* Toxicology & Environmental Research and Consulting, Building 1803, The Dow Chemical Company, Midland, Michigan 48674; and
BASF AG, Carl Bosch Strasse 38, DUP/PC-Z 470, 67056 Ludwigshafen, Germany
Received May 28, 2002; accepted October 4, 2002
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ABSTRACT |
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Key Words: ethylbenzene; inhalation exposure; tumorigenesis; mode of action; target tissues; S-phase DNA.
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INTRODUCTION |
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The subchronic toxicity and potential oncogenicity of inhaled EB vapor has been evaluated by the U.S. National Toxicology Program (NTP). In a subchronic inhalation toxicity study, both sexes of Fischer344 rats and B6C3F1 mice were exposed to up to 1000 ppm EB 6 h/day, 5 days/week, for up to 13 weeks (NTP, 1992). The weights of liver, kidneys, and lungs of both sexes of rats and liver of both sexes of mice exposed to 750 or 1000 ppm EB were increased, but not accompanied by histopathologic changes. In subsequent oncogenic evaluations, both sexes of Fischer 344 rats and B6C3F1 mice were exposed to 0, 75, 250, or 750 ppm vapor, 6 h/day, 5 days/week for 104 weeks (NTP, 1999
). Male and female rats exposed to 750 ppm EB had an increased incidence of renal effects, including greater severity of nephropathy, tubular hyperplasia, and renal tumors (primarily adenomas), relative to controls. Male mice exposed to 750 ppm had an increased incidence of metaplasia of pulmonary alveolar epithelium and lung tumors (primarily adenomas) while the high-exposure group female mice had increased incidences of hepatocellular adenomas and adenomas plus carcinomas. No tumors were observed at a 75-ppm exposure level.
EB has also been extensively evaluated in a wide variety of in vitro and in vivo genotoxicity assays. Despite its tumorigenicity in rodents, the weight of evidence indicates a general lack of genotoxic activity (IARC, 2000; and NTP, 1999
). Exposure of rats and mice to EB vapor also reportedly induces the activities of a number of mixed oxygenase enzymes (MFO) and glucuronosyl transferase (UGT). Elovaara et al. (1985)
observed increased activities of hepatic and renal MFO and UGT activities in rats exposed to
600 ppm EB for 5 days. Induction of hepatic MFO activity has also been reported by Pedersen and Schatz (1998)
in rats exposed to 300 ppm EB for 3 days while Fuciarelli (2000)
reported that hepatic cytochrome P450 levels were increased in mice, especially females, when exposed to 750 ppm EB for 112 days, relative to controls. Taken together, these findings suggest that EB may cause tumors in animals via a threshold promotion-based mode of action; however, significant data, especially on cell dynamics, was lacking.
The purpose of the present investigations was to evaluate treatment-related effects following short-term exposures to tumorigenic levels of EB that may suggest a plausible mode or modes of action.
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MATERIALS AND METHODS |
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Test material and exposures.
Ethylbenzene (99.9100% purity) was obtained from the Dow Chemical Company, Freeport, TX. Animals were singly housed and exposed, whole body, to EB vapor in 14.5 cubic meter inhalation chambers (2.4 x 2.4 x 2.4 meters, with a pyramidal top) under dynamic airflow conditions (approximately 12 air changes/h). EB atmospheres were generated using a glass J-tube method (Miller et al., 1980) in which liquid EB was metered into a stream of heated compressed air and subsequently diluted with filtered room air. EB concentrations were quantitated at least 6 times per exposure day in the one-week study and 1012 times per exposure day in the four-week study, using an automated sequential sampling system and gas chromatography (Hewlett Packard 5890 gas chromatograph with a flame ionization detector and interpolation to a standard curve). Instruments were calibrated daily using standards of known concentration. Nominal chamber concentrations were calculated based on the amount of test material used and total amount of air passed through the chamber during each exposure period. In addition, airflow, chamber temperatures and relative humidity were measured approximately once each hour. Chamber data were collected using a CAMILE® Data Acquisition and Control System (CAMILE Products LLC, Indianapolis, IN).
Test animals and husbandry.
Male and female Fischer 344 rats and B6C3F1 mice were obtained from Charles River Laboratories, Inc. (rats, Raleigh, NC; mice, Raleigh, NC or Portage, MI) and were 78 weeks of age at the initiation of exposure. Animal usage as part of this study was reviewed and approved by the laboratory Institutional Animal Care and Use Committee. The animals were housed in stainless steel cages under environmentally controlled conditions and acclimated to the laboratory for 7 days prior to the start of the study. LabDiet® Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO) and municipal water were provided ad libitum. Animals were randomly assigned to exposure groups based upon body weight and were uniquely identified via subcutaneously implanted transponders. Cageside health examinations were conducted twice daily. Animals dying spontaneously were necropsied on the day of death or as close to this as possible. Body weights were recorded pre-study, on the first day of study prior to exposure, weekly thereafter (four-week study), and on the day of scheduled necropsy.
Necropsy, serum chemistry, histopathology, and electron microscopy.
All test animals were sacrificed on the day following the fifth or twentieth exposure for the one- and four-week studies, respectively. All animals were anesthetized with methoxyflurane, weighed, blood samples obtained by orbital sinus puncture, serum harvested, and serum-chemistry parameters assayed for alanine transaminase, aspartate transaminase, alkaline phosphatase, creatinine, urea nitrogen, and -glutamyl transpeptidase. Animals were then decapitated, exsanguinated, and the kidneys of rats and the livers and lungs of mice excised and weighed. In the cell dynamics and histopathology subgroup, sections of target tissues from 3 animals/tissue were collected and preserved in a 2% glutaraldehyde-2% formaldehyde fixative for electron microscopy. All remaining target tissues from these subgroups of animals were immersion fixed in 10% neutral phosphate-buffered formalin. Mouse lungs were infused with fixative prior to immersion. In addition, a small section of duodenum of each animal was excised and preserved to serve as an internal control for S-phase DNA synthesis measurements. In the enzyme-activity subgroup, kidneys from rats and livers from 6 (one-week study) or 8 (four-week study) mice/sex/dose level were excised, snap frozen in liquid nitrogen and stored at 80°C. The lungs of mice from the enzyme groups were pooled to give 68 groups of 4 or 5 lungs each.
Histologic sections of preserved rat kidneys and mouse liver and lungs were prepared by standard methods, stained with hematoxylin and eosin, and examined using light microscopy. The kidneys of male rats were examined using fluorescence microscopy.
S-phase DNA synthesis.
Levels of S-phase DNA synthesis were determined on serial sections of paraffin-embedded organs using an immunohistochemical technique for identification of BrdU incorporation into nuclear DNA outlined by Eldridge et al. (1990). Animals (68/sex/group) were continuously infused with BrdU via osmotic minipumps (Model 2ML1 for rats and Model 2001 for mice, Alzet Corporation, Palo Alto, CA) implanted the day before initiation of exposures in the one-week study and six days prior to necropsy in the four-week study.
BrdU-labeled and unlabeled nuclei were counted from: (1) cortex (proximal convoluted tubules), outer medulla (inner and outer stripes), and inner medulla of rat kidneys; (2) hepatocytes from the centrilobular, midzonal, and periportal regions of mouse livers; and (3) epithelial cells of the lower airways and alveoli of mouse lungs. A labeling index (LI, the proportion of immunohistochemically stained nuclei to total nuclei), based upon a minimum total count of nuclei, was subsequently calculated. The minimum number of nuclei counted was 1000 in the cortex and outer stripe of the outer medulla of the kidney, 300 in the inner stripe of the outer medulla and inner medulla of the kidney, 2000 in each of the regions in the liver, and 1000 in the smaller airways and alveoli of the lung. Due to the focal nature of labeled nuclei in the renal cortex of the male rat, S-phase synthesis was subsequently reevaluated by counting five cortical foci ("hot spots") having the highest concentration of labeled cells (>1000 total nuclei for all rats) in a blinded manner. Liver sections were evaluated using the lobule-dependent zonal measurement method outlined by Bahnemann and Mellert (1997) by use of an ocular grid at 250x total magnification.
Apoptosis.
Organs from high dose and control animals were processed and immunohistochemically stained for identification of apoptotic cells using ApopTag® Plus Kit (Oncor, Inc., Gaithersberg, MD). Stained and unstained cortical and medullary (inner and outer stripe) kidney cells of the rat, centrilobular and periportal mouse liver hepatocytes, and epithelium of the lower airways and alveoli of the mouse lung were counted microscopically. An apoptosis index (AI, proportion of apoptotic cells) based upon a minimum total count of cells similar to those used for LI determination was calculated.
2u-globulin.
Deposition of 2u-globulin in the kidneys of male rats was evaluated by immunohistochemical staining at BASF Aktiengesellschaft (Ludwigshafen, Germany). Mounted, deparaffinized tissue was treated with 0.1% protease solution for antigen retrieval, reacted with mouse anti
2u-globulin followed by biotinylated antimouse antibody (secondary antibody) and alkaline phosphatase/strepavidin avidin-biotinylated horseradish peroxidase complex.
2u-globulin was then visualized by reaction with Fast Red. Quantitation was accomplished using an image analysis system (KS 400, Zeiss, Germany) at 200x magnification. Fifteen "hot spots" were analyzed, each including one field directly below the capsule and an adjacent field immediately underneath for examining the lower part of the cortical nephron, a total of 30 fields per animal.
Hepatic MFO and UGT activities.
Frozen samples of target tissues were thawed on ice and homogenized using a teflon pestle or a polytron (lungs). Microsomes were isolated using the method outlined by Guengerich (1982) at 05°C and stored at 80°C until assayed. Proteins were quantitated using the Pierce BCAä method (Pierce Chemical Co., Rockford, Illinois). CYP1A1, CYP1A2, and CYP2B1/2 activities were measured in vitro as ethoxyresorufin (EROD), methoxyresorufin (MROD, four-week study only), and pentoxyresorufin (PROD) O-dealkylase activities, respectively, using the fluorometric methods outlined by Kennedy and Jones (1994)
, Simmons and McKee (1992)
, and Burke et al. (1974)
. In addition, ethoxyfluorocoumarin-O-dealkylase (EFCOD) activity providing a net activity of several MFOs, including CYP2E1, CYP1A and CYP2B was measured using the fluorometric method outlined by DeLuca et al.(1988)
and Buters et al.(1993)
in the one-week study. CYP2E1 activity was measured as para-nitrophenol (p-NPH) hydroxylase activity using the spectrophotometric method outlined by Reinke and Moyer (1985)
, and UGT activity was measured using a modification of the spectrophotometric method of Stewart and McCrary (1987)
. All assays were modified for use in a 96-well format.
Statistical analysis.
All parameters examined statistically were first tested for equality of variance using Bartletts test (p = 0.01, Winer, 1971). If the results from Bartletts test were significant, then the data for the parameter were subjected to a transformation to obtain equality of variances. In the one-week study, final body weight, organ weight (absolute and relative), clinical chemistry parameters, LI, AI, and enzyme assay data were evaluated using a 2-way ANOVA with the factors of sex and dose. If the sex-dose interaction was significant, a one-way analysis was done separately for each sex. If the dose effect was significant, comparisons of individual dose groups to the control group were made with Dunnetts tests (Winer, 1971
). In the four-week study, exploratory data analysis was performed by a parametric or nonparametric ANOVA (Hollander and Wolfe, 1973
). If significant, the ANOVA was followed by Dunnetts test or the Wilcoxon rank-sum test (Hollander and Wolfe, 1973
) with a Bonferroni correction (Miller, 1966) for multiple comparisons to the control.
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RESULTS |
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Rat kidney.
No exposure-related clinical observations were noted in exposed groups of rats in either study. Rats exposed to 750 ppm EB weighed slightly less than controls; however, differences were statistically identified for both sexes only on study-day 8, and for males on study-day 27 and at four-week necropsy (Table 2). There were no effects from EB exposure that were considered to be of toxicological significance for any of the serum enzymes or analytes measured (data not shown).
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Analysis of apoptotic activity in kidneys of control and high-dose-group rats from the one-week study revealed a large degree of variability (data not shown), depending upon sex and kidney area examined. Mean AI values generally ranged from 0.17 to 0.37, with coefficients of variability generally >70%.
There were relatively minimal changes in renal enzyme activities of exposed rats in both studies relative to controls. Para-NPH activity in males, PROD activity in females, and UGT activity in both sexes of rats exposed to 750 ppm for one week were increased 89, 71, and 2930% of control levels, respectively (Fig. 5). A minimal, yet statistically identified 23% increase in the activity of p-NPH in females was also noted. The activities of renal PROD in male rats and EFCOD in both sexes of rats were below the detection limits of the assays utilized. There were no changes in enzyme activity levels of male or female rats exposed to 75 ppm EB for one week. Following four-week exposures, most enzyme activities were similar or somewhat lower than control values, with the only statistically identified changes being an approximate 30% decrease in the activities of MROD and PROD in exposed females (Fig. 5
).
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There were no gross pathologic effects identified at necropsy related to the inhalation exposure of mice to up to 750 ppm EB in either study. After both exposure periods, liver weights were increased for male and female mice exposed to 750 ppm EB relative to controls (Tables 4 and 5). Increases in absolute and relative liver weights ranged from approximately 612% and 1316% for males and females, respectively. Liver weight was not affected in mice of either sex exposed to 75 ppm EB for one week. Likewise, there was no effect of EB exposure upon lung weights in either sex of mouse at either necropsy time point
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Evaluation of apoptosis in liver and lungs from control and high-exposure groups from the one-week study revealed only infrequent apoptotic cells, usually 0 to 2 cells per anatomic region. The resulting AI calculated for either tissue ranged from only 0.06 to 0.25%, dependent upon site and sex (data not shown). Interanimal variability was high and no differences were statistically identified; however, AI levels by inspection were generally higher in lungs of exposed males relative to controls (means of 0.19 vs. 0.06).
Evaluation of enzyme activities revealed a number of treatment-related changes. In the mouse liver, following one week of EB exposure, EROD activity, reflecting primarily CYP1A subfamily activity, was minimally elevated 4060% in high-exposure-group males and females relative to controls (Fig. 7
). In male mice, similar increases were also noted in PROD activity, reflecting CYP2B subfamily activity, and in EFCOD activity, reflecting mixed CYP2E1, CYP1A, and CYP2B subfamily activities. The lack of change in the activity of hepatic p-NPH in male mice, a more specific measure of CYP2E1 activity, suggested that increased EFCOD activity reflected increases in CYP1A and CYP2B rather than in CYP2E1. The small increase in PROD activity of female mice, though statistically identified, was considered inconsequential. Treatment-related alterations of EFCOD and p-NPH were not observed in female mouse liver nor was altered UGT activity noted in the liver of any exposed mouse. At 75 ppm EB, the PROD and EFCOD activities were slightly, but statistically significantly, decreased for both sexes of mice.
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In the mouse lung, following one week of EB exposure, the in vitro activities of several MFO enzymes were decreased in a dose-related manner relative to controls (Fig. 8). The activities of EROD, PROD, and EFCOD were decreased 1733% in males and females inhaling 75 ppm EB and 2545% in both sexes of mice inhaling 750 ppm EB. No significant net changes in in vitro pulmonary p-NPH activity were observed in treated animals. After four weeks exposure, lung metabolic enzymes of males and females differed in their response to inhaled EB. In males, the activities of p-NPH and UGT were statistically identified as increased 73 and 51%, respectively, relative to controls. In females, the activities of EROD, MROD, and PROD were statistically identified as decreased 3350%.
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DISCUSSION |
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In male rats, exposure to a tumorigenic level of 750 ppm EB vapor appears to result in an initial 2u-globulin accumulation early in the exposure period, which may exacerbate EB-induced acceleration of chronic progressive nephropathy (CPN) typically observed in rats. This was evidenced by increased focal deposition of H&E staining hyaline droplets and
2u-globulin in the proximal tubule epithelial cells of the cortex and accompanying increased regenerative cell proliferation during the first week of exposure. Early changes were followed by a diminution in
2u-globulin deposition yet continued elevation of S-phase DNA synthesis and histopathological changes, suggesting CPN and a more chronic regenerative cell proliferation. This is consistent with the conclusions of Hard (2002)
who, upon extensive evaluation of renal histopathological changes occurring in EB-treated rats from NTP-sponsored studies, attributed tumor development to accelerated CPN. Both
2u-globulin nephropathy and CPN associated increases in cell turnover are recognized kidney tumor risk factor (Baetcke et al., 1991
; Hard, 1998
; Hard et al., 1997
; Swenberg et al., 1989
). The modest induction of MFO and UGT activities in males accompanying these changes, primarily following one-week exposure, were consistent with previous findings in rat kidney (Elovaara et al. (1985)
. and suggest an adaptive response to the metabolic load of EB and its metabolites.
In contrast to males, female rat kidneys did not display significant histopathological changes nor increased S-phase DNA synthesis. Indeed, a nearly 50% decrease in S-phase synthesis was noted following one-week exposure with no discernable change in apoptotic rates. Combined with the observed decreases, albeit minimal, in activities of all MFOs measured following four weeks exposure these data suggest an alteration or loss of MFO competent cells in female kidney with increasing exposure period. This change may serve to accelerate development of CPN at a level that does not elicit significant morphological changes nor measurable elevations in S-phase DNA synthesis over the time periods examined. It is noteworthy that in the NTP bioassay (NTP, 1999) kidney histopathology and tumor yield were less pronounced in females than males exposed to 750 ppm. Lack of treatment-related changes in the kidneys of either sex of rats exposed 75 ppm correlated to a lack of tumorigenic activity (NTP, 1999
).
Exposure of mice to 750 ppm EB vapor also caused sustained increases in the levels of cell proliferation as evidenced by increases in mitotic figures and S-phase DNA synthesis, indicating increased cell proliferation following both exposure periods. A greater incidence of mitotic figures was observed in females than males, consistent with the occurrence of liver tumors in this sex. Levels of S-phase synthesis were also higher in females than males at every location and exposure period; however, relative differences were not discernable, due to the unusually high background levels in controls relative to that measured in male controls (LI of 414% versus 13%, respectively). Significantly, a regiospecificity of increases in S-phase synthesis in both sexes was apparent with the greatest response in centrilobular hepatocytes. Chronic increase in cell proliferation has been identified as a critical step in numerous nongenotoxic mechanisms of tumorigenesis (as reviewed by Ashby et al., 1994; and Butterworth and Slaga, 1991
).
Consistent with their greater increase in liver weights, exposed female mice displayed greater induction, albeit still minimal in degree, of several MFOs and/or UGT than was observed in males. The known regiospecificity of these MFO enzymes within the liver lobule (Anderson et al., 1997; Leiber, 1997
; Omiecinski et al., 1990
) correlated well with the regional specificity of hypertrophic changes and increased cell proliferation in these mice, suggesting generation of a more toxic metabolite. Two of the enzymes affected in females, albeit to a minimal degree, CYP2E1 (measured as p-NPH), and UGT are responsible for the further metabolism of a metabolite of EB, 1-phenylethanol (Bestervelt et al., 1994
; Engstrom, 1984
). It is important to note that chronic induction of several MFOs, including those measured here, have also been associated with liver tumor formation in rodents (Grasso and Hinton, 1991).
Lungs of male and female mice exposed to 750 ppm EB vapor for one or four weeks displayed evidence of alterations in cell populations, despite a lack of histopathological changes, including at the ultrastructural level. Increases in epithelial S-phase DNA synthesis were accompanied, in both sexes of mice exposed for one week, by a possible increase in apoptosis in males and losses in EROD and PROD activities in both sexes. Loss of EFCOD activity (3536%) following one-week exposure likely also reflected the loss of CYP1A and 2B activities reflected in the EROD and PROD measurements, respectively. Following a four-week exposure, MFO activity appeared to have rebounded in male lung, even increasing significantly in the case of p-NPH, but remained depressed in females. In contrast, UGT activity was increased 51% over control levels in males following four weeks of exposure. These data, in toto, suggest an alteration in the metabolic potential or loss of Clara cells, the primary MFO-rich cells of the bronchiolar epithelium (Boyd, 1977; LaKritz et al., 1996
) early during EB exposure, followed by a more chronic, low-level turnover of cells. The lack of decreased activities of p-NPH, indicative of CYP2E1 activity, and UGT in both sexes of mice reflects the selective nature of the alteration or survival of a different, metabolically active subpopulation of cells. The low-level, yet persistent, changes may account for the lack of morphologically discernible changes over the exposure period employed. Chronic increases in S-phase DNA synthesis, combined with altered lung cell subpopulations and the genetic predisposition of male B6C3F1 mice to develop lung tumors relative to female B6C3F1 mice (Harling et al., 1996
; NTP, 1999
; Seilkop, 1995
; Ward et al., 1979
), suggest a plausible mechanism of tumor formation in EB-exposed males. Indeed, a reevaluation of mouse lung tissues from the NTP bioassay of EB has revealed the presence of multifocal occurrences of bronchiolar/parabronchiolar hyperplasia and altered tinctorial properties in mice chronically exposed to a tumorigenic level of EB (Brown, 2000
). These changes were similar to those observed in mice chronically exposed to styrene (Cruzan et al., 2001
) for which a selective metabolism/altered cell population of lung epithelium has been identified as responsible for lung tumor formation with this compound (Cruzan et al., 2002
). Significantly, human and rat lungs contain many fewer Clara cells than mice, suggesting a similar species-specific sensitivity (Plopper et al., 1980
).
We concluded that exposure to high levels of EB vapor can cause changes in male rat kidneys and mouse liver and lungs that, when combined with a general lack of EB genotoxicity, indicate a nongenotoxic mode of tumorigenic action dependent upon cell proliferation and altered cell population dynamics. Increases in regenerative cell proliferation in kidneys of male rats resulted from deposition of 2u-globulin, and in turn, accelerated CPN; both have been associated with tumor development. A similar response was not discerned in female rats under the conditions of the studies, suggesting that significant changes may appear only following longer-term exposure. In mouse liver, regiospecific hepatocellular hypertrophy, increases in mitotic figures and S-phase synthesis, and metabolic adaptation indicate formation of a toxic metabolite and subsequent regenerative cell proliferation. In mouse lungs, increases in S-phase DNA synthesis and loss/renewal of metabolic capacity in bronchiolar epithelium indicate a population shift, likely in Clara cells, again suggesting formation of a toxic metabolite and regenerative cell proliferation. Significantly, few changes were observed in these organs of rats or mice exposed to a nontumorigenic exposure level of 75 ppm EB vapor for one week.
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ACKNOWLEDGMENTS |
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NOTES |
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