Studies on the Mechanisms of Arsenic-Induced Self Tolerance Developed in Liver Epithelial Cells through Continuous Low-Level Arsenite Exposure

Elizabeth H. Romach*, Christopher Q. Zhao*, Luz María Del Razo{dagger}, Mariano E. Cebrián{dagger} and Michael P. Waalkes*,1

* Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at the National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina; and {dagger} Pharmacology and Toxicology, CINVESTAV-IPN, Mexico City, Mexico

Received September 8, 1999; accepted December 1, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Arsenic (As) is a human carcinogen. Our prior work showed that chronic (>18 weeks) low level (500 nM) arsenite (As3+) exposure induced malignant transformation in a rat liver epithelial cell line (TRL 1215). In these cells, metallothionein (MT) is hyper-expressible, a trait often linked to metal tolerance. Thus, this study examined whether the adverse effects of arsenicals and other metals were altered in these chronic arsenite-exposed (CAsE) cells. CAsE cells, which had been continuously exposed to 500 nM arsenite for 18 to 20 weeks, and control cells, were exposed to As3+, arsenate (As5+), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), antimony (Sb3+), cadmium (Cd2+), cisplatin (cis-Pt), and nickel (Ni2+) for 24 h and cell viability was determined by metabolic integrity. The lethal concentration for 50% of exposed cells (LC50) for As3+ was 140 µM in CAsE cells as compared to 26 µM in control cells, a 5.4-fold increase in tolerance. CAsE cells were also very tolerant to the acute toxic effects of As5+ (LC50 > 4000 µM) compared to control (LC50 = 180 µM). The LC50 for DMA was 4.4-fold higher in CAsE cells than in control cells, but the LC50 for MMA was unchanged. There was a modest cross-tolerance to Sb3+, Cd2+, and cis-Pt in CAsE cells (LC50 1.5–2.0-fold higher) as compared to control. CAsE cells were very tolerant to Ni2+ (LC50 > 8-fold higher). Culturing CAsE cells in As3+-free medium for 5 weeks did not alter As3+ tolerance, implicating an irreversible phenotypic change. Cellular accumulation of As was 87% less in CAsE cells than control and the accumulated As was more readily eliminated. Although accumulating much less As, a greater portion was converted to DMA in CAsE cells. Altered glutathione (GSH) levels were not linked with As tolerance. A maximal induction of MT by Zn produced only a 2.5-fold increase in tolerance to As3+ in control cells. Cell lines derived from MT normal mice (MT+/+) were only slightly more resistant (1.6-fold) to As3+ than cells from MT null mice (MT-/-). These results show that CAsE cells acquire tolerance to As3+, As5+, and DMA. It appears that this self-tolerance is based primarily on reduced cellular disposition of the metalloid and is not accounted for by changes in GSH or MT.

Key Words: arsenic; zinc; tolerance; metallothionein; cytotoxicity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Arsenic (As) is a common environmental toxicant and a known human carcinogen (IARC, 1987Go; NRC, 1999Go). A primary concern for chronic exposure of human populations to As is its carcinogenic potential. Exposure to As can also have profound adverse effects on the skin and peripheral vascular system (NRC, 1999Go). Organisms show a wide range of sensitivity to the toxic effects of As and tolerance to As toxicity can be acquired (Lee et al., 1989aGo,bGo, 1994; Ovelgonne et al., 1995Go; Vahter, 1983Go; Wang et al., 1996Go). Development of tolerance to As toxicity in humans may occur after chronic exposure, given the anecdotal reports of farmers in the Austrian Alps in the 19th century who consumed large amounts of As daily, amounts which could have been as large as 3–4 times the normally lethal dose (Vahter, 1983Go). Although As is a known human carcinogen, it also has anti-tumorigenic properties. In fact, As trioxide has recently been used with remarkable success in the treatment of acute promyelocytic leukemia (Look, 1998Go), and there is justifiable excitement about its chemotherapeutic potential. Therefore, understanding the mechanisms of As tolerance may not only aid in understanding its carcinogenic properties but could also have implications for its use in cancer chemotherapy.

Classified as a metalloid, As is thought to be directly toxic through its interaction with sulfhydryl groups in proteins (Aposhian, 1989Go; Sunderman, 1979Go). By direct binding to enzymes and other proteins, this metalloid is thought to perturb their function. It is also possible that As may be indirectly toxic through either the generation of reactive oxygen species (Chen et al., 1998Go) or through the disruption of cellular methylation reactions (Zhao et al., 1997Go). In biological systems, inorganic As exists as either arsenate (As5+) or arsenite (As3+) (Aposhian, 1989Go). Inorganic As is metabolized in many cells, via enzymatic methylation, to monomethylarsonic (MMA) and dimethylarsinic acids (DMA) (Abernathy et al., 1999Go; Styblo et al., 1995Go; Zakharyan et al., 1995Go). Generally speaking, methylated arsenicals are far less reactive than the inorganic forms of As, a fact which may account for their being far less acutely toxic in biological systems. Also, methylated arsenicals are more readily eliminated via the urine. For these reasons, methylation of inorganic arsenic is often thought to be a mechanism by which arsenic is detoxicated, although such a role is not entirely clear. For instance, there is now evidence that DMA may act as a DNA-damaging agent (Yamanaka et al., 1991Go) and can act to promote (Yamamoto et al., 1995Go) or cause tumors (Li et al., 1998Go). Methylation of arsenic also consumes glutathione (GSH) and S-adenosyl-methionine (SAM) (Styblo et al., 1995Go; Zakharyan et al., 1995Go) and therefore, As toxicity may partially result from the depletion of the cellular pools of either GSH or SAM (Shimizu et al., 1998Go; Zhao et al., 1997Go).

Several groups have shown that arsenic tolerance can develop in mammalian cells with a variety of treatment protocols (Lee et al., 1989aGo,bGo, 1994; Ovelgonne et al., 1995Go). However, these treatment protocols generally rely on harsh selection of tolerant cells by acute exposure to high levels of As or by exposure to progressively increasing levels of As (Lee et al., 1989aGo,bGo, 1994; Ovelgonne et al., 1995Go). As such, they do not simulate the low, but continuous, exposure that would be more typical of human exposures. In any event, the precise basis of As tolerance is not well defined but may involve both toxicokinetic and toxicodynamic elements (Lee et al., 1989aGo,bGo, 1994; Ovelgonne et al., 1995Go; Wang et al., 1996Go).

Previous studies in our laboratory showed that rat liver epithelial cells cultured in sub-toxic (125 to 500 nM) concentrations of As3+ for 18 or more weeks become malignantly transformed, producing tumors capable of metastasis upon inoculation into Nude mice (Zhao et al., 1997Go). These cells show a hyperexpressibility of metallothionein (MT), an observation that is frequently associated with metal tolerance (Zhao et al., 1997Go). MT is a soluble protein containing a very high portion of cysteinyl sulfhydryl residues. MT appears to function in the cellular defense against toxicity induced by many metals (Cherian, 1995Go) and, in some cases, can also act as a free-radical scavenger (Lazo et al., 1995Go; Sato and Bremner, 1993Go). Arsenic can enhance the expression of the MT gene, at least in vivo (Albores et al., 1992Go). Exposure to zinc, an effective inducer of MT synthesis (Cherian, 1995Go), prevents As toxicity in some instances (Kreppel et al., 1994Go). On the other hand, the activation of the MT gene prior to As3+ exposure in vitro appears to have little impact on the overt cytotoxicity or the molecular toxicity of the metalloid (Shimizu et al., 1998Go). Although the role of MT in preventing the toxic effect of many inorganics is clear, the role of MT in As tolerance is poorly defined.

The purpose of this study was to determine if adverse effects of acute exposure to inorganics were altered in cells that had been exposed to chronic, low levels of As3+ (Zhao et al., 1997Go), a treatment protocol that more closely simulates As exposure in humans. Specifically, studies were designed to determine if these chronic arsenite-exposed (CAsE) cells developed tolerance to the toxicity of arsenicals, and perhaps cross tolerance to other metals. Additional study was directed at defining the mechanisms, including altered disposition and enhanced cellular GSH or MT, by which these cells developed any such tolerance.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Sodium arsenite (As3+), sodium arsenate (As5+), dimethylarsinic acid (DMA; purity 98%), cadmium chloride (Cd2+), nickel chloride (Ni2+), cis-platinum (II) diammine dichloride (cis-Pt), and zinc chloride (Zn2+) were obtained from Sigma Chemical Company (St. Louis, MO). Monomethylarsonic acid (MMA; purity 99%) was obtained from ChemService (West Chester, PA). Antimony (III) oxide (Sb3+) was obtained from Aldrich Chemical Company (Milwaukee, WI).

Cell lines.
The main cell line used in these studies (TRL 1215) was originally derived from the liver of 10-day-old Fischer F344 rats (Idoine et al., 1976Go). These cells are diploid and normally nontumorigenic. The cells were cultured as previously described (Zhao et al., 1997Go). In all cases, cells, which had been grown in the continuous presence of 500 nM As3+ for 18 to 20 weeks (chronic arsenite exposed; CAsE), were compared to passage-matched control TRL 1215 cells.

In some studies, a cell line created from embryonic cells of transgenic mice with a targeted disruption of metallothionein I and II genes (MT-/-), along with the corresponding control cells (MT+/+) from normal mice, were graciously supplied by Dr. John Lazo, University of Pittsburgh, and were cultured as described previously (Lazo et al., 1995Go).

Determination of metabolic integrity.
A Promega Non-Radioactive Cell Proliferation Assay kit was used to determine acute cytotoxicity as defined by metabolic integrity. The assay measures the amount of formazan produced by metabolic conversion of Owen's reagent [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, MTS] by dehydrogenase enzymes found in the mitochondria of metabolically active cells. The quantity of formazan product, as measured by absorbance at 490 nm, is directly proportional to the number of living cells. A minimum of 4 replicates of 10,000 cells per well were plated in 96-well plates and allowed to adhere to the plate for 24 h, at which time the medium was removed and replaced with medium containing the test compounds (As3+, As5+, MMA, DMA, Cd2+, Ni2+, cis-Pt, Zn2+, Sb3+). Cells were then incubated for an additional 24 h, and toxicity was determined. CAsE cells were compared to time-matched controls in all cases. Data are expressed as metabolic integrity, using control as 100%.

Stability and acquisition time for arsenic tolerance.
To define the stability of acquired tolerance in CAsE cells, these cells were passaged in As3+-free medium for 5 weeks and then LC50 after As3+ was determined. In a separate series of experiments, control cells were continuously exposed to 500 nM As3+ for 5 weeks and then the LC50 after 24 h of As3+ exposure was determined, to help define the onset of acquired As tolerance.

Arsenite accumulation and efflux.
Control and CAsE cells were grown to 50% confluence in control medium. At that time the medium was removed and replaced with either fresh control medium or medium containing 10 µM As3+. Twenty-four hours later cells were harvested, counted, and the pellets digested in 50% perchloric:nitric acid, 2:1, overnight. These digests were used for determination of the amount of total As that accumulated after 24 h of exposure. Total As, which would include inorganic and organic forms, was determined using graphite furnace atomic absorption spectroscopy. Replicate sets of cells were washed with PBS at this time and allowed to incubate an additional 24 h in As-free medium in order to measure the amount of As efflux during this period. Triplicate determinations were used for each data point.

Determination of the ability of CAsE and control cells to metabolize arsenic.
CAsE cells, which had been grown in the continuous presence of 500 nM As3+ for 18 to 20 weeks, and control cells were treated with 500 nM of As3+ for 72 h. Cells were counted and then digested with 5 ml of 2 M HCl for 5 h at 80°C for analysis of arsenic metabolites. The digested samples were assayed using a method based on the generation of volatile arsine species: chromatographic separation and detection by hydride generation, coupled with atomic absorption spectroscopy (Crecelius et al., 1986Go). Depending on the boiling points, inorganic, methyl, and dimethyl arsenicals are reduced to their corresponding arsines at a pH of 1 to 2 (Crecelius et al., 1986Go). This method allows for the quantitation of total inorganic As (As3+ and As5+), total MMA (MMA3+ and MMA5+) and total DMA (DMA3+ and DMA5+). Triplicate measurements were made for each sample.

Determination of intracellular glutathione (GSH).
Untreated tolerant and control TRL 1215 cells were harvested, counted, and the cell pellets frozen. The level of intracellular GSH was determined in triplicate cell pellets using a fluorometric method (Hissin and Hilif, 1976). Values were normalized to cell number.

Studies defining the role of metallothionein (MT) in As tolerance.
Several studies were designed to help define the role of MT in acquired or native tolerance to As. Control TRL 1215 cells were grown to 50% confluence in control medium and the medium then was removed and replaced with medium containing from 0 to 175 µM Zn2+. Metabolic integrity was measured in Zn2+-treated control cells to define maximal induction of MT in the absence of significant toxicity, which occurred at 150 µM. After 24 h of exposure to non-toxic levels of Zn2+, cells were harvested by trypsinization, counted, and ruptured by sonication. MT protein levels were then estimated using the Cd saturation assay (Eaton et al., 1982) with Cd analyzed by atomic absorption in the graphite-furnace mode. Values are normalized to cell number.

Additional studies on the role of MT in As tolerance used the MT-/- (null for MT I and MT II) and the corresponding control (MT+/+) cell lines (Lazo et al., 1995Go). Cells were cultured in the presence and absence of various levels of As3+ for 24 h and metabolic integrity was assessed.

Statistics.
In all cases, a p value of <= 0.05 was considered to represent a significant difference. All data represent the mean ± SEM of 3 or more replications. Student's t-test was used to analyze differences between CAsE and control cells for cytotoxicity (after derivation of the mean LC50), As accumulation, and As metabolism data. The mean LC50 was determined from regression analysis of the linear portion of at least 4 separate metabolic integrity curves.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
When TRL 1215 cells were cultured in the presence of 500 nM sodium As3+ for 18 weeks or more, these chronic arsenite exposed (CAsE) cells clearly acquired tolerance to the acute toxic effects of As3+ when compared to control cells (Fig. 1Go). This is reflected as a major shift to the right of the cytotoxicity curve in CAsE cells compared to control. The LC50s were determined from analysis of the linear portion of the metabolic integrity curves and compared between CAsE and control cells. The LC50 for the CAsE cells, which had been chronically exposed to As3+ prior to the acute high dose, was 5.4-fold higher than the LC50 for As3+ in control cells (Table 1Go).



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 1. The acute cytotoxicity of As3+ in control and chronic As3+-exposed (CAsE) TRL 1215 cells as measured by metabolic integrity. CAsE cells had been exposed to 500 nM As3+ for 18 to 20 weeks and were compared to control TRL 1215 cells grown in As-free medium. The assay of cytotoxicity used measures the amount of formazan produced by metabolic conversion of MTS by dehydrogenase enzymes found in the mitochondria of metabolically active cells. Results are presented as the mean ± SEM, n = 4. Data are expressed as percent of untreated control, which is set at 100%.

 

View this table:
[in this window]
[in a new window]
 
TABLE 1 Cytotoxicity (LC50 in µM) of Arsenite, Arsenate, DMA, and MMA in Control and Chronically Arsenite-Exposed (CAsE) Cells
 
CAsE cells also became very tolerant to the acute toxicity of As5+ (Fig. 2Go). In this case the LC50 for an acute high dose of As5+ in CAsE cells was in excess of 4 mM as compared to a LC50 of 180 µM for As5+ in control cells (Table 1Go). Based on these results, the CAsE cells had a LC50 for As5+ that was 20-fold greater than that in control cells.



View larger version (16K):
[in this window]
[in a new window]
 
FIG. 2. The acute cytotoxicity of As5+ in control and chronic As3+-exposed (CAsE) cells as measured by metabolic integrity. CAsE cells had been exposed to 500 nM As3+ for 18 to 20 weeks and were compared to cells grown in control medium. The assay of cytotoxicity used measures the amount of formazan produced by metabolic conversion of MTS by dehydrogenase enzymes found in the mitochondria of metabolically active cells. Results are presented as the mean ± SEM, n = 4. Data are expressed as percent of untreated control, which is set at 100%.

 
Some tolerance to the toxicity of the methylated forms of arsenic occurred in CAsE cells. In general, both methylated forms (MMA and DMA) were much less toxic than the inorganic forms of As. The CAsE cells became tolerant to acute toxicity of DMA (Fig. 3Go). DMA is far less toxic to cells than inorganic forms of arsenic, with a LC50 of 13.9 mM in control cells and 61.2 mM in CAsE cells, a 4.4-fold increase (Table 1Go). Somewhat surprisingly, the CAsE cells, although tolerant to As3+, As5+, and DMA, did not develop any significant tolerance to MMA (Table 1Go). MMA, like DMA, required mM concentrations to generate significant cytotoxicity.



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 3. The acute cytotoxicity of DMA in control and chronic As3+-exposed (CAsE) cells as measured by metabolic integrity. CAsE cells had been exposed to 500 nM As3+ for 18 to 20 weeks and were compared to cells grown in control medium. The assay of cytotoxicity used measures the amount of formazan produced by metabolic conversion of MTS by dehydrogenase enzymes found in the mitochondria of metabolically active cells. Results are presented as the mean ± SEM, n = 4. Data are expressed as percent of untreated control, which is set at 100%.

 
CAsE cells passaged in As3+-free medium for 5 weeks maintained their tolerant phenotype when exposed to an acute high dose of As3+ (Fig. 4Go). There was no difference in LC50 for a 24-h As3+ exposure between CAsE cells that were maintained in As3+ containing medium throughout and those that were passaged in As3+-free medium for 5 weeks. In contrast, control cells that were passaged in As3+ (500 nM) containing medium for 5 weeks did not become tolerant to acute As3+ toxicity (Fig. 4Go). This indicates that longer time intervals of As3+ exposure are required for development of self-tolerance.



View larger version (26K):
[in this window]
[in a new window]
 
FIG. 4. Effects of As withdrawal from chronic As3+-exposed (CAsE) cells and short-term addition of As to control cells on acute As3+ cytotoxicity, as measured by metabolic integrity. CAsE cells were grown in As3+-free medium for 5 weeks prior to the determination of acute As3+ cytotoxicity and compared to CAsE cells maintained throughout the experiment in As3+-containing medium. Control cells were exposed to 500 nM As3+ for 5 weeks prior to the determination of acute As3+ cytotoxicity and compared to cells grown in control medium. The assay of cytotoxicity used measures the amount of formazan produced by metabolic conversion of MTS by dehydrogenase enzymes found in the mitochondria of metabolically active cells. Results are presented as the mean ± SEM, n = 4. Data are expressed as percent of untreated control,which is set at 100%.

 
Additional studies measured the amount of As that accumulated in CAsE cells versus control cells over a 24-h exposure period. The results showed that control cells accumulate approximately 8-fold more arsenic than tolerant cells (Table 2Go). As an indication of efflux, As exposed cells were placed in As3+-free medium for an additional 24 h. The control cells were able to eliminate only 74% of the total As that had accumulated. In the CAsE cells, 100% of the accumulated arsenic was eliminated during the same period.


View this table:
[in this window]
[in a new window]
 
TABLE 2 Arsenic Accumulation and Subsequent Loss in Control and Chronic Arsenite Exposed (CAsE) Cells
 
Studies were also directed at the determination of the comparative ability of CAsE and control cells to metabolize As (Fig. 5Go). CAsE cells and control cells were treated with 500 nM As3+ for 72 h and the levels of DMA generated and As accumulated, as reflected in the cellular content at the end of the test period, were analyzed. Over this exposure period, CAsE cells generated ~50% less DMA than control cells. CAsE cells again accumulated much less (~77% less) As (0.385 ± 0.028 ng/million cells) than control cells (1.57 ± 0.051 ng/million cells) over this 72-h period. Considering the fact that CAsE cells accumulated so much less As than control cells, these data may actually implicate a more efficient methylation in CAsE cells. In this regard, when values for DMA are adjusted by the total amount of inorganic As within cells at the end of the 72-h test period, the DMA/As ratio was over 2-fold higher in CAsE than in control cells (Fig. 5Go). Based on the magnitude of this increase in DMA/As ratio, the level of DMA generation in CAsE probably results from at least two competing factors. This includes depressed deposition, which would reduce the As available for DMA formation, and enhanced methylation rate, which would increase the relative amounts of DMA formed for a given amount of cellular As. MMA was not detectable in either cell type at the end of the exposure period (not shown).



View larger version (31K):
[in this window]
[in a new window]
 
FIG. 5. Arsenic methylation in control and chronic As3+ exposed (CAsE) cells. CAsE cells, which had been grown in the continuous presence of 500 nM As3+ for 18 to 20 weeks, and control cells were treated with 500 nM of As3+ for 72 h. Cellular DMA was analyzed after digestion by hydride generation coupled with flame atomic absorption spectroscopy. Results are presented as the mean ± SEM, n = 3. Left: cellular DMA at the end of the 72-h incubation period. Right: ratio of cellular DMA As to total cellular inorganic As at the end of the 72 h incubation period. MMA was not detected in either control or CAsE cells. An asterisk indicates a significant (p < 0.05) difference between control and CAsE cells.

 
The levels of intracellular glutathione did not differ between the sensitive control cells and arsenic-tolerant CAsE cells. CAsE cells had 1.22 ± 0.16 µg GSH/million cells and control cells had 0.83 ± 0.26 µg GSH/million cells. Although the levels tended to be higher in CAsE cells, they were not statistically different.

To help define the role of the metal-binding protein MT in acquired As tolerance, control TRL 1215 cells were cultured in several concentrations of Zn2+, an efficient inducer of MT expression, in order to determine the concentration that produced a maximal increase in the levels of the MT protein. When control TRL 1215 cells were cultured in the presence of 150 µM Zn2+ for 24 h, an 18.8-fold increase in cellular MT was seen, when compared to control. Higher concentrations of Zn2+ proved toxic to these cells. When cells with maximally induced MT protein levels were exposed to As3+, the LC50 for As3+ increased ~2.5-fold in the Zn2+ pretreated cells as compared to cells not pretreated (Fig. 6Go). Similar studies were performed with As5+ and DMA. Pretreatment with Zn2+ increased the LC50 for As5+ 1.6-fold over control. There was, however, no increase in the LC50 for DMA with Zn2+ pretreatment (not shown). Thus the extent of Zn2+-induced tolerance to inorganic As (1.6 to 2.5-fold) is much less than that acquired through CAsE (5.4- to >20-fold) and Zn2+ does not induce tolerance to DMA. A prior study showed that the basal levels of MT in CAsE cells are within 17% of the basal levels in control TRL 1215 cells (Zhao et al., 1997Go), indicating that major differences in basal MT do not account for tolerance in CAsE cells.



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 6. Effect of metallothionein on acute As3+ cytotoxicity in control cells as measured by metabolic integrity. Cells were grown in the presence of 150-µM zinc for 24 h, which resulted in a maximal (18.8-fold) induction of MT, prior to exposure to As3+. The assay of cytotoxicity used measures the amount of formazan produced by metabolic conversion of MTS by dehydrogenase enzymes found in the mitochondria of metabolically active cells. Results are presented as the mean ± SEM, n = 4. Data are expressed as percent of untreated control, which is set at 100%.

 
To further explore the issue of MT in As tolerance, mouse embryonic fibroblasts derived from MT I/II knockout animals (Lazo et al., 1995Go) were used. These were compared to analogous cells normally producing MT (MT+/+) derived in the same fashion (Lazo et al., 1995Go). Although cells that express MT normally had a higher LC50 for As3+ when compared to cells that do not express the protein, the differences were relatively small (LC50 of 16.3 ± 0.9 µM versus 10.2 ± 0.1 µM, a 1.6-fold increase in LC50). Similar results were obtained with As5+, DMA, and MMA (fold increases in LC50 of 1.6, 1.5, and 1.5, respectively). When compared to the increases in tolerance seen with acquired As tolerance (see Table 1Go), this indicates that MT cannot fully account for As3+-induced tolerance CAsE cells.

CAsE cells, which were tolerant to arsenicals, were also cross tolerant to the acute toxicity of Sb3+, Cd2+, and Ni2+, and to a lesser degree cis-Pt (Table 3Go). Cells tolerant to arsenicals had a LC50 for Cd2+ 2-fold higher than control cells. The As-resistant cells were also more tolerant to Sb3+ compared to control, as the LC50 was 1.9-fold higher. CAsE cells had a greater than 8-fold increase in the LC50 for Ni2+ when compared to control. CAsE cells also had a slight increase (1.5-fold) in the LC50 for cis-Pt as compared to control.


View this table:
[in this window]
[in a new window]
 
TABLE 3 Cross Tolerance to Other Metals in Chronically Arsenite-Exposed (CAsE) Cells
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of the present study indicate that acquired As tolerance can be induced in mammalian cells through chronic low-level, non-toxic exposure to As3+, which more properly duplicates human exposure situations. There are several reports in the literature of the induction of As tolerance using protocols where cells are exposed to progressively increasing concentrations of As, or through the use of step-down protocols where cells are at first exposed to a high, toxic dose of As and then subsequently to lower less toxic doses (Lee et al., 1989aGo,bGo, 1994; Ovelgonne et al., 1995Go). These protocols likely select for tolerant cells by killing off sensitive subpopulations from the total cell pool. This is drastically different from human exposure to As, where the most frequent form of exposure is to more constant, less-fluctuating, low levels of As in the environment. Thus, the system used to develop As-induced self tolerance in the present study provides an important model to study both the toxicological and pharmacological aspects of As tolerance and may prove helpful in defining mechanisms in chronic As toxicity.

There are several mechanisms that could explain the acquisition of As tolerance, including both toxicokinetic and toxicodynamic elements. Firstly, a toxicokinetic-based mechanism could involve reduced accumulation of As, possibly including reduced uptake or enhanced efflux of As or some combination of both. In this regard, in As-resistant bacteria, inducible operons have been identified that express energy-dependent pumps that efflux As3+, As5+, and Sb (Rosen et al., 1984; Silver et al., 1981Go). A plasmid-mediated As3+ efflux pump has been shown to occur in As resistance in E.coli, and this pump also appears to provide cross resistance to Sb (Tisa et al., 1990). Although an As pump has not been isolated in mammalian cells, there is evidence that such a system may exist. Increased As efflux was also reported in a line of As3+-resistant CHO cell variants (Wang et al., 1993; Lee et al., 1989bGo). Since the CAsE cells in the present study were also tolerant to As3+, As5+, and Sb3+, it seems reasonable that the tolerant cells may have enhanced expression of the putative As efflux pump. In fact, we found that the As-tolerant CAsE cells accumulate far less As than control cells. The difference in As accumulation may be primarily due to enhanced efflux, although reduced uptake cannot be excluded. In this regard, some human tumor-cell lines that are resistant to cis-Pt through reduced uptake show cross-tolerance to As3+ through a similar mechanism (Naredi et al., 1995Go). In any event, it appears that the biokinetics of As in tolerant cells favors accumulation of smaller amounts of As, a factor that would certainly reduce toxicity.

A toxicodynamic mechanism whereby cells enhance their ability to metabolize the more toxic inorganic arsenicals to the presumably less toxic, methylated forms could potentially induce tolerance to inorganic As. Some increase in the relative ability to methylate As appears to have occurred in CAsE cells. However, this mechanism would not account for the tolerance to DMA that occurred in concert with As3+ and As5+ tolerance. Furthermore this theoretical mechanism of As tolerance would place an additional burden on an already depleted SAM pool in these cells (Zhao et al., 1997Go), which could induce adverse effects outside the realm of acute cytotoxicity. Since these cells are As tolerant, despite depletion of methyl donor pools, this suggests that enhanced methylation of inorganic As is not the only mechanism whereby these cells acquire tolerance. Additionally these same cells are tolerant to Cd2+, Ni2+, and cis-Pt and this tolerance would clearly not involve methylation of these inorganics. In any event, defining the role of methylation in arsenic-induced self-tolerance requires additional research.

GSH is thought to play a role in the acquisition of As tolerance in some cases. It is thought that GSH may decrease As toxicity through several means, including (1) in its role as an antioxidant; (2) in its role as a co-factor in enzymatic methylation reactions; (3) by binding As directly thereby reducing toxic potential; or (4) through enhanced efflux of an As-GSH conjugate (Huang, et al., 1993Go; Rosen, 1995Go). There are several reports in the literature of As-resistant cells where resistance inversely correlates with intracellular GSH levels (Chang et al., 1991Go; Lee et al., 1989aGo,bGo). However, there are also reports of similar resistant cell lines where there is no difference in the amount of GSH between tolerant and control cells (Wang et al., 1996Go). The role of GSH in the efflux of As in mammalian cells is not clear although an As-GSH conjugate appears to be the species that is pumped out in some resistant bacteria (Rosen, 1995Go). However, it is also clear that some toxic manifestations of As are totally independent of GSH levels (Ramos et al., 1995Go). In the present study, As-tolerant CAsE cells did not have more intracellular GSH than controls. This does not, however, completely rule out a role for GSH in As tolerance in these cells, since rates of production and utilization may be altered independent of actual levels. More studies are needed in order to ascertain if GSH plays a role in the tolerant phenotype seen in CAsE.

Zinc pretreatment protects against the toxic effects of As in mice (Kreppel et al., 1994Go). Since Zn is a very effective inducer of MT, this raises the possibility that MT may play a role in protection against As toxicity. Metallothioneins are small proteins that bind various metals and are thought to play an important role in essential metal homeostasis and toxic metal detoxification (Cherian, 1995Go). MTs may also function as radical scavengers (Lazo et al., 1995Go; Sato and Bremner, 1993Go). However, Kreppel et al. (1994) determined that tolerance to As in exposed mice was not due to MT binding the metalloid in a fashion similar to that seen in MT reduction of Cd toxicity by sequestration. They concluded that the protective effects of Zn were due to some effect other than induction of MT. Our data in general support this conclusion. Control cells pretreated with Zn2+ to induce the MT protein did show a modest tolerance to As. However, the magnitude of this response was far less than expected, given the 19-fold increase in MT levels. With metals that bind to MT, like Cd, an induction of this magnitude would greatly reduce toxicity. Furthermore, other metals that effectively induce MT, like Cd, have little effect of the toxicity of As in vivo (Hochadel and Waalkes, 1997Go). These studies are also consistent with studies showing that the affinity of As for MT is quite low (Waalkes et al., 1984Go). Furthermore, As does not induce MT in vitro (Shimizu et al., 1998Go) unlike Cd or Zn, which both induce and bind to the protein. In addition, our results also showed that cells that do not express the major forms of the MT (MT-/- cells) do not have a marked increase in the relative sensitivity to As in comparison to cells normally expressing MT (MT+/+ cells). This further suggests that MT plays a very limited role in the induction of tolerance to As. However, the hypomethylation of DNA induced by chronic low-level exposure to As3+ makes the MT gene hyperinducible in these CAsE cells (Zhao et al., 1997Go). Thus, although CAsE cells may express more MT with appropriate stimulation, which may account for the cross-tolerance to Cd2+, and possibly cis-Pt, MT appears to have little to do with acquired or native As tolerance.

The As-tolerant cells in this study were also highly cross-tolerant to Ni2+. Nickel is a redox active metal and indirect damage due to generation of reactive oxygen species is probably important in Ni2+ toxicity and carcinogenesis (Kasprzak, 1991Go). The resistance to Ni2+ toxicity may therefore be due to a resistance to oxidative damage. The fact that the CAsE cells are highly cross-tolerant to Ni2+ suggests that they may have reduced sensitivity to reactive oxygen species. Arsenic compounds may generate active oxygen species because of their metabolism (Yamanaka et al., 1991Go) and the genotoxicity of arsenic has been hypothesized to be mediated through reactive oxygen species. However, more studies are needed to understand the mechanism of cross-tolerance to Ni2+ in As-resistant cells.

Recently Rossman et al. (1999) isolated and identified two cDNAs, asr1 and asr2, which confer As3+ resistance to As3+-sensitive cell lines. One of the cDNAs, asr1, shows almost complete homology with the rat fau gene, a tumor suppressor gene which functions in the ubiquitin system to signal protein degradation. Arsenite has been previously shown to inhibit the ubiquitin-dependent proteolytic pathway (Klemperer and Pickart, 1989Go) and this has been hypothesized to contribute to its toxicity and carcinogenicity by preventing the proteolysis of important proteins (Rossman et al., 1999). The ubiquitin system has been implicated in growth control and carcinogenesis, and therefore, may play a role in As carcinogencity. The precise nature by which this system could affect cross-tolerance to DMA, Ni, Cd, Sb, or cis-Pt, as seen in the present As-resistant cells, is unclear.

Previous studies showed that the CAsE cells have undergone malignant transformation and produce malignant, potentially metastatic, tumors upon inoculation into Nude mice (Zhao et al., 1997Go). Thus, the development of As-induced malignant transformation occurs concurrently with the development of tolerance to acute As toxicity. This creates an important question as to whether or not the molecular changes involved with adaption to As are also important to the process of As carcinogenesis. The As-tolerant phenotype persisted in the present study even after tolerant cells were grown in As-free medium for a protracted period. This suggests that tolerance is a permanent, genotypic change in CAsE cells which is consistent with the persistence of the malignant transformation seen in these same cells when grown in As-free medium (Zhao et al., 1997Go). Analysis of the genetic changes associated acquisition of As-induced self-tolerance and malignant transformation in these CAsE cells is currently underway, and should help substantiate any linkage between tolerance and transformation. It is quite possible that development of tolerance to As toxicity may give cells a selective growth advantage when confronted by continuing As exposure, thereby increasing their tumorigenic potential.

In summary, cells exposed to low concentrations of As3+ for 18 weeks or more were resistant to the acute toxicity of inorganic arsenicals and DMA but not MMA. These cells were also cross-tolerant to Ni, Cd, Sb, and cis-Pt. The induction of tolerance may be due, in part, to an increase in metal efflux and certainly has a toxicokinetic component. However, it is likely that this is not the only mechanism responsible for the increased tolerance.


    ACKNOWLEDGMENTS
 
The authors wish to thank and Dr. John S. Lazo for providing the MT null-cell line and Ms. Tammy Knowles for technical assistance. The authors also thank Drs. Jie Liu and Hua Chen for critically reviewing this manuscript.


    NOTES
 
1 To whom correspondence should be addressed at Inorganic Carcinogenesis Section, NCI at NIEHS, Mail Drop F0-09, 111 Alexander Drive, Research Triangle Park, NC 27709. Fax: (919) 541–3970. E-mail: waalkes{at}niehs.nih.gov. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abernathy, C. O., Liu, Y. P., Longfellow, D., Aposhian, H. V., Beck, B., Fowler, B., Goyer, R., Menzer, R., Rossman, T., Thompson, C., and Waalkes, M. (1999). Meeting Report, Arsenic: Health effects, mechanisms of actions, and research issues. Environ. Health Perspect. 107, 593–597.[Medline]

Albores, A., Koropatnick, J., Cherian, M. G., and Zelazowski, A. J. (1992). Arsenic induces and enhances rat hepatic metallothionein production in vivo. Chem. Biol. Interact. 85, 127–140.[ISI][Medline]

Aposhian, H. V. (1989). Biochemical toxicology of arsenic. Rev. Biochem. Toxicol. 10, 265–300.

Chang, W. C., Chen, S. H., Wu, H. L., Shi, G. Y., Murota, S. I., and Morita, I. (1991). Cytoprotective effect of reduced glutathione in arsenical-induced endothelial cell injury. Toxicology 69, 101–110.[ISI][Medline]

Chen, Y. C., Lin-Shiau, S. Y., and Lin, J. K. (1998). Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis. J. Cell. Physiol. 177, 324–333.[ISI][Medline]

Cherian, M. G. (1995). Metallothionein and its interactions with metals. In Handbook of Experimental Pharmacology; Toxicology of Metals, Biochemical Effects (R. A. Goyer and M. G. Cherian, Eds.), Vol. 115, pp. 121–138. Springer-Verlag, New York.

Crecelius, E. A., Bloom, N. S., Cowan, C. E., and Jenne, E. A. (1986). Determination of the arsenic species in limnological samples by hydride generation atomic absorption spectroscopy. In Speciation of Selenium and Arsenic in Natural Waters and Sediments, Volume 2: Arsenic Speciation, pp. 1–28. Electric Power Institute, Palo Alto, CA (EA-4641; Project 2020–2).

Eaton, D. L., and Toal, B. F. (1982). Evaluation of the Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues. Toxicol. Appl. Pharmacol. 66, 134–142.[ISI][Medline]

Hissin, P. J., and Hilf, R. (1976). A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal. Biochem. 74, 214–226.[ISI][Medline]

Hochadel, J. F., and Waalkes, M. P. (1997). Sequence of exposure to cadmium and arsenic determines the extent of toxic effects in male Fischer rats. Toxicology 116, 89–98.[ISI][Medline]

Huang, H., Huang, C. F., Wu, D. R., Jinn, C. M., and Jan, K. Y. (1993). Glutathione as a cellular defense against arsenite toxicity in cultured Chinese hamster ovary cells. Toxicology 79, 195–204.[ISI][Medline]

IARC (1987). International Agency for Research on Cancer Monographs on the Evaluation of the Carcinogenic Risks to Humans: Supplement 7, Overall evaluations—carcinogenicity: An updating of IARC monographs, Volumes 1 to 42. IARC Scientific Publications, Lyon, France.

Idoine, J. B., Elliott, J. M., Wilson, M. J., and Weisburger, E. K. (1976). Rat liver cells in culture: Effect of storage, long-term culture, and transformation on some enzyme levels. In Vitro 12, 541–553.[ISI][Medline]

Kasprzak, K. S. (1991). The role of oxidative damage in metal carcinogenicity. Chem. Res. Toxicol. 4, 604–615.[ISI][Medline]

Klemperer, N. S., and Pickart, C. M. (1989). Arsenite inhibits two steps in the ubiquitin-dependent proteolytic pathway. J. Biol. Chem. 264, 19245–19252.[Abstract/Free Full Text]

Kreppel, H., Liu, J., Liu, Y., Reichl, F. X., and Klaassen, C. D. (1994). Zinc-induced arsenite tolerance in mice. Fundam. Appl. Toxicol. 23, 32–37.[ISI][Medline]

Lazo, J. S., Kondo, Y., Dellapiazza, D., Michalska, A. E., Choo, K. H., and Pitt, B. R. (1995). Enhanced sensitivity to oxidative stress in cultured embryonic cells from transgenic mice deficient in metallothionein I and II genes. J. Biol. Chem. 270, 5506–5510.[Abstract/Free Full Text]

Lee, T. C., and Ho, I. C. (1994). Differential cytotoxic effects of arsenic on human and animal cells. Environ. Health Perspect. 102, 101–105.[Medline]

Lee, T. C., Ko, J. L., and Jan, K. Y. (1989a). Differential cytotoxicity of sodium arsenite in human fibroblasts and Chinese hamster ovary cells. Toxicology 56, 289–299.[ISI][Medline]

Lee, T. C., Wei, M. L., Chang, W. J., Ho, I. C., Lo, J. F., Jan, K. Y., and Huang, H. (1989b). Elevation of glutathione S-transferase activity in arsenic-resistant Chinese hamster ovary cells. In Vitro Cell. Dev. Biol. 25, 442–448.[ISI][Medline]

Li, W., Wanibuchi, H., Salim, E. I., Yamamoto, S., Yoshida, K., Endo, G., and Fukushima, S. (1998). Promotion of NCI-Black-Reiter male rat bladder carcinogenesis by dimethylarsinic acid, an organic arsenic compound. Cancer Lett. 134, 29–36.[ISI][Medline]

Look, A. T. (1998). Arsenic and apoptosis in the treatment of acute promyelocytic leukemia. J. Natl. Cancer Inst. 90, 124–133.[Abstract/Free Full Text]

Naredi, P., Heath, D. D., Enns, R. E., and Howell, S. B. (1995). Cross-resistance between cisplatin, antimony potassium tartrate, and arsenite in human tumor cells. J. Clin. Invest. 95, 1193–1198.[ISI][Medline]

NRC (National Research Council) (1999). Arsenic in the Drinking Water, pp. 101–129. National Academy Press, Washington, DC.

Ovelgonne, H. H., Wiegant, F. A., Souren, J. E., Van Rijn, H., and Van Wijk, R. (1995). Enhancement of the stress response by low concentrations of arsenite in arsenite-pretreated Reuber H35 hepatoma cells. Toxicol. Appl. Pharmacol. 132, 146–155.[ISI][Medline]

Ramos, O., Carrizales, L., Yanez, L., Mejia, J., Batres, L., Ortiz, D., and Diaz-Barriga, F. (1995). Arsenic increased lipid peroxidation in rat tissues by a mechanism independent of glutathione levels. Environ. Health Perspect. 103, 85–88.[ISI][Medline]

Rosen, B. P. (1995). Resistance mechanisms to arsenicals and antimonials. J. Basic Clin. Physiol. Pharmacol. 6, 251–263.[Medline]

Rosen, B. P., and Borbolla, M. G. (1984). A plasmid-encoded arsenite pump produces arsenite resistance in Escherichia coli. Biochem. Biophys. Res. Commun. 124, 760–765.[ISI][Medline]

Rossman, T. G. (1995). Metal mutagenesis. In Handbook of Experimental Pharmacology; Toxicology of Metals, Biochemical Effects (R. A. Goyer, and M. G. Cherian, Eds.), Vol. 115, pp. 373–406, Springer-Verlag, New York.

Rossman, T. G., Goncharova, E. I., Rajah, T., and Wang, Z. (1997). Human cells lack the inducible tolerance to arsenite seen in hamster cells. Mutat. Res. 386, 307–314.[ISI][Medline]

Rossman, T. G., and Wang, Z. (1999). Expression cloning for arsenite-resistance resulted in isolation of tumor-suppressor fau cDNA: Possible involvement of the ubiquitin system in arsenic carcinogenesis. Carcinogenesis 20, 311–316.[Abstract/Free Full Text]

Sato, M., and Bremner, I. (1993). Oxygen-free radicals and metallothionein. Free Radic. Biol. Med. 14, 325–337.[ISI][Medline]

Shimizu, M., Hochadel, J. F., Abshire, M. K., and Waalkes, M. P. (1998). Effects of glutathione depletion on cadmium-induced metallothionein synthesis, cytotoxicity, and proto-oncogene expression in cultured rat myoblasts. J. Toxicol. Environ. Health 51, 609–621, 1997.[ISI]

Silver, S., Budd, K., Leahy, K. M., Shaw, W. V., Hammond, D., Novick, R. P., Willsky, G. R., Malamy, M. H., and Rosenberg, H. (1981). Inducible plasmid-determined resistance to arsenate, arsenite, and antimony (III) in Escherichia coli and Staphylococcus aureus. J. Bacteriol. 146, 983–996.[ISI][Medline]

Styblo, M., Delnomdedieu, M., and Thomas, D. J. (1995). Biological mechanisms and toxicological consequences of the methylation of arsenic. In Handbook of Experimental Pharmacology; Toxicology of Metals, Biochemical Effects (R. A. Goyer and M. G. Cherian, Eds.), Vol. 115, pp. 407–434, Springer-Verlag, New York.

Sunderman, F. W. (1979). Mechanisms of metal carcinogenesis. Biol. Trace Elem. Res. 1, 53–86.

Tisa, L. S., and Rosen, B. P. (1990). Molecular characterization of an anion pump. The ArsB protein is the membrane anchor for the ArsA protein. J. Biol. Chem. 265, 190–194.[Abstract/Free Full Text]

Vahter, M. (1983). Metabolism of arsenic. In Biological and Environmental Effects of Arsenic (B. Fowler, Ed.), pp. 171–198. Elsevier, Amsterdam.

Waalkes, M. P., Harvey, M. J., and Klaassen, C. D. (1984). Relative in vitro affinity of hepatic metallothionein for metals. Toxicol. Lett. 20, 33–39.[ISI][Medline]

Wang, Z., Dey, S., Rosen, B. P., and Rossman, T. G. (1996). Efflux-mediated resistance to arsenicals in arsenic-resistant and -hypersensitive Chinese hamster cells. Toxicol. Appl. Pharmacol. 137, 112–119.[ISI][Medline]

Wang, Z., and Rossman, T. G. (1993). Stable and inducible arsenite resistance in Chinese hamster cells. Toxicol. Appl. Pharmacol. 118, 80–86.[ISI][Medline]

Yamamoto, S., Konishi, Y., Matsuda, T., Murai, T., Shibata, M. A., Matsui-Yuasa, I., Otani, S., Kuroda, K., Endo, G., and Fukushima, S. (1995). Cancer induction by an organic arsenic compound, dimethylarsinic acid (cacodylic acid), in F344/DuCrj rats after pretreatment with five carcinogens. Cancer Res. 55, 1271–1276.[Abstract]

Yamanaka, K., Hasegawa, A., Sawamura, R., and Okada, S. (1991). Cellular response to oxidative damage in lung induced by the administration of dimethylarsenic acid, a major metabolite of inorganic arsenics, in mice. Toxicol. Appl. Pharmacol. 108, 205–213.[ISI][Medline]

Zakharyan, R., Wu, Y., Bogdan, G. M., and Aposhian, H. V. (1995). Enzymatic methylation of arsenic compounds: Assay, partial purification, and properties of arsenite methyltransferase and monomethylarsonic acid methyltransferase of rabbit liver. Chem. Res. Toxicol. 8, 1029–1038.[ISI][Medline]

Zhao, C. Q., Young, M. R., Diwan, B. A., Coogan, T. P., and Waalkes, M. P. (1997). Association of arsenic-induced malignant transformation with DNA hypomethylation and aberrant gene expression. Proc. Natl. Acad. Sci. U S A 94, 10907–10912.[Abstract/Free Full Text]