KA, PH Peterman, BM Judy, JA Taylor, CE Orazio, RL Ruhlen, FS vom
Saal, and WV Welshons 2003. Bisphenol A is released from
used polycarbonate animal cages into water at room temperature.
Health Perspectives doi:10.1289/ehp.5993.
leaching out of water bottles causes aneuploidy
et al. report that significant amounts of bisphenol A leaches
into water that comes in contact with polycarbonate animal cages.
Their results raise the specter of widespread inadvertent
and hitherto unsuspected contamination of generations of animal
testing by an estrogenic compound, bisphenol
A. The result may be that many experiments have provided false
assurances of safety about hormonal impacts of contaminants.
results may also help explain inconsistencies between different
experiments, given that the type of animal housing differs among
labs and the rate of leaching is affected by the age of the polycarbonate
could this be? Most hormonal systems have maximal response levels
to exposure, demonstrating what scientists call an asymptote.
dose increases, there comes a point at which further increases
in dose no longer increase the effect. In hormone systems this
occurs by the time that all available hormone receptors have
bound to hormone molecules.
the following two circumstances (graphs below):
this hypothetical experiment, there is an asymptotic relationship
between the dose and the response. In the first version, to
the left, there is no inadvertent estrogenic contamination.
Thus controls have 0 response. Addition of an experimental
treatment provokes an increase in response. The difference
between E and C is the observed effect.
the graph to the right, the "control" has significant
estrogenic contamination. Hence "C", the control response,
is significantly greater than true 0 (although the experimenter
is unaware that they are not measuring "true 0") and
close to the maximum response, "E", that can be achieved
via experimental treatment.
difference between "E" and "C" is much smaller
than that between E and C (in the first graph) and more likely to
be insignificant, particularly with small sample sizes or in experiments
with statistical variability.
bisphenol A contamination has been shifting control animals to the
right along the dose curve, then experimenters are less likely to
have found the estrogenic effects they would have observed without
the bisphenol A contamination.
effect would be seen in experiments with compounds that share molecular
mechanisms with bisphenol A, e.g., an ability to bind with the estrogen
did they do? Howdeshell et al. put purified water
in different types of cages at room temperature and neutral pH,
let the water sit for one week, and then measured both the amount
of BPA in the water using high pressure liquid chromatography and
the estrogenicity of the water using a standard assay (the MCF-7
human breast cancer cell proliferation assay).
types were new vs. old polycarbonate, new polysulfone,
old polypropylene, and glass. Polycarbonate and polysulfone cages
are made with bisphenol A. They also compared the uterine weight
of mice reared shortly after weaning for one week in old polycarbonate
vs. old polypropylene cages. Animals in polycarbonate cages drank
water from old polycarbonate water bottles, while those in polypropylene
drank water from glass bottles.
cages were rinsed prior to the experiments to remove prior leaching,
and were not scrubbed for the duration. Thus any accumulation was
a result of passive leaching during the week of the experiment.
did they find? Old polycarbonate leached significantly
more bisphenol A than any other type of cage. New polycarbonate
and new polysulfone leached detectable amounts, approximately one-tenth
or less that of the old polycarbonate. No bisphenol A was detectable
in the water from the glass or polypropylene cages, although trace
amounts of nonylphenol, another estrogenic substance, were measured
in the latter.
the MCF-7 assay, all water samples from old polycarbonate cages
induced proliferation. By adding an anti-estrogen to these experiments,
they were able to block the response, indicating it was caused by
an estrogenic substance in the water. Additional chemical analysis
strongly indicated that the estrogenicity was due to BPA.
from new polycarbonate and new polysulfone cages did not stimulate
proliferation. Water from one of the old polypropylene cages, however,
did produce a slight elevation in response, most likely because
of the trace amounts of nonylphenol (noted above).
the mouse experiments, "prepubertal uterine wet weight was
approximately 16% heavier in females housed in the used polycarbonate
caging with used polycarbonate water bottles relative to mice housed
in used polypropylene cages, although the results were not statistically
significant (P =0.31). The authors note, however, that uterine weight
is not a very sensitive whole-animal assay for BPA exposure. Patterns
of mammary duct development are approximately 4000 times more
does it mean? According to the authors: "Our findings
here suggest that aquatic laboratory animals may be exposed to sufficient
BPA due to leaching from worn polycarbonate caging to result in
a significant impact on reproductive parameters. This prediction
is based on a number of recent reports of significant effects at
very low concentrations of BPA in frogs, fish and mollusks."
to mammals like rats and mice, the cornerstones of regulatory toxicology,
while the uterine results were not significant, it is likely other
assays such as in utero effects on prostate
weight or time
to puberty would have yielded significant results, as these,
like mammary duct development, are far more sensitive to BPA.
et al. also observe that polycarbonate is not the only
source of bisphenol A in the laboratory: "BPA migration into
human serum has been reported with the use of polycarbonate and
polysulfone plastic hemodialysis equipment. Another potential route
of BPA exposure in the laboratory is polyvinyl chloride (PVC) pipes
used in the supply of tap water; BPA is added as a stabilizer in
the production of PVC products.
recommend that researchers be aware of the potential confounding
effects of BPA contamination and take extra measures to minimize
this possibility, including filtering water.
key question, which they do not even begin to address, is how many
negative results now pervade the scientific and regulatory literature
because of BPA leaching from laboratory equipment. Their findings
make this a strong possibility.