American Health Foundation, One Dana Road, Valhalla, New York 10595
Received February 5, 2002; accepted May 15, 2002
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ABSTRACT |
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Key Words: ethyl benzene; Fischer 344 rat; carcinogenicity study; renal cancer; mode of action; chronic progressive nephropathy; risk assessment.
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INTRODUCTION |
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Ethyl benzene has been tested by the National Toxicology Program (NTP) for carcinogenicity in two-year inhalation studies with Fischer 344 (F344) rats and B6C3F1 mice (Chan et al., 1998; NTP, 1999
). In male rats, but not in females, there was a statistically significant increase (p < 0.01) in renal tubule tumors (adenomas and carcinomas combined) at the high dose of 750 ppm after evaluation of a standard single section of each kidney, but at the relatively low incidence of 14%. In mice, ethyl benzene was associated with significant increases in lung adenomas in males and of liver adenomas in females, but there were no increases in kidney tumors (Chan et al., 1998
; NTP, 1999
). Upon step-sectioning of the rat kidneys, additional adenomas were found that confirmed an increase in males and revealed an increase in female rats. Step-sectioning raised the incidences to 42 and 16% at the high dose, in male and female rats, respectively, compared with 6 and 0% in the respective control groups. Focal renal tubule hyperplasia, considered to be a precursor stage of renal tubule tumor, was also increased in incidence in the rat kidneys. Supplemental evaluation of kidney by step-sectioning, as opposed to reliance on standard single sections, is considered by NTP to be useful in conclusively demonstrating an association between chemical administration and renal tubule hyperplasia and/or adenoma, particularly in male rats (Eustis et al., 1994
).
The International Agency for Research on Cancer (IARC) recently evaluated the carcinogenic risk of ethyl benzene to humans (IARC, 2000). Based on the NTP rodent studies, IARC noted sufficient evidence in experimental animals for the carcinogenicity of this chemical, leading to the overall evaluation that ethyl benzene falls into group 2B (possibly carcinogenic to humans; IARC, 2000
). As the IARC Working Group noted that the positive mouse findings related to benign lung and liver tumors that were within the historical control range (IARC, 2000
), the impetus for considering sufficient evidence for carcinogenicity must have rested with the renal tumor increase in rats, particularly with the results based on step-sectioning.
The purpose of this work was to determine if a mechanistic basis could be proposed for the increase in renal tumors observed in the two-year NTP carcinogenicity study in F344 rats with ethyl benzene by reevaluating the histopathology of the rat kidney tissue. Included among the modes of action for which histopathologic evidence was sought were sustained cytotoxicity/cell regeneration, hyaline droplet accumulation indicative of an 2u-globulin (
2u-g)-related mechanism, and interaction with the spontaneous aging kidney disease of rats, chronic progressive nephropathy (CPN), all of which are pertinent to renal tumor induction in the rat kidney (Hard, 1998
, 1999
). Regarding genotoxicity, in vivo studies with ethyl benzene are all negative, and the in vitro studies are predominantly negative. Thus, ethyl benzene has consistently proved to be nonmutagenic in bacteria, yeast, and insects, has not caused chromosomal aberrations in mammalian cells, and was negative in in vivo test systems for micronucleus induction (IARC, 2000
). Therefore, it is considered that this chemical is not acting to cause renal tumors via genotoxic or oxidative stress mechanisms (Hard, 1998
).
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MATERIALS AND METHODS |
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Kidney examination.
For the two-year study, all hematoxylin and eosin (H&E)-stained kidneys from the standard, single sectioning procedure were examined, as well as all step sections that had been identified by NTP as having a renal proliferative lesion. The slides were evaluated in nonblinded fashion with knowledge of the exposure levels. In addition to examination by brightfield microscopy, a selection of H&E-stained kidneys were also examined under fluorescence at a wavelength of 450490 nm to evaluate lysosomal activity (Maunsbach, 1966) and/or hyaline droplet density (Hard and Snowden, 1990
) and to aid in the discrimination between types of tubule proliferative lesions. In this reevaluation, the zoning schema of Young and Wissig (1964) for rat kidney was used, comprising zone 1, the cortex, zone 2, the outer stripe of outer medulla (OSOM), zone 3, the inner stripe of outer medulla (ISOM), zone 4, the inner medulla, and zone 5, the papilla. From the renal tubule aspect, the first (S1) and second (S2) segments of convoluted proximal tubule are located in zone 1, and the third straight segment of proximal tubule (S3, pars recta) in zone 2. Radially disposed tracts of S3 also extend into the cortex as medullary rays. Zones 3, 4, and 5 consist mainly of thin descending and thick ascending limbs of Henle, and collecting ducts.
Histopathologic criteria.
Criteria used for diagnosing tubule basophilia, karyomegaly, CPN, and proliferative lesions such as tubule hyperplasia and renal tubule tumors were those described by the Society of Toxicologic Pathologists (Hard et al., 1995, 1999
) and/or the IARC (Alden et al., 1992
). Simple tubule hyperplasia represents an increase in the number of epithelial cells without increasing the tubule lining beyond a single layer of cells. Atypical tubule hyperplasia (ATH) represents complex internal proliferation of the epithelial lining of a tubule beyond the normal single layer into the lumen, but essentially retaining the integrity of the tubule outline. Tubule proliferation transcends from atypical hyperplasia to adenoma when it obliterates the integrity of a single tubule, and/or vascular ingrowth into the proliferative focus can be discerned. The distinction between adenoma and carcinoma is the presence of areas of necrosis and hemorrhage in the latter, usually accompanied by a trend to cellular pleomorphism. It is widely accepted that atypical tubule hyperplasia, adenoma, and carcinoma represent sequential stages in the progression from preneoplastic to malignant proliferation (Dietrich and Swenberg, 1991
; Hard, 1990
; Nogueira et al., 1993
). CPN is histologically characterized in the early stages by single tubule profiles or focal tubule aggregates of basophilia in the cortex associated with thickened basement membranes, often with hyaline cast formation involving the deeper medullary portion of the same tubule. The basophilic tubules represent simple tubule hyperplasia accompanied by sporadic single cell death and mitotic activity. With progression of the disease, the foci of tubule alteration enlarge into areas of affected tubules, ultimately involving almost all of the kidney bilaterally.
Grading of CPN.
CPN was graded according to a semiquantitative system devised by the author based on lesion pathogenesis, with 0 representing nil lesions, 1 minimal, 2 mild, 3 low-moderate, 4 mid-moderate, 5 high-moderate, 6 low-severe, 7 high-severe, and 8 end-stage. In this system, grades from minimal to high moderate represent a progressive increase in the number of CPN lesions as focal changes; low severe the point where foci begin to coalesce into areas of cortical tubule change; high severe where a majority of the cortical parenchyma is involved; and end stage where no, or almost no, normal parenchyma remains. It is in end-stage CPN that renal failure leading to death occurs. CPN of lower grades could not be assessed in some rats undergoing severe autolysis because of unscheduled death, but autolysis did not affect diagnosis and grading of advanced stages of CPN or recognition of neoplastic lesions.
Statistical analysis.
An analysis of variance was used to test for differences in grade of CPN. This was followed by a Dunnetts test to compare the treated groups with the controls at p = 0.05. A Pearson correlation coefficient was calculated to measure the association between the incidence of end-stage CPN and the incidence of proliferative lesions. A one-sided Fisher Exact test was used to compare the incidence of renal tumors in the control group to the incidence in each respective treated group at p = 0.05, with and without adjustment for end-stage CPN.
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RESULTS |
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Atypical Tubule Hyperplasia
The group frequencies of ATH (as well as renal tubule adenoma and renal tubule carcinoma) after evaluation of both the original and step sections of kidney are summarized in Table 3.
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In Table 3, it can be seen that there was an apparent increase in ATH in both high-dose males (13 rats, or 26%, with ATH) and females (6 rats, or 12%, with ATH) compared with the control groups (seven males, or 14%, and one female, or 2%, with ATH). These lesions occurred randomly at depths equivalent to zones 1 and 2, but some occurred immediately below the capsule in zone 1. Of the lesions listed in Table 3
, two rats each in the control and low-dose male groups had ATH of oncocytic type, that is, cells with "ground-glass," chromophobic to pale eosinophilic cytoplasm, and very small, dark, eccentric nucleus. One control male and one mid-dose female had ATH of familial type (see Discussion section) characterized by large eosinophilic cells, with prominent, large nucleoli, and a vacuolar appearance represented by numerous, clear, discrete round spaces.
Adenomas
As shown in Table 3, there was an apparent increase in the incidence of adenomas in both high-dose males (17, or 34%, with adenomas) and females (7, or 14%, with adenomas) compared with the control groups. (four males or 8%, and one female, or 2%, with adenomas). Other than the exceptions noted later in this section, adenomas were consistently basophilic proliferations of well-differentiated type (Fig. 5
). The majority were small, approximately the diameter of a 40x field (0.5 mm) or less (Fig. 6
), whereas some less-discrete lesions could be regarded as only borderline between ATH and adenoma (Fig. 7
). As with ATH, adenomas were distributed through the outer two zones of the kidney, including the subcapsular site. In each of the low-dose male and control female groups, a single adenoma was an oncocytoma, a distinct subtype of renal tubule adenoma that is considered benign and appears not to progress to carcinoma (Montgomery and Seely, 1990
), having the characteristic morphologic features described under ATH of "ground-glass" cytoplasm and very small, dark, eccentric nucleus (Fig. 8
). In the control and high-dose male and high-dose female groups, there were, respectively, one, one, and three rats with adenomas of the distinctive eosinophilic, vacuolar (familial) phenotype (Fig. 9
).
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Multiplicity of Proliferative Lesions
The number of rats bearing more than one proliferative tubule lesion (i.e., more than one focus of ATH, ATH plus a tumor, or multiple tumors) was greater in the high-dose groups for both males and females than in the respective control groups (Table 4). In the high-dose males, seven rats had two or more adenomas versus none in the control males. In addition, 12 high-dose male rats had more than one profile of ATH or one or more profiles of ATH plus adenoma. Only two control males had an adenoma together with one or more ATH profiles. Likewise, five high-dose females had either more than one profile of ATH or ATH plus an adenoma, whereas no control females had multiple lesions. All of the rats with proliferative lesions were affected by advanced, mainly end-stage CPN.
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The three carcinomas diagnosed in three high-dose males also occurred in rats with advanced CPN (one at grade 7, two at grade 8). However, the size of these lesions prohibited defining them as CPN-related in the precise way possible with ATH and small adenomas. Neither the oncocytic or eosinophilic type of proliferative lesion showed such a consistent association with high grade of CPN as was seen with the basophilic lesions.
In Table 5, the relationship between the highest grade of CPN and basophilic proliferative lesions is demonstrated by comparing lesion incidence in the end-stage kidney between groups. This table lists the number of rats in each group with end-stage CPN as well as the lesion types in those animals. The Pearson correlation coefficient calculated to measure the association between the incidence of end-stage CPN and the incidence of proliferative lesions was 0.99851 with a probability of < 0.0001, demonstrating a very high degree of correlation. Thus, when male rats with end-stage disease were compared for all three lesion types, the percent incidences for control and high-dose rats were the same (83 vs. 80%, respectively). In the female rats, the control group had no cases of end-stage CPN for comparison, but the percent of end-stage kidneys with proliferative lesions in the high-dose females was very similar to the male rats at 75%. When this type of analysis is extended to include the next highest grade of CPN, that is, considering grades 7 and 8 together, the equivalency in risk between groups for an association of advanced CPN with a renal proliferative lesion is constant. Thus, when the incidence of renal tumors is adjusted for end-stage CPN alone, or grades 7 and 8 together, there is no statistically significant increase in renal tumors in any treated group versus the controls by the Fisher Exact test.
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Thirteen-Week Toxicity Study
There was no evidence of compound-induced tubule cell injury, proliferative response, or karyomegaly involving proximal tubules in male or female rats exposed to ethyl benzene for 13 weeks.
Incidence of CPN.
Table 7 shows the frequency of CPN in the groups that were evaluated. In the males, the mode was shifted from grade 1 (minimal) in the control group to grade 2 (mild) in the 750 ppm males and to grades 2 (mild) and 3 (low-moderate) in the 1000 ppm males. The mean grade for each of these groups was significantly different from the control mean (see Table 7
). No statistically significant change was evident in the females at 13 weeks, the majority of them having grade 0 (nil) CPN in the 0, 750, and 1000 ppm groups.
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Granular casts.
Only one rat showed evidence of granular casts. This was a high-dose male rat with two tubular profiles containing granular casts in zone 4, probably involving different levels of the same tubule. As this was a single observation, and 2u-g granular casts are characteristically observed at the junction of zones 2 and 3, this was considered an incidental finding.
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DISCUSSION |
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The majority of the proliferative tubule lesions associated with ethyl benzene exposure were basophilic in nature, comprising either ATH foci or very small adenomas less than 0.5 mm in diameter. A number of the adenomas were considered to be marginal neoplastic lesions and borderline between ATH and adenoma. Only three carcinomas were diagnosed. These were basophilic tumors like the majority of adenomas, occurring in the high-dose males. Three of the high-dose females, as well as one control and one high-dose male, had renal tubule tumors of a very different, eosinophilic, and vacuolar phenotype. Renal tubule tumors composed exclusively of large eosinophilic cells associated with prominent vacuole-like, clear spaces (minilumens) represent a phenotype that has been associated only with familial or spontaneous occurrence to date. This tumor phenotype has been described as a spontaneous lesion in male and female conventional rat strains in 90-day studies where a familial association was suspected (Hard et al., 1994), as bilateral, multicentric carcinomas in untreated F344 littermates (Thurman et al., 1995
), and as one of the predominant phenotypes in the hereditary Long-Evans Eker rat model (Everitt et al., 1992
). To the authors knowledge, there has been no recorded instance of this phenotype having been induced de novo by a chemical agent. Thus, three of the high-dose female rats in the NTP ethyl benzene study had kidney tumors consistent with a familial or spontaneous type, which most likely would not have been related to test agent exposure.
To better meet the goals of this investigation, the additional results of this histopathological review are discussed below according to the possible modes of action that are candidates for representing the basis underlying the development of the basophilic proliferative lesions.
Sustained Cytotoxicity and Cell Regeneration
Nephrotoxic chemicals can lead to renal tumor formation by causing chronically sustained cytotoxicity accompanied by compensatory cell regeneration (Hard, 1998). Although the precise mechanism is not yet elucidated, it is generally accepted that persistent cell proliferation of a compensatory nature is a risk factor for tumor development (Butterworth and Goldsworthy, 1991
; Cohen, 1995
; Croy, 1993
; Foster, 1997
; Moore and Tsuda, 1998
; Preston-Martin et al., 1990
). Chloroform has become the classic nongenotoxic example of chemicals acting to produce renal tubule tumors in rats and mice via this mode of action (Butterworth et al., 1995
). In a recent reevaluation (Hard et al., 2000
) of the carcinogenicity bioassay in Osborne-Mendel rats (Jorgenson et al., 1985
), cortical proximal tubule changes found in association with chloroform exposure over the two-year period included cytoplasmic basophilia and vacuolation, single-cell death, increased mitotic activity, simple tubule hyperplasia, and karyomegaly, indicative of chronically sustained cytotoxicity and cell turnover.
In contrast to chloroform, it seems quite clear that sustained cytotoxicity and regenerative cell proliferation is not an operative mode of action for ethyl benzene. This is because reevaluation of the rat kidneys from both the 13-week toxicity study and the two-year carcinogenicity study with this chemical demonstrated an absence of all of the above morphological indicators of sustained compound-induced tubule cell injury.
Hyaline Droplet (2u-g) Nephropathy
Recent research has shown that ethyl benzene can induce a hyaline droplet response at a concentration of 750 ppm in male F344 rats, but not in female F344 rats or B6C3F1 mice, when administered for 1 or 4 weeks by inhalation (Stott et al., 2001). The hyaline droplet response was identified as focal
2u-g accumulation, accompanied by a 79% increase in S-phase DNA synthesis at the 4-week time point. In the reevaluation of the NTP 13-week study, there was also a modest increase in hyaline droplets in proximal tubules of male rats at the high dose of 1000 ppm. Of greater biological significance, however, was the absence of granular casts at the OSOM/ISOM junction in the subchronic studies of both Stott et al.(2001) and the NTP, and the absence of linear papillary mineralization in the NTP two-year study. These two facts suggest that the hyaline droplet-inducing property of ethyl benzene did not exceed the threshold of cytotoxicity needed for an association with renal tumor development through a mechanism of sustained cell loss and regeneration (Williams et al., 2001
). However, it cannot be ruled out that this effect contributed as an additive factor superimposed on another, primary mode of action. In any case, regulatory and authoritative agencies have determined that
2u-g nephropathy in the male rat, and the associated renal tumors, is a sex- and species-specific response having no relevance to human risk assessment (Baetcke et al., 1991
; Rice et al., 1999
).
Interaction with Chronic Progressive Nephropathy (CPN)
One of the most important observations resulting from this review, confirmatory of the NTP conclusions, is that ethyl benzene caused an exacerbation of age-related spontaneous renal disease, CPN, at the high dose of 750 ppm, markedly so in the male rats, and modestly in the females. What was not evident from the NTP reports were the numbers of high-dose rats that had end-stage CPN, that is, a terminal condition where the kidneys are so morphologically altered that renal failure (as well as secondary hyperparathyroidism) occurs. In the high-dose males, which had the highest incidence of renal proliferative lesions by far, 68% of the original group of 50 rats had end-stage CPN versus only 12% in the control males. Taken further, almost 80% of high-dose males had either grades 7 or 8 CPN, a reversal of the situation in the controls, where only 20% of rats had the two highest grades. A modest exacerbation of CPN was evidenced in the high-dose females by 37% having either grade 6, 7, or 8 CPN versus 0% with these grades in the control females.
A second finding of importance from the review was the fact that almost all of the basophilic ATH and adenomas occurred in rats with advanced grades of CPN (grades 7 and 8), as did the multiple lesions, and they were all located within CPN areas. A third revelation was the equivalent incidence of renal tubule proliferative lesions in control and treated rats with end-stage CPN. In the males, for example, 33% of the controls with end-stage kidneys had adenomas compared with 41% of high-dose animals. Likewise, the percent incidence of ATH, a precursor stage of adenoma, in control and high-dose males with end-stage kidney was 50 and 32%, respectively. If all types of renal proliferative lesion on the continuum from ATH to carcinoma are assessed together in this way, the percent incidences were 83% in control males with end-stage kidney and 80% in high-dose males. Consequently, after adjustment for end-stage CPN, there was no statistically significant increase in renal tumors with treatment in either males or females. The increase in renal tumors observed particularly after step-sectioning was therefore a function of the number of rats provoked by the chemical or attendant conditions into an advanced stage of CPN.
It has long been recognized that CPN is not merely a degenerative disease but highly regenerative, in that cell proliferation is increased in many of the affected tubules (Hard, 1998; Konishi and Ward, 1989
; Short et al., 1989
), that unusual forms of attempted tubule regeneration (STR) appear to occur in the end-stage kidney (Hard et al.,1997
), and that it is not unusual to find atypical tubules or foci of atypical hyperplasia in advanced stages of the disease (Blatherwick and Medlar, 1937
; Foley et al., 1964
; Hard et al., 1995
). Based on observations with hydroquinone, it has been proposed that some chemicals can interact with CPN to enhance the proliferative aspect of this spontaneous disease process, thus leading indirectly to a small increase in low-grade renal tubule tumors (Hard et al., 1997
; Hard, 1998
). In the ethyl benzene study, the occurrence of the majority of renal proliferative lesions in rats with the most severe grades (7 or 8) of CPN, the location of the lesions within CPN-affected areas, the high proportion of tumors that were small or marginal lesions borderline between ATH and adenoma, and the increased multiplicity of small proliferative lesions, are all characteristics predictive of an etiological association with CPN.
Eustis et al.(1994) reported that for each of the NTP studies in which step-sectioning helped to establish a relationship between chemical exposure in carcinogenicity bioassays and a positive renal tubule hyperplasia/adenoma response in male rats, there was a concomitant increase in the severity of CPN. In the light of the ethyl benzene review, this suggests that in studies where multiple sectioning of kidney appears to establish a positive renal carcinogenic effect for a test chemical, it may be revealing CPN-related renal tumor development.
Notwithstanding the above evidence that the apparent increase in ATH and adenomas was a function of the increased number of rats with advanced stages of CPN at the high dose, an interaction of ethyl benzene with CPN is also likely. Not only was ethyl benzene clearly associated with an exacerbation in severity and incidence of CPN, but a nonneoplastic proliferative state (STR) was observed in a complex form more frequently in the end-stage kidneys of high-dose male rats than in end-stage kidneys of control male rats. In this respect, the findings with ethyl benzene were similar to those with hydroquinone for which an interaction with CPN was proposed (Hard et al., 1997).
Conclusions
Histopathological reevaluation of the rat kidneys from the NTP carcinogenicity bioassay with ethyl benzene provided persuasive evidence that the apparent increase in renal tumors was strongly associated with an accompanying exacerbation by the chemical of CPN to advanced grades of severity. In the male rats, a mild induction of 2u-g nephropathy may also have been a minor contributing factor. In the high-dose females, three of seven adenomas were associated with advanced CPN as in the males, whereas three presented a morphology consistent with spontaneous or familial origin. Thus, the primary mode of action underlying the increased renal tumor incidence with ethyl benzene is considered to be an interaction of the chemical with CPN, a spontaneous age-related disease of rodents. Because CPN has no identical disease counterpart in humans, the kidney results from the carcinogenicity bioassay with ethyl benzene should be considered as having no relevance to humans for risk assessment purposes.
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ACKNOWLEDGMENTS |
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NOTES |
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