* Toxicology and Molecular Biology Branch,
Biostatistics Branch, and
Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, 1095 Willowdale Road, Morgantown West Virginia 265052888; and
§ Battelle Marine Sciences Laboratory,1529 West Sequim Bay Road, Sequim, Washington 983829099
Received October 17, 2000; accepted December 21, 2000
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ABSTRACT |
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Key Words: cancer causes; risk assessment; EPA levels; arsenic; cancer; mechanism..
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INTRODUCTION |
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Understanding the mechanism of action for arsenic carcinogenicity can be an important factor in establishing the shape of the dose-response curve and assessing cancer risk, particularly at low levels of exposure. The mechanism by which arsenic causes cancer has been under intense investigation, and progress has been made in elucidating this process. Although arsenic itself is not mutagenic at doses that are not cytotoxic, some deleterious effects on DNA have been observed including inhibition of DNA repair, potentiation of DNA damage by other agents, sister chromatid exchange, and gene amplification (Lee et al., 1988; Lerda, 1994
; Li and Rossman, 1989
). These effects do not adequately explain arsenic`s carcinogenic properties, and epigenetic mechanisms have been proposed. Central to an epigenetic process is evidence indicating that arsenic stimulates cell proliferation by affecting specific cell-signal-transduction pathways. Specifically, arsenic has been shown to activate the mitogen-activated protein kinase (MAPK) cascade (Chen et al., 1998
; Huang et al., 1999
; Liu et al., 1996
; Trouba et al., 2000
), ultimately resulting in the activation of transcription factors, such as the activating protein (AP)-1 family (Burleson et al., 1996
; Cavigelli et al., 1996
; Simeonova et al., 2000
). AP-1, which is one of several transcription factors that helps regulate the expression of diverse genes, is responsible for many of the biological effects of tumor promoters, including induction of transforming oncoproteins and growth-factor expression (Angel et al., 1991). A consequence of chronic cell proliferation would be an increased likelihood of neoplasia, by providing a microenvironment for increased proliferation of mutated cells. Consistent with the hypothesis are studies demonstrating increased numbers of papillomas in Tg:AC transgenic mice given sodium arsenite (Germolec et al., 1998
). Similarly, bladder tumors have been reported in diethynitrosamine-treated rats provided dimethyarsenic acid (DMA) in their drinking water (Yamamoto et al., 1995
).
On the assumption that AP-1 activation represents a "precursor" marker for arsenic-induced bladder cancer, its DNA binding activity was semi-quantified in urinary bladders of mice exposed to control and arsenic-containing drinking water in order to help define dose-response characteristics. Changes in AP-1 binding activity were compared to histopathological changes and arsenic accumulation in bladder tissue.
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MATERIALS AND METHODS |
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Histology.
Bladders were removed and fixed by immersion in 10% neutral-buffered formalin and processed for paraffin embedding. Each paraffin block was step-sectioned and stained with hematoxylin and eosin. Pathological assessments were performed in a blind fashion. The samples were fixed for transmission electron microscopy as previously described (Simeonova et al., 2000). Ultrathin sections were prepared and stained with uranyl acetate and lead acetate and examined by electron microscopy.
Arsenic determination in tissues.
Urinary bladders from control or arsenic-treated mice were quick-frozen in acid-free vials and stored at 70°C. The tissue samples were digested by addition of 6 N HCl at 80°C for 16 h in a specially designed reaction vessel. Analyses of arsenic tissue levels were performed by Battelle Marine Sciences Lab (Seqium, WA) using a complex atomic absorption method (Crecelius, 1998) as previously described (Simeonova et al., 2000
). Quality control was established through calibration and testing of the hydride generation, purging, and detection systems.
Nuclear extracts and electrophoresis mobility-shift assay (EMSA).
Nuclear proteins were prepared from aliquots of 1 x 107 cells or frozen samples of bladder tissue pooled from 3 identically treated mice as previously described (Schreiber et al., 1989). DNA binding reactions and EMSAs were performed as described previously (Simeonova et al., 1997
, 2000
). Briefly, the 5' ends of the double-stranded oligonucleotides were labeled with
32P-ATP (New England Nuclear/Dupont, Boston, MA), using 610 U of T4 polynucleotide kinase (USB/Amersham, Cleveland, OH). Binding reactions (30 µl) were performed on ice for 30 min in reaction mixtures containing 10 µg of nuclear proteins, 20 mM TrisHCl (pH 7.8), 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 5 mM dithiothreitol, 50 µg/ml bovine serum albumin, 2 µg of poly(dI-dC).poly(dI-dC), 10% glycerol and approximately 0.1 ng (2 x 105 cpm) of specified probe. For detection of AP-1 DNA-binding activity, an oligonucleotide was obtained from Santa Cruz (Santa Cruz, CA) containing an AP-1 consensus sequence: 5' -CGC TTG ATG ACT CAG CCG GAA-3'. Protein-DNA complexes were separated on a 5% non-denaturing polyacrylamide gel. Gels were electrophoresed at 125 V in 50 mM Tris-50 mM boric acid/1 mM EDTA, dried, and autoradiographed overnight. The autoradiograms were scanned with a computerized laser densitometer (Eagle Eye II Image Analysis System, Stratagene, La Jolla, CA) and the results were examined using the One Dscan gel-analysis software and the NIH Image 1.54 analysis software. The data are presented graphically as a ratio of the mean control to experimental values.
Statistical analysis.
All experiments were replicated and representative findings are shown. Analyses were conducted using JMP software (SAS Institute, Cary, NC). One-way analysis of variance was performed on the data and linear contrasts were determined using the least-squares means. The Jonckheere-Terpstra test was used to establish dose-response trends using one-side alternatives, which gives a priori hypothesis for the direction of the response.
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RESULTS AND DISCUSSION |
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When the data from 0.5 to 10 µg/ml exposure groups were pooled for comparison with the control group, the average AP-1 activity was significantly different from controls (p = 0.03). In an attempt to provide a more thorough examination of the dose-response relationship between arsenic exposure and AP-1 activity, the AP-1 values from the 2 experiments were normalized to the 50-µg/ml dose groups and then presented as the percent change from controls (Fig 1E). Since this represented pooled data, the response curve was not statistically modeled. However, features reflected in the combined dose-response curve suggest that measurements at and below 10 µg/ml may have a much shallower slope than measures above 10 µg/ml, and thus, reflect a non-linear response curve. Alternatively, the measurement error for the EMSA's in this dose range may be relatively high compared to the real impact of arsenic on induction of AP-1 activity enough so as to bias the dose-response curve to reflect a linear-type relationship. In any case, at arsenic exposure levels between 10 and 100 µg/ml, a dose-responsive, monotonic increase in AP-1 activity is noted.
Rodents subchronically administered sodium arsenite (Simeonova et al., 2000) or DMA (Arnold et al., 1999
; Yamamoto et al., 1995
) in the drinking water develop hyperplasia of the urinary bladder epithelium, manifested by structural irregularities and thickening of the transitional cell layer. In the present studies, multiple histopathological changes in the bladder epithelium were also evident in mice administered exposure levels of 50 and 100 µg/ml (Fig. 2B
) of sodium arsenite in the drinking water. The changes in the 50 and 100 µg/ml exposure groups were indistinguishable, consisting of simple hyperplasia and the appearance of eosinophilic, cytoplasmic inclusions. The latter reflect pathological degenerative changes. Urothelial cells from hyperplastic bladders did not form papillary structures but progressed toward the lumen of the bladder. Occasional squamous metaplasia, without keratinization, was observed in some hyperplastic areas. Transmission electron microscopy indicated the presence of pleomorphic projections representing microvilli formation on the intercellular surface (Fig. 2D
). There was no evidence of histopathological changes in mice exposed to levels below 50 µg/ml (data not shown), nor was there evidence of microcrystalluria, calculi, or amorphous precipitates in any of the tissues examined.
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A series of experiments were also conducted to help establish time-to-recovery. Groups of mice were provided drinking water containing 50 µg/ml of sodium arsenite for 8 weeks, and then allowed untreated water for varying periods. AP-1 DNA binding activity and arsenic bladder concentrations were examined at 2, 4, and 8 weeks following exposure cessation, and values were adjusted relative to their respective controls (Fig. 4). Mice treated for 8 weeks at the 50 µg/ml level served as the 0-time group. Pairwise contrasts indicated that arsenic levels remained statistically elevated until week 8 following removal of arsenic from the drinking water, at which time there was a significant decrease from time 0 (p < 0.03). A t-test between animals never exposed to arsenic and those allowed to recover from exposure for 8 weeks indicated that a significant amount of arsenic, however, remained in the tissue at this time (p = .003). AP-1 levels at each time point were also compared to the 0-time recovery point. AP-1 activity at 2 and 4 weeks remained elevated, being similar to the 0 time. A slight, but not significant difference (p = 0.07) between the 0-time group and the 8-week recovery time point was observed. There was a statistically significant dose-response decrease in arsenic tissue levels as a function of time (p = 0.037) but not in AP-1 activity. This would suggest that the arsenic bladder effects described in this study are fairly long lasting although eventually reversible.
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NOTES |
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