PHTHALATES AND
PHTHALATE
ALTERNATIVES: EFFECTS
ON PROLIFERATIVE AND
ESTROGENIC TARGET
GENES
 Endocrine disruption occurs when a synthetic chemical,
endocrine disrupting chemical (EDC), is absorbed into the
body an...
 Although the phthalates Di-n-butyl Phthalate (DBP) and
Diisononyl phthalate (DINP) have been banned in
production, phtha...
1. Phthalate Alternatives are being used extensively to replace
phthalates as plasticizers in commercial production, but
t...
 Observe and compare the effects of two
phthalates, DBP and DINP, and two
phthalate alternatives, DOTP and
BHT, (combined...
1. Phthalates will increase expression of proliferative
and estrogenic genes since they have shown such
effects in male re...
CHEMICAL SUMMARIES
[9]
CHEMICAL STRUCTURES
Di-Butyl Phthalate (DBP):
commonly used phthalate in
industrial production.
Diisononyl Phthalate (DINP...
CELL TREATMENT
Ishikawa Cells are starved for 24
hours
EXPERIMENT 1
DBP
BHT+E2
DOTP+E2
DINP+E2
DBP+E2
E2*
VEH
BHT
DINP
DOT...
DATA COLLECTION
Cells Directly Lysed for RNA Extraction
RNA Reverse Transcribed into cDNA
PCR Reaction performed
Comparati...
CONTROLLED VARIABLE:
1. Vehicle treatment with ethanol only at 10-5
concentration.
INDEPENDENT VARIABLE:
1. Ishikawa cells...
 We determined change in expression of the 7 target
genes. The housekeeping gene was approximately equal
in CT count for ...
1. C-myc is a regulatory transcription factor which
affects cell proliferation, cell growth, and
differentiation.
2. In co...
1. Cyclin D1 (CD1) works primarily during the G1 phase of
the cell cycle and is shown to be a marker of cell
proliferation...
1. WISP-2 is an estrogen mediated gene that
influences the mediation of the WNT proteins
which affect developmental proces...
1. Progesterone receptor is a major target gene
of estrogen action and plays an important
role of differentiation within t...
1. Estrogen receptor is a nuclear receptor that is
responsive to estrogenic compounds [2].
2. Expression of the Estrogen R...
Figure 1: The results from experiment 1 (no co -
treatment with estradiol). Fold change conveys
exponential increase or de...
Figure 3: The results from experiment 1 (no
co-treatment with estradiol) for the gene
progesterone receptor. The most sign...
The Hypothesis was proved partially correct.
1. Phthalates increased expression of proliferative and
estrogenic genes, the...
 DBP, DINP, DOTP, and BHT lead to upregulation in
proliferative and estrogen-mediated genesmaking
it possible to classif...
 The outcome of Hypothesis #3 may be attributed to
antagonistic effects of Endocrine Disruptors.
RESULT ANALYSIS
Antagoni...
1. Therefore, the total effects are likely to be more
prominent in leading to disease.
2. We can declare DBP, DINP, DOTP, ...
 Although this study showed results for the endocrine
disruptive effects of DBP, DINP, DOTP, and BHT only on
Ishikawa cel...
1. Endocrine disruptors, Natural resources defense council (1998).
2. European Commission, Environment (2013).
3. UNPE and...
The work in Dr. Serdar E. Bulun’s laboratory is funded in part by
grant number P01-H1O057877 given by the National Institu...
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PHTHALATES AND PHTHALATE ALTERNATIVES: EFFECTS ON PROLIFERATIVE AND ESTROGENIC TARGET GENES

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A novel study shows that phthalates and phthalate alternatives can be classified as potential endocrine disruptors. Read more to find out!

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PHTHALATES AND PHTHALATE ALTERNATIVES: EFFECTS ON PROLIFERATIVE AND ESTROGENIC TARGET GENES

  1. 1. PHTHALATES AND PHTHALATE ALTERNATIVES: EFFECTS ON PROLIFERATIVE AND ESTROGENIC TARGET GENES
  2. 2.  Endocrine disruption occurs when a synthetic chemical, endocrine disrupting chemical (EDC), is absorbed into the body and disrupts the body’s normal functions by mimicking or blocking estrogen.  Estrogen is produced in all vertebrates and it is important that estrogen is released at the right time and stage in the organism to maintain homeostasis.  Phthalates are chemicals commonly used as plasticizers in industrial production. INTRODUCTION Estrogen enters the cell, binds to Estrogen Receptor and causes transcription of certain genes to occur.
  3. 3.  Although the phthalates Di-n-butyl Phthalate (DBP) and Diisononyl phthalate (DINP) have been banned in production, phthalate alternatives such as Di-octyl Terephthalate (DOTP) and Butylated Hydroxytoluene (BHT) have replaced traditional phthalates.  Very little conclusive research has been done on the effects of these chemicals on the reproductive system. CURRENT STUDIES
  4. 4. 1. Phthalate Alternatives are being used extensively to replace phthalates as plasticizers in commercial production, but there is very little conclusive research done on the harmful effects of phthalates and phthalate alternatives. 2. If we are going to be constantly exposed to these chemicals and since over 1 million tons of these chemicals are used in production, it is important to know their effects. 3. Investigating EDCs is important in improving public health because presence of EDCs within the body can lead to reproductive diseases such as endometriosis, infertility, diabetes, metabolic syndrome, breast or prostate cancer, early puberty, and obesity. 4. This original study pioneers in testing the effects of certain phthalates and phthalate alternatives in a real-time in vitro environment. IMPORTANCE
  5. 5.  Observe and compare the effects of two phthalates, DBP and DINP, and two phthalate alternatives, DOTP and BHT, (combined with and without estradiol) in comparison to Vehicle (VEH) and 17β-estradiol (E2) treatments on target genes affecting proliferation and estrogenic function in Ishikawa cells. PURPOSE
  6. 6. 1. Phthalates will increase expression of proliferative and estrogenic genes since they have shown such effects in male reproductive system. 2. Phthalate Alternatives should not change expression of proliferative and estrogenic genes since they have replaced phthalates in production. 3. Results from Experiment 2 will magnify fold- changes seen in Experiment 1 because addition of E2 to each treatment should simply increase fold- change to a greater degree. We will add E2 to each treatment (in Exp. 2) in order to simulate real-time body conditions. HYPOTHESIS
  7. 7. CHEMICAL SUMMARIES [9]
  8. 8. CHEMICAL STRUCTURES Di-Butyl Phthalate (DBP): commonly used phthalate in industrial production. Diisononyl Phthalate (DINP): phthalate used in phthalate 17- Estradiol (E2): Naturally occurring within the human body. Butylated Hydroxytoluene (BHT): phthalate alternative, commonly used as a food additive Dioctyl Terephthalate (DOTP): phthalate alternative used as a plasticizer
  9. 9. CELL TREATMENT Ishikawa Cells are starved for 24 hours EXPERIMENT 1 DBP BHT+E2 DOTP+E2 DINP+E2 DBP+E2 E2* VEH BHT DINP DOTP VEH E2* EXPERIMENT 2 Cells are treated with compound for 24 hours at 10-5 M.* *Only E2 was 10-7 M because of standard procedures. Ishikawa cells are grown and cultured until 70-80% confluent
  10. 10. DATA COLLECTION Cells Directly Lysed for RNA Extraction RNA Reverse Transcribed into cDNA PCR Reaction performed Comparative CT setting was used to determine relative amounts of target gene expression in each sample Fold change calculated by comparing CT values of target genes to those of GAPDH, the housekeeping gene. *Cycle Threshold (CT) is the number of cycles required for the fluorescent signal (after PCR reaction) to exceed the threshold.
  11. 11. CONTROLLED VARIABLE: 1. Vehicle treatment with ethanol only at 10-5 concentration. INDEPENDENT VARIABLE: 1. Ishikawa cells are treated with various chemical compounds. 2. Cells are treated with compound in conjunction with or without estradiol (E2). DEPENDENT VARIABLE: 1. Change in expression of proliferative and estrogenic genes VARIABLES
  12. 12.  We determined change in expression of the 7 target genes. The housekeeping gene was approximately equal in CT count for each treatment in both experiments. DATA ANALYSIS PROCEDURES Gene Targets Proliferative Estrogenic C-Myc CD1 PS2WISP-2 PR ER SDF-1
  13. 13. 1. C-myc is a regulatory transcription factor which affects cell proliferation, cell growth, and differentiation. 2. In comparison to the vehicle, all treatments without addition of estradiol increased expression of C-myc. 3. Treatments with the target chemicals and estradiol together increased expression of C-myc in comparison to vehicle treatment, observing the same trend as observed when treating cells with compound only. RESULTS: PROLIFERATIVE GENES
  14. 14. 1. Cyclin D1 (CD1) works primarily during the G1 phase of the cell cycle and is shown to be a marker of cell proliferation. 2. There was an increase in expression of CD1 in cells treated with DBP, DINP, and DOTP while cells treated with BHT showed a decrease in expression. 3. This trend was for the most part consistent with the results observed with treatment of compound and estradiol in Experiment 2. 4. the addition of estradiol to the other compounds led to a more prominent increase in fold change (Experiment 2). RESULTS: PROLIFERATIVE GENES
  15. 15. 1. WISP-2 is an estrogen mediated gene that influences the mediation of the WNT proteins which affect developmental processes. 2. Combination of estradiol and compound (Experiment 2) decreased the expression of WISP2 more than if the cells had been treated with the compound only (Experiment 1). RESULTS: ESTROGENIC GENES
  16. 16. 1. Progesterone receptor is a major target gene of estrogen action and plays an important role of differentiation within the uterus. 2. Treatment with E2 heavily increased expression of PR, as expected since Estradiol is a known PR-mediator. 3. With the addition of E2 to chemical treatments in Experiment 2, upregulation was significantly influenced. RESULTS: ESTROGENIC GENES
  17. 17. 1. Estrogen receptor is a nuclear receptor that is responsive to estrogenic compounds [2]. 2. Expression of the Estrogen Receptor in E2 treated cells was slightly greater than that of the vehicle group. 3. In comparison with experiment 1, there was much less change in experiment 2, likely because of the mediating effects of Estradiol. RESULTS: ESTROGENIC GENES Pathway of ER activation through estrogen
  18. 18. Figure 1: The results from experiment 1 (no co - treatment with estradiol). Fold change conveys exponential increase or decrease in target gene expression. 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 CD1 C-MYC ER SDF1 PS2 WISP2 FoldChange Gene Target VEH DBP DINP DOTP BHT E2 Figure 2: The results from experiment 2 are shown above (compound and estradiol co - treatment) for target proliferative and estrogenic genes. 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 CD1 C-MYC ER SDF1 PS2 WISP2 FoldChange Gene Target VEH DBP+E2 DINP+E2 DOTP+E2 BHT+E2 E2 DATA
  19. 19. Figure 3: The results from experiment 1 (no co-treatment with estradiol) for the gene progesterone receptor. The most significant statistically significant increase in expression of PR occurred in cells treated with E2 (p<0.0001). 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25 V DBP DINP DOTP BHT E2 FoldChange Cell Treatment PR PR Figure 4: The results from experiment 2 (compound and estradiol co -treatment) for the gene Progesterone receptor. DBP+E2, DINP+E2, DOTP+E2, BHT+E2, and E2 significantly increased PR expression (p<0.0001). 0 2 4 6 8 10 12 V DBP+E2 DINP+E2 DOTP+E2 BHT+E2 E2 FoldChange Cell Treatment PR PR DATA
  20. 20. The Hypothesis was proved partially correct. 1. Phthalates increased expression of proliferative and estrogenic genes, therefore acting as potential endocrine disruptors. Therefore, Hypothesis #1 was correct. 2. Phthalate alternatives also increased expression of proliferative and estrogenic genes, also acting as potential endocrine disruptors. Therefore Hypothesis #2 was incorrect. 3. Hypothesis #3 was incorrect. Simply adding E2 to each treatment in Experiment 2 led to unforseen decreases in expression of target genes in comparison to Experiment 1. Therefore there must be some internal interaction between E2 and each phthalate and phthalate alternative. DISCUSSION
  21. 21.  DBP, DINP, DOTP, and BHT lead to upregulation in proliferative and estrogen-mediated genesmaking it possible to classify them as potential endocrine disrupting compounds.  Substances that have properties that may lead to endocrine disruption, such as abnormal increase in proliferation and heavy, abnormal increase or decrease in expression of estrogen-mediated genes. OUTCOMES
  22. 22.  The outcome of Hypothesis #3 may be attributed to antagonistic effects of Endocrine Disruptors. RESULT ANALYSIS Antagonistic Effects of EDCs Bonding to Cellular Receptor Binding to Transport Proteins Alters Metabolic Rates Prevents Estrogen from binding Affects degradation of Estrogen
  23. 23. 1. Therefore, the total effects are likely to be more prominent in leading to disease. 2. We can declare DBP, DINP, DOTP, and BHT as potential endocrine disruptors because they increase expression of proliferative and estrogen- mediated genes for the most part. 3. We think that DBP and DOTP may follow mechanisms similar to estradiol while DINP and BHT may follow mechanisms more similar to progesterone. IMPLICATIONS OF STUDY
  24. 24.  Although this study showed results for the endocrine disruptive effects of DBP, DINP, DOTP, and BHT only on Ishikawa cells, the claims and observations reported can be applied to healthy myometrium cells and even leiomyoma cells.  In this relatively unstudied field, our study produces novel results and proposes that man-made chemicals interact with natural hormones within the cell.  Therefore, it is important to control the use of these substances in industrial, medicinal, and household products because of the effects of these chemicals on the female reproductive system. APPLICATIONS
  25. 25. 1. Endocrine disruptors, Natural resources defense council (1998). 2. European Commission, Environment (2013). 3. UNPE and WHO, Inter-Organization Programme for the Sound Management of Chemicals (2012). 4. Tulane University, E.Hormone (2012). 5. McLachlan, J. A, Best Practice and Research Clinical Endocrinology and Metabolism. 20, 63 (2005). 6. Moody, S., et. al. Endocrinology, 154, 3460 (2013). 7. University of Massachusetts Lowell, Lowell Center for sustainable production (2011). 8. Kulak, J, et al., Reproductive Sciences. 20, 129 (2012). 9. Gui, Y, et al., MHR Basic Science of Reproductive Medicine. 5, 866 (1999). 10.Ohta, Y, et al. Ann Otol Rhinol Laryngol. 109, 1046 (2000). 11.Li, Y, et al., Environmental Health Perspectives. 121, 459 (2013). 12.Nishida, M. Hum Cell. 15, 104 (2002). 13.Evans, R. et al., Proceedings of the National Academy of Sciences of the United States of America. 77, 5856 (1980). REFERENCES
  26. 26. The work in Dr. Serdar E. Bulun’s laboratory is funded in part by grant number P01-H1O057877 given by the National Institutes of Health (NIH). ACKNOWLEDGEMENTS

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