Pyrrolidine dithiocarbamate augments Hg
2+-mediated induction of
macrophage cell death via oxidative stress-induced apoptosis and
necrosis signaling pathways
Chun-Fa Huanga,b, Shing-Hwa Liub, Shoei-Yn Lin-Shiauc,*
aSchool of Chinese Medicine, College of Chinese Medicine, China Medical University, 404 Taichung, Taiwan.
bInstitute of Toxicology, College of Medicine, National Taiwan University, 100 Taipei, Taiwan.
cInstitutes of Pharmacology, College of Medicine, National Taiwan University, 100 Taipei, Taiwan.
*To whom corresponding author should be addressed to Shoei-Yn Lin-Shiau Ph.D., Institute of Pharmacology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, 100 Taipei, Taiwan.
Tel.: +886 (2) 2312-3456 ext: 88313 Fax: + 886 (2) 2391-5297
E-mail: [email protected]
Exposure to mercury can lead to several injuries in mammals, including immune system dysfunction, and pyrrolidine dithiocarbamate(PDTC), as a metal chelator and antioxidant, has been indicated to increase the cytotoxic effects of toxic metals. However, the toxicological effects and possible mechanisms of mercury in combination with PDTC are most unclear. In this study, we showed that PDTC dramatically increase the cytotoxic effect of HgCl2 on cultured murine macrophages (RAW 264.7 cells). PDTC augmented HgCl2-induced cytotoxic effects by facilitating the entry of mercury into the cells. The Hg2+/PDTC complex significantly and rapidly increased the formation of reactive oxygen species (ROS) and decreased intracellular glutathione (GSH) levels in these cells. Flow cytometry analysis showed that the numbers of sub-G1 hypodiploid cells and annexin V-FITC binding cells increased after Hg2+/PDTC complex exposure, and several features of mitochondria -dependent apoptosis were also induced, including mitochondrial membrane depolarization, cytosolic cytochrome c release, poly(ADP-ribose) polymerase (PARP) and caspase 3/7 activation, and DNA fragmentation. Moreover, both apoptotic and necrotic cells were detected using acridine orange/ethidium bromide dual staining. Meanwhile, depleted intracellular ATP levels and increased lactate dehydrogenase (LDH) release were observed, suggesting the induction of necrotic cell death processes. These Hg2+/PDTC complex-induced cytotoxicity-related signals could be reversed by pretreatment with the antioxidant N-acetylcysteine. In conclusion, these results suggest that Hg2+/PDTC complex-induced oxidative stress causes macrophage cell death via both apoptosis and necrosis. These findings imply for the first time that PDTC dramatically increase the uptake and toxicological effects of Hg2+ instead of detoxification.
Keywords: HgCl2; PDTC; apoptosis; necrosis; reactive oxygen species; caspase 3/7
because of industrial pollution. Reports indicate that approximately 200 tons of mercuric compounds are introduced into the environment annually by industrial activities (Shaffi, 1981). Exposure to mercury can induce seriously toxic injuries in various mammalian cells and organ systems, including disruption of nerve system,
interference with lymphocyte function, and causing cell death (Clarkson and Magos, 2006; Jarup, 2003). The main sources of most human are exposed to mercury through consumption of contaminated fish and food, disinfectants, antiseptics, tooth pastes, lens solutions, and disk batteries (Clarkson et al., 2003; Geier et al., 2008).
Other routes of exposure include specific work-related environment, such as handling of mercuric salts by workers or accidents resulting in intoxication from
mercuric compounds (Akagi et al., 1995; Li et al., 2008). Mercury is known to produce toxic effects in mammalians, and the immune system, as well as nervous system, is the very sensitive target tissue for mercury (Lawrence and McCabe, 2002; Raszyk et al., 1997). Epidemiological study has reported that Hg exposure is associated with increased risk of immunotoxic effects (such as development of lupus and autoimmune diseases)(Cooper et al., 2004; Kubicka-Muranyi et al., 1993). Growing studies have indicated that mercuric chloride (HgCl2, inorganic form of mercury) has also been shown to affect immune function, including the induction of autoimmune-like pathology, the production of many abnormal inflammatory cytokines, the interference with immune function, and causing cytotoxicity (Gardner et al., 2010; Kim and Sharma, 2003; Pollard and Landberg, 2001; Silbergeld et al., 2005; Strenzke et al., 2001). However, little is known about the immunotoxic effects and the possible mechanisms of exposure to HgCl2.
dithiocarbamates (DCs) which are a class of metal-chelating and antioxidant compounds with various applications in agricultural pesticides, catalysts of the rubber manufacturing industry, and medicine for the treatment of fungal and bacterial infections (Liesivuori and Savolainen, 1994; Kang et al., 2008; Sunderman, 1981; WHO, 1988). Therefore, human may be exposed to PDTC from drugs, crop residues, contaminated groundwater and food, and occupational pollutants (Vettorazzi et al., 1995; WHO, 1988). In mammalian cells, PDTC has been shown to be a potent inhibitor of NF-B that is capable of preventing inflammatory responses and apoptosis (Jin et al., 2007; Li et al., 2010). PDTC also induced antiproliferative and pro-apoptotic effects in cancer cells (Malaguarnera et al., 2003; Morais et al., 2006 and 2009). However, Calviello et al. (2005) and Valentine et al. (2006) found that oral administration of PDTC in rat significantly increased copper levels and oxidative stress damage in the peripheral nerves, resulting in nervous system derangement and dysfunction. Other studies have reported that PDTC increases Cu2+-induced cell death, which is dependent on the increase in the intracellular levels of Cu2+ and Zn2+
but not of other metals (Cs+, Mn2+, Fe2+, Co2+, Bi2+, Pb2+, Ba2+, La3+, and Cr6+) that can induce ROS- or inhibit proteasomal activity-mediated apoptosis signaling pathways (Chen et al., 2000; Daniel et al., 2005; Milacic et al., 2008). Because PDTC is widely used in agriculture, industry, and medicine, and Hg2+ is presented throughout
environmental pollutants or contaminated food, exposure to both PDTC and Hg2+ in
human is likely. However, the cytotoxic effects and possible mechanisms of the combined action of PDTC and Hg2+ (Hg2+/PDTC complex) are not well understood.
In the immune system, macrophages play important roles in various inflammatory responses that prevent tissue or organ damage, including responses to invading microbial infections, chemical, or physical irritants (Morrissette et al., 1999).
Macrophages can induce the production of ROS to kill pathogens, infected cells, and invading microorganisms. Excesses ROS have detrimental effects in mammals and are involved in a variety of undesirable biological reactions and functional damage processes, such as neurodegenerative disease and inflammatory conditions after exposure to toxic agents (Finkel and Holbrook, 2000; Huang et al., 2008). Mercury can induce toxic effects by inducing oxidative stress that alters cellular function and eventually results in cell death and pathological injury (Buttke and Sandstrom, 1994; Koropatinck and Zalups, 1997). Furthermore, mercury is broadly sulfhydryl-reactive and regulates redox status; this can cause an imbalance in the pro-oxidant/antioxidant enzyme equilibrium and result in the alteration of cellular function and eventually induction of apoptosis and pathological injury in immune and brain cells (Close et al., 1999; Shenker et al., 2002). Although a change in redox homeostasis may play a role in mercury-induced cytotoxicity, the specific cellular processes and the downregulation of molecular signaling pathways induced by HgCl2 remain to be clarified.
In view of humans may be simultaneously exposed to PDTC and Hg2+, it is importance to elucidate the toxic effects of the Hg2+/PDTC complex and determine the pathways it activates since these can lead to the apoptosis and necrosis of murine macrophages. To this aim, we sought to investigate the effects of the Hg2+/PDTC complex in oxidative stress damage and explored whether intracellular GSH depletion, mitochondrial membrane potential (MMP) depolarization, cytosolic cytochrome c release, activation of poly(ADP-ribose) polymerase (PARP) and caspase-3/-7, and internucleosomal DNA fragmentation are implicated in Hg2+/PDTC complex-induced apoptosis in macrophages. Acridine orange/ethidium bromide dual staining, lactate dehydrogenase (LDH) release, and intracellular ATP depletion were
also analyzed in Hg2+/PDTC complex-induced necrosis. Treatment with a potent antioxidant N-acetylcysteine (NAC) at different stages confirmed the involvement of major signaling pathways in Hg2+/PDTC complex-induced macrophage damage.
2.1. Materials
Cell culture reagents were obtained from GIBCO BRL, Life Technologies, USA. Annexin-V-FITC and PI binding assay kit and LDH cytotoxicity assay kit were procured from BioVision (BioVision Research Products, CA, USA). Propidium iodide, N-acetyl-L-cysteine (NAC), 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT), and Adenosine 5`-triphosphate bioluminescent assay kit were purchased from Sigma Chemical company (Sigma-Aldrich, USA). 3,3`-Dihexyloxacarbocyanine iodide (DiOC6), and 2`,7`-dichlorofluorescein diacetate (DCFH-DA) (Molecular Probes, Eugene, OR, USA) are water insoluble and were dissolved in dimethyl sulfoxide (DMSO). Mouse- or rabbit-monoclonal antibodies specific for cytochrome c, PARP, caspase-3, caspase-7, and α-tubulin were purchased from Santa Cruz (Santa Cruz Biotechnology, Inc., CA, USA). All other reagents and chemicals used were purchased from Sigma-Aldrich.
2.2. Cell Culture
The mouse macrophage cell line-RAW264.7 cell was obtained from the American Type Culture Collection (ATCC, CRL-2278). Cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 2 mM glutamine, antibiotics (100 U/ml penicillin A and 100 U/ml streptomycin), and 10% heat-inactivated fetal bovine serum (Gibco BRL, Life Technologies, USA) and cultured in a 37 ℃ humidified incubator containing 5% CO2-95% air mixture.
2.3. Morphological features of RAW 264.7 cells
RAW 264.7 cells were seeded at 1 ×106 cells/well in a glass slide and incubated with HgCl2 and PDTC either alone or in combination at 37°C for 24 h. Photomicrographs were obtained with a 20× objective lens using a cooled CCD
camera (OlymPix 50 2500) adapted to a Zeiss Axiovert 135-TV microscope.
2.4. Cell viability
RAW 264.7 cells were seeded in 96 well plates (2× 104 cells/well) and allowed to adhere and recover overnight. The cells were transferred to fresh media and then treated with HgCl2 and PDTC either alone or in combination in the absence or present (1h pre-treatment) of NAC (1 mM) for 24 h. At the end of treatments, the medium was aspirated and cells were incubated with fresh medium containing 0.2 mg/mL 3-(4, 5-dimethyl thiazol-2-yl-)-2, 5-diphenyl tetrazolium bromide (MTT) at 37℃. After 4 h, the medium was removed and the blue formazan crystals were dissolved in 100 L DMSO. Following mixing, and an
enzyme-linked immunosorbent assay reader (Bio-Rad, model 550, Hercules, CA, USA) was used for measurement the absorption at 570 nm.
2.5. Determination of mercury content
Mercury content of the RAW 264.7 cell was detected as previously described (Chen et al., 2010). In brief, RAW 264.7 cells were seeded at 2 ×106 cells/dish in a 6 cm2 dish and allowed to adhere and recover overnight. The cells were transferred to fresh media and incubated with HgCl2-30 M and PDTC-10 M either alone or in
combination for 1, 4, and 24 h. At the end of treatments, cells were harvested, washed three times with PBS three times, and followed by addition of 0.15% nitric acid, and the mixture was vortexed and frozen at -20 °C for 2 h or overnight. Tubes were thawed at 37 °C for 20 mins and centrifuged at 1,000×g at 4 °C for 10 mins. The supernatant (0.5 mL) were placed in a 15 mL polyethylene tube, and 0.5 ml of a 3:1 mixture of hydrocholic acid (35%) and nitric acid (70%) was added. The tubes were
dilution buffer (0.3% nitric acid and 0.1% Triton X-100 in distilled water) was added to the digested material, and the total mercury content was determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The detection limit for mercury was ~0.1 ppb (g/L).
2.6. Determination of reactive oxygen species (ROS) production
ROS production was monitored by flow cytometry using the peroxide-sensitive fluorescent probe: DCFH-DA, as described Chen et al., 2006. In brief, cells were seeded at 2 ×105 cells/well in a 24-well plate and incubated with HgCl2-30 M and
PDTC-10 M either alone or in combination. At the end of various time course treatments, cells were incubated with medium containing 20M DCFH-DA for 15 min at 37℃. After incubation with the dye, cells were harvested and washed twice with PBS, and then re-suspended in ice-cold phosphate buffered saline (PBS). The intracellular peroxide levels were measured by a flow cytometer (FACScalibur, Becton Dickinson, Sunnyvale, CA, USA), that emitted a fluorescent signal at 525 nm.
2.7. Analysis of intracellular GSH contents
RAW 264.7 cells were seeded and incubated with HgCl2 and PDTC in the same manner as for ‘2.6. Determination of ROS production’ analysis. At the end of various time course treatments, cells were incubated medium containing with
60 M monochlorobimane (mBCL, a fluorescent probe for determining the intracellular GSH levels) for further 30 min at 37 ℃. After loading the cells with mBCL, the supernantants were discarded and cells were washed twice with PBS, and the measurement of the intracellular GSH levels were determined as described previously (Lu et al., 2011).
2.8. Flow cytometric analysis of apoptotic and necrotic cells.
2.8.1. Measurement of sub-G1 DNA content. RAW 264.7 cells were seeded and
incubated with HgCl2 and PDTC in the same manner as for ‘2.6. Determination of
ROS production’ analysis for 24 h at 37℃. At the end of treatments, cells were
harvested and washed twice with PBS, and then re-suspended in 1 mL of cold 70% (v/v) ethanol and stored at 4 ℃ for 24 h. Then, the fixed cells were washed twice with PBS and incubated at 37 ℃ for 30min with 1 mg/mL RNase-A dissolved in 0.5 mL of 0.2 % Triton X-100/PBS solution. Following the incubation, cells were washed with PBS, the cells were stained with 50 g/mL propidium iodide (PI) at 4 ℃ for 30 min in dark conditions. The stained cells were subjected to flow cytometry analysis of DNA content (FACScalibur, Becton Dickinson). Nuclei displaying hypodiploid, sub-G1 DNA contents were identified as apoptotic.
2.8.2. Determination of phosphatidyl serine (PS) externalization: Annexin-V fluorescein Isothiocyanate (FITC) and propidium iodide (PI) binding assay.
Apoptosis was detected using probe: annexin V, a protein that binds to phosphatidyl serine (PS) residues which is an important marker for exposed on the cell surface of apoptotic cells (Lu et al., 2011). Cells were seeded and incubated with HgCl2 and PDTC in the same manner as for ‘2.6. Determination of ROS production’ analysis for 24 h at 37 ℃. At the end of treatments, cells were harvested and washed twice with PBS, and then stained with annexin -V-FITC and PI binding assay kit for 15min at room temperature in dark conditions. The stained cells were analyzed by a flow cytometry(FACScalibur, Becton Dickinson). The quadrants mean of data: lower right- apoptosis, annexin V binding; upper right- necrosis after apoptosis, both of PI and annexin V binding; upper left- necrosis, PI staining; lower left- live cells.
orange/ethidium bromide assay. Discrimination between apoptotic and necrotic cell death was based on previously published diagrams and described (Johnson et al., 2005; Kern and Kehrer, 2002). In briefly, RAW 264.7 cells were seeded and incubated with HgCl2 and PDTC in the same manner as for ‘2.6. Determination of
ROS production’ analysis for 24 h at 37℃. At the end of treatments, cells were
harvested and washed twice with PBS, and then re-suspended and incubated with
1mL PBS containing 2L of acridine orange and 2L of ethidium bromide (final concentration 500 ng/mL for each) for 5 min in dark conditions. Two-parameter fluorescence was acquired from more than 10,000 individual cells per sample using a flow cytometer (FACScalibur, Becton Dickinson). Green emissions from acridine orange and red emissions from ethidium bromide were captured at 525 and 620 nm, respectively. Live, apoptotic, and necrotic populations were analyzed using CellQuest software.
2.9. Determination of Mitochondrial Membrane Potential (MMP)
Cells were seeded and incubated with HgCl2 and PDTC in the same manner as for ‘2.6. Determination of ROS production’ analysis for various time courses at 37℃. At the end of treatments, cells were incubated with medium with 100 nM 3,3’-dihexyloxacarbocyanine iodide (DiOC6) for 30 min at 37℃. After incubation with the dye, cells were harvested and washed twice with PBS, and then re-suspended in ice-cold phosphate buffered saline (PBS). MMP was analyzed by a flow cytometer (excitation at 475 nm and emission at 525 nm; FACScalibur, Becton Dickinson)(Chen et al., 2006).
2.10. Western blot analysis
with HgCl2 and PDTC in the same manner as for ‘2.6. Determination of ROS
production’ analysis for various time courses at 37℃. At the end of treatments,
the expressions of protein activation were analyzed by the protocol performance of Western blot (as previously described in Lu et al., 2011). In brief, equal amounts of proteins (50 g per lane) was subjected to electrophoresis on 10% (W/V)
SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 1 h in PBST (PBS, 0.05% Tween-20) containing 5% nonfat dry milk. After blocking, the membranes were incubated with specific antibodies against PARP, caspase-3, caspase-7, and
-tubulin in 0.1% PBST (1:1000) for 1 h at room temperature. After they were washed in 0.1% PBST followed by two washes (15 min each), the blots were subsequently incubated with goat anti-mouse or anti-rabbit IgG-HRP secondary antibody (1:1000) for 1 h. The antibody-reactive bands were revealed by the
enhanced chemiluminescence reagent kit (PerkinElmerTM, Life Sciences) and were used to expose them to Kodak radiographic film. When the detection of cytosol cytochrome c expression was performed as previously described in Chen et al. 2010. In brief, at the end of treatments, cells were detached, washed twice with PBS, and then homogenized with a pestle and mortar in the extract buffer (0.4 M mannitol, 25 mM MOPS (pH 7.8), 1 mM EGTA, 8 mM cysteine, and 0.1 % (w/v) bovine serum albumin). The cell debris was removed via centrifugation at 6,000g for 2 min. The supernatant was recentrifuged at 12,000g for 15 min to pellet the mitochondria. The supernatant (cytosolic fraction) was detected cytochrome c expression by western
blotting analysis.
DNA fragmentation analysis was based on that described by Chen et al. (2008). Briefly, cells were seeded and incubated with HgCl2 and PDTC in the same manner as for ‘2.10. Western blot analysis’ for various time courses at 37℃. Atthe end of treatments, cells were harvested and washed twice with PBS, and then lysed in 100 ml of lysis buffer (50 mM Tris (pH 8.0), 10 mM ethylenediaminetetraacetic acid (EDTA), 0.5 % sodium sarkosinate, and 1 mg/mL proteinase K) for 3 h at 56 ℃, and treated with 0.5 mg/ mL RNase A for another 1h at 56℃. DNA was extracted from phenol/chloroform/isoamyl alcohol (25/24/1) precipitation and then resuspended in TE buffer (Tris buffer, PH: 7.2 plus 1 mM EDTA). The loading buffer (50 mM Tris, 10 mM EDTA, 1% (w/v) low melting point agarose, 0.25% (w/v) bromophenol blue) and samples were loaded onto a presolidified, 2% agarose gel containing 0.1 mg/mL
ethidium bromide (Sigma, St. Louis, MO). The agarose gels were run at 50 V for 90 min in a TBE buffer. The gels were observed and photographed under UV light.
2.12. Analysis of intracellular ATP levels
The intracellular ATP content in RAW 264.7 cells was determined by
Adenosine 5’-triphosphate bioluminescent assay kit (FL-AA, Sigma-Aldrich, USA), as previously described (Yen et al., 2007). In brief, cells seeded and incubated with HgCl2 and PDTC in the same manner as for ‘2.6. Determination of ROS
production’ analysis for various time courses. At the end of treatments, cells were
harvested and washed twice with PBS, and then lysed with cell lysis buffer (Cell Signaling Technology, Inc., MA, USA) on ice. Cell lysates were centrifuged at 10,000×g for 10 min at 4℃, and then 50 L supernatant and ATP assay buffer
FL-AAB (PH 7.8) were mixed and immediately measured the amount of light with a luminometer (Gemini XPS Microplate Reader, Molecular Devices, USA).
2.13. Lactate dehydrogenase (LDH) release assay
The amount of LDH leaked from the cytosol of damage cells into the medium after exposure of RAW 264.7 cells (2 ×105 cells/well in a 24-well plate) to HgCl2 and
PDTC in the same manner as for ‘2.6. Determination of ROS production’ analysis for 24 h was detected. The amount of LDH release from cells was quantified by used the LDH cytotoxicity assay kit according to the manufacture’ instruction. The absorbance was measured with an enzyme-linked immunosorbent assay reader (Bio-Rad, model 550, Hercules, CA, USA) at 490 nm.
2.14. Statistical analysis
The values in the text are given as means ± standard errors of the mean (SEM). The significance of difference was evaluated by the paired Students‘t-test. When more than one group was compared with one control, significance was evaluated according to one way analysis of variance (ANOVA); the Duncan’s post-hoc test was applied to identify group differences. Probability values of < 0.05 were considered statistically significant.
3. Results
3.1. Effects of the Hg2+/PDTC complex on cell viability and ROS production in RAW 264.7 cells
To examine the cytotoxic effects of either native or complex forms of HgCl2 and PDTC on macrophages, morphological changes and cell viability were analyzed of RAW 264.7 cells were analyzed. As shown in Figure 1A, treatment of cells with PDTC (10 M) did not have any toxic, but slight cell shrinkage was observed after HgCl2 treatment (30 M). Significant cell detachment, shrinkage, and cell death-like morphological changes were observed in cells exposed to HgCl2 plus PDTC (Figure 1A). Cell viability assay indicated that exposure to high concentrations of HgCl2 reduced the number of viable cells (EC50 was 64.6 ± 3.2 M; viability of cells treated with 30 M HgCl2 was 72.7% ± 2.9% of control) and PDTC was not cytotoxic at concentrations of 1-10 M (Figure 1B). However, treatment with a combination of 10 M PDTC plus 30 M HgCl2 resulted in a dramatic reduction in the number of viable cells (10.9% ± 4.5% of control group, Figure 1C). Moreover, the intracellular Hg level in RAW 264.7 cells exposed to HgCl2 plus PDTC for 1 h was significantly higher than that in cells exposed to 30 M HgCl2 alone (1652.86 ± 263.65 vs 175.24 ± 12.02 ng Hg/mg protein; n ≧ 6, p < 0.05). This increase was greater apparent after
cells exposed to PDTC plus HgCl2 for 4 and 24 h (2884.11 ± 164.57 ng Hg/mg protein and 7206.07 ± 749.51 ng Hg/mg protein, respectively; n ≧ 6, p < 0.05 as compared to the HgCl2 group; Table 1). These results were consistent with the cytotoxicity assay data, suggesting that PDTC combined with HgCl2 can form the Hg2+/PDTC complex and augment the cytotoxic effects of HgCl2 by increasing intracellular Hg levels.
Hg2+/PDTC complex-induced cell death in RAW 264.7 cells. As shown in Figure 2A, cells treated with 30 M HgCl2 alone for 5-60 min showed increased ROS levels in a
time-dependent manner while those treated with 10 M PDTC alone did not. After cells exposed to the Hg2+/PDTC complex, ROS production was more rapid onset and
higher peak level than in cells treated with HgCl2 alone. Moreover, treatment of RAW 264.7 cells with HgCl2 (30 M) plus PDTC (10 M) rapidly and significantly depleted intracellular GSH levels to 58.3% ± 2.4%, and 47.4% ± 4.6% of control levels after 30 and 60 min, respectively (Figure 2B). By contrast, treatment with 30 M HgCl2 alone slightly increased intracellular GSH levels. These Hg2+/PDTC complex-induced responses could be reversed by pretreatment with an antioxidant, NAC (1 mM), a thiol-containing antioxidant capable of directly inactivating ROS as well as inducing GSH production (Figures 1C, 2A, and 2B).
3.2. Effects of HgCl2 and the Hg2+/PDTC complex on apoptosis, mitochondrial depolarization, cytochrome c release, activation of PARP and caspase 3/7, and induction of DNA fragmentation in RAW 264.7 cells
To investigate whether the cytotoxic effects of the Hg2+/PDTC complex on RAW 264.7 cells resulted in apoptosis, the sub-G1 hypodiploid cell population was first analyzed by flow cytometry with PI staining. As shown in Figure 3A, the sub-G1 hypodiploid cell population increased after treatment with 30 M HgCl2 alone for 24 h. Meanwhile, treatment with 30 M HgCl2 plus 10 M PDTC induced a more profound increase in the hypodiploid cell population than 30 M HgCl2 alone (p < 0.05). Next, externalization of phosphatidylserine (PS), an important event during apoptosis (Anderson et al., 2002), was determined using an annexin V-FITC binding assay by flow cytometry. As shown in Figure 3B, the cells in the lower right quadrant
were early apoptotic cells. HgCl2 (30 M) treatment for 24 h increased the annexin
V-positive RAW 264.7 cell population as compared to the control cell population (15.3% ± 1.1% vs. 1.6% ± 0.2%, p < 0.05). Treatment with Hg2+/PDTC complex
resulted in a markedly large annexin V-positive cell population than treatment with HgCl2 alone (35.5% ± 2.8% vs. 15.3% ± 1.1%, p < 0.05). Pretreatment with NAC (1 mM) effectively reversed both HgCl2- and Hg2+/PDTC complex-induced RAW 264.7 cell apoptosis. These results indicated that the exposure of RAW 264.7 cells to the Hg2+/PDTC complex enhanced apoptosis.
Increasing evidence indicates that mitochondrial dysfunction (including: MMP depolarization and cytochrome c release) might be linked to apoptotic cascades. Thus, to investigate whether Hg2+/PDTC complex-induced apoptosis was mediated by mitochondria-dependent pathways, we measured MMP by using the cationic dye DiOC6. Treatment of RAW 264.7 cells with HgCl2 (30 M) alone for 4 h, but not with PDTC (10 M) alone, slightly but significantly induced a decline in MMP (84.3% ± 2.1% of control group, p < 0.05). In contrast, the Hg2+/PDTC complex rapidly and markedly depolarized MMP in RAW 264.7 cells in a time-dependent manner (Figure 4A). We also investigated the release of cytochrome c from the mitochondria into the cytosol in Hg2+/PDTC complex-treated RAW 264.7 cellsby western blot analysis. Treatment with the Hg2+/PDTC complex for 0.5 h, but not with 30 M HgCl2 alone, effectively increased the level of cytochrome c levels in the cytosol of these cells (Figure 4C). These Hg2+/PDTC complex-induced responses were prevented by pretreatment with 1 mM NAC (Figure 4B and C).
To further examine whether PARP degradation and caspase-3/-7 protease activation were involved in Hg2+/PDTC complex-triggered apoptosis of RAW 264.7 cells, we investigated the expression of these proteins using western blot analysis. As
shown in Figures 4D and 4E, exposure of cells to the Hg2+/PDTC complex for 1-8 h, but not to HgCl2 (30 M) alone, significantly degraded the full-length PARP (116 kDa), full-length pro-caspase-3 (32 kDa), and full-length pro-caspase-7 (35 kDa). All of these responses could be reversed by pretreatment with 1 mM NAC.
Furthermore, we used agarose gel electrophoresis to detect internucleosomal DNA fragmentation. As shown in Figure 5A, cells treated with HgCl2 (30 M) for 4 h showed slight DNA fragmentation, but PDTC (10 M) alone did not observed any DNA fragmentation. Treatment with the Hg2+/PDTC complex significantly increased DNA fragmentation in a time-dependent manner (Figure 5A). Pretreatment with NAC (1 mM) prevented Hg2+/PDTC complex-induced DNA fragmentation production (Figure 5B).
3.3. Necrotic cell death is also involved in Hg2+/PDTC complex-induced cytotoxicity
To investigate this issue of necrosis involved in Hg2+/PDTC complex-induced cytotoxicity, we performed a PI (a cell-impermeant nuclear stain) binding assay. As shown in Figures 3B and 6A (quantitative PI-positive cells in the Annexin V/PI binding analysis in Figure 3B), treatment with 30 M HgCl2 resulted in a greater number of cells that had undergone necrosis after apoptosis or that had undergone necrotic cell death (upper right plus upper left quadrants) than was observed in control group (p < 0.05). Treatment with the Hg2+/PDTC complex resulted in a more significant number of necrotic cells than treatment with HgCl2 alone (67.7% ± 1.9% vs. 26.1% ± 1.1%, p < 0.05).
To further confirm whether Hg2+/PDTC complex-induced cytotoxicity in RAW 264.7 cells was associated with necrotic cell death, flow cytometry examination using
cell populations as compared to control (apoptotic (Ap) cells: 7.6% ± 0.3% vs. 0.9% ± 0.1%; necrotic (N) cells: 21.0% ± 0.6% vs. 2.8% ± 0.3%). It was resulted in more significant production of apoptotic and necrotic cells after treatment of cells with the Hg2+/PDTC complex than treatment with HgCl2 alone (apoptotic (Ap) cells: 26.9% ± 0.4%; necrotic (N) cells: 70.2% ± 0.3%; Figure 6B).
It has been reported that necrotic cell death is associated with an early loss of intracellular ATP levels (Kim et al., 2003). We next examined the intracellular ATP levels in RAW 264.7 cells after exposure to the Hg2+/PDTC complex. Exposure of cells to Hg2+/PDTC complex for 0.25-1 h markedly depleted intracellular ATP levels in a time-dependent manner (40.1%±5.7%, 21.7%±2.9 % and 13.8%±4.0 % of control levels after 0.25, 0.5, and 1 h, respectively, Figure 7A). However, after treatment with 30 M HgCl2 the intracellular ATP levels of cells slightly increased to 127.2 ± 5.5 % and 137.2 ± 9.9 % of control levels after 0.5 and 1 h, respectively. Moreover, LDH release, a biomarker of cell membrane damage and necrotic cell death (Chen et al., 2010), from RAW 264.7 cells after treatment with 30 M HgCl2 for 24 h was significantly increased than from control group, and it was more remarkably increased after exposure of cells to the Hg2+/PDTC complex (Figure 7B). These Hg2+/PDTC complex-induced necrotic cell death responses could also be reversed by pretreatment with 1 mM NAC (Figures 6 and 7).
4. Discussion
In this study, we examined the mechanisms underlying Hg2+/PDTC complex (30 M HgCl2 combined with 10 M PDTC)-induced cytotoxicity in RAW 264.7 cells. Our results first time showed that the Hg2+/PDTC complex induced dramatic cytotoxicity by significantly increasing intracellular Hg content, inducing ROS generation, and depleting intracellular GSH content to a greater degree than HgCl2 alone. Moreover, both ROS-mediated mitochondrial-dependent apoptosis and ATP depletion-associated necrosis were involved in Hg2+/PDTC complex-induced cell death.
ROS can elicit oxidative stress, which serves as a trigger for cell death and affects a number of pathological and physiological processes (Finkel and Holbrook, 2000; Huang et al., 2008). The deleterious effects of ROS include oxidative modification of DNA and gene mutation, and various apoptosis-related proteins are produced at different phases of the apoptotic pathway, including release of mitochondrial death-related proteins (such as cytochrome c and apoptosis-inducing factor), and activation of PARP and caspase cascades (Kim et al., 2009; Zhang et al., 2003). Moreover, GSH is the central regulator of cellular redox status, and is an important defense against ROS-induced damage and consumed by an excess of ROS generation. ROS-induced GSH depletion affects mitochondrial function and induces cell death via mitochondrial-mediated apoptosis signaling (Chandra et al., 2000; Naoi et al., 2009). Mitochondria are very sensitive to oxidative stress via different mechanisms and their dysfunction plays a critical role in apoptosis (Kowaltowski et
a complex with PDTC that increases copper-induced cytotoxicity by oxidative stress (Chen et al., 2000 and 2008; Milacic et al., 2008; Zhang et al., 2008). However, no previous report has indicated whether the Hg2+/PDTC complex can cause severe cytotoxicity. In this study, we found that the Hg2+/PDTC complex was capable of inducing more significant and rapid apoptosis than HgCl2 in RAW 264.7 cells by triggering intracellular GSH depletion, mitochondrial dysfunction, and PS externalization, and an increase in the number of sub-G1 hypodiploid cells. In addition, we found that the Hg2+/PDTC complex resulted in a marked activation of PARP that was associated with the activation of caspase-3/-7 proteases. Pretreatment with the antioxidant NAC prevented Hg2+/PDTC complex-induced cell apoptosis. These results indicate that the Hg2+/PDTC complex induces ROS-regulated cell apoptosis via the mitochondrial-dependent apoptotic pathway.
Oxidative stress caused by xenobiotic substances is capable of inducing both apoptotic and necrotic cell death (Marinho-Filho et al., 2010; Tan et al., 1998). Apoptosis and necrosis are distinct mechanisms of cell death with different characteristic. Apoptosis, also called programmed cell death, is a crucial and regulated protective process in multicellular organisms. It is characterized by cell shrinkage, condensation and margination of nuclear chromatin, DNA fragmentation, activation of caspase cascades, and production of apoptotic bodies (Higuchi, 2004; MacLellan and Schneider, 1997). In contrast, necrosis is a passive process that is characterized by cell swelling, spillage of intracellular contents into the extracellular milieu, and ultimately the rupture of cell and organelle membranes (Fiers et al., 1999; Higuchi, 2004). Previous studies have also shown that inorganic mercury-induced cell death occurs via both apoptosis and necrosis in several cell types (Kim et al., 2004; Lash et al., 2007). However, it is still unclear whether Hg2+/PDTC complex-induced
cytotoxicity mediated by apoptotic or necrotic pathways. Our results of acridine orange and ethidium bromide dual staining showed that the Hg2+/PDTC complex more effectively induced apoptosis and necrosis than HgCl2 alone, which were consistent with the results obtained for the annexin V-FITC/PI dual fluorescence
probe-binding assay. On the other hand, bioenergetic depletion by toxic agents, that results in a significant decrease the intracellular ATP levels under conditions of cell membrane damage, is also a biomarker for necrotic cell death (Kern and Kehrer, 2002; Skulachev, 2006). Moreover, it has been demonstrated that LDH release from cells is also an important indicator of cell membrane damage and necrotic cell death (Lopez et al., 2003). Here, we found that exposure of RAW 264.7 cells to the Hg2+/PDTC complex for 0.1–1 h resulted in significant intracellular ATP depletion and exposure for 24 h caused marked LDH release. However, the intracellular ATP levels were also increased after 0.5-1 h treatment with HgCl2 (30 µM) (Figure 7A), and that was decreased after 8 h treatment (27.2%±2.8% of control levels, p < 0.05, data not shown). These findings were due to increase the intracellular ATP levels at early stage of apoptosis and finally result in the intracellular ATP decrease (Skulachev, 2006). Moreover, pretreatment with NAC effectively reversed these Hg2+/PDTC complex-induced responses. These results implicate that Hg2+/PDTC complex-induced cell damage also activated the oxidative stress-mediated necrotic cell death pathway. Overall, the findings of the current study indicate that both apoptotic and necrotic cell death pathways are involved in Hg2+/PDTC complex-triggered RAW 264.7 cell cytotoxicity.
GSH, an important thiol-based antioxidant in mammalian cells, is directly involved in preventing ROS damage. GSH has been indicated to function as a hydrogen peroxide scavenger, cofactor of antioxidant enzymes, and modulator of
many important cellular processes (Griffith, 1999; Franco et al., 2007). In the present study, we have showed that GSH levels in RAW 264.7 cells exposed to HgCl2 (30 M) were slightly elevated after 0.25-1 h (an approximate 10-20 % increase) and decreased after 4 h (87.3% ± 4.8% of control levels, p < 0.05, data not shown).
This response suggests that an early adaptive response can be elicited from cells against mercury-induced oxidative stress damage (Wang et al., 2009). However, GSH depletion-induced chromosomal DNA fragmentation has also been associated with apoptosis and necrosis triggered by oxidative stress (Higuchi, 2003 and 2004). Here, our findings shown that the treatment of RAW 264.7 cells with the Hg2+/PDTC complex rapidly and significantly decreased intracellular GSH levels after 0.5 and 1 h. Agarose gel electrophoresis analysis also indicated that DNA fragmentation induced by the Hg2+/PDTC complex was much greater than that induced by HgCl2 alone. The antagonizing effects of NAC (a low-molecular-weight thiol and a precursor of GSH) effectively prevented the Hg2+/PDTC complex-induced decrease in intracellular GSH levels and reduce DNA fragmentation in RAW 264.7 cells, which might be due to its glutathione formation action. Thus, these results imply that oxidative stress-mediated GSH depletion is involved in the toxic mechanism of Hg2+/PDTC complex-induced apoptosis and necrosis.
HgCl2 is a toxic heavy metal that causes deleterious pathophysiological injuries, including neurological and immunological dysfunction, elicits autoimmunity, and induces cell death (Araragi et al., 2003; Gardner et al., 2010; Hu et al., 1999; Silbergeld et al., 2005). Several studies have shown that HgCl2 can induce cytotoxic responses in various immune cells at dosages between 30 and 100 M (Araragi et al., 2003; Kim and Sharma, 2003 and 2004; Shenker et al., 2000). In mercury-contaminated areas (such as Japan and Iraq), severe neuropathological deteriorations
in victims is associated with high Hg levels in the brain (above 12,000 g/L) and hair (above 17,000 g/L)(Amin-Zaki et al., 1976; Ekino et al., 2007). Moreover, epidemiological studies have generated a strong body of evidence that indicates the industrial release of mercuric compounds into rivers and oceans results in the accumulation of the toxicant in fish and food, which can contain as much as 3000 g/L or 2.53-1120 ng mercury/g wet weight (Kjellstrom et al., 1986; Li et al., 2010; Maramba et al., 2006). Thus, the concentration of mercury used in this study was likely equivalent to that ingested by humans who consume mercury-contaminated food. Furthermore, PDTC, a synthesized derivative of DCs, is known as an antioxidant and inhibitor of NF-B activation and exerts an apoptogenic effect on normal and cancer cells (Malaguarnera et al., 2003; Morais et al., 2006 and 2009; Parodi et al., 2005). PDTC exhibits a characteristic band at a 1412/cm-1 assignable to (N-CSS). This band defines a carbon-nitrogen bond order between a single
bound ( = 1250-1350/cm-1) and a double bond ( = 1640-1690/cm-1)(Herlinger et al., 1970; Lupien et al., 1965). After chelation with Cu2+, this band is observed the high energy shifts, indicating a strong chelating effect from PDTC to Cu2+ and a slight increase of the carbon-nitrogen double-bond character (Criado et al., 1989). Zhang et al. (2008) has reported the reaction of PDTC and Cu2+ to form a complex, which is determined the chemical structure of Cu(PDTC)2. The Cu(PDTC)2 complex significantly induced cytotoxicity in neuroblastoma cells, which was more potent than the effects of anticancer drug-cisplatin (Zhang et al., 2008). Growing evidence indicates that exposure to PDTC plus Cu2+ (form a PDTC-Cu2+ complex) can cause a significant oxidative stress damage and a potently inhibiting the proteasomal activity resulting in apoptosis in various cells, which is accompanied with the significant intracellular Cu2+ accumulation (Chen et al.,
2000 and 2008; Daniel et al., 2005; Malaguarnera et al., 2003; Milacic et al., 2008). Several studies have indicated that mercury can induce the production of ROS resulting in severe cellular damage and dysfunction, and PDTC can enhance copper toxicity, however, to the best of our knowledge, whether PDTC can enhance the toxicological effects of HgCl2 (as well as Cu2+ to form a complex) in murine
macrophage cell death remains unclear. Results of our study showed that exposure to HgCl2 increased intracellular Hg levels, and that was more markedly increased after 1, 4, and 24 h of exposure to PDTC plus HgCl2 (the ratio of intracellular Hg levels in the HgCl2 group to those in the PDTC plus HgCl2 group: 1:9.4 at 1 h; 1:12.4 at 4 h; and 1:20.7 at 24 h, respectively), suggesting the formation of the Hg2+/PDTC complex. Furthermore, the Hg2+/PDTC complex was capable of inducing more
significant oxidative stress-mediated apoptotic and necrotic events in RAW 264.7 cells. These findings indicate that the reaction of Hg2+ and PDTC forms a complex that induces oxidative stress-mediated apoptotic and necrotic pathways causing RAW 264.7 cell death.
5. Conclusion
In conclusion, our results provide strong toxicological evidence that the reaction of Hg2+ and PDTC forms an Hg2+/PDTC complex that has dramatically enhanced toxicological effects in murine macrophages. More importantly, this study for the first time demonstrates that PDTC enhances the entry of Hg2+ into the intracellular fraction and significantly induces cell death via oxidative stress-regulated apoptotic and necrotic signaling pathways.
Conflict of interest statement
Acknowledgments
This study was supported by a research grant from the National Science council, Taipei, Taiwan (NSC95-2320-B-002-102), and also supported in part by Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH101-TD-B-111-004).
References
Akagi, H., Malm, O., Branches, F.J.P., Kinjo, Y., Kashima, Y., Guimaraes, J.R.D., Oliveira, R.B., Haraguchi, K., Pfeiffer, W.C., Takizawa, Y., 1995. Human exposure to mercury due to goldmining in the Tapajos River basin, Amazon, Brazil: Speciation of mercury in human hair, blood and urine . Water, Air, and Soil Pollution 80, 85-94.
Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA 90, 7915-7922. Amin-Zaki, L., Elhassani, S., Majeed, M.A., Clarkson, T.W., Doherty, R.A.,
Greenwood, M.R., Giovanoli-Jakubczak, T., 1976. Perinatal methylmercury poisoning in Iraq. Am. J. Dis. Child. 130, 1070-1076.
Anderson, H.A., Englert, R., Gursel, I., Shacter, E., 2002. Oxidative stress inhibits the phagocytosis of apoptotic cells that have externalized phosphatidylserine. Cell Death Differ. 9, 616-625.
Araragi, S., Kondoh, M., Kawase, M., Saito, S., Higashimoto, M., Sato, M., 2003. Mercuric chloride induces apoptosis via a mitochondrial-dependent pathway in human leukemia cells. Toxicology 184, 1-9.
Calviello, G., Filippi, G.M., Toesca, A., Palozza, P., Maggiano, N., Nicuolo, F.D., Serini, S., Azzena, G.B., Galeotti, T., 2005. Repeated exposure to pyrrolidine-dithiocarbamate induces peripheral nerve alterations in rats. Toxicol. Lett. 158, 61-71.
1989. New PtS4 chromophores of dithiocarbamates derived from a-amino acids: synthesis, characterization and thermal behavior. Inorg Chim Acta 160, 37-42.
Chabicovsky, M., Prieschl-Grassauer, E., Seipelt, J., Muster, T., Szolar, O.H., Hebar, A., Doblhoff-Dier, O., 2010. Pre-clinical safety evaluation of pyrrolidine dithiocarbamate. Basic Clin. Pharmacol. Toxicol. 107, 758-767.
Chandra, J., Samali, A., Orrenius, S., 2000. Triggering and modulation of apoptosis by oxidative stress. Free Radic. Biol. Med. 29, 323-333.
Chen, S.H., Liu, S.H., Liang, Y.C., Lin, J.K., Lin-Shiau, S.Y., 2000. Death signaling pathway induced by pyrrolidine dithiocarbamate-Cu(2+) complex in the cultured rat cortical astrocytes. Glia 31, 249-261.
Chen, S.H., Lin, J.K., Liang, Y.C., Pan, M.H., Liu, S.H., Lin-Shiau, S.Y., 2008. Involvement of activating transcription factors JNK, NF-kappaB, and AP-1 in apoptosis induced by pyrrolidine dithiocarbamate/Cu complex. Eur J Pharmacol 594, 9-17.
Chen, Y.W., Huang, C.F., Tsai, K.S., Yang, R.S., Yen, C.C., Yang, C.Y., Lin-Shiau, S.Y., Liu, S.H., 2006. Methylmercury induces pancreatic beta-cell apoptosis and dysfunction. Chem. Res. Toxicol. 19, 1080-1085.
Chen, Y.W., Huang, C F., Yang, C.Y., Yen, C.C., Tsai, K.S., Liu, S.H., 2010. Inorganic mercury causes pancreatic beta-cell death via the oxidative stress-induced apoptotic and necrotic pathways. Toxicol. Appl. Pharmacol. 243, 323-331.
Clarkson, T.W., Magos, L., Myers, G.J., 2003. The toxicology of mercury--current exposures and clinical manifestations. N. Engl. J. Med. 349, 1731-1737. Clarkson, T.W., Magos, L., 2006. The toxicology of mercury and its chemical
Close, A.H., Guo, T.L., Shenker, B.J., 1999. Activated human T lymphocytes exhibit reduced susceptibility to methylmercury chloride-induced apoptosis. Toxicol. Sci. 49, 68-77.
Cooper, G.S., Parks, C.G., Treadwell, E.L., St Clair, E.W., Gilkeson, G.S., Dooley, M.A., 2004. Occupational risk factors for the development of systemic lupus erythematosus. J Rheumatol 31, 1928-1933.
Daniel, K.G., Chen, D., Orlu, S., Cui, Q.C., Miller, F.R., Dou, Q.P., 2005. Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells. Breast Cancer Res. 7, R897-908.
Ekino, S., Susa, M., Ninomiya, T., Imamura, K., Kitamura, T., 2007. Minamata disease revisited: an update on the acute and chronic manifestations of methyl mercury poisoning. J. Neurol. Sci. 262, 131-144.
Fiers, W., Beyaert, R., Declercq, W., Vandenabeele, P., 1999. More than one way to die: apoptosis, necrosis and reactive oxygen damage. Oncogene 18, 7719-7730.
Finkel, T., Holbrook, N.J., 2000. Oxidants, oxidative stress and the biology of ageing. Nature 408, 239-247.
Franco, R., Schoneveld, O.J., Pappa, A., Panayiotidis, M.I., 2007. The central role of glutathione in the pathophysiology of human diseases. Arch Physiol. Biochem. 113, 234-258.
Gardner, R.M., Nyland, J.F., Silbergeld, E.K., 2010. Differential immunotoxic effects of inorganic and organic mercury species in vitro. Toxicol Lett 198, 182-190.
Geier, D.A., King, P.G., Sykes, L.K., Geier, M.R., 2008. A comprehensive review of mercury provoked autism. Indian J Med Res 128, 383-411.
Griffith, O.W., 1999. Biologic and pharmacologic regulation of mammalian glutathione synthesis. Free Radic. Biol. Med. 27, 922-935.
Herlinger, A.W., Wenhold, S.L., Long, T.V., 2nd, 1970. Infrared spectra of amino acids and their metal complexes. II. Geometrical isomerism in bis (amino acidato) copper (II) complexes. J Am Chem Soc 92, 6674-6681.
Higuchi, Y., 2003. Chromosomal DNA fragmentation in apoptosis and necrosis induced by oxidative stress. Biochem. Pharmacol. 66, 1527-1535.
Higuchi, Y., 2004. Glutathione depletion-induced chromosomal DNA fragmentation associated with apoptosis and necrosis. J. Cell. Mol. Med. 8, 455-464.
Hu, H., Moller, G., Abedi-Valugerdi, M., 1999. Mechanism of mercury-induced autoimmunity: both T helper 1- and T helper 2-type responses are involved. Immunology 96, 348-357.
Huang, C.F., Hsu, C.J., Liu, S.H., Lin-Shiau, S.Y., 2008. Neurotoxicological mechanism of methylmercury induced by low-dose and long-term exposure in mice: oxidative stress and down-regulated Na+/K(+)-ATPase involved. Toxicol. Lett. 176, 188-197.
Jarup, L., 2003. Hazards of heavy metal contamination. Br. Med. Bull. 68, 167-182. Jin, X.H., Ohgami, K., Shiratori, K., Koyama, Y., Yoshida, K., Kase, S., Ohno, S.,
2007. Inhibition of nuclear factor-kappa B activation attenuates hydrogen peroxide-induced cytotoxicity in human lens epithelial cells. Br. J. Ophthalmol. 91, 369-371.
Johnson, V.J., Kim, S.H., Sharma, R.P., 2005. Aluminum-maltolate induces apoptosis and necrosis in neuro-2a cells: potential role for p53 signaling. Toxicol. Sci. 83, 329-339.
Kang, M.S., Choi, E.K., Choi, D.H., Ryu, S.Y., Lee, H.H., Kang, H.C., Koh, J.T., Kim, O.S., Hwang, Y.C., Yoon, S.J., Kim, S.M., Yang, K.H., Kang, I.C.,
2008. Antibacterial activity of pyrrolidine dithiocarbamate. FEMS Microbiol. Lett. 280, 250-254.
Kern, J.C., Kehrer, J.P., 2002. Acrolein-induced cell death: a caspase-influenced decision between apoptosis and oncosis/necrosis. Chem. Biol. Interact. 139, 79-95.
Kim, J.S., Qian, T., Lemasters, J.J., 2003. Mitochondrial permeability transition in the switch from necrotic to apoptotic cell death in ischemic rat hepatocytes. Gastroenterology 124, 494-503.
Kim, S.H., Sharma, R.P., 2003. Cytotoxicity of inorganic mercury in murine T and B lymphoma cell lines: involvement of reactive oxygen species, Ca(2+) homeostasis, and cytokine gene expression. Toxicol. In Vitro 17, 385-395. Kim, S.H., Sharma, R.P., 2004. Mercury-induced apoptosis and necrosis in murine
macrophages: role of calcium-induced reactive oxygen species and p38 mitogen-activated protein kinase signaling. Toxicol. Appl. Pharmacol. 196, 47-57.
Kim, W.H., Goo, S.Y., Lee, K.H., Park, S.J., 2009. Vibrio vulnificus-induced cell death of human mononuclear cells requires ROS-dependent activation of p38 and ERK 1/2 MAPKs. Immunol. Invest. 38, 31-48.
Kjellström , T . , Kennedy , P . , Wallis , S . , Mantell , C. , 1986. Physical and mental development of children with prenatal exposure to mercury from fish. Stage 1: Preliminary tests at age 4. Report 3080. Solna, Sweden: National Swedish Environmental Protection Board.
Koropatnick, J., Zalups, R.K., 1997. Effect of non-toxic mercury, zinc or cadmium pretreatment on the capacity of human monocytes to undergo lipopolysaccharide-induced activation. Br. J. Pharmacol. 120, 797-806.
transition and oxidative stress. FEBS Lett 495, 12-15.
Kubicka-Muranyi, M., Behmer, O., Uhrberg, M., Klonowski, H., Bister, J., Gleichmann, E., 1993. Murine systemic autoimmune disease induced by mercuric chloride (HgCl2): Hg-specific helper T-cells react to antigen stored in macrophages. Int J Immunopharmacol 15, 151-161.
Lash, L.H., Putt, D.A., Hueni, S.E., Payton, S.G., Zwickl, J., 2007. Interactive toxicity of inorganic mercury and trichloroethylene in rat and human proximal tubules: effects on apoptosis, necrosis, and glutathione status. Toxicol. Appl. Pharmacol. 221, 349-362.
Lawrence, D.A., McCabe, M.J., Jr., 2002. Immunomodulation by metals. Int Immunopharmacol 2, 293-302.
Li, P., Feng, X., Qiu, G., Li, Z., Fu, X., Sakamoto, M., Liu, X., Wang, D., 2008. Mercury exposures and symptoms in smelting workers of artisanal mercury mines in Wuchuan, Guizhou, China. Environ. Res. 107, 108-114.
Li, P., Feng, X., Qiu, G., 2010. Methylmercury exposure and health effects from rice and fish consumption: a review. Int. J. Environ. Res. Public. Health 7, 2666-2691.
Li, S., Zhong, S., Zeng, K., Luo, Y., Zhang, F., Sun, X., Chen, L., 2010. Blockade of NF-kappaB by pyrrolidine dithiocarbamate attenuates myocardial inflammatory response and ventricular dysfunction following coronary microembolization induced by homologous microthrombi in rats. Basic Res. Cardiol. 105, 139-150.
Liesivuori, J., Savolainen, K., 1994. Dithiocarbamates. Toxicology 91, 37-42.
Lopez, E., Figueroa, S., Oset-Gasque, M. J., and Gonzalez, M. P. (2003). Apoptosis and necrosis: two distinct events induced by cadmium in cortical neurons in
Lu, T.H., Hsieh, S.Y., Yen, C.C., Wu, H.C., Chen, K.L., Hung, D.Z., Chen, C.H., Wu, C.C., Su, Y.C., Chen, Y.W., Liu, S.H., Huang, C.F., 2011. Involvement of oxidative stress-mediated ERK1/2 and p38 activation regulated mitochondria-dependent apoptotic signals in methylmercury-induced neuronal cell injury. Toxicol. Lett. 204, 71-80.
Lupien, P.J., Hinse, C.M., Avery, M., 1969. Cholesterol metabolism and vitamin B6. I. Hepatic cholesterogenesis and pyridoxine deficiency. Can J Biochem 47, 631-635.
MacLellan, W.R., Schneider, M.D., 1997. Death by design. Programmed cell death in cardiovascular biology and disease. Circ. Res. 81, 137-144.
Malaguarnera, L., Pilastro, M.R., DiMarco, R., Scifo, C., Renis, M., Mazzarino, M.C., Messina, A., 2003. Cell death in human acute myelogenous leukemic cells induced by pyrrolidinedithiocarbamate. Apoptosis 8, 539-545.
Maramba, N.P., Reyes, J.P., Francisco-Rivera, A.T., Panganiban, L.C., Dioquino, C., Dando, N., Timbang, R., Akagi, H., Castillo, M.T., Quitoriano, C., Afuang, M., Matsuyama, A., Eguchi, T., Fuchigami, Y., 2006. Environmental and human exposure assessment monitoring of communities near an abandoned mercury mine in the Philippines: a toxic legacy. J. Environ. Manage. 81, 135-145.
Marinho-Filho, J.D., Bezerra, D.P., Araujo, A.J., Montenegro, R.C., Pessoa, C., Diniz, J.C., Viana, F.A., Pessoa, O.D., Silveira, E.R., de Moraes, M.O., Costa-Lotufo, L.V., 2010. Oxidative stress induction by (+)-cordiaquinone J triggers both mitochondria-dependent apoptosis and necrosis in leukemia cells. Chem. Biol. Interact. 183, 369-379.
Milacic, V., Chen, D., Giovagnini, L., Diez, A., Fregona, D., Dou, Q.P., 2008. Pyrrolidine dithiocarbamate-zinc(II) and -copper(II) complexes induce
apoptosis in tumor cells by inhibiting the proteasomal activity. Toxicol. Appl. Pharmacol. 231, 24-33.
Morrissette, N., Gold, E., Aderem, A., 1999. The macrophage--a cell for all seasons. Trends. Cell. Biol. 9, 199-201.
Morais, C., Gobe, G., Johnson, D.W., Healy, H., 2009. Anti-angiogenic actions of pyrrolidine dithiocarbamate, a nuclear factor kappa B inhibitor. Angiogenesis 12, 365-379.
Morais, C., Pat, B., Gobe, G., Johnson, D.W., Healy, H., 2006. Pyrrolidine dithiocarbamate exerts anti-proliferative and pro-apoptotic effects in renal cell carcinoma cell lines. Nephrol. Dial. Transplant. 21, 3377-3388.
Naoi, M., Yi, H., Maruyama, W., Inaba, K., Shamoto-Nagai, M., Akao, Y., Gerlach, M., Riederer, P., 2009. Glutathione redox status in mitochondria and cytoplasm differentially and sequentially activates apoptosis cascade in dopamine-melanin-treated SH-SY5Y cells. Neurosci. Lett. 465, 118-122. Parodi, F.E., Mao, D., Ennis, T.L., Bartoli, M.A., Thompson, R.W., 2005.
Suppression of experimental abdominal aortic aneurysms in mice by treatment with pyrrolidine dithiocarbamate, an antioxidant inhibitor of nuclear factor-kappaB. J. Vasc. Surg. 41, 479-489.
Pollard, K.M., Landberg, G.P., 2001. The in vitro proliferation of murine lymphocytes to mercuric chloride is restricted to mature T cells and is interleukin 1 dependent. Int Immunopharmacol 1, 581-593.
Raszyk, J., Toman, M., Gajduskova, V., Nezveda, K., Ulrich, R., Jarosova, A., Docekalova, H., Salava, J., Palac, J., 1997. Effects of environmental pollutants on the porcine and bovine immune systems. Vet Med (Praha) 42, 313-317.
freshwater teleosts. Toxicol. Lett. 8, 187-194.
Shenker, B.J., Guo, T.L., Shapiro, I.M., 2000. Mercury-induced apoptosis in human lymphoid cells: evidence that the apoptotic pathway is mercurial species dependent. Environ. Res. 84, 89-99.
Shenker, B.J., Pankoski, L., Zekavat, A., Shapiro, I.M., 2002. Mercury-induced apoptosis in human lymphocytes: caspase activation is linked to redox status. Antioxid. Redox. Signal. 4, 379-389.
Silbergeld, E.K., Silva, I.A., Nyland, J.F., 2005. Mercury and autoimmunity: implications for occupational and environmental health. Toxicol Appl Pharmacol 207, 282-292.
Skulachev, V.P., 2006. Bioenergetic aspects of apoptosis, necrosis and mitoptosis. Apoptosis 11, 473-485.
Strenzke, N., Grabbe, J., Plath, K.E., Rohwer, J., Wolff, H.H., Gibbs, B.F., 2001. Mercuric chloride enhances immunoglobulin E-dependent mediator release from human basophils. Toxicol Appl Pharmacol 174, 257-263.
Sunderman, F.W., Sr., 1981. Chelation therapy in nickel poisoning. Ann. Clin. Lab. Sci. 11, 1-8.
Tan, S., Wood, M., Maher, P., 1998. Oxidative stress induces a form of programmed cell death with characteristics of both apoptosis and necrosis in neuronal cells. J. Neurochem. 71, 95-105.
Valentine, H.L., Amarnath, K., Amarnath, V., Valentine, W.M., 2006. Dietary copper enhances the peripheral myelinopathy produced by oral pyrrolidine dithiocarbamate. Toxicol. Sci. 89, 485-494.
Vettorazzi, G., Almeida, W.F., Burin, G.J., Jaeger, R.B., Puga, F.R., Rahde, A.F., Reyes, F.G., Schvartsman, S., 1995. International safety assessment of pesticides: dithiocarbamate pesticides, ETU, and PTU--a review and update.
Teratog. Carcinog. Mutagen. 15, 313-337.
Wang, L., Jiang, H., Yin, Z., Aschner, M., Cai, J., 2009. Methylmercury toxicity and Nrf2-dependent detoxification in astrocytes. Toxicol. Sci. 107, 135-143. WHO, 1988. Dithiocarbamate Pesticides, Ethylenethiourea, and Propylenethiourea: a
General Introduction. Environmental Health Criteria 78, pp. 11–68. World Health Organization, Geneva.
Yen, C.C., Lu, F.J., Huang, C.F., Chen, W.K., Liu, S.H., Lin-Shiau, S.Y., 2007. The diabetogenic effects of the combination of humic acid and arsenic: in vitro and in vivo studies. Toxicol. Lett. 172, 91-105.
Zhang, J., Dong, M., Li, L., Fan, Y., Pathre, P., Dong, J., Lou, D., Wells, J.M., Olivares-Villagomez, D., Van Kaer, L., Wang, X., Xu, M., 2003. Endonuclease G is required for early embryogenesis and normal apoptosis in mice. Proc. Natl. Acad. Sci. USA 100, 15782-15787.
Zhang, H., Wu, J.S., Peng, F., 2008. Potent anticancer activity of pyrrolidine dithiocarbamate-copper complex against cisplatin-resistant neuroblastoma cells. Anticancer Drugs 19, 125-132.
Figure Legends
Fig. 1. The cytotoxic effects of HgCl2 and pyrrolidine dithiocarbamate (PDTC) on RAW264.7 cell. Cells were treated with HgCl2 and PDTC either alone or in combination (Hg2+/PDTC complex) for 24 h, and then cellular morphological changes were examined by an inverted phase contrast microscope (all ×400)(A). a.: control; b.: PDTC-10 M; c.: HgCl2-30 M; d.: HgCl2-30 M plus PDTC-10 M. The arrows indicated shrinkage and death cells. Cells were treated with HgCl2 (0.1 to 100 M), or PDTC (1, 3, 10 and 30 M) for 24 h (B) in the absence or presence (1h pre-treatment) of NAC (1 mM)(C), and cell viability was determined by MTT assay. All data are
determinations. *p < 0.05 as compared to the untreated control. #p < 0.05 as compared to the HgCl2 group. **p < 0.05 as compared to that in the absence of NAC group.
Fig. 2. Effects of HgCl2and Hg2+/PDTC complex on ROS generation and intracellular GSH depletion in RAW264.7 cells. Cells were exposed to 30 M HgCl2 and 10 M PDTC either alone or in combination in the absence or presence (1h pre-treatment) of NAC (1 mM) for various duration (5-60 minutes). (A) ROS was determined by flow cytometry, and (B) intracellular GSH content was determined by the fluorescence spectrometer as described in the Materials and Methods section (the GSH levels of control group was 0.42±0.01 nmoles-GSH/mg-protein). All data are presented as mean ± SEM for four independent experiments with triplicate determinations. *p < 0.05 as compared to the untreated control. #p < 0.05 as compared to the HgCl2 group. **p < 0.05 as compared to that in the absence of NAC group.
Fig. 3. Flow cytometry analysis showing the effects of HgCl2 and Hg2+/PDTC complex-induced apoptosis in RAW264.7 cell. Cells were treated with 30 M HgCl2, 10 M PDTC alone or Hg2+/PDTC complex for 24 h in the absence or presence (1h pre-treatment) of NAC (1 mM). (A) Sub-G1 hypodiploid cell population and DNA content were examined by flow cytometry. (B) Measurement of phosphatidylserine exposure on the outer cellular membrane leaflet by dual staining with Annexin V-FITC and propidium iodide negative cells. All data are presented as mean ± SEM for four independent experiments with triplicate determinations. *p < 0.05 as compared to the untreated control. #p < 0.05 as compared to the HgCl2 group alone. **p < 0.05 as compared to that in the absence of NAC group.
degradations of PARP, pro-caspase-3 and pro-caspse-7 in RAW 264.7 cells. Cells were treated with 30 M HgCl2 and 10 M PDTC alone or Hg2+/PDTC complex for 0.25-4 h (A and B, mitochondrial membrane potential), 0.25-1 h (C, cytochrome c), or 1-8 h (D, PARP; E, pro-caspase-3 and -7 degradation) in the absence or presence (1h pre-treatment) of 1 mM NAC. Data in A and B are presented as mean ± SEM for four independent experiments with triplicate determinations. Results shown in C, D, and E are typical representatives of at least three independent experiments, and the intensity of bands was analyzed by densitometry and expressed as fold change related to the control (mean ± SEM). *p < 0.05 as compared to the untreated control. #p < 0.05 as compared to the HgCl2 group alone; **p < 0.05 as compared to that in the absence of NAC group. Results shown in C, D, and E are representative of three independent experiments.
Fig. 5. Effects of HgCl2 and Hg2+/PPTC complex on the internucleosomal DNA fragmentation ladder in RAW 264.7 cells. Cells were treated with 30 M HgCl2 and 10 M PDTC alone or Hg2+/PDTC complex for 1-4 h in the absence (A) or presence (1h pre-treatment)(B) of 1 mM NAC. Agarose gel electrophoresis analysis of DNA fragmentation was performed as described in Materials and Methods section. M, marker.
Fig. 6. Induction of apoptosis and necrosis in RAW 264.7 cells by HgCl2 and Hg2+/PDTC complex. Cells were treated with 30 M HgCl2 and 10 M PDTC alone or Hg2+/PDTC complex for 24 h in the absence or presence (1h pre-treatment) of 1 mM NAC. (A) Quantitative PI-postive cells of Annexin V/PI dual staining analysis, and (B) discrimination of live, apoptotic, and necrotic cell population was determined
analysis as described in the Materials and Methods section. L = live cells; A = apoptosis; N = necrosis. All data are representative of four independent experiments with triplicate. Data in A are presented as mean ± SEM (n ≧ 6). *p < 0.05 as compared to the untreated control; #p < 0.05 as compared to the HgCl2 group alone; **p < 0.05 as compared to that in the absence of NAC group.
Fig. 7. Effects of HgCl2 and Hg2+/PDTC complex on intracellular ATP levels and LDH release in RAW 264.7 cells. Cells were treated with 30 M HgCl2 and 10 M PDTC alone or Hg2+/PDTC complex for 0.1~1h (intracellular ATP assay) and 24 h (LDH release assay) in the absence or presence (1h pre-treatment) of NAC (1 mM). The intracellular ATP level was measured by adenosine 5’-triphosphate bioluminescent assay kit (A; the ATP levels of control group was 74.98±2.38 nmoles-ATP/mg-protein) and LDH release was detected by LDH cytotoxicity kit (B) as described in the Materials and Methods section. Data are presented as means ± SEM for four independent experiments with triplicate determinations. *p < 0.05 as compared to the untreated control; #p < 0.05 as compared to the HgCl2 group alone; **p < 0.05 as compared to that in the absence of NAC group.
