Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: the case of bisphenol A.

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.

In vivo effects of bisphenol A in laboratory rodent studies.

Concern is mounting regarding the human health and environmental effects of bisphenol A (BPA), a high-production-volume chemical used in synthesis of plastics. We have reviewed the growing literature on effects of low doses of BPA, below 50 mg/(kg day), in laboratory exposures with mammalian model organisms. Many, but not all, effects of BPA are similar to effects seen in response to the model estrogens diethylstilbestrol and ethinylestradiol. For most effects, the potency of BPA is approximately 10-1000-fold less than that of diethylstilbestrol or ethinylestradiol. Based on our review of the literature, a consensus was reached regarding our level of confidence that particular outcomes occur in response to low dose BPA exposure. We are confident that adult exposure to BPA affects the male reproductive tract, and that long lasting, organizational effects in response to developmental exposure to BPA occur in the brain, the male reproductive system, and metabolic processes. We consider it likely, but requiring further confirmation, that adult exposure to BPA affects the brain, the female reproductive system, and the immune system, and that developmental effects occur in the female reproductive system.

Prenatal testosterone exposure permanently masculinizes anogenital distance, nipple development, and reproductive tract morphology in female Sprague-Dawley rats.

In mammals, abnormal increases in fetal androgens disrupt normal development of the female phenotype. Due to the recent concern regarding environmental androgen-active chemicals, there is a need to identify sources of fetal androgen variation and sensitive developmental markers for androgenic activity in female rats. Anogenital distances (AGD), nipple retention, reproductive tract, and external genitalia are morphological parameters organized by prenatal androgens and are predictive of altered masculinized/defeminized phenotype in adult female mice and rats. The objectives of this study were to (1) characterize the natural prenatal androgen environment of rats including the magnitude of the intrauterine position (IUP) effect, (2) characterize the permanent effects of prenatal androgen exposure on female rats, and (3) determine the ability of AGD and areolas to predict these permanent androgenic alterations in female rats. Untreated male fetal rats had higher tissue testosterone (T) concentrations than females in the amniotic fluid, reproductive tract, gonad, and fetal body. The intrauterine position (IUP) of male and female fetuses did not affect T concentrations or AGD in male or female rats at gestational day (GD) 22. Female offspring exposed to 0, 1.5, and 2.5 mg/kg/day testosterone propionate (TP) on GDs 14-18 displayed increased AGD at postnatal day (PND) 2 and decreased nipples at PND 13 and as adults. TP-induced changes in neonatal AGD and infant areola number were reliable indicators of permanently altered adult phenotype in female rats. Further, females in the two high-dose groups displayed increased incidences of external genital malformations and the presence of prostatic tissue, not normally found in female rats.

Developmental exposure to environmental estrogens alters anxiety and spatial memory in female mice.

Humans and wildlife are exposed to numerous anthropogenic drugs and pollutants. Many of these compounds are hormonally active, and recent evidence suggests that the presence of these endocrine disruptors permanently alters normal development and physiology in a variety of vertebrate species. Here, we report on the effects of developmental exposure to two common estrogenic pollutants, bisphenol A and ethinyl estradiol on sexually dimorphic, non-reproductive behavior. Mice (Mus musculus domesticus) were exposed to environmentally relevant levels of these chemicals (2 and 200 microg/kg/day for bisphenol A and 5 microg/kg/day for ethinyl estradiol) throughout prenatal and early postnatal development. As adults, the animals were observed in a variety of tests measuring sexually dimorphic behaviors including short-term spatial memory (in a radial-arm maze and a Barnes maze) and anxiety (in an elevated-plus maze and a light/dark preference chamber). Developmental exposure to ethinyl estradiol was found to masculinize behavior in all of the assays used. Bisphenol A increased anxious behavior in a dose-dependent fashion but had no effect on spatial memory. These results indicate that non-reproductive, sexually dimorphic behavior is sensitive to endocrine disruption. In addition, these experiments suggest that both humans and wildlife are being exposed to levels of these endocrine disrupting compounds that are sufficient to disrupt the development of the nervous system and that may have permanent consequences on sexually dimorphic behaviors.

Perinatal exposure to endocrine disrupting compounds alters behavior and brain in the female pine vole.

Endocrine-disrupting chemicals (EDCs) are synthetic chemicals that arise from sources such as pesticides and have the ability to mimic or inhibit gonadal steroid hormones. The objective of this research was to examine the effects of EDCs on the behaviors associated with monogamy and the expression of related neuropeptide receptors. Pine voles, a novel experimental mammal, were chosen because they display strong monogamous pair bonding. Female pine voles were orally administered estrogenic diethylstilbestrol (DES) and methoxychlor (MXC) or oil control throughout gestation and lactation of pups. Exposed pups were tested as adults. Preference for the mate and maternal behaviors were assessed. While the ability to form partner preferences was intact, DES-exposed females showed increased aggression toward a stranger, while MXC exposed females showed a strong trend toward spending more time alone. Oxytocin (OT) receptor binding in the brain was assessed for possible effects on this behaviorally important neuropeptide signaling system. The cingulate cortex showed a reduction in OT binding in the MXC group. These findings demonstrate that exposure to EDCs during pre- and neonatal development can alter female adult neural phenotype and behavior related to monogamous behavior traits.

The anogenital distance index of mice (Mus musculus domesticus): an analysis.

The anogenital distance (AGD) is sexually dimorphic in Mus musculus domesticus, with the male AGD approximately twice as long as that in female mice. Among female mice, the AGD varies as a function of prenatal androgen exposure. The anogenital distance index (AGDI) has been developed to serve as an indicator of prior androgen exposure due to intrauterine position (IUP). Concerns have been raised that the AGDI may not be an appropriate indicator of female IUP in mice. To further refine the AGDI, we have applied some commonly used and suggested transformations to the original data set of female CD-1 mice of known IUP, weaning body mass, and AGD. Our analysis suggests that the residual log transformation and untransformed body mass AGDIs are the most accurate means to predict the IUP of the pup. However, the IUP is only one mechanism by which a fetus may be exposed to hormonal variations in utero. Additional analyses revealed that the AGDI is influenced not only by the IUP of the female fetus but also by the identity of the dam (indicative of maternal influences) and the number of male fetuses found in the particular uterine horn. Therefore, the AGDI is not strictly a predictor of female IUP but of the intrauterine androgen environment in mice.

Animal models and studies of in utero endocrine disruptor effects.

The rate of organ and system development in mammals, including humans, is most rapid during the prenatal period. Perturbations of the endocrine system during this period can have profound effects on later anatomy, physiology, behavior, and the onset of disease. Endocrine-disrupting compounds can cause perturbations during fetal development by mimicking or blocking natural hormones. In experimental studies, compounds that mimic estrogens and those that block androgen action have been shown to have a number of long-term effects. Among these effects are the acceleration of puberty onset, increased incidence of adult cancers such as vaginal and prostate cancers, and alterations in sexually dimorphic anatomy, physiology, and behavior. Laboratory animal models continue to play a crucial role in identifying endocrine disruptors, determining their mode of action, and demonstrating their consequences.

Rickettsia rickettsii infection in the pine vole, Microtus pinetorum: kinetics of infection and quantitation of antioxidant enzyme gene expression by RT-PCR.

The pine vole, Microtus pinetorum, was evaluated as a laboratory animal model for infection with Rickettsia rickettsii. Voles demonstrated signs of acute disease, and 45% of infected animals died following intraperitoneal infection with 3 x 10(6) plaque forming units of R. rickettsii. Spleen, liver, kidney, lung, brain, testes and blood were analyzed for rickettsial burden by a quantitative PCR assay. The distribution of rickettsiae in tissues during the course of infection was determined by immunohistochemical staining and pathological changes in tissues were correlated with the clinical severity of infection. Quantitative RT-PCR assays were designed to measure the mRNA levels of the antioxidant enzyme genes for catalase, glutathione peroxidase, glutathione reductase, heme oxygenase, Cu-Zn superoxide dismutase (SOD) and Mn-SOD, and 2 housekeeping genes, actin and glyceraldehyde phosphate dehydrogenase. Tissues from acutely ill animals on days 2 to 6 of infection, convalescent animals, and uninfected control animals were studied. The number of transcripts of each enzyme gene was determined and compared to the degree of rickettsial infection present. These studies demonstrate that the pine vole is a valuable experimental model for studying infection with R. rickettsii. Our results provide the first experimental evidence that R. rickettsii causes alteration(s) of the anti-oxidant system in vivo.

Intrauterine position effects.

A review of the literature suggests that individual variability in sex-related traits may be influenced by variations in hormonal exposure during fetal development. In litter-bearing mammals, fetuses develop in utero and may be subjected to differing hormonal environments based upon the sex of neighboring fetuses. Female fetuses developing between two males tend to show masculinized anatomical, physiological and behavioral traits as adults. Female fetuses developing without adjacent males, on the other hand, tend to show more feminized traits as adults. These traits include permanently altered hormone levels, reproductive organs, aggressive behaviors, secondary sex ratios and susceptibility to endocrine disruption. This intrauterine effect is due to the transfer of testosterone from male fetuses to adjacent fetuses. While these effects have been most clearly demonstrated in mice, other rodents and swine also show intrauterine position (IUP) effects. Some of these effects are similar to the influence of prenatal stress on adult phenotypes. A few reports on human twins suggest that variability in some masculine and feminine traits may be due to intrauterine hormonal signals. IUP effects may impact a number of scientific fields of research such as endocrine disruption, toxicology, population biology, animal production and health.

Impaired olfactory behavior in mice deficient in the alpha subunit of G(o).

The ability to respond to chemical signals is essential for the survival and reproduction of most organisms. Olfactory signaling involves odorant receptor-mediated activation of G(olf), a homologue of G(s), on the dendrites of olfactory neurons. Olfactory receptor cells, however, also express Galpha(i2) and Galpha(o) on their axons, with all neurons expressing G(o) and a subset G(i2). Despite their abundance, possible contributions of G(o) and G(i2) to chemoreception remain unexplored. We investigated whether homologous recombinant mice deficient in the alpha subunit of G(o) are able to respond to odorants, whether possible olfactory impairments are dependent on genetic background, and whether formation of glomeruli in their olfactory bulbs is compromised. In an olfactory habituation/dishabituation test, G(o)-/- mice were unresponsive when exposed to odorants. Analysis of variance shows that performance of G(o)+/- mice crossed into the CD-1 background is also diminished in this test compared to their G(o)+/+ counterparts. Following food deprivation, G(o)-/- mice in the 129 Sv-ter/C57BL/6 genetic background were unable to locate a buried food pellet until they were approximately 10 weeks of age after which they performed as well as their litter mate controls. However, CD-1 G(o)-/- mice could locate a buried food pellet even when tested immediately after weaning. Despite their compromised olfactory responsiveness, histological examination did not reveal gross alterations in the olfactory bulbs of G(o)-/- mice. Thus, Galpha(o) is necessary for the expression of olfactory behavior under normal conditions and dependent on genetic background, but is not essential for the formation and maintenance of glomeruli.