Monograph on the Potential Human Reproductive and ... - OEHHA

356 CHAPIN ET AL.
concentrations used and time that cells were incubated.
There was no discussion of statistical procedures, and it
was not clear if statistical analyses were conducted for
some endpoints.]
Bisphenol A inhibited calcium ATPase activity in rat
testis microsomes. Mean7SEM median inhibitory concentration
(IC50) values were reported at 0.4070.15 mM
[91734 lg/L] for inhibition of calcium ATPase activity
and 2.571.0 mM [5717228 mg/L] for calcium uptake.
Exposure to 200 mM [47 mg/L] bisphenol A increased
intracellular calcium levels in TM4 cells. A viability study
was conducted to determine if increased intracellular
calcium levels resulted in cell death. Bisphenol A
exposure resulted in reduced TM4 cell viability (percent
viability compared to control cells was 93, 64, and 17% at
concentrations of 100, 300, and 600 mM). The study
authors concluded that these results provide evidence
that environmental estrogens may induce toxicity in male
reproductive development by disrupting calcium
homeostasis.
Strengths/Weaknesses: This interesting mechanistic
study examined the role of bisphenol A in modulating
intracellular calcium levels. It is difficult to interpret the
relationship between microsomal and intact cell effects of
bisphenol A given the large difference in concentrations
needed to produce an effect. Moreover, it is not clear if
bisphenol A caused cytotoxicity by a calcium-dependent
or non-calcium-mediated process.
Utility (Adequacy) for CERHR Evaluation Process:
This study was not considered useful for the evaluation
process.
Tabuchi et al. (2002), supported by the Japanese
Ministry of Education, Culture, Sports, Science, and
Technology and Takeda Science Foundation, examined
the effects of bisphenol A exposure on viability and gene
expression in TTE3 cells, a mouse Sertoli cell line. The
cells were incubated for 24 hr in media containing 0 or
24–400 mM [5.5–91 mg/L] bisphenol A (99.7% purity) in a
DMSO vehicle [culture ware not indicated]. Cell
viability was determined, and gene expression changes
were examined using microarray and PCR techniques.
Data were analyzed by Dunnett multiple conversion test
or Student t-test. Compared to values in control cells,
bisphenol A exposure reduced cell viability by 25% at
100 mM [23 mg/L], 33% at 200 mM [46 mg/L], and 96% at
400 mM [91 mg/L]. Based on the results of the cell-viability
studies, a bisphenol A concentration of 200 mM [46 mg/L]
was selected for the gene expression studies. Of 1081
genes examined by microarray, mRNA was downregulated
in 3 cases and upregulated in 10 cases. Six genes
were selected for evaluation of mRNA expression by
PCR, and of those genes, 1 was downregulated (ERa) and
5 were upregulated (iNOS, chop-10, odc, BipGRP78, and
osip). The study authors concluded that microarray
analysis is a useful tool for investing molecular mechanisms
of bisphenol A-induced toxicity in testicular cells.
Strengths/Weaknesses: This interesting mechanistic
study appears to have been well conducted, but it is
unclear from the data if bisphenol A-related changes in
chop-10 are a primary (or secondary) effect or are the
result of cytotoxicity.
Utility (Adequacy) for CERHR Evaluation Process:
This study is not useful in the evaluation.
Tabuchi and Kondo (2003), supported by Japanese
Ministry of Education, Culture, Sports, Science, and
Technology, Takeda Science Foundation, and Toyama
Daiichi Bank Foundation, conducted a series of experiments
to examine the effects of in vitro bisphenol A
exposure on gene expression in mouse Sertoli cells. The
experiments used TTE3 cells, an immortalized Sertoli cell
line established from transgenic mice expressing temperature-sensitive
simian virus large T-antigen. Cells
were exposed to bisphenol A (99.7% purity) in a DMSO
vehicle [culture ware not discussed]. The majority of
experiments were repeated 2–4 times, and data were
analyzed by Student t-test. [Statistical significance was
not reported in the results section of the study.]
Before conducting gene expression studies, cells were
exposed to 25–400 mM [5.7–91 mg/L] bisphenol A for 3–
24 hr, and viability was determined using a tetrazolium
compound. Cell viability was reduced at bisphenol A
concentrationsZ200 mM [46 mg/L], and reductions in
viability were increased with longer durations of
exposure. Intracellular calcium levels were measured
using a fluorescence imaging technique over a 15-min
period in cells exposed to 0–400 mM [0–91 mg/L] bisphenol
A, and a dose-related increase in calcium influx
was observed at Z100 mM [23 mg/L]. Based on results for
cell viability and calcium influx studies, a concentration
of 200 mM [46 mg/L] was selected for the gene-expression
experiments.
Using a PCR technique, it was determined that
expression of mRNA for transferrin was decreased and
glucose-regulated protein mRNA was increased by
bisphenol A exposure of up to 24 hr. Observations of
increased intracellular calcium concentration and upregulated
glucose-regulated protein mRNA expression led
the study authors to conclude that bisphenol A stresses
the endoplasmic reticulum. Gene expression was analyzed
by a cDNA microarray technique after exposure for
3, 6, 12, and 24 hr, and it was determined that 31 of 865
genes examined were upregulated by exposure to
bisphenol A; no downregulation of genes was observed.
The greatest change in gene expression was observed for
chop-10, a stress-response gene. Upregulation of 4 genes,
c-myc, fra-2, odc, and chop-10, were confirmed by
quantitative PCR. Chop-10 was determined to be the
most responsive gene. To determine if chop-10 was
required for development of endoplasmic reticulum
stress and cell injury, a stably transfected cell line
expressing chop-10 antisense RNA (chopR14) was developed.
Mock cells were used as negative controls in
studies where cells were exposed to 200 mM [46 mg/L]
bisphenol A for up to 24 hr. Production of chop-10
protein, as determined by Western blot analysis, was
reduced in the chopR14 cells compared to the mock cells
following exposure to bisphenol A. In contrast to the
mock cells, no reductions in cell viability or transferrin
mRNA expression were observed in the chopR14 cells
following bisphenol A exposure. There were no changes
in glucose-regulated protein mRNA expression in
chopR14 versus mock cells. The study authors postulated
that bisphenol A may disrupt the male reproductive
system by altering calcium homeostasis in Sertoli cell
endoplasmic reticulum without interacting with the ER
and that genes such as chop-10 may be involved in the
process.
Strengths/Weaknesses: This mechanistic study appears
to have been well conducted, but it is unclear
from the data if bisphenol A-related changes in chop-10
Birth Defects Research (Part B) 83:157–395, 2008