Evaluation of Potential Modes of Action of Inhaled Ethylbenzene in Rats and Mice

William T. Stott*,1, Keith A. Johnson*, Rainer Bahnemann{dagger}, Susan J. Day* and Randal J. McGuirk*

* Toxicology & Environmental Research and Consulting, Building 1803, The Dow Chemical Company, Midland, Michigan 48674; and {dagger} BASF AG, Carl Bosch Strasse 38, DUP/PC-Z 470, 67056 Ludwigshafen, Germany

Received May 28, 2002; accepted October 4, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Potential factors underlying the tumorigenic activity of ethylbenzene (EB) were examined in F344 rats and B6C3F1 mice inhaling 750 ppm EB vapor 6 h/day, 5 days/week, for one or four weeks. Target tissues (kidneys of rats and livers and lungs of mice) were evaluated for changes in organ weights, mixed function oxygenases (MFO), glucuronosyl transferase activities, S-phase DNA synthesis, apoptosis, {alpha}2u-globulin deposition, and histopathology. In male rats, kidney weight increases were accompanied by focal increases in hyaline droplets, {alpha}2u-globulin, degeneration, and S-phase synthesis in proximal tubules. In female rats, only decreased S-phase synthesis and MFO activities occurred. In mice, increased liver weights were accompanied by hepatocellular hypertrophy, mitotic figures, S-phase synthesis, and enzyme activities. S-phase synthesis rates in terminal bronchiolar epithelium were elevated and accompanied by loss of MFO activity. Exposure to a nontumorigenic level of 75 ppm for one week caused few changes. These data, considered with the general lack of EB genotoxicity, suggest a mode of tumorigenesis dependent upon increased cell proliferation and altered population dynamics in male rat kidney and mouse liver and lungs. A similar response in the kidneys of female rats appears to require a longer exposure period than was employed.

Key Words: ethylbenzene; inhalation exposure; tumorigenesis; mode of action; target tissues; S-phase DNA.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ethylbenzene (EB) is largely used for the production of styrene at the site of manufacture in closed systems, but it may also be found in hydrocarbon solvents (e.g., mixed xylenes), gasoline, and tobacco smoke. Tang et al. (2000)Go estimated exposure of the general populace to be approximately 1.8 µg/kg/day, with up to 99% coming via inhalation exposure and only 0.01 to 0.03 µg/kg/day from consumption of food packaged in materials having low residual levels of EB.

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, 1992Go). 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, 1999Go). 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, 2000Go; and NTP, 1999Go). 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)Go 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)Go in rats exposed to 300 ppm EB for 3 days while Fuciarelli (2000)Go reported that hepatic cytochrome P450 levels were increased in mice, especially females, when exposed to 750 ppm EB for 1–12 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Study design.
Two studies of differing exposure duration involving both sexes of rats and mice were undertaken, one for five days (one-week study) and one for four weeks (four-week study). Animals were divided into two subgroups within each study, one designated for cell dynamic and histopathology measurements and one for enzyme activity measurements. Cell dynamics and histopathology subgroups consisted of 6 rats and 6 mice/sex/exposure level from the one-week study and 8 rats and 8 mice/sex/exposure level from the four-week study. Enzyme activity subgroups consisted of 6 rats and 30 mice/sex/exposure level from the one-week study and 8 rats and 40 mice/sex/exposure level from the four-week study. Male and female Fischer 344 rats and B6C3F1 mice were exposed to 0, 75, or 750 ppm EB vapor 6 h/day for five consecutive days (one-week study) or to 0 or 750 ppm under the same regimen for four weeks (four-week study). General health and body weight data were collected during the exposure period. The cell dynamic and histopathology subgroup animals had miniosmotic pumps implanted for additional dosing of bromodeoxyuridine (BrdU) during the entire exposure period (one-week study) or the fourth week of exposure (four-week study). Following exposure, blood samples were collected from the latter subgroups of each study for serum-chemistry evaluations. All animals were then euthanized and necropsied, and rat kidneys and mouse liver and lungs were excised from 6–8 animals/sex/group. For enzyme subgroups, lungs from all surviving mice were pooled (4–5/sex/group). Antemortem data collected from one or both groups of animals in each study included selected serum chemistry parameters, organ weights, gross pathologic and histopathologic changes, S-phase DNA synthesis levels, {alpha}2u-globulin deposition (male rats only), and the in vitro activities of selected MFOs and UGT. Due to large interanimal variability, apoptosis measurements in all tissues were undertaken only in the one-week study.

Test material and exposures.
Ethylbenzene (99.9–100% 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., 1980Go) 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 10–12 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 7–8 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 {gamma}-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 6–8 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)Go. Animals (6–8/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)Go 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.

{alpha}2u-globulin.
Deposition of {alpha}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 {alpha}2u-globulin followed by biotinylated antimouse antibody (secondary antibody) and alkaline phosphatase/strepavidin avidin-biotinylated horseradish peroxidase complex. {alpha}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)Go at 0–5°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)Go, Simmons and McKee (1992)Go, and Burke et al. (1974)Go. 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)Go and Buters et al.(1993)Go 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)Go, and UGT activity was measured using a modification of the spectrophotometric method of Stewart and McCrary (1987)Go. 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 Bartlett’s test (p = 0.01, Winer, 1971Go). If the results from Bartlett’s 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 Dunnett’s tests (Winer, 1971Go). In the four-week study, exploratory data analysis was performed by a parametric or nonparametric ANOVA (Hollander and Wolfe, 1973Go). If significant, the ANOVA was followed by Dunnett’s test or the Wilcoxon rank-sum test (Hollander and Wolfe, 1973Go) with a Bonferroni correction (Miller, 1966) for multiple comparisons to the control.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposures.
All exposures and environmental conditions were within acceptable limits. In the one-week study, the mean concentrations for the 0-, 75-, and 750-ppm exposure groups were 0.0, 75.2 ± 0.3 ppm and 738.6 ± 56.0 ppm, respectively. Variability in the high-exposure level resulted from a pump malfunction in a single 30-min period during which the concentration fell to approximately 138 ppm, and then rebounded to 825 ppm for approximately 15 min. In the four-week study, the mean concentrations of EB vapors for the 0 and 750 ppm exposure groups were 0 and 761 ± 14 ppm, respectively.

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 2Go). 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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TABLE 2 Treatment-related Effects in F344 Rats in the Four-Week Study
 
The kidney weights of males and females exposed to 750 ppm EB were slightly increased approximately 5–8% following both exposure periods (Tables 1 and 2GoGo). The kidney weights of rats of either sex exposed to 75 ppm EB in the one-week study were not affected (Table 1Go).


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TABLE 1 Treatment-Related Effects in F344 Rats in the One-Week Study
 
Histopathological examination of rat kidneys revealed an increase in number and size of hyaline droplets that occurred in cells of the proximal convoluted tubules (PCT) of males exposed to 750 ppm EB relative to controls in the one-week study (Table 3Go, Fig. 1Go). Droplets were distinct, homogeneous, membrane-bound, and brightly eosinophilic structures that in some cases appeared to fill much of the cytoplasm. Associated minor degeneration of PCT was noted, with the associated epithelial cells sometimes having pyknotic nuclei or being sloughed into the tubule lumen. Ultrastructurally, the major alteration in the PCT of these animals involved the phagolysosomes, the principal storage site of {alpha}2u-globulin. These organelles had much greater variability in size than those of controls, with some being larger than the nucleus. Smaller phagolysosomes of exposed males were of irregular shape, often with a pale indentation, while others, particularly the largest ones, had linear sides suggestive of paracrystalline structure. Males exposed to 75 ppm EB had only an equivocal increase in hyaline droplets in the PCT.


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TABLE 3 Histopathologic Effects in Kidneys of F344 Rats Exposed to Ethylbenzene Vapor in the One- and Four-Week Studies
 


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FIG. 1. Photomicrographs of kidneys from male rats exposed to 0 (A) or 750 ppm EB vapor (B) for one week. The proximal convoluted tubular epithelium of exposed rats has more numerous and larger hyaline droplets (arrows) than controls. (H&E stained).

 
Following four weeks of exposure to 750 ppm EB, male rats had a subtle renal lesion described as nephropathy, which was characterized by nuclear-size and -staining variations and vacuolation or a decreased amount of cytoplasm (Table 3Go; Fig. 2Go). This effect was present in multiple foci that were located primarily in the midcortical region, similar to the site and distribution of the areas where hyaline droplet accumulation is typically present in F344 male rats. Hyaline droplets were prominent in the kidneys of males but no treatment-related accumulation was evident upon examination of either H&E-stained tissue or of autofluorescence upon evaluation by fluorescence microscopy. In contrast, no treatment-related renal changes were identified in females exposed to 750 ppm EB in either study (Tables 1–3GoGoGo).



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FIG. 2. Photomicrographs of kidneys from a male rat exposed to 0 (A) or 750 ppm EB vapor (B) for four weeks. The proximal convoluted tubular epithelium of exposed rats has variable cell and nuclear size (some larger nuclei are indicated by arrows) and the cytoplasm is not as uniformly eosinophilic. A mitotic figure is also present (*); H&E stained.

 
Evaluation of S-phase DNA synthesis and {alpha}2u-globulin deposition in high-exposure group males of both studies revealed localized focal effects in the cortical tubular epithelium, relative to controls (Tables 1 and 2GoGo). Significantly, changes coincided with foci of increased hyaline-droplet deposition and tubular epithelial degeneration (Figs. 1–4GoGoGoGo). Analysis of cortical "hot spots" revealed a 41% and 79% greater LI than controls in one- and four-week studies, respectively. Cortical "hot spots" also contained approximately 160% and 66% more {alpha}2u-globulin than controls following exposure for one week (Table 1Go, Fig. 4Go) and four weeks (Table 2Go), respectively. In contrast to cortical areas, BrdU labeled cells of the outer stripe of the outer medulla were uniformly distributed and displayed no evidence of a treatment-related change. In addition, the inner medulla had LI values of only 0.33 and 0.65% for control and exposed males, respectively (data not shown), which were not statistically identified as different. There was no effect upon cortical cell S-phase synthesis in males exposed to 75 ppm EB in the one-week study.



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FIG. 3. Photomicrographs of kidneys from a male rat exposed to 0 (A) or 750 ppm EB vapor (B) for four weeks and immunohistochemically stained for S-phase DNA synthesis. Labeled nuclei are not uniformly distributed in the cortex but focal areas with labeled nuclei are larger and more frequent in exposed rats.

 


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FIG. 4. Photomicrographs of kidneys from a male rat exposed to 0 (upper panel) or 750 ppm EB vapor (lower panel) for one week and immunohistochemically stained for {alpha}2u-globulin. Exposed rats have greater numbers of affected cells, particularly in the subcapsular area, and the size of stained droplets is larger.

 
The rate of S-phase DNA synthesis in the kidneys of female rats was lower than in males, and labeled cells appeared randomly distributed in all areas examined. In the one-week study, a nearly 50% decrease in LI was observed in cortical tubular epithelium of the kidneys of high-exposure-group females (Table 1Go). No effects upon S-phase synthesis were noted in other sites. In the four-week study, no change in S-phase synthesis was observed in cortical tubular epithelium of exposed females; however, a minimal (28%), yet statistically identified, decrease was noted in synthesis rate in the outer stripe of the outer medulla (Table 2Go).

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 29–30% of control levels, respectively (Fig. 5Go). 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. 5Go).



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FIG. 5. Rat kidney mixed function oxygenase (EROD, MROD, PROD, pNPH, EFCOD) and glucuronosyl transferase (UGT) activities following one or four weeks of exposure to EB vapor (% control calculated from means of 6–8 animals/sex/group). *Significantly different from controls by Dunnett’s test, p < 0.05.

 
Mouse liver and lung.
While mice generally tolerated exposure to EB well, some spontaneous deaths occurred early in each study. In the one-week study, three control females and two 75-ppm-group males died while seven females from the 750 ppm EB exposure group died in the four-week study, all within the first few days of exposures. These animals were diagnosed as cachexic, apparently a result of poor adaptation to the inhalation-chamber environment. There were no statistically identified differences in the body weights of any exposed groups relative to controls, nor were there any treatment-related changes in serum enzymes or analytes measured (data not shown).

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 5GoGo). Increases in absolute and relative liver weights ranged from approximately 6–12% and 13–16% 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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TABLE 4 Treatment-related Effects in B6C3F1 Mice in the One-week Study
 

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TABLE 5 Treatment-related Effects in B6C3F1 Mice in the Four-week Study
 
Histopathological examination of livers and lungs of mice exposed to 750 ppm EB revealed contrasting morphologies. Increased numbers of mitotic figures were observed in liver of a majority of exposed males and females (>6 per lobe), most in the midzonal to centrilobular areas, in both studies (Table 6Go). More mitoses were present in liver of control and 75 ppm EB exposure group females than males. In contrast, there were no histopathologic effects related to EB exposure noted in the lungs of mice of either sex or study. Additional evaluation of mouse liver and lung tissue using electron microscopy revealed no significant treatment-related changes at an ultrastructural level.


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TABLE 6 Histopathologic Effects in Liver of B6C3F1 Mice in the One- and Four-week Studies
 
The relative rate of S-phase synthesis in the liver of male mice of the one-week study exposed to 750 ppm EB progressively increased across the liver lobule from a 180% increase in periportal hepatocytes to a 479% increase in midzonal hepatocytes to a 1116% increase in centrilobular hepatocytes (Table 4Go). The differential anatomic effect was still evident, though less pronounced, after four weeks of EB exposure (Table 5Go). A similar regional progression was evident in high-exposure-group females from the one-week study (Table 4Go, Fig. 6Go). Although females had higher LI levels in all hepatic zones than males, the relative increase was less due to the much greater control LI levels. A relatively high LI was again observed in females in the four-week study (Table 5Go); however, a relatively high degree of interanimal variability confounded interpretation of the data. A maximal 56% increase in LI was noted in centrilobular hepatocytes of female mice exposed to 750 ppm EB. Only minimal, nonsignificant, changes in LI were noted in either sex of mice exposed to 75 ppm EB for 5 days.



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FIG. 6. Photomicrographs of livers from female mice exposed to 0 (A) or 750 ppm EB vapor (B) for one week and immunohistochemically stained for BrdU incorporation. Labeled nuclei are most numerous in the midzonal area of control mice and are markedly increased in both the centrilobular and midzonal areas of exposed mice. Portal areas (p) and central veins (c) are indicated.

 
In the lungs of mice exposed to 750 ppm EB, there appeared to be a consistent increase in BrdU-labeled cells in the smaller airways (terminal bronchioles) relative to controls. In the one-week study, LI were increased 180% and 149% over controls in males and females, respectively. After four weeks of exposure, the LI of high-exposure-group males and females was increased 82% and 115% over controls, respectively, although neither was statistically identified. There appeared to be no consistent effect upon S-phase synthesis in the alveoli. A statistically identified, 38% decrease in the S-phase synthesis rate of alveolar epithelium was found in the EB-exposed males after four weeks; however alveolar cell LI was not affected either after one week nor for exposed females at either time point.

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 ~40–60% in high-exposure-group males and females relative to controls (Fig. 7Go). 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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FIG. 7. Mouse liver mixed function oxygenase (EROD, MROD, PROD, pNPH, EFCOD) and glucuronosyl transferase (UGT) activities after one or four weeks of exposure to EB vapor (% control calculated from means of 6–8 animals/sex/group). *Significantly different from controls by Dunnett’s test; p < 0.05.

 
Following four weeks exposure to 750 ppm EB, liver PROD activity remained statistically identified as increased in EB-exposed males (81%) and females (130%) relative to controls. In females, EROD and UGT activities were also statistically identified as increased: 61 and 31%, respectively. Minimal 24–27% increases in the mean activities of p-NPH were also noted in both sexes. The latter changes were not statistically identified as different from controls.

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. 8Go). The activities of EROD, PROD, and EFCOD were decreased 17–33% in males and females inhaling 75 ppm EB and 25–45% 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 33–50%.



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FIG. 8. Mouse lung mixed function oxygenase (EROD, MROD, PROD, pNPH, EFCOD) and glucuronosyl transferase (UGT) activities after one or four weeks exposure to EB vapor (% control calculated from means of 6–8 groups, each containing 4–5 pooled lungs/sex/group). *Significantly different from controls by Dunnett’s test, p < 0.05.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure of male and female Fisher 344 rats and B6C3F1 mice to EB vapors for approximately one or four weeks resulted in a number of treatment-related changes in the kidneys of rats and the liver and lungs of mice. Several of these changes have been associated with nongenotoxic modes of tumorigenesis that, significantly, are reversible upon cessation of dosing and display clear thresholds.

In male rats, exposure to a tumorigenic level of 750 ppm EB vapor appears to result in an initial {alpha}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 {alpha}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 {alpha}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)Go who, upon extensive evaluation of renal histopathological changes occurring in EB-treated rats from NTP-sponsored studies, attributed tumor development to accelerated CPN. Both {alpha}2u-globulin nephropathy and CPN associated increases in cell turnover are recognized kidney tumor risk factor (Baetcke et al., 1991Go; Hard, 1998Go; Hard et al., 1997Go; Swenberg et al., 1989Go). 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)Go. 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, 1999Go) 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, 1999Go).

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 4–14% versus 1–3%, 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., 1994Go; and Butterworth and Slaga, 1991Go).

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., 1997Go; Leiber, 1997Go; Omiecinski et al., 1990Go) 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., 1994Go; Engstrom, 1984Go). 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 (35–36%) 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, 1977Go; LaKritz et al., 1996Go) 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., 1996Go; NTP, 1999Go; Seilkop, 1995Go; Ward et al., 1979Go), 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, 2000Go). These changes were similar to those observed in mice chronically exposed to styrene (Cruzan et al., 2001Go) 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., 2002Go). Significantly, human and rat lungs contain many fewer Clara cells than mice, suggesting a similar species-specific sensitivity (Plopper et al., 1980Go).

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 {alpha}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.


    ACKNOWLEDGMENTS
 
These studies were sponsored by the Styrenics Steering Committee, a sector group of the European Chemical Industry Council (Brussels, Belgium). We thank K. A. Jackson and V. A. Linscombe of Toxicology Environmental Research and Consulting, The Dow Chemical Company, Midland, MI for their expert technical assistance.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (989) 638-9863. E-mail: wtstott{at}dow.com. Back


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