FEATURE ARTICLE
Assessing Risks from Bisphenol-A
Evaluating human health risks from endocrine disruptors such as BPA is difficult, but animal studies suggest trouble is afoot
Heather Patisaul
Chasing Meaningful Effects
Within the brain, volumes of sexually dimorphic nuclei in the hypothalamus are commonly used as a biomarker to study the neurodevelopmental impacts of endocrine disruptors. In the rodent brain, one of the most frequently studied regions is the sexually dimorphic nucleus of the preoptic area, known as the SDN, which is considerably larger in males than in females. The SDN is sensitive to steroid hormones from gestational day 18 until four days after birth. Following this critical period, a phase of sexually dimorphic apoptosis, or programmed cell death, begins, ending on postnatal day 12. Early exposure to estrogen, derived from the conversion of testicular androgens, protects the male SDN from apoptosis. In females, because estrogen levels are low during this period, apoptosis in the SDN progresses unabated, resulting in a smaller SDN.
Attempts to evaluate the impact of BPA exposure on SDN volume, in both males and females, have yielded largely negative and somewhat inconsistent results. For example, we determined that administration of the relatively high dose of 500 micrograms BPA over two days, beginning the day after birth, failed to alter SDN volume in male rats. Other studies, using higher and lower doses of BPA and exposure periods spanning both the gestational and postnatal periods, have also largely failed to find evidence for BPA effects on SDN volume.
Although these results are initially reassuring, we’ve concluded that using the SDN as a biomarker for endocrine disruption is problematic for several reasons. First, it appears to be relatively resistant to most endocrine disruptors, particularly in the Sprague Dawley rat. In addition, the functional role of the SDN is not readily apparent. It has long been accepted to play a role in male sexual behavior, but its role appears to be minimal. A final caveat is that humans have no clear SDN, so it is not entirely evident which reproductive health effects might be predicted by disruption of this region in rodents. It may be a useful region, however, to demonstrate mode of action. For example, we have found that BPA significantly increases the number of SDN cells containing calbindin-D28k, a protein thought to protect against apoptosis. This effect is consistent with the hypothesis that BPA acts as an estrogen mimic. Collectively these data show that, although the SDN is a popular marker for endocrine disruption, it may not actually be a very good one.
Another sexually dimorphic brain region that we and others have examined is the anteroventral periventricular nucleus (AVPV). In contrast to the SDN, the AVPV is larger in females than in males, and neonatal estrogen exposure induces, rather than prevents, the apoptosis that produces the size difference. Also, the functional significance of the AVPV is very well understood. The AVPV is essential for the coordination of the hormonal and environmental signals that regulate the secretion of gonadotropin releasing hormone (GnRH). In normal adult females, a “surge” of GnRH, elicited though positive feedback by the preovulatory rise of estradiol, induces the release of luteinizing hormone and, consequently, ovulation. In male rodents, neonatal estrogen, aromatized from testicular androgen, acts to defeminize the male AVPV such that GnRH neurons are no longer capable of responding to elevated estrogen levels with a surge of GnRH. Similarly, neonatal estrogen administration can defeminize the female AVPV, resulting in the inability to generate a GnRH surge in adulthood. The clearly defined functional significance of this region, combined with its sensitivity to estrogen during a discrete neonatal window, makes it a potentially ideal target for examining the impact of BPA exposure on sex-specific brain organization.
We found that administering the relatively high dose of 500 micrograms of BPA to male rats for only two days within this critical neonatal window of estrogen sensitivity did not affect the overall size of the AVPV. It did, however, result in a more female-typical number of dopaminergic neurons within the AVPV. Beverly Rubin of Tufts University subsequently reported finding male-typical numbers of dopaminergic neurons in the AVPV of female mice exposed from gestation through lactation to lower, environmentally relevant doses but did not look at AVPV volume. Together, these studies suggest that the AVPV is likely more sensitive to endocrine disruption than the SDN. They also support the hypothesis that BPA exposure may disrupt sexual differentiation of the brain in general, and within the AVPV specifically.
Another group of neurons of interest within the AVPV is the newly discovered kisspeptin neurons. It is now apparent that the kiss1 gene, which codes for a family of proteins called kisspeptins (KISS), is vital for the regulation of GnRH secretion in many species, including humans. It appears to be essential for the timing of both pubertal onset and ovulation. People with a mutated form of the kisspeptin receptor never enter puberty and remain hypogonadal throughout life. In the rat, there are two major populations of KISS neurons, one in the AVPV and one in the arcuate nucleus, another sexually dimorphic nucleus in the hypothalamus. The density of KISS neurons appears only to be sexually dimorphic in the AVPV, with females having significantly more KISS neurons than males. This population is thought to be critical for initiating the surge of GnRH that precedes ovulation.
To establish whether neonatal exposure to BPA could disrupt development of the AVPV KISS system, we injected rats with either 50 milligrams or 50 micrograms of BPA per kilogram daily for four days starting at birth. In rats, this is when the KISS system is most sensitive to estrogen. (In humans, this sensitivity window likely occurs in the early part of pregnancy’s second trimester.) In female rats, only the higher dose of BPA affected AVPV KISS levels. No effect was observed in the males. Interestingly, however, we also found a slight, but statistically significant, reduction of KISS in the arcuate nucleus of females exposed to the higher BPA dose. In contrast to the AVPV, the number of KISS neurons in the arcuate nucleus is not thought to be sexually dimorphic, and this population appears to be important for the regulation of steroid negative (rather than positive) feedback. Collectively these findings are important because they demonstrate that permanent, sex-specific effects within the brain are possible at exposure levels approximately equivalent to the LOAEL. By definition, no effects should be observable at or below this level.
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