1

Hormone Disrupting Chemicals

1993: Sharpe and Skakkebæk publish the estrogen

There’s nothing like a cold dip

estrogen hypothesis

“We argue that the increasing incidence of reproductive abnormalities in the human male may be related to increased oestrogen exposure in utero, and identify mechanisms by which this exposure could occur.”

100

150

Sperm cells 106 per ml

1930 1940 1950 1960 1970 1980 19900

50

2000

Topari et al, 1996

Intersex in British Perch populations

Jobling et al. 1998Jobling et al. 1998

Intersex Intersex Roach and reproduction? (Jobling et al, 2002)

2

Intersex Roach and reproduction?(Jobling et al, 2002)

Xenoestrogens are chemically diverse

Xenoestrogen activity a result of 3D structure?

A Model of an intracellular receptor

H2N

COOH Hormone binding siteInhibitory protien complex

Transcription-activating domain

Hinge region

DNA-binding domain

H2N

COOH

A/B C ED F

The estrogen receptor domainsActivating function 1

Nuclear Localisationsignal

Activating function 2

DNA binding domain Hinge

Ligand binding domaindomain

Homology between Human and Rainbow trout ER

A/B: 20%

C: 92%

E: 60%

Hingeregion Agonist binding domain

Antagonist binding domain

Mathews et al. 2001

Hormone disruption mechanisms

• Hormone synthesis

• Hormone secretion

• Hormone transport• Hormone transport

• Receptor binding

• Receptor complex mediated transcription

• Hormone elimination

• -ve/+ve feedback loops

3

Possible sex homone disruption mechanisms:

EstrogenAndrogen

(Arukwe, 2001)

gAnti-EstrogenAnti-Androgen

Actually many of the really major stories in Ecotoxicology have in hindsight involved

endocrine disruption

Examples:

DDT• DDT• PCB’s• Dioxin• TBT

Cholesterol

Steroid Pathways Made Simple

P450 enzymesXenoandrogens

Progestogens Androgens

EstrogensCorticoids

Aromataseinhibitors

xenoestrogens

Imposex in the dogwhelk probably caused by elevated testosterone: a

result of aramatase inhibition?

Normal male Imposex female (4)

Imposex female (5/6)

Imposex Development in the Dog Whelk

Penis Penis Penis

Vas deferens

Prostate gland

Vas deferens

Capsule gland

Capsule gland

Nodule formation on the vas deferens

♀ Clean locality Egg capsules from plymouth bay

Stage 6 females are sterile

♀ From poluted location with aborted egg capsules

Egg capsules fused with mother shell

4

Why the elevated testosterone levels?

1. Aromatase inhibition hypothesis (Bettin et al 1996;

Morcillo et al, 1998 a,b; Santos et al 2002)

2. Esterification hypothesis (Gooding et al, 2001, 2003;) total tesosterone level unchanged but esterified fraction reduced

3. Imbalance in Phase I and phase II enzymes resulting in a reduction in conjugation products and an increase in active androgen fractions (Ronis and Mason, 1996; Morcillo and Porte (1999)

Or imposex a result of a regulatory peptide? Testosterone levels are then a secondary effect

Oberdörstera and McClellan-Green, 2000

PMF=Penis modulating factorASO=Accessory sex organT=TestosteroneE2=Estrogen

Normal female Partly masculinised female

AndrogenEffectsmosquitofish caught below an american Pulp mill

Female with advanced masculinisation

Normal male

Androgens or aramatase inhibition??

Parks et al,2001

Paper-millmill

Initial stages of female masculinisation Toft et al, 2004

Feminization is quantified by counting segments(Parks et al 2001)

Reference females

R f lP ll t d f l Reference malesPolluted females

5

Downstream sites caused greater AR-binding inhibition than 1micromolar flutamide

1nM

Parks et al, 2001

Downstream sites caused similar AR-mediated gene expression as DHT

Flutamide

Parks et al, 2001

The chemical induces AR migration into the nucleus

Dexamethasonepotent glucocorticoidreceptoragonist

Masculinization is not a result of corticosteroid receptor activity

Parks et al, 2001

Brain and ovarian aromatase activity is higher in fish exposed to pulp mill

effluent (Orlando et al., 2002)

BrainOvary

Anti-Oestrogen effects• Aromatase inhibition• Xenoandrogens• Receptor Crosstalk

Between two intracellular receptors:Arylhydrocarbon receptor (AhR) &ER receptor

AhR causes synthesis of P450 enzyme EROD

ER induces production of vitellogenin in fish

6

Vitellogenin production in juvenile Coho salmon

Ah receptor

PCB

Arukwe et al., 2001

Level oforganization Effect

Population

Organism

Number of offspringSex ratio

Time to maturationSexual behaviour

Organ

Cell

Molecule

ColourationTestis size

Gonopodial length

Sperm count

The Guppy Mating System

Changes in sexual behaviour

• Highly promiscuous

• Continuous reproduction• Continuous reproduction

• Viviparous, sperm-counts possible

• Female choice, male ornamentation –display / courtship behaviour. Handicap models.... Good gene models....

• Two different male mating strategies... Full display / ”Sneaky matings”

Colouration index

Gonopodial index

Female Male

The ”sigmoid display”

7

SædtællingEffects of antiandrogens

Vinclozolin

N

O

O

OCl

Cl Cl

Cl Cl

Clp,p’-DDE

N

Cl

Cl

O H

OO

OCl

Cl

N

O HO

M1 M2 Hydroxyflutamide

N

CF3

OH NO2

O

Effects of antiandrogens- exposure of juveniles

****

Gonadosomatic IndexSperm count Courtship Behaviour

***

*

VIN 10 μg/mg

DDE 0.01 μg/mg

DDE 0.1 μg/mg

FLU 0.01 μg/mg

FLU 1.0 μg/mg *

**

Foetus Juvenile Adult

Sperm cells(mill.)

0 1 2 3 4 5 6

Testis weight(%)

3.0 3.5 4.0 4.5

Number of sigmoid displays(10 min)

1 2 3 4 5

*Control

VIN 0.1 μg/mg *

Is Guppy Reproduction Affected ?

VirginVirgin♀♀ + + + ♂♂ (30)(30)•• male fitness traits (Sigmoid, sperm)male fitness traits (Sigmoid, sperm)

•• Size of virgin females first clutchSize of virgin females first clutch

VirginVirgin♀♀ (30)(30) + + + ♂♂•• Size of virgin females first clutchSize of virgin females first clutch

♀♀ (pregnancy)(pregnancy) + + + ♂♂•• Size of virgin females first clutchSize of virgin females first clutch

Vinclozolin

moi

d di

spla

yspe

r 10

min

.

4

6

8

10

12

*

Male sexual display

Vinclozolin dose μg/mg dry feed

0,0 0,1 1,0 10,0

Num

bers

ig

0

2

4

Foetus Juvenile Adultca. 30 days

(milli

oner

celle

r pr.

han)

2,00

2,50

3,00

3,50

**

The effect of vinclozolin on sperm count

Vinclozolin dosis μg/mg tør foder 0,0 0,1 1,0 10,0

Sæ

dtal

1,00

1,50***

Foetus Juvenile Adultca. 30 days

8

uven

iles

per a

kvar

ium

10

12

14

16

18

20

*

Effect of vinclozolin on male fitness

Vinclozolin dose μg/mg dry feed 0,0 0,1 1,0 10,0

Num

ber o

f ju

2

4

6

8**

Foetus Juvenile Adultca. 30 days

EDCs and Sexual competition?

Guppy paternity assessed using micro- satellites (Kristensen et al 2005)

• Hormones work at low concentrations which means completely changing our approach to the dose/response

bl

EDC research has completely changed ecotoxicology and environmental

management

problem

• There can now be a large time-gap between exposure and effect. Even across generations..

• A “new” concept becomes the prevailing dogma…. “windows of sensitivity”