* American Health Foundation, Valhalla, New York;
National Toxicology Program, NIEHS, Research Triangle Park, North Carolina; and
U.S. EPA, Research Triangle Park, North Carolina
Received July 1, 1999; accepted October 1, 1999
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ABSTRACT |
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Key Words: kidney; rat; adenoma; carcinoma; cytotoxicity; simple tubule hyperplasia; regeneration; karyomegaly; risk assessment..
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INTRODUCTION |
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The proposed U.S. Environmental Protection Agency (EPA) guidelines for carcinogenicity emphasize the incorporation of mechanistic data into the assessment process. Thus, intermediate endpoints related to the mode of action underlying cancer induction are an important consideration for current risk assessments (U.S. EPA, 1996; Wiltse and Dellarco, 1996
). There is a very strong correlation between the intermediate endpoints of chloroform-induced cytotoxicity and regenerative cell proliferation associated with liver and kidney tumors in mice. This correlation is based on studies investigating distribution, metabolism, and cell turnover in relevant strains. However, the correlation is not as strong in rats, leading to a conclusion that a mode of action for chloroform based on the available data from this species cannot be determined (Chiu et al., 1996
). One reason for this view is that studies investigating intermediate endpoints such as cytotoxicity and cell proliferation have used short-term studies in the Fischer-344 rat, a strain which did not develop renal tumors after chronic exposure by inhalation (Nagano et al., 1998
). The toxicity of chloroform in the Osborne-Mendel rat has not been as thoroughly investigated because pathogen-free animals are not commercially available. Further, there is little data on chloroform-induced cytotoxicity in the rat kidney after prolonged exposure. To address this question, the kidneys from a previously published 2-year study in Osborne-Mendel rats (Jorgenson et al., 1985
) were re-examined to determine whether or not there were histological alterations consistent with sustained cytotoxicity associated with chronic chloroform administration.
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MATERIALS AND METHODS |
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In the re-evaluation of the 104-week phase of this study, the kidneys stained with hematoxylin and eosin (H&E) were examined from approximately 50 rats in the untreated controls, and all animals exposed to 200, 400, 900, or 1800 ppm (19, 38, 81, and 160 mg/kg/day, respectively). In addition, kidneys from rats at interim time points of 6, 12, and 18 months, from the untreated and water-matched controls, and from the 1800 (high-dose) and 900 (high mid-dose) ppm groups were examined, as well as the 18-month time points at 400 and 200 ppm. It should be noted that not all kidney slides in the study were available for examination. For example, water-matched controls at 2 years and the low-dose (200 ppm) group at 2 years were not available for re-evaluation (Table 1).
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An earlier carcinogenicity bioassay sponsored by the National Cancer Institute (NCI) was also held in the NTP Archives. In this study, chloroform had been administered in corn oil by oral gavage at doses of 0, 90, and 180 mg/kg to male and female Osborne-Mendel rats for 78 weeks, with sacrifice of surviving animals at 111 weeks (NCI, 1976). Very poor durability of the H&E stain, drying of the mountant, and a high incidence of tissue autolysis precluded quantitative evaluation of kidneys in this study. However, a very limited assessment involving only the untreated male controls and high-dose males was carried out.
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RESULTS |
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The range of renal changes described above were approximately equivalent in rats treated with the high dose (1800 ppm) after 2 years or 18 months, but were of a lower grade after 6 and 12 months of treatment. For the 900-ppm-dose group, similar changes of lower grade (i.e., faint basophilia, vacuolation, nuclear crowding, or karyomegaly) were observed in 50% of rats after 2 years of treatment, and in 58% after 18 months, but less than half the rats treated for 6 or 12 months (Table 1). In contrast, none of these changes were observed at the lower doses of 400- and 200-ppm after 2 years and/or 18 months, or in the untreated controls at any time point, or the water-matched controls at 18, 12, or 6 months (the 2-year water-matched controls were unavailable for examination) (Table 1
).
In contrast to the changes described above, CPN lesions were distinguished as foci or areas characterized by more intense tubule basophilia associated with thickened basement membranes (Figs. 7 and 8). Furthermore, CPN-affected tubules did not show lumen reduction from hyperplasia by way of nuclear crowding, but sometimes by tubule contraction and/or collapse associated with tubule atrophy (Fig. 8
). In all groups evaluated, the severity of CPN decreased with increasing doses of chloroform (Table 1
).
Renal tubule tumors (of the chromophilic type) were confirmed in 7 of 49 high-dose male rats available for examination, which had been treated for 2 years. The average cross-sectional diameter of these tumors was 3.6 x 3.3 mm with only one tumor exceeding 1-cm diameter. In the case of very small tumors (1 mm or less), it was possible to determine that the site of origin was the mid- to deep cortex. In addition, several foci of atypical tubule hyperplasia, a preneoplastic lesion, were observed at the high dose. All but one of these precursor lesions were also located in the mid- to deep cortex, the odd one occurring in the outer cortex. An eighth renal tumor, an adenoma, was found in an 18-month interim necropsy rat treated with 1800-ppm chloroform, which had not previously been reported. One focus of atypical tubule hyperplasia was found in a high-dose rat treated for 12 months, and one in a rat treated with 900-ppm chloroform for 18 months. These additional lesions were also located in the mid- to deep cortex.
A systematic evaluation of the kidneys of rats treated with chloroform in corn oil by gavage in the NCI bioassay was not possible because of poor durability of staining, desiccation of the cover slip mountant, and frequent autolytic change. In the few cases where tissue preservation and staining permitted, renal tubule changes similar to those recorded in the drinking water bioassay, indicative of chronic cytotoxicity, were seen in the mid- to deep cortex in high-dose (180-mg/kg) male rats, but were absent from the control males. Twelve renal tubule tumors were confirmed in 11 high-dose males in this study. In general, the renal tumors resulting from gavage administration were conspicuously larger than those occurring in the drinking water bioassay. The average cross-sectional diameter was 8.9 x 6.1 mm (an underestimate because the sections in some cases represented incomplete portions of tumors), with 5 tumors exceeding 1 cm in diameter.
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DISCUSSION |
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Toxic renal injury was observed in all high-dose (1800 ppm) males and approximately one half of animals receiving the second highest dose (900 ppm). The toxic change was characterized by slight cellular basophilia, cytoplasmic vacuolation, pyknosis, nuclear crowding, mitosis, and karyomegaly. These chloroform-associated renal alterations were relatively subtle, and would not necessarily be recognized during routine microscopic examination. Moreover, this represents the first report identifying the constellation of changes described (focal nuclear crowding without thickened basement membranes but with basophilia, nucleolar hypertrophy, and apparent obscuring of the tubule lumen) as a histologic marker of chronically sustained, simple renal tubule hyperplasia. Cytoplasmic vacuolation as a manifestation of toxic injury was also seen in proximal convoluted tubules in studies examining the acute and subacute effects of chloroform in rats (Larson et al., 1995a; Templin et al., 1996b
). Nuclear crowding reflects a local increase in cell number consistent with simple tubule hyperplasia (Hard et al., 1995
). With increase in number tubule cells are compacted, giving the false impression, due to tangential sectioning, of obliterating the tubule lumen. Nucleolar hypertrophy in foci of hyperplasia is an indicator of cells actively progressing through the cell cycle. These were relatively mild changes but, nonetheless, signifying the presence of sustained renal tubule cytotoxicity and compensatory regeneration. The chronic form of regenerative hyperplasia presented here differs from the renal lesion described in previous rat studies on acute and subchronic exposure to chloroform, which was typical of regenerative hyperplasia seen after acute nephrotoxicity (e.g., Larson et al., 1995b; Templin et al., 1996b) The regenerative tubule cell response to acute nephrotoxicity, while also lacking thickened basement membrane, is characterized by a single layer of short, cuboidal proximal tubule epithelium, with prominently basophilic cytoplasm and normal to slightly hypochromic nuclei without obfuscation of the tubule lumen. With its crowded nuclei sometimes obscuring the tubule lumen, the chronic form of tubule regeneration appears to represent a simple hyperplastic response beyond that needed for cell replacement.
In this study, which was conducted in male rats only, Jorgenson et al. (1985) recorded renal tubule tumor incidences of 1.3%, 2%, 1.3%, 2.7%, 6.3%, and 14% for the control, water-matched control, and 19-, 38-, 81-, and 160-mg/kg exposure groups, respectively. In the present re-assessment of renal histopathology from this study, evidence of persistent cytotoxicity and regenerative hyperplasia was found in all rats at the 160-mg/kg-dose level, the dose at which renal tubule tumors were statistically increased. Similar changes were also observed in rats at 81 mg/kg, but at a much lower incidence and grade consistent with a non-statistical renal tumor frequency in this group. Additional evidence that these proximal tubule changes in the high-dose group are linked to neoplasia is their concordance to the zonal distribution of lesions. Cytotoxic tubule lesions, occasional foci of atypical tubule hyperplasia (the precursor of renal tubule adenoma), and incipient renal tubule tumors were located exclusively in the mid- to deep cortex. Such site concordance is critical to any linkage between sustained cell replication via cytotoxicity, and tumor induction.
Special care was taken to discriminate between the toxicity-related changes and spontaneous age-related nephropathy, CPN. The tubule alterations described above, associated with chloroform exposure, were distinct from the spectrum of lesions comprising CPN, and occurred in areas of cortex not involved with this spontaneous disease process. Kidneys from rats necropsied after 6, 12, or 18 months of treatment had lesions indicative of chronically sustained cytotoxicity/compensatory regeneration that were morphologically different from the early stages of CPN. The earliest histopathological manifestation of CPN is a single cortical tubule profile showing distinct basophilia and thickened basement membrane, and is associated with hyaline cast formation at some part of the same nephron (Hard et al., 1999). In the chloroform bioassay, there was an inverse relationship between CPN and increasing dosage of chloroform, which was undoubtedly a secondary effect dependent in some way on the poor palatability of the test agent that resulted in decreased water and food consumption. It is well known that caloric overload exacerbates and dietary restriction protects from spontaneous nephropathy in old rats. It might be argued that the greater severity of CPN in control rats prevented recognition of the changes described here as being linked to cytotoxicity/cell regeneration. This was not the case because such changes were not present in any control animal that had a low grade of CPN, but they were observed in all high-dose and some high-mid-dose rats that had severe grades of CPN. In addition, none of the alterations associated with chronic toxicity were present in any control or vehicle-control animals from the interim time points, regardless of severity of CPN, and these were only present in the high-dose (1800 ppm) and high-mid-dose (900 ppm) groups of animals.
These findings are important in the context of chloroform-risk assessment because they provide new information that extends the substantial database relating to mode of action underlying the renal tumor response (Golden et al., 1997). This database is particularly comprehensive for mice. In keeping with the sex specificity of the two positive carcinogenicity bioassays in Osborne-Mendel rats, chloroform has produced statistically significant increases in renal tubule tumor incidence in mouse bioassays, but in males only. These have included a 2-year study in which chloroform was administered by inhalation, resulting in a 25% incidence in male BDF1 mice (Nagano et al., 1998
), and a 2-year study where the compound was given orally in a toothpaste formulation, producing a 21% incidence in ICI males (Roe et al., 1979
). Chloroform has been tested in other mouse strains but with negative or non-significant results.
In the mouse, there is a very strong correlation between the levels of the enzyme CYPIIE1 in cortical proximal tubules, which is responsible for conversion of chloroform to toxic metabolites (Brady et al., 1989, Guengerich et al., 1991
), binding of radiolabeled compound to cortical microsomal proteins, metabolism of chloroform, induction of proximal convoluted tubule cytotoxicity, and compensatory cell regeneration. Various studies in mice have demonstrated that only the male is susceptible to chloroform-induced renal cytotoxicity (Eschenbrenner and Miller, 1945
; Klaassen and Plaa, 1967
; Smith et al., 1983
). This marked sex difference response parallels the much higher levels of CYPIIE1 in male compared to female mice (Henderson et al., 1989
; Henderson and Wolf, 1991
). Consequently, there is a much greater accumulation of chloroform in male mouse renal cortex than in females (Taylor et al., 1974
). Furthermore, there is a correlation between the extent of covalent binding of chloroform or its metabolite to renal proteins and microsomes and the degree of renal tubule necrosis, providing evidence of a cause-and-effect relationship linking covalent binding with tissue necrosis (Ilett et al., 1973
) mediated by the metabolism of chloroform (Pohl et al., 1984
; Smith et al., 1983
, Smith and Hook, 1983
).
Male DBA mice metabolize chloroform in the kidney at twice the rate of male C57BL mice (Pohl et al., 1984), demonstrating a higher level of covalent binding of chloroform to renal microsomes (Clemens et al., 1979
), and developing a much greater extent of renal injury (Ahmadizadeh et al., 1984
; Hill et al., 1975
). There is ample evidence that, in turn, the proximal tubule cytotoxicity induced by chloroform causes increased proximal tubule cell proliferation, representing regenerating tubules (Gemma et al., 1996
; Templin et al., 1996a
). All of this evidence correlates with the positive renal-tumor response observed in male, but not female BDF1 mice (Nagano et al., 1998
), which strain is derived from the susceptible DBA strain, and the negative tumor response in the B6C3F1 mouse (NCI, 1976
), as well as its parental C57BL strain (Roe et al., 1979
). Consequently, a strong case exists for a mode of action for chloroform-induced renal neoplasia in the mouse based on cytotoxicity and sustained tubule-cell regeneration.
In the rat kidney, according to published data, the correlation appears not to be as strong as in the mouse. Conventional carcinogenicity bioassays have been conducted only in the Osborne-Mendel, Fischer 344 (F-344) and Sprague-Dawley rat. The latter two strains were negative for renal tumor induction in contrast to the Osborne-Mendel (Jorgenson et al., 1985; Nagano et al., 1998
; NCI, 1976
; Palmer et al., 1979
). However, almost all of the recent studies investigating intermediate end-points associated with chloroform have been conducted in the F-344 rat. In addition, there appears to be little difference in the levels of CYPIIE1 between male and female F-344 rats (Wilke et al., 1994
), and one earlier study (Smith et al., 1985
) concluded that intrarenal bioactivation of chloroform by cytochrome P-450 did not appear to play a major role in nephrotoxicity in this species. Although male and female rats are generally regarded as being equally susceptible to chloroform-induced renal injury, some gender differences have been observed in subacute studies if doses required to elicit damage are compared. Thus, in F-344 rats, a lowest-effect level for males was 34 mg/kg/day (Larson et al., 1995a
) and for females, 200 mg/kg/day (Larson et al., 1995b
). A further data gap relates to the limited information on whether tubule damage can be sustained throughout a chronic period of exposure of rats to chloroform. Prior to this report, the longest period for which observations had been recorded was in an unspecified strain of rat, where renal tubule injury persisted through 6 months of exposure in males, with females again being less affected (Torkelson et al., 1976
).
The sole investigation of intermediate endpoints induced by chloroform in the Osborne-Mendel strain was a 2-day study involving a single gavage dose in males (Templin et al., 1996b). This study confirmed that chloroform induced regenerative cell proliferation in a renal tumor-responding strain, at least as an acute response. The lack of any further data on this strain, particularly concerning the persistence of renal injury and cell turnover through prolonged periods of exposure, has hampered the risk assessment process for chloroform. These concerns have been expressed recently by Chiu et al (1996), who stated that there is no apparent correlation between cytotoxicity and renal cancer in male rat studies, and that a mode of action for chloroform carcinogenesis could not be determined from the available data. Specifically, this view was based on the conclusions that in the bioassay with Osborne-Mendel rats, the tumors were recorded in test groups for which no observable cytotoxicity had been reported, and that there was no correlation between cytotoxicity and cancer in the kidneys of individual male rats at necropsy (Chiu et al., 1996
). However, distinction of chemically induced tubule injury from lesions of spontaneous CPN was not taken into account in this viewpoint. Moreover, subtle changes indicative of chemically induced cytotoxicity were not usually recorded in bioassays conducted at the time of the chloroform tests.
An incidental finding in this histopathological review was the striking difference in the dimensions of renal tumors induced by chloroform administered by corn-oil gavage versus those arising from exposure via drinking water. On average, tumors associated with the gavage mode of administration were at least twice the size of those produced by chloroform in drinking water. Such tumor size disparity can be matched with the differences recorded in the extent of hepatic and renal toxicity and cell proliferation induced by the two different modes of administration (Larson et al, 1995a; Pereira, 1994
), gavage exposure producing the more severe toxic response in each organ. Once-daily bolus delivery of chloroform is thought to result in higher target tissue levels than occurs following exposure to the same total dose consumed via the drinking water in fractionated quantities spread over a 24-h period. Such differences in tissue dosimetry would be expected to correlate with the magnitude of tissue responses, including the parameters of rate of induced tumor growth and size.
In conclusion, a histopathological re-evaluation of the original kidney tissue preparations from the various time points of the 2-year chloroform-in-drinking-water carcinogenicity bioassay with male rats has established the presence of renal tubule cell alterations consistent with chronically persistent cytotoxicity and cell turnover at dose-levels associated with renal tubule tumor induction. Such changes were confirmed also in the NCI oral gavage bioassay. Coupled with the preponderance of data showing that chloroform is non-genotoxic, the finding reported in this study adds to the weight of evidence that the carcinogenicity of chloroform occurs via a secondary mechanism, with the obligatory key events of sustained cellular toxicity and regenerative hyperplasia.
Under the new proposed EPA guidelines for carcinogen risk assessment (U.S. EPA, 1996), which provide the opportunity to apply sound mechanistic data in the place of default assumptions, this study provides more evidence that chloroform-induced renal cancer in rats occurs via a cytotoxic/regenerative hyperplasia mode of action. Thus, the carcinogenic risk associated with such events would be expected to be nonlinear (Foster, 1997
), and best represented by a margin-of-exposure (MOE) risk assessment approach.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at American Health Foundation, One Dana Road, Valhalla, N.Y. 10595. Fax: (914) 592-6317. E-mail: gordonhard{at}msn.com.
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