Cancer Research AACR Membership Advances in Breast Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, S.-X.
Right arrow Articles by Hei, T. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, S.-X.
Right arrow Articles by Hei, T. K.
[Cancer Research 65, 3236-3242, April 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Mitochondrial Damage Mediates Genotoxicity of Arsenic in Mammalian Cells

Su-Xian Liu1, Mercy M. Davidson2, Xiuwei Tang3, Winsome F. Walker2, Mohammad Athar3, Vladimir Ivanov1 and Tom K. Hei1,4

1 Center for Radiological Research, Departments of 2 Neurology and 3 Dermatology, College of Physicians & Surgeons, and 4 Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, New York, New York

Requests for reprints: Tom K. Hei, Center for Radiological Research, Columbia University, VC11-205, 630 West 168th Street, New York, NY 10032. Phone: 212-305-8462; Fax: 212-305-3229; E-mail: tkh1{at}columbia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Arsenic is an important environmental carcinogen that affects millions of people worldwide through contaminated water supplies. For decades, arsenic was considered a nongenotoxic carcinogen. Using the highly sensitive AL mutation assay, we previously showed that arsenic is, indeed, a potent gene and chromosomal mutagen and that its effects are mediated through the induction of reactive oxygen species. However, the origin of these radicals and the pathways involved are not known. Here we show that mitochondrial damage plays a crucial role in arsenic mutagenicity. Treatment of enucleated cells with arsenic followed by rescue fusion with karyoplasts from controls resulted in significant mutant induction. In contrast, treatment of mitochondrial DNA–depleted ({rho}0) cells produced few or no mutations. Mitochondrial damage can lead to the release of superoxide anions, which then react with nitric oxide to produce the highly reactive peroxynitrites. The mutagenic damage was dampened by the nitric oxide synthase inhibitor, NG-methyl-L-arginine. These data illustrate that mitochondria are a primary target in arsenic-induced genotoxic response and that a better understanding of the mutagenic/carcinogenic mechanism of arsenic should provide a basis for better interventional approach in both treatment and prevention of arsenic-induced cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Inorganic arsenic is naturally occurring and ubiquitously present in the environment. Contamination of drinking water by arsenic, either by natural means or through industrial pollution, represents the major route of human exposure (1, 2). It is a serious environmental problem worldwide because of the large number of contaminated sites that have been identified and the hundreds of millions of people at risk, particularly in developing countries, such as West Bengal in India and Bangladesh (3, 4). Although the water supplies in the United States are generally low in arsenic, there have been reports of arsenic contamination of ground water in the Southwest with levels in the hundreds and, in few cases, >1,000 µg/L (1). In the United States, arsenic contamination of the environment is mainly through the use of chromated copper arsenate–preserved timbers in the construction industry (5). Leaching of arsenic and other preservative components from wood used in aquatic and residential situations poses serious threat to the environment with some 140 million pounds of chromated copper arsenate solution being used annually.

Epidemiologic data have shown that chronic exposure of humans to inorganic arsenical compounds is associated with liver injury, peripheral neuropathy, and an increased incidence of cancer of the lung, skin, bladder, and liver (6, 7). However, the mechanism(s) underlying its carcinogenicity remains poorly understood. For decades, arsenic has been considered by many to be a nongenotoxic carcinogen. Although it has been shown to induce chromosomal aberrations and inhibit DNA repair, it is only weakly active or, more often, completely inactive in bacterial and mammalian cell mutation assays (8–10). Using the AL human-hamster hybrid cell assay, we showed that this could be a result of the poor recovery of multilocus mutations that are prevalently induced by arsenic so that arsenic is, in fact, a potent mutagen (11, 12). We further showed that hydroxyl radicals, generated through a superoxide-mediated process involving hydrogen peroxide, play an important role in mediating the genotoxic effects of arsenic (13). These findings are consistent with the reports stipulating the functional role of oxidants in mediating the biological effects of arsenite by linking glutathione content (14), heme oxygenase (15, 16), oxidative DNA damage (12, 17), and metallothionine (18) to the end points being examined. However, the origin of these oxyradical species and the pathways involved in the generation of other secondary radical species are not known.

Synthesis of ATP in mitochondria requires oxidative metabolism and the consumption of oxygen, which results in the production of various reactive oxygen species including hydroxyl radicals and superoxide anions as by-products. It is likely that arsenic-induced mitochondrial membrane damage results in the leakage of superoxide anions into the cytosol of cells, a possibility that is consistent with our previous observation based on electron spin resonance (ESR) spin trap studies (13). There is evidence that arsenic up-regulates intracellular nitric oxide concentration in mammalian cells (19, 20). A possible consequence of mitochondrial damage is, therefore, the secondary production of peroxynitrites, a strong oxidant formed by the diffusion-controlled reaction of superoxides and nitric oxide (21). In this regard, there is recent evidence that arsenic induces peroxynitrite production in bovine endothelial cells and may exacerbate the inflammatory process typical of atherosclerosis (22). Whereas arsenic has been shown to alter mitochondrial membrane potential and induce apoptosis in various human cancer cells (23, 24), the role of mitochondria as a genotoxic target of arsenic has yet to be established. Using enucleation and fusion techniques and the human-hamster hybrid (AL) cells, we show here that mitochondria are a direct target of arsenic-induced genotoxicity in mammalian cells and that peroxynitrite anions are an important mediator in the process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. The AL hybrid cells, which contain a standard set of CHO-K1 chromosomes and a single copy of human chromosome 11, were used. Chromosome 11 encodes cellular surface markers that render AL cells sensitive to killing by specific monoclonal antibodies in the presence of rabbit serum complement (HPR, Inc., Denver, PA). Antibody E7.1 specific to the CD59 antigen was produced from hybridoma culture as described (25, 26). Cells were maintained in Ham's F-12 medium, supplemented with 8% heat-inactivated fetal bovine serum, 25 µg/mL gentamicin, and 2x normal glycine (2 x 10–4 mol/L), at 37°C in a humidified 5% CO2 incubator and passaged as described (11, 13, 27).

Chemical treatment. Stock solution (1 mg/mL) of sodium arsenite (Sigma Chemical, St. Louis, MO) was prepared and used as described (11, 12). Working concentrations were prepared by diluting the stock with complete F-12 medium. Enucleated cytoplasts and mitochondrial DNA (mtDNA)–deficient cells were treated with graded doses of arsenite for various periods of time as indicated. After treatment, cells were washed twice with plain F-12 medium and processed for the various end points as described. In experiments involving the nitric oxide synthase inhibitor, both the active NG-methyl-L-arginine (L-NMMA) and the inactive enantiomer NG-methyl-D-arginine (D-NMMA; Molecular Probes, Inc., Eugene, OR), dissolved in F-12 medium and filter sterilized, were added to the cultures 1 hour before arsenic treatment and remained in the medium throughout the treatment period.

Enucleation of AL cells. Exponentially growing AL cells (~2 x 105 cells per dish) grown in either 35-mm-diameter tissue culture plastic dishes or 35 mm glass bottom microwell dishes ({Delta}TC3 dishes, BiopTechs, Butler, PA) were enucleated as described (28, 29). Briefly, cells were treated with cytochalasin B (1 µg/mL) for 60 minutes and the dishes containing the cells were centrifuged upside down inside a 250 mL Sorvall centrifuge bottle (Nalge-Nunc International, Binghamton, NY) using a GSA rotor at 6,700 rpm at 37°C for 20 minutes. After centrifugation, the cultures were washed thrice with complete medium to remove excess cytochalasin B. The enucleated cells were then incubated at 37°C for 30 minutes to recover before treatment with arsenite. The efficiency of enucleation was evaluated by the absence of nuclear staining with a 50 nmol/L solution of Hoechst 33342 (Polysciences, Warington, PA) for 20 minutes. The cytoplasts were then treated with arsenite for a period of 3 to 4 hours as described above.

Preparation of karyoplast from AL cells. The nuclei that were centrifuged out of the AL cells, containing a small amount of cytoplasm (<10% v/v of controls), would remain at the bottom of the centrifuge tubes along with some whole cells that escaped enucleation. Karyoplasts were collected and pelleted by centrifugation at 1,500 rpm for 5 minutes at 37°C. After washing with medium twice to remove remnants of cytochalasin B, the karyoplasts were resuspended in complete F-12 medium until fusion with cytoplasts. The percentage of whole cells in the karyoplast preparation was evaluated by staining with Hoechst dye as described above. Karyoplast preparations with <5% whole cells were used for cytoplast fusion. Viability of karyoplasts obtained was >95% as determined by trypan blue staining.

Fusion of cytoplasts with karyoplasts. Control or arsenite-treated cytoplasts grown in 35-mm-diameter dishes (~2 x 105 cells per dish) were washed twice with F-12 medium and fused with karyoplasts in a ratio of 3:1 using 50% polyethylene glycol (PEG-1450, American Type Culture Collection, Manassas, VA) as described (29, 30). Briefly, the cytoplast-karyoplast mixture was incubated in a 5% CO2 incubator at 37°C for 2 hours before fusion. To initiate the fusion, medium was aspirated, and 1 mL of prewarmed, sterile 50% PEG containing 10% DMSO (pH 7.3) was added drop-wise over 1 minute. Immediately, complete F-12 medium was added and the fusion mixture was returned to the incubator. After 4 to 5 hours of incubation, the fusion cells were replenished with fresh F-12 medium containing 50 µg/mL uridine. This step was necessary to provide cells with diminished respiratory chain function an additional source of pyrimidine for better recovery (31). Fusion cells were incubated for 5 to 6 days before being trypsinized and expanded for clonogenic survival and mutagenesis assay as described below.

Determination of reactive oxygen species formation in arsenite-treated cytoplasts. To quantify the level of reactive oxygen species in arsenic-treated live cells, cytoplasts and, in untreated controls, exponentially growing AL cells and freshly enucleated cytoplasts (2 x 105 cells) grown on 35 mm glass bottom microwell dishes ({Delta}TC3 dishes, BiopTechs), were pretreated for 40 minutes at 37°C with 1 µmol/L fluorescent probe, 5',6'-chloromethyl-2',7'dichlorodihydro-fluorescein diacetate (CM-H2DCFDA, Molecular Probes; refs. 13, 32). Graded doses of arsenite, with or without the radical scavenger DMSO (0.1%), were then added to the cultures. Cultures were examined using a confocal microscope and a semiquantitative estimation of the reactive oxygen species–associated fluorescent signal, expressed as percent control, was obtained using the composite images generated by Adobe Photoshop (Adobe Systems, Inc., San Jose, CA). A total of 50 to 60 individual cells per experiment were selected randomly and the fluorescent images quantified. On average, over 300 cells were measured per treatment group as described (13).

Generation of mitochondrial DNA–deficient {rho}0AL cells. Exponentially growing AL cells grown in 100-mm-diameter tissue culture dishes were treated with freshly prepared chemotherapeutic drug ditercalinium (gift sample from Dr. Robert Schultz, National Cancer Institute, NIH, Bethesda, MD) in F-12 medium at a dose of 1.5 µg/mL for 3 to 4 months in the presence of uridine (50 µg/mL) as described (33, 34). During this time period, colonies of drug-resistant cells were cloned and continued to be passaged in drug-containing medium. Fresh medium was replenished every 3 to 4 days. Periodically, expression of mtDNA in ditercalinium-treated clonal isolates was determined by PCR amplification of genomic DNA using the peptidyl transferase encoding region of the mitochondrial 16S rRNA gene as primer (33). When the mtDNA was found to be >95% depleted in the population, these {rho}0 cells were rinsed twice with HBSS and used in the mutagenesis experiments.

Determination of mitochondrial membrane potential. To determine the mitochondrial membrane potential of {rho}0AL cells, a membrane potential–sensitive fluorescent probe JC-1 (Molecular Probes) was used (35). JC-1 is a cationic carbocyanine dye that presents itself as green fluorescent monomers at low concentration (i.e., in cells with low mitochondrial function or membrane potential). In contrast, in cells with normal mitochondrial function, membrane potential–driven accumulation of these dyes resulted in the formation of yellowish-red fluorescent J-aggregates. Briefly, control and {rho}0 cells were plated into 35 mm glass bottom microwell dishes as described above. After overnight incubation, the cells were loaded with 10 µmol/L JC-1 for 30 minutes at 37°C, washed and viewed using a epifluorescence confocal microscope with either a green excitation filter at 488 nm or a blue excitation filter at 543 nm, which allows the visualization of the green and red fluorescence, respectively, on a heated 37°C stage.

Western blotting of 3-nitrotyrosine–containing protein. Control and arsenite-treated cells were harvested and proteins extracted as described (36). The samples were then centrifuged and the protein content of the supernatant was determined by Bradford assay (37). Thirty micrograms of total cell lysate from each treatment group was fractionated by SDS-PAGE gel, transferred onto Hybond membranes, and immunoblotted with rabbit antinitrotyrosine antibody (0.5 µg/mL, Upstate Biotechnologies, Lake Placid, NY) at 4°C. Peroxidase-conjugated anti-rabbit IgG (1:10,000, The Jackson Laboratory, Bar Harbor, ME) was used to detect 3-nitrotyrosine–containing proteins by the enhanced chemiluminescence (ECL) procedure (Amersham, Arlington Heights, IL). Cells treated overnight with peroxynitrite (80 µmol/L, Upstate Biotechnologies) were used as positive controls.

Mutation assay. After treatment of either whole cells or cytoplasts with arsenite, followed by rescue fusion with karyoplasts to reconstitute AL cells in the latter case, cultures were replated into T25 flasks and cultured for 5 to 7 days. This expression period is needed to permit surviving cells to recover from the temporary growth lag caused by arsenite and loss of expression of CD59 surface antigen in mutant cells. To determine mutant fractions, 5 x 104 cells were plated into each of six 60 mm dishes as described (11, 13, 27). The mutant fraction at each dose (MF) was calculated as the number of surviving colonies divided by the total number of cells plated after correction for any nonspecific killing due to complement alone.

Statistical analysis. Data were presented as mean and standard derivations. Comparisons of fluorescent intensity and mutant fractions between treated groups and controls were made by Student's t test. P ≤ 0.05 between groups was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of reactive oxygen species by arsenite in cytoplasts. To ascertain if the nucleus is a necessary target for arsenite-induced genotoxicity in mammalian cells, enucleated cytoplasts were exposed to arsenite followed by rescue fusion with normal karyoplasts from untreated cells to determine whether gene mutations can be induced in the absence of direct nuclear damage by arsenite. First, we determined if cytoplasts, upon arsenic treatment, could generate reactive oxygen species. Figure 1 shows representative confocal images of fluorescent signals from control (A-C) and enucleated AL cells (D-F) pretreated with the fluorescent probe, CM-H2DCFDA, for 40 minutes and subsequently treated with sodium arsenite (2 µg/mL) for 5 minutes before the pictures were taken. The nonfluorescent dye passively diffuses into cells where the acetates are cleaved by intracellular esterase (32, 38). The resulting diol is more polar and is better retained inside the plasma membrane. The diol can then be oxidized by oxyradical species to the fluorescent form with absorbency at 504 nm. The fluorescent signals obtained were quantified as a surrogate index for the production of reactive oxygen species using Adobe Photoshop image analysis software as described (13). A 2 µg/mL dose of arsenite (15.4 µmol/L) increased the average fluorescent intensity in whole cells by 2.5-fold above control levels (Fig. 1B versus A). Likewise, enucleated cells treated with sodium arsenite (Fig. 1E) were able to generate radical species in the absence of nuclei. Similar to the whole cells, cytoplasts treated with arsenite had an average fluorescent intensity 2.4-fold above nontreated counterparts (Fig. 1E versus D). Arsenite-treated cytoplasts showed a fluorescent intensity ~77% that of comparatively treated whole cells (55.7 ± 3.8 versus 72.3 ± 7.1; Fig. 1E versus B). In the presence of 0.1% DMSO, a radical quencher, the fluorescent signals in both arsenite-treated control culture (Fig. 1C) and enucleated cells (Fig. 1F) were reduced to essentially background levels, thus confirming the oxyradical nature of the fluorescent signals.



View larger version (34K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Representative fluorescent signals generated from composite images obtained by confocal microscopy of either whole (A-C) or enucleated AL cells (D-F) pretreated with the fluorescent probe, CM-H2DCFDA, for 40 minutes with or without subsequent sodium arsenite treatment (2 µg/mL or 15.4 µmol/L). A, control AL cells treated with only the fluorescent probe; B, 5 minutes after the addition of arsenite; C, treatment as in (B), but with concurrent 0.1% DMSO; E, enucleated AL cells treated with only the fluorescent probe; E, 5 minutes after the addition of arsenite to the cytoplasts; F, treatment as in (E), but with 0.1% DMSO. Magnification, x100.

 
Mutagenicity of arsenite-treated cytoplasts. To determine if cytoplasts can initiate signaling pathways resulting in genotoxic damage upon arsenite treatment, enucleated cells were treated with arsenite (2 µg/mL for 3 hours) and immediately fused with karyoplasts at a ratio of 3:1. This relatively short treatment period was necessary because the viability of cytoplasts decreased rapidly after 4 to 5 hours postenucleation (data not shown; ref. 39). When cytoplasts were fused with karyoplasts under the conditions used in the present study, three fusion outcomes were possible. First, a karyoplast could fuse with another karyoplast to produce an unstable doublet. A second possible outcome was that a cytoplast fused with another cytoplast to produce another nonviable cytoplasmic doublet. The only viable outcome would be the fusion of a cytoplast with a karyoplast. Whereas the fusion efficiency was only 15% to 20%, the successfully fused cells had a high viability index (~80%) as determined by colony-forming capacity. Cultures formed by fusion of nontreated cytoplasts with nuclei in a similar manner were used as controls. As shown in Fig. 2, treatment of cytoplasts alone with arsenite resulted in a total mutant fraction of 297 ± 29 CD59– mutants per 105 survivors. Given the background mutation incidence from the fusion of nontreated cytoplasts to nuclei was 124 ± 17, the induced mutant fraction due to arsenite-induced cytoplasmic damage was 173 ± 23 CD59– mutants per 105 survivors.



View larger version (14K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Incidence of CD59– mutations in AL cells reconstituted from arsenite-treated cytoplasts (2 µg/mL or 15 µmol/L for 3 hours) using karyoplasts from nontreated control cells. Similarly, reconstituted cells without arsenite exposure were used as controls. Data are pooled from four to five experiments. Bars, SD.

 
Inactivation of mitochondrial membrane potential in {rho}0 cells. Our next approach was to ascertain the contribution of mitochondria in mediating the genotoxic response of AL cells to arsenite treatment. First, we determined if {rho}0AL cells have normal mitochondrial membrane potential. {rho}0AL cells were generated by long-term treatment with the DNA-intercalating agent ditercalinium in the presence of uridine. The degree of mtDNA depletion depends on the treatment period. After treatment for 3 to 4 months, several clonal isolates were completely depleted of their endogenous mtDNA, as determined by PCR assay (Fig. 3A). Mitochondrial function in control and {rho}0AL was determined using JC-1 stain as shown in Fig. 3B and C. In cells with normal mitochondrial function, membrane potential–driven accumulation of these dyes results in the formation of red fluorescent J-aggregates as shown in control AL (Fig. 3B). In {rho}°AL cells with depressed mitochondrial membrane potential, the mitochondria seem green when stained with JC-1 dye for a comparable period of 30 minutes at 37°C (Fig. 3C). Immunohistochemical staining of mtDNA encoded cytochrome c activities in {rho}0AL cells was found to be negative whereas in control AL cells, there was perinuclear clustering of mitochondrial staining (data not shown).



View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Characterization of {rho}0AL cells. A, PCR amplification of genomic DNA from {rho}0AL cells using the peptidyl transferase encoding region of the mitochondrial 16S rRNA gene as primer, which yield a PCR product of 308 bp (65). B, localization of JC-1 by epifluorescence confocal microscopy in control AL cells under green excitation filter where red J-aggregates was detected. In {rho}0AL cells, the depressed mitochondrial membrane potential resulted in few J-aggregates and the staining was mostly green under blue excitation filter (C). Magnification, x100.

 
Mutagenicity of arsenite in {rho}0AL cells. {rho}0AL cells with diminished mitochondrial membrane potential (cultures from Fig. 3C) were treated with graded doses of sodium arsenite for 18 hours. As shown in Fig. 4, arsenite treatment of {rho}0AL cells resulted in a dose-dependent increase in cytotoxicity. The plating efficiency of nontreated {rho}0AL cells had a plating efficiency similar to that of the wild-type controls. The CD59– mutant fraction of the {rho}0AL cells was ~462 ± 87, a value that was six times higher than the corresponding wild-type controls. In contrast, {rho}0AL cells treated with graded doses of sodium arsenite responded poorly to the mutagenic challenge of arsenite and showed little or no mutations above the background levels. Likewise, {rho}0AL cells that were panned (40) to reduce the preexisting mutant fraction similarly showed no mutant induction upon treatment with arsenic (data not shown). These data strongly suggest that mitochondria function is necessary for arsenic-induced genotoxicity.



View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Mutation frequency at the CD59– locus in {rho}0AL cells treated with graded doses of sodium arsenite for 18 hours. The survival fraction of the various treatment groups is shown above each column. Data are pooled from three experiments. Bars, SD. P.E., plating efficiency; S.F., surviving fraction when compared with nontreated {rho}0AL cells.

 
Role of peroxynitrites in arsenic-induced genotoxicity. Figure 5 shows the toxicity and incidence of CD59– mutations in wild-type AL cells treated with graded doses of arsenite for 24 hours with or without concurrent treatment with L-NMMA (1 mmol/L). The methyl arginine was applied 1 hour before arsenite treatment and remained throughout the exposure period. Arsenite induced a dose-dependent increase in toxicity as well as CD59– mutation in AL cells after 24 hours of treatment (columns 2 and 3) that was partially blocked by L-NMMA treatment (columns 4 and 5). The suppression by L-NMMA on arsenic-induced mutant fraction at either doses was highly significant (P < 0.05). In contrast, the inactive enantiomer, D-NMMA, was totally inactive in suppressing the mutagenicity of arsenic (column 6 versus 4). Treatment with L-NMMA alone was neither toxic nor mutagenic to AL cells (column 7).



View larger version (29K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Incidence of CD59– mutations in wild-type AL cultures treated with graded doses of sodium arsenite for 24 hours with or without cotreatment with L-NMMA. The survival fraction of the various treatment groups is shown above each column. Data are pooled from three to four experiments. Bars, SD.

 
Arsenite induces 3-nitrotyrosine protein modification in AL cells. Because peroxynitrite promotes nitration and oxidation of phenolic compounds, such as tyrosine, formation of 3-nitrotyrosine–containing proteins has often been used as a biomarker of peroxynitrite induction in mammalian cells (41, 42). Figure 6 shows the dose response induction of 3-nitrotyrosine modification of a 110 kDa protein band in arsenite-treated (24 hours) AL cells. Peroxynitrite (80 µmol/L overnight treatment) was used as positive control in the Western blot. Addition of L-NMMA (1 mmol/L) to the arsenite-treated group (1 µg/mL) reduced the level of protein modification to essentially control level and attested to the peroxynitrite origin of the reaction.



View larger version (33K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. Representative Western blot of 3-nitrotyrosine–modified protein, a marker of peroxynitrite anion formation, from either control or arsenite-treated (24 hours exposure) AL cells. Peroxynitrite-treated cells (80 µmol/L) were used as a positive control and ß-actin was used as housekeeping control. Image was quantified using a densitometer and normalized to the ß-actin level. Proteins containing 3-nitrotyrosine were detected using a polyclonal 3-nitrotyrosine antibody. A protein band with a size of 110 kDa was consistently identified. Treatment with L-NMMA reduced the level of protein modification in arsenite-treated cells (1 µg/mL) to essentially control levels.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The metalloid arsenic, while a natural component of the earth's crust, is a serious environmental concern and a global health problem affecting millions of people. A better understanding of the mutagenic/carcinogenic mechanism of arsenic should provide a basis for better interventional approach in both treatment and prevention.

Using the highly sensitive human-hamster hybrid (AL) cell assay that is proficient in recovering multilocus deletions, we dispelled the earlier notion that arsenic is a nongenotoxic carcinogen (11–13). Using ESR spin trap, we further showed that superoxide-driven hydroxyl radicals are an important mediator in the genotoxicity of arsenic (13). The obvious questions that follow are "What is the origin of these reactive oxygen species?" and "Can there be any other secondary radical species involved in the process?" Using enucleation and cell fusion techniques, we show here that mitochondria are an important target of arsenic-induced genotoxicity. The generation of superoxide anions is consistent with the downstream induction of peroxynitrites, the effect of which can be reduced by an inhibitor of nitric oxide synthase (43). Thus, the genotoxicity of arsenic is mediated by a combination of both reactive oxygen and nitrogen species.

An important corollary of the present study is that the nucleus is not necessarily the only and sufficient target for environmental carcinogens. Treatment of cytoplasts with arsenic, in the absence of the nucleus, initiates oxyradical production, as detected by using CM-H2DCFDA. This induction, however, is only ~77% of the level measured in whole cells and is likely to be a result of the loss of a small amount of cytoplasm during the enucleation process, either as free vesicles or as ruminants attached to the karyoplasts. These data suggest that cytoplasm plays an essential role in the initiation of oxidative damage, an observation previously made with targeted cytoplasmic irradiation by {alpha} particles (44).

Mitochondria, being the metabolic center of a cell, are intimately involved in the production of reactive oxygen species, mainly superoxides and hydrogen peroxides. Approximately 2% to 4% of the oxygen consumed by mitochondria is converted to superoxides by the electron transport system (45). Mitochondrial structures, on the other hand, are also very susceptible to oxidative damage through membrane lipid peroxidation, protein oxidation, and mtDNA damage. Evidence implicating mitochondria as a possible target of arsenic toxicity has been obtained mainly through the induction of apoptosis in various leukemia and cancer cell lines (46, 47). Our findings that AL cells with diminished mitochondrial function are unable to respond to arsenic-induced genotoxic damage are corroborated with the observation that the antioxidant, N-acetyl-cysteine, completely suppressed arsenic-induced apoptosis in HeLa cells by preventing mitochondrial membrane depolarization (23).

In the present study, the role of mitochondria in mutant induction by arsenic was evaluated by two complementary approaches: (a) with reconstituted cells made by the fusion of arsenic-treated cytoplasts with karyoplasts from controls to show that the nucleus need not be present at the time of arsenic treatment and (b) {rho}0AL cells without mtDNA and diminished mitochondrial membrane potential responded poorly to arsenite-induced mutagenesis. Although treatment with ditercalinium toward the generation of {rho}0 status increased the background mutant fraction of the AL cells, the clonogenic efficiency of the {rho}0AL cells was not much different from the wild-type cells. In addition, reducing the preexisting background mutant fraction of {rho}0AL cells by the panning technique does not alter the sensitivity of the {rho}0AL cells to the mutagenicity of arsenic (data not shown). However, compared with wild-type cells, {rho}0AL cells are ~40% more susceptible to the cytotoxic effects of arsenic (Fig. 4; refs. 11–13). Although increases in intracellular oxidant levels can also be mediated by other cytosolic components including lysosomes, endoplasmic reticulum, and membrane-bound NADPH oxidase (48), our findings using {rho}0AL cells clearly defined mitochondria as a primary target in mediating the DNA-damaging effect of arsenic in mammalian cells. In this regard, our current data are consistent with our previous observations that Rhodamine 6G–treated AL cells with diminished mitochondrial membrane potential show essentially no mutations upon exposure to arsenic.5

Peroxynitrite anion is a strong oxidant and nitrating species resulting from the near diffusion–controlled reaction of superoxide with nitric oxide (49). There is evidence that, in tissues, peroxynitrites not only escape the scavenging by most low molecule weight antioxidants (50), but can also activate stress responsive pathways and kinases of the src family to modulate cellular signal transduction cascade (49, 51). Because mitochondria constitute a primary locus for the intracellular formation and reactions of peroxynitrite, which has a much longer half life compared with hydroxyl radicals and can readily diffuse across biomembranes (43), it is likely that multiple radical species are involved in the genotoxic response of arsenic. Our present findings are consistent with the observation that nitric oxide, an upstream molecule in the biosynthesis of peroxynitrite, has been implicated in endothelial cell damages associated with arsenic exposure (52).

Although formation of 3-nitrotyrosine has been used as marker of endogenous peroxynitrite formation (41, 42), there is recent indication that other reactions, such as myeloperoxidase with hydrogen peroxide, can also lead to nitrotyrosine formation (53). As a result, an increase in 3-nitrotyrosine content, as detected by Western blot in the present study, should be viewed as an indicator of an increase in nitrosative damage, an interpretation that does not alter the conclusion of the present findings.

The CD59 locus of the AL cells used in this study has been used successfully in detecting mutagens that induce predominately large deletions, such as radiation (54) and asbestos fibers (55), findings that have subsequently been confirmed using other mutagenic assay systems that are equally proficient in recovering multilocus deletions (56, 57). There is no evidence that the AL cell is intrinsically hypermutable (25, 54, 58) and its mutability at the HPRT locus (located on the X-chromosome of the hamster gene) is no different from that of other human or rodent cell lines (11, 25, 55). Whereas the AL cell assay is a useful model for mechanistic studies, precise carcinogenic mechanism for arsenic in specific target tissues, such as the skin and lung, where compounding factors may be present, need to be further evaluated.

Although epidemiologic studies have firmly established that arsenic is a human carcinogen, the mechanism whereby arsenic induces human cancer remains unclear and is likely to involve more than one mechanism (see refs. 59, 60 for review). The doses of arsenite used in the present study (1-2 µg/mL or 1-2 ppm) are very high compared with the arsenic level generally found in drinking water supplies in the United States, with the exception of some Southwest states where arsenic in the level of 200 to 300 ppb and, in few cases, >1 ppm, have been identified (1, 61). However, in certain parts of Bangladesh and West Bengal in India, as many as 5% of the drinking wells that were sampled had arsenic level exceeding 1 ppm and that 17% of the wells had levels exceeding 200 µg/L (4, 62), 20 times higher than the current U.S. maximum contaminant level of 10 µg/L. Thus, the dose used in the present study, while high according to the U.S. standard, is still environmentally relevant in other parts of the world. Furthermore, the treatment period used in the present study is only a matter of hours compared with chronic exposure in epidemiologic studies. There is evidence that arsenic may function as a cocarcinogen with UV light in the induction of skin cancers (63). However, arsenic has been shown to induce chromosomal aberrations and sister chromatid exchanges in the lymphocytes of individuals living in highly contaminated areas (64). Our present findings that mitochondria is an important target for arsenic-induced genotoxicity provide an additional impetus in focusing on mitochondria as an important cellular target in interventional remedy for arsenic-induced diseases.


   Acknowledgments
 
Grant support: NIH grants ES 05786, ES 11804, HD32062, and NS11766; Superfund grant P42 ES 10349; and Environmental Center grant ES10349.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Charles Waldren for critical reading of the manuscript.


   Footnotes
 
5 Liu et al., unpublished observation. Back

Received 5/10/04. Revised 2/ 7/05. Accepted 2/11/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Chan PC, Huff J. Arsenic carcinogenesis in animals and in humans: mechanistic, experimental and epidemiological evidence. Environ Carcinog Ecotoxicol Rev 1997;C15:83–122.
  2. Gebel T. Confounding variables in the environmental toxicology of arsenic. Toxicology 2000;144:155–62.[CrossRef][Medline]
  3. Dahr RK, Biswas BK, Samanta G, et al. Groundwater arsenic calamity in Bangladesh. Curr Sci 1997;73:48–59.
  4. Nickson R, McArthur J, Nurgess W, Ahmed KM, Ravenscroft P, Rahman M. Arsenic poisoning of Bangladesh groundwater. Nature 1998;395:338–9.[CrossRef][Medline]
  5. Katz SA, Salem H. Chemistry and toxicology of building timbers pressure treated with chromated copper arsenate: a review. J Appl Toxicol 2005;25:1–7.[CrossRef][Medline]
  6. Taylor PR, Qiao YL, Schatzkin A, et al. Relation of arsenic exposure to lung cancer among tin miners in Yunnan Province, China. Br J Ind Med 1989;46:881–6.[Medline]
  7. Chiou HY, Hsueh YM, Liaw KF, et al. Incidence of internal cancers and ingested inorganic arsenic: a seven year follow up studies in Taiwan. Cancer Res 1995;55:1296–300.[Abstract/Free Full Text]
  8. Lee TC, Oshimura M, Barrett JC. Comparison of arsenic induced cell transformation, cytotoxicity, mutation and cytogenetic effects in Syrian hamster embryo cells in culture. Carcinogenesis 1985;6:1421–6.[Abstract/Free Full Text]
  9. Hughes MF. Arsenic toxicity and potential mechanisms of action. Toxicol Lett 2002;133:1–16.[CrossRef][Medline]
  10. Moore M, Harrington-Brock K, Doerr CL. Relative genotoxic potency of arsenic and its methylated metabolites. Mutat Res 1997;386:279–90.[CrossRef][Medline]
  11. Hei TK, Liu SX, Waldren C. Mutagenicity of arsenic in mammalian cells: role of reactive oxygen species. Proc Natl Acad Sci U S A 1998;95:8103–7.[Abstract/Free Full Text]
  12. Kessel M, Liu SX, Xu A, Santella R, Hei TK. Arsenic induces oxidative DNA damage in mammalian cells. Mol Cell Biochem 2002;234:301–8.
  13. Liu SX, Athar M, Lippai I, Waldren CA, Hei TK. Induction of oxyradicals by arsenic: implications for mechanisms of genotoxicity. Proc Natl Acad Sci U S A 2001;98:1643–8.[Abstract/Free Full Text]
  14. Styblo M, Serves SV, Cullen WR, Thomas DJ. Comparative inhibition of yeast glutathione reductase by arsenicals and arsenothiols. Chem Res Toxicol 1997;10:27–33.[CrossRef][Medline]
  15. Menzel DB, Rasmussen RE, Lee E, et al. Human lymphocytes heme oxygenase I as a response biomarker to inorganic arsenic. Biochem Biophys Res Commun 1998;250:653–6.[CrossRef][Medline]
  16. Kitchin KT, Del Razo LM, Brown JL, Anderson WL, Kenyon EM. An integrated pharmacokinetic and pharmacodynamic study of arsenic action. 1. Heme oxygenase induction in rats. Teratog Carcinog Mutagen 1999;19:385–402.[CrossRef][Medline]
  17. Shen J, Wanibuchi H, Salim EI, et al. Liver tumorigenicity of trimethylarsine oxide in male Fisher 344 rats—association with oxidative DNA damage and enhanced cell proliferation. Carcinogenesis 2003;24:1827–35.[Abstract/Free Full Text]
  18. Jiang G, Gong Z, Li XF, Cullen WR, Le XC. Interaction of trivalent arsenicals with metallothionein. Chem Res Toxicol 2003;16:873–80.[Medline]
  19. Kao YH, Yu CL, Chang LW, Yu HS. Low concentrations of arsenic induce vascular endothelial growth factor and nitric oxide release and stimulate angiogenesis in vitro. Chem Res Toxicol 2003;16:460–8.[CrossRef][Medline]
  20. Yuan SS, Hou MF, Chang HL, et al. Arsenic induced nitric oxide generation is cell cycle dependent and aberrant in NBS cells. Toxicol In Vitro 2003;17:139–43.[CrossRef][Medline]
  21. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol 1996;271:1424–37.
  22. Bunderson M, Coffin JD, Beall HD. Arsenic induces peroxynitrite generation and cyclooxygenase-2 protein expression in aortic endothelial cells: possible role in atherosclerosis. Toxicol Appl Pharmacol 2002;184:11–8.[CrossRef][Medline]
  23. Woo SH, Park IC, Park MJ, et al. Arsenic trioxide induces apoptosis through a reactive oxygen species-dependent pathway and loss of mitochondrial membrane potential in HeLa cells. Int J Oncol 2002;21:57–63.[Medline]
  24. Ivanov V, Hei TK. Arsenic sensitizes human melanomas to apoptosis via TNF{alpha} mediated pathways. J Biol Chem 2004;279:22747–58.[Abstract/Free Full Text]
  25. Waldren CA, Jones C, Puck TT. Measurement of mutagenesis in mammalian cells. Proc Natl Acad Sci U S A 1979;76:1358–62.[Abstract/Free Full Text]
  26. McGuinness SM, Shibuya SM, Ueno AM, Vannais D, Waldren CA. Mutant quantity and quality in mammalian cells exposed to {gamma}-rays: effects of caffeine. Radiat Res 1995;142:247–55.[Medline]
  27. Hei TK, Wu LJ, Liu SX, Vannais D, Waldren CA. Mutagenic effects of a single and an exact number of {alpha} particles in mammalian cells. Proc Natl Acad Sci U S A 1997;94:3765–70.[Abstract/Free Full Text]
  28. King MP, Koga Y, Davidson M, Schon EA. Defects in mitochondrial protein synthesis and respiratory chain activity segregate with the tRNA(Leu(UUR)) mutation associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes. Mol Cell Biol 1992;12:480–90.[Abstract/Free Full Text]
  29. Davidson M, Zhang L, Schon EA, King MP. Genetic and functional complementation of deleted mitochondrial DNA. Neurology 1995;45:381–7.
  30. Smith R III, Huston MM, Jenkins RN, Huston DP, Rich RR. Mitochondria control expression of a murine cell surface antigen. Nature 1983;306:599–601.[CrossRef][Medline]
  31. Sobreira C, Davidson M, King MP, Miranda AF. Dihydrorhodamine 123 identifies impaired mitochondrial respiratory chain function in cultured harboring mtDNA mutations. J Histochem Cytochem 1996;44:571–9.[Abstract]
  32. LeBel CP, Ischiropoulos H, Bondy SC. Evaluation of the probe 2'7'-dichlorodihydrofluorescent-diacetate as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol 1992;5:227–31.[CrossRef][Medline]
  33. Clark KM, Brown TA, Davidson MM, Papadopoulou LC, Clayton DA. Differences n nuclear gene expression between cells containing monomer and dimer mitochondrial genomes. Gene 2002;286:91–104.[CrossRef][Medline]
  34. Inoue K, Takai D, Hosaka H, et al. Isolation and characterization of mitochondrial DNA less lines from various mammalian cell lines by application of an anticancer drug, ditercalinium. Biochem Biophys Res Commun 1997;239:257–60.[CrossRef][Medline]
  35. Reers M, Smiley ST, Mottola-Hartshorn C, Chen A, Lin M, Chen LB. Mitochondrial membrane potential monitored by JC-1 dye. Methods Enzymol 1995;260:406–17.[Medline]
  36. Piao CQ, Zhao YL, Hei TK. Analysis of p16 and p21 expression in tumorigenic human bronchial cells induced by asbestos. Oncogene 2001;20:7301–6.[CrossRef][Medline]
  37. Bradford M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–53.[CrossRef][Medline]
  38. Szejda P, Parce JW, Seeds MS, Bass DA. Flow cytometric quantification of oxidative product formation by polymorphonuclear leukocytes during phagocytosis. J Immunol 1984;133:3303–7.[Abstract]
  39. Wigler MH, Weinstein IB. A preparative method for obtaining enucleated mammalian cells. Biochem Biophys Res Commun 1975;63:669–74.[CrossRef][Medline]
  40. Zhu LX, Waldren CA, Vannais D, Hei TK. Cellular and molecular analysis of mutagenesis induced by charged particles of defined LET. Radiat Res 1996;145:251–9.[Medline]
  41. Pryor WA, Squadrito GL. The chemistry of peroxynitrite: a product from reaction of nitric oxide with superoxide. Am J Physiol 1995;268:699–722.
  42. Olken NM, Osawa Y, Marletta MA. Characterization of the inactivation of nitric oxide synthase by NG-methyl-L-arginine: evidence for heme loss. Biochemistry 1994;33:14784–91.[CrossRef][Medline]
  43. Radi R, Peluffo G, Alvarez MN, Naviliat M, Cayota A. Unraveling peroxynitrite formation in biological systems. Free Radic Biol Med 2001;30:463–88.[CrossRef][Medline]
  44. Wu LJ, Randers-Pehrson G, Xu A, et al. Targeted cytoplasmic irradiation with {alpha} particles induces mutations in mammalian cells. Proc Natl Acad Sci U S A 1999;96:4959–64.[Abstract/Free Full Text]
  45. Chance N, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 1979;59:527–605.[Free Full Text]
  46. Miller WH Jr, Hyman MS, Lee JS, Singer J, Waxman S. Mechanisms of action of arsenic trioxide. Cancer Res 62:3893–903.
  47. Shen ZY, Shen WY, Chen MH, Shen J, Cai WJ, Zeng Y. Mitochondria, calcium and nitric oxide in the apoptotic pathway of esophageal carcinoma cells induced by As2O3. Int J Mol Med 2002;9:385–90.[Medline]
  48. Smith KR, Klei LR, Barchowsky A. Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells. Am J Physiol Lung Cell Mol Physiol 2001;280:442–9.
  49. Klotz L, Schroeder P, Sies H. Peroxynitrite signaling: receptor tyrosine kinases and activation of stress signaling pathways. Free Radic Biol Med 2002;33:737–43.[CrossRef][Medline]
  50. Lymer S, Hurst JK. Rapid reaction between peroxynitrite ions and carbon dioxide: implications for biological activity. J Am Chem Soc 1995;117:8867–8.[CrossRef]
  51. Minetti M, Mallozzi C, Michela Di Stasi AM. Peroxynitrite activates kinases of the SRC family and upregulates tyrosine phosphorylation signaling. Free Radic Biol Med 2002;33:744–54.[CrossRef][Medline]
  52. Lee MY, Jung BI, Seung MC, et al. Arsenic induced dysfunction in relaxation of blood vessels. Environ Health Perspect 2003;111:513–7.[Medline]
  53. Eiserich JP, Hristova M, Cross CE, et al. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 1998;391:393–7.[CrossRef][Medline]
  54. Hei TK, Waldren CA, Hall EJ. Mutation induction by neutrons as determined by an antibody-complement mediated cell lysate system. I. Experimental observations. Radiat Res 1988;115:281–91.[CrossRef][Medline]
  55. Hei TK, Piao CQ, He ZY, Vannais D, Waldren CA. Chrysotile fiber is a potent mutagen in mammalian cells. Cancer Res 1992;52:6305–9.[Abstract/Free Full Text]
  56. Both K, Henderson DW, Turner DR. Asbestos and erionite fibers can induce mutations in human lymphocytes that resulted in loss of heterozygosity. Int J Cancer 1994;59:538–42.[Medline]
  57. Evans HH. Failla Memorial lecture: The prevalence of multilocus lesions in radiation induced mutants. Radiat Res 1994;137:131–44.[CrossRef][Medline]
  58. Zhu LX, Waldren CA, Vannais D, Hei TK. Cellular and molecular analysis of mutagenesis induced by charged particles of defined LET. Radiat Res 1996;145:251–9.
  59. Rossman TG. Mechanism of arsenic carcinogenesis: an integrated approach. Mutat Res 2003;533:37–65.[Medline]
  60. Hei TK, Filipic M. Role of oxidative damage in the genotoxicity of arsenic. Free Radic Biol Med 2004;37:574–81.[CrossRef][Medline]
  61. Warner ML, Moore LE, Smith MT, Kalman DA, Fanning E, Smith AH. Increased micronuclei in exfoliated bladder cells of individuals who chronically ingest arsenic-contaminated water in Nevada. Cancer Epidemiol Biomarkers Prev 1994;3:583–90.[Abstract]
  62. Van Geen L, Ahsan H, Hornemna AH, et al. Promotion of well switching to mitigate the current arsenic crisis in Bangladesh. Bull WHO 2002;80:732–7.[Medline]
  63. Rossman TG, Uddin AN, Burns FJ, Bosland MC. Arsenite cocarcinogenesis: an animal model derived from genetic toxicology studies. Environ Health Perspect 2002;110:749–52.[Medline]
  64. Mahata J, Basu A, Ghoshal S, et al. Chromosomal aberrations and sister chromatid exchanges in individuals exposed to arsenic through drinking water in West Bengal, India. Mutat Res 2003;534:133–43.[Medline]
  65. Hashiguichi K, IKushima T. Nucleotide changes in mitochondrial 16s rRNA gene from different mammalian cell lines. Genes Genet Syst 1998;73:317–21.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Toxicol PatholHome page
A. Wang, D. C. Wolf, B. Sen, G. W. Knapp, S. D. Holladay, W. R. Huckle, T. Caceci, and J. L. Robertson
Dimethylarsinic Acid in Drinking Water Changed the Morphology of Urinary Bladder but Not the Expression of DNA Repair Genes of Bladder Transitional Epithelium in F344 Rats
Toxicol Pathol, June 1, 2009; 37(4): 425 - 437.
[Abstract] [Full Text] [PDF]

Home page
 Evid Based Complement Alternat MedHome page
S. C. Kim, S. J. Park, J. R. Lee, J. C. Seo, C. H. Yang, and S. H. Byun
Cytoprotective Activity of Glycyrrhizae radix Extract Against Arsenite-induced Cytotoxicity
Evid. Based Complement. Altern. Med., June 1, 2008; 5(2): 165 - 171.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
E. Dopp, U. von Recklinghausen, L. M. Hartmann, I. Stueckradt, I. Pollok, S. Rabieh, L. Hao, A. Nussler, C. Katier, A. V. Hirner, et al.
Subcellular Distribution of Inorganic and Methylated Arsenic Compounds in Human Urothelial Cells and Human Hepatocytes
Drug Metab. Dispos., May 1, 2008; 36(5): 971 - 979.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. Zhou, V. N. Ivanov, Y.-C. Lien, M. Davidson, and T. K. Hei
Mitochondrial Function and Nuclear Factor-{kappa}B-Mediated Signaling in Radiation-Induced Bystander Effects
Cancer Res., April 1, 2008; 68(7): 2233 - 2240.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. Vujcic, M. Shroff, and K. K. Singh
Genetic Determinants of Mitochondrial Response to Arsenic in Yeast Saccharomyces cerevisiae
Cancer Res., October 15, 2007; 67(20): 9740 - 9749.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. A. Partridge, S. X.L. Huang, E. Hernandez-Rosa, M. M. Davidson, and T. K. Hei
Arsenic Induced Mitochondrial DNA Damage and Altered Mitochondrial Oxidative Function: Implications for Genotoxic Mechanisms in Mammalian Cells
Cancer Res., June 1, 2007; 67(11): 5239 - 5247.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
A. Poonepalli, L. Balakrishnan, A. K. Khaw, G. K. M. Low, M. Jayapal, R. N. Bhattacharjee, S. Akira, A. S. Balajee, and M. P. Hande
Lack of Poly(ADP-Ribose) Polymerase-1 Gene Product Enhances Cellular Sensitivity to Arsenite
Cancer Res., December 1, 2005; 65(23): 10977 - 10983.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, S.-X.
Right arrow Articles by Hei, T. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, S.-X.
Right arrow Articles by Hei, T. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online