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Sarah Mae Pope
ENV 499: Prospectus
November 10, 2011
Does Exposure to 4-tert-octylphenol Cause Genetic Alterations Similar to Estrogen?
The Environmental Protection Agency has established that certain synthetic
chemicals may alter normal endocrine processes in both human and non-human animals
(U.S Environmental Protection Agency, 2003). These substances are collectively known
as endocrine disrupting chemicals (EDC’s) and are connected to a variety of
physiological pathologies including type II diabetes, decrease in gamete quality,
autoimmune disease, and infertility (Van, 2011). Of the many products listed as EDC’s,
the class of chemicals known as alkylphenols are particularly notorious for their
connection to reproductive dysfunction.
Alkylphenols such as 4-tert-octylphenol (OP) are synthetic detergents used in a
variety of commercially available cleaning products, as well as for industrial purposes as
dispersants and agents to maintain emulsion (Bonefeld- Jorgensen, Long, Hofmeister, &
Vinggaard, 2007). Alkylphenols have the potential to accumulate within the environment,
especially in aqueous reservoirs such as sewage containments, streams, rivers and lakes
(Funabashi, Nakamura, & Kinura, 2004). The potential for drinking water contamination
is substantial with levels of OP as high as 0.084 ppb being found in some water systems
(Mayer, Dyer, & Propper, 2003). Previous studies have demonstrated that exposure to
octylphenol can lead to female sex biases among developing amphibians (Kloas, Lutz, &
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Einspanier, 1999). Long-term exposure has also been shown to cause a number of
unusual reproductive characteristics among phenotypically male Xenopus laevis, such as
the presence of serum vitellogenin, decreased sperm count, as well as the growth of
oviducts (Porter et al., 2011). The purpose of this study is to better understand the
mechanisms by which OP disturbs normal endocrine processes.
The feminization of genetically male amphibians suggests that OP may be
operating as an estrogen agonist- either by actively binding to estrogen receptors or
through the activation of processes (such as gene transcription) that are normally estrogen
dependent. Previous research on the potential for octylphenol estrogen receptor (ER)
binding has shown that OP has the capacity to bind to ER’s within fish (Andreasse &
Korsgaard, 2000), rats (Gregory et. al, 2009) and human cells (Paris et al., 2002).
However, the affinity of OP for estrogen receptor binding is nearly 1,000 fold less than
the binding potential for 17--estradiol (Andreassen, Skjoedt, & Korsgaard, 2005). The
binding of OP to estrogen receptors strongly suggests estrogenic activity of this chemical.
If OP is indeed acting through estrogenic pathways, along with ER binding, we
may also expect there to be alterations in transcription levels of genes normally
influenced by the presence of estrogen, such as cytochrome P450 aromatase (CYP19) and
steroidogenic factor-1. The biosynthesis of estrogens requires the aromatization of
testosterone; this rate-limiting step is catalyzed by cytochrome P450 aromatase (Lange,
Hartel, & Meyer, 2002). Studies in vertebrates have shown that estrogens often act as upregulators of cytochrome P450 aromatase (CYP19) activity (Kinoshita & Chen, 2003;
Sretarugsa & Wallace, 1997). Therefore, we may expect that EDC’s that mimic the
activity of estrogen would also cause an increase in CYP19 action.
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Steroidogenic factor-1 (SF-1) is an orphan nuclear receptor that functions
as a co-activator for many genes involved in the manufacture of steroid hormones from
cholesterol (Busygina, Vasiliev, Klimova, Ignatieva, & Osadchuk, 2005). In particular,
SF-1 regulates the activity of CYP19 during steroidogenesis (Ikeda, Shen, Ingraham, &
Parker, 1994) and exhibits predicable patterns of expression during vertebrate gonadal
development (Ozisik, Achermann, Meeks, & Jameson, 2003). A study by Mayer, Dyer,
and Propper (2002), demonstrated that SF-1 displays sex-specific transcription levels
during gonadogenesis in the American bullfrog. They found that the expression of SF-1
increases immediately prior to the growth of ovarian tissue in female members of this
species. In male bullfrogs, the observed SF-1 transcription levels were found to decrease
during the biological manifestation of testes. Like CYP19, the presence of estrogen has
been shown to influence the normal expression of SF-1 in vivo (Fleming & Crews, 2001).
The effects of OP exposure on sexual differentiation result in biological
consequences that are characteristic of estradiol induction (e.g. female sex-bias, unusual
female-like anatomy in males). The affinity of OP to estrogen receptors, further suggests
that this EDC may be mimicking the normal activity of endogenous estrogens. If OP was
indeed acting through estrogenic mechanisms, we would expect to see similar
transcription levels of CYP19 and SF-1 induced by both OP and estrogen. Given that OP
has been shown to influence developmental processes similar to estradiol, we hypothesize
that concurrent exposure to OP and estrogen will cause similar expression of CYP19 and
SF-1.
To test this hypothesis, oocytes from the model species Xenopus laevis will
receive acute exposure to both 17- estradiol (EE2) and OP. Afterward, transcription
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levels of CYP19 and SF-1 will be examined using real-time polymerase chain reaction
(RT-PCR). In order to assess gene transcription products, protein concentrations of both
estradiol and progesterone will be recorded via enzyme-linked immunosorbent assay
(ELISA).
Materials and Methods
Animals, Laboratory Supplies and Culture Media
Female African clawed frogs (Xenopus laevis) will be obtained from a
commercial supplier. The animals will be housed in glass containers filled with reverse
osmosis treated water. Room temperature will be maintained at 24.5 F and animals will
be fed frog brittle three times a week. All biochemicals, hormones and media products
will be purchased from business suppliers.
A non-nutrient saline media for X. laevis oocytes will be formulated using
Leibovitz (L-15) and gentamicin. The L-15 will be prepared from a powder according to
the instructions provided upon purchase. Alterations to standard instructions will include
diluting the L-15 to 70% and adjusting to a pH of 7.4. The media solution will be
sterilized via Millipore filtration then stored in a refrigerator ( 3 C) pending use.
Oocyte Collection and Treatment
An adult female X. leavis will be euthanized by immersion in 0.02%
methanesulfonate-222 solution. Once killed, oocytes of combined stage groups will be
collected and transferred to the L-15 media solution. Each oocyte sample will receive 1
of 14 treatments. Treatment groups include OP exposure samples (10-9, 10-8, 10-9 M), as
well as 10-9, 10-8, 10-9 M E2 exposed conditions both with and without 10-10 M human
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chorionic gonadotropin hCG added. There will also be a sample of oocytes incubated
only with 10-10 hCG and one that will not be given any treatment.
Once oocytes are assigned to a particular sample, they will be incubated with
treatment for 12 hours in a Dubnoff incubator. The Dubnoff machine will be set at 80
oscillations per minute. Upon completion of incubation, the samples will be transferred to
RNA later stabilization reagent to preserve gene expression and kept frozen (-18 C) until
ready to analyze.
Gene Transcription and Protein Analysis
Prior to genetic analysis, RNA for each sample will be acquired using Purelink
Purification System instructions for extraction. The purified RNA will be resuspended in
40 L RNAse free water. Nanodrop ND- spectrophotometer will be used to assess the
concentration and purity of the nucleic acids within each sample. Synthesis of cDNA will
be accomplished by adding 11L of RNA extract with 1 µL of oligo (dT) primer, then
allowing solution to run through the Thermocyler program cDNA-BTS.
Once the cDNA has been prepared, real-time PCR (RT-PCR) will be run using
the Biorad iQ5 PCR machine cDNA amplification protocol. Dilution series of 1:4 to
1:128 will be used for CYP19 and SF-1 genes. Annealing temperatures for CYP19 and
SF-1 are 62°C and 58°C respectively. Once PCR is complete, all PCR data will be
analyzed using the comparative CT method.
Concentration of steroids will be determined through the use of an immuno
evaluation. Estrogen and progesterone will be assayed with a commercial ELISA kit
using standards provided within the manufacturers instructions. Spectrophotometric
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readings will be converted to concentrations through employment of a best fit and linear
curve.
Projected Results
We anticipate that OP will exert molecular alterations similar to estradiol. That is,
exposure to OP should influence the activity of genes that are normally responsive to
estrogen binding and are essential to the process of steroidogenesis- namely CYP19 and
SF-1. If oocytes incubated with OP display transcription levels of CYP-19 and SF-1 that
are similar to that produced by estradiol, the supposition that OP disrupts endocrine
processes by operating as an estrogenic agonist will be supported. By strengthening the
hypothesis originally stated within this paper, we may suspect that OP could also
contribute to a number of physiological maladies commonly associated with exogenous
exposure to estrogen. In particular, estradiol has been recognized as a potent carcinogen
in both laboratory mammals and humans (International Agency for Research on Cancer,
1999). Exposure to estrogen has been shown to increase the presence of breast, pituitary,
lymphatic, testicular, uterine and cervical malignancies in rodents (Huseby,1980; Inoh,
Kaiya, Yokoro, 1985; Noble, Hochachka, & King, 1975). If OP was initiating the same
genetic cascade as estradiol, it may be inferred that the same processes that manifest
carcinogenesis through this sex steroid would be applicable for OP as well.
As mentioned previously, OP is found within many U.S. water sources
(Mayer, Dyer, & Propper, 2003). In light of its ability to disrupt reproductive
organogenesis, the potential for this chemical to corrupt drinking water is disturbing.
However, aqueous contamination may be especially traumatic if it is found that genetic
expression normally associated with malignant tumor development results from the
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influence of OP. Better understanding of the potential biological effects initiated by OP
exposure will allow us to more fully comprehend the risk associated with environmental
contact.
Timeline
To date: Oocytes have been collected and transferred to RNA later stabilization reagent.
November 20, 2011- January 15, 2012: RNA from oocytes will be extracted and
synthesis of cDNA will be completed.
January 15, 2012- March 15, 2012: PCR analysis of CYP19 and SF-1 transcription
levels from oocyte tissues will be finalized.
March 15, 2012- May 1, 2012: ELISA will be used to assess protein levels of P and E2.
May 1, 2012- May 15, 2012: All data will be analyzed for statistical significance and
results reported.
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