Endocrine Disrupting Effects of Fresh OSPW:
Mechanisms of Action and the
Usefulness of Ozonation for Treating OSPW
Steve Wiseman
Toxicology Centre
University of Saskatchewan
Endocrine Disruption in the Oil Sands
 Decreased plasma testosterone (T) and estradiol (E2) in
goldfish (Lister et al., 2008) and yellow perch (van den
Heuvel et al., 1999) exposed to OSPW.
 Reduced synthesis of T and E2 by ovarian and testicular
tissues from goldfish (Lister et al., 2008).
 Impaired reproduction of fathead minnows exposed to
OSPW (Kavanagh et al., 2011).
- decreased fecundity in females.
- 2o sex characteristics less pronounced in males.
- males had less T.
- females had less E2.
Steroid-Like ‘Naphthenic Acids’ in OSPW
Some naphthenic acids have structures similar to sex steroid hormones.
Rowland et al., 2011
Estradiol (E2)
Testosterone (T)
Endocrine disruption by ozonated OSPW?
O3
Production
of new NAs
78% degradation
of NAs
Parent NAs
CH3
O3
O - + H+
O2 +
OH
O-
Mimic?
OH
OH
OH
Parent NA ?
CH3
New NA ?
estradiol
OH
Effects of OSPW and Ozonated OSPW on Sex Steroid
Synthesis in the H295R Cell Line
He, Y., Wiseman, S.B., Zhang, X., Hecker, M., Jones, P.D., El-Din, M.G., Martin, J.W., Giesy, J.P. 2010.
Ozonation attenuates the steroidogenic disruptive effects of sediment free oil sands process water
in the H295R cell line. Chemosphere. 80:578-584.
Testosterone
Estradiol
5000
4000
3000
2000
1000
0
*
*
*
Media estradiol (ng/ml)
Media testosterone (ng/ml)
Sex steroid synthesis
350
300
*
*
250
200
150
100
50
0
 OSPW decreased synthesis of T and increased synthesis of E2.
 Ozonation attenuates the effects.
 Dose-response effect of ozonation.
Cyp19a transcript
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Cyp19a
*
*
*
Sulfotransferase activity
Mechanism of effect
1.2
Sulfotransferase
1.0
*
0.8
0.6
*
0.4
0.2
0.0
 Greater E2 results from greater CYP19a transcript abundance and less
metabolism of E2.
 Incomplete attenuation of effects by ozonation of OSPW.
Effects of OSPW and Ozonated OSPW on Sex Steroid
Receptor Signaling in Vitro
He, Y., S.B. Wiseman, M. Hecker, X. Zhang, N. Wang, L.A. Perez, P.D. Jones, M. Gamal El-Din,
J.W. Martin, and J. P Giesy. 2011. Effect of Ozonation on the Estrogenicity and Androgenicity of
Oil Sands Process-Affected Water. Environ Sci. Technol. 45:6268–6274
Hormone signaling - estrogenicity
Estrogenic response
3.0
b
 OSPW and ozonated OSPW are
estrogenic.
b
Ozonation neither attenuated nor
potentiated estrogenicity of OSPW.
2.5
2.0
1.5
a
a
a
1.0
0.5
0.0
dia
e
M
W
ICI
ICI
P
S
+
+
-O
W
W
3
P
P
O
S
OS
-O
3
O
P
OS
W
 Chemical(s) in OSPW and
ozonated OSPW bind to the ER.
 Neither OSPW nor ozonated OSPW
are anti-estrogenic.
Rowland et al., 2011
Hormone signaling – anti-androgenicity
Androgenic response
1.2
OSPW + T
O3-OSPW + T
*
1.0
0.8
*
 Neither OSPW nor ozonated
OSPW are androgenic.
*
 OSPW and ozonated OSPW are
anti-androgenic.
0.6
0.4
 Ozonation attenuated but did
not abolish the anti-androgenicity.
0.2
0.0
0.01
0.05
[T] ng/ml
0.10
Effects of OSPW and Ozonated OSPW on the
Sex Steroid Synthesis and Signaling in Sexually
Maturing Fathead Minnows
Sex Steroid Synthesis and Signaling
Hormone production
Hormone action
Hypothalamus
Liver
KISS-R
KISSpeptin
VTG
CHG
GnRH
Pituitary
SPG
GnRH-R
LH / FSH
Testes
LH / FSH
ER
Ovaries
LH-R / FSH-R
LH-R / FSH-R
Estradiol
Testosterone
AR
Pituitary - Females
mRNA Abundance
(Relative to Freshwater)
Freshwater
OSPW
O3-OSPW
b
8
6
a
4
2
a
b
a
a
ab
b
a
0
GnRH-R
FSH-B
LH-B
 Greater synthesis of LH but not FSH in OSPW.
- LH promotes final oocyte maturation and stimulates E2 synthesis by ovaries
 Ozonation attenuates effects of OSPW on LH-B but stimulates FSH-B synthesis.
Gonad - Females
Gonadotropin Receptors
a
Steroidogenic Enzymes
3.0
a
1.5
1.0
b
0.5
b b
0.0
b
mRNA Abundance
(Relative to Freshwater)
mRNA Abundance
(Relative to Freshwater)
2.0
2.5
2.0
a
1.5
LH-R
aa
a
1.0
0.5
b
0.0
FSH-R
Freshwater
OSPW
O3-OSPW
R
StA
P1
CY
1A
b
a
b
b
a
7
P1 bHSD bHSD P19
Y
C
3
CY
17
 Significantly less abundance of transcripts of FSH-R , LH-R and CYP19a (aromatase)
in OSPW and ozonated OSPW
- could result in impaired synthesis of E2.
 Ozonation does not attenuate effects on transcript abundances.
Liver - Females
Sex Hormone Receptors
1.4
Egg Envelope Proteins
a
a
a
2.0
1.2
1.0
0.8
0.6
0.4
0.2
b
b
b b
b b
0.0
mRNA Abundance
(Relative to Freshwater)
mRNA Abundance
(Relative to Freshwater)
1.6
Freshwater
OSPW
O3-OSPW
1.5
a
a
a
1.0
0.5
b b
b b
b b
CHG L
CHG H
0.0
AR
ER-a
ER-B
VTG
 Impaired E2 synthesis could lead to less ER expression.
 Alternatively, estrogen-like chemicals (naphthenic acids) in OSPW and ozonated
OSPW could down regulate ER signaling pathways by negative feedback
 Could explain the decreased fecundity in female fathead minnows (Kavanagh et al.,
2011).
Pituitary - Males
b
mRNA Abundance
(Relative to Freshwater)
5
b
Freshwater
OSPW
O3-OSPW
4
3
2
1
a
a
a
a
a
b b
0
GnRH-R
FSH-B
LH-B
 Greater synthesis of FSH and LH in OSPW.
- FSH promotes early maturation of testes.
- LH stimulates final maturation of testes and spermatogenesis.
- Both stimulate synthesis of T by testis.
 Ozonation of OSPW attenuates the effect of OSPW.
Gonad - Males
Gonadotropin Receptors
b
4
3
Steroidogenic Enzymes
b
a
a
2
a
1
LH-R
b
8
c
6
b
4
2
0
0
FSH-R
b
10
b
mRNA Abundance
(Relative to Freshwater)
mRNA Abundance
(Relative to Freshwater)
5
Freshwater
OSPW
O3-OSPW
a
a
a
A
R
17 HSD HSD
19
StA YP11 CYP
P
B
CY
3B
C
17
 Greater abundance of FSH-R and LH-R may be a response to greater FSH and LH
Released from the pituitary in response to OSPW.
 Activation of FSH-R and LH-R may be stimulating greater sex steroid (T) production.
 Ozonation of OSPW partially attenuates effects of OSWP.
Liver - Males
Sex Hormone Receptors
8
mRNA Abundance
(Relative to Freshwater)
mRNA abundance
(Relative to Freshwater)
Egg Envelope Proteins
b
5
4
3
2
a
Freshwater
OSPW
O3-OSPW
a
1
b
b
6
b
4
a
a
2
a
a
a
a
0
0
AR
ERa
ERB
VTG
CHG-L
CHG-H
 Greater abundance of ER-a transcripts could be due to greater concentrations of E2
secreted from ovaries or due to estrogenic chemicals in OSPW.
 Effect of OSPW on VTG and CHG could be due direct stimulation of ER by either
estrogenic compounds in OSPW (as shown in cell line reporter assay) or E2.
 Could explain the less prominent male secondary sexual characteristics in male
fathead minnows (Kavanagh et al., 2011).
- Could also be a result of anti-androgenic effects of OSPW.
Conclusions
 OSPW has endocrine disrupting effects on fathead
minnows.
 OSPW has different effects in male and female fathead minnows.
- effects may provide mechanistic basis for results from other studies.
 Endocrine disrupting effects appear to be at all levels of the HPG-L axis.
 Effects of ozonation on endocrine disrupting effects of OSPW less clear
than in other studies.
- Ozonation attenuates affects of OSPW on some endocrine endpoints but
effect is more prominent in males than in females.
John Giesy
Yuhe He
Julie Anderson
Markus Hecker
Paul Jones
Karsten Liber
Warren Zubot
Jon Martin
Mohamed Gamal El-Din
Leo Perez-Estrada
Ahmed Moustafa