



Kubo, K, O Arai, M Omura, R Wantanabe, R Ogata, and S Aou. 2003. Low dose effects of bisphenol A on sexual differentiation of the brain and behavior in rats. Neuroscience Research 45: 345-356.

Kubo et al. report that at levels beneath the 'safe' level established by the EPA, bisphenol A (BPA) exposure in the womb alters sexual differention of the brain and behavior in rats. Their study adds to the weight of evidence that health standards for exposure to this molecule, used to make polycarbonate plastic and now a ubiquitous contaminant found in many people, need to be strengthened dramatically.

Exposure to BPA is widespread. BPA has been detected in human tissues from Japan, Germany and in 95% of human urine samples in the US.

Most recent research on BPA's potential for causing harm has focused on reproductive endpoints, and many studies now document reproductive changes at low levels of exposure. Kubo et al.'s startling findings expand the range of endpoints into a new domain, brain development and behavior.

What did they do?

Kubo et al. exposed rats while pregnant and while nursing to BPA,at two different doses, 30 µg/kg/day and 300 ug/kg/day (referred to below as BPA-L and BPA-H, respectively). Current EPA standards identify 50 µg/kg/day as the “safe” level (“reference dose”). Kubo et al. then compared the offspring of the BPA-exposed rats to the offspring of unexposed controls, as well as to the offspring of a positive control group exposed to DES, and also to the offspring of rats exposed to another estrogenic chemical called resveratrol (RVT) naturally present in grapes and other fruits.

After birth, they examined the reproductive development, brain structure and behavior of the rats that had been exposed in the womb:

  Reproductive development: They measured body weight and anogenital distance on the first day after birth, and tracked offspring for timing of testicular descent and vaginal opening. At the age of 12 weeks male and female rats were killed and their reproductive organs dissected to observe various reproductive system endpoints, including sperm counts, the weight of reproductive organs and hormone levels.  

Brain structure: Some of the offspring were sacrificed at the age of 14 weeks to examine the size of two areas of the brain known to be sexually dimorphic:

  • the locus coeruleus (LC), believed to be a key brain center for anxiety and fear. Normally this is larger in females than in males
  • an area in the hypothalamus called the sexually dimorphic nucleus of the preoptic area (SDN-POA), which plays a role in regulating sexual behavior and has been proposed, in humans, to be important for determining sexual orientation. The SDN-POA is normally larger in males than in females.


Behavior: When the rats exposed in the womb reached the age of 6 weeks, they tested their response to a novel setting using the standard "open field test." This test is used to quantify both their exploratory behavior and their fear response. Previous studies have demonstrated that males and females typically perform differently on this test, and that exposure to sex hormones (testosterone or estradiol) during a critical period soon after birth can eliminate gender differences in performance.

Kubo et al. also examined the sexual behavior of male offspring by observing the number of times a male mounted a female, the number of mounts with vaginal penetration that preceded ejaculation, the proportion of mounts with vaginal penetration that preceded ejaculation, and various measures of the time it took for the males to ejaculate. Female sexual behavior was measured by the number of ear wiggles, the proportion of mounts where females took a sexually receptive position (called lordosis), and by a previously-developed score showing whether and how much females rejected male rats (from 0 (no rejection) to 3 (maximum rejection)



What did they find?

Kubo et al. found that exposure to BPA reversed the sex differences in the size of the LC area of the brain and eliminated the usual sex differences in the open field behavior test, without affecting sexual development of the reproductive system.

In control animals, LC volume is greater in females than in males (graph to right).

Animals treated with BPA at both low and high doses and with DES all showed a reversal of this normal sexual dimorphism.

Males differed significantly from females within all treatments ( p <0.05).


In addition, all treated animals differed from controls of the same sex except males receiving high bisphenol A and females treated with DES ( p < 0.05).

Exposure to RVT eliminated the normal sexual dimorphism in LC volume (not shown).

They also found that exposure to bisphenol A and DES reversed the normal sexual dimorphism in cell number in the LC (not shown). Normally females have more neurons in the LC than males. RVT eliminated the normal dimorphism.

BPA had no effect on the size of the SDN-POA.

Exposed animals behaved differently in the open field tests compared to controls.


In untreated animals, females moved significant more, reared more frequently (graph to left), and stayed longer in the center area than males.

These sex differences were reduced or eliminated following in offspring exposed to BPA.

As one example (above), while male and females differed in the amount of rearing behavior in the control group ( p<0.05), this sexual dimorphism was absent in animals treated with bisphenol a (but present in animals treated with DES). Males treated with low doses of BPA and low doses of DES differed significantly from males in the control group ( p <0.05).

Differences in the two kinds of exploratory behavior (rearing, length of stay in the center area) disappeared, but there was a still a difference between the sexes in how much they moved. For some behaviors observed in this test (length of stay in the center area), the males shifted toward more female-like behavior and females shifted toward more male-like behavior (suggesting that BPA caused both demasculinization of males and defeminization of females).

For rearing and locomotion, male behavior became more female-like but female behavior did not change. DES had similar but not identical effects as BPA, whereas the offspring of RVT-exposed rats behaved the same as controls in this test. The results in the open-field test are summarized in the table below.



BPA: low dose

BPA: high dose



Distance moved

Same as controls

Sex difference eliminated (males behave like control females)

Sex difference eliminated (males behave like control females)

Same as controls

Exploring: rearing

Sex difference eliminated (males behave like control females)

Sex difference eliminated (males behave like control females)

Sex difference eliminated (males behave like control females)

Same as controls

Exploring: time in center

Sex difference eliminated (males and females behave intermediate between control males and females)


Sex difference eliminated and both males and females stayed in center more than controls

Same as controls

At the doses used in this study, BPA had no effect on hormone levels, sexual development (as defined above), sperm counts, female sexual behavior, and little effect on male sexual behavior. The proportion of mounts with vaginal penetration that preceded ejaculation (out of the total number of mountings) decreased in the offspring of animals exposed to the low-dose of BPA, but not in the high-dose group, compared to controls.

BPA had no effect on the weight of female reproductive organs (uterus and ovaries), but did increase the weight of the testis in male offspring of animals exposed to the high dose of BPA.

What does it mean?

Previous work by Kubo et al. had shown that higher doses of BPA given to pregnant and nursing rats interfered with the normal gender differences in the LC and behavior (in the open field test).

The current study adds to a growing body of evidence showing adverse impacts of BPA beneath levels considered to be safe by government standards. It is the first indication that exposure to BPA before birth and during nursing at these low levels can disrupt the normal sexual differentiation of both the brain and behavior, indicating that the government health standard for BPA may be inadequate. Other effects observed at low doses include altered reproductive tract development and function, increased rate of sexual maturation and changes in maternal behavior.







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