Endocrine disruption of personal care products &

related mechanisms

Kyungho Choi

August 19, 2015

School of Public Health, Seoul National University

Contents

• Introducing

– SNU, School, Department

– My laboratory and research interests

• Endocrine disrupting chemicals (EDCs)

– Importance in public health

– EDCs

– Case introduction and future studies

To me and my Korean fellows

• Thailand – Thai smile

– King Rama 9

– Korean War of 1950-3

• Mahidol University – No 1 in medicine and public health

– Great international program

– Smart students

Seoul National University in Winter

SNU in wikipedia

• A national research university located in Seoul, Korea. Founded in 1946, SNU has served as a model for many national and public universities in Korea. Today SNU comprises sixteen colleges and six professional schools, and about 30,000 students.

• QS World University Rankings (2014/15) considered it 31st in the world and 3rd in Asia, whilst it was 4th in the independent regional QS Asian University Rankings (2013).The university was 4th in Asia and 50th in the world by the 2014-15 Times Higher Education World University Rankings when its World Reputation Rankings considered it to be 26th globally. In 2009, the Ecole des Mines de Paris - MINES Paris Tech reported that Seoul National is ranked 5th in the world in terms of the number of alumni holding CEO positions in Fortune 500 enterprises.

https://en.wikipedia.org/wiki/QS_World_University_Rankingshttps://en.wikipedia.org/wiki/QS_Asian_University_Rankingshttps://en.wikipedia.org/wiki/QS_Asian_University_Rankingshttps://en.wikipedia.org/wiki/Times_Higher_Education_World_University_Rankingshttps://en.wikipedia.org/wiki/Times_Higher_Education_World_University_Rankingshttps://en.wikipedia.org/wiki/World_Reputation_Rankingshttp://en.wikipedia.org/wiki/Fortune_500

School of Public Health

Small but strong school!

• Alumni

– Shin, Young-soo (Regional Director WPRO)

– Kim, Hwa-joong (former Minister Ministry of Health)

Environmental Health within SPH

Bioinformatics

Health

Statistics

Demography

Environ

Health

Nutrition

Epidemiology

Mol/Genetic

Epidemiology

Disease

Epidemiology

Biostatistics Epidemiology

Hlth Behavior

Public Hlth

Policy

Dept of Environmental Health

Environ Toxicology

Exposure Sci Indoor Environ Qual Occup

Hygiene

Environ/Occup Medicine

Environ Hlth Microbiology

Environ Chem & Monitoring

Air Pollution & Climate Change

Biomonitoring

Environ Hlth Engineering

Environmental Health within Dept

Envion Monit Exp Assessmt

Biomonitoring

Risk Assessmt

Toxicology

Epidemiology

Health effect

Chemicals and

Microbials

Environmental Toxicology Laboratory (ETL)

• 1 Research Prof, 1 postdoc

• 4 PhD students, 6 MPH students

• 1 Researcher

• 1 Administrative staff

Research interest

• Environmental toxicology – Fish and cell lines of human and animal origins

– Endocrine, genotoxicity, oxidative stress related mechanisms

– POPs, EDCs, pharmaceuticals

• Exposure and risk assessment, epidemiology – Endocrine disrupting chemicals

– Pharmaceuticals and personal care products

Current research interest

• Linking chemicals and adverse health effects using – Observational studies in human population

– Laboratory toxicological studies using cells and animals

Adding biological plausibility on the observed associations

EDCs and Environmental Health

Environmental impacts on health

• Environmental factors cause >25% of global burden of disease

• About 3 million children under 5 die due to environmentally linked diseases

15 Source Neira M, WHO

Environmental impacts on health

16 Source Neira M, WHO

Environmental burden of disease is highest in the poorest countries

Major global environ health risks

17 Source Neira M, WHO

• Poor hygiene and sanitation

• Household water insecurity

• Disease vectors

• Chemical hazards

• Injuries and accidents

• Air pollution indoor/outdoor

• Emerging issues Global climate change Ozone depletion Persistent organic pollutants Obesity Electrical and electronic wastes Endocrine disruption

Endocrine disrupting chemicals

• WHO/IPCS 202

• Exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse effects in an intact organism, or its progeny, or (sub)populations

EDCs are global concern

20

(WHO/UNEP, 2012)

Chapter 1. General aspects on endocrine disruption Chapter 2. Evidence for endocrine disruption in humans and wildlife

• Female/Male reproduction health • Sex ratio • Thyroid-related disorders • Neurodevelopmental disorders in children and wildlife • Hormone-related cancers • Adrenal disorders in humans and wildlife • Bone disorders • Metabolic disorders • Immune function and diseases in humans and wildlife • Population declines

Chapter 3. Human and wildlife exposures to EDCs

Concerns on endocrine disrupting effects of chemicals are of worldwide concern

Endocrine related diseases

• Sperm quality decrease in 40% male (several EU countries) -> infertility?

• Malformation of male genitals – Cryptorchidism, hypospadias

• Pregnancy and delivery

• Endocrine cancers

• Precocious puberty

• Obesity and T2 diabetes

EDCs

• Phenols-APs, BPA • Phthalates • POPs

– DDTs, OCPs, PCBs – PFCs, PBDEs

• Metals – Pb, Hg, Cd, As

• Pharmaceuticals – NSAIDs

• and more

Regulating sex hormone balances

Modes of action - nuclear receptor mediated

effect

- hormone regulation

Regulating thyroid hormone balances

2015-08-24 24

(1) DDT, PCBs (2) PCBs,FRs (3) PBDEs,chlordane (4) PCBs, HCB, FRs (5) (6) PCBs

(1) Synthesis of THs

(2) Transport proteins

(3) Cellular uptake

(4) The TH receptor

(5) Iodothyronine deiodinases

(6) Metabolism in the liver

(Lyn et al., 2009; Boas et al., 2012)

Case studies on consumer chemicals

• Sex hormone – disruption and reproduction effects of

benzophenone 3

• Thyroid hormone – disruption by alternative flame retardants

• Epidemiological observations – of thyroid hormone disruption by consumer

chemicals

Sex hormone disruptions by environmental chemicals and their

mechanisms Benzophenone-3

(Kim et al., Aquat Toxicol, 2014)

Benzophenone-3 (BP-3)

• Widely used UV filter - Employed in sunscreens and various cosmetic products

- To protect products or human skins from the deleterious effects of UV

light

Benzopheone-3 (BP-3)

BP-3 in aquatic environment

• Surface waters - Both direct (recreational activities) and indirect (via WWTPs) inputs

- Up to 125 ng/L in a recreational lake in Switzerland (Poiger et al., 2004)

• Wastewater influent and effluent

• Sediments and soil

• Biota like fish

(Diaz-Cruz et al. Trends Anal Chem. 2008)

Direct input and indirect input of UV filters such as BP-3

BP-3 in human samples

• Frequently detected in urines worldwidebreastmilk, and

cord blood

(Kim et al. Environ Int, in revision)

Endocrine disrupting potential of BP-3

• in vitro - MCF-7 cell proliferation

- YES assay: weak estrogenic, strong anti-

estrogenic and anti-androgenic activities

(Schlumpf et al. Environ Health Perspect. 2001) (Coronado et al. Aquat Toxicol. 2008) (Fent et al. Chimia. 2008)

• In fish - VTG induction: varies among species

- Damage on reproduction and hatchability in Japanese medaka following

21 d exposure

Metabolism of BP-3

• Mainly metabolized into benzophenone-1 (BP-1)

- In rat, fish, and human (Bluthgen et al., 2012; Okereke et al., 1993; Wang and Kannan, 2013)

• BP-1 has greater estrogenic receptor binding activity than BP-3 in vitro (Kunz et al., 2006; Molina-Molina et al., 2008)

2-hydroxy-4-methoxybenzophenone (BP-3) 2,4-dihydroxybenzophenone (BP-1)

-50.00

0.00

50.00

100.00

150.00

-3.00 -1.00 1.00

% E

2-m

ax

Log μM

BP3

BP1

E2

(BP-3 and BP-1 in MVLN assay, preliminary data)

Research Gap

• Ecotoxicological information of long-term exposure to BP-3 and its metabolites is limited

• Underlying mechanism of the effects is still unclear - Currently available mostly on endocrine disrupting potential of BP-3

based on the receptor binding affinities

•

•

Study design

• Test organism: Japanese medaka - 28 d exposure of adult fish pairs

- F1 early life stage (ELS) fish exposure until 30 dph

• Observation/Endpoints

14 d adult fish exposure

study

- plasma vitellogenin (VTG) and sex hormones (E2, T)

- liver and gonadal mRNA expression

14 d mating study - # of eggs/female/day

F1 ELS toxicity study - hatchability

- weight, length, and condition factor

Measured concentrations

• Low recovery of BP-3 (17 ~ 36% nominal conc) • low solubility (0.005% DMSO); metabolism

• Metabolism to BP-1 • both adult and juvenile fish can biotransform BP-3 to BP-1

Nominal

conc.

Adult Japanese medaka exposure F1 early life stage exposure

BP-3 BP-1 BP-3 BP-1

0 h 48 h Median 0 h 48 h Median 0 h 48 h Median 0 h 48 h Median

Water ctrl

14 d adult fish exposure (Day 0-14)

• Plasma E2 • Decreasing trend but no

significance

• Plasma T • Significant increase in

males

• E2/T ratio • Significant decrease in

both male and female

0 4.7 8.4 26 90

Te

sto

ste

ron

e

(co

ntr

ol=

1)

0

1

2

3

4

5

6

7

Male

0 4.7 8.4 26 90

17e

str

ad

iol

(co

ntr

ol=

1)

0.0

0.5

1.0

1.5

2.0

2.5

Female

0 4.7 8.4 26 90

0 4.7 8.4 26 90

0 4.7 8.4 26 90

E2

/T r

atio

(co

ntr

ol=

1)

0.0

0.5

1.0

1.5

2.0

2.5

0 4.7 8.4 26 90

*

= -0.005p = 0.191

= -0.009p = 0.075

= 0.033p = 0.019

= 0.006p = 0.120

= -0.008p = 0.014

= -0.011p = 0.027

*

(A)

(B)

(C)

Nominal conc. 0 15 50 150 500 0 15 50 150 500

Measured conc. g/L

g/L

g/L

14 d adult fish exposure (Day 0-14)

• gonadal mRNA expression • overall down-regulations of several steroidogenic genes

• including cyp19a (E2 → T) and hsd17b3 (sex hormone production) genes

0 4.7 8.4 26 90

cyp17 m

RN

A fold

change

0.0

0.5

1.0

1.5

2.0

2.5

Male

0 4.7 8.4 26 90

sta

r m

RN

A fold

change

0.0

0.5

1.0

1.5

2.0Female

0 4.7 8.4 26 90

0 4.7 8.4 26 90

0 4.7 8.4 26 90

hsd17b3

mR

NA

fold

change

0.0

0.5

1.0

1.5

2.0

0 4.7 8.4 26 90

= -0.004p = 0.122

= -0.010p = 0.000

= -0.008p = 0.014

= -0.005p = 0.004

= -0.003p = 0.190

= -0.006p = 0.001

*

g/L

0 4.7 8.4 26 90

hsd3b m

RN

A fold

change

0.0

0.5

1.0

1.5

2.0

Male

0 4.7 8.4 26 90

cyp11a m

RN

A fold

change

0.0

0.5

1.0

1.5

2.0

Female

0 4.7 8.4 26 90

0 4.7 8.4 26 90

0 4.7 8.4 26 90

cyp19a

mR

NA

fold

change

0.0

0.5

1.0

1.5

2.0

0 4.7 8.4 26 90

= -0.007p = 0.032

= -0.005p = 0.029

= -0.006p = 0.007

= -0.009p = 0.004

= -0.005p = 0.034

= -0.007p = 0.001

g/L

*

*

g/L

g/L g/L

g/L

***

*

*******

(A) (B)

(C) (D)

(E) (F)

Nominal conc. 0 15 50 150 500 0 15 50 150 500

Measured conc.

Nominal conc. 0 15 50 150 500 0 15 50 150 500

Measured conc.

14 d adult fish exposure (Day 0-14)

• steroidogenesis pathway

Cholesterol

Pregnenolone

17α-OH-pregenolone

DHEA

Progesterone

17α-OH-Progesterone

Androstendeione

11-Deoxy-

corticosterone

11-Deoxycortisol

Testosterone (T)

Estreone

Corticosterone

Cortisol 17β-estradiol (E2)

Aldosterone

STAR,

CYP11A

CYP17

CYP17

CYP17

HSD17B

CYP19A

CYP17

CYP17

HSD3B

CYP11B

CYP11B

HSD17B

CYP21 CYP21

HSD3B

CYP11B

HSD3B

CYP19A

14 d adult fish exposure (Day 0-14)

• plasma vitellogenin (VTG) level - significant VTG induction

in male fish

- VTG : an estrogen dependent protein

- same results in transcripts in liver of male fish (data not shown)

• increase of VTG conc? - vs E2/T ratios significantly decreased

- biotransformation of BP-1 being responsible? BP-1 with greater ER binding affinity

Male

0 4.7 8.4 26 90

pla

sm

a V

TG

(conto

l=1)

0

3000

4000

5000

Female

0 4.7 8.4 26 90

= 34.22p = 0.000

= 0.007p = 0.132*

g/L

Nominal conc. 0 15 50 150 500 0 15 50 150 500

Measured conc.

Mating study (Day 15-28)

• Egg production per female during Day 22-28 - significantly reduced at 26 BP-3 μg/L (day 22 to 28 d)

- 20 fold lower than those reported in Coronado et al. (620 μg/L, 2008)

• Cumulative number of eggs during Day 22-28 - significant decreases at 26 μg/L observed from Day 26

22 23 24 25 26 27 28

Cu

mu

lative

eg

g n

um

be

r

0

10

20

30

40

50

60 solvent ctrl

4.7 mg/L BP-3

8.4 mg/L BP-3

26 mg/L BP-3

0 4.7 8.4 26 90

No

. o

f e

gg

s/f

em

ale

/da

y

0

2

4

6

8

10= -0.087p = 0.011

g/L

**

day

***

*

(A) (B)

†

Nominal conc. 0 15 50 150 500

Measured conc.

At 90 μg/L, serious mortality was observed in both sex fish and no eggs were produced.

4.7 ug/L BP-3

8.4 ug/L BP-3

26 ug/L BP-3

F1 ELS toxicity study

• Hatching of F1 eggs - At 30 μg/L (nominal 150 μg/L) BP-3, relatively lesser hatchability

(88.3 %)

• Growth following subsequent 30 d exposure of F1 fish - Condition factor in significant decreasing trend

- Later life stage effects?

†

Measured

Conc.

Nominal

Conc. n

Hatchability

(%)

Solvent ctrl 5 96.7± 2.0

5.4 µg/L 15 7 96.4 ± 1.7

12 µg/L 50 7 98.8 ± 1.2

30 µg/L 150 5 88.3 ± 5.7

n Number of replicates; n=12 eggs per each replicate

Hatchability = Number of hatched eggs/total eggs x 100

condition factor, K=100ⅹ[total weight (g)]/total length (cm3)]

0 5 12 30

9

10

11

12

13

14

g/L

Juvenile

fis

h length

(m

m)

Juvenile

fis

h d

ry w

eig

ht

(mg)

0 5 12 30

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

0 5 12 30

0.14

0.16

0.18

0.20

0.22

0.24

0.26

Conditio

n F

acto

r

= 0.037

r2 = 0.216

p for trend = 0.216

= 0.001

r2 = 0.000

p for trend = 0.982

= -0.002

r2 = 0.292

p for trend = 0.014

(A) (B) (C)

Nominal conc. 0 15 50 150

Measured conc.

0 15 50 150 0 15 50 150

Length Dry weight Condition Factora

Summary of BP-3 study

• Biotransformation to BP-1 in both adult and fry fish • BP-3 is transformed into BP-1 in both adult and fry Japanese medaka

• Sex hormone and reproduction effects • BP-3 (and its metabolite BP-1) affects sex endocrinology (in both

transcripts and hormones) and reproduction of fish at as low as 26

μg/L

• Lowered condition factor of F1 juveniles • Two generation exposure led to lowered condition factors of F1

juvenile fish

• Future studies • Implication in other organisms including humans

• Longer term exposure in later life stages of F1 fish including

reproduction and growth performances

Thyroid hormone disruptions by environmental chemicals

Alternative flame retardants

Alternative chemicals replacing PBDEs, flame retardants

• After phaseout of commercial mixture of PBDEs, organophosphate (OP) flame retardants, and other chemicals (FireMaster 550, Dechlorane plus etc) have replaced

Experimental models

in vivo organism

Zebrafish embryo/larva

0 hpf embryo 168 hpf larva 48-72 hpf

(hpf, hours post-fertilization)

- Developmental toxicity - Hormone and gene analysis

Pituitary gland GH3 cell (Rat) Central regulation

Thyroid gland FRTL-5 cell (Rat) Thyroid hormone Synthesis/Regulation

TSH

T3, T4

in vitro cell lines

- Gene analysis

Triphenyl phosphate (Kim et al., Aquat Toxicol, 2015)

• Triphenyl phosphate (TPP)

– Flame retardants added to carpets, furniture, and electronics

– Thyroid hormone disrupting potentials

• similar compounds : PBDEs, TDCPP

• In vitro experimental studies – Gene expression changes in GH3 and FRTL-5 cells

45

tshβ trα trβ dio1 dio2 nis tg tpo tshr pax8 nkx2.1

GH3 cells FRTL-5 cells

Zebrafish larvae after TPP exposure

46

• TH synthesis increased; genes for hormone synthesis are upregulated

• Malformation rate was increased T4 T3

TBB and TBPH (Jung JE., MPH Thesis, 2015)

• Alternative flame retardants replacing PBDEs

– Wire, cable insulation, carpet, furniture foam, wall coverings, coated fabrics, and adhesive

• Endocrine disruption potentials – TBPH structurally similar to phthalates

– House dust TBPH is positively associated with

human plasma T3 (Johnson et al., 2013)

– Sex and thyroid hormone disruption by TBB or

TBPH in experimental studies (Patisaul et al., 2012, Saunders et al., 2013, Mankidy et al., 2014)

47

– Endocrine disruption potential (Johnson et al., 2013; Saunders et al., 2013)

GH3 and FRTL5 cells after TBB and TBPH exposure

• TBB and TBPH act like T3 or TSH in both cell lines

48

tshβ trα trβ dio1 dio2

Fold

ch

an

ge

0.0

0.5

1.0

1.5

2.08.0

8.5

9.0

9.5

10.0 T3

Solvent control

0.05 mg/L

0.5 mg/L

5 mg/L

50 mg/L

*

*

*

**

*

*

tshβ trα trβ dio1 dio2

Fold

ch

an

ge

0.0

0.5

1.0

1.5

2.08.0

8.5

9.0

9.5

10.0T3

Solvent control

0.1 mg/L

1 mg/L

10 mg/L

100 mg/L

*

*

*

*

*

*

*

*

*

*

nis tg tpo pax8 nkx2.1

Fold

ch

an

ge

0

1

2

3

4

20

22

24

26

28TSH

Solvent control

0.5 mg/L

5 mg/L

50 mg/L*

*

*

**

* * * **

nis tg tpo pax8 nkx2.1

Fold

ch

an

ge

0

1

2

28

TSH

Solvent control

0.01 mg/L

0.1 mg/L

1 mg/L TBPH

*

*

* **

**

*

TBB TBPH

GH3

FRTL-5

Zebrafish larvae after TBB and TBPH exposure

• Different response – TBB increases T3 concentrations

– TBPH decreases both T4 and T3 concentrations

• Mechanisms involving targets other than pituitary and thyroid gland? Or metabolites?

49

T4 c

on

ten

t (n

g/g

)

0

10

20

30

40

T3 c

on

ten

t (n

g/g

)

0

1

2

39

10

11

12

*

0

*

= -0.017

r2 = 0.185p = 0.066

0.05 0.5 5 50 0.05 0.5 5 50

= -0.241

r2 = 0.188p = 0.058

0

T4 c

on

ten

t (n

g/g

)

0

10

20

30

40

T3 c

on

ten

t (n

g/g

)

0

2

4

6

8

10

12= 0.071

r2 = 0.170p = 0.079

0 0.05 0.5 5 50 0.05 0.5 5 500

TBB exposure TBPH exposure

Dechlorane Plus (DP) (Kang et al. in review)

• Replacement for usage of decaBDE

– Electric devices, textile, building, and construction

• Previous information on toxicity of DP

– Oxidative stress and oxidative DNA damage in liver (Li et al., 2013; Wu et al., 2012)

– Evidence of thyroid hormone disruption from an epidemiology study (Ben et al., 2014)

50

Exposure design

• Male adult zebrafish

– Low water solubility of DP (4.4x10-8 mg/L)

→ oral gavage feeding

51

oral gavage feeding Corn oil, 0.3, 1, or

3 μg/g of DP-25

0 day

6 day

sampling

2 day

Blood (serum) Hormone concentration (T4, T3)

Brain, liver, and testis

mRNA: thyroid and sex steroid hormone related gene

Results of PO exposure to DP

• Increase of T4 – Increase of plasma T4

– Genes stimulating thyroid hormone synthesis were upregulated

52

DP-25 (g/g zebrafish wet weight)

0 0.3 1 3

rela

tiv

e g

en

e e

xp

ress

ion

0.0

0.5

1.0

1.5

2.0

0 0.3 1 3n=3 n=3 n=3 n=3 n=3 n=3 n=3 n=3

T4 T3

(H. Kang et al. in preparation)

DP-25 (g/g zebrafish wet weight)

0 0.3 1 3

rela

tive g

ene e

xpre

ssio

n

0

1

2

3

4

5

0 0.3 1 3n=5 n=5 n=5 n=5 n=5 n=5 n=5 n=5

crh tsh

*

*

Summary

• Many alternative flame retardants are thyroid disrupting chemicals

• Using both in vitro and in vivo approaches, thyroid disrupting potential of alternative chemicals can be effectively screened

• Further observations in human population to follow

53

– Confirmation in long term low dose exposure should follow

Thyroid hormone disruptions by environmental chemicals among

general population Epidemiological observations

Previous studies

• Environmental phenols and thyroid in NHANES 2007-2008

55

BP-3 and thyroid hormones

• Statistical analysis

– Multiple regression model

– Covariates : age, sex, race and ethnicity, BMI, urinary cotinine, iodine, and creatinine

56

ln-Free T3 ln-Free T4 ln-TSH

β (95% CI) p-

value

β (95% CI) p-

value

β (95% CI) p-

value

-0.0011

(-0.0036 to 0.0013)

0.37 -0.0044

(-0.0083 to -0.00053)

0.026 0.014

(-0.0010 to 0.030)

0.067

Total T3 Total T4 ln-Tg

β (95% CI) p-

value

β (95% CI) p-

value

β (95% CI) p-

value

-0.36872

(-0.89 to 0.16)

0.17 -0.070

(-0.11 to -0.033)

0.0002 0.00022

(-0.024 to 0.024)

0.99

ln-BP-3*ln-Free T3 ln-BP-3*ln-Free T4 ln-BP-3*ln-TSH

NHANES 2007-2008

Our cohort

• Children’s Health and Environmental Chemicals in Korea Cohort

• to construct an exposure profile for newborns and their mothers through time. • to assess the adverse effects on birth outcome and onward Participants

57

• 352 pairs of pregnant women and fetuses

• Seoul, Anyang, Ansan, and Jeju (2011-2013)

– Study team

• ~20 professors from 3 universities and 6 university hospitals

Phthalates and thyroid hormone levels in CHECK panel mothers

• Individual model – adjusted for maternal age, BMI, gestational

age, delivery mode, parity

58

Thyroid hormone

Phthalates β p

Free T3 MEHP -0.033

Phthalates and thyroid

• Cross-sectional study

59

In children who had phthalate-tainted food, TSH were inversely associated with intake amount (Wu et al. 2013)

MEHP, MEP, and MBP was negatively associated with total T3 and free T3 in children (Boas et al. 2010)

Total T3 and free T4 were negatively associated with DEHP metabolites, but no relationships with TSH, total T4 and free T3 (Meeker et al. 2007)

• Possible mechanism in Zebrafish study

- Dio (Deiodinases) gene induction - UGT (conjugation and excretion) induction

(Zhai et al., 2014)

(MEHP exposure)

POPs and thyroid hormones

60

Matrix n POPs Thyroid hormone measurement Reference fT3 TT3 fT4 TT4 TSH matrix

Cord serum 104 PCBs - ↑ - - - / ↓s Cord serum & bloodspot

This study

PBDEs - - - - ↑ / ↑s OCPs - ↓ - ↓ - / ↑s Cord serum 108 PBDEs - - Cord serum Kim et al. (2009b) Cord serum 297 PBDEs - ↓ / -s - Cord serum &

bloodspot

Herbstman et al. (2008b)

PCBs ↓ / -s - / ↓s -

Cord serum 39 DDTs - ↓ - Cord serum Asawasinsopon et al. (2006) Cord serum 92 PCBs - - - Cord blood Takser et al. (2005)

Cord serum 70 HCB, PCBs, p,p’-DDE

-s bloodspot

Ribas-Fito et al. (2003)

β-HCH ↑ Cord plasma 410, 260 PCBs - - - Cord serum

Dallaire et al. (2008) HCB - ↑ - Cord plasma 198 PCBs, HCB ↓ ↓ -

Cord plasma Maervoet et al. (2007)

p,p’-DDE ↓ - Cord blood 90 PBDEs - - ↓ - - Cord blood Kim et al. (2011) Cord blood 50 PCBs - ↓ -

Cord blood Zhang et al. (2010)

PBDEs - - - Cord blood 54 PBDEs ↓ ↓ - - - Cord blood Lin et al. (2010) Maternal serum

104 PCBs - - - - - / -s Cord serum & bloodspot This study PBDEs - - - - - / ↑s

OCPs ↓ ↓ ↓ - - / ↑s (Kim et al., 2015)

Possible topics for study

• Identifying important chemicals of concern – Sensitive human populations: Pregnant women or

young children

– Susceptible population: E-waste workers

• Associating health effects – Hormones, birth outcomes, diseases

• Tracing exposure sources

Promising topics of study: Alternatives

• Bisphenol A vs S

• PBDEs vs TDCPP

• DEHP vs DEHT

ขอบคุณ

Acknowledgement ETL, SPH, SNU (SM Kim, SW Lee, SJ Kim, JE Jung, H-B Kang) CHECK Study Team (Prof SK Kim, H-B Moon, J Park) CHECK participants KFDA, NRF

Department of Environmental Health Sciences and Collaborations

Environmental Monitoring

Environ Hlth Microbiology

Biomonitoring

Environ Chem & Monitoring

Air Pollution & Climate Change

Occup Hygiene

Exposure Sci Indoor Environ Qual

Environ/Occup Medicine

Environ Hlth Engineering

Environ Toxicology

Environmental Monitoring

• Fate and transport of chemicals

– Hg, pharmaceuticals and personal care products, perfluorinated compounds, etc

• Exposure modelling of pollutants in air and water

Exposure assessment/Biomonitoring

Environ Hlth Microbiology

Biomonitoring

Environ Chem & Monitoring

Air Pollution & Climate Change

Occup Hygiene

Exposure Sci Indoor Environ Qual

Environ/Occup Medicine

Environ Hlth Engineering

Environ Toxicology

Exposure assessment

• Exposure assessment in indoor

– PM2.5, SHS, VOC, nano, HAPs

– dust mite, bioaerosol in home

• Workplace exposure assessment

– welding fume, nano, asbestos, EDCs, PBDEs

• Exposure assessment in humans

– BDEs, PCBs, POPs/EDCs

Biomonitoring

• Designing National Environmental Health Biomonitoring Program

• Biomarkers of exposure and effect

• Construction and validation of PBPK and reverse dosimetry

Toxicology and risk assessment

Environ Hlth Microbiology

Biomonitoring

Environ Chem & Monitoring

Air Pollution & Climate Change

Occup Hygiene

Exposure Sci Indoor Environ Qual

Environ/Occup Medicine

Environ Hlth Engineering

Environ Toxicology

Toxicology and risk assessment

• Environmental toxicology of POPs, EDCs, pharmaceuticals

– Endocrine disruption potentials of EDC alternative chemicals

• Exposure and risk assessment of chemicals among susceptible humans

– Risks of phthalate exposure through breastfeeding

Microbiology and health effects

Environ Hlth Microbiology

Biomonitoring

Environ Chem & Monitoring

Air Pollution & Climate Change

Occup Hygiene

Exposure Sci Indoor Environ Qual

Environ/Occup Medicine

Environ Hlth Engineering

Environ Toxicology

Microbiology and health effects

• Microbiome in twins and health implications

• Norovirus and public health

• Exposure assessment of dust mite, bioaerosol in home

Health effect and epidemiology

Environ Hlth Microbiology

Biomonitoring

Environ Chem & Monitoring

Air Pollution & Climate Change

Occup Hygiene

Exposure Sci Indoor Environ Qual

Environ/Occup Medicine

Environ Hlth Engineering

Environ Toxicology

Health effects and epidemiology

• Health effect assessment

– Community Health Survey Near Industrial Complex Study on Reproductive Health Problems among Hospital Workers

– Health Risk Assessment on Semiconductor (Fab) Workers

– Laboratory health issues

• Cohort studies

– EDCs and health effects among mother-baby pairs

Hormones Hormones are molecules produced by

specialized cells in a large variety of glands and tissues

These molecules travel through the blood to produce effects at sometimes distant target tissues

송과선

부갑상선

시상하부

뇌하수체

갑상선

흉선

부신

생식선

Experimental measures

• Thyroid hormone disruption In vitro In vivo

Thyroid gland FRTL5 (Rat)

Thyroid hormone Synthesis/Regulation

Pituitary gland GH3 (Rat)

Central regulation

(hpf, hours post-fertilization)

0 hpf embryo 120 hpf larva 48-72 hpf 1

Development - Hatchability - Malformation rate - Larval survival

Sampling

T3, T4 Gene transcription

Early life stage of zebrafish

Thyroid hormones (THs)

• Functions – Increase basal metabolic rate – Essential to proper development and

differentiation of all cells – Regulate protein, fat, and carbohydrate

metabolism

– Changes of THs even within normal range may cause significant effects on development of cognitive function of fetus (Berbel et al., 2009; Haddow et al., 1999; Pop et al., 2003; Porterfield, 1994)

• Regulated by TSH secreted from

pituitary gland Thyroid hormones: Triiodothyronine (T3) and its prohormone, thyroxine (T4)

wikipedia

2015-08-24 79

– Growing body of evidence on chemicals and thyroid toxicity, but still limited direct evidence in human populations (WHO/UNEP, 2012)

(Laurberg, 2009)

Chemicals on thyroid hormone homeostasis

Materials and methods

2015-08-24 80

• Subjects and sampling – IRB of SPH SNU – 105 pregnant women participating CHECK Panel study – Questionnaire and blood sampling just before delivery

• Target POPs with > 60% detection – OCPs 19: dichlorodiphenyl trichloroethanes (DDTs), chlordanes (CHDs), hexachlorocyclohexanes (HCHs),

hexachlorobenzene (HCB), heptachlor, heptachlor epoxide, mirex – PCBs 19: PCB18, 28, 33, 44, 52, 70, 101, 105, 118, 128, 138, 153, 170, 180, 187, 194, 195, 199 and 206 – PBDEs 19: BDE17, 28, 47, 49, 66, 71, 77, 85, 99, 100, 119, 126, 138, 153, 154, 156, 183, 184 and 191

• Thyroid related hormones – tT3, fT3, tT4, fT4, TSH

Seoul

Ansan

Anyang

Jeju

Materials and methods

• Subjects and sampling – Pregnant women and matching fetuses pair participating in CHECK Panel (n=104) – Questionnaire, blood sample collection from mother before delivery and from cord during

delivery

• Thyroid hormones

– tT3, fT3, tT4, fT4, TSH from mother and her infant – TSH in bloodspot at day 2-3 after birth

• Target POPs with > 60% detection

– Use of proxy values with the same method as that for maternal blood measurement

• Statistical analysis

– Natural regression model – Covariates: maternal age, prepregnancy BMI,

gestation, number of pregnancy, mode of delivery

• Maternal thyroid hormone levels were also added in the regression model

2015-08-24 81

Interaction between thyroid hormones and sex hormones

• Regulation of hormone dynamics

– Hypothalamic-pituitary-thyroid (HPT) axes : thyroid system

– Hypothalamic-pituitary-gonad (HPG) axes : reproductive system

– Hypothalamic-pituitary-adrenal (HPA) axes : adrenal system

82

● T ○ E2

○ T4 ● T3

Reported interaction between thyroid and sex hormone in fish

• THs may inhibit the production or secretion of sex hormones – T4 treatment in female catfish decreased both tissue or serum E2 level (Supriya et al.,

2005)

– In female zebrafish, concentrations of FSH and LH were negatively correlated with concentrations of T4 and T3 (Liu et al., 2011)

• THs also seem to directly act on fish oocytes – T4 treatment in female catfish increased vitellogenic/post-vitellogenic follicles,

suggesting rapid oocyte growth (Supriya et al., 2005) – T3 administered along with gonadotrophin (GTH) increased E2 production in

ovarian follicles isolated from adult Japanese medaka (Soyano et al. 1993)

• THs play an important role in testicular development and function (Wagner et al.,

2008)

83

• E2 can suppress T3 production in fish

– E2 treatment reduced plasma T3 levels in immature rainbow trout (Letherland, 1985; Mercure et al., 2001) and in salmon (Yamada et al., 1993)

– In female fish when plasma E2 levels are high, thyroid hormones tend to be low (Cyr et al., 1988; Norberg et al., 1989)

Sex hormone and thyroid hormone changes by TDCPP or

TPP • In adult male and female zebrafish

84

Related underlying mechanisms

• Cross-talk : central hormone regulation 변화 – E2 decrease by prochloraz (PCZ) down-regulated the CRH gene expression and T4 decreased by

PTU up-regulated the FSH and LH gene expression in adult female zebrafish (Liu et al., 2011)

• E2 증가에 의한 deiodination activity 변화 – E2 decreased T4-ORD activity and rT3-ORD activity in liver, but increased kidney T3-IRD activity

in both sexes (Wiens and Eales, 2005) ; hepatic T3 감소 또는 renal T3 degradation 증가

• 갑상선 호르몬 변화와 steroidogenesis 간의 연관성 – PTU, an antithyroid medicine, decreased T4 cyp11a and star gene expression and production of

testosterone in rat leydig cells (Chiao et al., 2002)

85

PCZ exposure PTU exposure

Atrazine disrupts sex of frogs

• An atrazine-induced female frog (a genetic male) is shown (bottom) copulating with an unexposed male sibling. This union produced viable eggs and larvae that survived to metamorphosis and adulthood. Yet, because both animals were genetic males, the offspring were all males. (Tyrone Hayes photo)

PNAS 2010 107, 4613

Japanese medaka (Oryzias latipes)

• A suitable model for evaluating the estrogenicity of EDCs and reproductive effects

• Recommended for testing of chemicals - OECD TG 203 (Fish, Acute Toxicity Test)

- OECD TG 204 (Fish, Prolonged Toxicity Test: 14-day Study)

- OECD TG 210 (Fish, Early-life Stage Toxicity Test)

- easy to control the spawning

- good to obtain a number of eggs

Regulating thyroid hormone balances

Human observations- POPs and thyroid hormones in serum

(Kim et al., 2013)

Human observations- POPs and thyroid hormones in serum

2015-08-24 90

Matrix n POPs Thyroid hormone measurement Reference fT3 TT3 fT4 TT4 TSH matrix

Cord serum 108 PBDEs - - Cord serum Kim et al. (2009b) Cord serum 297 PBDEs - ↓ / -s - Cord serum &

bloodspot

Herbstman et al. (2008b)

PCBs ↓ / -s - / ↓s -

Cord serum 39 DDTs - ↓ - Cord serum Asawasinsopon et al. (2006)

Cord serum 92 PCBs - - - Cord blood Takser et al. (2005)

Cord serum 70 HCB, PCBs, p,p’-DDE

-s bloodspot

Ribas-Fito et al. (2003)

β-HCH ↑ Cord plasma

410, 260

PCBs - - - Cord serum Dallaire et al. (2008)

HCB - ↑ - Cord plasma

198 PCBs, HCB ↓ ↓ - Cord plasma

Maervoet et al. (2007)

p,p’-DDE ↓ - Cord blood 90 PBDEs - - ↓ - - Cord blood Kim et al. (2011) Cord blood 50 PCBs - ↓ -

Cord blood Zhang et al. (2010)

PBDEs - - - Cord blood 54 PBDEs ↓ ↓ - - - Cord blood Lin et al. (2010)

(Kim et al., 2015)

Questions

• EDCs and thyroid hormones among sensitive human

populations

– Pregnant women and newborn infants

• Experimental approach to screen thyroid disruption of

substituting chemicals

– Flame retardants- TPP, Firemaster, Dechlorane Plus

91

Study population

• Children’s Health and Environmental Chemicals in Korea Panal

• to construct an exposure profile for newborns and their mothers through time.

• to assess the adverse effects on birth outcome and onward

– Participants

2015-08-24 92

• 352 pairs of pregnant women and fetuses

• Seoul, Anyang, Ansan, and Jeju (2011-2013)

– Study team

• ~20 professors from 3 universities and 6 university hospitals

Association between several persistent organic pollutants and thyroid hormone

levels in serum among the pregnant women of Korea

(Kim SM et al., Environ Int 2013)

POPs

versus

T3, T4, TSH

Maternal Blood

Materials and methods

• Statistical analysis

– Use of proxy values for nondetects

• LOQ ( limit of quantification) / √2 for chemicals >75% detection

• Only detected values were used for statistical analysis for chemicals 60-75% detection

– Effects of maternal blood serum POPs on thyroid hormones

• Multiple linear regression

• Natural log transform measured POPs data

• Covariates: maternal age, pre-pregnancy BMI, gestation period, number of pregnancies, mode of delivery

2015-08-24 94

Association between POPs and THs in maternal serum

2015-08-24 95

Free T3 Total T3 Free T4 Total T4 TSH

β 95% CI β 95% CI β 95% CI β 95% CI β 95% CI

PCB

ΣPCB -0.053^ -0.108, 0.002 -0.117* -0.198, -0.036 -0.029 -0.084, 0.027 -0.056* -0.109, -0.002

0.195 -0.167, 0.556

PCB28+ -0.015 -0.057, 0.027 -0.025 -0.100, 0.050 -0.068* -0.117, -0.019 -0.030 -0.081, 0.020 0.172 -0.165, 0.510

PCB52+ -0.092* -0.159, -0.026

-0.113* -0.223, -0.003 -0.011 -0.067, 0.044 -0.023 -0.081, 0.035 0.192 -0.146, 0.530

PCB118+ -0.020 -0.091, 0.051 -0.114* -0.223, -0.005 -0.049 -0.136, 0.038 -0.047 -0.134, 0.040 0.389 -0.183, 0.960

PBDE

ΣPBDE -0.049* -0.088, -0.009

-0.112* -0.170, -0.054 0.058* 0.016, 0.100 0.007 -0.032, 0.046 -0.055 -0.318, 0.209

BDE47 -0.021 -0.049, 0.007 -0.042* -0.084, -0.000 0.028 -0.001, 0.057 0.006 -0.021, 0.033 0.024 -0.154, 0.203

OCP

tNCHD -0.033 -0.095, 0.029 -0.085 -0.188, 0.018 -0.049 -0.118, 0.021 -0.059^ -0.128, 0.010 0.225 -0.160, 0.609

ΣDDT -0.052^ -0.104, 0.000 -0.096* -0.179, -0.013 -0.045 -0.106, 0.016 -0.035 -0.093, 0.022 0.299 -0.072, 0.670

p,p’-DDT 0.002 -0.035, 0.039 0.004 -0.053, 0.061 -0.045* -0.083, -0.008 -0.015 -0.051, 0.021 0.145 -0.090, 0.380

HCB+ -0.012 -0.061, 0.037 -0.035 -0.108, 0.038 -0.047* -0.095, -0.000 -0.023 -0.071, 0.026 0.091 -0.183, 0.365 Signs * and ^ indicate statistical significance at p=0.05, and 0.1, respectively. All POPs concentrations are in ng/g lipid and were natural log –transformed. Results of association were adjusted for age, gestation period, mode of delivery, parity, and pre-pregnancy BMI. Chemicals that were detected >=75% of the population at concentrations greater than the limit of quantification, a proxy value of ‘limit of quantification divided by square root 2’ was used. For chemicals that were detected in =60%, statistical analysis was conducted with detected values only. Such chemicals are indicated by ‘+’.

(n=105)

Significant association with TH remains for PCBs

2015-08-24 96

• Are effects of PBDEs real? – Significance correlation in

Spearman analysis

• between PCB52 and BDE47, and

• among BDE47, PCB52, and PCB118

– When all were entered in the model, only the effect of PCB52 remains

• DDTs, HCB, CHDs were generally associated with decrease of T3 or T4

Variables free T3

β 95% CI

BDE47 -0.039^ -0.081, 0.003

PCB52 -0.072* -0.141. -0.004

total T3

β 95% CI

BDE47 -0.006 -0.076, 0.063

PCB52 -0.114 -0.305, 0.076

PCB118 -0.078 -0.259, 0.104

Adjusted associations with significant PCBs as covariates between serum PBDEs and free or total T3 in pregnant women

Significant results from multiple regression analysis using each individual congener (Table 3) were reanalyzed by adding PCB52 for testing relationship between BDE47 and free T3, and PCB52 and 118 in model for testing relationship between BDE47 and total T3 as covariates.

Association between POPs exposure and thyroid hormones among newborn infants

(Kim SM et al., PlosOne 2015)

POPs

versus

T3, T4, TSH

Cord Blood

Challenges in studying thyroid hormones in newborns

• Cord thyroid hormones fluctuate significantly

2015-08-24 98

– Especially around delivery due to many causes (Herbstman et al., 2008)

– On average 30-60% of cord blood serum T4 is from mother (Thorpe-Beeston et al., 1991; Vulsma et al., 1989)

Vein (to baby)

Artery (to mother)

• Not feasible to sample blood from newborns • Bloodspot collected at days 2 and 18 after birth to measure T4

(Herbstman et al. 2008)

• May better reflect stable thyroid hormone levels of newborn

-> Measure thyroid hormones in bloodspot of newborn

-> Correct cord blood serum data with maternal serum TH levels

Association between POPs and THs in newborn infant serum

2015-08-24 99

Signs * and ^ indicate statistical significance of regression parameter at p=0.05, and 0.1, respectively. # indicates no significance of model in F test (p for ANOVA). For chemicals that were detected in =60%, statistical analysis was conducted with detected values only. Such chemicals are indicated by ‘+’.

Cord serum POPs (ng/g lipid)

Cord blood Bloodspot

Free T3 (pg/mL) Total T3 (ng/mL) Free T4 (ng/dL) Total T4 (μg/dL) TSH (μlU/mL) TSH (μlU/mL)

β 95% CI β 95% CI β 95% CI β 95% CI β 95% CI β 95% CI

PCB ΣPCB 0.012 (-0.03, 0.05) 0.034^ (0.00, 0.07) -0.002 (-0.02, 0.02) 0.004 (-0.03, 0.04) 0.012 (-0.10, 0.13) -0.160^ (-0.33, 0.01)

PBDE BDE-47+ -0.016 (-0.09, 0.06) -0.017 (-0.09, 0.06) -0.015 (-0.06, 0.03) -0.002 (-0.07, 0.06) 0.077 (-0.15, 0.31) 0.327* (0.03, 0.62)

BDE-99+ 0.008 (-0.06, 0.07) 0.013 (-0.05, 0.08) 0.002 (-0.04, 0.04) 0.013 (-0.05, 0.07) 0.210* (0.00, 0.42) 0.037 (-0.24, 0.32)

OCP β-HCH+ -0.023 (-0.11, 0.06) -0.002 (-0.09, 0.08) -0.019 (-0.07, 0.03) -0.016 (-0.10, 0.07) 0.106 (-0.16, 0.37) -0.270^ (-0.57, 0.03)

ΣCHD 0.018 (-0.04, 0.07) 0.029 (-0.02, 0.08) 0.005 (-0.02, 0.03) 0.021 (-0.03, 0.07) 0.149^ (0.00, 0.30) 0.197 (-0.05, 0.45)

p,p’-DDE -0.033 (-0.07, 0.01) -0.038^ (-0.08, 0.00) -0.002 (-0.02, 0.02) -0.016 (-0.05, 0.02) 0.082 (-0.04, 0.21) 0.208* (0.03, 0.39)

HCB+ -0.019 (-0.05, 0.01) -0.016 (-0.05, 0.02) -0.004 (-0.02, 0.01) -0.029* (-0.06, 0.00) 0.059 (-0.04, 0.16) -0.109 (-0.28, 0.06)

Maternal serum POPs (ng/g lipid)

Free T3 (pg/mL) Total T3 (ng/mL) Free T4 (ng/dL) Total T4 (μg/dL) TSH (μlU/mL) TSH (μlU/mL)

β 95% CI β 95% CI β 95% CI β 95% CI β 95% CI

β 95% CI

PBDE BDE-47 -0.006 (-0.04, 0.03) -0.010 (-0.04, 0.02) 0.004 (-0.01, 0.02) -0.008 (-0.03, 0.02) -0.028 (-0.13, 0.07) 0.130^ (-0.04, 0.29)

OCP β-HCH -

0.044* (-0.08, -0.01)

-

0.039* (-0.08, 0.00) -0.017 (-0.04, 0.00) -0.028 (-0.06, 0.01) 0.066 (-0.06, 0.19)

0.041 (-0.12, 0.26)

ΣCHD

-0.005 (-0.08, 0.07) -0.013 (-0.08, 0.05) -

0.038* (-0.08, -0.01)

-

0.056^ (-0.12, 0.00) 0.009 (-0.21, 0.23)

-0.221 (-0.41, 0.16)

ΣDDT -0.027 (-0.10, 0.05) -0.023 (-0.10, 0.05) -0.017 (-0.06, 0.02) 0.004 (-0.06, 0.07) -0.013 (-0.25, 0.22) 0.345* (0.06, 0.81)

p,p’-DDE -

0.036^ (-0.08, 0.01) -0.028 (-0.07, 0.01)

-

0.020^ (-0.04, 0.00)

-

0.034^ (-0.07, 0.01) 0.001 (-0.13, 0.13)

0.264* (0.11, 0.50)

HCB -0.005 (-0.04, 0.03) -0.015 (-0.05, 0.02) 0.001 (-0.02, 0.02) -0.011 (-0.04, 0.02) 0.089^ (-0.02, 0.19) -0.095 (-0.26, 0.09)

30% ↑ per BDE47 IQR ↑

5, 4% ↓ per β-HCH IQR ↑

25, 19% ↑ per DDT and p,p’-DDE IQR ↑

17% ↑ per p,p’-DDE IQR ↑

With all chemicals with strong correlation with each other, effects of PBDEs and OCPs remain

significant in the model

2015-08-24 100

Cord POPs Cord or bloodspot Thyroid hormones

β (95% CI) n

ΣPCB TT3 0.02 (-0.02, 0.06) 89

p,p’-DDE TT3 -0.04^ (-0.09, 0.00)

BDE-99 Cord TSH 0.22* (0.00, 0.42) 57

ΣCHD Cord TSH 0.13 (-0.06, 0.33)

ΣPCB bloodspot TSH 0.07 (-0.13, 0.27) 64

BDE-47 bloodspot TSH 0.38* (0.08, 0.67)

ΣCHD bloodspot TSH 0.04 (-0.20, 0.28) 72

p,p’-DDE bloodspot TSH 0.54* (0.25, 0.82)

Maternal POPs Cord or bloodspot Thyroid hormones

β (95% CI) n

β-HCH fT3 -0.04^ (-0.08, 0.01) 94

p,p’-DDE fT3 -0.02 (-0.07, 0.03)

ΣCHD fT4 -0.03 (-0.07, 0.01) 87

p,p’-DDE fT4 -0.01 (-0.05, 0.02)

BDE-47 bloodspot TSH 0.02 (-0.13, 0.18)

88 ΣDDT bloodspot TSH -0.13 (-0.58, 0.32)

p,p’-DDE bloodspot TSH 0.45* (0.14, 0.76)

Newborn infants

• POPs associated with thyroid hormones – BDE47, BDE99, p,p’-DDE, ΣDDT, β-HCH,

ΣCHD, HCB

• More reliable measurement of association – Adjusting cord blood serum hormones with

those of mother’s

– Employing bloodspot TSH measurements

2015-08-24 101

BP-3 and thyroid hormones

• 1,852 subjects in NHANES 2007-2008

– 971 males and 881 females

– Ages from 12 to 80 yrs

• Urinary environmental phenol conc. (ng/mL)

– 4-tert-octyl phenol, BP-3, BPA, TCS

– Parabens (MP, EP, PP, and BP)

• Serum thyroid measures

– TSH (μIU/mL)

– Free T3 (pg/mL) and T4 (ng/dL)

– Total T3 (ng/dL) and T4 (μg/dL)

– TPO antibody (IU/mL)

102 NHANES 2007-2008