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 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.

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

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 eff ects 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 eff ects.

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 eff ects of certain phthalates and phthalate alternatives in a real-time in vitro environment.

IMPORTANCE

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

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

CHEMICAL SUMMARIES

[9]

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

CELL TREATMENT

Ishikawa Cells are starved for 24 hours

EXPERIMENT 1

DBP

BHT+E2

DOTP+E2

DINP+E2

DBP+E2

E2*

VEH BHT

DINPDOT

P

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

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.

CONTROLLED VARIABLE: 1. Vehicle treatment with ethanol only at 10 -5

concentration.INDEPENDENT VARIABLE: 2. Ishikawa cells are treated with various

chemical compounds.3. Cells are treated with compound in

conjunction with or without estradiol (E2). DEPENDENT VARIABLE:4. Change in expression of proliferative and

estrogenic genes

VARIABLES

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

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

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

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

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

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

Figure 1: The resul ts f rom exper iment 1 (no co -treatment wi th estradio l ) . Fo ld

change conveys exponent ia l increase or decrease in target gene express ion.

CD1 C-MYC ER SDF1 PS2 WISP20

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

VEHDBPDINPDOTPBHTE2

Gene Target

Fold

Change

Figure 2: The resul ts f rom exper iment 2 are shown above (compound and estradio l co -treatment) for target

pro l i ferat ive and estrogenic genes.

CD1 C-MYC ER SDF1 PS2 WISP20

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

VEHDBP+E2DINP+E2DOTP+E2BHT+E2E2

Gene Target

Fold

Change

DATA

Figure 3: The resul ts f rom exper iment 1 (no co -treatment wi th estradio l ) for the gene progesterone receptor. The most

s ignifi cant stat ist ica l ly s ignifi cant increase in express ion of PR occurred in

ce l ls t reated with E2 (p<0.0001) .

V DBP DINP DOTP BHT E20

2.5

5

7.5

10

12.5

15

17.5

20

22.5

25

PR

PR

Cell Treatment

Fold

Change

Figure 4: The resul ts f rom exper iment 2 (compound and estradio l co -treatment)

for the gene Progesterone receptor. DBP+E2, DINP+E2, DOTP+E2, BHT+E2,

and E2 s ignifi cant ly increased PR express ion (p<0.0001) .

V

DBP+E2

DINP+

E2

DOTP+E2

BHT+E2 E2

0

2

4

6

8

10

12

PR

PR

Cell Treatment

Fold

Change

DATA

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

DBP, DINP, DOTP, and BHT lead to upregulation in proliferative and estrogen-mediated genesmaking 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

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

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

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

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

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