* Laboratory of Comparative Carcinogenesis, NCI at NIEHS, Mail Drop F0-09, Research Triangle Park, North Carolina 27709;
University of Kansas Medical Center, Kansas City, Kansas; and
Office of the Director, NIEHS, Research Triangle Park, North Carolina
Received December 2, 1999; accepted February 9, 2000
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ABSTRACT |
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Key Words: arsenite; arsenate; chronic exposures; metallothionein-I/II null mice; nephrotoxicity; serum cytokines; hepatotoxicity; glutathione.
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INTRODUCTION |
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The toxicity of arsenicals is highly dependent on the chemical forms. For instance, arsenite [As(III)] has an LD50 in mice of ~11 mg/kg, while the LD50 of arsenate [As(V)] is 34 times higher. Both As(III) and As(V) are metabolized to the organic forms, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) in many species, including humans. These organic arsenicals, (i.e., MMA and DMA), are 50100 times less acutely toxic than inorganic As(III) (ATSDR, 1998; Kreppel et al., 1993; Peoples, 1974
).
Metallothionein (MT) is a low-molecular-weight, cysteine-rich, metal-binding protein. MT has been proposed as playing an important role in the homeostasis of essential metals, in the detoxication of heavy metals, and in the scavenging of free radicals (Kägi, 1993; Sato and Bremner, 1993
). Moreover, MT is a small protein and easily induced by heavy metals, hormones, acute stress, and a variety of chemicals (Kägi, 1993
). Four major isoforms of MT have been identified. These include MT-I and MT-II, which existed in all tissues examined, and are the predominant forms in liver and kidney. On the other hand, MT-III and -IV are relatively minor forms and have very limited distributions since MT-III is located primarily in the brain (Palmiter et al., 1992
), and MT-IV is located in stratified squamous epithelia of the gastro-intestinal tract (Quaife et al., 1994
). MT is important in detoxication of many transition metals such as cadmium and mercury (Kägi, 1993
) but its role in As toxicity is poorly defined.
The synthesis of a variety of proteins, especially acute-phase proteins, is stimulated following As exposure. For instance, As(III) is an effective inducer of heat-shock protein and heme oxygenase both in vitro (Bauman et al., 1993; Keyse et al., 1990
) and in vivo (Brown and Rush, 1984
). MT, which is thought to be part of the acute-phase response, is also induced by arsenicals (Albores et al., 1992
; Hochadel and Waalkes, 1997
; Kreppel et al., 1993
; Maitani et al., 1987
). The induction of these proteins has been proposed as playing an adaptive role in As tolerance. However, little is known about the precise role of MT in As toxicity, especially during chronic exposure. To help examine the potential functions of MT, MT-I/II knockout (MT-null) mice have been produced (Masters et al., 1994
; Michalska and Choo, 1993
). These mice are essentially "normal" except for lack of the 2 predominant forms of the MT protein (MT-I and MT-II) (Iszard et al., 1995b
; Masters et al., 1994
). MT-null mice have provided a good tool for elucidation of the role of MT in chronic cadmium toxicity (Liu et al., 1998
). Therefore, the goal of the present work was to determine the role of MT in chronic As toxicity, using MT-I/II null mice as a model to help define any such role.
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MATERIALS AND METHODS |
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Animals and treatments.
MT-I/II null mice and corresponding wild-type (WT) mice (129 background) were obtained from Jackson Laboratories (Bar Harbor, ME), and maintained at 22 ± 1°C with a 12-h light/dark cycle. Mice were fed standard rodent chow (Harlan Teklad 7001) and allowed free access to tap water. Male and female mice aged 68 weeks were assigned in equal numbers to all groups (n = 46). For chronic oral exposure, mice were provided drinking water containing As(III) at concentrations of 7.5, 22.5, or 45 ppm, or As(V) at concentrations of 37.5 or 75 ppm. Control mice received tap water. For the chronic injection study, mice were injected sc in the dorsal thoracic midline with As(III) at doses of 10 and 30 µmol/kg, or As(V) at a dose of 100 µmol/kg, once daily, 5 days/week for 15 weeks. Control mice were injected sc with the same volume of saline (10 ml/kg). The injection route was chosen in an attempt to avoid the potential confounding factors in As absorption from the gastrointestinal tract, and to compare parenteral As effects with previous work in the literature.
Data and tissue collection.
Animal body weights were recorded weekly. At the end of the experiment (15 weeks for sc exposure; 48 weeks for po exposure), mice were killed by decapitation. Blood was collected and serum was prepared for blood biochemistry assays. Liver and kidney were removed and weighed, and portions were fixed in 10% neutral formalin, processed by the standard histological techniques, and stained with hematoxylin and eosin for light microscopic examination. Pathological examinations were conducted in "blind" fashion.
Biochemical assays.
Serum cytokine levels were determined using the ELISA kits (R&D Systems, Minneapolis, MN). ALT activity and BUN levels were assayed using kits from Sigma (St. Louis MO), according to manufacturer's instructions. In liver, reduced glutathione (GSH) was determined by the enzymatic method of Tietze (1969), cytosolic GSH peroxidase activity was assayed using hydrogen peroxide as a substrate, and glutathione reductase was assayed with oxidized glutathione as substrate by the method described by Iszard et al. (1995a). Hepatic MT concentrations were determined by the Cd/hemoglobin assay (Eaton and Toal, 1982). For determination of hepatic caspase-3, portions of livers were homogenized in 20 mM TrisHCl (pH 7.4) containing 1% Triton-100 and 150 mM NaCl, and were centrifuged at 12,000 g. A caspase-3 assay in the resulting supernatant was determined by measuring the cleavage reactions of AC-DEVD-AFC, resulting in the production of 7-amino-4-trifluoromethyl coumarin (AFC), by fluorescence spectrophotometer at
ex 400 nm and
em 500 nm (Gurtu et al., 1997
).
Statistics.
The means and standard error were calculated. The Student's t-test was used to analyze the difference between the wild-type mice and MT-null mice. In multiple comparisons to the matched control (WT or MT-null), an ANOVA analysis followed by Dunnett's test were employed. In all cases, a 2-sided p < 0.05 was considered to indicate a significant difference.
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RESULTS |
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The liver weights of the animals were not significantly altered by chronic As exposures in either the WT or MT-null mice. However, the kidneys were enlarged in both WT and MT-null mice, as reflected by increased kidney/body weight ratios (Fig. 1). Higher kidney/body weight ratios were seen in MT-null mice than in WT mice after repeated As injections at the 10 and 30 µmol/kg doses of As(III) and after chronic oral As(III) to the 22.5 ppm, suggesting MT-null mice are more sensitive to As(III) than WT mice.
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DISCUSSION |
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As-induced liver injury in humans, either from the long-term use of As-containing Fowler's solution in the treatment of psoriasis (Morris et al., 1974; Nevens et al., 1990
), or from As exposure in the drinking water (Mazumder et al., 1998
) or food (Zhou et al., 1993), typically is manifested initially as jaundice, and may progress to noncirrhotic portal hypertension and cirrhosis. Such lesions sometimes progress to liver neoplasia in humans, a potential target site of As carcinogenesis (NRC, 1999
). Our recent work indicates hepatocellular proliferative lesions (foci of alteration and adenoma) are increased by repeated As(V) exposure in mice (Waalkes et al., 1999
). Additional liver lesions in experimental animals include swollen mitochondria and altered liver function in rats given As(V) (2060 ppm) for up to 6 weeks (Fowler et al., 1977
). Fatty infiltration of the liver becomes evident after 12 months of oral As exposure in mice, which progresses to liver fibrosis by 15 months (Santra et al., 1997
). Although overt hepatocellular necrosis was not observed in the present study, the fatty changes, degenerative alterations, area of focal necrosis, and the increases in hepatic caspase-3 activities are all indicative of hepatic injury following chronic As(III) and As(V) exposures. Most importantly, this is the first report to show that the inability to produce MT in MT-null mice causes an increased sensitivity to chronic, As-induced hepatotoxicity.
Chronic renal injury, as evidenced by cloudy urine, proteinuria, glucouria, and renal tumors, is not uncommon in As-exposed humans (NRC, 1999; Mazumder et al., 1998
; Zhou et al., 1993). The kidney is the major organ for As elimination from the body, making renal exposure to As a major issue. Acute renal failure can be produced by As(III) in hamsters simultaneously given a GSH-depleting agent (Hirata et al., 1990
), while chronic exposure of mice to As(V) produces histopathological lesions within the kidney as well as in the liver, spleen, and thymus (Kerkvliet et al., 1980
). The present study showed that degenerative changes in kidneys, including tubular cell vacuolation, interstitial nephritis, and glomerular swelling occurred following chronic As exposure. The kidney is a known target site for As carcinogenesis in humans (NRC, 1999
), so As-induced nephrotoxicity could also potentially progress to kidney neoplasia under the appropriate circumstance, probably through compensatory cell proliferation. The increases in BUN, an indicator for kidney dysfunction, were pronounced in MT-null mice, further supporting the notion that MT-null mice were more sensitive than WT mice to chronic As-induced toxicity, including nephrotoxicity.
One of the mechanisms by which arsenicals produce toxic effects is through their interaction with cellular sulfhydryl groups in proteins or elsewhere (Aposhian, 1997). As(III) and As(V) have been shown to react with GSH to form As-GSH complexes (Scott et al., 1993
). Arsenicals and arsenothiols have been shown to inhibit glutathione reductase in vitro (Thomas, 1998
). Acute arsenical administration produces a significant depletion (up to 30%) of hepatic GSH (Ahmad et al., 1999
), while chronic exposure to As via injections (Flora et al., 1997
) or via drinking water (Santra et al., 1997
; Flora, 1999
) causes up to a 35% depletion in hepatic GSH, along with liver injury. Consistent with the literature, the present study observed up to a 35% depletion in cellular GSH following repeated As injections. In this regard, MT-null mice were more susceptible than WT mice to As(V) depletion of cellular GSH. GSH depletion may have important implications in chronic As toxicity in that: (1) GSH is thought to be an essential cofactor for inorganic As methylation, a possible detoxication mechanism (Aposhian, 1997
); and (2) GSH is an important cellular antioxidant, and depletion of GSH enhances As-induced cytotoxicity and aberrant gene expression (Barchowsky et al., 1996
; Shimizu et al., 1998
). In fact, it is thought that generation of reactive oxygen species may be important in As carcinogenicity (Snow, 1992
). Furthermore, many cells become tolerant to As toxicity through increased GSH (Chang et al., 1991
; Lee et al., 1989a
,b
). However, the role of GSH depletion in chronic As toxicity requires further definition.
This study also shows that chronic As exposures increased production of various proinflammatory cytokines as reflected by increased serum levels of IL-1ß, IL-6 and TNF-. These proinflammatory cytokines could play an important role in As-induced liver and kidney injury, as well as neoplastic changes. Increases in the levels of cytokines and growth factors such as CS-GMF, TGF-
, and TNF-
have been implicated in As-induced skin lesions and skin tumors (Germolec et al., 1998
). Similarly, it appears TNF-
is critical to the hepatotoxicity of other inorganics (Dong et al., 1998
), although its role in As-induced hepatotoxicity is as yet undefined. The cytotoxic effects of As in murine macrophages are associated with marked increase in the production of TNF-
(Sakurai et al., 1998
). Although the exact source of these proinflammatory cytokines cannot be defined in the present study, it is postulated that these serum cytokines could come from the inflammatory cells located in liver, kidney, spleen, or blood, and could play a role in As-induced hepatotoxicity and nephrotoxicity.
Arsenicals are moderately effective inducers of MT in mice (Kreppel et al., 1993; Maitani et al., 1987
) and rats (Albores et al., 1992
; Hochadel and Waalkes, 1997
). The potency of different arsenicals for MT induction varies markedly: As(III) is a relatively potent MT inducer, while it takes approximately 3 times more As(V), 50 times more MMA, and 120 times more DMA to induce similar levels of MT (Kreppel et al., 1993
). With regard to efficacy, MMA is the most effective MT inducer (80-fold), followed by As(III) (30-fold), As(V) (25-fold), and DMA (10-fold) at maximum tolerated doses (Kreppel et al., 1993
). In contrast to marked MT induction following acute high dose of arsenicals, induction of MT following chronic exposures in the present study was only 25-fold. It is quite clear that MT induction is one of the most important adaptive mechanisms for production of Cd tolerance (Liu et al., 1998
). In the present study, MT-null mice were more susceptible than WT mice to chronic As toxicity, suggesting that the induction of MT could be one of the cellular mechanisms affording protection against chronic As toxicity as well. However, the mechanism by which MT protects against chronic arsenic toxicity is unclear. The affinity of arsenic for MT in vitro is very low compared to heavy metals like zinc (Zn) or cadmium (Cd) (Waalkes et al., 1984
). In intact animals, only a small portion of the total As dose is found to be associated with the MT fraction in the liver (Albores et al., 1992
; Kreppel et al., 1994
; Maitani et al., 1987
). Additionally, As does not induce MT synthesis in vitro (Kreppel et al., 1993
; Shimizu et al., 1998
), suggesting the in vivo inductions are indirect. Thus, unlike the proposed detoxication mechanism for Cd by sequestration with MT (Liu et al., 1998
), MT probably does not protect against As toxicity by direct binding of the arsenical. Because of its high sulfhydryl content, it has been suggested that MT may also react with free radicals and electrophiles (Basu and Lazo, 1990
; Sato and Bremner, 1993
). In this regard, MT-null cells are more sensitive to oxidative-inducing agents (Lazo et al., 1995
), and also more sensitive to As-induced cytotoxicity (Romach et al., 2000
). As(III) caused increased formation of fluorescent dichlorofluorescein by producing oxidative stress in human fibroblasts (Lee and Ho, 1995
), and inorganic As(III) produced more reactive oxygen species and oxidative damage than did As(V) in mice receiving toxic doses of inorganic arsenicals (Liu et al., 2000
), indicating that oxidative stress is a critical step in As toxicity. Therefore, the primary function of MT in As tolerance may be as an antioxidant against As-induced oxidative injury. The exact role MT plays in reducing As toxication, however, requires further investigation.
In summary, chronic inorganic arsenicals exposure produced elevation of serum cytokines, the depletion of cellular GSH, and renal and hepatic injury. MT-null mice were more susceptible than WT mice to these toxic effects, suggesting that MT may play a role in decreasing As toxicity after chronic exposure to As.
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ACKNOWLEDGMENTS |
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NOTES |
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