complex mixtures of anti-androgens at concentrations …€¦ · complex mixtures of anti-androgens...
TRANSCRIPT
Complex mixtures of anti-androgens at concentrations below individual chemical effect levels produces reproductive tract
malformations in the male rat
Justin ConleyORD/NHEERL/TAD/RTB
SOT RASS-MixSS Webinar September 13, 2017
No conflicts of interest to disclose
This presentation does not necessarily reflect the views or policy of USEPA
Disclosure/Disclaimer
Declines in male reproductive health
Skakkebaeck et al. 2016
Pumarega et al. 20162003/2004 Cohortn = 4,793OCs, PCBs, PBDEs, PCDDs/Fs, PFCs
17%
Exposures are mixtures
Examples of multiple residue detections in CDC-NHANES
Qian et al. 20152007/2008 Cohortn = 2,604DEHP, DiBP, DBP, DINP, BBP, DIDP
Chemical mixtures research and risk assessment in federal laws
Food Quality Protection Act Safe Drinking Water Act
How do we group chemicals for cumulative risk assessment?
Androgen receptor
antagonism
Reduced AR dependent
mRNA/protein synthesis
Suppressed development of
gubernacular cords
Undescended testes
Reduced androgen dependent
tissue weights
Abnormal cell apoptosis/
proliferation
Reduced sperm
production
Reproductive tract
malformations
Reproductive tract cancers
Infertility
Molecular Initiating Events Key Events
Adverse Outcomes
Anti-androgen AOP
Key EventsAdverse
Outcomes
Androgen receptor
antagonism
Inhibition of CYP450
steroidogenic enzymes
Unknown MIE
Reduced AR dependent
mRNA/protein synthesis
Reduced testosterone
synthesis
Suppressed development of
gubernacular cords
Undescended testes
Reduced androgen dependent
tissue weights
Abnormal cell apoptosis/
proliferation
Inhibition of HMG-CoA reductase
Reduced cholesterol synthesis
Reduced sperm
production
5α-reductaseinhibition
Reduced DHT synthesis
Reproductive tract
malformations
Reproductive tract cancers
Infertility
“Anti-androgen” AOP network
Molecular Initiating Events Key Events
Adverse OutcomesKey Events
Adverse Outcomes
Complex mixtures of many chemicals with multiple “anti-androgenic” molecular initiating events act additively at low doses to disrupt male reproductive tract development
“Low”: below individual chemical effect levels (LOAEL or NOAEL)
Hypothesis
Linuron330-55-2
Prochloraz67747-09-5
Vinclozolin50471-44-8
p,p’-DDE72-55-9
dipentyl phthalate131-18-0
diethylhexyl phthalate117-81-7
diisobutyl phthalate84-69-5
diisoheptyl phthalate71888-89-6
dihexyl phthalate84-75-3
diheptyl phthalate3648-21-3 chemspider.com
dicyclohexyl phthalate84-61-7
Procymidone32809-16-8
Pyrifluquinazon337458-27-2
dibutyl phthalate84-74-2
benzyl butyl phthalate85-68-7
Simvastatin79902-63-9
Flutamide13311-84-7
Finasteride98319-26-7
Ultramix
Androgen receptor
antagonism
Inhibition of P450
steroidogenic enzymes
Unknown MIE
Reduced AR dependent
mRNA/protein synthesis
Reduced testosterone
synthesis
Suppressed development of
gubernacular cords
Undescended testes
Reduced androgen dependent
tissue weights
Abnormal cell apoptosis/
proliferation
Inhibition of HMG-CoA reductase
Reduced cholesterol synthesis
Reduced sperm
production
5α-reductaseinhibition
Reduced DHT synthesis
Reproductive tract
malformations
Reproductive tract cancers
Infertility
“Anti-androgen” AOP networkFinasteride
Molecular Initiating Events Key Events
Adverse OutcomesKey Events
Adverse Outcomes
p’p’-DDEPyrifluquinazon
ProcymidoneVinclozolinFlutamide
ProchlorazLinuron
Simvastatin
Phthalates
ChemicalLOAEL
(mg/kg/d) Effect Reference
Finasteride 0.03 AGD red., Nipple ret. Clark 1993
Flutamide 2.0 Hypospadias Welsh 2009
Vinclozolin 6.0 VP wt., AGD red., Nipple ret. Gray 1999; Hellwig 2000
Diethylhexyl phth. 11.0 Various malf. Gray 2009; Blystone 2010
Linuron 12.5 Testis & epididymal malf. McIntyre 2000
Procymidone 25.0 AGD red., Nipple ret., Various malf., Ostby 1999
Pyrifluquinazon 25.0 Various malf. Unpublished
Prochloraz 31.3 Testis malf., T prod. Blystone 2007; Noriega 2005
Dipentyl phth. 33.0 T prod. Hannas 2011; Furr 2014
Simvastatin 62.5 Testis malf., T prod. Beverly 2014
Dibutyl phth. 100.0 Nipple ret. Mylchreest 2000
p,p’-DDE 100.0 AGD red., Nipple ret., PPS delay Kelce 1995
Diisoheptyl phth. 227.0 Red. sperm count McKee 2006
Dicyclohexyl phth. 250.0 AGD red. Saillenfait 2009
Benzylbutyl phth. 250.0 AGD red. Tyl 2004
Diisobutyl phth. 250.0 AGD red. Saillenfait 2008
Dihexyl phth. 250.0 AGD red.; Nipple ret. Saillenfait 2009
Diheptyl phth. 500.0 AGD red. Saillenfait 2011
Individual chemical effect levels
ChemicalLOAEL
(mg/kg/d)100%
(mg/kg/d)50%
(mg/kg/d)25%
(mg/kg/d)12.5%
(mg/kg/d)6.3%
(mg/kg/d)
Finasteride 0.03 0.006 0.003 0.001 0.0007 0.0003
Flutamide 2.0 0.5 0.2 0.1 0.06 0.03
Vinclozolin 6.0 1.2 0.6 0.3 0.1 0.07
Diethylhexyl phth. 11.0 2.2 1.1 0.5 0.2 0.1
Linuron 12.5 2.5 1.2 0.6 0.3 0.1
Procymidone 25.0 5.0 2.5 1.2 0.6 0.3
Pyrifluquinazon 25.0 5.0 2.5 1.2 0.6 0.3
Prochloraz 31.3 7.5 3.7 1.8 0.9 0.4
Dipentyl phth. 33.0 6.6 3.3 1.6 0.8 0.4
Simvastatin 62.5 12.5 6.2 3.1 1.5 0.7
Dibutyl phth. 100.0 20.0 10.0 5.0 2.5 1.2
p,p’-DDE 100.0 20.0 10.0 5.0 2.5 1.2
Diisoheptyl phth. 227.0 45.4 22.7 11.4 5.6 2.8
Dicyclohexyl phth. 250.0 50.0 25.0 12.5 6.2 3.1
Benzylbutyl phth. 250.0 50.0 25.0 12.5 6.2 3.1
Diisobutyl phth. 250.0 50.0 25.0 12.5 6.2 3.1
Dihexyl phth. 250.0 50.0 25.0 12.5 6.2 3.1
Diheptyl phth. 500.0 100.0 50.0 25.0 12.5 6.2
1/5 1/10 1/20 1/40 1/80
Postnatal reproductive development
F0
F1
GD0 GD2 GD14 GD18 GD22
PND2
DOSING
AGD
PND13
NR
PND23
F0 Necropsy
PND34
VO PND41
PPS
PND90
F1 Necropsy
PND120
F1 Necropsy
-Charles River Sprague-Dawley-Oral gavage administration-5 dams/litters per dose group-Corn oil negative control
AGD = anogenital distanceNR = nipple retentionVO = vaginal openingPPS = preputial separation
Masculinizing window
Co
ntr
ol
LO
AE
L/8
0
LO
AE
L/4
0
LO
AE
L/2
0
LO
AE
L/1
0
LO
AE
L/5
1
2
3
4
5
AG
D (
mm
) * * * * * * *
Co
ntr
ol
LO
AE
L/8
0
LO
AE
L/4
0
LO
AE
L/2
0
LO
AE
L/1
0
LO
AE
L/5
0
2
4
6
8
1 0
1 2
# n
ipp
les
pe
r m
ale
* *
* *
* * * *
Early postnatal endpoints
Adult reproductive tissue weightsC
on
tro
l
LO
AE
L/8
0
LO
AE
L/4
0
LO
AE
L/2
0
LO
AE
L/1
0
LO
AE
L/5
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
Ve
ntr
al
pr
os
tate
wt
(mg
)
* *
p = 0 . 0 6 3
Co
ntr
ol
LO
AE
L/8
0
LO
AE
L/4
0
LO
AE
L/2
0
LO
AE
L/1
0
LO
AE
L/5
6 0
8 0
1 0 0
1 2 0
1 4 0
1 6 0
Gla
ns
pe
nis
wt
(mg
)
* *
* * * *
Co
ntr
ol
LO
AE
L/8
0
LO
AE
L/4
0
LO
AE
L/2
0
LO
AE
L/1
0
LO
AE
L/5
2
3
4
5
Pa
ire
d t
es
tis
wt
(g)
* ** *
* *
p = 0 . 0 5 5 p = 0 . 0 6 1
Co
ntr
ol
LO
AE
L/8
0
LO
AE
L/4
0
LO
AE
L/2
0
LO
AE
L/1
0
LO
AE
L/5
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
1 6 0 0
1 8 0 0
Pa
ire
d e
pid
idy
mis
wt
(mg
) * * * * **
p = 0 . 0 5 2 p = 0 . 0 6 4
Co
ntr
ol
LO
AE
L/8
0
LO
AE
L/4
0
LO
AE
L/2
0
LO
AE
L/1
0
LO
AE
L/5
0 . 8
1 . 2
1 . 6
2 . 0
2 . 4
2 . 8
Pa
ire
d s
em
ina
l v
es
icle
wt
(g)
* ** * * * * * *
p = 0 . 0 6 1 p = 0 . 0 6 0
Co
ntr
ol
LO
AE
L/8
0
LO
AE
L/4
0
LO
AE
L/2
0
LO
AE
L/1
0
LO
AE
L/5
0 . 5
1 . 0
1 . 5
2 . 0
LA
BC
wt
(g)
* * * * **
* *
* * * *
Ultramix Summary• Cumulative effects of many “anti-androgens”
• Effects appeared to conform to dose addition
• Adverse reproductive tract effects occurred low doses (LOAEL/80)
• Concerns:
• No doses in ultra low dose range (non-monotonic effects)
• Very high potency of drugs – finasteride/flutamide
• Additional questions:
• Accuracy of mixture models (Dose Addition vs. Response Addition)
• Benchmark Dose Modeling
Linuron330-55-2
Prochloraz67747-09-5
Vinclozolin50471-44-8
p,p’-DDE72-55-9
dipentyl phthalate131-18-0
diethylhexyl phthalate117-81-7
diisobutyl phthalate84-69-5
diisoheptyl phthalate71888-89-6
dihexyl phthalate84-75-3
diheptyl phthalate3648-21-3 chemspider.com
dicyclohexyl phthalate84-61-7
Procymidone32809-16-8
Pyrifluquinazon337458-27-2
dibutyl phthalate84-74-2
benzyl butyl phthalate85-68-7
Supermix
100% Dose
50% Dose
25% Dose
12.5% Dose
6.25% Dose
3.3% Dose
0.5% Dose
0.05% Dose
NOAELx2
NOAEL
NOAEL/2
NOAEL/4
NOAEL/8
NOAEL/15
NOAEL/100
NOAEL/1000
[Chemical]Dose
Supermix dosing
Co
ntr
ol
NO
AE
L/1
00
0
NO
AE
L/1
00
NO
AE
L/1
5
NO
AE
L/8
NO
AE
L/4
NO
AE
L/2
NO
AE
L
NO
AE
Lx
2
1
2
3
4
5
AG
D (
mm
)
* * * ** * * *
* * * *
Co
ntr
ol
NO
AE
L/1
00
0
NO
AE
L/1
00
NO
AE
L/1
5
NO
AE
L/8
NO
AE
L/4
NO
AE
L/2
NO
AE
L
NO
AE
Lx
2
0
2
4
6
8
1 0
1 2
# n
ipp
les
pe
r m
ale
* * * ** * * ** * * *
* * * *
Early postnatal endpoints
Adult reproductive tissue weightsC
on
tro
l
NO
AE
L/1
00
0
NO
AE
L/1
00
NO
AE
L/1
5
NO
AE
L/8
NO
AE
L/4
NO
AE
L/2
NO
AE
L
2x
NO
AE
L0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
Ve
ntr
al
pr
os
tate
wt
(mg
)
* * * *
* * * *
Co
ntr
ol
NO
AE
L/1
00
0
NO
AE
L/1
00
NO
AE
L/1
5
NO
AE
L/8
NO
AE
L/4
NO
AE
L/2
NO
AE
L
2x
NO
AE
L
0
5 0
1 0 0
1 5 0
2 0 0
Gla
ns
pe
nis
wt
(mg
)
N / AN / A
Co
ntr
ol
NO
AE
L/1
00
0
NO
AE
L/1
00
NO
AE
L/1
5
NO
AE
L/8
NO
AE
L/4
NO
AE
L/2
NO
AE
L
2x
NO
AE
L
0
1
2
3
4
5
Pa
ire
d t
es
tis
wt
(g)
* * * *
* * * *
Co
ntr
ol
NO
AE
L/1
00
0
NO
AE
L/1
00
NO
AE
L/1
5
NO
AE
L/8
NO
AE
L/4
NO
AE
L/2
NO
AE
L
2x
NO
AE
L
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
1 6 0 0
Pa
ire
d e
pid
idy
mu
s w
t (m
g)
* * * *
* * * *
Co
ntr
ol
NO
AE
L/1
00
0
NO
AE
L/1
00
NO
AE
L/1
5
NO
AE
L/8
NO
AE
L/4
NO
AE
L/2
NO
AE
L
2x
NO
AE
L
0 . 0
0 . 4
0 . 8
1 . 2
1 . 6
2 . 0
2 . 4
2 . 8
Pa
ire
d s
em
ina
l v
es
icle
wt
(g)
* * * *
* * * *
Co
ntr
ol
NO
AE
L/1
00
0
NO
AE
L/1
00
NO
AE
L/1
5
NO
AE
L/8
NO
AE
L/4
NO
AE
L/2
NO
AE
L
2x
NO
AE
L
0 . 0
0 . 5
1 . 0
1 . 5
2 . 0
LA
BC
wt
(g)
* * * *
* * * *
* * * *
Adult external malformations
Co
ntr
ol
NO
AE
L/1
00
0
NO
AE
L/1
00
NO
AE
L/1
5
NO
AE
L/8
NO
AE
L/4
NO
AE
L/2
NO
AE
L
NO
AE
Lx
2
0
2 0
4 0
6 0
8 0
1 0 0
Hy
po
sp
ad
ias
(%
of
litt
er
)
* * * ** * * ** * * *
Co
ntr
ol
NO
AE
L/1
00
0
NO
AE
L/1
00
NO
AE
L/1
5
NO
AE
L/8
NO
AE
L/4
NO
AE
L/2
NO
AE
L
NO
AE
Lx
2
0
2 0
4 0
6 0
8 0
1 0 0
% m
ale
s w
ith
nip
ple
s
* * * ** * * ** * * *
*
Adult internal malformations
ControlSupermix 50% and 100% doses
(Chemicals at NOAEL and 2xNOAEL)
Seminal vesicles
Testes
Epididymis Ventral prostate
Vas deferens
*Male sex accessory tissues absent in: 95% of males at 100% dose 54% of males at 50% dose
Mixture models of AGD
0 . 0 1 0 . 1 1 1 0 1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
T r e a t m e n t ( % o f t o p d o s e )
AG
D (
% o
f c
on
tro
l)
S u p e r m i x m a l e o b s e r v e d
E D 5 0 : 3 2 . 6 - 4 4 . 2 % ( 9 5 % C I )
D o s e a d d i t i o n m o d e l
E D 5 0 : 4 1 . 3 %
0
R e s p o n s e a d d i t i o n m o d e l
E D 5 0 : 5 2 . 4 %
0 . 0 1 0 . 1 1 1 0 1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
T r e a t m e n t ( % o f t o p d o s e )
Hy
po
sp
ad
ias
(%
of
litt
er
)
0
S u p e r m i x m a l e o b s e r v e d
E D 5 0 : 2 2 . 8 - 2 5 . 9 % ( 9 5 % C I )
D o s e a d d i t i o n m o d e l
E D 5 0 : 3 8 . 4 %
R e s p o n s e a d d i t i o n m o d e l
E D 5 0 : 6 2 4 . 8 %
0 2 0 4 0 6 0 8 0 1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
T r e a t m e n t ( % o f t o p d o s e )
Hy
po
sp
ad
ias
(%
of
litt
er
)
Mixture models of Hypospadias
0 . 0 1 0 . 1 1 1 0 1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
T r e a t m e n t ( % o f t o p d o s e )
AG
D (
% o
f c
on
tro
l)
0
Benchmark Dose estimation
BMD90 = 8.9 %top
BMDL90 = 6.2 %top ≈ NOAEL/8
BMDBMDL
Summary
• Chemicals with multiple “anti-androgenic” molecular initiating events act cumulatively to disrupt male reproductive tract development in the male rat
• Effects occur when component chemicals are at low individual doses• Significant reductions in tissue weights in Ultramix at LOAEL/80• Significant reductions in fetal T production in Supermix at NOAEL/8
• Utility of AOPs for grouping chemicals that affect similar biological pathway• Grouping based on common MIE is too restrictive• Overlap of critical KEs can identify chemicals for cumulative risk assessment
• Dose addition model more accurate than Response addition model
• Some of the most complex developmental studies on reproductive toxicants to date• Very few researchers/laboratories are studying in utero exposure to mixtures
• Exposure to environmental chemicals that disrupt androgen signaling may be contributing to adverse male reproductive human health effects at doses lower than previous estimated
Research Team
Hunter Sampson*
Vickie Wilson
Mary Cardon
PhillHartig
Nicki Evans
Christy Lambright
Earl Gray
Johnathan Furr*
Elizabeth Medlock-Kakaley
*no longer at USEPA
Questions?
Image ReferencesSlide 1 & 14: Sprague Dawley rat image from http://www.criver.com/products-services/basic-research/find-a-model/cd-igs-rat?loc=US
Slide 3: Skaekkebaek NE, Rajpert-De Meyts E, Louis GMB, Toppari J, Andersson AM, Eisenberg ML, Jensen TK, Jorgensen N, Swann SH, Sapra KJ, Ziebe S., Priskorn L, Juul, A. (2016) Male reproductive disorders and fertility trends: Influences of environment and genetic susceptibility. Physiol Rev 96: 55-97
Slide 4: Pumarega J, Gasull M, Lee DH, Lopez T, Porta M. (2016) Number of persistent organic pollutants detected at high concentrations in blood samples ofthe United States population. PLoS One 11(8):e0160432
Slide 4: Qian H, Chen M, Kransler KM, Zaleski RT (2015) Assessment of chemical coexposure patterns based upon phthalate biomonitoring data within the 2007/2008 National Health and Nutrition Examination Survey. J Exposure Sci Environ Epidemiol 25: 249-255
Slide 22: Noriega NC, Ostby J, Lambright C, Wilson VS, Gray Jr LE. (2005) Late gestational exposure to the fungicide prochloraz delays the onset of parturition and causes reproductive tract malformations in male but not female rat offspring. Biol of Reproduction 72:1324-1335
Slide 29: Sprague Dawley rat image from http://www.criver.com/products-services/basic-research/find-a-model/sprague-dawley-rat?loc=US