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ABSTRACT: Mono-(2-ethylhexyl) phthalate (MEHP), the biologically active metabolite of the plasticizer di-(2-ethylhexyl) phthalate, is a member of a class of chemical compounds with known adverse effects on the male reproductive system. Recent studies showed that oxidative stress and mitochondrial dysfunction in germ cells may contribute to phthalate-induced disruption of spermatogenesis. To determine whether the redox-protein mitochondrial thioredoxin-dependent peroxidase, peroxiredoxin 3 (Prx3), may be a component of germ cell homeostasis mechanisms, this study first examined the physiologic relevance of Prx3 in the rodent testis by determining its cell-specific expression. Our findings show that prx3 mRNA is expressed in a developmental, cell-specific manner in rat Leydig cells, Sertoli cells, and germ cells; among mouse germ cells, prx3 expression was highest in spermatocytes, findings consistent with those in rat. In mouse meiotic spermatocytes, Prx3 was strikingly localized at the nuclear perimeter and cytoplasm, findings suggestive of a direct role for Prx3 in determining spermatocyte response to toxicants. To better define the mechanisms involved in male germ cell dysfunction following phthalate exposure, an immortalized mouse spermatocyte-derived germ cell line, GC-2spd(ts), was exposed to MEHP (24 hours; 100 and 200 μM). We determined whether Prx3 and cyclooxygenase-2 (COX-2), pivotal proteins involved in oxidative stress responses in spatially restricted subcellular domains, were affected. Mitochondrial Prx3 and mitochondrial and cytosolic COX-2 significantly increased following 200 μM MEHP treatment; proliferation was inhibited without inducing cell death. Using this germ cell model, the data suggest that changes in cellular oxidation-reduction (redox) homeostasis in the germline can accompany MEHP exposure, disrupting mitochondrial antioxidant defenses, despite absence of phthalate-induced apoptosis.
Consumer and industrial demand for polyvinyl chloride (PVC)–containing products has resulted in increased commercial synthesis of plasticizers, such as the commonly used phthalate, di-(2-ethylhexyl) phthalate (DEHP). Exposure to phthalates is ubiquitous—for example, in food containers, toys, baby bottles, intravenous tubing, blood storage bags, PVC flooring, and household dust (Graham, 1973; Rock et al, 1986; Bornehag et al, 2004). Phthalates can leach out of products, increasing human exposure levels of phthalates (Rock et al, 1986). Raising further health concerns, phthalates have been found in human breast milk (Main et al, 2006).
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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Investigations in vivo and in vitro from multiple laboratories over the last 25 years have established that LCs and SCs are the primary targets of phthalate action in the testis (Gray and Butterworth, 1980; Foster et al, 1983, 2001; Sjoberg et al, 1986; Sharpe, 2001). Based on several studies, the major mode of action of MEHP on male germ cells is believed to be indirect (ie, through SC and LC dysfunction). However, DEHP metabolites have been found in germ cells, findings consistent with direct effects (Ono et al, 2004). Furthermore, at levels that induced apoptosis in germ cells and resulted in atrophy of the testis, DEHP treatment provoked oxidative stress, as measured by increases in ROS in subsequently isolated spermatocytes, but not SCs (Kasahara et al, 2002). Thus, the mitochondrion and redox homeostasis in male germ cells may represent a new target of phthalates, both organelle and stress-response mechanism directly and/or indirectly affected by MEHP exposure. Herein, this study investigated whether a mitochondrial redox protein, Prx3, is expressed in the rodent testis, with specific focus on developing male germ cells and the spermatocyte. Spermatogonia, pachytene spermatocytes, and round spermatids differentially express prx3. The differential expression of Prx3 between mitotic, meiotic, and postmeiotic germ cells may afford a defense against environmental stressors to some developmental stages while rendering others more susceptible to those stresses.
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-α (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.