TRIAZINE-MEDIATED DISRUPTION OF BLTK1 LEYDIG CELL STEROIDOGENESIS
By
Agnes Forgacs

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Biochemistry and Molecular Biology – Environmental Toxicology – Doctor of Philosophy
2013

ABSTRACT
TRIAZINE-MEDIATED DISRUTPION OF BLTK1 LEYDIG CELL STEROIDOGENESIS
By
Agnes Forgacs

Triazines are broad spectrum herbicides implicated in the etiology of testicular
dysgenesis eliciting perturbations of gonad development and reproductive function. In rodents
triazines alter the onset of puberty, cause reproductive senescence, and disrupt reproductive
tract development including dysregulation of androgen-dependent tissue development.
Disruption of testosterone biosynthesis is thought to underlie the effects of triazines in males
across vertebrate species. However, the current approaches used to identify developmental
and reproductive toxicants involve in vivo studies evaluating apical endpoints that are generally
descriptive and do not contribute to the elucidation of mechanism of action. As such, the
mechanism by which triazines disrupt steroidogenesis remains unknown. The objectives of this
study were to establish a model for the evaluation of triazines effects on steroidogenesis that is
amenable to mechanism determination and to identify a possible mode of action for triazineelicited disruption of steroidogenesis.
BLTK1 Leydig cells were characterized and demonstrated to be a viable in vitro model
for the evaluation of endocrine disruptor-mediated effects on steroidogenesis. BLTK1 cells
express all necessary steroidogenic enzymes essential for hormone biosynthesis and maintain
low basal levels of testosterone (T) production, inducible by recombinant human chorionic

gonadotropin (rhCG) and forskolin (FSK). The time- and concentration-dependent effects of
triazine herbicides, atrazine (ATR), propazine (PRO), simazine (SIM) and terbuthylazine (TBA),
and their chlorometabolites, desethylatrazine (DEA), desisopropylatrazine (DIA) and diamino-schlorotriazine (DACT), were evaluated in BLTK1 Leydig cells. Triazines and their
chlorometabolites induced concentration-dependent increases in basal progesterone (P) and T
levels. Triazines also elicited the differential gene expression of several steroidogenic enzymes
required for steroidogenesis. These results were consistent with the cumulative risk assessment
“Common Mechanism Group” designation that PRO, SIM, DEA, DIA and DACT have ATR-like
effects. Using ATR as the representative triazine, whole-genome microarrays identified
differential gene expression at later time points (> 12 hr) with affected genes associated with
steroidogenesis and cholesterol metabolism. Finally, the effects of ATR on rhCG-mediated
induction of steroidogenesis revealed antagonism of P and T levels, despite potentiation of
intracellular cAMP levels. The inhibition of cAMP-specific phosphodiesterases was identified as
underlying increases in cAMP levels. However, the ATR-mediated super-induction of cAMP
levels was not causative of T antagonism in the presence of rhCG. Disruption of
phosphorylation cascades likely contribute to ATR-mediated effects on steroidogenesis, with
observed effects on protein kinase A (PKA) target proteins. This study has established BLTK1
cells as a novel in vitro steroidogenic model for the evaluation of endocrine disrupting
chemicals and the evaluation of triazines in BLTK1 cells has expanded our knowledge of
triazine-mediated disruption of steroidogenesis.

ACKNOWLEDGEMENTS

Achieving a PhD and the graduate school experience would not be possible without the
encouragement, support and guidance of many people. I owe many more thanks than are
possible here, and I am forever grateful to all those who have helped me through the years. I
have grown both scientifically and personally in ways I never could have imagined.
First and foremost, I would like to thank my mentor Dr. Tim Zacharewski. He took a
chance bringing me in as an undergraduate co-operative education student eight years ago, and
fought for me five years ago when he invited me to pursue my PhD in his lab. I cannot express
how grateful I am for his mentorship. None of my success would be possible without him and I
wouldn’t be the scientist I am today without his support and guidance. Thank you for letting me
aim high, at times taking on more than I should, for always providing direct honest opinions and
feedback, and for teaching me how to obtain anything you set your mind to. This thank you also
needs to be extended to Jana, Lara and Nic for letting me into your family, thanks to you I never
felt alone in all my years from East Lansing, MI to Cary, NC.
Second, I need to thank to my committee members: Dr. John LaPres, Dr. Bill Henry, Dr.
Steve Bursian and Dr. Claire Vieille. I walked into my first committee meeting with two very
different projects and every meeting was as if I had a new project. Despite the evolving
projects, they were always available to meet with me and provided insight. Thank you for your
support and flexibility.

iv

I would also like to acknowledge the Zacharewski lab members, past and present. To Dr.
Darrell Boverhof and Dr. Cora Fong who introduced me to the lab, Dr. Lyle Burgoon and Dr.
Jeremy Burt for all the fun times in the bioinfo office. Thank you to Dr. Qi Ding, without whom
my dissertation would not have been possible, I am very grateful for your hard work with the
cells. Thank you to Dr. Anna Kopec for looking out for me in all facets of science and life, I miss
you in the lab and our workouts! Finally, thank you to Dr. Ed Dere, Dr. Josh Kwekel, Dr. Suntae
Kim, Dr. Michelle Angrish, Bryan Mets, Daniel Wright, Michelle D’Souza, Claudia Dominici, Rosie
Jaremba, Rance Nault, and Kelly Fader for their friendship and helping me in the lab.
I could not have completed all my projects nor a dissertation in reproductive toxicology
without the support of many collaborators and scientists I met at conferences. Thank you to Dr.
Bob Chapin for his enthusiasm and for personally introducing me to the RDTSS community. I am
very grateful for the SOT Colgate-Palmolive award to go to EPA/NCCT under the mentorship of
Dr. Keith Houck and to the ILSI North America Food and Chemical Safety Committee. Thank you
also to Dr. Ray Tice for all the one-on-one time for always encouraging me.
I’d also like to extend a thank you to my classmates and friends. Thanks to all my friends
back home who spend countless hours on the phone with me: I moved away over ten years
ago, but so many people visit and still keep regular phone-dates which keep me motivated. To
“Best friends plus Jeff”, our trivia team has been the highlight of my weeks this past year.
Finally, to my family who are my inspiration and motivation. I could never have endured
this graduate school experience without your love, support and encouragement. Thank you
Thomas for proving to me that anything can be achieved no matter the odds or others opinions,
and for encouraging me to always go for it and to stay true to myself. Thank you mom for the
v

many hours of listening to me go on, and on, and on about my never-ending drama both
personal and scientific, for always picking me up when I was down, and for instilling in me the “I
can have it all” mentality. Thank you dad for all the “Hungarian jokes” and honest advice, you
keep me balanced. To my fiancé Peer: you were the highlight of my graduate experience, thank
you for putting up with all my quirks and the chaos that my over-achieving workaholic
personality comes with, for always bringing me back to sanity – I look forward to many more
years of traveling, home renovations, and science together.

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TABLE OF CONTENTS
LIST OF TABLES ................................................................................................................. x
LIST OF FIGURES .............................................................................................................. xi
LIST OF ABBREVIATIONS .............................................................................................. xviii
CHAPTER 1 ....................................................................................................................... 1
REVIEW OF THE LITERATURE: TRIAZINE-MEDIATED TOXICITY AND STEROIDOGENESIS....... 2
INTRODUCTION .............................................................................................................. 2
TRIAZINE HERBICIDE APPLICATION AND EXPOSURE ...................................................... 2
TRIAZINE-MEDIATED TOXICITY ....................................................................................... 7
BIOLOGY OF TESTICULAR STEROIDOGENESIS .............................................................. 11
IN VITRO STEROIDOGENIC MODELS ............................................................................. 14
CONCLUSIONS .............................................................................................................. 17
REFERENCES ................................................................................................................. 19
CHAPTER 2 ..................................................................................................................... 30
RATIONALE, HYPOTHESIS AND SPECIFIC AIMS ................................................................. 31
RATIONALE ................................................................................................................... 31
HYPOTHESIS .................................................................................................................. 31
SPECIFIC AIMS............................................................................................................... 32
CHAPTER 3 ..................................................................................................................... 33
BLTK1 MURINE LEYDIG CELLS: A NOVEL STEROIDOGENIC MODEL FOR EVALUATING THE
EFFECTS OF REPRODUCTIVE AND DEVELOPMENTAL TOXICANTS ................................ 34
ABSTRACT ..................................................................................................................... 34
INTRODUCTION ............................................................................................................ 35
MATERIALS AND METHODS ......................................................................................... 36
CELL CULTURE AND TREATMENT ........................................................................ 36
MTT CYTOTOXICITY ASSAY .................................................................................. 39
WESTERN BLOTTING............................................................................................ 39
ENZYME IMMUNOASSAYS ................................................................................... 40
RNA ISOLATION AND GENE EXPRESSION ............................................................ 41
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DOSE-RESPONSE MODELING AND STATISTICAL ANALYSES ................................ 43
RESULTS ........................................................................................................................ 43
STEROIDOGENIC ENZYME EXPRESSION IN BLTK1 CELLS ..................................... 43
INDUCTION OF STEROIDOGENESIS BY FSK A ND rhCG ....................................... 46
rhCG- AND FSK-ELICITED STEROIDOGENIC GENE EXPRESSION........................... 48
EFFECTS OF EDCs ON TESTOSTERONE LEVELS .................................................... 54
DISCUSSION .................................................................................................................. 60
REFERENCES ................................................................................................................. 67
CHAPTER 4 ..................................................................................................................... 76
TRIAZINE HERBICIDES AND THEIR CHLOROMETABOLITES ALTER STEROIDOGENESIS IN
BLTK1 MURINE LEYDIG CELLS .................................................................................... 77
ABSTRACT ..................................................................................................................... 77
INTRODUCTION ............................................................................................................ 78
MATERIALS AND METHODS ......................................................................................... 81
CELL CULTURE AND TREATMENT ........................................................................ 81
MTT CYTOTOXICITY ASSAY .................................................................................. 81
ENZYME IMMUNOASSAYS ................................................................................... 82
RNA ISOLATION AND QRTPCR ............................................................................. 82
MICROARRAY ANALYSIS ...................................................................................... 83
STATISTICAL ANALYSES AND DOSE-RESPONSE MODELING ................................ 84
RESULTS ........................................................................................................................ 84
EFFECT OF TRIAZINES AND THEIR CHLOROMETABOLITES ON P AND T LEVELS . 84
TRIAZINE EFFECTS ON STEROIDOGENIC ENZYME GENE EXPRESSION ................ 93
WHOLE-GENOME EVALUATION OF ATR-MEDIATED DIFFERENTIAL GENE
EXPRESSION ......................................................................................................... 98
DISCUSSION ................................................................................................................ 111
REFERENCES ............................................................................................................... 116
CHAPTER 5 ................................................................................................................... 124
ATRAZINE-MECHANISM DISRUPTION OF STEROIDOGENESIS IN BLTK1 LEYDIG CELLS ..... 125
ABSTRACT ................................................................................................................... 125
INTRODUCTION .......................................................................................................... 126
MATERIALS AND METHODS ....................................................................................... 128
CHEMICALS ........................................................................................................ 128
CELL CULTURE AND TREATMENT ...................................................................... 129
ENZYME IMMUNOASSAYS ................................................................................. 129
WESTERN BLOTTING.......................................................................................... 129
viii

PHOSPHODIESTERASE INHIBITION ASSAY ......................................................... 130
DOSE-RESPONSE MODELING AND STATISTICAL ANALYSES .............................. 131
RESULTS ...................................................................................................................... 131
ATR-MEDIATED DISRUPTION OF rhCG-ELIDITED INDUCTION OF cAMP, P AND T
LEVELS................................................................................................................ 131
ATR EFFECTS ON cAMP INDUCTION .................................................................. 133
ATR-MEDIATED CHANGES IN PROTEIN PHOSPHORYLATION ............................ 139
DISCUSSION ................................................................................................................ 141
REFERENCES ............................................................................................................... 148
CHAPTER 6 ................................................................................................................... 155
CONCLUSIONS AND FUTURE RESEARCH ........................................................................ 156
BLTK1 MURINE LEYDIG CELL MODEL FOR STEROIDOGENESIS ................................... 157
TRIAZINE EFFECTS ON PROTEIN PHOSPHORYLATION ................................................ 157
REFERENCES ............................................................................................................... 159

ix

LIST OF TABLES
TABLE 1. PRIMER SEQUENCES FOR QRTPCR .................................................................... 42
TABLE 2. REPORTED EVALUATIONS OF EDCS THAT ALTER T LEVELS.................................. 55
TABLE 3. ENRICHED FUNCTIONAL ANNOTATION TERMS FOR ATR-ELICITED
DIFFERENTIALLY REGULATED GENES ............................................................... 109
TABLE 4. IPA PREDICTED TRANSCRIPTION FACTOR ACTIVATION/DEACTIVATION ........... 110

x

LIST OF FIGURES
FIGURE 1. STRUCTURE OF THE CHLORO-s-TRIAZINE BACKBONE ......................................... 3
FIGURE 2. STRUCTURES OF THE CMG TRIAZINES AND CHLOROMETABOLITES ................... 4
Simazine (SIM), Atrazine (ATR) and Propazine (PRO) are dealkylated in the environment
by photodegradation, microbial, or fungal degradation, and in mammalian systems by
cytochrome P450 enzymes (shown as black arrows) predominantly into the
chlorometabolites desisopropylatrazine (DIA), desethylatrazine (DEA) and diamino-schlorotriazine (DACT). These six chemicals comprise the triazine common mechanism
grouip (CMG).
FIGURE 3. STRUCTURE OF TERBUTHYLAZINE .................................................................... 5
Structure of terbuthylazine (TBA) is used as an ATR alternative in the European Union,
and as an algicide in the United States.
FIGURE 4. THE MALE HYPOTHALAMIC-PITUITARY-GONADAL (HPG) AXIS ........................... 9
The HPG axis is the endocrine regulatory framework consisting of stimulation and
feedback inhibitory hormone activity between the hypothalamus, pituitary and gonads.
Briefly, the hypothalamus produces gonadotropin-releasing hormone (GnRH), which
stimulates the anterior pituitary to secrete follicle-stimulating hormone (FSH) and LH.
LH stimulation of Leydig cells promotes T production, which both stimulates Sertoli
cells and feedback inhibits (-) GnRH and LH production. T can also be metabolized in
Leydig cells by aromatization into 17β-estradiol (E2) or by 5α-reduction into 5αdihydrotestosterone (DHT).
FIGURE 5. STIMULATION OF STEROIDOGENESIS IN LEYDIG CELLS .................................... 13
Leydig cell steroidogenesis can be stimulated upon activation of the G-protein-coupled
LHCGR (green) by LH or rhCG or stimulation of adenylyl cyclase by forskolin (FSK;
purple). Subsequent increases in intracellular cAMP (yellow) activate protein kinase A
(PKA; blue) resulting in the phosphorylation of steroidogenic enzymes (grey) and other
factors (ie. transcription factors; orange) eliciting increased expression and activation
of steroidogenic enzymes ultimately resulting in the production of testosterone.
FIGURE 6. OVERVIEW OF DE NOVO STEROIDOGENESIS PATHWAYS ................................. 16
Only steroidogenic cells of the testes (Leydig cells), ovaries (theca and granulose), and
adrenal gland (adrenal cortex) express the P450 side chain cleavage (P450scc) enzyme
required to cleave cholesterol forming pregnenolone, the commitment step for steroid
hormone biosynthesis. Metabolism to various classes of steroid hormone including
progestagens (green), glucocorticoids (yellow), androgens (blue), and estrogens (red) is
carried out by specific cell types in various tissues.

xi

FIGURE 7. THE STEROIDOGENIC PATHWAY ..................................................................... 44
Steroid hormones are biosynthesized from cholesterol. Cholesterol is mobilized and
delivered to the inner mitochondrial membrane by StAR, where it is cleaved by P450scc
to produce pregnenolone. Subsequent enzymatic events in the smooth endoplasmic
reticulum include metabolism by P450c17 and 3βHSD to produce progestagens (i.e.,
progesterone (P), 17α-hydroxypregnenolone and 17α-dhydroxyprogesterone).
Androgens (i.e., dehydroepiandrosterone and androstenedione) are produced by
P450c17, then converted to testosterone (T) by 17βHSD and finally reduced to 5αdihydrotestosterone (DHT) by 5αR1. Androgen metabolism by P450arom also yields
estrogens (i.e., estrone and 17β-estradiol (E2)).
FIGURE 8. STEROIDOGENIC ENZYME AND RECEPTOR EXPRESSION IN BLTK1 CELLS .......... 45
Expression of steroidogenic enzymes and receptors in untreated BLTK1 cells was
evaluated by RT-PCR and western blotting. mRNA and/or protein expression was
detected for all steroidogenic enzymes. PPARα, PPARγ, PXR, AhR, ERα, AR and SF-1
expression was also confirmed. NA: antibody not available
FIGURE 9. TEMPORAL rhCG- AND FSK-INDUCED cAMP, P AND T LEVELS .......................... 47
Intracellular cAMP (A) as well as P (B) and T (C) levels in media were evaluated in
response to 3 ng/ml rhCG (light bars) or 10 μM FSK (dark bars) relative to DMSO vehicle
controls (white bars). Both treatments elicited similar temporal profiles consisting of
maximal cAMP levels reached early at 0.5-1 hr followed by decrease and was
undetectable by 8 hr, while P reached maximum induction at 2 hrs followed by a
gradual decrease over time. In contrast, T levels steadily increased throughout the time
course. Data represent mean ± standard error of 3 replicates, *p < 0.05 vs. DMSO
controls.
FIGURE 10. CONCENTRATION-DEPENDENT INDUCTION OF cAMP, P, T AND E2 BY rhCG
AND FSK ....................................................................................................... 49
Intracellular cAMP concentrations (A) and secreted P (B) and T (C) levels in media
induced by rhCG (ng/ml; light bars) and FSK (μM; dark bars) at 4 hrs. E2 (D) levels in
media were measured 48 hrs after treatment. EC50 values were calculated by the
ToxResponse modeler. Data represent mean ± standard error of 3 replicates, *p < 0.05
vs. DMSO controls.
FIGURE 11. EFFECT OF THE PDE INHIBITOR IBMX ON rhCG- AND FSK-ELICITED
INDUCTION OF T ........................................................................................... 50
Concentration-dependent induction of cAMP (A, B) and T (C, D) by rhCG (0.1 – 100
ng/ml) or FSK (0.1 – 100 μM) alone (dark bars) or in the presence of 0.2 mM IBMX (light
bars) for 4 hrs. IBMX did not affect T induction, cAMP levels were increased, and the
potency of rhCG and FSK were enhanced. Data represent mean ± standard error of 3
independent replicates, *p < 0.05 vs. DMSO controls.

xii

FIGURE 12. DIFFERENTIAL GENE EXPRESSION OF PROGESTAGEN BIOSYNTHETIC
ENZYMES...................................................................................................... 51
The concentration-dependent (24 hrs, left column) and temporal (right column) effects
of rhCG (ng/ml, light bars) and FSK (μM, dark bars) on Star and Cyp17a1 mRNA levels
examined using QRTPCR. Cyp11a1 mRNA expression was not affected by treatment
(data not shown). Data represent mean ± standard error of 3 replicates, *p < 0.05 vs.
DMSO controls.
FIGURE 13. DIFFERENTIAL GENE EXPRESSION OF ANDROGEN PRODUCING ENZYMES ...... 52
The concentration-dependent (24 hrs, left column) and temporal (right column) effects
of rhCG (ng/ml, light bars) and FSK (μM, dark bars) on Hsd3b6 and Hsd17b3 mRNA
levels examined using QRTPCR. Data represent mean ± standard error of 3 replicates,
*p < 0.05 vs. DMSO controls.
FIGURE 14. DIFFERENTIAL GENE EXPRESSION OF T METABOLIZING ENZYMES .................. 53
The concentration-dependent (24 hrs, left column) and temporal (right column) effects
of rhCG (ng/ml, light bars) and FSK (μM, dark bars) on Srd5a1 and Cyp19a1 mRNA
levels examined using QRTPCR. Data represent mean ± standard error of 3 replicates,
*p < 0.05 vs. DMSO controls.
FIGURE 15. MTT EVALUATION OF EDC CYTOTOXICITY ..................................................... 56
BLTK1 cell viability following 24 hr treatment with rhCG (A), FSK (B), prochloraz (C), ATR
(D), glyphosate (E), triclosan (F), MEHP (G) and TCDD (H) using MTT assay. Cells
exhibited >90% viability at concentrations used for subsequent effects on
steroidogenesis. Data represent mean ± standard error of 3 independent replicates.
FIGURE 16. EFFECTS OF EDCs ON T LEVELS....................................................................... 58
The concentration-dependent effects of Prochloraz, Atrazine, glyphosate, Triclosan,
MEHP and TCDD on T levels were evaluated after 4 hrs (top row, black bars) relative to
DMSO controls (white bars). The effect of 30 μM Prochloraz, 300 μM Atrazine, 300 μM
glyphosate, 30 μM Triclosan, 100 μM MEHP and 100 nM TCDD on rhCG-induced (3
ng/ml rhCG) T levels 4 hrs after treatment (bottom row). RDX had no effect on basal or
rhCG induced T levels (data not shown). Data represent mean ± standard error of 3
replicates, *p < 0.05 vs. DMSO controls, #p < 0.05 vs. rhCG.
FIGURE 17. EVALUATION OF TRIAZINE AND CHLOROMETABOLITE CYTOTOXICITY ............ 86
BLTK1 cells were treated for 24 hrs with 1-600 ng/ml rhCG or 1-600 μM of PRO, ATR,
SIM, TBA, DEA, DACT or DIA followed by MTT reagent. No significant cytotoxicity was
detected for any treatment group, cells exhibited >90% viability even at the highest
doses. Data are plotted as % control of means ± standard error, n=3.

xiii

FIGURE 18. CONCENTRATION-RESPONSE EFFECTS OF TRIAZINES AND
CHLOROMETABOLITES ON P LEVELS.............................................................. 87
Concentration-dependent induction of P levels measured in the media by EIA 24 hrs
post-treatment using DMSO as the vehicle control (open bars), 0.1-100 ng/ml rhCG or
1-600 μM PRO, ATR, SIM, TBA, DEA, DACT or DIA (solid bars). All triazines and
chlorometabolites elicited dose-dependent induction of P levels, albeit with lower
efficacy compared to rhCG. Data are plotted as means ± standard error and were
analyzed using ANOVA followed by Dunnett’s post hoc test, n=3, *p<0.05.
FIGURE 19. CONCENTRATION-RESPONSE EFFECTS OF TRIAZINES AND
CHLOROMETABOLITES ON T LEVELS .............................................................. 89
Concentration-dependent induction of T levels measured in the media by EIA 24 hrs
post-treatment using DMSO as the vehicle control (open bars), 0.1-100 ng/ml rhCG or
1-600 μM PRO, ATR, SIM, TBA, DEA, DACT or DIA (solid bars). All triazines and
chlorometabolites elicited dose-dependent induction of T levels, albeit with lower
efficacy compared to rhCG. Data are plotted as means ± standard error and were
analyzed using ANOVA followed by Dunnett’s post hoc test, n=3, *p<0.05.
FIGURE 20. TIME-DEPENDENT EFFECTS OF TRIAZINES AND CHLOROMETABOLITES ON
P AND T LEVELS ............................................................................................ 92
BLTK1 cells were treated with 3ng/ml rhCG or 300 μM of ATR, PRO, SIM or TBA (A and
B), or chlorometabolite (i.e., DEA, DACT, or DIA) (C and D) and evaluated for effects on
P and T levels relative to time-matched DMSO (vehicle control). P (A and C) and T (B
and D) levels were measured at each time point by EIA. P levels in media increased
within 2 hr of treatment followed by a gradual decrease over time. In contrast, T levels
exhibited a concomitant time-dependent increase following treatment. Data were
plotted as mean ± standard error. Statistical significance was determined using ANOVA
followed by Tukey’s post hoc test, n=3.
FIGURE 21. CONCENTRATION-RESPONSE EVALUATION OF TRIAZINE- AND rhCGMEDIATED GENE EXPRESSION ...................................................................... 94
The concentration-dependent effects of rhCG (black striped bars), PRO (medium grey
bars), ATR (white bars), SIM (light grey bars), or TBA (black solid bars) on steroidogenic
enzyme gene expression examined 24 hr post-treatment using QRTPCR. Data are
plotted as mean ± standard error of three independent replicates, *p < 0.05 vs DMSO
controls.
FIGURE 22. TIME-DEPENDENT EFFECTS OF TRIAZINES AND rhCG ON STEROIDOGENIC
GENE EXPRESSION ........................................................................................ 99
Temporal gene expression profiling of steroidogenic enzymes in response to 3 ng/ml
rhCG (black striped bars) or 300 μM of PRO (medium grey bars), ATR (white bars), SIM
(light grey bars) or TBA (black solid bars), respectively, was examined by QRTPCR. Data
are plotted as mean ± standard error of three independent replicates, *p < 0.05 vs.
time-matched DMSO controls.
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FIGURE 23. TEMPORAL WHOLE-GENOME EVALUATION OF ATR-MEDIATED
DIFFERENTIAL GENE EXPRESSION................................................................ 103
Gene expression elicited by 300 μM ATR was evaluated using Agilent whole-genome
microarrays. Filtering of microarray data (A), and summary of the number of genes
exhibiting differential expression per time point (B; |fold change| > 1.5 and P1(t) >
0.999). Evaluation of Star expression by microarray (line) and QRTPCR (bars) (C).
QRTPCR validation of Star plotted as mean ± standard error of three independent
replicates, *p < 0.05 vs. time-matched DMSO controls. Microarray data are plotted as
mean fold change of three independent replicates, #P1(t) > 0.999.
FIGURE 24. K-MEANS CLUSTERING OF ATR-ELICITED TEMPORAL WHOLE-GENOME
GENE EXPRESSION ...................................................................................... 106
ATR (300 uM) elicited the differential expression (|fold change| > 1.5 and P1(t) > 0.999)
of 797 genes. K-means clustering using a Pearson correlation distance metric, identified
three distinctive profiles: increasing down-regulation, increasing up-regulation and
intermediated up-regulation. Light grey lines illustrate the profile of each gene within
the cluster, while the dark grey line illustrates the average trend for the group.
FIGURE 25. BLTK1 CELL MORPHOLOGY IN RESPONSE TO rhCG AND ATR TREATMENT .... 111
Cells were photographed using a phase/contrast microscope at 100x magnification 24
and 48 hrs after treatment with DMSO, 3 ng/ml rhCG or 300 µM ATR.
FIGURE 26. TEMPORAL EFFECTS OF ATR ON rhCG-INDUCTION OF STEROIDOGENESIS .... 132
Intracellular cAMP (A), P (B) and T (C) levels in media were evaluated upon treatment
with DMSO vehicle (white bars), 300 μM ATR (grey bars), 3 ng/ml rhCG (black bars), or
their co-treatment (striped bars) from 1-24 hrs. Data are plotted as mean ± standard
error, n=3, and were analyzed by ANOVA followed by Tukey’s post hoc test where
a

b

*p<0.05 vs. time-matched DMSO, p<0.05 vs. time-matched ATR, p<0.05 vs. timematched rhCG. ND: not detected.
FIGURE 27. CONCENTRATION-DEPENDENT EFFECTS OF ATR ON rhCG-MEDIATED
INDUCTION OF STERIODOGENESIS .............................................................. 134
Intracellular cAMP (A) and T levels in media (B) were evaluated after 4 hrs of
treatment. EC50 determined by sigmoidal dose-response curves fit by the ToxResponse
Modeler for the co-treatment of 3 ng/ml rhCG and 1-600 μM ATR (in shaded area).
Data are plotted as mean ± standard error, n=3, and were analyzed by ANOVA followed
by Dunnet’s post hoc test where #p<0.05 vs. rhCG. The ToxResponse modeler was used
to determine EC50 values based on sigmoidal dose-response curve modeling.

xv

FIGURE 28. ATR-MEDIATED EFFECTS ON rhCG- OR FSK-INDUCED STERIODOGENESIS ..... 135
Intracellular cAMP (A), and T levels in media (B) were evaluated after 4 hrs with DMSO,
rhCG (3 ng/ml), FSK (10μM), ATR (300 μM), or the combinations of these treatments as
indicated. Data are plotted as mean ± standard error, n=3, and were analyzed by
ANOVA followed by Tukey’s post hoc test where *p<0.05 vs. DMSO, #p<0.05 vs. rhCG or
FSK as indicated.
FIGURE 29. ATR AND IBMX EFFECTS ON rhCG-INDUCTION OF cAMP AND T ................... 136
Intracellular cAMP (A), and T levels in media (B) were evaluated after 4 hrs of
treatment with DMSO, rhCG (3 ng/ml), IBMX (0.2 mM), ATR (300 μM), or the
combinations of these treatments as indicated. Data are plotted as mean ± standard
error, n=3, and were analyzed by ANOVA followed by Tukey’s post hoc test where
*p<0.05 vs. DMSO, #p<0.05 vs. rhCG.
FIGURE 30. ATR-MEDIATED INHIBITION OF cPDE ........................................................... 138
Temporal effects on PDE activity (A) were assessed by quantification of 5’-AMP
produced upon incubation of PDE enzyme and 3’,5’-cAMP substrate alone (black
circles) or with the addition of 300 μM ATR (dark grey triangles) or 30 μM IBMX (light
squares) for up to 60 min. Dose-dependent effects of 0.01 – 300 μM ATR (dark grey
triangles) or 0.01 – 200 μM IBMX (light grey squares) were evaluated after 10 min (B),
with amount of 5’-AMP produced with enzyme alone (ie. 100% enzyme activity)
depicted as the black hashed line. The ToxResponse modeler was used to determine
IC50 values based on sigmoidal dose-response curve modeling.
FIGURE 31. EFFECT OF db-cAMP ON T LEVELS ................................................................ 139
Concentration-dependent induction of T levels by db-cAMP at 4 hrs in BLTK1 cells
demonstrates that high mM db-cAMP concentrations do not antagonize T production.
Data are plotted as mean ± standard error, n=3, and were analyzed by ANOVA followed
by Dunnett’s post hoc test where *p<0.05 vs. DMSO.
FIGURE 32. ATR EFFECTS ON PKA SUBSTRATE PHOSPHORYLATION LEVELS .................... 140
Western blot detection of phosphorylated PKA motif phospho-Ser/Thr residue with Arg
at -3 and -2 positions ((K/R)(K/R)X(S*/T*)) from whole cell BLTK1 extracts after 1 hr of
treatment with DMSO or 3-300 μM ATR. Protein size (kDa) is labeled on the right. n=1.
FIGURE 33. EFFECTS OF U0126 AND EVALUATION OF ERK1/2 PHOSPHORYLATION ........ 142
(A) The effects of MEK1/2 inhibitor U0126 on T in BLTK1 cells was measured by EIA at 4
hrs (grey bars), dose-dependent inhibition of rhCG-elicited T induction (hashed bars).
Western blot detection of phospho-ERK1/2 (42/44 kDa) from whole cell extracts after 1
hr of treatment. Data are plotted as mean ± standard error, n=3, and were analyzed by
ANOVA followed by Dunnet’s post hoc test where *p<0.05 vs. rhCG. (B) Comparison of
DMSO, 3 ng/ml rhCG, 300 μM ATR and rhCG/ATR co-treatment with and without 10
μM U0126. (C) Dose-response evaluation of 3-300 μM ATR with 3 ng/ml rhCG.
Membranes were stripped and probed for actin (37 kDa) as a loading control. N=1.
xvi

FIGURE 34. SIMPLIFIED SCHEME OF PKA ACTIVATION AND PKA INTERACTING
PROTEINS ................................................................................................... 146
Inactive PKA, comprised of two regulatory (R) and two catalytic (C) subunits, exists in
the cytosol where it is in complex with AKAP which serves as a scaffolding protein. PKA
is activated upon binding of cAMP to (R) and subsequent release of (C) which
phosphorylates target proteins including key steroidogenic regulators including CREB,
SF-1, StAR and cPDEs. PKA-mediated phosphorylation of PDE4, also known to interact
with the AKAP scaffold, activates PDE4 resulting in the turnover of cAMP and
deactivation of PKA. PDE4 can also be phosphorylated by phospho-ERK1/2 at an
alternate site, resulting in inhibition of its activity.

xvii

LIST OF ABBREVIATIONS
ANOVA

analysis of variance

AR

androgen receptor

ATR

atrazine

cAMP

3,5’-cyclic adenosine monophosphate

cGMP

3,5’-cyclic guanosine monophosphate

CMG

common mechanism group

CO2

carbon dioxide

CREB

cAMP response element binding protein

DACT

diamino-s-chlorotriazine

DAVID

database for annotation, visualization and integrated discovery

DEA

desethylatrazine

DEHP

diethylhexyl phthalate

DIA

desisopropylatrazine

DHT

dihydrotestosterone

DMEM

Dulbecco’s modified eagle medium

DMSO

dimethyl sulfoxide

E2

17β-estradiol

EC50

effective concentration causing 50% of the maximal response

EDC

endocrine disrupting compound

EIA

enzyme immunoassay

EPA

Environmental Protection Agency (United States)

xviii

ER

estrogen receptor

FBS

fetal bovine serum

FSK

forskolin

GPCR

G-protein-coupled receptor

HPG

hypothalamic-pituitary-gonadal

IBMX

3-isobutyl-1-methylxanthine

IPA

Ingenuity® pathway analysis

LH

luteinizing hormone

LHCGR

luteinizing hormone/chorionic gonadotropin receptor

MEHP

monoethylhexyl phthalate

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

P

progesterone

PCR

polymerase chain reaction

PDE

phosphodiesterase

cPDE

cAMP-specific phosphodiesterase

PKA

protein kinase A

PRO

propazine

QRTPCR quantitative real-time PCR
rhCG

recombinant human chorionic gonadotropin

RNA

ribonucleic acid

SIM

simazine

StAR

steroidogenic acute regulatory protein

T

testosterone

TBA

terbuthylazine
xix

CHAPTER 1

1

CHAPTER 1
REVIEW OF LITERATURE: TRIAZINE-MEDIATED TOXICITY AND STEROIDOGENESIS

INTRODUCTION
Chloro-s-triazines are broad-spectrum herbicides gaining public concern due to their
endocrine disruptive effects in aquatic and mammalian species. While the mode of action by
which triazines elicit their effects remains unknown, it is accepted that they target the
hypothalamic-pituitary-gonadal (HPG) axis eliciting altered steroid hormone levels, disruption
of gonadal morphometry and compromised reproduction. Establishing a physiologically
relevant model to evaluate de novo steroid hormone biosynthesis (steroidogenesis) that is
amenable to elucidating a mode of action is necessary to characterize how triazines disrupt
hormone levels.

TRIAZINE HERBICIDE APPLICATION AND EXPOSURE
Chloro-s-triazines, referred to herein as triazines, are broad-spectrum herbicides
characterized by a conserved chlorinated triazine ring backbone (Figure 1). Triazines are used to
control broadleaf and grassy weeds as well as algae through inhibition of photosynthesis (1, 2).
More specifically, high affinity, irreversible binding to the plastoquinone binding site on
photosystem II in the thylakoid membrane of chloroplasts inhibits electron transfer, effectively
blocking photosynthesis thereby eliminating target plants and algae (1, 2).

2

FIGURE 1. STRUCTURE OF THE CHLORO-S-TRIAZINE BACKBONE.

Several triazines are registered for use in the United States including atrazine (ATR) and
simazine (SIM) for use on agricultural crops, propazine (PRO) for indoor grown ornamental
plants, and terbuthylazine (TBA) for use as an algicide (Figure 2) (3). ATR was introduced by
Ciba-Geigy (then Novartis and currently Syngenta) in 1958, and is currently the most widely
used triazine with nearly 80 million pounds applied annually, predominantly on corn crops in
the Midwestern United States (4, 5). ATR is highly mobile, readily transported from
groundwater to streams and lakes, and is persistent in both soil and water with a half-life
ranging from 146 to 742 days in aerobic soil and water, respectively (6). It was the most
common pesticide contaminant detected in water in the United States between 1992 and 2001,
often detected above the U.S. Envionmental Protection Agency’s (EPA) suggested maximum
contamination level (MCL) of 3 ppb (6). In the Midwestern United States where use of ATR is
the greatest, storm runoff can result in peaks of ≥20 μg/L ATR in watersheds (7). Similarly, SIM

3

FIGURE 2. STRUCTURES OF THE CMG TRIAZINES AND CHLOROMETABOLITES.
4

FIGURE 2 (cont’d)
Simazine (SIM), Atrazine (ATR) and Propazine (PRO) are dealkylated in the environment by
photodegradation, microbial, or fungal degradation, and in mammalian systems by cytochrome
P450 enzymes (shown as black arrows) predominantly into the chlorometabolites
desisopropylatrazine (DIA), desethylatrazine (DEA) and diamino-s-chlorotriazine (DACT). These
six chemicals comprise the triazine common mechanism grouip (CMG).

is also a groundwater contaminant found above its MCL of 4 ppb with a soil half-life of 91 days
and water half-life of 664 days (6, 8). Ubiquitous groundwater contamination caused the
European Union to cancel ATR registration in 2003 ceasing its use in most of Europe with TBA
(Figure 3) having been introduced in its place (9). However, based on a risk assessment carried
out by the EPA in 2006 the re-registration of ATR was approved, and 60 other countries also
continue to use ATR (10, 11). Given the new body of scientific information, the EPA initiated a
new comprehensive re-evaluation of ATR, including more accurate methods to monitor human
exposure (12, 13).

FIGURE 3. STRUCTURE OF TERBUTHYLAZINE.
Structure of terbuthylazine (TBA) is used as an ATR alternative in the
European Union, and as an algicide in the United States.
5

Dealkylation of ATR and SIM in the environment by microbial and fungal degradation, as
well as photodegradation, form three major chlorinated metabolites (chlorometabolites):
desethylatrazine (DEA), desisopropylatrazine (DIA) and diaminotriazine (DACT; Figure 2), which
are commonly found with ATR and SIM in groundwater (6, 14, 15). As such, triazine exposure
estimations require measurements of not only the parent triazine, but also the
chlorometabolites (16).
In mammals, triazines are widely distributed throughout the body and rapidly
metabolized primarily by cytochrome P450s, namely CYP1A1/2, into DEA, DIA and DACT (1619). Evaluation of human urine by mass spectrometry also identified hydroxylated, dealkylated,
and glutathione-derived mercapturic acid metabolites of ATR, but the dealkylated
chlorometabolites DEA, DIA and DACT were the most abundant, consistent with rodent studies
(16). More specifically, urinary ATR metabolite composition was up to ~70% DACT, ~20% DEA,
and ~5% DIA at environmental exposure levels below the MCL (16). Maximum plasma levels in
mice are achieved within 1 hr, with maximum DACT plasma concentrations within 2 hrs postdose (18). Chlorometabolite concentrations of ~2 μM are reported in serum with less than 1
μM of parent compound 3 hrs following administration of 200 mg/kg ATR or an equimolar dose
of SIM or PRO in male Wistar rats (20). Most ATR and chlorometabolite dose is excreted within
24 hrs in mice and rats, with the exception of substantial DACT detected in urine up to 72 hrs
post-dose (18, 19). Estimations of human daily doses from occupational exposure to ATR during
mixing, loading and application ranged from 1.8 to 6.1 μg/kg/day, suggesting exposures can be
as high as 6.1 ppb (21).

6

Exposure to triazines is of concern due to their endocrine disruptive effects (20, 22, 23).
The EPA has grouped ATR, SIM and PRO as well as their major chlorometabolites (DEA, DIA and
DACT) into a “Triazine Common Mechanism Group” for cumulative risk assessment. Common
mechanism groups (CMGs) are defined for cumulative risk assessment as two or more
chemicals that cause a common toxic effect(s) using the same sequence of major biochemical
events, interpreted as mode of action (24). The triazine CMB was established based on the
conserved disruption of the HPG axis by all chemicals. More specifically, luteinizing hormone
(LH) surge was suppressed by all compounds in female rodents (24-26). The inclusion of the
chlorometabolites assumes toxic equipotency to the parent triazines (25), highlighting the need
to study the effects of chlorometabolites independent of the parent triazines.

TRIAZINE-MEDIATED TOXICITY
Many studies on triazine-mediated toxicity have focused on ATR as the representative
triazine since it is most widely used. Despite few studies evaluating PRO, SIM, TBA or
chlorometabolites, effects elicited by ATR are considered significant for all chemicals in the
triazine CMG. Due to the accumulation of ATR in water systems, the effects of ATR on a variety
of vertebrate classes, including aquatic species such as fish and frogs, have been widely
evaluated and compared to effects in mammalian species, namely rodents (27, 28).
Due to the structural similarities between the specific site of ATR action at
plastoquinone binding sites on photosystem II in plants and mammalian electron transport
chain (ETC) complex I and III quinone binding sites, the potential for ATR to interfere with
mitochondrial respiration has been investigated (29, 30). Mitochondrial dysfunction
7

characterized by decreased ETC complex III activity has been documented in rats, although it
was only observed in one study with chronic administration of ATR in drinking water for 5
months, and only in skeletal muscle (30). It is generally accepted that ATR does not target
mitochondrial ETC complexes in mammalian systems (13).
Species-specific effects of ATR exposure including immunomodulation (31-33), oxidative
stress (34) and tumor promotion (35) have been documented. For example, ATR-mediated
immunomodulation includes reduced ability of silver catfish fingerlings to withstand pathogenic
microorganism challenge (32), but increased T cell cytolytic activity in Balb/c mice (31).
Meanwhile, ATR increases hepatic lipid peroxidation and oxidative stress with concomitant
elevation of antioxidant enzyme expression only in fish (34, 36, 37). Mammary tumors resulting
from perturbations of LH surge have only been identified in female Sprague-Dawley rats (35,
38, 39). However, tumor promotion is not considered significant to human risk assessment, and
ATR is not categorized as a human carcinogen, due to differences in hormonal control of estrus
in Sprague-Dawley rats mediated by LH surge compared to mediation by estrogen in humans
(40, 41). Despite the lack of relevance of mammary tumor promotion to other species, the most
consistent and widely reported effects of ATR are on hormone-mediated reproductive
development and function.
Reproductive toxicity elicited by ATR includes changes in hormone levels and altered
reproductive function in many vertebrate species with generally ‘estrogenic effects’ despite
negligible interaction on estrogen receptor activity (17, 37, 38, 42-45). As such, the mode of
action by which ATR elicits endocrine disruptive effects remains unknown. In female rodents
ATR suppresses LH surge (46), alters estrous cycling (47), delays vaginal opening, uterine growth
8

and puberty (48-50), and disrupts steroidogenesis (51). Meanwhile, in male rodents ATR delays
puberty, alters testicular morphology and impairs androgen-dependent organ development
(52-57). Most of these ATR-mediated effects are attributed to changes in steroid hormone
levels associated with disruption of the HPG axis (Figure 4) (40, 58).

FIGURE 4: THE MALE HYPOTHALAMIC-PITUITARY-GONADAL (HPG) AXIS.
The HPG axis is the endocrine regulatory framework consisting of stimulation and feedback
inhibitory hormone activity between the hypothalamus, pituitary and gonads. Briefly, the
hypothalamus produces gonadotropin-releasing hormone (GnRH), which stimulates the
anterior pituitary to secrete follicle-stimulating hormone (FSH) and LH. LH stimulation of Leydig
cells promotes T production, which both stimulates Sertoli cells and feedback inhibits (-) GnRH
and LH production. T can also be metabolized in Leydig cells by aromatization into 17β-estradiol
(E2) or by 5α-reduction into 5α-dihydrotestosterone (DHT).
9

ATR-elicited reproductive and developmental effects in aquatic wildlife species are
consistent with rodent studies including disruption of steroidogenesis and gonadal
development, reduced fertility, and reproductive senescence (40, 44, 59-62). For example, in
male goldfish ATR exposure decreased plasma androgen concentrations and altered the
morphology of the testes, while elevated ovary atresia was evident in females (63). Gonad
abnormalities in both sexes of ATR-treated fathead minnow resulted in reduced egg production
and ultimately decreased reproduction (61). Meanwhile, ATR-elicited reproductive toxicity is
characterized by feminization comprising elevated estrogen levels, demasculinization
characterized by decreased androgen concentrations, and hermaphroditism in male Xenopus
laevis (27, 40, 44, 60, 64, 65). All these effects noted in frogs can be attributed to disruption of
steroidogenesis. For example, hermaphroditism in frogs is linked to a lack of 5α-reduced
androgens such as 5α-dihydrotestosterone (DHT), which is produced from testosterone (T) by
5α-reductase enzymes in the testes (60). Furthermore, both T and 5α-reduced androgens are
essential during male gonad development and for progression of spermatogenesis in Sertoli
cells (66). In fact, ATR elicits disruption of androgen levels and reproductive development (52,
53, 55) as well as decreased sperm number and motility in rodents and humans (56, 67, 68).
Consequently, the leading hypothesis proposed in the literature is that ATR’s endocrine
disruptive effects in males are due to alterations in steroidogenesis, namely affecting
progesterone (P) and T levels (45, 52, 53, 57).

10

BIOLOGY OF TESTICULAR STEROIDOGENESIS
Despite the consensus that ATR is an endocrine disruptor that dysregulates
steroidogenesis and alters reproductive function, the underlying mechanism and intracellular
target(s) of ATR remain unknown. Several fungicides, pesticides and phthalates are known to
elicit reproductive tract abnormalities including compromised reproductive fitness and increase
cancer incidence due to altered steroid hormone levels (69-72). As such, it is recognized that
steroidogenesis is a potential target for drugs, chemicals, natural products and environmental
contaminants that adversely impact reproductive development and fertility (73, 74). The
mechanisms involved in endocrine disrupting chemical (EDC)-mediated changes in steroid levels
remain largely unknown but could involve disrupting steroidogenic enzyme expression,
activation and/or activity (69). Developmental exposure to high doses of EDCs has been
associated with testicular dysgenesis syndrome (TDS) in humans, comprised of adverse effects
including cryptorchidism, hypospadias, low sperm count and testicular cancer, similar effects
have been characterized in rodents and wildlife species with similar etiology (75-78).
While both the adrenal glands and the testes/ovaries have the capacity to carry out
steroidogenesis, in males the testes are the primary organ producing T and may be a target for
ATR. In mammals, the male gonads contain terminally differentiated adult Leydig cells that are
the primary source of circulating T in the body. Leydig cells have an intracellular organelle
structure adapted to serve in steroidogenesis, including abundant smooth endoplasmic
reticulum and many mitochondria and lipid droplets, which provide the precursor to steroid
synthesis: cholesterol (79). The primary function of Leydig cells is the production of androgens

11

including DHT and T, which are essential for the descent of the testes during development and
for progression of spermatogenesis (66).
Leydig cell steroidogenesis is stimulated by the chief gonadotropin tropic stimulator, LH
secreted from the anterior pituitary activating the G-protein-coupled LH/chorionic
gonadotropin receptor (LHCGR; Figure 5) (66). Alternatively, human chorionic gonadotropin
(hCG), produced by the placenta during pregnancy, with 85% sequence similarity to LH (80), can
also stimulate the LHCGR and is often used for research due to a longer half-life (~24 hrs
compared to ~4 hrs for LH (81)). High affinity binding of either gonadotropin to LHCGR
expressed on the surface of Leydig cells, results in dissociation of the G-protein alpha
stimulatory subunit, which in turn activates adenylyl cyclase (66). The stimulation of adenylyl
cyclase activity, converting ATP into cAMP, rapidly increases intracellular cAMP levels (82, 83).
The cAMP levels are a function of both synthesis and degradation, with cyclic nucleotide
phosphodiesterase enzymes responsible for the turnover of cAMP (66). cAMP binding activates
protein kinase A (PKA), resulting in the phosphorylation and activation of steroidogenic
enzymes promoting the synthesis of steroid hormones (83). Leydig cell steroidogenesis can be
divided in to two phases: (1) an acute phase comprising LHCGR activation and induction of
intracellular cAMP and PKA signaling events, and (2) a longer term effect involving differential
gene expression and increased steroidogenic enzyme expression that sustains steroidogenesis
(66). While the acute phase induces immediate T production, sustained hormone biosynthesis
requires both phases. Therefore, any studies examining disruption of steroidogenesis should
examine both phases.

12

FIGURE 5: STIMULATION OF STEROIDOGENESIS IN LEYDIG CELLS.
13

FIGURE 5 (cont’d)
Leydig cell steroidogenesis can be stimulated upon activation of the G-protein-coupled LHCGR
(green) by LH or rhCG or stimulation of adenylyl cyclase by forskolin (FSK; purple). Subsequent
increases in intracellular cAMP (yellow) activate protein kinase A (PKA; blue) resulting in the
dissociation of the PKA regulatory subunit (dark blue) from the catalytic subunit (light blue). The
released catalytic subunit phosphorylates steroidogenic enzymes (grey) and other factors (ie.
transcription factors; orange) eliciting increased expression and activation of steroidogenic
enzymes ultimately resulting in the production of testosterone. For interpretation of the
references to color in this and all other figures, the reader is referred to the electronic version of
this dissertation.

IN VITRO STEROIDOGENIC MODELS
Current approaches to identify developmental and reproductive toxicants involve
examining offspring after administering pregnant animals with maternally toxic doses (84).
Apical endpoints such as nipple retention, ano-genital distance, vaginal opening and balanopreputial separation are assessed in addition to evaluating reproductive fitness (85, 86). Such
animal-based testing is expensive, time consuming, generally descriptive, and does not
significantly contribute to the elucidation of mechanisms (87-92). In vitro evaluation of
steroidogenesis is important for elucidating mechanisms of potential reproductive and
developmental toxicants (73, 74).
Steroidogenesis model development has focused on Leydig cells as they are the only cell
type to synthesize T and E2 directly from cholesterol (72). Ovarian steroidogenesis is a two-cell
system with both theca and granulosa cells producing P, but only theca cells express enzymes
to metabolize P to T, and subsequent aromatization of T to estrogens or 5α reduction to DHT
occurs only in granulosa cells (93). Steroidogenic theca cell line development has not been

14

successful, but there are several rodent models of Leydig cell origin including mouse MA-10 and
mLTC-1 cells as well as rat R2C cells. MA-10 and mLTC1 cells, established from transplantable
M548OP Leydig tumors carried in C57Bl/6 mice exhibit P induction by rhCG but lack the
enzymes required for subsequent metabolism to T (94-96). Recent studies report mLTC-1 cells
can produce low levels of androgens when stimulated with very high concentrations of rhCG
(97), however, the high concentration of rhCG required to elicit even minimal T levels limits the
physiological relevance of this system. The rat R2C Leydig cell line exhibits high basal
steroidogenic activity but lacks functional LHCGR expression (98), making R2C cells not a
suitable model system to study EDC-mediated effects on gonadotropin-stimulated
steroidogenesis since they are not inducible.
In addition to Leydig cells or ovarian theca and granulosa cells, the adrenal gland is the
only other organ expressing the cytochrome side chain cleavage enzyme (P450scc) capable of
de novo steroidogenesis in the mammalian system. The adrenal glands produce mainly
glucocorticoids, and subsequently mineralocorticoids, from progestagens (Figure 6). The
adrenal cortex, comprised of three different cell types (called zones), normally only produces
dehydroepiandrosterone (DHEA) sulfate and androstenedione (precursors of T) and does not
express sufficient levels of the 17-beta-hydroxysteroid dehydrogenase necessary for T
biosynthesis (99). Human H295R cells (ATCC CLR-2128), isolated from a pluripotent
adenocortical carcinoma, produce detectable basal cortisol (glucocorticoid), aldosterone
(mineralocorticoid), androgens and E2 (100). H295R cells are used as the steroidogenesis assay
in the U.S. EPA’s Endocrine Disruptor Screening Program to screen for effects of chemicals on T
and E2 biosynthesis (101). However, H295R cells do not express functional ACTH receptors (a
15

FIGURE 6: OVERVIEW OF DE NOVO STEROIDOGENESIS PATHWAYS.
16

FIGURE 6 (cont’d)
Only steroidogenic cells of the testes (Leydig cells), ovaries (theca and granulose), and adrenal
gland (adrenal cortex) express the P450 side chain cleavage (P450scc) enzyme required to
cleave cholesterol forming pregnenolone, the commitment step for steroid hormone
biosynthesis. Metabolism to various classes of steroid hormone including progestagens (green),
glucocorticoids (yellow), androgens (blue), and estrogens (red) is carried out by specific cell
types in various tissues.

member of the cell surface 7-transmembrane domain superfamily of G-protein coupled
receptors), and therefore steroidogenesis must be stimulated using FSK. Furthermore, the
relatively high T and E2 production, in addition to the glucocorticoid and mineralocorticoid
production suggest that H295R cells have the characteristics of zonally undifferentiated fetal
adrenal cells (100).

CONCLUSIONS
Although ATR-mediated endocrine disruption has been widely evaluated demonstrating
targeted effects on the HPG axis, most studies are descriptive in nature and the mechanism by
which the CMG triazines elicit these effects remains unknown. Furthermore, data suggest that
steroidogenesis may specifically be a target of triazines but sufficient models for
mechanistically evaluating steroidogenesis are lacking. Collectively, these data highlight the
need for mechanistic evaluation of CMG triazines and chlorometabolites in a physiologically
relevant steroidogenesis model that is amenable to mechanism determination. Establishing a

17

Leydig cell model for the evaluation of ATR-mediated disruption of steroidogenesis will provide
important insight to help understand the mechanism of triazine-elicited endocrine disruption.

18

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CHAPTER 2

30

CHAPTER 2
RATIONALE, HYPOTHESIS AND SPECIFIC AIMS

RATIONALE
Triazines are broad spectrum herbicides implicated in eliciting endocrine disruptive
effects including perturbation of male reproductive development function. Current approaches
used to evaluate reproductive toxicants involve evaluating apical endpoints that are generally
descriptive and do not contribute to the elucidation of mechanism of action. Incidentally, the
mechanism of action for triazines remains unknown. However, it has been suggested that
disruption of the HPG, specifically steroidogenesis in the gonads, may underlie the effects of
triazines across vertebrate species. The objectives of this study are to establish a model for the
evaluation of triazines effects on steroidogenesis that is amenable to mechanism determination
and to identify a possible mode of action for triazine-elicited disruption of steroidogenesis.

HYPOTHESIS
Triazines alter testosterone biosynthesis in BLTK1 Leydig cells by disrupting cAMP-mediated
steroidogenic gene expression.

31

SPECIFIC AIMS
In order to test the hypothesis, a Leydig cell model is required that will allow the
comprehensive examination of triazine-elicited steroidogenic disruption. This model will be
amenable to testing the hypothesis with the use of gene expression microarrays and enzymelinked immunoassays and will include the following aims:

1. Establish and characterize BLTK1 murine Leydig cells as a novel model for the
evaluation of steroidogenesis.

2. Characterize the time- and concentration-dependent effects of triazine herbicides
and their major chlorometabolites on steroidogenesis in BLTK1 Leydig cells.

3. Determine a mode of action by which Atrazine disrupts steroidogenesis.

32

CHAPTER 3

Forgacs AL, Ding Q, Jaremba RG, Huhtaniemi IT, Rahman NA and Zacharewski TR: BLTK1 murine
Leydig cells: A novel steroidogenic model for evaluating the effects of reproductive and
developmental toxicants. Toxicol Sci 2012, 127(2): 391-402.

33

CHAPTER 3
BLTK1 MURINE LEYDIG CELLS: A NOVEL STEROIDOGENIC MODEL
FOR EVALUATING THE EFFECTS OF REPRODUCTIVE AND
DEVELOPMENTAL TOXICANTS

ABSTRACT
Leydig cells are the primary site of androgen biosynthesis in males. Several
environmental toxicants target steroidogenesis resulting in both developmental and
reproductive effects including testicular dysgenesis syndrome. The aim of this study was to
evaluate the effect of several structurally diverse endocrine disrupting compounds (EDC) on
steroidogenesis in a novel BLTK1 murine Leydig cell model. We demonstrate that BLTK1 cells
possess a fully functional steroidogenic pathway that produces low basal levels of testosterone
(T), and express all the necessary steroidogenic enzymes including Star, Cyp11a1, Cyp17a1,
Hsd3b1, Hsd17b3 and Srd5a1. Recombinant human chorionic gonadotropin (rhCG) and
forskolin (FSK) elicited concentration- and time- dependent induction of cAMP, progesterone
(P) and T, as well as the differential expression of Star, Hsd3b6, Hsd17b3 and Srd5a1 mRNA
levels. The evaluation of several structurally diverse male reproductive toxicants including
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), atrazine, prochloraz, triclosan, monoethylhexyl
phthalate (MEHP), glyphosate and RDX in BLTK1 cells suggest different modes of action perturb
steroidogenesis. For example, prochloraz and triclosan anti-fungals reduced rhCG-induction of

34

T, consistent with published in vivo data, but did not alter basal T levels. In contrast, atrazine
and MEHP elicited modest induction of basal T but antagonized rhCG-mediated induction of T
levels, whereas TCDD, glyphosate and RDX had no effect on basal or rhCG-induction of T in
BLTK1 cells. These results suggest that BLTK1 cells maintain rhCG-inducible steroidogenesis and
are a viable in vitro Leydig cell model to evaluate the effects of EDCs on steroidogenesis. This
model can also be used to elucidate the different mechanisms underlying toxicant-mediated
disruption of steroidogeneis.

INTRODUCTION
Steroidogenesis is a potential target for drugs, chemicals, natural products and
environmental contaminants that adversely impact reproductive development and fertility (1,
2). The mechanisms involved in altering steroid levels remain largely unknown but could involve
disrupting enzyme expression and/or activity that affects testosterone (T) biosynthesis (3). For
example, fungicides, pesticides and phthalates elicit reproductive tract abnormalities,
compromise reproductive fitness, and cancer as a result of altering steroid levels (3-6).
Developmental exposure to high doses of endocrine disrupting chemicals (EDCs) has been
associated with testicular dysgenesis syndrome (TDS) in humans, comprised of adverse effects
including cryptorchidism, hypospadias, low sperm count and testicular cancer, such effects
have been characterized in rodents and wildlife species with similar etiology (7-10).
Current approaches to identify developmental and reproductive toxicants involve
examining offspring after dosing pregnant animals to maternally toxic levels (11). Apical
endpoints such as nipple retention, ano-genital distance, vaginal opening and balano-preputial
35

separation are assessed in addition to evaluating reproductive fitness (12, 13). To comply with
the European Commission’s Registration, Evaluation, Authorization, and Restriction of
Chemicals (REACH) initiative, approximately $2.9B and 41 million animals are required for
reproductive toxicity testing alone using current approaches (14-17). Such animal-based testing
is expensive, time consuming, generally descriptive, poses ethical concerns, and does not
significantly contribute to the elucidation of mechanisms (18-23). Furthermore, they are
contrary to the replacement, reduction and refinement of animal testing. Alternative shortterm assays such as the sliced testes assay and the human H295R adenal corticocarcinoma cellbased assay used in Tier 1 of the Endocrine Disruptor Screening Program, still require animals
or is based on adrenal steroidogenesis, which is not the primary site of sex hormone production
(24-27). Therefore, in vitro gonadal-based steroidogenesis assays that support ranking and
prioritization for further investigation are needed.
The “Toxicity Testing in the 21st Century: a Vision and a Strategy” report proposed
testing should be less reliant on whole animal studies with more effort devoted to systemsoriented, computational models developed using in vitro and in vivo data that could screen
chemicals, metabolites and mixtures for potential toxicity (19). In response, the U.S.
Environmental Protection Agency (USEPA), the National Institute of Environmental Health
Sciences National Toxicology Program, and the U.S. Food and Drug Administration have
initiated ToxCast, Tox21 and EDSP21 programs in collaboration with the National Institute of
Health Chemical Genomics Center, as well as other high-throughput service providers, to use in
silico and in vitro assays to screen chemicals for potential toxicity (28-30). These programs use
informatic platforms to integrate data from thousands of chemicals that are robotically
36

screened in diverse assays to develop predictive computational models of toxicity. Similarly, the
European Union has established ChemScreen and ReProTect to develop simple, rapid in vitro/in
silico screening systems for reproductive toxicants (www.chemscreen.eu; www.reprotect.eu)
(31). Pathways and over-represented gene functions identified in dose-response genomic and
high-throughput screening studies may also be used to determine points of departure in
provisional peer reviewed toxicity values (PPRTVs; http://hhpprtv.ornl.gov) for chemicals not
comprehensively

evaluated

by

the

Integrated

Risk

Information

System

(IRIS;

http://www.epa.gov/iris/index.html) or where there is minimal safety data. Despite this broad
spectrum of programs, which includes assays for cell viability, apoptosis, mitochondrial
function, receptor-mediated activity and DNA damage in human, mouse and rat cell lines, as
well as cell-free systems, none assess steroidogenesis in a high-throughput format (32).
In mammals, the induction of steroidogenesis has been thoroughly studied. Luteinizing
hormone (LH) secreted from the anterior pituitary induces gonadal steroidogenesis. LH binds Gprotein-coupled LH-hCG receptors (LHCGR) on the surface of Leydig cells. Subsequent
dissociation of the Gαs subunit stimulates adenylyl cyclase activity producing cAMP that induces
gene expression and enzyme activity increasing steroid biosynthesis. BLTK1 cells, isolated from
a testicular tumor that developed in a transgenic mouse expressing the mouse inhibin α
promoter/simian virus 40 T-antigen fusion gene (33, 34), retain functional LHCGR-mediated
steroidogenesis. They are stable over multiple passages, easily transfected and exhibit excellent
growth characteristics. In this study, recombinant human chorionic gonadotropin (rhCG) and
forskolin (FSK) are used as positive controls for the induction of steroidogenesis, as measured
by increases in progesterone (P), T and 17β-estraidiol (E2) levels in media. We investigate
37

murine BLTK1 Leydig cells as a novel model for evaluating the effects of chemicals on
steroidogenesis. Our results demonstrate that BLTK1 cells can be used to screen substances
that alter intracellular cAMP, steroidogenic gene expression and sex steroid levels.

MATERIALS AND METHODS
CELL CULTURE AND TREATMENT
Mouse Leydig BLTK1 (BLT-1 cells, clone K1) cells were isolated from a testicular tumor
that developed in a transgenic mouse expressing the mouse inhibin α promoter/simian virus 40
T-antigen fusion gene (34). Cells were maintained in phenol-red free Dulbecco’s modified eagle
medium nutrient mixture F-12 (DMEM/F-12 media; Invitrogen, Carlsbad, CA) with 10% fetal
bovine serum (FBS; Thermo Scientific HyClone, Logan, UT), 100 U/mL penicillin and 100 μg/mL
streptomycin (Invitrogen) and incubated under standard conditions (5% CO2, 37⁰C). For the
evaluation of steroidogenic enzyme and receptor expression, cells were grown to 80%
confluency and harvested without any treatment. For cAMP, progesterone (P), testosterone (T)
and estradiol (E2) determination, cells were grown to 80% confluency, transferred into 24-well
tissue culture plates (Sarstedt, Newton, NC) and incubated overnight. Cells were treated with
DMSO (Sigma, St. Louis, MO) vehicle, or 0.1, 0.3, 1, 3, 10, 30 or 100 ng/ml rhCG (obtained from
A.F. Parlow, NIDDK’s National Hormone & Peptide Program, Harbor-UCLA Medical Center,
Torrance, CA) or 0.1, 0.3, 1, 3, 10, 30 or 100 μM FSK (Sigma) and media was collected at
indicated times. Time course studies were conducted with DMSO vehicle, 3 ng/ml rhCG, or 10

38

μM FSK and media was collected at 1, 2, 4, 8, 12, 24 or 48 hrs. Gene expression studies used the
same study design, concentrations and time points with cells seeded into T-25 flasks (Sarstedt).
MTT CYTOTOXICITY ASSAY
BLTK1 cells in 96 well plates (Starstedt) were treated with 1, 3, 10, 30, 100, 300 or 600
ng/ml rhCG (positive control), 1, 3, 10, 30, 100, 300 or 600 μM FSK (positive control) or 1, 3, 10,
30, 100, 300 or 600 μM of test compound in triplicate, respectively. Media was aspirated after
24 hrs and replaced with 50 μL of fresh MTT reagent (5 mg/mL thiazolyl blue tetrazolium
bromide (Sigma) in phosphate buffered saline). Following a 3 hr incubation, MTT reagent was
aspirated and replaced with 150 μL DMSO. Cells were incubated for 2 hrs followed by
absorbance measurements at 595 and 650 nm (A595-A650) using an Emax precision microplate
reader (Molecular Devices, Sunnyvale, CA). Results are reported as percentage of control
calculated from the relative absorbance of treated versus DMSO controls where 100% indicates
no cytotoxicity.
WESTERN BLOTTING
Lysates were prepared in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM
EDTA, 0.5% Triton and protease inhibitor tablet (Roche Diagnostics, Indianapolis, IN)). Protein
concentration was determined spectrophotometrically by Bradford assay (Bio-Rad Laboratories,
Hercules, CA). Extracts (50 µg) were resolved on a 10% sodium dodecyl sulfate/polyacrylamide
gel and transferred to Immobilon membrane (Millipore, Billerica, MA). Membranes were
incubated with 1 µg/ml primary antibody [rabbit anti-StAR (Santa Cruz Biotechnology, Santa
Cruz, CA; Catalog No. 25806), rabbit anti-Cyp11a1 (Millipore; Catalog No. AB1244), goat anti-

39

Cyp17a1 (Santa Cruz; Catalog No. SC-46081), goat anti-3β-HSD (Santa Cruz; Catalog No. SC30820), goat anti-Srd5a1 (Santa Cruz; Catalog No. SC-20399) and rabbit anti-Cyp19a1 (Abcam,
Cambridge, MA; Catalog No. ab51924)] at 4oC overnight. Membranes were washed and
incubated with donkey anti-rabbit or donkey anti-goat IgG conjugated with horseradish
peroxidase (HRP; Santa Cruz; Catalog No. SC-2077 and SC-2056, respectively) for 1 hr. Enhanced
Chemiluminescence (Thermo Scientific Pierce, Rockford, IL) was used to visualize detected
protein.
ENZYME IMMUNOASSAYS
Enzyme immunoassay (EIA) kits for T (Cayman Chemical Company, Ann Arbor, MI; limit
of detection 6 pg/ml, cross-reactivity 27% for 5α-dihydrotestosterone and 19% for 5βdihydrotestosterone), E2 (Cayman Chemical Company; limit of detection 20 pg/ml, crossreactivity 14% for estradiol-3-glucuronide, 12% for estrone and 10% for estradiol 17glucuronide), and P (ALPCO Diagnostics, Salem, NH) were used according to the manufacturer’s
instructions. Intracellular cAMP was measured by EIA (Cayman Chemical Company; limit of
detection 3 pmol/ml cAMP, cross-reactivity 1.5% for cGMP) following hydrochloric acid
extraction. All kits consisted of 96-well pre-coated antibody plates, for which samples compete
for binding with conjugated hormone. Following incubation, plates were washed and measured
at 420 nm for cAMP, T and E2, or 405 nm for P, using an Emax precision microplate reader
(Molecular Devices). Standards (0.3 – 750 pmol/ml cAMP, 0 - 60 ng/ml P, 3.9 - 500 pg/ml T or
6.6 – 4,000 pg/ml E2) were used to generate a standard curve for quantification.

40

RNA ISOLATION AND GENE EXPRESSION
Total RNA was extracted from cell pellets using RNeasy Mini Kits (Qiagen, Valencia, CA)
according to the manufacturer’s protocol with an additional RNase-free DNase (Qiagen)
digestion. RNA was quantified at 260 nm (A260) and purity assessed using the A260/A280 ratio,
as well as by denaturing gel electrophoresis. First-strand cDNA was synthesized from RNA (1 µg)
using SuperScript II reverse transcriptase (Invitrogen) and anchored oligo-dT primer (Invitrogen)
as described by the manufacturer. For RT-PCR evaluation of steroidogenic enzyme and receptor
expression, the cDNA was used as template for PCR amplification with gene specific primers
(Table 1). Quantitative real-time PCR (QRTPCR) was used to quantify concentration- and timedependent expression of specific genes. Reactions in 96-well plates consisted of 30 µl, including
1 µl of cDNA template, 0.1 µM forward and reverse gene-specific primers, 3 mM MgCl2, 1.0
mM dNTPs, 0.025 IU AmpliTaq Gold, and 1 X SYBR Green PCR Buffer (Applied Biosystems,
Foster City, CA) using an Applied Biosystems PRISM 7500 Sequence Detection System.
Dissociation curve analysis assured single product amplification. To control for differences in
RNA loading, quality and cDNA synthesis, samples were standardized to the geometric mean of
three housekeeping genes: Actb, Gapdh and Hprt (35). Results were quantified using a standard
curve generated on the same 96-well plate and amplified by using purified cDNA product as
template specific for each gene (serial 10X dilutions from 108 to 101 copies). The slope of the
standard curve was used to assess amplification efficiency as described by the manufacturer
with all amplification efficiencies >90%. Fold changes were calculated relative to time-matched
vehicle. Relative expression was scaled such that time-matched vehicle control expression
equaled one for graphing purposes.
41

TABLE 1. PRIMER SEQUENCES FOR QRTPCR
Gene Symbol
Receptors

Steroidogenic
Enzymes

Housekeeping
Genes

RefSeq

Ahr
Ar
Era
Lhcgr
Pgr
Ppara
Pparg
Nr3c1 (Gr)
Nr5a1 (sf-1)
Nr5a2(Lrh-1)

Product
Size
155
117
1733
78
1604
107
198
112
166
730

Forward Primer

Reverse Primer

NM_013464
NM_013476
NM_007956
NM_013582
NM_008829
NM_011144
NM_011146
NM_008173
NM_139051
NM_030676

ACCAGAACTGTGAGGGTTGG
AAGCAGGTAGCTCTGGGACA
AGGGAAATCTTGAGCCCCTA
GACCAAAAGCTGAGGCTGAGA
ACGTCTTAGCCCCAGGTTTT
GTCCTCAGTGCTTCCAGAGG
TTTTCAAGGGTGCCAGTTTC
ACCTGCTGGGTAAACTGTGG
TACCCAGGGAGGAGGAGTCT
GCATGCCAAAAGAGCCTAAG

TCTGAGGTGCCTGAACTCCT
GAGCCAGCGGAAAGTTGTAG
AGCAAAATTAGCTGCCCTGA
CAATGTGGCCATCAGGGTAGA
CTGGAAGTCGCCGTAAAGAG
GGTCACCTACGAGTGGCATT
AATCCTTGGCCCTCTGAGAT
TTGCTATCGCACCACTGAAG
TGTCTCCTGCCCACTTCTCT
AAGAGGGTTACTGCCCGTTT

Cyp11a1
Cyp17a1
Cyp19a1
Hsd3b1
Hsd3b6
Hsd17b3
Star
Srd5a1

NM_019779
NM_007809
NM_007810
NM_008293
NM_013821
NM_008291
NM_011485
NM_175283

CCCATCCTCTTCAACTTCCA
GATCGGTTTATGCCTGAGCG
GACAGGCACCTTGTGGAAAT
GGTGCAGGAGAAAGAACTGC
TTTTTTTGAGGTATTGACAAGTATTTATTG
GAGTTGGCCAGACATGGACT
CTCGGTGCTTTAAGGTGAGC
ACCTGGTGTGTCCTGAAAGG

ACCTGGGCAGGTAATCACAG
CTCCGAAGGGCAAATAACTG
CGGATAAGTAATGCCCCAGA
ACAGCAGCAGTGTGGATGAC
TCCCCATTCAGAGCATGTATAGC
AGCTTCCAGTGGTCCTCTCA
GATTCCCGGGATGGTTCTAT
CCTGGGTTCCAACTTTAGCA

172
82
102
186
89
97
64
115

Hprt
Gapdh
Actb

NM_013556
NM_008084
NM_007393

AAGCCTAAGATGAGCGCAAG
GTGGACCTCATGGCCTACAT
GCTACAGCTTCACCACCACA

TTACTAGGCAGATGGCCACA
TGTGAGGGAGATGCTCAGTG
TCTCCAGGGAGGAAGAGGAT

104
125
123

42

DOSE-RESPONSE MODELING AND STATISTICAL ANALYSES.
ToxResponse uses particle swarm optimization to identify the best-fit across five model
classes: sigmoidal, exponential, linear, quadratic and Gaussian (36). The best fitting model was
then used to calculate EC50 values. All statistical analyses were carried out using SAS v9.1 (SAS
Institute, Cary, NC) by analysis of variance (ANOVA), with Dunnett’s or Tukey’s post hoc tests
for concentration-response and time course data, respectively. Differences between treatment
groups were considered significant when p < 0.05 relative to time-matched DMSO control.

RESULTS
STEROIDOGENIC ENYZME EXPRESSION IN BLTK1 CELLS
Steroidogenesis involves the conversion of cholesterol to progestagens (pregnenolone,
progesterone (P), 17α-hydroxypregnenolone and 17α-hydroxyprogesterone), followed by
further metabolism to androgens (dehydroepiandrosterone, androstenedione, testosterone (T)
and 5α-dihydrotestoeterone) and estrogens including estrone and 17β-estradiol (Figure 7).
Steroidogenic enzyme mRNA and protein were detected in BLTK1 cells by RT-PCR and/or
western blotting with all required steroidogenic enzymes expressed (Figure 8). In addition,
mRNA for several potential regulatory factors including luteinizing hormone G-coupled receptor
(LHGCR, data not shown), estrogen receptor (ER), androgen receptor (AR) and steroidogenic
factor 1 (SF-1), peroxisome proliferator-activated receptors (PPARα and PPARγ), the pregnane X

43

FIGURE 7. THE STEROIDOGENIC PATHWAY
Steroid hormones are biosynthesized from cholesterol. Cholesterol is mobilized and delivered
to the inner mitochondrial membrane by StAR, where it is cleaved by P450scc to produce
pregnenolone. Subsequent enzymatic events in the smooth endoplasmic reticulum include
metabolism by P450c17 and 3βHSD to produce progestagens (i.e., progesterone (P), 17αhydroxypregnenolone
and
17α-dhydroxyprogesterone).
Androgens
(i.e.,
dehydroepiandrosterone and androstenedione) are produced by P450c17, then converted to
testosterone (T) by 17βHSD and finally reduced to 5α-dihydrotestosterone (DHT) by 5αR1.
Androgen metabolism by P450arom also yields estrogens (i.e., estrone and 17β-estradiol (E2)).

44

FIGURE 8. STEROIDOGENIC ENZYME AND RECEPTOR EXPRESSION IN BLTK1 CELLS
Expression of steroidogenic enzymes and receptors in untreated BLTK1 cells was evaluated by RT-PCR and western blotting. mRNA
and/or protein expression was detected for all steroidogenic enzymes. PPARα, PPARγ, PXR, AhR, ERα, AR and SF-1 expression was
also confirmed. NA: antibody not available

45

receptor (PXR), and the aryl hydrocarbon receptor (AhR) were also detected. However, mRNA
for progesterone receptor (PR), glucocorticoid receptor (GR) or the liver receptor homologue 1
(LRH-1) was not detected in BLTK1 cells despite verification of RT-PCR primer specificity and
functionality in mouse Hepa1c1c7 cells (data not shown).
INDUCTION OF STEROIDOGENESIS BY FSK AND rhCG
Temporal profiles of intracellular cAMP as well as P and T levels in media were
evaluated in response to 3 ng/ml rhCG or 10 μM FSK by EIA (Figure 9). Intracellular cAMP was
induced by FSK at 30 min (~120 pmol/ml, ~10-fold) and by 1 hr in response to rhCG (635
pmol/ml, 60-fold). However, levels quickly diminished such that no intracellular cAMP was
detected by 8 hrs. Maximum P levels (200 ng/ml, 8-fold) were observed at 2 hrs in response to
rhCG and FSK, followed by a steady decline due to metabolism to androgens and estrogens. In
contrast, T levels gradually increased reaching a maximum of ~200 pg/ml (7-fold) at 48 hrs, with
significant increases detected as early as 1 hr post-treatment.
Concentration-dependent induction of intracellular cAMP and secreted P and T was
evaluated at 4 hrs when cAMP can still be detected (Figure 10). 17β-Estradiol (E2) was
evaluated at 48 hr (Figure 10), as it was not consistently detected at 4 hrs (data not shown).
cAMP, P and T were induced 25-, 10- and 4-fold, respectively, at 4 hrs, while E2 was induced ~4fold by 48 hr. The EC50 for cAMP induction was >24 ng/ml for rhCG and >29 μM for FSK.
Meanwhile, EC50 values of 1 ng/ml rhCG and 9 μM FSK were conserved for both P and T
induction, while E2 EC50s were 10 ng/ml for rhCG and 9 μM for FSK.

46

A

800

cAMP (pmol/ml)

600

*
*

DMSO
3 ng/ml rhCG
10 M FSK

*

400
200
150

*

*

*

100

*

50

*

0
0.5

1

2

4

8

12

24

48

Time (hrs)

B
Progesterone (ng/ml)

250

**

200

*
*

150

*
*

*

*

100

*
*

50

*

*

0
0.5

1

2

4

8

12

24

48

Time (hrs)

C
Testosterone (pg/ml)

250

*

*

200

*

150

*

50

*

*

*

100

*

*

*

*
*

*

0
0.5

1

2

4

8

12

24

48

Time (hrs)

FIGURE 9. TEMPORAL rhCG- AND FSK-INDUCED cAMP, P AND T LEVELS.
47

FIGURE 9 (cont’d)
Intracellular cAMP (A) as well as P (B) and T (C) levels in media were evaluated in response to 3
ng/ml rhCG (light bars) or 10 μM FSK (dark bars) relative to DMSO vehicle controls (white bars).
Both treatments elicited similar temporal profiles consisting of maximal cAMP levels reached
early at 0.5-1 hr followed by decrease and was undetectable by 8 hr, while P reached maximum
induction at 2 hrs followed by a gradual decrease over time. In contrast, T levels steadily
increased throughout the time course. Data represent mean ± standard error of 3 replicates, *p
< 0.05 vs. DMSO controls.

Intracellular cAMP levels are not only regulated by synthesis but also degradation, which
is regulated by cyclic nucleotide phosphodiesterase enzymes (PDEs) (37). The PDE inhibitor
IBMX maximizes cAMP levels in order to further induce steroidogenesis. However, IBMX cotreatment with rhCG or FSK did not increase T levels further, albeit rhCG and FSK potencies
were greater (Figure 11; T EC50 with and without IBMX by FSK: 0.1 μM vs. 9.4 μM and rhCG: 0.1
ng/ml vs. 0.9 ng/ml, respectively).
rhCG- AND FSK-ELICITED STEROIDOGENIC GENE EXPRESSION
The effect of rhCG and FSK on the gene expression of steroidogenic enzymes including
steroidogenic acute regulatory protein (Star) were evaluated by QRTPCR at 24 hrs. rhCG elicited
concentration-dependent induction of Star, Hsd3b6, Hsd17b3 and Srd5a1 mRNA levels (Figures
12-14). In contrast, only Star and Cyp17a1 mRNA exhibited concentration-dependent induction
by FSK at 24 hrs. Star, carrying out the rate-limiting step in steroidogenesis, was induced ~20fold by rhCG and ~10-fold by FSK. Temporal gene expression (Figures 12-14) also revealed
differences in the induction kinetics of Star, Hsd3b6 and Cyp19a1 by rhCG and FSK. Most
notably were differences in the induced levels of Hsd17b3 and Srd5a1. These differences likely
48

B

500

rhCG (EC 50: >24.0 ng/ml)
FSK (EC 50: >29.1 M)

400

*

300

*

*

200

*

*

*

100

*

*
0

*

Progesterone (ng/mL)

cAMP (pmol/ml)

A

0

0.1 0.3

1

3

10

250

FSK (EC 50: 8.5 M)

200

rhCG (EC 50: 9.9 ng/ml)

*

FSK (EC 50: 9.4 M)

200
150

*

D

*

100
50
0

0

0.1 0.3

1

3

*

*

50
0

0.1 0.3

*

1

3

10

30 100

Dose

Testosterone (pg/mL)

Estradiol (pg/ml)

250

*

*

100

Dose

C

**

*

*

*

150

0

30 100

rhCG (EC 50: 1.0 ng/ml)

10

250

FSK (EC 50: 9.3 M)

200

*

*

100

*

*

**

*

*

150

*

*

50
0

30 100

rhCG (EC 50: 1.0 ng/ml)

0

0.1 0.3

Dose

1

3

10

30 100

Dose

FIGURE 10. CONCENTRATION-DEPENDENT INDUCTION OF cAMP, P, T AND E2 BY rhCG AND FSK
Intracellular cAMP concentrations (A) and secreted P (B) and T (C) levels in media induced by rhCG (ng/ml; light bars) and FSK (μM;
dark bars) at 4 hrs. E2 (D) levels in media were measured 48 hrs after treatment. EC 50 values were calculated by the ToxResponse
modeler. Data represent mean ± standard error of 3 replicates, *p < 0.05 vs. DMSO controls.
49

B
*

*

400

*

300
200
0

Testosterone (pg/mL)

*

*

*

100

C

800

- IBMX
+ IBMX

cAMP (pmol/ml)

1500
1400
1300
1200
500

0

0.1

1

10

rhCG (ng/ml)

250
200

*

*

150

*

*
*

100
50
0

0

0.1

1

*

150

10

50

*
0

rhCG (ng/ml)

0.1

1

10

100

Forskolin (M)
- IBMX (EC 50 9.4 M)
+ IBMX (EC 50 0.09 M)

200
150

*
*

100

* *

* *

10

100

*

50
0

100

*

100

250

*

*

600
200

D

- IBMX (EC 50 0.9 ng/ml)
+ IBMX (EC 50 0.06 ng/ml)

*

- IBMX
+ IBMX

700

0

100

Testosterone (pg/mL)

cAMP (pmol/ml)

A

0

0.1

1

Forskolin (M)

FIGURE 11. EFFECT OF THE PDE INHIBITOR IBMX ON rhCG- AND FSK-ELICITED INDUCTION OF T
Concentration-dependent induction of cAMP (A, B) and T (C, D) by rhCG (0.1 – 100 ng/ml) or FSK (0.1 – 100 μM) alone (dark bars) or
in the presence of 0.2 mM IBMX (light bars) for 4 hrs. IBMX did not affect T induction, cAMP levels were increased, and the potency
of rhCG and FSK were enhanced. Data represent mean ± standard error of 3 independent replicates, *p < 0.05 vs. DMSO controls.
50

25

15

*

20

rhCG (ng/ml)

15

FSK (M)

*
*

10

*

*

30

100

* *
*

5
0
0.1

0.3

1

3
10
Dose

*

*
*

*
5

*
*

*

*

1

2

4
8
12
Time (hrs)

Fold Change

*

*

*

*

*

48

*

10

*
5

*
*

*

0
0.3

24

Cyp17a1

4

0.1

*

15

Cyp17a1
Fold Change

10 M FSK *

10

0

6

2

Star
3 ng/ml rhCG

*

Fold Change

Fold Change

Star

1

3
10
Dose

30

*
*

0

100

1

2

4
8
12
Time (hrs)

24

48

FIGURE 12. DIFFERENTIAL GENE EXPRESSION OF PROGESTAGEN BIOSYNTHETIC ENZYMES
The concentration-dependent (24 hrs, left column) and temporal (right column) effects of rhCG (ng/ml, light bars) and FSK (μM, dark
bars) on Star and Cyp17a1 mRNA levels examined using QRTPCR. Cyp11a1 mRNA expression was not affected by treatment (data
not shown). Data represent mean ± standard error of 3 replicates, *p < 0.05 vs. DMSO controls.
51

6

6

Hsd3b6

4

*

*

*
*

*

*

2

Fold Change

Fold Change

Hsd3b6

0
0.1

0.3

1

3
10
Dose

30

1

6

*
Fold Change

*
*

4
2

* *

2

30

Hsd17b3

*

*

0

100

8
Fold Change

4

2

Hsd17b3

4
8
12
Time (hrs)

24

48

*
*

20

*

10

*
*

0
0.1

0.3

1

3
10
Dose

30

0

100

1

2

4
8
12
Time (hrs)

24

48

FIGURE 13. DIFFERENTIAL GENE EXPRESSION OF ANDROGEN PRODUCING ENZYMES
The concentration-dependent (24 hrs, left column) and temporal (right column) effects of rhCG (ng/ml, light bars) and FSK (μM, dark
bars) on Hsd3b6 and Hsd17b3 mRNA levels examined using QRTPCR. Data represent mean ± standard error of 3 replicates, *p < 0.05
vs. DMSO controls.
52

6

6

*

Srd5a1

*

4

*

Fold Change

Fold Change

Srd5a1
*

2

*

4

*
*
2

*
*

0

0.1

0.3

1

3
10
Dose

30

0

100

6

Fold Change

Fold Change

2

0.3

4
8
12
Time (hrs)

24

48

Cyp19a1

4

0.1

2

6

Cyp19a1

0

1

1

3
10
Dose

30

4

*
*
2

0

100

1

2

4
8
12
Time (hrs)

24

48

FIGURE 14. DIFFERENTIAL GENE EXPRESSION OF T METABOLIZING ENZYMES
The concentration-dependent (24 hrs, left column) and temporal (right column) effects of rhCG (ng/ml, light bars) and FSK (μM, dark
bars) on Srd5a1 and Cyp19a1 mRNA levels examined using QRTPCR. Data represent mean ± standard error of 3 replicates, *p < 0.05
vs. DMSO controls.
53

result from different pathway activation. FSK directly induces cAMP levels by activating adenylyl
cyclase whereas rhCG induction via the LHCGR activates adenylyl cyclase and can also activate
alternate signal transduction pathways such as the release of arachidonic acid and its
metabolites (38, 39). Furthermore, Cyp17a1 induction was only seen with FSK, while rhCG
inhibited expression at 2 and 24 hrs. There were negligible effects on Cyp11a1 and Hsd3b1 in
both concentration-response and temporal evaluation (data not shown). More detailed studies
are warranted to elucidate the mechanisms responsible for the differential effects of rhCG and
FSK on steroidogenic gene expression.
EFFECTS OF EDCs ON TESTOSTERONE LEVELS
The effect of selected EDCs reported to elicit reproductive toxicity (Table 2) was
assessed in BLKT1 cells by evaluating T levels. They include the triazine herbicide Atrazine (40,
41), the broad spectrum herbicidal active ingredient glyphosate used in commercially available
Round-up (42, 43), the conazole anti-fungal Prochloraz (44, 45), the anti-fungal/anti-bacterial
Triclosan (46, 47), and the diethylhexyl phthalate (DEHP) metabolite monoethylhexyl phthalate
(MEHP) (48-50). None of these chemicals elicited significant cytotoxicity in the concentration
range examined (Figure 15). Compounds were evaluated for concentration-dependent effect on
basal T levels and at a single co-treatment concentration to investigate potential interactions
with rhCG-mediated stimulation of T levels (Figure 16).
T levels were induced 2-fold (60 pg/ml) and 10-fold (250 pg/ml) by Atrazine and MEHP,
respectively. No other examined chemical altered basal T levels. However, steroidogenesis in
Leydig cells is stimulated by LH via the LHCGR during late gestation first measurable at
gestational day (GD) 15 and peaking at GD 18 in the mouse (51), and throughout gestation in
54

TABLE 2. REPORTED EVALUATIONS OF EDCs THAT ALTER T LEVELS

Common Name

Atrazine

Chemical Name

2-chloro-4-ethylamino6-isopropylamino-striazine

CAS #

1912-24-9

Molecular
Weight
(g/mol)

Reproductive Toxicity
(References)

215.68

decreased serum T,
dysregulation of
steroidogenesis
(40, 41)

Triclosan

2,4,4’-trichloro-2’hydroxydiphenyl ether

3380-34-5

289.54

Prochloraz

N-propyl-N-(2,4,6trichlorophenoxy)ethylimidazole-1carboxamide

67747-095

376.67

anti-androgenic,
decreased T production by
Leydig cells, decreased
serum T levels
(46, 47)
decreased serum T levels,
AR antagonism, altered
androgen-dependent gene
expression
(44, 45)

321.97

decreased intratesticular T,
inhibition of steroidogenic
gene expression
(52-54)

1071-83-6

169.07

inhibition of steroidogenic
enzymes expression,
decreased T production
(42, 43)

2-(2ethylhexoxycarbonyl)
benzoic acid

4376-20-9

278.35

decreased serum T levels,
Leydig cell hyperplasia
(49, 50)

1,3,5-trinitroperhydro1,3,5-triazine

121-82-4

222.12

altered reproductive
capacity (55-57)

Dioxin
(TCDD)

2,3,7,8tetrachlorodibenzo-pdioxin

Glyphosate

N-(phosphonomethyl)
glycine

Monoethylhexyl
Phthalate
(MEHP)

RDX

1746-01-6

55

150

Triclosan

120
90
60

150

mEHP

120
90
60

100
300
600

1
3

Atrazine (M)

% of Control

60

0
Prochloraz (M)

% of Control

90

60

0

10
3
0
100
300
600

1
3
% of Control

TCDD

120
90
60

Glyphosate (M)

mEHP (M)

Triclosan (M)

FIGURE 15. MTT EVALUATION OF EDC CYTOTOXICITY

56

100
300
600

10
30

100
300
600

100
300
600

0

10
30

0
1
3

0
10
30

0

1
3

30

100
300
600

30

10
30

30
1
3

30

1
3

% of Control

150

90

30

FSK (M)

Glyphosate

120

60

120

30

rhCG (ng/ml)
150

90

Atrazine

10
30

0
100
300
600

0
10
30

30
1
3

30

% of Control

60

120

100
300
600

60

90

150

Prochloraz

10
30

90

120

% of Control

120

150

FSK

1
3

150

rhCG

% of Control

% of Control

150

TCDD (nM)

FIGURE 15 (cont’d)
BLTK1 cell viability following 24 hr treatment with rhCG (A), FSK (B), prochloraz (C), ATR (D),
glyphosate (E), triclosan (F), MEHP (G) and TCDD (H) using MTT assay. Cells exhibited >90%
viability at concentrations used for subsequent effects on steroidogenesis. Data represent
mean ± standard error of 3 independent replicates.

humans, with a similar programming window for androgen-dependent masculinization
extrapolated between humans and rodents (58)). LH stimulation is also crucial during puberty
and adulthood to stimulate T biosynthesis to support Sertoli cell spermatogenesis.
Consequently, co-treatment studies with 3 ng/ml rhCG were conducted to mimic Leydig cell
stimulation. Atrazine (300 μM) inhibited rhCG-induced T levels ~30% (rhCG alone ~100 pg/ml T
vs. Atrazine + rhCG ~70 pg/ml T). Similarly, 100 μM MEHP inhibited rhCG-elicited T induction
~20% (rhCG alone ~100 pg/ml vs. MEHP co-treatment ~80 pg/ml). In contrast, Prochloraz did
not significantly alter basal T levels, but co-treatment decreased rhCG induced T to control
levels (rhCG ~100 pg/ml vs. prochloraz + rhCG ~50 pg/ml vs. DMSO ~40 pg/ml). Similarly, 30 μM
Triclosan had no effect, yet co-treatment decreased rhCG-elicited T induction ~25% (rhCG alone
~100 pg/ml vs. triclosan + rhCG co-treatment ~75 pg/ml). Glyphosate, TCDD and RDX did not
induce, or alter rhCG induction of T.

57

100

100

100

*#

50

#

0

*#

50

58

C
G

C
G

rh
+
R

N
TC

PC

FIGURE 16. EFFECTS OF EDCS ON T LEVELS

rh

rh
C
G
+

rh

C
G

N
TC

SO
D
M

G

*#

0

LZ

+

rh
C

rh
C
G

LZ
PC

SO

0

*

100

AT

0

*

300 M Atrazine

150

SO

#
#

30  M Triclosan

150

D
M

100

Dose (M)

Testosterone (pg/mL)

*

D
M

20

10
30

3

1

0
0.
1
0.
3

10
30

3

1

0.
1
0.
3

30  M Prochloraz

50

40

Dose (M)

Testosterone (pg/mL)

Testosterone (pg/mL)

Dose (M)
150

* *

*

0

0

0

0

20

*

3
10
30
10
0
30
0
60
0

20

40

60

R

40

60

80

AT

60

80

Atrazine

1

80

Triclosan

Testosterone (pg/mL)

Prochloraz

Testosterone (pg/mL)

Testosterone (pg/mL)

100

59
G

100

rh
C

0
50

+

#

G

*

LY

*

rh
C

150 100 nM TCDD

G

Dose (nM)

LY

3
10
30
10
0
30
0
60
0

0

1

20

G

40

0

60

M
SO

80

Testosterone (pg/mL)

TCDD

Testosterone (pg/mL)

10
10
0

1

0.
1

100

D

0
50

G

*#
100

rh
C

*#

G

*

+

150 100 M mEHP

D
D

Dose (M)

rh
C

0

D
D

20

TC

40

0. 0
00
1
0.
01

60

Testosterone (pg/mL)

80

SO

0

0

200
100

Testosterone (pg/mL)

30

10

30

*

M

G

C

100

rh

G

mEHP

D

+

rh
C

10

3

300

TC

EH
P

50

m

P

EH

0

Testosterone (pg/mL)
250

m

SO

M

D

Testosterone (pg/mL)

FIGURE 16 (cont’d)
100

Glyphosate

80

60

40

20
0
Dose (M)

150 300 M Glyphosate

*
*

#

0

FIGURE 16 (cont’d)
The concentration-dependent effects of Prochloraz, Atrazine, glyphosate, Triclosan, MEHP and
TCDD on T levels were evaluated after 4 hrs (top row, black bars) relative to DMSO controls
(white bars). The effect of 30 μM Prochloraz, 300 μM Atrazine, 300 μM glyphosate, 30 μM
Triclosan, 100 μM MEHP and 100 nM TCDD on rhCG-induced (3 ng/ml rhCG) T levels 4 hrs after
treatment (bottom row). RDX had no effect on basal or rhCG induced T levels (data not shown).
Data represent mean ± standard error of 3 replicates, *p < 0.05 vs. DMSO controls, #p < 0.05 vs.
rhCG.

DISCUSSION
The in vitro evaluation of steroidogenesis is important for screening potential
reproductive and developmental toxicants (1, 2). Current models include human H295R
adrenacarcinoma cells and several rodent Leydig cell lines including MA-10s, mLTCs and R2Cs.
Human H295R cells (ATCC CLR-2128), isolated from a pluripotent adenocortical carcinoma in a
48 year old black female, produce detectable basal aldosterone, cortisol, androgens and E2
(59), and are part of the Endocrine Disruptor Screening Program to screen for effects on T and
E2 biosynthesis (60). However, H295R cells do not express functional ACTH receptors (a
member of the cell surface 7-transmembrane domain superfamily of G-protein coupled
receptors), and therefore steroidogenesis must be stimulated using FSK. Moreover, besides
glucocorticoids and mineralocorticoids, the adrenal cortex normally only produces
dehydroepiandrosterone (DHEA) sulfate and androstenedione (precursors of testosterone) and
does not express the 17-beta-hydroxysteroid dehydrogenase necessary for T biosynthesis (61).
As such it has been suggested that H295R cells have the characteristics of zonally
undifferentiated human fetal adrenal cells (59). Only Leydig cells synthesize T and E2 directly
60

from cholesterol (6). In ovarian steroidogenesis, theca and granulosa cells produce P, while only
theca cells metabolize P to T, and subsequent aromatization to estrogens or 5α reduction to
DHT occurs in granulosa cells (62). Steroidogenic theca cell line development has not been
successful and as such rodent models are of Leydig cell origin such as mouse MA-10 and mLTC-1
models as well as rat R2C cells. MA-10 and mLTC1 cells were established from transplantable
M548OP Leydig tumors carried in C57Bl/6 mice that exhibit P induction by rhCG, but lacked the
enzymes required for subsequent metabolism to T (63-65). However, recent studies report
mLTC-1 cells can produce low levels of androgens, with 100 IU/ml hCG eliciting a maximum of
600 pmol/well P and 10 pmol/well T in a similar 24-well plating format as used herein (66).
These levels are lower than those observed in resting, unstimulated BLTK1 cells. Rat R2C cells
lack functional LHCGR expression and are not rhCG inducible, but exhibit high basal
steroidogenesis activity (67).
In contrast, BLTK1 cells produce progestagens, androgens and E2 in response to trophic
hormone stimulus via LHCGR, the 7-transmembrane G-protein coupled cell surface receptor,
similar to T biosynthesis in vivo following LH secretion from the anterior pituitary (37). Although
human steroidogenesis prefers the ∆5 pathway over the ∆4 pathway in mice, the enzymatic
steps are highly conserved facilitating extrapolation of murine BLTK1 results to humans (6).
Gestation is the critical period of exposure to EDCs that targets fetal Leydig cells causing
reproductive and development toxicity. Fetal Leydig cells are observed as early as GD12.5 in
mice or week 6 in humans (34, 51). Murine fetal Leydig cells do not express measurable LHCGR
until GD 15.5, and murine fetal T production is LH-independent (68). However, human fetal
Leydig cells do express LHCGR and are dependent on LH- and hCG-stimulation of
61

steroidogenesis for masculinization during development (51). Though BLTK1 cells are not
murine fetal Leydig cells, they retain rhCG-inducible steroidogenesis characteristic of adult
murine Leydig cells and both fetal and adult human Leydig cells. The ability to evaluate rhCGinducible steroidogenesis provides a unique functional feature that is important for screening
EDC interactions that may occur during human fetal and adult exposure.
During gestation, LH stimulates Leydig cell proliferation and T production, while during
puberty and adulthood LH stimulates T biosynthesis to indirectly support spermatogenesis by
contributing to Sertoli cell stimulation. rhCG also promotes cell proliferation or apoptosis, but
no such effects were observed on BLTK1 cells (data not shown). rhCG is used clinically as an LH
substitute to treat LH deficiency and in research to stimulate steroidogenesis (69), due to its
longer half-life (~24 hrs compared to ~4 hrs for LH) (70). Like LH, rhCG-induces steroidogenesis
via the LHCGR, and initiates the same signaling pathways resulting in a robust in vivo and in
vitro steroidogenic response (69, 71, 72). Interestingly, LH/rhCG levels required for maximum
cAMP synthesis are 15-fold higher than the level needed for maximal T production (37),
suggesting that minimal changes in cAMP can affect steroidogenesis. Our results demonstrate
that IBMX co-treatment increased rhCG potency consistent with the potentiation of cAMP
induction with no effect on maximum T induction, suggesting maximum steroidogenic capacity
has been achieved. It also highlights the important role of phosphodiesterases in the regulation
of steroidogenesis. For example, Leydig cells isolated from phosphodiesterase 8A (PDE8A) null
mice are more sensitive to LH induction but induced T levels were comparable in wild type and
PDE8A null mice (73). These data are consistent with IBMX-treated BLTK1 cells and the
enhanced potency of rhCG and FSK but comparable induced T levels.
62

The acute effect of LH/rhCG is activation of cAMP signaling that induces rapid
mobilization of cholesterol stores and induction of steroidogenic enzyme activity. However,
long term effects include the induction of steroidogenic gene expression (37). Differential gene
expression is necessary as increased steroidogenesis in response to trophic hormone requires
de novo protein synthesis, such as StAR induction (67). Consequently, in addition to effects on T
biosynthesis, comprehensive screening should also include effects on cAMP as well as changes
in steroidogenic gene expression.
BLTK1 cells express all the necessary enzymes required for T biosynthesis and
metabolism, as well as LHCGR and several other regulators of steroidogenesis. rhCG and FSK, a
direct activator of adenylyl cyclase, induce steroidogenesis in BLTK1 murine Leydig cells as
indicated by time-dependent increases in cAMP, P, T, and E2 levels, and steroidogenic gene
expression. Robust induction of intracellular cAMP occurred rapidly after rhCG and FSK
treatment. Overall, rhCG and FSK elicited comparable induction of intracellular cAMP,
steroidogenic enzyme mRNA expression, and altered P, T and E2 levels (with and without
IBMX). Furthermore, BLTK1 cells exhibited robust and consistent induction of steroidogenesis
for up to 30 passages (data not shown) while H295R cell assays are limited to passages 5-10 (26,
60).
Intracellular cAMP, as well as P, T and E2 levels in media, exhibited concentrationdependent induction following rhCG and FSK treatment in the absence and presence of IBMX.
Selected EDCs also altered steroidogenesis in BLTK1 cells consistent with reports in other
models. For example, prochloraz, a residential and commercial imidazole fungicide that inhibits
C17-20 lyase (Cyp17a1) and aromatase (Cyp19a1), as well as antagonizes AR and ER activity (3,
63

44, 74), completely blocked T induction by rhCG. However, prochloraz alone did not alter basal
T levels most likely due to the low basal level of production in BLTK1 cells. In contrast,
prochloraz reduced basal T levels in H295R adrenal cells, which have relatively high basal T
levels (26). Similarly, ATR, which decreases circulating T levels and antagonizes rhCG-induced T
production in primary Leydig cells (40, 75-78), inhibited rhCG-induced T levels ~30% in BLTK1
cells. However, atrazine alone induced modest increases in T levels, consistent with results in
rat primary Leydig cell cultures (40). This modest induction of basal T is consistent with atrazine
acting as a phosphodiesterase inhibitor (79). Triclosan did not alter basal levels when
administered alone but decreased rhCG-induced T levels in co-treatment studies in agreement
with its reported anti-androgenic activity (46, 47, 80). Despite the confirmed expression of the
aryl hydrocarbon receptor (AhR) in BLTK1 cells, and previous studies in vivo suggesting AhRmediated inhibition of steroidogenic expression and reduction of fetal intratesticular T levels
(52-54), TCDD had no effect on T levels in BLTK1 cells. A recent study in mouse primary Leydig
cells has also demonstrated that TCDD does not alter Leydig cell steroidogenesis (81).
Collectively, these differences in agonist and antagonist profiles suggest EDC-elicited effects on
steroidogenesis use various modes of action that warrant further investigation. Moreover,
these results highlight the need for more comprehensive screens that not only monitor effects
on T and E2 levels, but also examine effects on steroidogenic enzyme expression and activity, or
even alternative signaling cascades in order to elucidate EDC-specific adverse outcome
pathways that are relevant to humans and wildlife.
BLTK1 cells can also be used to evaluate the concentration-dependent effects of specific
metabolites. The mono-ester metabolite, MEHP, is thought to be responsible for DEHP-elicited
64

in vivo reproductive and developmental toxicity in rodents at high doses. MEHP increased T
levels in BLTK1 cells, consistent with results obtained in mouse mLTC-1 Leydig cells (49).
Furthermore, the inhibition of rhCG-induced T levels by MEHP is also reported in mouse MA-10
Leydig cell line, consistent with the proposed antagonistic effect of phthalates on T levels (48,
82). Although, RDX elicits reproductive and developmental effects in zebra fish, earthworms
and crickets, no effects on T levels were observed in BLTK1 cells suggesting RDX may not elicit
steroidogenesic effects in mammalian systems or that other indirect interactions may be
involved (55, 56). Glyphosate also had no effect on T levels in BLTK1 cells, despite previous
studies showing effects on steroidogenesis in rat and mouse models (42, 43). However, these
studies evaluated Roundup formulations, which are complex mixtures with glyphosate as the
active pesticide ingredient. Glyphosate alone only has marginal effects compared to the
Roundup formulation (43), suggesting formulations as well as active and inert components
should be examined. Nevertheless, more comprehensive temporal and concentration-response
follow-up studies are required that include assessing cAMP, P, T, E2 levels and gene expression
prior to any conclusions regarding the steroidogenic effects of these chemicals.
In summary, current protocols and models are inadequate to screen the universe of
chemicals, metabolites and mixtures that may alter steroidogenesis (1, 2, 4). BLTK1 cells are a
novel complementary rhCG-inducible, Leydig-based model that can be used to assess effects on
steroidogenic gene expression, intracellular cAMP, and P, T and E2 levels in media. Their
consistent response characteristics and inducibility over 30 passages also make this cell line
attractive for high-throughput screening. Comprehensive characterization of effects on
intermediate steroid biosynthesis, including pregnenolone, 17-hydroxyprogesterone, DHEA,
65

androstenedioine, estrone and DHT, as well as the differential expression of steroidogenic
enzymes will also facilitate the elucidation of modes of action relevant to adverse outcome
pathways in humans and other relevant species.

66

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67

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CHAPTER 4

Forgacs AL, D’Souza ML, Huhtaniemi IT, Rahman NA and Zacharewski TR: Triazine herbicides
and their chlorometabolites alter steroidogenesis in BLTK1 murine Leydig cells. Toxicol Sci
2013, 134(1): 155-167.

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CHAPTER 4
TRIAZINE HERBICIDES AND THEIR CHLOROMETABOLITES ALTER
STEROIDOGENESIS IN BLTK1 MURINE LEYDIG CELLS

ABSTRACT
The triazine herbicides, Atrazine (ATR), Simazine (SIM), Propazine (PRO) and
Terbuthylazine (TBA) as well as their chlorinated metabolites have been implicated in the
etiology of testicular dysgenesis by altering steroidogenesis. To further investigate their effects
on testosterone biosynthesis, BLTK1 cells were used to evaluate steroid hormone levels as well
as genome-wide gene expression. BLTK1 cells are a novel murine Leydig cell line possessing an
intact steroidogenic pathway with constitutive low basal testosterone (T) levels that can be
induced by recombinant human chorionic gonadotropin (rhCG). Triazines (ATR, SIM, PRO and
TBA) and their chlorometabolites (DEA, DIA and DACT) induced concentration-dependent (1, 3,
10, 30, 100, 300 and 600 µM) increases in progesterone (P) and T levels relative to DMSO at 24
hrs. Temporal analysis (300 µM at 1, 2, 4, 8, 12, 24 or 48 hrs) elicited comparable P and T
profiles by all compounds with varying efficacies (ATR > TBA > PRO > DEA > DIA > DACT >> SIM)
that were similar to rhCG. ATR and TBA elicited time- and concentration-dependent induction
of Star, Hsd3b6 and Hsd17b3 mRNA levels while Hsd3b1, Cyp17a1 and Srd5a1 mRNA expression
was repressed. PRO elicited similar, albeit weaker effects while SIM had negligible effects
consistent with their induction of P and T levels. Whole-genome microarrays identified 797
differentially regulated genes elicited by 300 µM ATR, primarily at later time points (> 12 hr)
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with over-represented functions associated with steroidogenesis and cholesterol metabolism.
These results indicate that changes in P and T levels can be partially attributed to triazineelicited alterations in steroidogenic gene expression.

INTRODUCTION
Chloro-s-triazines are widely used broad-spectrum herbicides that block photosynthesis
by inhibiting electron transfer at the reducing site of photosystem II in the chloroplasts of
broadleaf and grassy weeds (1, 2). Atrazine (ATR), simazine (SIM), propazine (PRO) and
terbuthylazine (TBA) are triazine herbicides registered for use in the United States: ATR for
agricultural crops and grasses, SIM for grasses, fruit and agricultural crops, PRO for indoor
greenhouse use, and TBA as a slimicide for ornamental fountains (3-6). PRO and TBA use is
restricted in the U.S., and therefore human exposure is negligible (3, 7). In contrast, ATR and
SIM are widely distributed agricultural herbicides with potential exposure to both the parent
triazine and its major metabolites (7). ATR and SIM are biotransformed in the environment by
microbial and fungal degradation, as well as photodegradation, to form three major chlorinated
metabolites (chlorometabolites): des-ethyl-s-Atrazine (DEA), des-isopropyl-s-Atrazine (DIA) and
diaminochlorotriazine (DACT; Figure 2) (8, 9). Furthermore, cytochrome P450 metabolism
produces the same chlorometabolites detectable in urine, serum and various tissues of exposed
animals (10-12).
ATR, SIM, DEA, DIA and DACT persist in the environment and are mobile in soil and
ground water (13). ATR and SIM have been reported above the U.S. maximum contaminant
levels in groundwater (7, 14). For example, ATR and its metabolites were the most common
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pesticide contaminant detected in water, found in 75% of the streams and 40% of the
groundwater samples tested in agricultural and urban areas in the U.S. between 1992 and 2001
(15). Groundwater contamination resulted in the cancelation of ATR and SIM registrations and
cessation of their use in most of Europe (16-18) with the European Union approving the use of
TBA as an agricultural alternative (19). In contrast, the Environmental Protection Agency (EPA)
has approved the continued use of triazines in the U.S. (6).
ATR, SIM and PRO as well as their major chlorometabolites (DEA, DIA and DACT) are
categorized as a Common Mechanism Group (CMG), based on their disruption of the
hypothalamic-pituitary-gonadal (HPG) axis (7, 20). CMGs are defined for cumulative risk
assessment as two or more chemicals that cause a common toxic effect(s) using the same
sequence of major biochemical events, interpreted as mode of action (21). Moreover, the
major chlorometabolites are classified as toxicologically equipotent to the parent triazines by
the CMG (7). Therefore, all triazines and their major metabolites require further examination
regarding potential endocrine disrupting activities.
The endocrine disrupting effects of triazines and their metabolites have been
extensively studied in mammalian models and aquatic species. In amphibians, reptiles, and fish,
ATR reduced reproductive capacity, disrupted steroidogenesis and altered gonadal
development resulting in hermaphroditism and male feminization (22-26). Effects on
steroidogenesis in amphibians and fish suggest disruption of testosterone (T) biosynthesis may
underlie the effects of ATR in males (23, 24, 27). Rodent studies also report that ATR disrupts
male and female reproductive tract development and function by altering steroid levels that
affect estrus, androgen-dependent tissue development, puberty onset, reproductive
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senescence and overall fertility (28-31). PRO, SIM, DEA, DIA and DACT elicit similar effects in
rodents that are generally characterized as ATR-like with a limited number of studies examining
TBA (32-35). Collectively, these results suggest that triazines and their chlorometabolites target
the HPG axis (30, 36).
This study examines the effects of ATR, SIM, PRO and TBA as well as their primary
chlorometabolites (DEA, DIA and DACT) on steroidogenesis in BLTK1 murine Leydig cells. Leydig
cells are the primary site for T biosynthesis required for proper male development and
reproduction. Briefly, luteinizing hormone (LH) stimulation induces the expression and
activation of steroidogenic enzymes that increases progesterone (P) levels and metabolism to T
(37, 38). BLTK1 cells, a subclone of a murine Leydig cell line isolated from a testicular tumor in a
inhibin /SV40Tag transgenic mouse, produce low basal P and T levels which are inducible by
recombinant hCG (rhCG) and forskolin (39, 40). Unlike other murine Leydig cell lines such as
BLTC-1 (41), MA-10 (42) and mLTC-1 (43) that do not proceed beyond P biosynthesis, BLTK1
cells produce appreciable basal levels of T which can be induced by rhCG, an LH analog (40). In
this study, we have evaluated the time- and concentration-dependent effects of ATR, SIM, PRO
and TBA and their major chlorometabolites on P and T production and gene expression in
BLTK1 cells. Our results confirm that changes in P and T levels can be partially attributed to
triazine-elicited alterations in steroidogenic gene expression.

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MATERIALS AND METHODS
CELL CULTURE AND TREATMENT
BLTK1 cells were maintained in phenol-red free Dulbecco’s modified eagle medium
nutrient mixture F-12 (DMEM/F-12 media; Invitrogen, Carlsbad, CA) with 10% fetal bovine
serum (FBS; Thermo Scientific HyClone, Logan, UT), 100 U/mL penicillin and 100 μg/mL
streptomycin (Invitrogen) and incubated under standard conditions (5% CO2, 37⁰C). Cells were
seeded at 80% confluency in 24 well tissue culture plates (Sarstedt, Newton, NC) for
measurement of P and T levels in media. Concentration-response studies at 24 hrs were
conducted with DMSO (vehicle; Sigma, St. Louis, MO), 0.1, 0.3, 1, 3, 10, 30 or 100 ng/ml rhCG
(obtained from A.F. Parlow, NIDDK’s National Hormone & Peptide Program, Harbor-UCLA
Medical Center, Torrance, CA) or 1, 3, 10, 30, 100, 300 or 600 μM of ATR (Sigma, St. Louis, MO),
DIA, DEA, DACT, PRO, SIM or TBA (Accustandard, New Haven, CT). Time course studies were
conducted with DMSO (vehicle), 300 μM ATR, DIA, DEA, DACT, PRO, SIM, or TBA and rhCG (3
ng/ml) as a positive control. The entire volume of media from wells was collected at 1, 2, 4, 8,
12, 24 or 48 hrs, with different wells used for each time point, such that cumulative steroid
production was evaluated for the time points indicated. Cells were grown to 80% confluency in
T-25 flasks (Sarstedt) for RNA isolation.
MTT CYTOTOXICITY ASSAY
BLTK1 cells, plated into 96 well tissue culture plates (Starstedt), were treated with 1, 3,
10, 30, 100, 300 or 600 ng/ml rhCG or 1, 3, 10, 30, 100, 300 or 600 μM of ATR, PRO, SIM, TBA,
DEA, DIA or DACT. After 24 hrs, media was aspirated and 50 μL of freshly prepared MTT reagent

81

(5 mg/mL thiazolyl blue tetrazolium bromide (Sigma) in phosphate buffered saline) was added
to each well. Cells were incubated for 3 hrs, then MTT reagent was aspirated and 150 μL DMSO
was added to each well for 2 hrs before spectrophotometric measurement of product at 595
and 650 nm (A595-A650) using an Emax precision microplate reader (Molecular Devices,
Sunnyvale, CA). Results are reported as percentage of control calculated from relative
absorbance of treated versus controls where 100% indicates no cytotoxicity.
ENZYME IMMUNOASSAYS
T (Cayman Chemical Company, Ann Arbor, MI) and P (ALPCO Diagnostics, Salem, NH) EIA
kits were used following manufacturer’s instructions, as previously described (40). Briefly,
conjugated hormone and sample compete for antibody on 96-well pre-coated plates. Following
incubation, the plates were washed and a reaction

was initiated to yield a

spectrophotometrically detectable product (412 nm for T, 450 nm for P) using an Emax
precision microplate reader. A standard curve (3.9 - 500 pg/ml T and 0 - 60 ng/ml P) was
generated for P and T quantification. All chemicals were confirmed to have no cross-reactivity
with the EIA kits (data not shown).
RNA ISOLATION AND QRTPCR
Treated cells (T-25 flasks) were trypsinized (Invitrogen) and stored as pellets at -80˚C.
RNA was extracted using RNeasy Mini Kits (QIAGEN, Valencia, CA) according to the
manufacturer’s protocol with an additional RNase-free DNase (QIAGEN) digestion. RNA was
spectrophotometrically quantified at 260 nm (A260) and purity was assessed using the
A260/A280 ratio as well as denaturing gel electrophoresis.

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RNA (1 µg) was reverse transcribed by SuperScript II (Invitrogen) using an
anchored oligo-dT primer (Invitrogen) as described by the manufacturer. QRTPCR reactions
consisted of 30 µl reaction volumes that included 1 µl of cDNA template, 0.1 µM forward and
reverse gene-specific primers, 3 mM MgCl2, 1 mM dNTPs, 0.025 IU AmpliTaq Gold, and 1 X SYBR
Green PCR Buffer (Applied Biosystems, Foster City, CA) carried out in 96 well plates and
amplified using an Applied Biosystems PRISM 7500 Sequence Detection System. Dissociation
curve analysis assessed single product amplification. To control for differences in RNA loading,
quality and cDNA synthesis, samples were standardized to the geometric mean of three
housekeeping genes (Actb, Gapdh and Hprt) (44). Primer sequences for all genes evaluated are
available in Table 1. Results were quantified using a standard curve generated on the same 96
well plate used for samples by amplifying a purified cDNA template specific for each gene
(serial 10X dilutions from 108 to 101 copies). Standard curve slope was used to assess
amplification efficiency, as described by the manufacturer, with all amplification efficiencies
between 80-100%. Fold changes were calculated relative to time-matched vehicle. Relative
expression levels were scaled such that the expression level of the time-matched vehicle
control group equaled one for graphing purposes.
MICROARRAY ANALYSIS
The temporal changes in gene expression elicited by 300 μM ATR were evaluated using
Agilent mouse 4 X 44K oligonucleotide microarrays according to the manufacturer’s protocol
(Agilent Manual: G4140-90050 v5.0.1; array product number: G4122F; Agilent Technologies,
Santa Clara, CA). Briefly, 1 μg total RNA from ATR-treated BLTK1 cells and time-matched DMSO
controls was labeled independently with Cy3 and Cy5 dye and co-hybridized in an independent
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reference design. This design involves comparing treated samples to controls with two
independent labelings per sample (dye swap) for a total of 14 arrays per replicate (7 time points
with dye swap). Three replicates of this design were performed for a total of 42 arrays.
Microarrays were scanned at 532 nm (Cy3) and 635 nm (Cy5) using a GenePix 4000B scanner
(Molecular Devices, Union City, CA), using GenePix Pro 6.0 to extract features and background
intensities. All images and data were loaded into the TIMS dbZach data management system
(45). All microarray data passed our quality assurance protocols (46) and were normalized in
SAS v9.1 (SAS Institute) using a semi-parametric approach (47). Posterior probabilities were
determined on a per gene and per time point basis (48) to determine model-based t-values
(SAS v9.1) which were then used to determine P1(t) values by an empirical Bayes method using
R (R, Institute for Statistics and Mathematics, WU Wien). Data were filtered and prioritized in a
MySQL v5.1.40 database using a filtering criteria of |fold change| > 1.5 and P1(t) > 0.999 to
identify differentially expressed genes.
Functional annotation was carried out using Ingenuity Pathway Analysis (IPA, Ingenuity
Systems, Redwood City, CA; www.ingenuity.com) and the Database for Annotation,
Visualization and Integrated Discovery (DAVID; http://david.abcc.ncifcrf.gov/). Using IPA, genes
were evaluated on a per time point basis to identify significantly enriched (p-value < 0.05)
functions. The IPA Transcription Factor tool was used to identify predicted activation or
inhibition of transcription factors. K-means clustering was conducted using the TM4
MultiExperiment Viewer v.4.6.0 (49). DAVID was used to identify significantly enriched (p-value
< 0.05) Gene Ontology (GO) biological process functional annotation terms (GOTERM_BP_FAT).

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STATISTICAL ANALYSES AND DOSE-RESPONSE MODELING
EIA and QRTPCR concentration-response data were analyzed using SAS v9.1 (SAS
Institute, Cary, NC) by analysis of variance (ANOVA) followed by Dunnett’s post hoc test.
Differences between groups were considered significant when p < 0.05. EIA and QRTPCR data
from time course studies were also analyzed by SAS v9.1 by ANOVA, but followed by Tukey’s
post hoc test, using a significance cutoff of p < 0.05. Dose-response modeling was carried out
using a particle swarm optimization algorithm (ToxResponse modeler (50)) to identify the bestfit across five model classes (sigmoidal, exponential, linear, quadratic and Gaussian). All EC 50
values included in this study were determined from sigmoidal concentration-response curves.

RESULTS
EFFECT OF TRIAZINES AND THEIR CHLOROMETABOLITES ON P AND T LEVELS
The effect of triazine herbicides and their primary chlorometabolites on P and T levels in
media were assessed after 24 hrs of treatment with non-cytotoxic concentrations of triazines
and chlorometabolites (Figure 17). ATR, PRO, SIM or TBA as well as DEA, DIA, and DACT, elicited
concentration-dependent increases in P levels at 24 hrs, albeit with lower efficacy and potency
relative to rhCG (Figure 18). Each triazine and chlorometabolite induced P at 100-600 μM, with
a maximum induction of ~3.5-fold (90 ng/nl) in response to 600 μM ATR relative to vehicle
controls. In comparison, P levels were induced up to 170 ng/ml (~6-fold) following treatment
with 100 ng/ml rhCG. The EC50 for P induction by rhCG was 0.8 ng/ml while triazine and
chlorometabolite EC50s were ~33 μM. Difference in efficacy was more evident on T (Figure 19),
85

100
50

100
300
600

DIA

100
50

DACT (M)

100
300
600

10
30

0

1
3

100
300
600

DEA (M)

10
30

1
3

100
300
600

150

DACT

0

10
30

50

SIM (M)

% of Control

50

1
3

100
300
600

10
30

TBA (M)

10
30

1
3

100

0

1
3

0

150

DEA

100
300
600

50

100

ATR (M)

% of Control

% of Control

100

100
300
600

1
3

100
300
600

10
30

1
3
% of Control

150

TBA

SIM

0

PRO (M)

rhCG (ng/ml)
150

50
0

10
30

0

0

% of Control

50

100

10
30

50

100

150

ATR

1
3

100

150

PRO

% of Control

150

rhCG
% of Control

% of Control

150

DIA (M)

FIGURE 17. EVALUATION OF TRIAZINE AND CHLOROMETABOLITE CYTOTOXICITY.
BLTK1 cells were treated for 24 hrs with 1-600 ng/ml rhCG or 1-600 μM of PRO, ATR, SIM, TBA, DEA, DACT or DIA followed by MTT
reagent. No significant cytotoxicity was detected for any treatment group, cells exhibited >90% viability even at the highest doses.
Data are plotted as % control of means ± standard error, n=3.
86

* * *
*

150
100

* *

*

0
1
3
10
30
100
300
600

rhCG
(ng/ml)
EC50

0
1
3
10
30
100
300
600

* * *

0
1
3
10
30
100
300
600

50
0

*

* * *

*

0
0.1
0.3
1
3
10
30
100

Progesterone (ng/ml)

200

PRO
(M)

ATR
(M)

SIM
(M)

0.8 ng/ml

31.7 M

30.4 M

34.4 M

FIGURE 18. CONCENTRATION-RESPONSE EFFECTS OF TRIAZINES AND CHLOROMETABOLITES ON P LEVELS.
Concentration-dependent induction of P levels measured in the media by EIA 24 hrs post-treatment using DMSO as the vehicle
control (open bars), 0.1-100 ng/ml rhCG or 1-600 μM PRO, ATR, SIM, TBA, DEA, DACT or DIA (solid bars). All triazines and
chlorometabolites elicited dose-dependent induction of P levels, albeit with lower efficacy compared to rhCG. Data are plotted as
means ± standard error and were analyzed using ANOVA followed by Dunnett’s post hoc test, n=3, *p<0.05.

87

FIGURE 18 (cont’d)

*

*

*

80

*

*

*

*

*

60

*

*

*
*

*

40

0
1
3
10
30
100
300
600

DEA
(M)
EC50

0
1
3
10
30
100
300
600

0

0
1
3
10
30
100
300
600

20

0
1
3
10
30
100
300
600

Progesterone (ng/ml)

100

DACT
(M)

DIA
(M)

TBA
(M)

35.0 M

36.9 M

34.7 M

33.3 M

88

*
* * * *
*
* *

* * *

60

*

*

* *

40

0
1
3
10
30
100
300
600

0
1
3
10
30
100
300
600

rhCG
(ng/ml)
EC50

0
1
3
10
30
100
300
600

20

0
0.1
0.3
1
3
10
30
100

Testosterone (pg/ml)

250
170
90
80

PRO
(M)

ATR
(M)

SIM
(M)

0.7 ng/ml

32.3 M

29.2 M

40.4 M

FIGURE 19. CONCENTRATION-RESPONSE EFFECTS OF TRIAZINES AND CHLOROMETABOLITES ON T LEVELS.
Concentration-dependent induction of T levels measured in the media by EIA 24 hrs post-treatment using DMSO as the vehicle
control (open bars), 0.1-100 ng/ml rhCG or 1-600 μM PRO, ATR, SIM, TBA, DEA, DACT or DIA (solid bars). All triazines and
chlorometabolites elicited dose-dependent induction of T levels, albeit with lower efficacy compared to rhCG. Data are plotted as
means ± standard error and were analyzed using ANOVA followed by Dunnett’s post hoc test, n=3, *p<0.05.

89

250
170
90
80

*
* *

60

*

*

*

*

*

* *

40

0
1
3
10
30
100
300
600

0
1
3
10
30
100
300
600

DEA
(M)
EC50

0
1
3
10
30
100
300
600

20

0
1
3
10
30
100
300
600

Testosterone (pg/ml)

FIGURE 19 (cont’d)

DACT
(M)

DIA
(M)

TBA
(M)

44.8 M

-

41.9 M

33.1 M

90

where 100 ng/ml rhCG induced levels ~4-fold (170 pg/ml) while ATR only elicited a ~2-fold (75
pg/ml) increase. SIM was the least efficacious with a modest 1.3-fold induction of T. EC50 values
for T induction were 0.7 ng/ml for rhCG, and 30-45 μM for the triazines and chlorometabolies.
DACT only had T induction at 600 μM and therefore an EC50 value could not be determined
using the ToxResponse modeler. Overall, each compound elicited concentration-dependent
induction of P and T levels, with ATR exhibiting the greatest efficacy.
Based on the 24 hr concentration-response analysis, 300 μM was used to examine the
time-dependent effects of the parent triazines and their primary chlorometabolites on the
induction of P and T levels. ATR, TBA and PRO elicited comparable temporal P and T profiles,
albeit with lower potency compared to rhCG (Figure 20) with efficacy similar to levels in the
concentration-response studies. Like rhCG, triazines and their chlorometabolites induced
maximum P levels by 2 hrs, which subsequently decreased, while rhCG maintained P levels for
2-4 hrs before decreasing. Concomitantly, T increases were inversely proportional to the timedependent decreases in P levels. However, rhCG-mediated efficacy was significantly greater
compared to any triazine or chlorometabolite. Although SIM and DACT elicited comparable
temporal profiles, the effects on P and T levels were not statistically significant. Overall, ATR,
SIM, PRO, TBA, DEA, DIA and DACT elicited comparable temporal effects on P and T levels with
differences in efficacy.

91

Progesterone (ng/ml)

150
100
50
1

2

4

8

12

24

D

250

DMSO
rhCG
DEA
DACT
DIA

200
150
100
50
1

2

4

8

12

24

200
150
100

Time (hrs)

1

2

4

8

12

24

48

24

48

Time (hrs)
200

DMSO
rhCG
DEA
DACT
DIA

150
100
50
0

48

DMSO
rhCG
PRO
ATR
SIM
TBA

50
0

48

Time (hrs)

0

Testosterone (pg/ml)

DMSO
rhCG
PRO
ATR
SIM
TBA

200

0

C

B

250

Testosterone (pg/ml)

Progesterone (ng/ml)

A

1

2

4

8

12

Time (hrs)

FIGURE 20. TIME-DEPENDENT EFFECTS OF TRIAZINES AND CHLOROMETABOLITES ON P AND T LEVELS.
92

FIGURE 20 (cont’d)
BLTK1 cells were treated with 3ng/ml rhCG or 300 μM of ATR, PRO, SIM or TBA (A and B), or
chlorometabolite (i.e., DEA, DACT, or DIA) (C and D) and evaluated for effects on P and T levels
relative to time-matched DMSO (vehicle control). P (A and C) and T (B and D) levels were measured
at each time point by EIA. P levels in media increased within 2 hr of treatment followed by a gradual
decrease over time. In contrast, T levels exhibited a concomitant time-dependent increase following
treatment. Data were plotted as mean ± standard error. Statistical significance was determined
using ANOVA followed by Tukey’s post hoc test, n=3.

TRIAZINE EFFECTS ON STEROIDOGENIC ENZYME GENE EXPRESSION
The expression of steroidogenic enzymes involved in the metabolism of cholesterol to
progestagens, androgens and estrogens was evaluated by QRTPCR for parent triazines and rhCG.
ATR, PRO and TBA elicited concentration-dependent differential expression of several steroidogenic
genes at 24 hrs (Figure 21). Compared to the ~20-fold rhCG-mediated induction of Star, only ATR,
PRO and TBA induced Star mRNA levels (4-, 3-, and 3.5-fold, respectively), while SIM had negligible
effects. Meanwhile, Hsd3b6 mRNA induction was comparable (~3-fold) for ATR, PRO and TBA,
similar to rhCG. Interestingly, Hsd3b1 was repressed 10-, 10- and 4-fold in response to 600 μM ATR,
TBA and PRO, respectively, while rhCG had no significant effect on the expression of this gene.
Although Cyp17a1 was also repressed (8-, 7- and 4-fold decrease by ATR, PRO and TBA,
respectively), rhCG and SIM elicited negligible effects. Srd5a1 was induced ~3-fold by rhCG, but
repressed ~2-fold by ATR, PRO and TBA. Hsd17b3 mRNA levels were induced ~6-fold by both rhCG
and ATR treatment, while PRO, SIM and TBA had negligible effects. Only TBA repressed Cyp19a1 (5fold). None of the treatments altered Cyp11a1 expression.

93

30

Fold Change

20
10
8

Star

*
*

*

rhCG

PRO

ATR

SIM

TBA

*

6
4

*

2

**

*
*

*

*

*

*
**

*

0.1
0.3
1
3
10
30
100
1
3
10
30
100
300
600
1
3
10
30
100
300
600
1
3
10
30
100
300
600
1
3
10
30
100
300
600

0

Dose
(ng/ml)

Fold Change

2.5

Dose
(M)

Cyp11a1

2.0
1.5
1.0
0.5

0.1
0.3
1
3
10
30
100
1
3
10
30
100
300
600
1
3
10
30
100
300
600
1
3
10
30
100
300
600
1
3
10
30
100
300
600

0.0

Dose
(ng/ml)

Dose
(M)

FIGURE 21. CONCENTRATION-RESPONSE EVALUATION OF TRIAZINE- AND RHCG-MEDIATED
GENE EXPRESSION.
The concentration-dependent effects of rhCG (black striped bars), PRO (medium grey bars), ATR
(white bars), SIM (light grey bars), or TBA (black solid bars) on steroidogenic enzyme gene
expression examined 24 hr post-treatment using QRTPCR. Data are plotted as mean ± standard
error of three independent replicates, *p < 0.05 vs DMSO controls.
94

5

3

0.1
0.3
1
3
10
30
100
1
3
10
30
100
300
600
1
3
10
30
100
300
600
1
3
10
30
100
300
600
1
3
10
30
100
300
600

Fold Change

0.1
0.3
1
3
10
30
100
1
3
10
30
100
300
600
1
3
10
30
100
300
600
1
3
10
30
100
300
600
1
3
10
30
100
300
600

Fold Change

FIGURE 21 (cont’d)

2.5

Hsd3b1

2.0

1.5

1.0

0.5

*

*
** * *

2

*
*

0.0

Dose
(ng/ml)

Hsd3b6

Dose
(ng/ml)

95

*

*

*

** *
**

*

Dose
(M)

**

Dose
(M)

*

4

*

1

0

0.1
0.3
1
3
10
30
100
1
3
10
30
100
300
600
1
3
10
30
100
300
600
1
3
10
30
100
300
600
1
3
10
30
100
300
600

Fold Change
3

10

8

6

0.1
0.3
1
3
10
30
100
1
3
10
30
100
300
600
1
3
10
30
100
300
600
1
3
10
30
100
300
600
1
3
10
30
100
300
600

Fold Change

FIGURE 21 (cont’d)

Cyp17a1

2

1

**

0

*
**
*

Dose
(ng/ml)

**

Dose
(ng/ml)

96

*
*

Dose
(M)

*
*

Dose
(M)

Hsd17b3

*

*

4

2
1

0

7

5

3

0.1
0.3
1
3
10
30
100
1
3
10
30
100
300
600
1
3
10
30
100
300
600
1
3
10
30
100
300
600
1
3
10
30
100
300
600

Fold Change

0.1
0.3
1
3
10
30
100
1
3
10
30
100
300
600
1
3
10
30
100
300
600
1
3
10
30
100
300
600
1
3
10
30
100
300
600

Fold Change

FIGURE 21 (cont’d)

3

Cyp19a1

2

1

*

0

Dose
(ng/ml)

*
*

1

*
*

Dose
(ng/ml)

Dose
(M)

97

**

Dose
(M)

Srd5a1

*

2

*

0

The temporal effects of ATR, PRO, SIM and TBA on steroidogenic mRNA levels were also
examined (Figure 22). Despite some differences in triazines- vs. rhCG-elicited concentrationresponse profiles at 24 hrs, time-dependent ATR-, PRO- and TBA-elicited steroidogenic gene
expression profiles were comparable to rhCG, albeit with differing efficacies. In general,
Cyp11a1 and Cyp19a1 were unaffected by treatment while Star, Hsd3b6, Hsd17b3 and Cyp17a1
were induced by rhCG and triazines, in agreement with the 24 hr dose-response profiles. For
example, Star was induced 2-4-fold by ATR, PRO, SIM and TBA and 10-fold by rhCG with
maximum expression achieved at 8-12 hrs. Similarly Cyp17a1 also showed peak induction at ~8
hrs with repression evident by 24-48 hrs for both rhCG and ATR. Furthermore, Srd5a1 exhibited
divergent regulation with rhCG inducing expression 5-fold from 8-24 hrs while ATR, PRO and
TBA elicited >80% repression from 24-48 hrs. Overall, SIM elicited negligible effects on the
expression of the steroidogenic genes examined.
WHOLE-GENOME EVALUATION OF ATR-MEDIATED DIFFERENTIAL GENE EXPRESSION
Due to the similarities among the triazines and chlorometabolites only ATR was investigated for
genome-wide differential gene expression as it had greater efficacy. Agilent microarrays contain
~41,000 features, representing 21,308 unique genes, with 797 differentially expressed (|fold
change| > 1.5 and P1(t) > 0.999) by 300 μM ATR (Figure 23a). Differential gene expression
increased over the course of the study with 23 genes at 1 hr, 18 at 2 hr, 117 at 4hr, 134 at 8 hr,
284 at 12 hr, 377 at 24 hr, and 493 at 48 hrs (Figure 23B). Star induction measured by
microarray was validated by QRTPCR, demonstrating comparable time-dependent profiles and
levels of induction from 4-48 hrs (Figure 23C). K-means analysis of the 797 differentially
expressed genes identified three distinct clusters with 317 induced, 362 repressed and
98

16

Fold Change

12

Star

PRO

ATR

SIM

TBA

**

8
4

hCG

*

*

*
*

2

*

*
*

**

**

*

*

*

*

***

1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48

0

Time (hrs)

Fold Change

2.5

Cyp11a1

2.0
1.5
1.0
0.5

1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48

0.0

Time (hrs)
FIGURE 22. TIME-DEPENDENT EFFECTS OF TRIAZINES AND rhCG ON STEROIDOGENIC
GENE EXPRESSION.
Temporal gene expression profiling of steroidogenic enzymes in response to 3 ng/ml rhCG
(black striped bars) or 300 μM of PRO (medium grey bars), ATR (white bars), SIM (light grey
bars) or TBA (black solid bars), respectively, was examined by QRTPCR. Data are plotted as
mean ± standard error of three independent replicates, *p < 0.05 vs. time-matched DMSO
controls.

99

1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48

Fold Change
2.5

5

3

2

1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48

Fold Change

FIGURE 22 (cont’d)

Hsd3b1

2.0

1.5

1.0

*

0.5

**
*
*

*
*

*
*

Time (hrs)

100

*

0.0

Time (hrs)

Hsd3b6
*

4

*

*
**

**
**

1

0

1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48

Fold Change
3

15

12

9

6

3

1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48

Fold Change

FIGURE 22 (cont’d)

Cyp17a1

2

*

*

*

*

*

*
*

*
*

101

*

1

*
*

0

Time (hrs)

Hsd17b3

**
*

Time (hrs)
*

*

1

0

1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48

Fold Change
4

15

1

1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48
1
2
4
8
12
24
48

Fold Change
FIGURE 22 (cont’d)

Cyp19a1

3

*

2

1

0

Time (hrs)

10

Srd5a1

5

* **

2

*
**
*

102

*

0

Time (hrs)
*
*

FIGURE 23. TEMPORAL WHOLE-GENOME
DIFFERENTIAL GENE EXPRESSION.

EVALUATION

OF

ATR-MEDIATED

Gene expression elicited by 300 μM ATR was evaluated using Agilent whole-genome
microarrays. Filtering of microarray data (A), and summary of the number of genes exhibiting
differential expression per time point (B; |fold change| > 1.5 and P1(t) > 0.999). Evaluation of
Star expression by microarray (line) and QRTPCR (bars) (C). QRTPCR validation of Star plotted as
mean ± standard error of three independent replicates, *p < 0.05 vs. time-matched DMSO
controls. Microarray data are plotted as mean fold change of three independent replicates,
#
P1(t) > 0.999.

103

FIGURE 23 (cont’d)

118 exhibiting induction from ~4-24 hrs that tapered by 48 hrs (Figure 24). The Ingenuity®
Transcription Factor Tool predicted enrichment of androgen receptor (AR) activity (24 and 48
hr, p-value 7.59e-03) based on 19 differentially expressed AR target genes at 24 and 48 hrs, of
which 10 were repressed and 9 exhibited induction. The AR target genes contributing to the
104

prediction of AR signaling activation were all differentially expressed at later time points,
coinciding with increasing T levels detected at 24 and 48 hrs (Figure 20). However, AR mRNA
expression levels were not altered by ATR, suggesting that the activation of AR signaling was a
result of androgen binding rather than increased expression.
Functional annotation was conducted using DAVID and Ingenuity® Pathway Analysis
(IPA) to identify enriched gene ontology (GO) terms and over-represented functions,
respectively (Table 3). Enriched GO terms were associated with hormone metabolism and
regulation (including steroidogenic gene expression evaluated by QRTPCR) as well as
cholesterol biosynthesis and metabolism. Examples included cholesterol de novo biosynthesis
related genes such as Hmcgr, Hmgcs1, Lss, Sqle and Cyp51 (all showing 1.5 to 2-fold induction
at 8-12 hrs), cholesterol transport and uptake (Star up to 3-fold from 4-48 hrs and Ldlr 2-fold at
8 and 12 hrs), and metabolism into steroids (as demonstrated by QRTPCR of steroidogenic
enzyme expression, Figure 22). IPA transcription factor analysis also predicted the activation of
Srebf1 (Table 4) and Srebf2 (p-value 1.70e-05), which regulate gene expression associated with
sterol homeostasis. The predicted activation of Srebf1 and Srebf2 was consistent with the
intermediate up-regulation of their target genes as identified in K-means clustering. In addition,
Lhcgr, the cell surface receptor for LH/rhCG, was up-regulated 1.6-fold at 24 hrs.

105

FIGURE 24. K-MEANS CLUSTERING OF ATR-ELICITED TEMPORAL WHOLE-GENOME
GENE EXPRESSION.
ATR (300 uM) elicited the differential expression (|fold change| > 1.5 and P1(t) > 0.999) of 797
genes. K-means clustering using a Pearson correlation distance metric, identified three
distinctive profiles: increasing down-regulation, increasing up-regulation and intermediated upregulation. Light grey lines illustrate the profile of each gene within the cluster, while the dark
grey line illustrates the average trend for the group.

106

FIGURE 24 (cont’d)

107

FIGURE 24 (cont’d)

108

TABLE

3.

ENRICHED FUNCTIONAL ANNOTATION
DIFFERENTIALLY REGULATED GENES

TERMS

FOR

ATR-ELICITED

p-value

Functional Annotation Terms
Top 5 GO Terms (DAVID)
Regulation of Hormone Levels (GO:0010817)
Hormone Metabolic Process (GO:0042445)
Cholesterol Biosynthetic Process (GO:0006695)
Cellular Hormone Metabolic Process (GO:0034754)
Cholesterol Metabolic Process (GO:0008203)

Number of
Genes

5.73e-05
6.28e-05
2.37e-04
2.09e-04
1.05e-04

17
14
7
10
12

Top 5 Functional Terms (IPA)
Cancer
1.6e-17 – 1.4e-03*
276
Reproductive System Disease
1.3e-11 – 1.3e-03*
167
Reproductive System Development & Function
1.3e-11 – 1.5e-03*
102
Tissue Development
1.5e-09 – 1.4e-03*
178
Tissue Morphology
2.5e-08 – 1.3e-03*
192
*p-value range provided by IPA for each time point the function is enriched.
IPA also identified functions associated with reproduction as over-represented. This

included the induction of Rad18, Insl6 and Prnp (1.5-fold at 48 hrs), required for
spermatogenesis, as well as the repression of Srd5a1 and Srd5a3 (2-fold at 48 hr) required for
male reproductive development. Tissue development and morphology was also overrepresented and included several actin regulatory proteins genes such as Avil (induced 2-fold at
48 hr), Fmn1 (induced ~2-fold 8-12 hr), and Cald1 (down-regulated ~2-fold 8-48 hrs). The
predicted activation of Creb1 and Atf4 (Table 4) suggest cAMP-mediated gene expression, and
included the induction of Vegfa, Gadd45a, and Bcat1 consistent with changes in cell
morphology and growth. Induction of the phosphodiesterase (Pde) genes Pde4b (up 1.8-fold
12-48 hr), Pde4dip (up 1.6-fold at 24 hr), and Pde10a (up to 3-fold 4-48hr) are also indicative of

109

cAMP signaling. The predicted activation of CCAAT/enhancer-binding proteins (Cebp), Cebpa (pvalue 4.38e-05) and Cebpb (Table 4), as well as ATR-mediated differential expression of Cebpd
(repressed 1.5-fold at 12 hr) and Cebpg (up-regulated 1.5-fold at 24 hrs) further suggest ATRmediated changes in cellular growth and differentiation. Interestingly, BLTK1 cells exhibited
morphological changes (e.g., contracted cell bodies, increased pseudopod length) in response
to rhCG at 24 hrs (Figure 25). MTT (Figure 17) and trypan blue staining (data not shown)
indicated no significant rhCG- or ATR-elicited cytotoxicity with no effects on cell growth or
doubling time over the 48 hr time course based on cell counting (data not shown).

TABLE 4. IPA PREDICTED TRANSCRIPTION FACTOR ACTIVATION/DEACTIVATION
Transcription
Time Points
Predicted Activation*
Number of
p-value
Factor
Enriched
(time point in hrs)
Genes*
Atf4
1.68e-12
4, 8, 12, 24, 48 Activated (4, 12, 24, 48)
25
Foxo4
1.54e-07
4, 8,12, 24, 48
Cebpb
5.31e-07
4, 8,12, 24, 48
Creb1
5.29e-06
4, 8, 12, 24, 48
Activated (4, 48)
27
Srebf1
4.87e-06
4, 8, 12
Activated (8, 12)
14
* Transcription factors are predicted to be activated or deactivated based on the differential
expression of known target genes. Time point of activation/deactivation (in parentheses) and
the number of genes contributing to prediction are provided.

110

FIGURE 25. BLTK1 CELL MORPHOLOGY IN RESPONSE TO rhCG AND ATR TREATMENT.
Cells were photographed using a phase/contrast microscope at 100x magnification 24 and 48
hrs after treatment with DMSO, 3 ng/ml rhCG or 300 µM ATR.

DISCUSSION
The endocrine disruptive effects of triazines have been extensively studied in vivo (24,
28, 30, 32-35, 51), however their mechanism remains unresolved. ATR, SIM, PRO, TBA, and
DACT all induce mammary gland tumors in female Sprague-Dawley rats, which was the
common mode of action used to define them as a common mechanism group (CMG) (20, 30).
The CMG also includes the chlorometabolites DEA, DIA and DACT, which are found in drinking
water and food and elicit comparable HPG axis disruption (20, 33, 36). At the time, only ATR,
SIM and PRO were deemed to have uses that result in exposure of the general public thus
excluding TBA from the CMG. ATR and SIM exposure was of greatest risk due to widespread use
111

and persistence in ground and surface water (15). Estimations of human ATR exposure based
on urine levels of turf applicators is ~2 μM ATR equivalents (including metabolites) while acute
exposure is approximately half this amount (12). ATR metabolite profiles in human urine consist
of 50% DACT, 30% DEA and 5% DIA (12). Rodent studies suggest similar ATR metabolism,
primarily by CYP1A1/2 (10, 11). Maximum plasma levels in mice are achieved within 1 hr, with
maximum DACT plasma concentrations within 2 hrs post-dose (11). Chlorometabolite
concentrations of ~2 μM are reported in serum with less than 1 μM of parent compound
following administration of 200 mg/kg ATR or an equimolar SIM or PRO dose in male Wistar
rats (36). Although, these human and rodent urine and serum levels encompass the
concentrations used in our concentration-response studies, the effects on steroidogenesis
appear to occur at triazine levels not encountered by humans, even in occupational
environments.
Responses in BLTK1 cells are consistent with in vivo studies indicating ATR, PRO, DEA,
DIA and DACT induce dose-dependent increases in serum P levels in male Wistar rats within 15
min to 3 hrs with no significant induction in response to SIM (36). SIM treatment for 41 days
delayed vaginal opening and the onset of puberty in female rats (35) suggesting more modest
disruption of steroidogenesis compared to other triazines and chlorometabolites in agreement
with its effects in BLTK1 cells. ATR and DIA were reported to be equally efficacious on eliciting
changes in plasma progesterone levels in male Wistar rats (36), consistent with their equipotent
induction of P and T levels in BLTK1 cells. Additionally, DACT was described to be the least
efficacious in male Wistar rats (36), also consistent with T induction in the current study where
it had no effects at concentrations <600 μM. In addition, the CMG categorization of ATR, PRO,
112

SIM, DEA, DIA and DACT is in agreement with their P and T induction effects in BLTK1 cells as
demonstrated by their similar EC50 values (33). Furthermore, TBA elicits comparable effects
including P and T induction as well as differential steroidogenic gene expression. Moreover, the
effects of all triazines and their cholormetabolites, including TBA, are similar to effects reported
in rat primary Leydig cells at doses as low as 20 μM ATR (52). Despite differences in the
differential expression of specific steroidogenic genes, the effects on P and T levels were
comparable across all treatments suggesting ATR, PRO, SIM and TBA as well as their major
chlorometabolites alter the regulation of steroidogenesis using a common mode of action.
Activation of Leydig cell steroidogenesis exhibits (1) an acute phase initiated by
gonadotropin binding to LHCGR and induction of intracellular cAMP and PKA signaling events,
and (2) a longer term effect involving differential gene expression and increased steroidogenic
enzyme expression that affects the steroidogenic maintenance (53). While the acute phase
induces immediate T levels, sustained production requires both phases. ATR alters basal T levels
in BLTK1 cells and alters Star, Hsd3b, Cyp17a1 and Hsd17b expression in rat primary Leydig cells
(51). The current study extends the effects on P and T levels to other triazines and their major
chlorometabolites to include time- and dose-dependent effects on the long-term phase of
steroidogenesis. Most ATR-elicited differential gene expression occurred after 12 hrs suggesting
effects on long-term steroidogenesis. For example, while Star and Cyp17a1 showed maximum
induction at ~8 hrs, repression at later time points (24-48 hrs) is consistent with the feedback
inhibition exerted by androgens mediated by the AR (54, 55). These genes contributed to the
prediction of AR activation at later time points (24-48 hr), most likely due to increasing T levels
at these time points. In addition, the induction of several cholesterol biosynthetic genes and
113

predicted activation of Srebf1 and Srebf2 are consistent with steroid biosynthesis and longer
term maintenance of steroidogenesis.
The early induction of Star also coincides with immediate increases in P and T levels.
StAR mediates the rate-limiting transport of cholesterol to the inner mitochondrial membrane
required for steroidogenesis and is one of the few genes with significant induction at earlier
time points. Star expression is largely cAMP-mediated, with cAMP levels regulated not only by
production but also by phosphodiesterase (PDE) turnover (56). ATR is reported to inhibit PDE4
in rat Leydig and anterior pituitary cells (57), human recombinant PDE4A1 (58), and cAMPspecific PDEs in swine (59, 60). Biologically Multiplexed Activity Profiling (BioMAP; a primary,
human, cell-based assays covering a diverse array of regulatory networks with a broad range of
target mechanisms relevant to human toxicity) also suggests ATR, SIM and PRO increase cAMP
levels, consistent with PDE inhibition (61). These data suggest that the late induction of Pde10a,
Pde4b and Pde4dip in BLTK1 cells at 48 hrs may be a compensatory response to PDE inhibition
that warrants further investigation.
Collectively, the current study demonstrates the effects of the CMG triazines, as well as
TBA, and their primary chlorometabolites on steroidogenesis in BLTK1 Leydig cells. Our data
suggest that altered gene expression may underlie the long-term steroidogenic effects of
triazines and their chlorometabolites, although additional studies are needed to elucidate their
acute effects on P and T levels. Although our results are consistent with other reports of
triazines inducing P and/or T levels (34, 36, 52, 62), other in vivo studies show triazines
antagonize serum T levels (63-66). Preliminary studies suggest triazines antagonize rhCGstimulated steroidogenesis (40), and ongoing co-treatment studies with triazine and rhCG are
114

examining potential triazine targets that underlie the disruption of steroidogenesis in BLTK1
Leydig cells.

115

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CHAPTER 5

124

CHAPTER 5
ATRAZINE-MEDIATED DISRUPTION OF STEROIDOGENESIS
IN BLTK1 LEYDIG CELLS

ABSTRACT
Atrazine (ATR), a widely used broad-spectrum triazine herbicide, is an endocrine
disruptor altering steroidogenesis resulting in reproductive and developmental toxicity. BLTK1
Leydig cells were used as a steroidogenesis model to investigate the effects of ATR on
testosterone (T) biosynthesis. Induction of steroidogenesis by 3 ng/ml recombinant human
chorionic gonadotropin (rhCG) induced intracellular 3’,5’ cyclic adenosine monophosphate
(cAMP) ~20-fold and T ~3-fold at 4 hrs. Co-treatment with 300 μM ATR potentiated cAMP levels
100-fold yet antagonized rhCG-induction of T by ~20% at 4 hrs, the same time point. ATR
inhibited cAMP-specific phosphodiesterase (cPDE) with an EC50 of ≥98 μM, suggesting
potentiation of cAMP levels is a result of cPDE inhibition. However, concentrations of up to 3
mM db-cAMP did not antagonize T levels suggesting super-induction of cAMP is not sufficient
to disrupt T steroidogenesis. Evaluation of cAMP-activated protein kinase A (PKA) activity by
western blot revealed ATR dose-dependent changes in target protein phosphorylation. These
results suggest ATR inhibits cPDEs, but that decreases in rhCG-induced T levels are likely due to
changes in phosphorylation of key steroidogenic regulatory proteins.

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INTRODUCTION
Atrazine (ATR; 2-chloro-4,6-bis(isopropylamino)-s-triazine), a broad-spectrum herbicide
used on agricultural crops and grasses for pre-emergent weed control, is one of the most
widely distributed herbicides in North America since the early 1960s. ATR exposure remains a
concern as high volume use and mobility in soil and groundwater result in ATR equivalent levels
(comprised of ATR and its chlorinated metabolites) above U.S. maximum contaminant levels in
75% of streams and 40% of groundwater samples tested in agricultural and urban areas in the
United States between 1992 and 2001 (1-4).
ATR-elicited reproductive and developmental toxicity has been consistently observed
across fish, amphibian and mammalian species (5-8), with endocrine disruptive effects targeting
the hypothalamic-pituitary-gonadal (HPG) axis (7, 8). In male rodents these effects include
altered testicular morphology with instances of testicular atrophy (9), decreased androgendependent organ development such as decreased seminal vesicle and ventral prostate weights
(10), delayed perputial separation (11), and decreased sperm number and motility (12). These
effects have been attributed to ATR-mediated decreases in testosterone (T) levels in the
testicular interstitium and in serum (9-15). Several studies also noted increases in circulating
17β-estradiol (E2) levels in the serum of male rodents (9, 13). These results have contributed to
the hypothesis that ATR-mediated effects are estrogenic; however, there is negligible
interaction of ATR with the estrogen receptor (ER) and on ER activity across species (16-19).
To better understand ATR’s effects on steroid hormone biosynthesis and metabolism, in
vitro evaluation ATR effects on steroidogenesis in H295R adrenocortical carcinoma cells has
been conducted. H295R cells, unlike adult differentiated adrenal cells, have the characteristics
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of zonally undifferentiated fetal adrenal cells producing T and E2 in addition to glucocorticoids
and mineralocorticoids (20). In H295R cells ATR increases E2 production (21-24). However,
reports are inconsistent regarding T production being increased (24) or decreased (21, 22).
Consequently, the induction of aromatase (P450c19), the enzyme responsible for the
aromatization of T to make E2, is a proposed mechanism for ATR (13, 21, 22, 25).
Leydig cells are the primary site for T biosynthesis and are a more physiologically
relevant system in which to evaluate the effects of ATR on steroidogenesis. Leydig cells are
required for proper male development and reproduction and are capable of de novo
steroidogenesis, producing both T and E2 from cholesterol. In rat primary Leydig cells ATR
induces basal T production, and increases cAMP levels (26). The secondary messenger cAMP
was also increased in ATR-treated H295R cells (21, 23), leading to the investigation of ATR as a
cAMP-specific phosphodiesterase (cPDE) inhibitor (27-29). While cPDE inhibition by ATR has
been demonstrated (21, 27-29), it remains to be elucidated how elevated cAMP levels relate to
disruption of hormone production in Leydig cells.
This study examines the mode of action by which ATR disrupts steroidogenesis in BLTK1
murine Leydig cells. BLTK1 cells are a subclone of BLT-1 murine Leydig cells isolated from a
testicular tumor in a inhibin /SV40Tag transgenic mouse (30, 31). Unlike other murine Leydig
cell lines such as BLTC-1 (32), MA-10 (33) and mLTC-1 (34), BLTK1 cells produce low basal P and
T levels, which are inducible by recombinant human chorionic gonadotropin (rhCG, a luteinizing
hormone analog) and forskolin (31). Previous studies in BLTK1 cells have demonstrated ATRelicits partial agonism of steroidogenesis relative to rhCG (35). The effects of ATR on rhCGinduced steroidogenesis were evaluated, revealing antagonism of T induction, consistent with
127

in vivo studies reporting decreased T in serum. Furthermore, our results support ATR-mediated
inhibition of cPDE, but are the first to demonstrate that elevated intracellular cAMP levels do
not underlie antagonism of rhCG induction of T production. Evaluation of protein
phosphorylation reveals that ATR likely affects T production in BLTK1 Leydig cells by altering the
phosphorylation status of key steroidogenic regulatory proteins.

MATERIALS AND METHODS
CHEMICALS
Culture medium composed of phenol-red free Dulbecco’s modified eagle medium
nutrient mixture F-12 (DMEM/F-12 media; Invitrogen, Carlsbad, CA) was supplemented with
10% fetal bovine serum (FBS; Thermo Scientific HyClone, Logan, UT), 100 U/mL penicillin and
100 μg/mL streptomycin (Invitrogen). Recombinant human chorionic gonadotropin (rhCG) was
obtained from A.F. Parlow, NIDDK’s National Hormone & Peptide Program, Harbor-UCLA
Medical Center (Torrance, CA). Dimethyl sulfoxide (DMSO), Atrazine (ATR), 3-isobutyl-1-methylxanthine (IBMX), forskolin (FSK), and dibutyryl-3’,5’ cyclic adenosine monophosphate (dbcAMP) were obtained from Sigma-Aldrich (St. Louis, MO). Primary antibodies for
phosphorylated PKA substrate (RRXS*/T*; #9624) and phosphorylated ERK1/2 (p44/42 MAPK
phospho-Thr202/Tyr204; #9101) were obtained from Cell Signaling Technology (Danvers, MA),
and for actin (sc-1616) from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibody
goat anti-rabbit IgG (#170-6515) and donkey anti-goat (sc-2056) conjugated with horseradish

128

peroxidase were obtained from Bio-Rad Laboratories (Hercules, CA) and Santa Cruz,
respectively.
CELL CULTURE AND TREATMENT
Cell culture and treatment were conducted as previously described (35, 36). Briefly,
BLTK1 murine Leydig cells were maintained under standard conditions (5% CO2, 37⁰C), grown to
80% confluency, transferred into 24-well tissue culture plates (Sarstedt, Newton, NC), and
incubated overnight prior to treatment. Following 4 hr treatment, media was collected for
progesterone (P) and testosterone (T) measurement while cells were harvested for intracellular
cAMP measurement and protein quantification.
ENZYME IMMUNOASSAYS
Intracellular cAMP was measured by EIA (Cayman Chemical Company, Ann Arbor, MI)
following hydrochloric acid extraction as described by vendor. Levels of P and T from media
samples were also measured by commercially available EIA kits for T (Cayman Chemical
Company) and P (ALPCO Diagnostics, Salem, NH), used according to the manufacturer’s
instructions. Spectrophotometric detection was conducted at 420 nm for cAMP and T or 405
nm for P using an Emax precision microplate reader (Molecular Devices, Sunnyvale, CA).
Standards (0.3 – 750 pmol/ml cAMP, 0 - 60 ng/ml P, or 3.9 - 500 pg/ml T) were used to generate
a standard curve allowing quantification.
WESTERN BLOTTING
Lysates were prepared in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM
EDTA, 0.5% Triton) containing protease inhibitor (Roche Diagnostics, Indianapolis, IN). Protein
129

concentrations were determined spectrophotometrically using Bradford reagent (Cayman). Ten
µg of protein was resolved by 12% sodium dodecyl sulfate/polyacrylamide gel electrophoresis
(SDS-PAGE; Bio-Rad Laboratories) and transferred to Immobilon membrane (Millipore, Billerica,
MA). Membranes were incubated with primary antibody at 4oC overnight, washed in Trisbuffered saline supplemented with 0.1% Tween-20 (TBST; Sigma) and incubated with secondary
antibody for 1 hr at room temperature. Enhanced Chemiluminescence (Thermo Scientific
Pierce, Rockford, IL) was used to visualize detected protein.
PHOSPHODIESTERASE INHIBITION ASSAY
PDE inhibition was evaluated using a commercially available kit (Enzo Life Sciences,
Farmingdale, NY) with purified PDE enzyme from bovine brain. Briefly, 3’,5’-cAMP substrate is
metabolized by cAMP-specific PDE (cPDE) into 5’-nucleotides and subsequent cleavage into
nucleoside and phosphate by a 5’-nucleotidase is detected using BIOMOL GreenTM reagent.
Assays are conducted in 96-well plates with a 50 μL reaction volume containing 200 μM 3’,5’cAMP as substrate, 20 U PDE enzyme, 50 kU 5’-nucleotidase and 0.03 – 300 μM ATR or 0.02 –
200 μM IBMX. Plates were incubated at 30⁰C, reactions were stopped by the addition of
BIOMOL GreenTM reagent followed by 20 min incubation at room temperature to allow color to
develop, and measured at 620 nm using an Emax Precision microplate reader. Quantification
was carried out using a standard curve generated with known quantities of 5’-AMP substrate
incubated with 5’-nucleotidase alone. Controls were included using 5’-AMP as substrate to
ensure that ATR did not inhibit the activity of the 5’-nucleotidase (data not shown). The cPDEspecific inhibitor, IBMX, was used as positive control. All determinations were conducted in
triplicate.
130

DOSE-RESPONSE MODELING AND STATISTICAL ANALYSES
Dose-response modeling was performed using the ToxResponse modeler (37) whereby
particle swarm optimization identifies the best-fit across five model classes: sigmoidal,
exponential, linear, quadratic and Gaussian. Data fitting sigmoidal dose-response curves were
used to calculate EC50 values. All statistical analyses were carried out using SAS v9.1 (SAS
Institute, Cary, NC) by analysis of variance (ANOVA), with Dunnett’s or Tukey’s post hoc tests for
concentration-response and time course data, respectively. Differences between treatment
groups were considered significant when p < 0.05 relative to time-matched DMSO control.

RESULTS
ATR-MEDIATED DISRUPTION OF rhCG-ELICITED INDUCTION OF cAMP, P AND T LEVELS
The temporal effects of 300 μM ATR, 3 ng/ml rhCG and their co-treatment on cAMP, P
and T levels were evaluated in BLTK1 cells (Figure 26). Upon stimulation of Leydig cells by rhCG,
a rapid ~200-fold induction of intracellular cAMP was observed within 1hr and persisted to
4hrs. Meanwhile, ATR only resulted in a maximal 2-fold induction at 1hr. Interestingly, cotreatment of ATR and rhCG potentiated the induction of intracellular cAMP levels to ~400-fold
at 1 hr with similar potentiation at all time points where intracellular cAMP was detected (≤ 4
hrs). P and T levels were induced by both ATR and rhCG although ATR only resulted in partial
agonism relative to rhCG. Concomitantly, co-treatment resulted in antagonism of rhCGmediated induction of both P and T at all time points where these steroid hormones were
detected (≥2 hrs).

131

A
cAMP (pmol/ml)

10000

*
*a

1000

ab

*a

200

24 hr

12 hr

*a

50

*

*b

4hr

2hr

b

*a

2 hr

*a
*b

*
6hr

100

4 hr

6 hr

*a
*b

*
12 hr

*b

*
24h

*a

12hr

150

0

6 hr

ND
24h

4 hr

ND
12hr

2 hr

6hr

4hr

1 hr

2hr

ND

1 hr

24 hr

200
150

2 hr

4 hr

*

6 hr

*

12 hr

*b

*

*b

24h

*

*ab

*a
*ab

*a

*a

12hr

1 hr

*

*a

6hr

0

* * *
1 hr

50

*a
*b

4hr

100

2hr

Progesterone (ng/ml)

*a

10

1 hr

Testosterone (pg/ml)

DMSO
300 M ATR
3 ng/ml rhCG
3 ng/ml rhCG + 300 M ATR

ab

*

1 hr

C

*

100

1

B

* ab

24 hr

FIGURE 26. TEMPORAL EFFECTS OF ATR ON rhCG- INDUCTION OF STEROIDOGENESIS.
Intracellular cAMP (A), P (B) and T (C) levels in media were evaluated upon treatment with
DMSO vehicle (white bars), 300 μM ATR (grey bars), 3 ng/ml rhCG (black bars), or their cotreatment (striped bars) from 1-24 hrs. Data are plotted as mean ± standard error, n=3, and
were analyzed by ANOVA followed by Tukey’s post hoc test where *p<0.05 vs. time-matched
a

b

DMSO, p<0.05 vs. time-matched ATR, p<0.05 vs. time-matched rhCG. ND: not detected.

132

Dose-response evaluation of ATR’s effects on rhCG-mediated induction of cAMP and T
levels was evaluated at 4 hrs (Figure 27). Only cAMP and T were evaluated as cAMP is an
essential secondary messenger required for the stimulation of steroidogenesis and T is the
primary product of Leydig cells; P was not pursued as treatment effects were comparable to T.
Concentration-dependent potentiation of rhCG-induced intracellular cAMP levels was observed
at ≥100 μM ATR with a maximal ~10-fold super-induction at 600 μM ATR. Meanwhile, ATR cotreatment resulted in up to ~35% antagonism of rhCG-elicited induction of T. EC50 values of
~100 μM were consistent for both cAMP potentiation and T antagonism with ATR /rhCG cotreatment.
ATR EFFECTS ON cAMP INDUCTION
Elevated intracellular cAMP levels can be the result of increased production or
decreased turnover. To evaluate whether ATR affects G-protein-coupled receptor (GPCR)
signaling underlying adenylyl cyclase activation, and hence cAMP production, the effect of ATR
on rhCG- and FSK-mediated cAMP induction and T production was compared (Figure 28).
Induction of steroidogenesis with rhCG is initiated by GPCR signaling via LHCGR to activate
adenylyl cyclase, whereas FSK-mediated induction of steroidogenesis directly targets adenylyl
cyclase (Figure 5). Both rhCG and FSK induced cAMP and T levels (Figure 28). ATR co-treatment
resulted in potentiation of cAMP levels ~10-fold on both rhCG- and FSK-mediated induction and
decreased T levels ~20% relative to rhCG or FSK induction alone. These data suggest that
disruption of GPCR signaling likely does not underlie ATR’s effects on cAMP or T levels since the
same effects were observed when inducing steroidogenesis with and without LHCGR.

133

B
1000
100

#

#

#

#

10

1
3
10
30
10
0
30
0
60
0

30 D
M
3 0  M SO
ng
/m AT
lr R
hC
G

1
M ATR
+ 3 ng/ml rhCG

100

EC50: 104 M

80
#

60

#

#

40
20
0

1
3
10
30
10
0
30
0
60
0

EC50: 111 M

30 D
M
3 0  M SO
ng
/m AT
lr R
hC
G

cAMP (pmol/ml)

10000

Testosterone (pg/ml)

A

M ATR
+ 3 ng/ml rhCG

FIGURE 27. CONCENTRATION-DEPENDENT EFFECT OF ATR ON rhCG-MEDIATED INDUCTION OF STEROIDOGENESIS.
Intracellular cAMP (A) and T levels in media (B) were evaluated after 4 hrs of treatment. EC50 determined by sigmoidal doseresponse curves fit by the ToxResponse Modeler for the co-treatment of 3 ng/ml rhCG and 1-600 μM ATR (in shaded area). Data are
plotted as mean ± standard error, n=3, and were analyzed by ANOVA followed by Dunnet’s post hoc test where #p<0.05 vs. rhCG.
The ToxResponse modeler was used to determine EC50 values based on sigmoidal dose-response curve modeling.

134

#

*

100

*
*

10

#

*

120
90

*
*

*

*

60
30
0

D

M

SO
AT
R

R FS
+ K
FS
K

AT

AT

R rhC
+ G
rh
C
G

AT

D

M

SO
AT
R

1

#

R FS
+ K
FS
K

*

1000

150

AT

B

#

R rhC
+ G
rh
C
G

10000

Testosterone (pg/ml)

cAMP (pmol/ml)

A

FIGURE 28. ATR-MEDIATED EFFECTS ON rhCG- OR FSK-INDUCED STEROIDOGENESIS.
Intracellular cAMP (A), and T levels in media (B) were evaluated after 4 hrs with DMSO, rhCG (3
ng/ml), FSK (10μM), ATR (300 μM), or the combinations of these treatments as indicated. Data
are plotted as mean ± standard error, n=3, and were analyzed by ANOVA followed by Tukey’s
post hoc test where *p<0.05 vs. DMSO, #p<0.05 vs. rhCG or FSK as indicated.

Inhibition of cAMP turnover by cPDE could also lead to elevated cAMP levels, thus the
effects of ATR on intracellular cAMP levels were compared to the effects elicited by the cPDEspecific inhibitor IBMX in BLTK1 cells (Figure 29). The potentiation of rhCG-induced intracellular
cAMP levels was comparable with ATR or IBMX co-treatment. However, the IBMX/rhCG cotreatment also resulted in increased T levels in contrast to the antagonism observed with
ATR/rhCG co-treatment. The co-treatment of IBMX with ATR did not change T levels compared
to ATR alone (Figure 29). These data suggest that cPDE inhibition elicits comparable effects as
ATR on rhCG-induction of intracellular cAMP levels. However, the super-induction of

135

intracellular cAMP in response to rhCG co-treatment with the cPDE inhibitor IBMX does not to
correlate with the antagonism of T levels observed with ATR co-treatment.

B

10000

*# *#

1000

*
100

#

150

Testosterone (pg/ml)

cAMP (pmol/ml)

A

#

10

50

*#
#
*# *

M
D

D

M

SO
A
TR AT
+ R
IB
M
X
IB
rh
M
C
X
+ G
A
TR rhC
+ G
rh
C
G

0

SO
A
TR AT
+ R
IB
M
X
IB
rh
M
C
X
+ G
A
TR rhC
+ G
rh
C
G

1

*

100

*#

FIGURE 29. ATR AND IBMX EFFECTS ON rhCG-INDUCTION OF cAMP AND T.
Intracellular cAMP (A), and T levels in media (B) were evaluated after 4 hrs of treatment with
DMSO, rhCG (3 ng/ml), IBMX (0.2 mM), ATR (300 μM), or the combinations of these treatments
as indicated. Data are plotted as mean ± standard error, n=3, and were analyzed by ANOVA
followed by Tukey’s post hoc test where *p<0.05 vs. DMSO, #p<0.05 vs. rhCG.

136

To confirm ATR inhibits cPDEs purified bovine PDEs were incubated with ATR or IBMX
positive control (Figure 30). Temporal evaluation using 300 μM ATR or 30 μM IBMX for 5-60 min
in the presence of cAMP substrate revealed ATR-mediated inhibition of PDE activity is most
robust at early time points inhibiting cAMP turnover nearly entirely. However, at ≥30 min ATR
only elicited ~60% inhibition while IBMX inhibited cAMP turnover >90% at all time points with
nearly no 5’-AMP produced throughout the time course. Dose-dependent inhibition of PDE was
conducted with 0.01-300 μM ATR or 0.01-200 μM IBMX for 10 min. This early time point was
selected based on ATR having more robust inhibition at early time points in the time course.
IC50s for PDE inhibition by ATR and IBMX at 10 min were ≥98 μM and 21 μM, respectively. The
highest concentration of ATR did not maximally inhibit cAMP turnover, as seen with ≥100 μM
IBMX. However greater concentrations were not evaluated due to solubility limit of ATR.
To confirm that super-induction of cAMP does not underlie antagonism of T production,
BLTK1 cells were treated with db-cAMP up to millimolar concentrations, comparable to
intracellular cAMP levels achieved with ATR/rhCG co-treatment. Concentration-dependent dbcAMP-mediated induction of T levels reached 5-fold at 3 mM, comparable to the levels
achieved with 3 ng/ml rhCG (Figure 31). These results demonstrate that millimolar
concentrations of cAMP are not sufficient to antagonize T production.

137

A

PDE Enzyme
300 M ATR
30 M IBMX

5'-AMP (nmol)

1.0

0.5

0.0
5

15

30

45

60

Time (min)

B
ATR IC50 : 98 M

5'-AMP (nmol)

1.0

IBMX IC50 : 21 M

0.5

0.0
0.01

0.1

1

3

10
10
10

30

100 300

Dose (M)

FIGURE 30. ATR-MEDIATED INHIBITION OF cPDE.
Temporal effects on PDE activity (A) were assessed by quantification of 5’-AMP produced upon
incubation of PDE enzyme and 3’,5’-cAMP substrate alone (black circles) or with the addition of
300 μM ATR (dark grey triangles) or 30 μM IBMX (light squares) for up to 60 min. Dosedependent effects of 0.01 – 300 μM ATR (dark grey triangles) or 0.01 – 200 μM IBMX (light grey
squares) were evaluated after 10 min (B), with amount of 5’-AMP produced with enzyme alone
(ie. 100% enzyme activity) depicted as the black hashed line. The ToxResponse modeler was
used to determine IC50 values based on sigmoidal dose-response curve modeling.

138

Testosterone (pg/mL)

120

*

*

100

*

80
60

* *

40

*

20
3

0.3
1

0.1

0.003
0.03

DMSO
3 ng/ml rhCG

0

db-cAMP
(mM)

FIGURE 31. EFFECT OF db-cAMP ON T LEVELS.
Concentration-dependent induction of T levels by db-cAMP at 4 hrs in BLTK1 cells demonstrates
that high mM db-cAMP concentrations do not antagonize T production. Data are plotted as
mean ± standard error, n=3, and were analyzed by ANOVA followed by Dunnett’s post hoc test
where *p<0.05 vs. DMSO.

ATR-MEDIATED CHANGES IN PROTEIN PHOSPHORYLATION
Many regulators of steroidogenesis including the transcription factors steroidogenic
factor 1 (SF-1) and cAMP response element binding protein (CREB), the cholesterol transporter
StAR, and PDEs are activated by PKA phosphorylation (38). Given the critical role of PKA in the
induction of steroidogenesis, the effect of ATR on PKA activity was visualized by western blot
using an antibody that detects PKA consensus motif phosphorylation. Whole cell extracts from
BLTK1 cells treated with DMSO, 3, 30, or 300 μM ATR for 1 hr were evaluated (Figure 32). The
139

results illustrate the promiscuity of PKA revealing many proteins with this phosphorylated
motif. While the identities of the proteins visualized on the western blot are unknown, ATR
resulted in dose-dependent effects on phospho-protein abundance. For example at ~35 kDa
and ~25 kDa the abundance of phospho-proteins appears to dose-dependently increase or
decrease, respectively.

FIGURE 32. ATR EFFECTS ON PKA SUBSTRATE PHOSPHORYLATION LEVELS.
Western blot detection of phosphorylated PKA motif phospho-Ser/Thr residue with Arg at the 3 and -2 positions ((K/R)(K/R)X(S*/T*)) from whole cell BLTK1 extracts after 1 hr of treatment
with DMSO or 3-300 μM ATR. Protein size (kDa) is labeled on the right. n=1.

140

The mitogen-activated protein kinase (MAPK) phosphorylation cascade interacts with
the PKA phosphorylation network, with many shared target proteins including PDEs and StAR
(39-45). More specifically, the MAPK ERK1/2 has been implicated in the regulation of
steroidogenesis, by phosphorylation that activates StAR (41, 42, 44) and inhibits PDEs (40, 43).
To evaluate the role of ERK1/2 phosphorylation in BLTK1 T biosynthesis, the MAPK kinase
(MEK1/2) responsible for activating ERK1/2 was inhibited using U0126 (Figure 33A). U0126
completely abolished rhCG-induced T production at ≥10 μM, confirming the importance of the
MEK/ERK phosphorylation cascade in BLTK1 steroidogenesis. Levels of phospho-ERK1/2 in
BLTK1 cells treated with 3 ng/ml rhCG or 300 μM ATR were similar to DMSO, while the
rhCG/ATR co-treated BLTK1 cells had significantly more phospho-ERK1/2 (Figure 33B). The
induction of phospho-ERK1/2 in co-treated BLTK1 cells further evaluated upon co-treatment of
3-300 μM ATR with 3 ng/ml rhCG (Figure 33C). Although no dose-dependent effect was
observed, it is possible that lower concentrations of ATR need to be evaluated.

DISCUSSION
Atrazine is a reproductive toxicant shown to disrupt androgen-mediated reproductive
development and antagonize serum T levels in male rodents (9-15). With Leydig cells as the
primary site of androgen steroidogenesis, the current study characterized the effects of
Atrazine on BLTK1 murine Leydig T biosynthesis. The in vitro BLTK1 cell line is a novel model
system amenable to mechanistic evaluation of steroidogenesis (36). Upon stimulation of Leydig
cells by gonadotropin (ie. rhCG or LH) rapid increases in the intracellular secondary messenger
cAMP (within an hour) and in P (~1-2 hrs) are expected, followed by increased T at later time
141

FIGURE 33. EFFECTS OF U0126 AND EVALUATION OF ERK1/2 PHOSPHORYLATION.
142

FIGURE 33 (cont’d)
(A) The effects of MEK1/2 inhibitor U0126 on T in BLTK1 cells was measured by EIA at 4 hrs
reveal no effects on basal T (grey bars) but dose-dependent inhibition of rhCG-elicited T
induction (hashed bars). Western blot detection of phosphorylated ERK1/2 (42/44 kDa) from
whole cell BLTK1 extracts after 1 hr of treatment. Data are plotted as mean ± standard error,
n=3, and were analyzed by ANOVA followed by Dunnet’s post hoc test where *p<0.05 vs. rhCG.
(B) Comparison of DMSO, 3 ng/ml rhCG, 300 μM ATR and rhCG/ATR co-treatment with and
without 10 μM U0126. (C) Dose-response evaluation of 3-300 μM ATR with 3 ng/ml rhCG.
Membranes were stripped and probed for actin (37 kDa) as a loading control. N=1.

points (>4 hrs) (46). These kinetics were observed in response to rhCG treatment in BLTK1 cells,
as well as in response to ATR albeit with lower efficacy, as shown previously (35). Interestingly,
cAMP levels were potentiated when rhCG-stimulated cells were treated with ATR while both P
and T levels were antagonized. The induction of P and T levels in un-stimulated (non-rhCG
treated) BLTK1 cells is similar to other in vitro studies such as induction of T in H295R cells (23,
24, 26). H295R cells do not have functional GPCR-inducible steroidogenesis, as such evaluating
the effect of ATR on gonadotropin stimulated steroidogenesis is not possible. Though our
results suggest ATR does not target GPCR-mediated activation of adenylyl cyclase, the ability to
make such determinations is unique to BLTK1 cells. In BLTK1 cells, antagonism of gonadotropininduced T biosynthesis is consistent with most in vivo reports describing ATR-mediated
decreases in interstitial testicular and circulating T levels (9, 13).
Several studies have suggested ATR is a cPDE inhibitor (26-29, 47). We have confirmed
ATR-mediated cPDE inhibition using isolated bovine PDEs. Furthermore, in BLTK1 cells the
effects on intracellular cAMP levels were comparable with ATR/rhCG and IBMX/rhCG cotreatments suggesting ATR elicits a similar cAMP induction profile as the cPDE inhibitor IBMX.
143

ATR-mediated cAMP accumulation in rat primary Leydig cells treated ex vivo was shown to be
PDE4-specific (27). PDE4, along with PDE8, is the highest abundance cPDE in Leydig cells (40). In
BLTK1 cells the gene expression of PDE4 isoform B (Pde4b) and PDE4 isoform D interacting
protein (Pde4dip) were induced nearly 2-fold after 12-48 hrs of ATR treatment, supporting ATRmediated effects on PDE4 in BLTK1 cells (35).
The results in BLTK1 cells suggest potentiated intracellular cAMP levels do not antagonize T
steroidogenesis. This was confirmed by IBMX/rhCG-elicited cAMP potentiation and by db-cAMP
treatment both increasing T production. Thus, accumulation of cAMP does not underlie ATRmediated antagonism of T levels in BLTK1 Leydig cells. These data are contradictory to previous
studies in primary Leydig cells where cPDE inhibition was assumed to cause decreases in T
production (26, 27). The study by Pogrmic-Majkic et al. (26) noted cAMP accumulation and
altered T production with ATR treatment; they abolished this effect with the addition of a PKA
inhibitor (H-89). Since PKA activation requires cAMP binding to the regulatory subunit, the
authors assumed cAMP signaling, via PKA, caused the changes in T levels. PKA activity was
affected by ATR in BLTK1 cells as shown by western blot, consistent with the previous study
suggesting a critical role of PKA in ATR-mediated disruption of Leydig cell steroidogenesis (26).
However, more studies are needed to identify the targets of PKA involved. Furthermore, not all
phosphorylated PKA substrate peptides increased in abundance as a function of ATR
concentration, rather there appears to be at least one peptide that decreases. These data
demonstrate that ATR dose-dependently affects PKA activity, and that evaluating changes in
the phospho-proteome is likely important for understanding how ATR disrupts steroidogenesis.
predicted activation of CREB as well as the differential expression of several SF-1 target genes
144

using whole-genome microarrays (35), supporting the hypothesis that PKA target protein
activity is affected by ATR in BLTK1 cells.

Of particular interest is the interaction between PKA and PDE4, as these two enzymes have
both been previously implicated in ATR-elicited disruption of steroidogenesis in Leydig cells (26,
27). In fact, both PKA and PDE4 are found bound to the same scaffold protein, A kinase anchor
protein (AKAP; Figure 34) (40-43, 48, 49). Scaffolding by AKAP keeps PKA near its target proteins
at specific subcellular locations to optimize spatiotemporal regulation of signaling pathways in
one complex (41, 42, 48). Furthermore, MAPK association with AKAP complexes, namely MEK
and ERK association, has also been demonstrated (39, 41, 48, 50). As a result, the tightly
controlled activity of regulatory enzymes is accomplished by a complex of AKAP-scaffolded
proteins. For example, PDE4 is phosphorylated at two unique sites: activated by PKA-mediated
phosphorylation in the up-stream conserved region near the N-terminus but inhibited by
ERK1/2-mediated phosphorylation in the catalytic region near the C-terminus (40, 43, 45, 49,
51). Additionally, the rate limiting step of steroidogenesis, mobilization of cholesterol to the
mitochondria by StAR, also requires phosphorylation by PKA, which is proposed to be facilitated
by interactions between PKA, StAR and AKAP at the mitochondria in Leydig cells (42, 52).
Activation of several other important steroidogenic regulatory proteins such as CREB, SF-1, and
StAR are also mediated by PKA and/or MAPK phosphorylation (41, 44, 52-54). We have shown

145

FIGURE 34. SIMPLIFIED SCHEME OF PKA ACTIVATION AND PKA INTERACTING PROTEINS.
Inactive PKA, comprised of two regulatory (R) and two catalytic (C) subunits, exists in the cytosol where it is in complex with AKAP
which serves as a scaffolding protein. PKA is activated upon binding of cAMP to (R) and subsequent release of (C) which
phosphorylates target proteins including key steroidogenic regulators including CREB, SF-1, StAR and cPDEs. PKA-mediated
phosphorylation of PDE4, also known to interact with the AKAP scaffold, activates PDE4 resulting in the turnover of cAMP and
deactivation of PKA. PDE4 can also be phosphorylated by phospho-ERK1/2 at an alternate site, resulting in inhibition of its activity.
146

In summary, the current study presents the novel hypothesis that ATR elicits disruption
of steroidogenesis in Leydig cells by altering PKA- and MAPK-mediated phosphorylation.
Specific target proteins of interest include StAR and PDE4, both of which can be phosphorylated
by PKA and ERK1/2 (42, 44, 45, 51). Our results support previous suggestions of ATR-mediated
cPDE inhibition and consequently accumulation of intracellular cAMP. However, the results
presented herein demonstrate that potentiation of cAMP levels does not cause antagonism of T
production in BLTK1 Leydig cells. Future studies will seek to identify the target proteins of PKA
showing altered phosphorylation status in an ATR-dependent manner and evaluate AKAPinteracting proteins that may affect PKA and ERK activity contributing to the disruption of
steroidogenesis.

147

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148

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US-EPA. (2006) Triazine Cumulative Risk Assessment, (EPA, U. S., Ed.), Office of
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Gilliom, R. J., Barbash, J.E., Crawford, C.G., Hamilton, P.A., Martin, J.D., Nakagaki, N.,
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154

CHAPTER 6

155

CHAPTER 6
CONCLUSIONS AND FUTURE RESEARCH

The preceding studies describe the establishment, characterization, and application of a
novel BLTK1 murine Leydig cell model for the evaluation of EDC-mediated effects on
steroidogenesis. BLTK1 Leydig cells offer a physiologically relevant, gonadotropin-inducible in
vitro system amenable to mechanism determination. Evaluation of the triazine CMG chemicals
ATR, PRO, SIM, DEA, DIA and DACT confirmed their equipotency for sterodogenic disruption,
but highlighted differences in their efficacy. Furthermore, evaluation of ATR as the
representative triazine revealed effects on both the short and long-term phases of
steroidogenesis. The short-term effects include potentiation of rhCG-induced intracellular
cAMP levels resulting from inhibition of cPDEs and changes in PKA-mediated phosphorylation.
The long-term effects include the differential expression of genes associated with cholesterol
metabolism, regulation of hormone levels and tissue morphology as well as the predicted
activation of AR and CREB. The work embodied in this dissertation presents a new model for
the evaluation of steroidogenesis that can be of great value to screen for reproductive and
developmental toxicants and novel findings regarding a suggested mode of action for triazinemediated disruption of steroidogenesis.

156

BLTK1 MURINE LEYDIG CELL MODEL FOR STEROIDOGENESIS
BLTK1 Leydig cells were characterized as a novel steroidogenic model for the evaluation
of reproductive and developmental toxicants. BLTK1 cells maintain the enzymes and regulatory
proteins necessary for steroidogenesis, biosynthesizing androgens and estrogen from
cholesterol in response to gonadotropin stimulus. Having a physiologically relevant in vitro
model for evaluating steroidogenesis allows for mechanism of action elucidation by using
molecular approaches that are not feasible in vivo. Though in vitro models are routinely used to
investigate mechanism of action, endocrine disruption is rarely limited to one cell type or
tissue. For example, triazines target the HPG axis by not only altering gonadal steroidogenesis
but also by disrupting LH surge in rodents. Studies evaluating ATR-mediated effects in the
hypothalamus or pituitary should also be evaluated to further characterize the effects of
triazine on the HPG. Similar cAMP- and MEK-mediated phosphorylation cascades regulate both
GnRH and LH release(1, 2), with PDE4 expression throughout the brain ATR may elicit
potentiation of cAMP in hypothalamus, however PDE10 is the only characterized cPDE in the
pituitary (3).

TRIAZINE EFFECTS ON PROTEIN PHOSPHORYLATION
Triazines may alter T biosynthesis in BLTK1 Leydig cells by disrupting PKA and MAPK
phosphorylation. However, the specific target proteins involved have not yet been identified.
The delineation between cAMP accumulation and disruption of T production described in
Chapter 5 is a novel finding that requires further examination. The hypothesis that ATR
interferes with AKAP-tethered protein interaction complexes affecting the phosphorylation of
157

interacting proteins can be evaluated as antibodies are readily available for specific phosphoproteins including PDE4. Additionally, mass spectrometry-based proteomic approaches can be
used to identify the PKA-targeted proteins. For example, the antibody used to visualize proteins
with PKA motif phosphorylation by western blot in Chapter 5 could be used for
immunoprecipitation to isolate target proteins with PKA phosphorylation. The extracted
proteins can be analyzed by mass spectrometry to attempt protein identification. Comparison
of proteins found in ATR-treated BLTK1 extracts compared to controls or rhCG-treatment can
reveal treatment-mediated effects. The integration of protein phosphorylation data with the
whole-genome microarray data in Chapter 4 and the characterized time- and dose-dependent
effects on P and T levels measured by EIA in Chapter 5 would provide a more complete
characterization of ATR’s mechanism underlying disruption of BLTK1 steroidgenesis.

158

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