Exposure to DEPEs increased activation of JNK, ER chaperone GRP78, and

ER stress has shown to be associated with apoptosis. To examine whether ER stress plays an important role in DEPEs-induced apoptosis in H9c2 cells, cells were treated with 3, 15, and 25 μg/mL DEPEs for 24 hours. There was an increase in expression of GRP78 and CHOP after 25 μg/mL DEPEs treatment for 24 hours (Figure 6). H9c2 cells were then exposed to 25 μg/mL DEPEs for various time periods (3, 6, and 24 hours),

and the protein levels of GRP78 and CHOP were elevated after incubation for 24 hours (Figure 7A). Many studies have demonstrated that JNK is related to ER stress (Schröder

& Kaufman, 2005b; Szegezdi et al, 2006). And a short-term exposure to DEPEs found that JNK was phosphorylated within 1 hour (Figure 7B). These results showed that DEPEs activated JNK and induced the expression of ER stress markers GPR78 and CHOP.

2.4 P-JNK mediated DEPEs-induced upregulation of GRP78 and CHOP.

DEPEs activated of JNK prior to activation of ER stress markers, GRP78 and CHOP. In order to further identify the signal transduction pathway involved in the mechanism behind DEPEs-induced cytotoxicity, H9c2 cells were pretreated with SP600125 (JNK inhibitor) for 1 hour and followed by the administration of DEPEs (25 μg/mL) for 1 hour (Figure 8A) and 24 hours (Figure 8B). The cells were lysed and subsequently subjected to western blot assay to assess the effects of the JNK inhibitor on DEPEs-induced ER stress markers. The results showed that SP600125, JNK inhibitor, completely suppressed DEPEs-induced expression of GRP78 and CHOP, suggesting that DEPEs induced ER stress through JNK pathway.

2.5 Necrosis is also involved in DEPEs-induced cytotoxicity.

In figure 5, treatment of 25 μg/mL DEPEs also caused an increase in PI-positive H9c2 cell population. To confirm if necrosis is involved in DEPEs-induced cytotoxicity, H9c2 cells were treated with 3, 15, and 25 μg/mL DEPEs for 24 hours. LDH leakage, as a marker of cell membrane damage due to necrosis, was measured. And LDH released from H9c2 cells was significantly increased at 24 hours after DEPEs administration (Figure 9A). Loss of intracellular ATP level is another characteristic of necrotic cell

death, and it was decreased after DEPEs treatment (Figure 9B). These results indicated that DEPEs induced necrosis in H9c2 cells.

CHAPTER IV Discussion

Part I: Effects of DEPEs on neonatal rat cardiomyocytes

Cardiac hypertrophy is a common endpoint of many cardiovascular diseases and is characterized by an increase in cardiomyocyte size and protein content. Because of the small sizes of DEPs, they can penetrate the epithelium and vascular walls, and enter the bloodstream. Therefore, the toxicity of organic and inorganic constituents of the particles is important. The present study demonstrated that DEPEs induced cardiomyocyte hypertrophy, in which PKC-mediated MAPK pathway might be involved.

In this study, 1 to 15 μg/mL DEPEs had no significant effects on neonatal rat cardiomyocyte viability. Under this condition, the toxic effects of DEPEs on cardiomyocytes were investigated. DEPEs (5 μg/mL) increased cardiomyocyte size and the ratio of total protein/cell, which are characteristics of cardiac hypertrophy.

Meanwhile, the mRNA levels of hypertrophic markers BNP and β-MHC were enhanced.

These data suggested that DEPEs can induce cardiomyocyte hypertrophy. This finding is consistent with the observation that traffic exposure is associated with higher left ventricular mass in adults (Van Hee et al, 2009).

Hypertrophy can cause alterations in contractile protein compositions, including reactivation of β-MHC, skeletal α-actin, and myosin light chain-2 genes (Chien et al, 1991). It can also result in induction of non-contractile protein genes such as ANP and BNP (Cameron & Ellmers, 2003; Rohini et al, 2010). ANP and BNP defend against

increased hemodynamic load by decreasing blood pressure, regulating fluid homeostasis by increasing salt and water excretion, and regulating several hormones, such as angiotensin II, endothelin-1, and vasopressin. ANP and BNP are predominantly located in the cardiac atria and ventricle, respectively. BNP is also found in atria. Both ANP and BNP decrease plasma volume and blood pressure in response to an increased tension of the respective cardiac chamber (Krishnaswami, 2008). Activation of ERK and p38 MAPKs by hypertrophic agonist or by activated upstream kinase was reported to lead to phosphorylation of GATA-4, and analysis of the ANP and BNP promoter regions has also revealed binding sites for GATA-4 (Kerkela et al, 2002). Besides, full induction of β-MHC in pressure-overloaded rat hearts requires intact GATA binding elements in the

promoter regions (Pikkarainen et al, 2004). And Induction of ERK and JNK pathways could induce BNP promoter activity independently of GATA-4 binding (Kerkela et al, 2002). Thus, MAPKs are able to induce gene expression of hypertrophic markers, ANP, BNP, and β-MHC. In the present study, DEPEs induced the activation of MAPKs and

these hypertrophic markers in neonatal rat cardiomyocytes, which indicated that DEPEs might elevate gene expression levels of the hypertrophic markers through MAPKs pathway.

It has been hypothesized that ER stress is a pathogenic factor responsible for inducing cardiac hypertrophy. In most cell types, phosphorylation of eIF2α is involved in the initiation of the UPR, leading to a general inhibition of protein synthesis (Schröder & Kaufman, 2005a); however, dephosphorylation of eIF2α via ATF4 induced expression of CHOP and induction of GADD34 reverses the inhibition (Wek et al, 2006). ER stress may induce protein synthesis directly in cardiomyocytes, and thereby cause cell enlargement and cardiac hypertrophy (Dickhout et al, 2011), but the mechanisms by which ER stress induces protein synthesis in cardiomyocytes are still

unknown. On the other hand, angiotensin II, a known mediator of cardiac hypertrophy, has been shown to induce ER stress in rat cardiomyocytes by increased ER chaperone and CHOP expression (Okada et al, 2004). Therefore, ER stress can be involved in DEPEs-induced cardiomyocyte hypertrophy.

A variety of organic and inorganic constituents of DEPs have the potential to cause ROS generation. Inhaled particles that deposit within the distal lung have the potential to generate ROS, depending on the surface area of the particle and the reactive composition of the particle. Many transition metals present on particles serve as catalysts for ROS production. DEPs and lipopolysaccharide from gram negative bacteria have also been shown to stimulate generation of ROS in alveolar macrophages (Bonner, 2007). Besides, angiotensin II increases β-MHC gene expression in part via the generation of ROS (Shih et al, 2001). It was reported that DEPEs decreased neonatal rat cardiomyocytes viability and it might be due mainly to ROS formation (Okayama et al, 2006). And ROS has shown to participate in activation of p38 and JNK via apoptosis signal-regulating kinase 1 (Saitoh et al, 1998). Collectively, it suggested that ROS might be involved in DEPEs-induced cardiomyocyte hypertrophy.

Part II: Effects of DEPEs on H9c2 cells

Many studies have reported that DEPs induce oxidative stress, which played a key role for cascade activation during DEPEs-induced cellular injury (Bonner, 2007;

Okayama et al, 2006). In the present study, 15 and 25 μg/mL DEPEs significantly reduced H9c2 cell viability, where apoptosis and necrosis coexist.

After exposure to DEPEs extracted at different times, sometimes apoptotic and necrotic cell population both could be observed, and sometimes only necrotic cell population could be detected. DEPEs extracted at different times may vary in contents of organic compounds. Although the procedures of extraction were standardized as possible, there still may be some inaccuracies. Components of DEPEs should be standardized after every extraction, and this is the disadvantage of extraction.

Compared with DEPEs-induced cytotoxicity in neonatal rat cardiomyocytes at same dose, DEPEs seemed to cause more severe cytotoxicity in H9c2 cells. Although H9c2 cells and neonatal rat cardiomyocytes were reported to show similar hypertrophic responses in vitro (Watkins et al, 2010), they are still different in some ways. First, H9c2 cells are premature cardiac myoblasts, whereas neonatal rat cardiomyocytes are mature. Second, H9c2 cells have good proliferation ability but neonatal rat cardiomyocytes do not. Third, neonatal rat cardiomyocytes have contractile characteristics, and H9c2 cells are not contractile. The reason that DEPEs have more cytotoxic effects on H9c2 cells than that on neonatal rat cardiomyocytes may be that the latter have encountered some stress before being isolated from rats. But the exact differences of cytotoxicity between neonatal rat cardiomyocytes and H9c2 cells should be determined under the same dose treatment.

The present study showed that DEPEs induced not only ER stress but also apoptosis in H9c2 cells. Previous study has demonstrated that JNK inhibition prevented the

UPR-dependent human aortic smooth muscle cell death induced by 7-ketocholesterol (Pedruzzi et al, 2004). However, lead induces ER stress, but the induction of GRP78 and GRP94 expression via the JNK-AP-1 pathway functions as a defense mechanism against lead-induced cytotoxicity in vascular endothelial cells (Shinkai et al, 2010).

Therefore, ER stress induced by DEPEs in H9c2 cells may be the cause of apoptosis, or it may play a protective role. Besides, 7-ketocholesterol and lead both could induce ER stress marker GRP78 through the activation of JNK, which is consistent with the responses of H9c2 cells to DEPEs in this study.

In mouse macrophage cell line, DEPs induced apoptosis via p53 and Mdm2 (Yun et al, 2009). DEPs also produced DNA damage in the Chinese hamster lung fibroblast (V79) cell line (Gu et al, 2005). In neonatal rat cardiomyocytes, DEPEs extracted from PBS containing 0.05% Tween 80 induced cytotoxicity via ROS production. And in this study, DEPEs extracted from organic solvent induced neonatal rat cardiomyocyte hypertrophy and H9c2 cell death.

CHAPTER V

Conclusions and future applications

The present study demonstrated that low concentrations of DEPEs prompted neonatal rat cardiomyocyte hypertrophy. On the other hand, high concentrations of DEPEs induced apoptosis as well as necrosis in myocardial cells. These data provide a better understanding of potential mechanisms that link DEPs to toxicological regulation of cardiomyocytes. And the understanding of DEPEs-induced cardiomyocyte toxicity can help to find the ways to decrease the risks of DEPs-induced heart diseases.

Figures and Tables

Table 1. Certified Concentrations for Selected PAHs in SRM 2975

Table 2. Reference Concentrations for Selected PAHs in SRM 2975

Table 3. Concentrations for Selected Nitro-Substituted PAHs in SRM 2975

Table 4. Primers used in this study

Figure 1. Exposure to DEPEs had no effects on cardiomyocyte viability. Neonatal rat cardiomyocytes were treated with DEPEs (1, 5, 10, 12.5 and 15 μg/mL) and incubated for 24 hours. The cytotoxicity was analyzed by MTT assay as described under Materials and Methods. DMSO was used as a control. Data are presented as mean values ± S.E.M. from three independent experiments.

A.

B.

C.

Figure 2. DEPEs induced cardiomyocytes hypertrophy. Cultured cardiomyocytes were exposed to vehicle control and various concentrations of DEPEs for 24 hours. (A) DEPEs-induced morphologic changes of cardiomyocytes. Cardiomyocytes were immunostained with an anti-desmin antibody (green), and the nucleus was stained with Hoechst 33258 (blue). The bar graph represents the relative cellular size to control. (B) Dose response effects of DEPEs with detection of the hypertrophy marker total protein/cell number. (C) Pathologic hypertrophy marker ANP, BNP, and β-MHC mRNA expression measured by real-time RT-PCR after 24 hours DEPEs treatment. The mRNA expression was standardized to expression of GAPDH. DMSO was used as a control. Data are presented as mean values ± S.E.M. from three independent experiments. * P<0.05 versus control; ** P<0.01 versus control

Figure 3. DEPEs induced phosphorylation of PKC, p38, ERK and JNK in cardiomyocytes. Cells were treated with 5 μg/mL DEPEs for the indicated times.

Whole cell lysates were processed for western blot analysis to detect changes in P-PKC, P-p38, P-ERK, and P-JNK. β-actin was used as an equal loading control. DMSO was used as a control. Data are presented as mean values ± S.E.M. from three independent experiments. * P<0.05 versus control; ** P<0.01 versus control

Figure 4. DEPEs reduced H9c2 cell viability. H9c2 cells were treated with DEPEs (3, 15 and 25 μg/mL) for 24 hours. To determine the cell viability, cells were subjected to the MTT assay as described under Materials and Methods. DMSO was used as a control.

Data are presented as mean values ± S.E.M. from three independent experiments. * P<0.05 versus control; ** P<0.01 versus control

Figure 5. DEPEs induced apoptosis in H9c2 cells determined by annexin V-FITC/PI staining. Cells were treated with DEPEs (15 and 25 μg/mL) for 24 hours, and cell apoptosis was measured by annexin V-FITC binding. Histograms show the percentages of early (annexin V-FITC positive and PI-negative) and late (annexin V-FITC positive and PI-positive) apoptotic cells in control and DEPEs-treated cells.

DMSO was used as a control. Data are presented as mean values ± S.E.M. from three independent experiments. * P<0.05 versus control; ** P<0.01 versus control

Figure 6. DEPEs induced expression of GRP78 and CHOP proteins. H9c2 cells were exposed to DEPEs (3, 15 and 25 μg/mL) for 24 hours, and total cell lysates were subjected to western blot analysis using the indicated antibodies. β-actin was used as internal standard. DMSO was used as a control. Data are presented as mean values ± S.E.M. from three independent experiments. * P<0.05 versus control; ** P<0.01 versus control

A.

B.

Figure 7. DEPEs induced phosphorylation of JNK and expression of GRP78 and CHOP proteins. H9c2 cells were exposed to DEPEs (25 μg/mL) for various time periods, and total cell lysates were subjected to western blot analysis using the indicated antibodies. β-actin was used as internal standard. DMSO was used as a control. Data are presented as mean values ± S.E.M. from three independent experiments. * P<0.05 versus control; ** P<0.01 versus control

A.

B

Figure 8. JNK inhibitor reversed DEPEs-induced expression of ER stress markers.

H9c2 cells were pre-incubated with 15 μM JNK inhibitor, SP600125, for 1 hour, and treated with 25 μg/mL DEPEs for 1 hour (A) and 24 hours (B). Lysates were then immunoblotted for P-JNK, GRP78, and CHOP. β-actin was used as internal standard.

DMSO was used as a control. Data are presented as mean values ± S.E.M. from three independent experiments. * P<0.05 versus control; ** P<0.01 versus control; ## P<0.01 versus DEPEs treatment alone without JNK inhibitors

A.

B.

Figure 9. Intracellular ATP levels and LDH release in H9c2 cells after DEPEs treatment. Cells were exposed to DEPEs for 24 hours, and intracellular ATP levels (A) and LDH release (B) were determined. DMSO was used as a control. Data are presented as mean values ± S.E.M. from three independent experiments. * P<0.05 versus control;

** P<0.01 versus control

.

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