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Background: In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world., Objectives: We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes., Discussion: Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., "good science"). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research., Conclusions: Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.

This review assesses the effects of environmental concentrations of bisphenol-A (BPA) on wildlife. Water concentrations of BPA vary tremendously due to proximity to point and non-point sources, but reported concentrations in stream/river water samples are less than 21 microg/L, and concentrations in landfill leacheate are less than 17.2mg/L. Extensive evidence indicates that BPA induces feminization during gonadal ontogeny of fishes, reptiles, and birds, but in all cases the amount of BPA necessary to cause such ontogenetic disruption exceeds concentrations in the environment. Extensive evidence also exists that adult exposure to environmental concentrations of BPA is detrimental to spermatogenetic endpoints and stimulates vitellogenin synthesis in model species of fish. Most of the reported effects of BPA on vertebrate wildlife species can be attributed to BPA acting as an estrogen receptor agonist, but mechanisms of disruption in invertebrates are less certain. A comparison of measured BPA environmental concentrations with chronic values suggests that no significant margin of safety exists for the protection of aquatic communities against the toxicity of BPA. Further studies should examine the most vulnerable vertebrate and invertebrate species.

Wound healing in crustaceans preserves the integrity of the integument and prevents entry of pathogens. We studied the interaction between the moulting hormones (ecdysteroids) and the cellular events under the wound during wound healing with or without bacteria infection. Wounding of the carapace by abrasion induced a rapid increase in circulating ecdysteroid levels to a low sustained plateau level for about 12 days, followed by a sharp premoult peak and moulting. Within 48h of wounding, the nuclear receptor for ecdysteroids (EcR) appeared in the nuclei of haemocytes (hyaline, semigranular and granulocytes), visualized by confocal laser scanning microscopy and anti-EcR. Hyaline haematocytes aggregated in layers below the wound site and granulocytes engaged in phagocytosis. Therefore, the immune system responds directly and rapidly to ecdysteroids. Epidermal cells developed EcR only several days after the haemocytes and only under intact carapace, not under the wound where they appeared apoptotic. At the wound margin, EcR-positive epidermal cells and fibroblasts proceeded to migrate across the wound between the layers of haemocytes. Epidermis was fully regenerated by day 15; at this time the ecdysteroid titre began rising towards a premoult peak and EcR disappeared from the nuclei of epidermal cells suggesting that high amounts of ecdysteroids exert negative control on EcR. When bacteria were injected at the time of wounding, both the plateau level of ecdysteroid titre and the cellular events of wound healing were prolonged by 5-7 days, showing that healing of the wound is slower and that the duration of the plateau phase of the titre depends on the degree of assault on the animal. We conclude that the low levels of ecdysteroids induced by wounding activate the immune system to begin healing below the wound and also stimulate adjacent epidermal cells to commence the process of wound repair.

Adult male crayfish Procambarus clarkii exist in two morphotypes. They continue to molt as adults, switching between Form Is and Form IIs. Form Is are primary reproductive types, with large chelae and spines on the ischiopodites of the third and fourth pair of walking legs. Form IIs are non-reproductive types with smaller chelae and no spines on the ischiopodites. We investigated the hormonal control of these transitions in two ways, by eyestalk ablation and by methyl farnesoate (MF) treatments. Eyestalk ablation accelerates molting and increases MF levels in the blood. MF is a hormone that regulates both reproduction and morphogenesis. MF concentrations were determined in two ways. The hemolymph samples were extracted first, then purified, using normal phase HPLC. The fractions containing MF were collected and analyzed for MF concentration, utilizing both internal and external standards by GC/MS. The other hemolymph samples were analyzed from individual animals by HPLC. The concentrations of ecdysteroids were determined by radioimmunoassay. In the control animals, 4 out of 4 untreated Form I males molted into Form II, while 6 out of 7 Form IIs molted into Form Is. Eight of 8 ablated Form Is molted into Form IIs as expected, while 5 of 5 ablated Form IIs molted into Form IIs, instead of Form Is. MF treatment of intact animals resulted in 6 of 7 Form Is becoming Form IIs and 5 of 6 Form IIs becoming Form IIs. These results were highly significant in comparison of Form I and IIs in each treatment (eyestalk intact, eyestalk ablated and eyestalk intact with MF) by a chi square analysis, P = 0.006, P < 0.0005, and P = 0.013, respectively. MF premolt blood levels suggested that Form IIs were produced in the presence of 1.3 ng/ml MF, while Form Is result from MF levels less than 0.5 ng/ml. Since both eyestalk ablation and MF treatment resulted in the failure of Form IIs becoming Form Is, it was concluded that the control of morphogenesis of primary reproductives (Form Is) depends on a low level of MF prior to the molt, while Form IIs are formed in the presence of increased levels of MF.

We have identified, by gas chromatography/mass spectrometry, four alkylphenols that are present in the hemolymph and tissues of the American lobster Homarus americanus and in marine sediments. These alkylphenols are used industrially in antioxidant formulations for plastic and rubber polymer manufacturing, and are similar in structure to a known endocrine disruptor, bisphenol A. The compound 2-t-butyl-4-(dimethylbenzyl)phenol was present at concentrations of 0.02 to 1.15 microg/ml in hemolymph and 8.95 to 21.58 microg/g in sediments. A second compound, 2,4-bis-(dimethylbenzyl)phenol, was present at concentrations between 0.07 and 19.78 microg/ml in hemolymph and 138.94 to 224.89 microg/g in sediment, while a third compound, 2,6-bis-(t-butyl)-4-(dimethylbenzyl)phenol, was found at concentrations between 0.01 and 13.00 microg/ml in hemolymph, 2.55 and 6.11 microg/g in hepatopancreas, and 47.85 and 74.66 microg/g in sediment. A fourth compound, 2,4-bis-(dimethylbenzyl)-6-t-butylphenol, was found at concentrations of 0.20 to 70.71 microg/ml in hemolymph, 23.56 to 26.89 microg/g in hepatopancreas, and 90.68 to 125.58 microg/g in sediment. These compounds, along with bisphenol A, 4-dimethylbenzylphenol, and nonylphenol, display high juvenile hormone activity in bioassays. Alkylphenols at high concentrations are toxic to crustaceans and may contribute significantly to lobster mortality; at lower concentrations, they are likely to have endocrine-disrupting effects.

The effect of methyl farnesoate (MF) on the ovaries of female red swamp crayfish, Procambarus clarkii, undergoing vitellogenesis was determined both in vivo and in vitro. The in vivo assay showed a positive effect of MF on oocyte growth when injected alone and in combination with 17 beta-estradiol, but not in combination with JHIII or 17 alpha-hydroxyprogesterone. A higher level of incorporation of labeled leucine was induced by MF on isolated pieces of ovary. The same effect was seen when ovary and mandibular organ (MO) were coincubated. These results suggest that MF stimulated the synthesis of vitellin in the ovary of crayfish. In vitro, 17 alpha-hydroxyprogesterone completely suppressed the stimulatory action of the MO on the ovary, suggesting a competitive inhibition between 17 alpha-hydroxyprogesterone and MF on the ovary and/or a negative feedback by that steroid on the MO.