FUNDAMENTAL AND APPLIED TOXICOLOGY 39, 89–100 (1997)ARTICLE NO. FA972362

Toxicity of Divinylbenzene-55 for B6C3F1 Micein a Two-Week Inhalation Study

Daniel L. Morgan,* Joel F. Mahler,* Ralph E. Wilson,* Michael P. Moorman,*Herman C. Price, Jr.,† and Robert W. O’Connor†

*National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709;and †Man Tech Environmental Technology, Inc., Research Triangle Park, North Carolina 27709

Received February 10, 1997; accepted August 12, 1997

DVB a highly reactive crosslinking agent and may also con-Toxicity of Divinylbenzene-55 for B6C3F1 Mice in a Two-Week tribute to its toxicity. Occupational exposure to DVB occurs

Inhalation Study. Morgan, D. L., Mahler, J. F., Wilson, R. E.,primarily by inhalation and dermal contact and consequently

Moorman, M. P., Price, H. C., Jr., and O’Connor, R. W. (1997).DVB is an irritant to the eyes and respiratory system (Clay-Fundam. Appl. Toxicol. 39, 89–100.ton and Clayton, 1981). The current Occupational Safety

Divinylbenzene (DVB) is a crosslinking monomer used primar- and Health Administration threshold limit value (8-hr time-ily for copolymerization with styrene to produce ion-exchange weighted average) for DVB is 10 ppm (ACGIH, 1994).resins. The toxicity of inhaled DVB was investigated because of Divinylbenzene monomer is manufactured by dehydroge-the potential for worker exposure and the structural similarity of

nation of mixed isomeric diethylbenzenes. After removal ofDVB to styrene, a potential carcinogen. Male and female B6C3F1light by-products, the product is recovered as a mixture ofmice were exposed to 0, 25, 50, or 75 ppm DVB for 6 hr/day, 5m- and p-DVB and m- and p-ethylvinylbenzene (EVB), thedays/week for up to 2 weeks. Six mice/sex/dose group were killedpartial dehydrogenation product. Because the DVB mono-after 3, 5, and 10 exposures and six mice/sex in the 75 ppm groupmer readily polymerizes to a brittle, insoluble resin, it iswere killed 7 days after 10 exposures. The most severe effects

occurred in the nasal cavity and liver, with less severe effects heavily inhibited with tert-butyl catechol and diluted withoccurring in the kidneys. In the nasal cavity olfactory epithelium EVB to minimize this reaction. Three commercial grades ofacute necrosis and inflammation were present at early time points DVB are produced containing approximately 22% (DVB-followed by regeneration, architectural reorganization, and focal 22), 55% (DVB-55), and 80% (DVB-80) DVB.respiratory metaplasia by 7 days after the last exposure. Olfactory

By far, the greatest use of DVB is as a cross-linkingepithelial changes were concentration-dependent with extensivemonomer for copolymerization with styrene, or with acrylicinvolvement at 75 ppm and peripheral sparing at 25 ppm. Thereand methacrylic acids to produce ion-exchange resins usedwas also necrosis and regeneration of olfactory-associated Bow-in water treatment and in the chemical and pharmaceuticalman’s glands as well as the lateral nasal (Steno’s) glands. Hepato-industries (Coulter and Kehde, 1970; Kirk-Othmer, 1981,cellular centrilobular (CL) necrosis was observed only in the 75

ppm dose group and was similar to that caused by styrene. A 1983). Copolymerization with styrene results in resins withtime-dependent progression was observed, characterized by CL reduced solubility in most solvents, increased heat-distortiondegeneration after 1 exposure, necrosis after 3 and 5 exposures, temperatures, increased surface hardness, and improved im-and chronic inflammation with CL karyomegaly after 10 expo- pact and tensile strengths (Kirk-Othmer, 1983). Divinylben-sures and 7 days after the 10th exposure. Hepatic GSH levels were

zene is also used in styrene-butadiene rubber to improve thedecreased in a dose-dependent manner. In the kidneys, transient

swelling, shrinkage, and extrusion properties of the producttubular damage was observed in some male mice exposed to 75(Kirk-Othmer, 1983). The DVB monomer has been used asppm, and appeared to be a response to DVB-induced tubulara sustained release agent, as a dental filling component, andepithelial injury.as an insecticide stabilizer (Clayton and Clayton, 1981).

Divinylbenzene is structurally similar to styrene (Fig. 1)and is likely biotransformed by the same metabolic pathways.Divinylbenzene (DVB; CAS No. 1321-74-0) is a specialtyStyrene is metabolized in animals and humans by cytochromemonomer used for producing vinyl polymers. The toxicityP450 to styrene-7,8-epoxide, a direct-acting carcinogenof inhaled DVB was investigated because of the potential(IARC, 1987), and because DVB is likely oxidized to a simi-for worker exposure and the structural similarity of DVB tolar epoxide or diepoxide, there is concern about the potentialstyrene, a potential human carcinogen (IARC, 1987). The

bifunctionality conferred by the two vinyl groups makes carcinogenicity of this chemical. Because DVB has two reac-

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TABLE 1Relative Liver Weights of B6C3F1 Mice Exposed

to 75 ppm Divinylbenzene

DVB Exposure daysconcentration

(ppm) 1 3 5 10

MalesFIG. 1. Chemical structures of meta- and para-divinylbenzene (m-, p-0 4.84 { 0.36 4.98 { 0.33 4.87 { 0.24 4.51 { 0.40DVB), meta- and para-ethylvinylbenzene (m-, p-EVB), and styrene.

75 5.18 { 0.4 5.77 { 0.65* 6.90 { 0.61* 4.90 { 0.32Females

0 5.05 { 0.66 5.06 { 0.54 5.01 { 0.38 5.09 { 0.2875 5.69 { 0.46 6.56 { 0.67* 6.73 { 0.43* 5.23 { 0.19tive vinyl groups it may be metabolized to a toxic epoxide

more readily than styrene. In addition, commercial formula-Note. Male and female B6C3F1 mice were exposed to 75 ppm DVB ortions of DVB contain a significant amount of EVB (Fig. 1) air (controls) 6 hr/day, 5 days/week for 2 weeks. Relative liver weights (%

which could also be metabolized to a reactive epoxide. body weight) were significantly increased after three and five exposures toThere have been no carcinogenicity studies and few muta- DVB-55. Values represent means { SD, six mice/group.

* Significant greater than air-exposed controls (p õ 0.05).genicity studies conducted on DVB. Zeiger et al. (1987)reported that DVB-55 was negative for mutagenicity in Sal-monella. In an initial inhalation study, DVB-55 was found

Inhalation exposure. Liquid DVB-55 was vaporized in the exposureto be a weak genotoxicant in B6C3F1 mice, causing a dose-chamber air supply, and the vapor was mixed with conditioned air (HEPA-dependent increase in sister chromatid exchange, with a lessfiltered, charcoal-scrubbed, temperature- and humidity-controlled) and de-

pronounced increased frequency of chromosomal aberrations livered to the Hazleton 1000 exposure chambers (Lab Products, Maywood,in splenocytes and micronuclei in polychromatic erythro- NJ) at nominal concentrations of 0, 0.25, 0.5, and 0.75 mg/L DVB-55

(equivalent to 0, 25, 50, or 75 ppm DVB and 0, 20, 40, or 60 ppm EVB).cytes (Kligerman et al., 1996). In unpublished reportsChamber concentrations of DVB were measured at 30-min intervals by gas(Nitchke et al., 1984, 1986), inhalation of DVB-55 was re-chromatography with flame ionization detection. Actual mean DVB cham-ported to be toxic to the kidneys, liver, and olfactory epithe-

lium in rats and mice. The objectives of the current studywere to compare the toxicity of DVB-55 to that of styrene

TABLE 2under similar experimental conditions, and to better charac-Summary of Major Nasal Cavity Effects in Miceterize the inhalation toxicity of DVB-55 for B6C3F1 mice.

Exposed to DVB-55The B6C3F1 mouse was used in these studies because thisspecies/strain was previously found to be a sensitive model DVB concentration (ppm)for investigating the hepatotoxicity caused by styrene (Mor-

Nasal cavity lesion 0 25 50 75gan et al., 1993a,b,c). In an initial range-finding study (un-published data), male mice were exposed to 0, 50, or 100

5 Exposuresppm DVB (0, 0.5, or 1.0 mg/L DVB-55) for 6 hr/day. AllOlfactory epithelium necrosis/mice in the 100 ppm dose group were found dead or were

degeneration 0 / // ///killed in moribund condition after one exposure. MarkedLateral gland necrosis 0 0 // ///degeneration/necrosis was present in the livers of all sched-

uled sacrifice mice, and slightly less extensive changes were10 Exposures

present in the moribund mice. No deaths occurred at 50Olfactory epitheliumppm. For this reason mice were exposed to 0, 25, 50, or 75

Degeneration 0 / / ///ppm DVB in this 2-week study.Respiratory metaplasia 0 // // /

MATERIALS AND METHODS 10 Exposures / 7 days recovery

Olfactory epitheliumChemical. Divinylbenzene-55 is a major commercial formulation ofDVB, and was utilized in this study. Divinylbenzene is synthesized from Respiratory metaplasia 0 NE NE ///mixed isomers of ethylbenzene, and the product is recovered as a mixtureof m- and p-DVB, and m- and p-EVB. Divinylbenzene-55 (Aldrich Chemi- Note. Nasal cavity lesions in male and female (combined) B6C3F1 mice

after exposure to DVB-55 6 hr/day, days/week for up to 2 weeks. Six mice/cal Co., Milwaukee, WI) consists of approximately 55% DVB (16% paraand 39% meta isomers), 43% EVB (11% para and 32% meta isomers), sex were necropsied 7 days after the last exposure to evaluate the fate of

the lesions. Relative severity scores; 0 (not present) to ///; NE, notand small amounts of unreacted diethylbenzene. The inhibitor 4-tert-butyl-catechol was present at 1.08 mg/ml. examined.

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FIG. 2. Olfactory region of an air-exposed control mouse (A) and a mouse exposed to 75 ppm DVB for 5 days (B). In the control animal, the thickdark-staining olfactory epithelium covers the nasal septum (S), dorsal meatus (M), and turbinates (T). In the DVB-exposed animal there is diffuse lossof olfactory cells, and cellular debris and exudate in the lumen (arrows). 271.

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FIG. 3. Higher magnification of the dorsal meatus from a control mouse (A) and a mouse exposed to 75 ppm DVB for 5 days (B). In the controlanimal, note the tall olfactory epithelium (E) and the underlying lamina propria containing Bowman’s glands (B) and olfactory nerve bundles (N). Inthe DVB-exposed mouse the epithelium is diminished to two or three layers of flattened cells and there is loss of Bowman’s gland cells. Note the celldebris in the lumen. 1601.

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FIG. 4. Steno’s glands of an air-exposed control mouse (A) and a mouse exposed to 75 ppm DVB for 5 days (B). The normal glands surround themaxillary sinus (*) and consist of tightly packed acini and ducts. In the DVB-exposed animal there is coagulative necrosis of the acini leaving onlygranular cell debris with interspersed ducts. 1201.

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FIG. 5. Olfactory epithelium from a mouse exposed to 25 ppm DVB for 5 days. There is degeneration and necrotic cell debris in the dorsal meatus(M) but the epithelium of the more peripheral turbinate (T) is intact. 1001.

ber concentrations were within {10% of the target concentrations for the obtained (after removal of gall bladders). Sections of the left hepatic lobes(2–3 mm in thickness) were removed, fixed in 10% neutral-buffered forma-2-week study (27 { 4, 54 { 2, 75 { 7 ppm DVB).lin, embedded in paraffin, sectioned, and stained with hematoxylin andAnimals. Six-week-old male B6C3F1 mice (Charles River, Portage,eosin. Slides were evaluated by light microscopy. After 5 and 10 exposures,MI) were acclimated to laboratory conditions for 2 weeks prior to the startand 7 days after the last exposure (75 ppm dose group only), samples ofof the study. During acclimation, mice were weighed and randomized tospleen, kidney, and lung were collected and immersion-fixed in 10% neu-treatment groups. Mice were acclimated to the Hazleton inhalation exposuretral-buffered formalin. Following removal of the brain, the nasal cavity waschambers for 3 days prior to exposure. Animals were fed NIH 07 diet andflushed with fixative through the nasopharyngeal duct and the skulls werechlorinated, filtered, UV-disinfected tap water was provided ad libitum.immersion-fixed. After fixation, skulls were decalcified and three transverseFood was removed during the exposures. Mice were housed individuallysections of the anterior, middle, and posterior nasal cavity were obtained.in the Hazleton 1000 chambers and exposed to nominal concentrations ofAll tissue samples were paraffin-embedded, sectioned at 6 mm, stained with25, 50, or 75 ppm DVB for 6 hr/day (approximately 7 AM to 1 PM), for uphematoxylin and eosin, and examined by light microscopy.to 5 days/week for 2 weeks. Control animals breathed conditioned air. All

Glutathione analysis. Immediately after obtaining blood samples formice were observed twice a day for clinical signs of toxicity. Groups ofclinical chemistry and liver sections for histopathology, the remaining liversix mice/sex/dose group were killed after 1, 3, 5, or 10 exposures and antissues from six mice/sex/dose/time point were frozen at 0707C until ana-additional group of six mice/sex in the 75 ppm dose group was killed 7lyzed for glutathione (GSH). Kidneys were collected from mice after 1, 3,days after the 10th exposure to examine the potential reversibility of DVB5, and 10 exposures and stored at 0707C until analyzed for GSH. Totaltoxicity.GSH was measured in liver and kidney samples by the GSH recyclingClinical chemistry. Immediately after 1, 3, 5, or 10 exposures, six mice/method of Griffith (1980). Animals were consistently euthanized and tissuessex/strain/dose group were weighed, then anesthetized by CO2:O2 (70:30)collected at the same time of day to avoid differences in tissue GSH dueinhalation. Blood was collected by cardiac puncture, transferred to serumto diurnal changes.separation vials devoid of anticoagulant, allowed to clot for about 15 min,

Statistics. Statistically significant differences between means were de-and then centrifuged at 3000 rpm for 10 min at 57C. The serum samplestermined by one-way analyses of variance and Dunnett’s multiple compari-were stored at 0707C until all samples had been collected. Samples wereson test (Sokal and Rohlf, 1969).then analyzed for alanine aminotransferase (ALT) and sorbitol dehydroge-

nase (SDH), creatinine, and blood urea nitrogen (BUN) using an automated RESULTSanalyzer (Monarch System 2000, Instrumentation Laboratory, Lexington,

MortalityMA) and commercially available reagents.After two exposures 5/30 males and 1/30 females in theHistopathology. After collection of blood for clinical chemistry, six

mice/sex/strain/dose group/time point were necropsied and liver weights 75 ppm dose group died or were euthanized in moribund

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FIG. 6. Olfactory epithelium from a mouse exposed to 75 ppm DVB for 10 days and allowed to recover for 7 days. Note the metaplastic ciliatedcells on the surface (arrow) and nests of regenerating Bowman’s gland cells (*) in the lamina propria. 4001.

condition. One male was euthanized in moribund condition Body and Organ Weightsafter one exposure, and 1 female and 4 males were either

Divinylbenzene exposure had no significant effect onfound dead or euthanized in moribund condition after twoexposures. body weights of male or female mice throughout the study

TABLE 3Incidence and Severity of Liver Lesions in BC3F1 Mice Exposed to 75 ppm Divinylbenzene

Exposure days

Liver lesion 1 3 5 10 10 / recovery

MalesDegeneration 6/6a (2.2)b

Necrosis 6/6 (2.3) 6/6 (2.5) 1/6 (1.0)Chronic inflammation 5/6 (1.8) 2/6 (1.5)Karyomegaly 6/6 (1.0) 6/6 (1.0)

FemalesDegeneration 6/6 (2.5)Necrosis 6/6 (2.7) 5/6 (1.8)Chronic inflammation 1/6 (2.0) 4/6 (1.7)Karyomegaly 6/6 (1.0) 6/6 (1.0)

Note. Liver lesions in male and female B6C3F1 mice after exposure to 75 ppm DVB 6 hr/day, 5 days/week for up to 2 weeks. Six mice/sex werenecropsied 7 days after the last exposure to evaluate the fate of the lesions.

a Incidence Å number affected/number examined.b Severity scores (in parentheses) are averages based on the number of animals with lesions; severity of necrosis was scored on a scale of 1 to 5 based

on the extent of involvement seen in a standard section from the left hepatic lobe.

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TABLE 4(data not shown). Relative liver weights of male and femaleKidney Lesions in B6C3F1 Mice Exposed to 75 ppmmice were significantly increased after 3 and 5 exposures to

Divinylbenzene75 ppm (Table 1). By day 10 liver weights were no longersignificantly greater than controls. Relative and absolute

Exposure daysspleen weights were significantly decreased in male and fe-Kidney

male mice in all dose groups after 5 exposures and after 10 lesions 5 10 10 / Recoveryexposures to 50 and 75 ppm (data not shown).

MalesRegeneration 4/6a (2.2)b 3/6 (1.3) 1/6 (1.0)HistopathologyCasts 2/6 (2.0) 0/6 0/6

FemalesTreatment-related changes were present in the nasal cav-Regeneration 1/6 (3.0) 0/6 0/6ity, liver, and kidneys. There were no remarkable findingsCasts 1/6 (3.0) 0/6 0/6in the spleen or lung of DVB-exposed mice at any time pointNecrosis 1/6 (3.0) 0/6 0/6

examined.Note. Kidney lesions in male and female B6C3F1 mice after exposureNasal cavity. A spectrum of time- and dose-dependent

to 75 ppm DVB 6 hr/day, 5 days/week for up to 2 weeks. Six mice/sexchanges was present in the nasal cavity of male and femalewere necropsied 7 days after exposure day 10 to evaluate the fate of themice affecting primarily the olfactory region and associated lesions.

glands (Table 2). The most severe and extensive changes a Incidence Å number affected/number examined.were seen in the 75 ppm dose group at the earliest time b Severity scores (in parentheses) are averages based on the number of

animals with lesions; severity of necrosis was scored on a scale of 1 to 5point examined (after five exposures). In these animals, mostbased on the extent of involvement seen in a standard section.of the olfactory epithelium was degenerative or necrotic (Fig.

2), with replacement of the normal epithelial architecture bya thin lining consisting of two to three layers of flattenedcells (Fig. 3). In the few areas where olfactory mucosa was tory cells by tall columnar ciliated cells, was evident in somestill recognizable, there was marked necrosis of single cells mice. Regeneration of Bowman’s glands was evidenced bywithin the epithelium. Exudate composed of serous fluid and scattered clusters of cells in the lamina propria and promi-necrotic cell debris was present in the lumen, usually in the nent duct openings in the epithelium. Regenerative hypercel-dorsal meatus and within the scrolls of the turbinates. lularity of the lateral nasal glands was also seen. At the lower

There was diffuse necrosis of Bowman’s glands character- concentrations, a morphologically normal neuroepitheliumized by loss of gland tissue and scattered pyknotic cells in with interspersed foci of respiratory metaplasia covered mostthe lamina propria (Fig. 3). In addition, necrosis of the lateral of the olfactory region after 10 exposures, with the exceptionnasal (Steno’s) glands was present and characterized by co- of the dorsal meatus, where a poorly organized epitheliumagulative change of the acini with sparing of the ducts (Fig. remained. Cytoplasmic hyaline change of sustentacular cells4). A concentration-dependent difference in nasal toxicity was frequently seen in peripheral areas.after five exposures was evidenced by less extensive necrosis In mice receiving 10 exposures to 75 ppm and allowedand degenerative change at the lower concentrations. In par- to recover for 7 days, the most noteworthy change of theticular, the more peripheral olfactory epithelium lining the olfactory mucosa was respiratory metaplasia, most consis-turbinates and lateral wall tended to be spared at lower con- tently seen in the dorsal meatus (Fig. 6). At this time pointcentrations, while the epithelium and Bowman’s glands of there was prominent regeneration of Bowman’s glands asthe dorsal meatus were consistently affected at all concentra- evidenced by clusters of cells in the lamina propria (Fig. 6).tions (Fig. 5). At 50 ppm, necrosis of the lateral nasal glands There were no appreciable effects on the respiratory epithe-was more focal and less severe than that at 75 ppm, and at

lium of the anterior and middle nasal cavity at any of the25 ppm no necrosis of this gland was evident.

concentrations or time points examined.After 10 exposures, degenerative changes of the olfactory

Liver. In the liver, chemical-related changes were ob-epithelium were still evident, although of much less severityserved in both sexes but only in the 75 ppm exposure groupand extent than after 5 exposures, suggesting that these(Table 3). A time-dependent progression was observed, char-changes were indicative of regenerative hyperplasia. In ani-acterized by centrilobular (CL) degeneration after 1 expo-mals exposed to 75 ppm, a thickened, multilayered epithe-sure, necrosis after 3 and 5 exposures, and chronic inflam-lium with variable degrees of architectural reorganizationmation and karyomegaly after the 10th exposure and up tohad been restored in most areas of the olfactory mucosa7 days after the last exposure to 75 ppm. Marked hepatocel-with more advanced restoration present in peripheral areaslular necrosis was found in all mice which died early, andcompared to the dorsal meatus. Focal respiratory metaplasia

of the epithelium, characterized by the replacement of olfac- was considered to be the cause of death.

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TABLE 6Kidneys. In the kidneys, tubular regeneration was pres-Serum Chemistry in Female B6C3F1 Mice Exposed to DVB-55ent in some mice, mostly males exposed to 75 ppm DVB

(Table 4). This change consisted of foci of tubules withDVBepithelial cell basophilia and karyomegaly. Rare mitotic fig-

concentration ALT SDH Creat Bunures were present in these foci. Many mice with regenerative (ppm) (IU/L) (IU/L) (mg/dL) (mg/dL)foci also had dilated tubules with protein casts. In males,

1 Exposurethe incidence and severity of regenerative change decreased0 19 { 2 19 { 1 0.5 { 0.1 17 { 1with time, suggesting a transient, acute tubular injury fol-

25 38 { 3* 22 { 1 0.5 { 0.1 17 { 1lowed by resolution. Only a single female examined after50 41 { 4* 25 { 1 0.6 { 0.1 20 { 1

five exposures had evidence of renal injury. 75 69 { 4* 50 { 5* 0.8 { 0.1* 23 { 1*3 Exposures

0 18 { 1 15 { 1 0.4 { 0.1 12 { 1Clinical Chemistry25 22 { 2 19 { 1 0.5 { 0.1 23 { 150 24 { 2 19 { 1 0.6 { 0.1* 26 { 1*

Serum ALT and SDH activity were significantly elevated 75 760 { 127* 1473 { 216* 0.7 { 0.1* 23 { 3in most dose groups after one exposure and in the 75 ppm 5 Exposures

0 22 { 3 21 { 3 0.4 { 0.1 16 { 1dose groups of male and female mice at most time points25 21 { 1 19 { 1 0.3 { 0.1 14 { 2(Tables 5 and 6). Serum enzyme activities in males and50 24 { 2 20 { 1 0.4 { 0.1 24 { 1*females were only slightly elevated when examined after the75 79 { 13 43 { 5 0.5 { 0.1* 28 { 4*

last exposure. Exposure to 75 ppm DVB caused significant 10 Exposuresincreases in serum creatinine in males and females at all 0 18 { 2 18 { 1 0.2 { 0.1 14 { 1

25 19 { 1 17 { 1 0.3 { 0.1 13 { 1time points. Slight, but statistically significant, increases in50 22 { 2 17 { 1 0.3 { 0.1* 14 { 1blood urea nitrogen levels were detected in both males and75 28 { 2* 23 { 1* 0.4 { 0.1* 22 { 1*females primarily in the 50 and 75 ppm dose groups.

Note. Female B6C3F1 mice were exposed to DVB-55 6 hr/day, 5 days/week for up to 2 weeks. Blood was collected and analyzed after 1, 3, 5,and 10 exposures. Values represent means { SEM; n Å 6.

TABLE 5 * Significantly greater than air-exposed controls (p õ 0.05).Serum Chemistry in Male B6C3F1 Mice Exposed to DVB-55

DVB Glutathioneconcentration ALT SDH Creat BUN

Hepatic glutathione levels were decreased in a dose-de-(ppm) (IU/L) (IU/L) (mg/dL) (mg/dL)pendent manner in male and female mice (Fig. 7). Depletion

1 Exposure of hepatic GSH appeared to decrease as the study progressed.0 23 { 2 20 { 1 0.5 { 0.1 19 { 1 There were no apparent gender differences in hepatic GSH

25 44 { 4* 33 { 2* 0.5 { 0.1 19 { 1levels. No significant chemical-related changes were ob-50 47 { 3* 32 { 2* 0.6 { 0.1 19 { 1served in kidney GSH levels (data not shown).75 62 { 4* 45 { 2* 0.7 { 0.1* 23 { 1

3 Exposures0 23 { 2 18 { 1 0.4 { 0.1 16 { 1 DISCUSSION

25 23 { 5 21 { 3 0.5 { 0.1 27 { 350 30 { 3 22 { 1 0.6 { 0.1* 31 { 2* Short-term inhalation studies were conducted to compare75 2083 { 883* 2180 { 458* 0.8 { 0.1* 30 { 7*

the toxicity of DVB-55 with that of styrene using a similar5 Exposuresstudy design. Divinylbenzene is structurally similar to sty-0 22 { 3 25 { 4 0.5 { 0.1 19 { 2

25 21 { 3 23 { 2 0.3 { 0.1 15 { 2 rene and was found to have similar toxicological properties.50 24 { 1 24 { 1 0.4 { 0.1 24 { 1 Both chemicals are significantly more toxic for mice than75 791 { 280* 590 { 177* 0.7 { 0.1* 26 { 1* rats (Nitchke et al., 1986; Roycroft et al., 1992), both cause

10 Exposuresnasal toxicity and hepatotoxicity in mice (Roycroft et al.,0 16 { 1 21 { 1 0.2 { 0.1 15 { 11992; Morgan et al., 1993a), and both have relatively steep25 26 { 3* 19 { 1 0.3 { 0.1 16 { 1

50 17 { 2 18 { 1 0.4 { 0.1* 21 { 2* dose–response curves (Morgan et al., 1993b, 1995). Divi-75 27 { 1* 25 { 2 0.5 { 0.1* 25 { 1* nylbenzene-55 appeared to be more acutely toxic than sty-

rene. Inhalation exposure of mice to 100 ppm DVB (1 mg/Note. Male B6CF1 mice were exposed to DVB-55 6 hr/day, 5 days/week

L of DVB-55) caused 100% mortality, whereas exposure offor up to 2 weeks. Blood was collected and analyzed after 1, 3, 5, and 10B6C3F1 mice to 200 ppm styrene was not lethal (Morganexposures. Values represent means { SEM; n Å 6.

* Significantly greater than air-exposed controls (p õ 0.05). et al., 1995). Because DVB-55 is a mixture of chemicals,

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ppm DVB, whereas these lesions were observed in miceexposed to 25 ppm DVB (Nitchke et al., 1986). Similarly,isoprene, an aliphatic vinyl compound, causes lesions in theolfactory epithelium after inhalation exposure (Melnick etal., 1990b). As with DVB and styrene, mice were consider-ably more susceptible to isoprene-induced nasal toxicity thanrats. However, inhalation exposure to similar concentrationsof the vinyl substituted compounds, a-methylstyrene (Mor-gan et al., unpublished data), 1,3-butadiene (Melnick et al.,1990a), and chloroprene (Melnick et al., 1996), did not causenasal cavity toxicity in B6C3F1 mice. Chloroprene, a struc-tural analogue of butadiene, did not cause nasal toxicityin mice; however, lesions of the olfactory epithelium wereobserved in rats (Melnick et al., 1996).FIG. 7. Hepatic glutathione (GSH) depletion in male (- - -) and female

The dorsal medial areas of the olfactory mucosa were(—) mice exposed to 25 (j), 50 (m), or 75 ppm (l) DVB-55 for 1, 3, 5,or 10 days. Values represent means of six mice/group and are expressed most affected by DVB inhalation and may reflect an airflow-as percentage of control. dependent component. The nasal lesions in DVB-exposed

mice were confined to the lamina propria of the olfactoryepithelium and the lateral nasal glands. This site-specific

the potential toxicity of the other constituents must be con-distribution of the lesions indicates that DVB undergoes site-

sidered in evaluating the toxicity of DVB-55. The DVB-55specific metabolism to a toxic metabolite. The olfactory epi-

used in this study contained a significant amount of EVBthelium of rodents has considerable metabolic capability

which may have contributed to the overall toxicity; 1 mg/L(Dahl and Hadley, 1991), thus in situ metabolism of DVB

of DVB-55 contains 79 ppm EVB in addition to 100 ppmand styrene to epoxides and the subsequent reaction with

DVB. Although published toxicity data for EVB could notessential macromolecules in the olfactory epithelium maybe found, the presence of a vinyl group on EVB suggestsbe the cause of the nasal toxicity of these vinyl compounds.that it may similarly be metabolized to a reactive epoxide.Similar site-specific metabolism and nasal toxicity of numer-The reactivity and toxicity of DVB and styrene can beous indirect-acting chemicals such as nitrosamines, dibasicattributed to oxidative metabolism of their vinyl substituents.esters, nitriles, acetaminophen, methyl bromide, 3-methylin-Oxidative metabolism of olefinic double bonds to the epox-dole, and 3-methylfuran has been attributed to a high P450ide by cytochrome P450 is recognized as a critical determi-enzyme activity in the olfactory epithelium (Gaskell, 1990).nant to the toxic responses and to the metabolic fate of vinylIn contrast, inhalation exposure to 1,2-epoxybutane, a direct-compounds. Styrene-7,8-epoxide is the reactive metaboliteacting, short-chain epoxide, caused inflammatory, degenera-of styrene (Nordqvist et al., 1985) and the correspondingtive, and proliferative lesions in both the respiratory and theepoxides of DVB are likely the toxic metabolites of thisolfactory epithelium of rats and mice (Dunnick et al., 1988).chemical. Divinylbenzene may be more reactive and toxic

Divinylbenzene-55 inhalation also caused hepatocellularthan styrene because it can potentially be metabolized toCL necrosis in mice similar to that caused by styrene. Theeither an epoxide or a diepoxide (meta and para). The pres-progression of this lesion was similar for both chemicalsence of two reactive epoxides per molecule may increasewith initial degeneration of CL cells followed by necrosis,the likelihood that DVB will react with essential cellularchronic inflammation, and karyomegaly. For both chemicalscomponents.regeneration and repair of hepatocellular necrosis occurredThe nasal cavity is a primary target site of both inhaledin spite of continued chemical exposure. In addition, severeDVB-55 and styrene, and mice were more susceptible tohepatocellular necrosis was present in mice found dead orthis lesion than rats. DVB-55 caused more severe nasal cav-in moribund condition after exposure to either DVB-55 ority toxicity than styrene and caused necrosis of the nasalstyrene, and was likely the cause of death. As in the olfactorycavity epithelium at the lowest concentration (25 ppm) usedepithelium, hepatotoxicity is likely a result of site-specificin this study. Styrene inhalation for up to 90 days causedmetabolism of DVB (and styrene) to reactive metabolites.mild degeneration of the olfactory epithelium of rats andLocalization of lesions in the CL region is indicative ofmice exposed to 1000 and 62.5 ppm, respectively (Roycrofttoxic metabolites produced by oxidative P450 metabolismet al., 1992). A no-effect level for styrene was not attainedof indirect-acting toxicants (Rappaport, 1979).for the nasal cavity toxicity in mice; 62.5 ppm was the lowest

Mice were more susceptible than rats to the hepatocellularconcentration used. Minimal to mild olfactory degenerationor necrosis was observed in rats exposed to 190 and 470 necrosis caused by DVB-55 inhalation. Hepatotoxicity was

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99INHALATION TOXICITY OF DVB-55 IN MICE

not observed in rats after inhalation exposure to up to 470 the Teflon bag. If this degradation was not detected, theanalytical instruments would have been standardized incor-ppm DVB for 2 weeks (Nitchke et al., 1986) or 1500 ppmrectly, resulting in an overestimation of the chamber concen-styrene for 13 weeks (Roycroft et al., 1992). This speciestrations. Thus, it is possible that the chamber concentrationsdifference in susceptibility to styrene has been attributed toreported by Nitchke et al. were actually lower than whatgreater epoxidase activity and less epoxide hydrolase activitywas measured by the analytical equipment.in mice relative to rats (Glatt and Oesch, 1987). Because

This study demonstrated that the nasal cavity, liver, andDVB is likely metabolized by the same pathways as styrene,kidneys are the target tissues of inhaled DVB-55 in B6C3F1the same species difference in susceptibility would be ex-mice. DVB-55 was toxic for mice at lower concentrationspected.than styrene, possibly because DVB is more readily metabo-Inhalation of DVB-55 caused a dose-dependent depletionlized to epoxides or forms a more reactive epoxide or diepox-of hepatic GSH in males and females mice, indicating theide, or possibly because of the toxicity contributed by otherformation and conjugation of reactive, electrophilic metabo-components of DVB-55. Additional studies are currentlylites. Styrene inhalation causes a similar dose-dependentunderway to investigate the metabolism and toxicokineticsdepletion of hepatic GSH in mice; this GSH depletion hasof inhaled DVB-55 in B6C3F1 mice and F344 rats.been attributed to glutathione-S-transferase-catalyzed and

nonenzymatic conjugation of GSH with styrene-7,8-oxideACKNOWLEDGMENTS(Morgan et al., 1993b). Styrene caused a greater hepatic

GSH depletion in female mice (Morgan et al., 1993b); how- Inhalation exposures were performed at the NIEHS inhalation facilityever, a gender difference in GSH depletion was not observed under contract to ManTech Environmental Technology, Inc. (Research Tri-

angle Park, NC). The authors acknowledge the technical support of D.after DVB-55 inhalation.Crawford, P. Dixon, N. Gage, L. Goods, J. Kinlaw-Parker, A. Miller, K.DVB-55 inhalation appeared to cause injury to the tubularPatrick, S. Philpot, J. Richards, P. Rydell, W. Stephens, P. Tedesco, andepithelium in the kidneys of mice. A transient tubular dam-T. Ward. The authors thank Dr. R. Melnick and Dr. M. Cunningham for

age was observed in some male mice in the 75 ppm dose critical review of the manuscript, and Dr. K. Morgan for consultations ongroup. Slight, but statistically significant, increases in blood nasal lesions.urea nitrogen were detected in male and female mice in the

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