Characterization of Estrogenic Characterization of Estrogenic - - PowerPoint PPT Presentation

Characterization of Estrogenic Characterization of Estrogenic Endocrine Disrupting Endocrine Disrupting Chemicals in Source Waters Chemicals in Source Waters

• f Central Arizona
• f Central Arizona

Nikole Fales, Absar Alum, and Morteza Abbaszadegan Arizona State University

What is an Endocrine What is an Endocrine Disruptor? Disruptor?

• As defined by the EDSTAC

As defined by the EDSTAC

• “An exogenous substance that

“An exogenous substance that changes endocrine function and changes endocrine function and causes adverse effects at the level of causes adverse effects at the level of the organism, its progeny, and/or the organism, its progeny, and/or (sub) populations of organisms” (sub) populations of organisms”

Partial List of Synthetic and Natural Partial List of Synthetic and Natural Compounds With Suspected/Known Compounds With Suspected/Known Estrogenic Activity Estrogenic Activity

Phenolics Phenolics Nonyl Nonyl-

• and octylphenols

and octylphenols Alkylphenol Alkylphenol polyethoxycarboxylates polyethoxycarboxylates Bisphenol A Bisphenol A Phytoestrogens Phytoestrogens Organochlorine Compounds Organochlorine Compounds Chlordecone, Endosulfan, Chlordecone, Endosulfan, Sex steroids Sex steroids E2 E2 Synthetic hormones Synthetic hormones EE2 EE2

Natural Compounds Natural Compounds Synthetic Compounds Synthetic Compounds

Effects of EDCs Effects of EDCs

• Mimic natural hormones

Mimic natural hormones

• Alters natural hormonal balance

Alters natural hormonal balance

• Block hormone receptors

Block hormone receptors

• Prevents action of normal hormones

Prevents action of normal hormones

• Alter the synthesis and metabolism of

Alter the synthesis and metabolism of natural hormones natural hormones

• Affects the concentration of natural hormones

Affects the concentration of natural hormones in the body in the body

Mechanism Animation Mechanism Animation

Normal Hormonal Function Normal Hormonal Function

CELL RECEPTOR HORMONE

REACTION

Mechanism Animation Mechanism Animation

Hormone mimicking Hormone mimicking

CELL RECEPTOR EDC

Endocrine disruptor gives a stronger than normal signal

REACTION

Mechanism Animation Mechanism Animation

Hormone mimicking Hormone mimicking

CELL EDC

Endocrine disruptor gives a weaker than normal signal

REACTION

Mechanism Animation Mechanism Animation

Hormone blocking Hormone blocking

CELL EDC HORMONE

Endocrine disruptor prevents natural hormone from sending signal

NO REACTION

Potential Sources Potential Sources

• Activities in Watershed

Activities in Watershed

• Agriculture

Agriculture

• Recreation

Recreation

• Indigenous Flora and Fauna

Indigenous Flora and Fauna

Endocrine Disruption is a Endocrine Disruption is a complicated science . . . complicated science . . .

Metabolites formed upon incubation Metabolites formed upon incubation with human flora under aerobic and with human flora under aerobic and anaerobic conditions (72h) anaerobic conditions (72h)

No metabolites No metabolites Estriol Estriol 15a 15a-

• Hydroxyestrone

Hydroxyestrone 15a 15a-

• -
• Hydroxyestrone

Hydroxyestrone 16 16-

• Epiestriol

Epiestriol 16 16-

• Oxoestradiol

Oxoestradiol Estriol Estriol 16a 16a-

• Hydroxyestrone

Hydroxyestrone Estrone Estrone Estradiol Estradiol Estradiol Estradiol Estrone Estrone Metabolite Metabolite Substrate Substrate

Scubert 1964

Chemical structures of Chemical structures of Bisphenol Bisphenol A and the chlorinated A and the chlorinated derivatives derivatives

Fukazawa, et al., 2001

BPA BPA and its chlorinated

and its chlorinated derivatives

derivatives

3 3 1500 1500 3,3’,5,5’ 3,3’,5,5’-

• tetraCIBPA

tetraCIBPA 20 20 210 210 3,3’,5 3,3’,5-

• triCIBPA

triCIBPA 38 38 110 110 3,3’ 3,3’-

• diCIBPA

diCIBPA 8 8 520 520 3,5 3,5-

• diCIBPA

diCIBPA 8 8 550 550 3 3-

• CIBPA

CIBPA 1 1 4200 4200 Bisphenol A Bisphenol A Relative Relative Activity Activity EC ECx10

x10

(nM) (nM) Compound Compound ECx10 = Concentration at which ratio of signal of sample to that of blank was 10

Fukazawa, et al., 2002

Objectives Objectives

• Investigate occurrence of estrogenic

Investigate occurrence of estrogenic endocrine disrupting chemicals in source endocrine disrupting chemicals in source water in central Arizona water in central Arizona

• Develop an estrogenicity profile of source

Develop an estrogenicity profile of source waters waters

• Evaluate effect of chlorination on raw

Evaluate effect of chlorination on raw water water

Sampling Sites Sampling Sites

• Salt River

Salt River

• Verde River

Verde River

• CAP canal

CAP canal

• Water Treatment Plant 1

Water Treatment Plant 1

• Water Treatment Plant 2

Water Treatment Plant 2

Sampling Sites Sampling Sites

Verde River at its intersection with SR-87 in the Fort McDowell indian reservation Verde River Salt River N Salt River just before the confluence of the Salt and Verde Rivers

Sample Collection and Assay Sample Collection and Assay Strategy Strategy

Concentration by Liquid-Liquid Extraction Sample from Phoenix area source water Site Selection Cell Culture E-Screen Assay MCF-7 Cell Line CellTiter96 Aqueous One (Promega)

E E-

• Screen Assay

Screen Assay

Day 1 Day 2 Day 3 Day 4 Day 5

Extra Cells

Day 6 Day 7

Dose Response Curve of Dose Response Curve of 17 17β β-

• Estradiol Used to Determine

Estradiol Used to Determine Detection Limit (DL) Detection Limit (DL)

500 1000 1500 2000 2500 3000 3500 4000 4500 5 E

• 1

4 1 E

• 1

3 4 E

• 1

3 6 E

• 1

3 8 E

• 1

3 1 E

• 1

2 2 E

• 1

2 4 E

• 1

2 6 E

• 1

2 8 E

• 1

2 1 E

• 1

1 5 E

• 1

1 1 E

• 1

. 1 . 1 C

• n

t r

• l

2 4 h r s

Concentration Num ber of Cells

Data Analysis Data Analysis

• Graphs use Relative Proliferation Effect, or

Graphs use Relative Proliferation Effect, or the percent difference from control the percent difference from control

• 0 –

– 25% : Not highly estrogenic 25% : Not highly estrogenic

• 25

25 – – 70% : Moderately estrogenic 70% : Moderately estrogenic

• >70%: Highly estrogenic

>70%: Highly estrogenic

Seasonal variations in the Seasonal variations in the estrogenicity of Verde River water estrogenicity of Verde River water and effect of chlorination and effect of chlorination

2ppm for 30 minute

20 40 60 80 100 Feb March April May June July RPE Verde River Non-Chlorinated Verde River Chlorinated

Seasonal variations in the Seasonal variations in the estrogenicity of Salt River water estrogenicity of Salt River water and effect of chlorination and effect of chlorination

2ppm for 30 minute 20 40 60 80 100 Feb March April May June July Salt River Non-Chlorinated Salt River Chlorinated

Seasonal variations in the Seasonal variations in the estrogenicity of CAP Canal water estrogenicity of CAP Canal water and effect of chlorination and effect of chlorination

20 40 60 80 100 Feb March April May June July RPE CAP Canal Non-Chlorinated CAP Canal Chlorinated

2ppm for 30 minute

Summary of Results Summary of Results

Percent Decrease in Percent Decrease in Estrogenicity Estrogenicity After Chlorination After Chlorination

43.5 % 43.5 % CAP canal CAP canal 44.5 % 44.5 % Salt River Salt River 51.2 % 51.2 % Verde River Verde River

Summary of Results Summary of Results

Concentration of estrogenic chemicals as EEQ Concentration of estrogenic chemicals as EEQ 0.12 0.12 – – 12.0 12.0 ng ng/L /L Verde River Verde River 0.09 0.09 – – 11.0 11.0 ng ng/L /L Salt River Salt River 0.17 0.17 – – 12.0 ng/L 12.0 ng/L CAP canal CAP canal

Conclusions Conclusions

• In general, estrogenic potency in Verde River,

In general, estrogenic potency in Verde River, Salt River, and CAP canal was higher in summer Salt River, and CAP canal was higher in summer months months

• Chlorination of raw water from Verde River, Salt

Chlorination of raw water from Verde River, Salt River, and CAP canal resulted in significant River, and CAP canal resulted in significant decrease in estrogenicity (most cases <DL) decrease in estrogenicity (most cases <DL)

• Chlorination gave 51.2, 44.5, and 43.5 %

Chlorination gave 51.2, 44.5, and 43.5 % removal, respectively removal, respectively

• Chlorination byproducts were not highly

Chlorination byproducts were not highly estrogenic in nature estrogenic in nature

Acknowledgements Acknowledgements

• Funding for this research was provided by

Funding for this research was provided by NSF Water Quality Center at ASU NSF Water Quality Center at ASU

• We appreciate the help extended by the

We appreciate the help extended by the Salt River Project (SRP), Central Arizona Salt River Project (SRP), Central Arizona Project (CAP), and the two water Project (CAP), and the two water treatment plants. treatment plants.

Questions? Questions?

Equations for EDCs Equations for EDCs Calculations Calculations

PE = Cell # Max(Sample)/Cell # Negative PE = Cell # Max(Sample)/Cell # Negative control control RPE=[(PE RPE=[(PE sample

sample –

–1)/(PE 1)/(PEE2

E2 –

–1)]X100 1)]X100 EEQ= (EC EEQ= (EC50

50E2

E2)/(EC

)/(EC50

50Sample)

Sample)