but not to ovalbumin, as analyzed by the Bio-imaging Analyzer System (BAS, Fuji Photo Film, Tokyo). Affinity crosslinking of [3H]heparin was then used to detect heparin-binding proteins on the surface of intact platelets. Several discrete protein bands were detected by the BAS-imaging of SDS-PAGE.

Acute haloperidol attenuates the hypomotility induced with 7-hydroxy-DPAT.

Dopamine D3 receptors have been implicated in pathophysiological substrates of schizophrenia, and neuroleptic drugs which are antagonists primarily at D2 receptors possess therapeutic activity in this disorder. In the present study, rats tested for hypomotility induced by 7-hydroxy-DPAT (7OH, a selective D3 agonist) were pretreated with the neuroleptic haloperidol. These animals showed an attenuated agonist-induced suppression of behavior compared with rats receiving 7OH alone. The drug combination also 'normalized' dopamine metabolism in the frontal cortex, as turnover ratios which are typically enhanced by acute neuroleptic administration were no longer significantly increased when 7OH was also given. These observations suggest that the effects of haloperidol in cortical regions regulating limbic locomotor systems may be important for therapeutic efficacy in schizophrenic symptoms generated from a D3 substrate.

Effects of morphiceptin in the medial preoptic area on male sexual behavior.

Morphiceptin, a selective mu opioid agonist, injected into the medial preoptic area (MPOA), delayed the onset of copulation in male rats, but did not affect genital reflexes, sexual motivation or general motor activity. In a dose-dependent manner, morphiceptin (100 ng and 1000 ng) injected into the MPOA increased mount and intromission latencies. Similar injections of morphiceptin into the ventromedial hypothalamus had no effect on any parameter of copulation. The increase in copulatory latencies following the injection of the highest dose of morphiceptin was blocked by pretreatment with the opioid antagonist naloxone. In the X-maze task, morphiceptin had no effect on sexual motivation, as measured by the percentage of trials on which the male chose the female's chamber, but it increased the number of trials in which the subject did not select a chamber within 60 s and the latency to the female the first time he chose her chamber. Similar to the copulation task, the mount and intromission latencies were also increased in the X-maze, after the male reached the female. Morphiceptin in the MPOA had no effect on ex copula genital reflexes, tested in restrained supine males, or on motor activity, tested in a grid box. These results suggest that morphiceptin disrupts either the specific copulatory somatomotor pattern or a more general motivational component.

Influence of fetal and maternal genotype for circulating insulin-like growth factor I on fetal growth in mice.

The role of insulin-like growth factor I in the regulation of fetal growth was investigated in two lines of mice selected for high or low concentrations of this factor in plasma. In Expt 1, females from each line were mated with males of the reciprocal line to generate fetuses of equivalent genotype. Females with low concentration of the factor in plasma exhibited the typical negative relationship between mean fetal mass and litter size (b = -0.032 +/- 0.006 g per fetus, P < 0.01). However, dams of the line with high concentrations of the factor did not exhibit this relationship (b = -0.004 +/- 0.006 g per fetus), despite the fact that they had 26% larger litters (P < 0.05) at a common maternal body mass. This difference in maternal constraint apparently reflects a greater capacity for nutrient transfer to the fetuses in the dams with more insulin-like growth factor I in plasma, as suggested by the absence of a relationship between mean placental mass and mean fetal mass in that line. In Expt 2, the effect of fetal genotype for insulin-like growth factor I was investigated by transferring embryos of the two lines into females of an unrelated strain. Fetuses from the line with high concentrations of the factor in plasma were heavier at term (1.51 versus 1.37 g, pooled SE = 0.05 g, P < 0.05) than fetuses from the line with low concentrations in plasma. It is therefore concluded that fetal growth is influenced by both the maternal and fetal genotypes for insulin-like growth factor I, but in qualitatively different manners.

Role of testosterone in regulating the growth of mice from lines selected for low vs high plasma insulin-like growth factor-I concentrations.

A study was undertaken to investigate the role of testosterone in regulating growth and circulating levels of insulin-like growth factor-I in male mice from lines divergently selected on the basis of plasma IGF-I. Controls of each lines were sham-operated at 10 days of age and treated with peanut oil from day 14 to day 70. A second group, which was castrated at 10 days and treated with testosterone enanthate (0.5 micrograms.(g body weight)-1.day-1) from day 14 to 70, did not differ from controls in body weight but had higher plasma IGF-I concentrations. Delaying testosterone therapy until day 42 in a third group retarded growth, with body weights being significantly lower than those of other two groups from days 35 to 56. However, plasma IGF-I levels in this group were not different from those of controls. Effects of line and treatment were additive. It is concluded that the greater pubertal growth of high-line compared to low-line males is not due to greater stimulation of circulating IGF-I by testosterone. Furthermore, testosterone does not appear to influence pubertal growth by acting on circulating levels of IGF-I.

Reproductive performance and fetal growth in female mice from lines divergently selected on the basis of plasma IGF-1 concentrations.

Reproductive performance, mammary gland weight and plasma concentrations of insulin-like growth factor-1 (IGF-1) were examined in 18-day-pregnant mice from lines divergently selected on the basis of plasma IGF-1 concentration. Females of the high IGF-1 (H) line were 14% heavier than those of the low IGF-1 (L) line at mating but did not differ in conception rate during a 15-day mating period. H-line females produced significantly larger litters by an average of 1.5 fetuses (19%), heavier fetuses (7%), greater total fetal weight (30%), heavier placental discs (15%), greater total placental weight (35%) and heavier mammary glands (18%). Plasma IGF-1 values were 12% greater in H-line than L-line females at Day 19 of gestation but the line difference was not significant. It is concluded that differences between the lines in litter size and mammary gland weight are most likely due to differences in maternal bodyweight (which are in turn a consequence of selection for plasma IGF-1 at puberty). Whether the difference in fetal weight is a function of fetal capacity to grow in utero or ability of the dam to provide nutrients for fetal growth is yet to be determined.

Responses to divergent selection for plasma concentrations of insulin-like growth factor-1 in mice.

A divergent selection experiment with mice, using plasma concentrations of insulin-like growth factor-1 (IGF-1) at 42 days of age as the selection criterion, was undertaken for 7 generations. Lines were not replicated. To obtain sufficient plasma for the IGF-1 assay, blood from four individuals was volumetrically bulked to obtain a litter mean IGF-1 concentration. This necessitated the use of between family selection. Although inbreeding accumulated in a linear fashion in each of the high, control and low lines, the rates were different for each line (3.6, 1.6 and 5.3% per generation for the high, control and low lines, respectively). As a consequence, the effects of selection and inbreeding are confounded in this experiment. Divergence between the high and low lines in plasma concentrations of IGF-1 continued steadily until generation 5. In generations 6 and 7, there was a reduced degree of divergence and this contributed towards the low realized heritability value of 0.15 +/- 0.12. Six-week liveweight showed a steady positive correlated response to selection for or against plasma concentrations of IGF-1 until generation 4 (high-low difference = 1.7 g = 12%). In generation 5, a substantial drop in 6-week liveweight in the low line relative to both the high and control lines occurred (high-low difference, 3.9; g, 25%). This difference was maintained until generation 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic selection for insulin-like growth factor-1 in growing mice is associated with altered growth.

Substantial responses in the 6-week and mature body-weights of mice occurred after 7 generations of selection for or against plasma levels of Insulin-like Growth Factor-1 (IGF-1). Plasma levels of IGF-1 were also significantly different after 7 generations of selection (high line = 85 +/- 2 ng/ml, low line = 58 +/- 2 ng/ml). The average 6-week weight in the line selected for high plasma IGF-1 was 22.5 +/- .2 g compared with 18.5 +/- .2 g in the low plasma IGF-1 line, after 7 generations of selection. The difference between lines was maintained at 20 weeks of age. These data provide further evidence for the roles of IGF-1 in the regulation of somatic growth and as a mediator of a genetic component of growth.

Variation in plasma concentration of insulin-like growth factor-1 and its covariation with liveweight in mice.

Three experiments were undertaken to examine the degree and causes of variation in plasma concentrations of insulin-like growth factor-1 (IGF-1) in mice. The relationship between IGF-1 concentrations and liveweight was also examined. In all three experiments, a number of non-genetic factors were found to contribute significantly to the variation in IGF-1 concentrations, the most important of these being sex and litter size. In one experiment, where pups from 16 litters were cross-fostered to avoid the confounding of maternal and direct genetic effects, a heritability of 0.40 +/- 0.27 was estimated for plasma IGF-1 concentration at 35 days of age. To examine further the existence of genetic variation in plasma concentrations of IGF-1 and the genetic covariation between plasma IGF-1 levels and other body traits, a selection experiment with mice has been initiated. Moderate to strong phenotypic correlations between IGF-1 concentrations and weight at an early age have been found in all three experiments.

Transcription of bovine satellite DNA by purified RNA polymerase III.

Purified calf thymus RNA polymerase III synthesizes, from calf thymus DNA template, RNA which hybridizes to the major repeated sequence of Eco R1-digested calf thymus DNA. Similar results are obtained with RNA transcribed from calf thymus chromatin. It is suggested that this DNA sequence, which is derived from bovine satellite DNA, may be genetically active.