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From the * Population Council and
The Rockefeller University, New York, New
York.
| Correspondence to: Dr Patricia L Morris, 1230 York Ave, New York, NY 10021 (e-mail: p-morris{at}popcbr.rockefeller.edu). |
| Received for publication May 11, 2007; accepted for publication October 18, 2007. |
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Abstract |
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Key words: COX-2, mitochondria, spermatocytes, Leydig, Sertoli
Phthalates are endocrine disruptors and peroxisome proliferators. Mono-(2-ethylhexyl) phthalate (MEHP), the biologically most active metabolite of DEHP, has deleterious effects on the male reproductive system, especially in neonatal and prepubertal males (Oishi, 1990; Li et al, 2000). Investigations over the last 3 decades indicate that Sertoli cells (SCs) and Leydig cells (LCs) represent the primary direct testicular targets for MEHP (Dostal et al, 1988; Heindel and Chapin, 1989; Thysen et al, 1990; Akingbemi et al, 2001; Mylchreest et al, 2002; Foster, 2005; Mahood et al, 2005; Ge et al, 2007) However, MEHP induces dramatic changes in germ cells, which until recently were thought to be mediated indirectly by somatic cell effects; these untoward effects include oxidative stress, mitochondrial dysfunction, cytochrome c release, and apoptosis (Richburg and Boekelheide, 1996; Kasahara et al, 2002). Induction of oxidative stress may represent a common mechanism in endocrine disruptor–mediated dysfunction, specific to certain testicular cells (Latchoumycandane et al, 2002). Recent evidence suggests that mitochondria are targets of phthalates. After administration of DEHP to rats, mitochondria isolated 6 hours later from the testis show reduced respiratory function (Oishi, 1990). In primary rat SCs treated with MEHP (24 hours), an increase in glycolysis, reduction of ATP levels, and a decrease in succinate dehydrogenase activity are observed (Chapin et al, 1988). Moreover, after in vivo or in vitro treatment with DEHP or MEHP, respectively, mitochondrial swelling in LCs has been observed (Jones et al, 1993). Collectively, these findings suggest that alterations in mitochondrial structure and function are cellular signatures of phthalate-induced testicular toxicity.
Given that mitochondria represent an intracellular target for MEHP and that increases in testicular reactive oxygen species (ROS) and oxidative stress follow MEHP exposure, we hypothesized that MEHP would affect levels of mitochondrial proteins involved in regulating cellular oxidation-reduction (redox) homeostasis.
Among oxidative stress–related genes altered in the testes of male rat fetuses exposed to phthalates, peroxiredoxin3 (Prx3) is reduced, as assessed using DNA microarray analysis (Liu et al, 2005). Increases in Prx3 protein are cytoprotective, maintaining mitochondrial integrity (Shibata et al, 2003; Matsushima et al, 2006); a significant reduction in Prx3 can potentially sensitize a cell to an apoptotic stimulus (Chang et al, 2004). Therefore, Prx3 may represent one target of MEHP-mediated oxidative stress in the male germline.
Induction of cyclooxygenase-2 (COX-2), arachidonic acid utilization, and prostaglandin production are critical regulators in response to cellular redox status and extracellular proapoptotic conditions (Feng et al, 1995; Jiang et al, 2004). Pertinent to this study in a germline model and further attesting to the overall biologic significance of this mechanism, a recent study shows that inhibition of COX-2 augments hydrogen peroxide–induced apoptosis in mouse embryonic stem cells (Liou et al, 2007).
Therefore, we investigated the effect(s) of short-term MEHP exposure on cellular Prx3 and COX-2 levels in male germ cells using the SV-40 immortalized mouse spermatocyte-derived cell line model, GC-2spd(ts) (Hofmann et al, 1994; Wolkowicz et al, 1996). Our findings indicate that MEHP disrupts spermatocyte cellular redox mechanisms.
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Materials and Methods |
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For reverse transcriptase–polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR (Q-PCR) analyses, rat SCs were prepared as in Jenab and Morris, 1998; and Morris et al, 1988; LCs were prepared as in Kanzaki and Morris, 1998, 1999; and germ cells were prepared as in Jenab and Morris, 1998. Total RNA was extracted from isolated testicular cells in TRIzol reagent (Invitrogen Life Technologies Inc, Grand Island, New York), according to the manufacturer's instructions. RNA concentration was measured using a Lambda35 UV/Vis Spectrophotometer (Perkin-Elmer Corp, Norwalk, Connecticut).
For bioimaging studies, whole testes were obtained from 7-week-old and 1-year-old male C57BL/6 mice, and cells were isolated and prepared as previously described (Page et al, 1998). Briefly, dissected testicles were placed in a freshly prepared solution of 2% formaldehyde in 1x calcium- and magnesium-free Dulbecco phosphate-buffered saline, pH 7.1 (PBS) with 0.05% Triton-X for 5–10 minutes at room temperature. Seminiferous tubules were liberated by removal of the tunica albuginea and dispersion into fixative. Tissue (3–5 mm) was placed in a small droplet of fixative on a glass microscope slide rinsed in 100% ethanol with 1 drop of concentrated hydrochloric acid. Tubules were minced to release cells, covered with a 22x22-mm cover slip, tapped gently with a pencil eraser to squash cells, and immersed into liquid nitrogen until the bubbling stopped. After freezing, the cover slip was removed, and the slide was immediately placed in PBS for three 5-minute washes and allowed to air dry. Dry slides were immediately used for immunolabeling or stored at –80°C.
RT-PCR Analysis
Total RNA (4 µg) was reverse transcribed for 15 minutes at 42°C in a
mixture containing 5 mM MgCl2, 1x PCR buffer II, 4 mM each of
deoxy-NTP, 1 U/µL ribonuclease inhibitor, and 2.5 mM random hexamers.
Samples were denatured at 99°C for 5 minutes. PCR was performed using 2
µL of each RT product as a template. The following primers were used: Prx3
forward primer, 5'-GCTGAGTCTCGACGACTTTAAGGG-3'; Prx3 reverse
primer, 5'-CTTGATCGT AGGGGACTCTGGTGT-3'; S16 ribosomal gene
forward primer, 5'-TCCGCTGCAGTCCGTTCAAGTCTT-3'; and S16 ribosomal
gene reverse primer, 5'-GCCAAACTTCTTGGATTCGCAGCG-3'. AmpliTaq DNA
polymerase (Applied Biosystems, Foster City, California) was used. The PCR
mixture (25 µL) contained 2 mM MgCl2, 1x PCR buffer II,
and each primer at 0.2 µM. Amplification was performed in a programmable
thermal controller (model PTC-100; MJ Research Inc, Watertown, Massachusetts).
The samples were first denatured at 95°C for 2 minutes, followed by 30 PCR
cycles; the temperature profile was 95°C (30 seconds), 60°C (30
seconds), and 72°C (1.5 minutes). After the last cycle, additional
extension incubation at 72°C (7 minutes) was performed. After
amplification, PCR products were separated on a NuPAGE-Novex polyacrylamide
gel (4%–20% Tris/boric acid/EDTA; Invitrogen). The bands were visualized
by ultraviolet fluorescence after staining with ethidium bromide (1 µg/mL)
for 15 minutes, and the image was recorded using a computer-assisted camera
(Eastman Kodak Co, Rochester, New York).
Q-PCR
Using a standard curve method of analysis, fluorescence-monitored Q-PCR
assays were conducted to quantitatively determine the levels of Prx3 mRNA in
each sample. For each experimental sample, 18S ribosomal RNA was used as an
endogenous control to normalize the Prx3 data. Reactions (25 µL total
volume) were set up in quadruplicate in optical quality 96-well reaction
plates containing 1x Power SYBR Green PCR Master Mix (Applied
Biosystems) and 200 nM primers for Prx3, 1x Q-PCR Master Mix Plus
(Eurogentec, Philadelphia, Pennsylvania), and VIC dye-labeled probe (Applied
Biosystems) for 18S ribosomal RNA. The experimental cDNA sample, 2 µL of a
sixfold dilution of the RT product, was subsequently added to each well. Q-PCR
was performed using an Applied Biosystems model 7700 Sequence Detection
System. The temperature profile for the reactions was 50°C (2 minutes),
95°C (10 minutes), and 40 cycles of 95°C (15 seconds) and 60°C (1
minute). Using the manufacturer's software, a threshold above noise was
chosen, and the cycle number (CT) at which fluorescence exceeded
the threshold was determined. For each real-time RT-PCR assay, a standard
curve was generated using cDNA corresponding to the control and based on 7
twofold serial dilutions ("control" cDNA in water). The mean
CT value for each cDNA sample was expressed as an arbitrary value
relative to the standard curve after linear regression analysis. Experimental
samples were diluted sixfold for comparison with the standard curve. A
no-template control, in duplicate, was performed for each reaction. Prx3 data
were normalized using the corresponding 18S values, and results are expressed
as arbitrary units relative to control, set as a value of 1.
Bioimaging and Immunofluorescence
Mouse testes from young (7-week-old) and older adult (1-year-old) mice were
used to obtain "squash preparations" of testicular cells. For
bioimaging and immunofluorescence, the previously air-dried slides were placed
in a 0.04% PhotoFlo solution (Eastman Kodak Co) for 2 minutes, drained, and
allowed to air dry. Once-dried slides were hydrated in 1x antibody
dilution buffer (ADB) containing 1% normal donkey serum (Sigma-Aldrich Corp,
St Louis, Missouri), 0.3% BSA, and 0.005% Triton-X (in PBS) for 30 minutes at
room temperature. Slides were incubated overnight with 60 mL of the primary
antibody cocktail, which consisted of Synaptonemal Complex Protein 3 (SCP3;
1:100; Novus, Littleton, Colorado), a standard marker of early meiotic cells,
and Prx3 (Santa Cruz Biotechnology, Santa Cruz, California) (1:100) in
1x ADB at 37°C in a humid chamber. Following the primary antibody
incubation, slides were washed once in 1x ADB for 20 minutes, then a
second wash in 1x ABD at 4°C for at least 5 hours. Slides were then
incubated for 1 hour with 60 mL of the secondary antibody cocktail
(fluorescein donkey anti-rabbit and rhodamine donkey anti-goat [1:100 in
1x ADB; Jackson ImmunoResearch, West Grove, Pennsylvania]) at 37°C
in a humid chamber. Slides were washed 3 times in 1x PBS for 10 minutes
each and counterstained with 4,6-diamidino-2-phenylindole dihydrochloride
(DAPI) in Antifade (BioRad Laboratories, Hercules, California), and a cover
slip was applied. Cells were located using a wide-field fluorescence
microscope (Zeiss, Oberkochen, Germany), and images were collected and
analyzed with the MetaVue acquisition software (Universal Imaging,
Downingtown, Pennsylvania) using a Hamamatsu Orca ER B/W digital camera.
MEHP
MEHP (TCI America, Portland, Oregon) was dissolved in DMSO to a
concentration of 400 mM and stored in single-use aliquots
(–20°C).
Cell Culture and MEHP Treatment
The GC-2spd(ts) cell line, herein abbreviated as GC-2, was purchased from
the American Type Culture Collection (Manassas, Virginia). This cell line
expresses a temperature-sensitive mutant of the mouse p53 tumor suppressor
gene, encoding an Ala135 to Val135 change that affects protein folding. Cells
were cultured at 37°C, at which they express both wild-type and mutant p53
(Hofmann et al, 1994;
Wolkowicz et al, 1996), and
were maintained in log-phase growth in flasks (75 cm2; BD Falcon,
Bedford, Massachusetts) at 5% CO2 with Dulbecco modified Eagle
medium (Invitrogen) containing 10% FBS (JRH Biosciences, Lenexa, Kansas) and 4
µg/mL Gentamycin (Invitrogen).
Cells were seeded in replicate culture plates (as indicated below) 24 hours prior to dosing. MEHP was added to a final concentration of either 100 or 200 µM MEHP. Cells were treated with either 0.05% or 0.1% DMSO as the matched vehicle control, as indicated. Following treatment, cells were maintained for 24 hours until harvested.
Cell Proliferation
Cells were seeded at a density of 2 x 105 cells/well in
6-multiwell plates (Becton Dickinson, Franklin Lakes, New Jersey), and
harvested using trypsin (0.25%; Invitrogen). Cell number was determined using
a hemocytometer, and viability was assessed by trypan blue exclusion. For cell
cycle studies, cells were harvested by trypsinization, rinsed, an aliquot
counted, and the remaining cells fixed in 70% ethanol, then stored at
–20°C until use. On the day of the flow cytometric assay, cells were
washed twice (PBS, 1% FBS), labeled with Isotype or Ki67 FITC (BD Pharmingen,
San Diego, California), and incubated for 30 minutes at room temperature in
the dark. Cells then were washed once, samples were aspirated, and cells were
resuspended in PI Master Mix (propidium iodide, RNAase, and PBS 1% Triton-X).
The cells were incubated in the dark (30 minutes, room temperature) and then
analyzed using a FACS Calibur (BD Biosciences, San Jose, California).
Preparation of Whole-Cell Lysates
Cells were seeded at a density of 1 x 106 in
75-cm2 flasks, rinsed in ice-cold PBS, and harvested with a 25-cm
cell scraper (Sarstedt Inc, Newton, North Carolina). Whole-cell lysates were
obtained (Ishikawa et al,
2005). Protein concentration was determined with BSA as a standard
using the Bio-Rad protein assay reagent (Bio-Rad Laboratories). Absorbance was
read at 590 nm in a Lambda35 UV/Vis Spectrophotometer (Perkin-Elmer Corp).
Preparation of Mitochondrial-Enriched and Cytosolic Fractions
Cells were seeded at a density of 3 x 106 in
150-cm2 flasks (Corning Costar Corp, Cambridge, Massachusetts).
After rinsing with ice-cold PBS, cells were harvested using a 38-cm cell
scraper (Sarstedt) with PBS (5 mL) twice, pooled, pelleted by centrifugation
at 650 x g (5 minutes, room temperature), suspended in 1 mL
PBS, and centrifuged at 960 x g (5 minutes; 4°C;
microcentrifuge [model 5417R; Eppendorf, Westbury, New York]). The wash was
repeated and pellets resuspended in 1 mL of sucrose mitochondrial isolation
buffer (SMIB; 0.25 M sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM NaCl, 1 mM EDTA,
1 mM EGTA, pH 7.5, containing 1 mM dithiothreitol, 1 mM sodium orthovanadate,
0.1 mM phenylmethylsulfonylfluoride, 2 µg/mL aprotinin, 2 µg/mL
pepstatin, and 2 µg/mL leupeptin) and incubated on ice (10 minutes). Cells
were lysed by 25 up-and-down passes through a 27.5-gauge G needle fitted to a
1-mL syringe (Becton Dickinson). Homogenates were centrifuged at 1020 x
g (10 minutes), supernatants centrifuged at 1020 x g
(10 minutes) to remove unbroken cells and nuclei, and then centrifuged at 15
000 x g (10 minutes) to pellet mitochondria. The
postmitochondrial supernatant was taken as the "cytosolic"
fraction. The mitochondrial-enriched pellet was suspended in 200 µL of SMIB
and centrifuged at 12 200 x g (10 minutes); this wash was
repeated once. The final mitochondrial-enriched pellet was suspended in SMIB,
and samples were stored at –80°C. Protein concentration was
determined using Bio-Rad protein assay reagent; absorbance was read at 590 nm
using an MRX plate reader with Revelation software, version 3.2 (DYNEX
Technologies Inc, Chantilly, Virginia).
Western Blot Analysis
Whole-cell lysates (15 µg), mitochondrial-enriched extracts (15 µg),
or cytosolic extracts (15 µg) were subjected under reducing conditions to
sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 10%
Tris/Bis NuPAGE gels (Invitrogen) and were transferred using the Bis/Tris
transfer system (Invitrogen) to a 0.45-µm nitrocellulose membrane
(Schleicher & Schuell, Keene, New Hampshire). Membranes were hybridized
sequentially with rabbit anti-mouse Prx3 antiserum (1:2000; a gift from Dr Chi
V. Dang, Johns Hopkins University, Baltimore, Maryland), rabbit polyclonal
anti–COX-2 antibody (1:3000; Cayman Chemical Co, Ann Arbor, Michigan),
mouse monoclonal anti–voltage-dependent anion channel (VDAC) antibody
(1:1000; clone 89-173/016; EMD Biosciences, San Diego, California), and mouse
monoclonal anti–β-actin antibody (1:12 000; Sigma-Aldrich). Blots
were developed with the ECL Western blotting system (Amersham Biosciences,
Arlington Heights, Illinois) and exposed to X-ray film (Eastman Kodak).
Densitometric analysis was performed using NIH ImageJ, version 1.33u
(http://rsb.info.nih.gov/ij/index.html).
Signals were normalized to VDAC (mitochondrial-enriched) or β-actin
(whole-cell or cytosolic) protein lysates.
Statistical Analysis
Densitometric analyses are expressed in arbitrary units. All results are
the mean ± SEM derived from the number of individual different
experiments (3–7), unless noted. For cell line studies, the vehicle
"control" was used for normalization to a value of
"one" for each experiment. For cell-specific expression studies,
data were normalized as indicated in the figure legend. Statistical analyses
to determine significant differences used 1-way ANOVA analysis with Dunnett
multiple comparison test. A P value 
.05 was considered
significant.
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Results |
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RT-PCR analyses were performed to determine the physiologic expression of Prx3 in the rodent testis. Since rat and mouse models are used primarily to investigate the effects of phthalates on male reproductive biology, primers that recognize both rat and mouse Prx3 mRNA transcripts in freshly isolated testicular cells were used in this study. Our studies indicate that prx3 mRNA is expressed in spermatogonia, pachytene spermatocytes, and round spermatids of both rat and mouse testis (Figure 1). Interestingly, pachytene spermatocytes from both rat and mouse testis show the highest expression levels of prx3 mRNA comparatively among these germ cells (Figure 1A).
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Pachytene spermatocytes show the highest levels of prx3 among the male germ cells examined. Therefore, we next determined Prx3 in mouse meiotic spermatocytes. For the immunofluorescent detection of Prx3, we used cells obtained from squash preparations of whole testes obtained from 7-week-old and 1-year-old adult mice (Figure 3). Pachytene spermatocytes were identified using an antibody against SCP3, the linear structures representing the meiosis-specific synaptonemal complexes that tether duplicated homologous chromosomes for recombination (Figure 3A and B). As illustrated, pachytene spermatocytes were abundant in the mouse testes of both young adult and older males (Figure 3A, inset). Prx3 was strikingly localized at the perimeter of the nucleus and in the cytoplasm of the meiotic germ cells. These bioimaging observations were also confirmed using a second antibody against Prx3 (data not shown).
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To determine whether short-term MEHP exposure directly affects proliferation, GC-2 cells were treated with MEHP for 24 hours. Compared with matched vehicle-treated control cells, those exposed to 100 or 200 µM MEHP were significantly reduced in numbers by 20% and 40%, respectively (Figure 4). Cell viability, as assessed by trypan blue exclusion, was unaffected by MEHP treatment (Figure 4, inset), indicating no induction of cell death. No change in adherence or anoikis (death by detachment) was noted. Cell viability data are consistent with our cell cycle studies indicating that there were no changes in cell cycle distributions (data not shown). Our findings indicate that MEHP (doses up to 200 µM) did not induce cell death or cell cycle arrest in GC-2 cells.
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To determine the mechanisms involved in the response of GC-2 cells to MEHP,
total and mitochondrial protein levels of the Prx3 antioxidant protein were
evaluated. MEHP treatment (24 hours) did not alter total Prx3 protein levels
(Figure 5A). Next, the
mitochondrial-enriched fraction was isolated from MEHP-treated GC-2 cells.
MEHP (200 µM) significantly increased mitochondrial Prx3 levels (
50%;
Figure 5B), whereas increases
at 100 µM MEHP were not significant (40%;
Figure 5B). These data show
that mitochondrial Prx3 is increased in response to MEHP.
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Both MEHP (Ledwith et al, 1997) and oxidative stress (Feng et al, 1995; Li et al, 2002) have been reported to regulate COX-2 levels. Therefore, we next investigated whether MEHP induces COX-2 in GC-2 spermatocytes.
MEHP Induces COX-2 Expression in GC-2 Cells
In situ hybridization studies to determine cellular expression in the
testis of normal adult rats showed basal levels of COX-2 mRNA in
spermatogonia, spermatocytes, and SCs
(Winnall et al, 2007).
Previous studies from our laboratory show low, basal expression of COX-2 in
rat LCs and SCs (Walch and Morris, 2002;
Ishikawa et al, 2005). The
current study shows that COX-2 is constitutively expressed in the
SV-40–transformed GC-2 cell line, similar to those heightened levels
shown in several immortalized cancer-derived and non–cancer-derived cell
lines (Lee et al, 2002;
Liou et al, 2005;
Richardson et al, 2005). MEHP
(100 or 200 µM; 24 hours) did not significantly further increase these
steady-state cellular COX-2 levels (Figure
6A).
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Because COX-2 is predominately located in the nuclear envelope as well as
endoplasmic reticulum, substantive subcellular domain changes could be masked
in whole-cell lysates. Therefore, cellular fractionation was performed to
determine whether subcellular COX-2 levels may be affected following acute
MEHP exposure. Mitochondrial COX-2 significantly increased following 200 µM
MEHP treatment (40%; Figure
6B). Subsequently, the postmitochondrial supernatant containing
lighter-weight membrane-bound organelles and cytosolic proteins was collected
as the cytosolic fraction and analyzed by Western blot for COX-2.
Additionally, cytosolic COX-2 levels significantly increased following 200
µM MEHP (60%; Figure 6C).
However, MEHP did not affect nuclear COX-2 levels (data not shown). Increases
in mitochondrial and cytosolic COX-2 levels are significant and contribute to
the nonsignificant increase observed in the overall cellular COX-2 levels
(Figure 6A). Taken together,
the data are indicative of increased mitochondrial COX-2 levels in GC-2 cells
in response to MEHP treatment.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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As both LCs and SCs have been shown to be primary targets of phthalate action, we determined prx3 expression in the somatic cells of the rat testis. Our findings of differential expression for Prx3 between LCs and SCs, as well as between their immature relative to their differentiated cell types, may have physiologic implications during development. For example, expression in a given cell may provide a protective mechanism with which to respond to such a stress, or its relative deficiency could underlie vulnerability or sensitivity to low toxicant levels at various stages of maturation, perhaps as an underlying contributory factor to the differential effects of phthalates on neonatal and adult testis.
Both analyses by RT-PCR and bioimaging show that pachytene spermatocytes express Prx3. Therefore, we focused on Prx3 in the spermatocyte as a novel target for the direct action of phthalates on the germ cell. Since exposure to MEHP (50–200 µM) and not DEHP induces oxidative stress in germ cells but not SCs in cultures of testicular cells isolated from DEHP-treated rats (Kasahara et al, 2002), the Prx3-positive, spermatocyte-derived germ cell line GC-2 was employed to model the effects of short-term MEHP exposure on cellular redox parameters in the spermatocyte. Our findings that MEHP does not induce cell death in GC-2 cells are in agreement with a recent study that showed that exposure to MEHP (200 µM; 24 hours) does not induce apoptosis in GC-2 cells (Chandrasekaran et al, 2006). However, we did observe a reduction in cell number after 24-hour exposure to both 100 and 200 µM MEHP without changes in cell cycle parameters. Similar inhibitory effects of phthalates on cell growth have been reported using other nontesticular cell lines. For example, dimethoxyethyl phthalate inhibits growth of mouse fibroblast cells (Dillingham and Autian, 1973). The present findings are consistent with a MEHP effect on cell proliferation.
Here we report that 24-hour exposure to MEHP increases mitochondrial Prx3.
Oxidative stress and mitochondrial insult can increase levels of Prx3. For
instance, the bovine homolog of Prx3 increases in cow aortic endothelial cells
after 24-hour exposure to various oxidative stresses and 12-hour exposure to
actinomycin A, a mitochondrial respiratory chain inhibitor
(Araki et al, 1999). Increases
in the antiapoptotic Prx3 in the GC-2 cells are consistent with the findings
that 24-hour exposure to MEHP does not induce apoptosis. Our data suggest that
the ability to induce Prx3 is protective, since reducing Prx3 sensitizes cells
to oxidative stress and apoptosis. For example, acute myeloid leukemia cells
showing heightened sensitivity to oxidative stress have an approximately
threefold decrease in Prx3 expression (Oh
et al, 2004). Prx3 levels are downregulated in motor neuron
disease, a disease characterized by oxidative stress
(Wood-Allum et al, 2006). Both
acute and chronic oxidative injuries can lead to reduced Prx3 levels
(Hattori et al, 2003;
Wood-Allum et al, 2006).
Furthermore, depleting Prx3 not only increases intracellular levels of ROS in
unstimulated HeLa cells, but also increases ROS generation and subsequent
apoptosis induced by staurosporine (mitochondrial-dependent pathway) and by
tumor necrosis factor-
(death receptor–mediated pathway;
Chang et al, 2004). Prx3
knockout mice have reduced body weight and are more susceptible to
lipopolysaccharide-induced oxidative stress than their wild-type littermates
(Li et al, 2007). Taken
together, these data indicate that the loss of Prx3 results in diminished
protective responsiveness and increased susceptibility to oxidative stressors.
Given the role of Prx3 in maintaining cellular redox and mitochondrial
homeostasis, as well as preventing mitochondrial-dependent apoptosis, the
increases in mitochondrial Prx3 steady-state levels seen in GC-2 cells reflect
changes in cellular redox homeostasis and/or inhibition of apoptosis following
short-term MEHP exposure. By extension, our studies suggest that individual
germ cell sensitivity to untoward effects of phthalates may be based in part
on Prx3 levels.
Prx3 is not only important in maintaining both mitochondrial and cellular redox homeostasis, but also influences cell growth. In this study, increases in steady-state levels of endogenous Prx3 accompany the reduction in GC-2 cell number after short-term exposure to MEHP. In a previous study, Prx3 overexpression in mouse WEHI7.2 thymoma cells slowed cell proliferation without alteration in apoptosis relative to the vector control (Nonn et al, 2003). However, another study reported that stable transfection with Prx3 antisense DNA of the Rat1a fibroblast cell line overexpressing the transcription factor c-Myc (R1a-myc) and a human breast cancer epithelial cell line (MCF7/ADR) increased doubling times compared with vector controls (Wonsey et al, 2002). These reports demonstrate the ability of Prx3 to affect cell growth, and they suggest collectively that the effects of Prx3 on cell growth are cell type dependent. The slowed growth rate observed in GC-2 cells in response to MEHP could be, in part, a consequence of the effects of increased mitochondrial Prx3.
Given the effect of MEHP on Prx3 in GC-2 cells, we investigated whether short-term exposure to MEHP would affect steady-state levels of COX-2. Various stimuli can alter redox homeostasis and induce COX-2 (Feng et al, 1995; Kiritoshi et al, 2003). COX-2 protects mouse embryonic stem cells from oxidative stress–induced apoptosis (Liou et al, 2007). MEHP has been shown to potently induce COX-2 in an immortalized mouse hepatocyte cell line (Ledwith et al, 1997). First, we found COX-2 to be constitutively expressed in GC-2 cells, consistent with basal levels of expression in several immortalized cancer-derived and non–cancer-derived cell lines (Lee et al, 2002; Liou et al, 2005; Richardson et al, 2005), unlike primary rat SCs and human fibroblasts, which have low to nondetectable basal COX-2 levels (Ishikawa et al, 2005; Liou et al, 2005). In situ hybridization studies to determine cellular expression in the testis of normal adult rats showed basal levels of COX-2 mRNA in spermatogonia, spermatocytes, and SCs (Winnall et al, 2007). The constitutive expression of COX-2 observed in the GC-2 cell model is reminiscent of that in normal testicular physiology. Second, COX-2 is detected in the mitochondrial-enriched fractions of GC-2 cells, and the mitochondrial levels increase following 24 hours of MEHP exposure, changes that may afford resistance to MEHP-induced apoptosis. In agreement with these findings, in several cancer cell lines, COX-2 localizes to mitochondria and confers resistance to apoptosis induced by oxidative stress (Liou et al, 2005). Third, the effect of MEHP exposure overall is an increase in COX-2 in GC-2 spermatocytes.
Induction of COX-2 or stabilization of its protein may partially represent underlying mechanisms responsible for the slowed growth of GC-2 cells observed. For example, overexpression of COX-2 induces cell cycle arrest by a prostanoid-independent mechanism in various immortalized cell lines, including human umbilical endothelial vein cell–derived ECV-304, mouse fibroblast NIH3T3, African green monkey kidney fibroblast-like COS7, and human embryonic kidney HEK293, as well as in primary bovine microvascular endothelial cells (Trifan et al, 1999). Interestingly, 100 µM MEHP, a dose that potently induces COX-2 protein levels, does not significantly affect prostaglandin E2 synthesis in immortalized mouse hepatocytes (Ledwith et al, 1997). Whether COX-2 effects on GC-2 cell growth involve the activation of a specific prostaglandin cascade or subsequent peroxidase activity remains to be determined in ongoing studies.
Rat testis removed 6–24 hours after administration of DEHP (2 g/kg) showed increased ROS, as measured by superoxide and hydrogen peroxide generation. Exposure to doses up to 50–200 µM MEHP but not DEHP for 30 minutes increases ROS generation in primary rat germ cells but not SCs obtained from DEHP-treated rats (Kasahara et al, 2002). Our present study identified spermatocyte Prx3 and COX-2 as potential cellular MEHP sensors and indicates that increased steady-state levels of both Prx3 and COX-2 could result from early redox signaling events following MEHP exposure.
In summary, Prx3 is differentially expressed in mouse and rat testicular cells. Under normal physiologic conditions in the rodent testes, the expression of both stressor responders Prx3 and COX-2 in spermatocytes validates the utilization of this mouse spermatocyte cell line model to further identify the effects of MEHP on cellular redox homeostatic mechanisms. The present study identified 2 direct germ cell phthalate responses; that is, increases in 2 redox-sensitive proteins, mitochondrial Prx3 and COX-2. Short-term exposure to MEHP negatively affects cell proliferation, an effect accompanied by increases in Prx3 and COX-2, despite the absence of cell death. Further understanding of cellular redox status following transient exposure of the male germline to MEHP will facilitate our understanding of the male reproductive health risks of chronic, low level, and/or long-term exposure to phthalates in our environment.
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Acknowledgments |
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Footnotes |
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98% and progenitor, 90%–93% purity) to determine prx3 mRNA
expression. (A) A representative gel image of the RT-PCR cDNA products.
(B, C) Q-PCR values for prx3 mRNA and 18S are expressed
as a ratio, normalized as arbitrary units relative to the immature Sertoli
cell level, which is set as "1". All samples (n = 4) were analyzed
simultaneously in the same prx3 mRNA and 18S assays. Error bars
represent SEM, *p < .05; **p < .01 indicate
significance differences of the means from that of the immature SCs using
ANOVA with Dunnett multiple comparison, n = 4. Note the difference in scales,
Panel B vs Panel C.
