Francesca Farabollini

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.

Perinatal Exposure to the Estrogenic Pollutant Bisphenol A Affects Behavior in Male and Female Rats

Bisphenol A (BPA) is an environmental estrogen with potentially aversive effects on public health. In rats, we studied the effects of perinatal exposure to BPA on nonsocial behaviors partly influenced by gonadal hormones. BPA was administered orally to one group of mother rats at a concentration within the range of human exposure from 10 days before mating until the weaning of the pups. In a second group, BPA was given at a higher dosage during a critical period for brain organization, i.e., from day 14 of gestation until day 6 after birth. The offspring of the treated mothers were tested in the holeboard and the elevated plus-maze at 85 days of age. Various aspects of nonsocial behavior were affected by BPA, differently in males and females, confirming that exposure to a weak environmental estrogen in the period of sexual differentiation of the brain can influence adult behavior. However, contrary to our expectation, a clear masculinization of females was not observed. In general, the factor analysis indicated that in treated males both the motivation to explore and anxiety are reduced, while in females, motor activity and motivation to explore are depressed. Because there were no substantial differences between the two modalities of BPA administration, we suggest that the prolonged treatment with the low dosage compensates for the higher dosage given during a shorter steroid-sensitive period. This may be a cause of concern for public health, given the greater incidence of prolonged exposure of humans to low concentrations released into the environment.

Bisphenol-A exposure during pregnancy and lactation affects maternal behavior in rats.

In mammals, endogenous estrogens are crucial for sexual differentiation during the perinatal period, and the modulation in adulthood of many neuroendocrine and behavioral functions involved in reproduction. In rats, the estrogenic environment during pregnancy and lactation affects directly maternal behavior. This experiment was aimed to test whether the exposure to the estrogenic compound bisphenol-A (BPA; 0.040 mg/kg/die, orally) of adult female rats, from mating to weaning of the pups, could alter maternal behavior. An appropriate methodology was applied to reveal differences in the behavior of dams directed to male and female pups, testing the dams on postnatal days 3-4 and 8-9. Results show different maternal behavioral patterns towards male and female pups of control mothers, with more ano-genital licking to males than to females. Exposure of mothers to BPA modified their behavior, reducing specific components of maternal behavior, both active and passive, irrespective of the sex of pups and the period of observation. This experiment shows that maternal behavior is affected by a prolonged exposure to a low dose of BPA during pregnancy and lactation, thus suggesting an effect on neural circuits in adulthood.

Early exposure to a low dose of bisphenol A affects socio-sexual behavior of juvenile female rats

Play behavior is affected by alteration of the hormonal environment during development. In fact, congenital adrenal hyperplasia or early administration of diethylstilbestrol are able to modify female play behavior in mammals. In this research, play behavior of female rats was used to explore the effects of perinatal exposure to low, environmentally relevant dose of bisphenol A (BPA), a xenoestrogen widely diffused in the environment. We used 18 females born to mothers exposed to 40 microg/kg/day BPA during pregnancy and lactation, and 18 control females. The subjects were observed in a heterosexual social situation from 35 to 55 days of age. Six main behaviors were identified by principal component analysis (PCA): exploration, defensive behavior to males, play behavior with males, play behavior with females, low-intensity mating behavior, social grooming. Early administration of BPA was responsible for a significant increase of exploration (including social investigation) (p<0.001), as well as a decrease of play with males (p<0.02) and social grooming (p<0.01) at 45 days of age, indicating a general decrease of playful interactions. In general our results suggest that BPA does not induce a clear masculinization of female behavior, but is able however to defeminize some aspects of female behavior. This result is compatible with the estrogenic properties of BPA, and suggests caution in the use of a chemical that, in the range of human exposure, is able to influence the development of the brain during a critical period, resulting in long-term effects on behavior.

Sex-dependent effects of aversive stimulation on holeboard and elevated plus-maze behavior

The behavioral effects of exposure to inescapable shocks (IS) were studied in both the holeboard and the elevated plus-maze, 24 and 72 h after IS in male and female Wistar rats. The following effects were observed at the 24-h interval. In both sexes, head-dipping in the holeboard was reduced by IS, whereas general activity (ambulation and rearing) was reduced in males and not in females. Furthermore, the results of a correlation analysis indicate that previous exposure to IS disrupts the dissociation observed in control groups between exploratory activity directed at the holes (head-dipping) and general activity in the holeboard (ambulation and rearing). Effects of IS on plus-maze behavior could be observed in a clear suppression of rearing in males and not in females. IS did not affect time spent on the open arms. At the 72-h interval, IS affected head-dipping in the holeboard only in males and not in females. The present findings show that the effect of IS on specified behavioral elements is sex-dependent, with stronger and longer-lasting effects in males than in females.

Sex-dependent effects of restraint on nociception and pituitary-adrenal hormones in the rat.

The sex-dependent effects of acute restraint (RT) on nociceptive and pituitary-adrenal responses were investigated in the rat. In a first experiment, the effect of 30 min RT on pain sensitivity was evaluated through repeated use of the tail withdrawal test during and after treatment. RT induced an increase in the nociceptive threshold, i.e., analgesia, in males and females, but the duration and time-course of this effect varied between sexes. The latencies returned to approximately control values in females in the second half of RT, but in males they remained higher for the whole period of RT and immediately afterwards. Twenty-four hours later, males displayed longer latencies than controls in response to simple reexposure to the environment. In a second experiment, ACTH and corticosterone plasma levels were measured immediately after 15 or 30 min of RT. ACTH and corticosterone were higher in restrained animals than in controls after both periods of treatment, and in both sexes; however, females showed higher basal and stress corticosterone levels than males. The role played by corticosteroids in the nociceptive responses of the two sexes is discussed.

Exposure to the estrogenic pollutant bisphenol A affects pain behavior induced by subcutaneous formalin injection in male and female rats.

We investigated the effects of perinatally administered bisphenol A (BPA), an environmental contaminant with estrogenic activity, on formalin-induced nociceptive responses. Male and female offspring of mother rats treated with BPA or oil were cross-fostered after birth to obtain three homogeneous groups: BPA-prenatal, receiving BPA via the placenta; BPA-postnatal, receiving BPA through suckling; OIL, control, from mothers receiving only peanut oil (vehicle). All groups underwent a pain test with s.c. formalin injection (50 microl, 10%) or were sham injected (pricking with a syringe needle) in the dorsal hind paw. They were immediately placed in an open field apparatus where pain responses (licking, flexing and paw-jerk) were recorded for 60 min. Corticosterone, testosterone and estradiol serum levels were determined in blood obtained at the end of the experiment. BPA-prenatal treatment induced an increase in licking duration in females and in flexing duration in both sexes in the first half of the test (0-30 min after formalin injection). BPA-postnatal treatment induced a decrease in paw-jerk frequency in males and females during the second part of the test (30-60 min after formalin injection). Plasma concentrations of corticosterone, estradiol and testosterone did not differ significantly between groups. These results indicate that exposure to BPA modified the activity of neural pathways and/or centers involved in nociception and pain in a sex-related and exposure-related manner.

Pubertal exposure to estrogenic chemicals affects behavior in juvenile and adult male rats

In this paper, we tested the hypothesis that exposure to estrogens of different source and estrogenic potency at early puberty could affect the development of socio-sexual behavior in the male rat. Puberty is regarded as a second stage of the ontogenetic period, in the sexual maturation of mammals, particularly sensitive to gonadal hormone milieu. We treated animals orally, from postnatal day 23 to 30, with an environmentally compatible dose of bisphenol A (BPA, 40 microg/kg/day) and with a dosage of ethinylestradiol (EE, 0.4 microg/kg/day) comparable to the human oral contraceptives. Exposure to EE altered the temporal pattern of male sexual activity, reducing performance, in the adult animals; slight modifications, in the same direction, were observed with BPA. Short-term behavioral effects were observed in the treated animals, both with BPA and EE: the exploratory drive, directed to a stimulus object and to the environment, as well as to conspecifics, was reduced in the juveniles. Modifications in the circulating T levels were observed after treatments: T was reduced in the juveniles, both with BPA and EE. The decrement persisted in the adult animals but reached significance only in the BPA group. On the whole, effects of pubertal exposure on behavior are more marked with EE than BPA. This can be due to the much higher estrogenic potency of EE; the direction of the behavioral effects of BPA, compared with EE, is however indicative of an estrogenic mechanism.

Bisphenol-A differently affects estrogen receptors-α in estrous-cycling and lactating female rats

The effect of long-term exposure to bisphenol-A (BPA) on estrogen receptor-alpha (ER) immunoreactivity was studied in the medial preoptic area, arcuate nucleus and the ventromedial nucleus of the hypothalamus of estrous cycling and lactating female rats. Pregnant/lactating or estrous cycling rats were exposed to BPA (40 mg/Kg/day) or peanut oil. Lactating females showed fewer ER-immunoreactive cells than non-lactating females in the medial preoptic area and ventromedial nucleus of the hypothalamus. BPA induced an increase in ER-immunoreactive cells in the medial preoptic nucleus irrespective of lactation. BPA only induced a decrease in ER-immunoreactive cells in the arcuate nucleus of the lactating group; oil induced an increase in ER-immunoreactive cells in the lactating with respect to non-lactating group. The results demonstrate that exposure of adult females to BPA modifies the number of ERs.

Effects of pain, morphine and naloxone on the duration of animal hypnosis

Previous research has shown that animal hypnosis (tonic immobility) in the rabbit may be elicited in a condition of prolonged nociceptive stimulation. These experiments show that long-lasting irritative pain, produced within 15 min of formalin injection, potentiates the duration of hypnosis. Morphine, in the absence of painful stimuli, also potentiates hypnosis duration and this effect is antagonized by naloxone. Naloxone reduces hypnosis duration, but only at high doses (15 mg/kg). In a condition of irritative pain, the potentiation of hypnosis duration is abolished by naloxone (5 mg/kg). Hypnosis response is abolished in 6 out of 7 morphine-tolerant rabbits, but prolonged pain restores the response. The hypothesis that an opioid mechanism may be activated during animal hypnosis is discussed.