* School of Biological and Chemical Sciences, Deakin University, 221 Burwood Highway, Victoria, Australia 3125; and
Nelson Institute of Environmental Medicine, New York University School of Medicine, 57 Old Forge Rd., Tuxedo, New York 10987
Received May 17, 2002; accepted August 16, 2002
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ABSTRACT |
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Key Words: arsenite; glutathione; glutathione reductase; keratinocytes; gene expression.
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INTRODUCTION |
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Regulation of cellular redox status by low dose iAs may therefore play a critical role in its pathology. The single most abundant reducing agent within cells is the sulfhydryl tripeptide glutathione. Glutathione is synthesized from its three constituent amino acids by the combined activities of glutamate-cysteine lyase (GCL; also known as, -glutamylcysteine synthetase [
-GCS]) and glutathione synthetase. Glutathione and enzymes related to GSH synthesis comprise a system that maintains the intracellular reducing environment and acts as a primary defense against excessive generation of harmful ROS (Morales et al., 1998
; Ochi et al., 1994
). The oxygen radical scavenging activity of reduced GSH directly facilitates ROS detoxification and the repair of ROS-induced damage. Indirectly, reduced GSH acts as a substrate for glutathione peroxidase (GPx) and glutathione S transferase (GST), enzymes involved in other ROS detoxification reactions. Glutathione reductase (GR) also plays a critical role by regenerating reduced GSH from the oxidized form (GSSG or GSH disulfide).
The GSH system would also seem to play a large role in protecting cells against exposure to iAs. This is supported by studies showing that resistance to iAs in mammalian cells is correlated with higher levels of intracellular GSH and higher activities of GSH-related enzymes (Lee and Ho, 1995; Lee et al., 1989
). Apart from improving the redox balance of cells, GSH would be in direct competition with protein thiols to form complexes with sulfhydryl-binding arsenic(III) species. Protein thiols are susceptible to oxidation by trivalent arsenicals, and may be critical targets in arsenic poisoning (Lin et al., 1999
; Styblo et al., 1997
).
Acute exposure to iAs is known to induce a cellular stress response in mammalian cells that involves increases in heatshock proteins, heme-oxygenase, and GSH (Deaton et al., 1990; Lee and Ho, 1995
; Ochi, 1997
). Depending on the species involved, an increase in the synthesis of GSH in mammalian cells exposed to arsenic could either be due to an increase in the rate of cystine uptake or to an increase in the enzyme activity of GCL (Ochi, 1997
). It is still unclear what regulatory mechanisms are associated with increased levels of GSH in human cells exposed to iAs. Also, very little is known about the effects of iAs on the activities of other GSH related enzymes in vivo. The activities and gene expression of GCL, GR, and GST were assessed in this study that examines the response of the GSH system in human cells exposed to sublethal concentrations of AsIII. AG06 and HaCaT human keratinocyte cell lines were primarily utilized in this study because skin is one of the most susceptible organs to chronic arsenic exposure. WI-38 fibroblast and PMC42 breast tumor cell lines were also included to evaluate tissue specificity. This study is novel in that AsIII modulation of the GCL and GR was assessed at an enzymatic and pretranslational level.
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MATERIALS AND METHODS |
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Materials and chemicals.
Sodium arsenite (NaAsO2), reduced GSH, oxidized GSH (GSSG), baker's yeast GR, ß-nicotinamide adenine dinucleotide phosphate (NADPH) tetrasodium salt, sulfosalicylic acid, 5,5-dithio-bis(2-nitrobenzoic acid; DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), neutral red (NR), L-buthioninesulfoximine (BSO), L-cystine, monoclonal anti ß-actin IgG, and oligonucleotide primers for polymerase chain reaction (PCR) were obtained from Sigma. Anti-GR polyclonal antibodies were kindly provided by R. H. Schirmer (University of Heidelberg, Germany), whereas anti-rabbit and mouse IgG horseradish peroxidase (HRP) conjugated secondary antibodies (raised in sheep) were obtained from Silenus. Media and sera for cell culture were obtained from Trace Elements (Australia). Hybond-N nylon membranes and Hybond-P PVDF membranes were obtained from Amersham. [35S]-Cystine and [32P]-dCTP were supplied by NEN research products (DuPont) and Amersham respectively. 2-Vinylpyridine was obtained from Aldrich Chemical Co.
Cell culture.
The SV40 transformed AG06 and immortalized HaCaT (Henseleit et al., 1996) keratinocyte cell lines were obtained from Dr. Mark Steinberg (City College of NY) and Prof. Nobert E. Fusenig (German Cancer Research Centre, Germany), respectively. Initial experiments were done using AG06 cells; however, later experiments used the analogous HaCaT cells due to lack of continuing availability of the AG06 cell line. Our results showed that there was minimal difference between these two keratinocyte cell lines with respect to their response to arsenic. Normal diploid WI-38 and immortalized GM847 human fibroblasts were obtained from CSL (Geelong, Australia) and the Murdoch Institute (Melbourne, Australia), respectively. PMC42 breast tumor cells were obtained from Dr. Leigh Ackland (Deakin University, Australia). Cells were maintained in either basal medium Eagles (WI-38), Dulbeccos modified Eagles medium with high sodium bicarbonate content (AG06 and HaCaT cells) or RPMI medium supplemented with 10% v/v FBS (for PMC42 cells), 2 mM glutamate, 100 Units-penicillin and 100 µg/ml streptomycin at 37°C. For cystine uptake studies, cystine-free medium was used. Keratinocyte and breast tumor cell lines were maintained in a humidified atmosphere of 5% CO2. The fibroblasts were routinely split at a 1:4 ratio whereas the other cell lines were split at a 1:6 ratio. Sodium arsenite in PBS (103-fold concentrated preparations) was added to medium of preconfluent cultures in the logarithmic phase of growth 24 h after seeding. In toxicity experiments where cells were pretreated with BSO and chloroethanol (CHE), AsIII was added 48 h after seeding.
Neutral red assay.
The viability of cells grown in 96 well microtiter plates was assessed by the uptake of NR dye according to the protocol of Babich and Borenfreund (1987). Immediately after AsIII treatment, cells were maintained for 3 h in 0.2 ml complete medium containing NR (50 µg/ml). The wells were then washed with a solution of 1% v/v formaldehyde, 1% w/v CaCl2 (0.2 ml) before a 50% v/v ethanol, 1% v/v acetic acid solution (0.2 ml) was added. After a further 30 min, the plates were placed in a Bio-Rad microplate reader and shaken vigorously before absorbance values at 540 nm were determined.
Glutathione assays.
The total intracellular GSH content of AsIII treated cells grown in microtiter plates was determined using the protocol described by Clarke et al.(1996). Briefly, treated cells were washed with phosphate buffered saline (PBS) immediately before the addition of 25 µl 5% w/v sulfosalicylic acid. Microtiter plates were then subjected to two freeze-thawing steps. A 10 µl aliquot from each well was transferred to a clean well of another microtiter plate with 165 µl of 0.1 M sodium phosphate buffer (pH 7.5) containing 0.2 mM NADPH (final concentration), 0.52 mM DTNB, and 0.15 mM EDTA. After incubation at 37°C for 15 min, 40 µl of bakers yeast GR (0.56 U/well) was added. The plates were then immediately shaken for 10 s before OD values at 405 nm were determined at fixed time intervals. The GSH content of samples was determined using standard curves generated with known amounts of GSH. For the determination of GSSG, 2-vinylpyridine (final concentration of 25 mM) was added to wells to bind any inactive reduced GSH before the addition of GR (Davies et al., 1984
).
Enzyme assays.
Cells grown in 6 well plates or 25 cm2 tissue culture flasks were harvested then homogenized in PBS using a Labsonic U (B. Braum) microtip sonicator. Sonicates were centrifuged at 12,000 x g (4°C) for 30 min before the supernates (cell lysates) were transferred and kept on ice until assayed. Aliquots of cell lysate were stored at 20°C for protein determination using the DC protein assay kit (Bio-Rad). The GST enzyme assay was based on that described by Lee et al.(1989). Reactions, conducted at room temperature, were initiated by adding 50 µl of cell lysate to 950 µl of 0.1 M sodium phosphate buffer (pH 6.8) with 1 mM EDTA, 1 mM reduced GSH, and 1 mM CDNB. The formation of conjugates of GSH and CDNB were monitored spectrophotometrically at 340 nm. The GR enzyme assay was based on that described previously by Styblo and associates (1997), and involved monitoring the reduction in NADPH absorbance at 340 nm. Cell extracts were preincubated in 0.15 M sodium phosphate buffer (pH 7) containing 6 mM EDTA and 0.1 mM oxidised GSH at 37°C. Reactions were initiated by the addition of 0.23 mM NADPH. The enzyme activity of GCL (
GCS) was measured by HPLC, as previously described (Yan and Huxtable, 1995
).
Cystine uptake.
Cystine uptake was measured as described by Ochi (1997). Briefly, AsIII treated cells maintained in six-well plates were washed twice with cold PBS and incubated in medium containing 12.7 µM [35S] L-cystine for 30 min. Cells were then washed twice with cold PBS, collected by trypsin-EDTA, and incubated at 37°C for 10 min. The radioactivity in the cells was measured by scintillation counting using a Beckman LS-9800.
Northern blot probes.
The DNA probes utilized for Northern analysis were PCR amplified regions of human cDNA. The sequences of the oligonucleotide primers for GR and the catalytic heavy chain of GCL (GCLC) are described below (Table 1), whereas the sequences for the glyceraldehyde-3-phosphate dehydrogenase (GADPH) primers have been published elsewhere (Tan et al., 1999
). Amplification of WI-38 cDNA was performed in a Corbett Research PC-960G thermal gradient cycler, using Taq DNA polymerase (Roche).
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Northern analysis.
Total cellular RNA was isolated from harvested cells using the QIAGEN RNeasy kit according to the manufacturer's instructions. The procedures for the fractionation of RNA samples/RNA molecular weight markers (Promega) using agarose-formaldehyde gels and blot transfer using nylon membranes were adapted from Sambrook et al.(1989). After blotting, transfer of total RNA was visualized by staining with Blot Stain Blue Reversible Northern Blot Staining Solution (Sigma). PCR probes used to detect mRNA fixed to oven baked membranes were labeled with radioactive
[32P]-dCTP using random oligonucleotides as primers. Random primed DNA labeling, obtained using High Prime kits, and the purification of radiolabeled nucleic acids using Mini Quick Spin columns were conducted according to the manufacturer's instructions (Roche). Probe hybridization and post hybridization washes were essentially the same as those described by Sambrook et al.(1989)
. Hybridized blots were exposed to X-ray film kept at 70°C. After exposure, blots were stripped by treatment with boiling 0.5% w/v SDS, and then rehybridized with a PCR probe for GADPH. GADPH is encoded by a housekeeping gene, and was used as a loading control. All values for mRNA concentrations were calculated relative to the amount of GAPDH in the same lane. Developed X-ray films were scanned using a Bio-Rad G710 densitometer, and analyzed using Bio-Rad Quantity One software.
Statistical analysis.
Results are expressed as the mean and SEM of at least 3 separate experiments. Statistical analyses were performed using the Students t-test. Values of p < 0.05 were considered to represent statistically significant differences.
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RESULTS |
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DISCUSSION |
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This study shows that the expression of various components of the GSH system are significantly upregulated in cultured human fibroblast and keratinocyte cell lines by treatment with sublethal concentrations of iAs. In agreement with previous studies utilizing other mammalian cell lines, treatment of keratinocytes with AsIII induced an increase in the level of intracellular GSH (Lee and Ho, 1995; Ochi, 1997
). The increase in GSH levels in PMC42 breast cells after exposure to AsIII is much less pronounced. Higher GSH levels in the keratinocytes were primarily attributed to an increase in the enzyme activity of GCL. The upregulation of GCL enzyme activity by AsIII parallels a corresponding increase in the level of GCL mRNA. However, only the mRNA levels for the catalytic subunit of GCL (GCLC) were assayed. It remains to be determined whether both the catalytic and regulatory subunits are upregulated by arsenic, as has been seen with other forms of oxidative stress (Tian et al., 1997
). In Chinese hamster ovary cells, a significant increase in the enzyme activity of GCL has been shown to occur upon exposure to dimethyl arsenic acid (DMA), but not AsIII (Ochi, 1997
). An increase in the rate of cystine uptake also occurs (Fig. 4
), but does not appear to be a major contributing factor to the higher levels of GSH observed in keratinocyte cells after treatment with AsIII. The relative ratio of reduced GSH to oxidized GSSG is unchanged despite the large rise in GSH levels, presumably due a combination of increased oxidative stress and a concomitant rise in glutathione reductase activity, as noted below.
Our experiments, using low dose arsenic treatment for a period of 24 to 48 h, resulted a dose dependent increase in GSH levels in all (epithelial) cell types examined. Thomas et al. (2001) have reported transient down regulation of GSH levels in primary rat hepatocyte cells after a short (30 min) exposure to arsenic. Other investigators (Liu et al., 2000
; Maiti and Chatterjee, 2000
; Santra et al., 2000
) have also reported down regulation of GSH after acute and chronic in vivo exposures to fairly high dose arsenic. It has been our experience that the effects of arsenic are quite tissue specific and also dose dependent. High doses of iAs and long-term exposures often cause a down-regulation of genes that we find to be upregulated after short-term (6 to 48 h) exposure to subtoxic As. This dual response is typical of transcription factor activity as well as gene expression (Hu et al., 2002
). The response seen by Thomas et al.(2001)
, although seen with As concentrations similar to those we have used, is not typical of the response we see in other cell types and may be limited to very short exposure times or may be specific for rat hepatocytes.
In addition to an increase in GSH levels, exposure to subtoxic AsIII also upregulates the activity of glutathione reductase in all cell types examined, especially fibroblasts. Northern analysis indicated that AsIII-induced upregulation of GR enzyme activity, as with GCL, occurs predominantly at the level of increased mRNA. In contrast, the enzyme activity of GST, another important component of the GSH system, was only upregulated in the keratinocyte cell lines and only at concentrations of AsIII higher than those required to upregulate GR and GCL. Although GST species have been found to be upregulated by arsenic and other forms of oxidative stress in some cell types, such as Chinese hamster ovary cells (Vallis and Wolf, 1996) and hepatocytes (Tchounwou et al., 2001
), this is not necessarily a characteristic of all cell types.
There is a variety of evidence showing that intracellular GSH and GSH-related enzyme activities protect mammalian cells against acute exposure to iAs (Lee and Ho, 1995; Ochi, 1997
; Wang et al., 1996
). The cytoprotective effect of GSH would in part be attributable to its antioxidant properties as exposure to iAs induces a rapid burst in harmful ROS (Barchowsky et al., 1999a
; Liu et al., 2001
). More specifically, reduced GSH is involved in the metabolism and maintenance of the thiol moieties of proteins that may otherwise be susceptible to oxidation by trivalent arsenic (Anderson, 1998
). Glutathione may also play a role in the metabolic processing of iAs by methylation in the liver (Styblo et al., 1996
). However, both keratinocytes and fibroblasts exhibit very low levels of arsenic methyltransferase activity compared to the liver (Styblo et al., 1999
). The presence of monomethyl arsenic acid and dimethyl arsenic acid in the skin (Yu et al., 2000
) is therefore, likely due to initial methylation in the liver and subsequent transport of the methylated derivatives in the blood. The recent discovery that trivalent forms of organic, arsenic monomethyl arsenous acid (MMAsIII) or dimethyl arsenous acid (DMAsIII), can be more toxic than iAsIII (Styblo et al., 2001
) is not relevant here because arsenic methylation does not appreciably occur in these cell types (Styblo et al., 2001
). Changes in GSH levels in keratinocytes and fibroblasts are probably not related to any increase in metabolic detoxification, or toxification, of iAs by methylation.
It is likely that increases in GSH and the activities of related enzymes are part of a multifaceted adaptive response offering cells protection against the acute toxic effects of iAs and that cellular responses to sublethal concentrations of iAs are mediated by redox sensitive signaling events and not by the stoichiometric chemical interactions that can occur between trivalent arsenicals and other nonprotein and protein sulfhydryls such as glutathione (GSH) or certain enzymes. However, long-term changes to the GSH system resulting from chronic exposure to iAs may dysregulate redox-sensitive cell signaling events, having important consequences for tumor growth and progression. Alterations in the levels of GSH and GSH-related enzymes have been observed to occur at different stages of tumor development (Lusini et al., 2001; Perquin et al., 2001
). It has been proposed that increased expression of GSH and the activities of related enzymes, including GR, improve malignant cell resistance to oxidative stress, facilitating cell proliferation and aggressiveness (Perquin et al., 2001
).
The mechanism by which iAs regulates the activities of the GCL and GR enzymes in vivo remains unclear. Previous studies have shown iAsIII does not affect yeast GR (nor equine GST) enzyme activity in cell free systems at the low physiologically relevant concentrations that were utilized in this study (Chouchane and Snow, 2001; Styblo et al., 1997
). However, it remains to be determined if critical cysteine residues in GR or GCL are influenced by redox changes affected by iAs in vivo. Further investigations are also required to determine if ROS mediates the changes in the expression of GR and GCL after exposure to iAs. Previous studies have shown that gene expression of GSH-related enzymes in human cells is increased under conditions of oxidative stress (Bergelson et al., 1994
; Shi et al., 1994
). For instance, increased ROS mediates the upregulation of GCL in HepG2 cells by UVA irradiation (Morales et al., 1998
). A 5 upstream AP-1 consensus binding site in the GCL promoter is primarily involved in this process. In hepatoma cells, there is a correlation between quinone-mediated production of harmful ROS, the induction of AP-1 binding activity, and GST-Ya gene expression (Pinkus et al., 1996
). Arsenite has also been shown to modulate the expression of the GST-Ya gene by an increase in AP-1 binding activity at a 5 upstream regulatory element within the GST-Ya promoter (Pinkus et al., 1996
). Other studies have shown that iAsIII induces AP-1 activation, although there is conflicting evidence about which mitogen activated protein (MAP) kinase pathway is involved (Barchowsky et al., 1999b
; Huang et al., 1999
; Parrish et al., 1999
). We have recently shown that short exposures (24 h or less) to very low dose iAs can significantly upregulate both AP-1 and nuclear factor-
B (NF-
B) DNA binding activity in GM847 fibroblasts (Hu et al., 2002
). This increased DNA binding activity is due in part to an increase in the relative amounts of the cJun and cFos proteins, but with no significant increase in mRNA levels. Perhaps more importantly, these low levels of AsIII also upregulate two key redox activators of AP-1 and NF-
B, Ref-1 and thioredoxin (Hu et al., 2002
). Barchowsky et al. have shown that low dose AsIII also induces AP-1 activation in porcine endothelial cells (Barchowsky et al., 1999b
). Both NF-
B and AP-1 are implicated in the inducible expression of a wide variety of genes involved in oxidative stress and cellular response mechanisms (Allen and Tresini, 2000
).
Overall this investigation shows that iAs, a known human carcinogen, upregulates the activities of multiple GSH-related enzymes in human keratinocyte and fibroblast cells. This would have to be considered a protective response, as acute exposure to iAs induces a rapid, but transient, burst in harmful ROS. The upregulation of the GSH-related enzyme activities by iAs observed in this study produces higher GSH levels (i.e., by induction of GCL) and promotes the antioxidant properties of GSH (i.e., through increased GR and GST). These increases in GCL and GR activity are mediated by increased steady-state mRNA levels and are produced by nonlethal concentrations of iAs that are likely to be relevant to arsenic-related diseases such as skin cancer. As we have found with respect to changes in transcription factor binding (Hu et al., 2002), long-term modulation of the GSH system by chronic exposure to sublethal iAs may not be the same as the acute changes we have noted here, but may also have a strong influence on cellular redox signaling events. In particular, this might play an important role in arsenic carcinogenesis.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed. Fax: +61 3 9251 7328. E-mail: esnow{at}deakin.edu.au.
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