Toxicology 206 (2005) 383–388

Comparison of the depletion of glutathione in mouse liver and lungfollowing administration of styrene and its metabolites styrene

oxide and 4-vinylphenol

Meredith Turner, Nancy A. Mantick, Gary P. Carlson∗

School of Health Sciences, Purdue University, West Lafayette, IN 47907, USA

Received 2 June 2004; received in revised form 14 July 2004; accepted 22 July 2004Available online 8 September 2004

Abstract

Styrene is hepatotoxic and pneumotoxic in mice. Its major metabolite styrene oxide and its minor, but potent, metabo-lite 4-vinylphenol cause similar toxicities. Styrene and styrene oxide cause decreases in reduced glutathione levels in tis-sues. The current studies examined styrene and styrene oxide in a time and dose-dependent manner and 4-vinylphenol ina time dependent fashion. Styrene (600 mg/kg, 5.8 mmol/kg ip) caused decreased GSH levels in both liver and lung withinone hour. A maximum was seen at three hours with return to control levels by 12 h. Lower doses also caused changesin a dose-dependent fashion. For styrene oxide, similar findings were observed with a dose of 300 mg/kg (2.5 mmol/kg).GSH levels in liver, but not lung, returned to control by 6 h. Again a dose response was found for both tissues. While 4-v umotoxict styrene ands est the pos-s in theset©

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inylphenol (100 mg/kg, 0.83 mmol/kg) was administered at a dose known to be more hepatotoxic and more pnehan styrene or styrene oxide and it caused decreased GSH levels, the degree of depletion was less compared totyrene oxide. In general the lung was more affected by these agents than was liver. The decreases in GSH suggibility that the toxicity of styrene in lung and liver may be related to a profound but reversible oxidative stressissues.

2004 Elsevier Ireland Ltd. All rights reserved.

eywords:Styrene; Styrene oxide; 4-Vinylphenol; Glutathione

. Introduction

Styrene is widely utilized for a number of purposesith significant human exposure possible, especially

∗ Corresponding author. Tel.: +765 494 1412; fax: +765 494 1414.E-mail address:gcarlson@purdue.edu (G.P. Carlson).

for workers in the reinforced plastics industry (Milleret al., 1994; Cohen et al., 2002). In rodents, styrencauses both liver and lung damage with the mobeing a more sensitive species than the rat (Morganet al., 1993a, 1993b; Gadberry et al., 1996; Cruet al., 1997). The greatest concern, however, is whestyrene causes cancer in humans. IARC (2002) cl

300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.tox.2004.07.013

384 M. Turner et al. / Toxicology 206 (2005) 383–388

fies it as a possible human carcinogen. It causes lungtumors in mice (Cruzan et al., 2001) but not in rats(Cruzan et al., 1998).

Styrene oxide formed from styrene may be detoxi-fied via microsomal epoxide hydrolase to give styreneglycol, which is further metabolized to mandelic acidand phenylglyoxylic acid (Bond, 1989; Sumner andFennell, 1994). Particularly in rats and mice, glu-tathione conjugation of styrene oxide also occurs. Fol-lowing inhalation exposure approximately 60% of themetabolites recovered from rodents are formed via theepoxide and hydrolysis pathway, and about 40% arevia glutathione conjugates.

It is not entirely clear how styrene causes toxicityin the liver and lung. One possibility is through ox-idative stress. Styrene administration has been shownto decrease glutathione levels in liver.Vainio andMakinen (1977)administered ip doses of styrene rang-ing from 150 to 1000 mg/kg to hamsters, guinea pigs,rats and mice, and found dose-dependent decreasesin nonprotein sulfhydryl content (primarily GSH) 3 hlater. The mouse appeared to be the most sensitivespecies.Srivastava et al. (1983)administered single ipdoses of styrene ranging from 50 to 600 mg/kg to ratsand found a dose-dependent decrease in GSH threehours later.Beiswanger et al. (1993)injected rats ipwith styrene oxide (250 mg/kg) and found a decreasein hepatic GSH after 2 h, which rebounded to controllevel by 8 h. They found a similar pattern in the cere-bellum, cerebral cortex, and hippocampus.

ep-a )a , 450a asei entsws p tor useds t 2 ha 0 h.

e orie ione e-c e inl velsrp k for

2 weeks and found significant decreases in non-proteinsulfhydryl content immediately after the last exposure.They also found that single ip injections of styrene(400 mg/kg) or styrene oxide (200 mg/kg) resulted insignificant decreases after 2 h. Interestingly, they foundno changes when rats were given up to 400 mg/kg/dayfor 3 days and measurements were made 24 h afterthe last dose. They also found decreases in pulmonary(−40%) and hepatic (−35%) nonprotein sulfhydrylcontent immediately following inhalation of 300 ppmstyrene, 5 days per week, for 2 weeks (Coccini et al.,1998). Other studies have demonstrated that mice aremore sensitive than rats to pulmonary GSH depletionwith mice showing a significant decline when exposedby inhalation to 80 ppm styrene 6 h/day for two dayswith rats requiring 300 ppm to obtain a significant de-crease (Filser et al., 2002; Csanady et al., 2003).

The toxicity of styrene is generally associated withits biotransformation to styrene oxide (Bond, 1989).However, another metabolite of interest is the ring hy-droxylated metabolite 4-vinylphenol, which has beenfound in the urine of both rats (Bakke and Scheline,1970; Pantarotto et al., 1978) and humans (Pfaffliet al., 1981; Manini et al., 2003). Although only a smallpercentage of styrene forms this metabolite, it is muchmore potent as both a hepatotoxicant and pneumotox-icant than is styrene or styrene oxide (Carlson et al.,2002; Vogie et al., 2004). There is no published infor-mation available on the metabolism of 4-vinylphenolalthough it has been hypothesized that it may involveap dosed f re-d unga litess

2

2

aso H).S mi-c A)w %)a re

A number of studies suggest that recovery of htic glutathione occurs fairly rapidly.Das et al. (1981dministered styrene orally to rats at doses of 270nd 900 mg/kg daily for 7 days and found a decre

n GSH only at the highest dose when measuremere made 24 h later.Katoh et al. (1989)administeredtyrene (300 mg/kg) or styrene oxide (300 mg/kg) iats 3 times per week for 1 week. Both chemicals caignificant decreases in hepatic GSH and GSSG after administration with recovery between 10 and 2

Few studies have examined the effect of styrents metabolites on glutathione content in lung.Elovaarat al. (1990)exposed rats in a single 24 h inhalatxposure to 500 cm3/m3 styrene and found a 66% drease of GSH in lung and only a 16% decreasiver one hour after exposure. Tissue glutathione leeturned to normal by 24 h.Coccini et al. (1997)ex-osed rats to 300 ppm styrene 6 h/day, 5 days/wee

ring-opened pathway (Boogaard et al., 2000). Theurpose of the current study was to determine theependence and time course for the depletion ouced glutathione by styrene in mouse liver and lnd compare it to the effects of the two metabotyrene oxide and 4-vinylphenol.

. Materials and methods

.1. Animals and chemicals

4-VP (10% in propylene glycol; purity >95%) wbtained from Lancaster Synthesis (Windham, Ntyrene and styrene oxide were from Aldrich Cheal Co. (Milwaukee, WI). Trichloroacetic acid (TCas from Fisher (Fair Lawn, NJ). Glutathione (>98nd 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) we

M. Turner et al. / Toxicology 206 (2005) 383–388 385

from Sigma Chemical Co. (St. Louis, MO). All otherchemicals were reagent grade or better.

CD-1 [Crl:CD-1 (ICR) BR] mice were obtainedfrom Charles River Laboratories (Wilmington, MA).They were housed in group cages in environmentallycontrolled rooms on a 12 h light:12 h dark cycle. Ro-dent laboratory chow (No. 5001, Purina Mills, Inc., St.Louis, MO) and tap water were allowed ad libitum. Allanimals were allowed a minimum of one week to adaptto the animal facilities and diet before being used in anyexperiment.

2.2. Experimental design

Groups of 4 to 6 male mice (22–28 grams) wereadministered styrene (600 mg/kg, 5.8 mmol/kg) incorn oil or corn oil as control vehicle ip. To determinethe time course for GSH depletion, they were sacrificedat 1, 3, 6, 12 or 24 h. For examining the effect of dose,mice were treated with 0, 200, 400 or 600 mg/kg (0,1.9, 3.8 or 5.8 mmol/kg) ip styrene and sacrificed after3 h. Similar experiments were carried out with styreneoxide. It was administered ip at a dose of 300 mg/kg(2.5 mmol/kg), and measurements were made at 1, 2,6 and 12 h after dosing. For the dose response study,the doses selected were 0, 75, 150 and 300 mg/kg(0.63, 1.25, and 2.5 mmol/kg), and the time was 2 h.For 4-vinylphenol, which is water soluble, the vehiclewas saline. The mice were administered 100 mg/kg ipand were sacrificed after 1, 2 or 6 h. Control animalsr ionsw tiona dyw

2

to0 e re-m atesw l and3 ateb alfv geda int y wast -t tion

of DTNB in 0.1 M potassium phosphate buffer giv-ing a final concentration of 10 mM DTNB. Sampleswere mixed by vortexing, and the absorbance was de-termined at 464 nm. A standard curve was generatedranging from 0 to 625 nmol GSH and was used to cal-culate the tissue GSH levels.

2.4. Statistical analysis

Values are expressed as mean± S.E. The numbersof animals in each group are indicated in the tables.In comparing between two groups, Student’st test wasused. In comparing multiple values, an ANOVA wasutilized followed by Student Newman–Keuls’ test todetect differences among the groups. In each case thelevel of significance selected wasP < 0.05.

3. Results

When styrene was administered to mice at the rela-tively high dose of 600 mg/kg ip, there was a very rapidand dramatic decrease in reduced glutathione levels inboth liver and lung by one hour (Table 1). The greatesteffect was observed after three hours, and there wassome recovery evident by 6 h. By 12 h, there was nodifference between the control and treated groups foreither liver or lung.

To determine the dose response relationship be-tween styrene and reduced glutathione levels, groups ofm renei e ofm slys cantdt evelsi

abo-l oser tabo-l ide( reasei nda ar roupt ass elsr

eceived saline. In all cases, the dosing solutere prepared immediately before administrand were given in a volume of 1 ml/100 g boeight).

.3. Assay for reduced glutathione

Entire lungs and livers (approximately 0.20.28 grams and 1.2 to 1.9 grams, respectively) weroved and weighed, and deproteinized homogenere prepared by homogenizing the tissues in 2 mml, respectively of cold 0.1 M potassium phosphuffer, pH 7.5, followed by the addition of one-holume of 10% TCA. Homogenates were centrifut 10,000× g in a refrigerated centrifuge for 5 m

o prepare the supernatants for assay. The assahat ofSpeisky et al. (1986, 1988). Briefly, the deproeinized supernatants were combined with a solu

ice were administered 200, 400 or 600 mg/kg styp, and measurements were made after 3 h, the tim

aximum effect in the 600 mg/kg study. As previouhown, the highest dose of styrene caused signifiecreases in GSH in both liver and lung (Table 2). The

wo lower doses also caused decreases in GSH ln both tissues in a dose-dependent manner.

Since styrene oxide is considered to be the metite responsible for its hepatotoxicity, time and desponse relationships were evaluated for this meite. As with the parent compound, styrene ox300 mg/kg ip) also caused a rapid and severe decn GSH levels with significant effects noted by 1 h amaximum effect at 2 h (Table 3). For liver, there was

ecovery of the GSH concentration in the treated go control levels by 6 h, but significant reduction wtill present in the lung at that time. Lung GSH leveturned to normal by 12 h.

386 M. Turner et al. / Toxicology 206 (2005) 383–388

Table 1Glutathione depletion in CD-1 mouse liver and lung following styrenea administration

Hours Lung Liver

No. Control No. Styrene No. Control No. Styrene

Glutathione concentrationb

1 4 9.64± 0.41 5 2.84± 0.37c 4 3.07± 0.06 5 2.34± 0.11c

3 5 8.13± 0.29 5 1.80± 0.18c 5 1.69± 0.07 5 0.66± 0.03c

6 5 6.08± 0.45 6 2.30± 0.22c 5 2.73± 0.15 6 1.81± 0.08c

12 5 6.81± 0.42 5 7.06± 0.54 5 4.71± 0.24 5 4.90± 0.2424 5 8.81± 0.40 6 7.98± 0.60 5 2.48± 0.11 6 3.05± 0.13c

a 600 mg/kg, ip.b �mol/g liver or lung; value is mean± S.E.c Significantly different from control,P < 0.05.

Table 2Dose response for glutathione depletion by styrenea in the CD-1mouse

Dose Number Liver Lung

Glutathione concentrationb

Control 5 7.05± 0.38 a 2.01± 0.07 a200 mg/kg 5 4.85± 0.28 b 1.23± 0.10 b400 mg/kg 5 2.30± 0.33 c 0.48± 0.05 c600 mg/kg 5 1.51± 0.34 c 0.51± 0.05 c

Within each column, values with different letters (a–c) are signifi-cantly different from each other,P < 0.05.

a Administered ip to groups of 5 mice 3 h prior to termination.b �mol/g liver or lung; value is mean± S.E.

In the dose response studies, styrene oxide at300 mg/kg ip produced the expected response (Table 4).When the dose was decreased to 150 mg/kg there werestill significant differences between treated and con-trol groups in both liver and lung. However, when thelowest dose of 75 mg/kg was administered there wasonly a decrease in lung GSH with liver GSH being nodifferent from control.

Table 3Glutathione depletion in CD-1 mouse liver and lung following styrene oxidea administration

Hours Liver Lung

No. Control No. Styrene oxide No. Control No. Styrene oxide

Glutathione concentrationb

1 4 8.64± 0.67 4 1.21± 0.16c 4 1.56± 0.22 4 0.65± 0.32c

2 4 7.77± 0.55 4 1.76± 0.12c 4 1.40± 1.00 4 06 8 6.35± 0.90 8 4.85± 0.53 4 1.41± 0.07 4 0.51± 0.10c

12 4 6.02± 1.16 3 5.48± 0.31 4 2.67± 0.14 3 2.35± 0.11a 300 mg/kg, ip.b �mol/g liver or lung; value is mean± S.E.c Significantly different from control,P < 0.05.

Table 4Dose response for glutathione depletion by styrene oxidea in theCD-1 mouse

Dose Number Liver Lung

Glutathione concentrationb

Control 4 5.70± 0.39 a 0.92 + 0.13 a75 mg/kg 4 5.02± 0.85 a 0.68± 0.09 a150 mg/kg 4 4.20± 0.43 a 0.32± 0.11 b300 mg/kg 4 1.07± 0.13 b 0 c

Within each column, values with different letters (a–c) are signifi-cantly different from each other,P < 0.05.

a Administered ip to groups of 4 mice 2 h prior to termination.b �mol/g liver or lung; value is mean± S.E.

Since the styrene metabolite 4-vinylphenol is muchmore potent in producing liver and lung toxicity thanis either styrene or styrene oxide (Carlson et al., 2002;Vogie et al., 2004), it was administered to mice at a doseof 100 mg/kg ip, which is known to be both more hepa-totoxic and pneumotoxic than styrene or styrene oxide.Although there were significant decreases in hepaticGSH levels at 1 and 2 h, these were very minimal com-

M. Turner et al. / Toxicology 206 (2005) 383–388 387

Table 5Glutathione depletion in CD-1 mouse liver and lung following 4-vinylphenola administration

Hours Liver Lung

No. Control No. 4-VP No. Control No. 4-VP

Glutathione concentrationb

1 4 9.48± 0.88 4 6.26± 0.28c 4 0.92± 0.09 4 0.61± 0.112 4 9.97± 0.25 4 8.81± 0.19c 4 2.37± 0.19 4 1.86± 0.03c

6 4 4.33± 0.34 4 5.62± 0.28c 4 0.95± 0.38 4 1.33± 0.41a 100 mg/kg, ip.b �mol/g liver or lung; value is mean± S.E.c Significantly different from control,P < 0.05.

pared to styrene and styrene oxide (Table 5). SimilarlyGSH depletion in lung was observed, but again this wasless than that observed with styrene and styrene oxide.Higher doses could not be used because they are lethal.

4. Discussion

Styrene has been shown in a number of studies tocause a decrease in reduced glutathione levels, possi-bly resulting in oxidative induced stress in both liverand lung (Vainio and Makinen, 1977; Srivastava et al.,1983; Beiswanger et al., 1993). This may be related toa major portion of their metabolism through conjuga-tion with glutathione. Interestingly, the effect appearsto occur very rapidly but with a return of GSH to con-trol levels between 12 and 24 h (Katoh et al., 1989;Beiswanger et al., 1993). This could explain why (Daset al., 1981) found that 900 mg/kg caused GSH deple-tion in liver at 24 h but not with lower doses.

Of particular interest is the lung as a potential tar-get tissue, especially since it is the site of tumors inmice (Cruzan et al., 2001). Limited studies have alsoindicated that glutathione levels are also decreased inthis organ in rats (Elovaara et al., 1990; Coccini et al.,1997) and mice (Filser et al., 2002; Csanady et al.,2003).

The current studies support the results of the previ-ous studies in that styrene administration caused a dose-d ffectw ationw hortl ox-i issuesw ed,e oses

than with the parent compound. In both cases the de-gree of GSH depletion and the length of time it wasdecreased were greater in lung than in liver. This find-ing is in agreement with the studies ofElovaara et al.(1990) who exposed rats to a single 24 h inhalationexposure of 500 cm3/m3 styrene and found a 66% de-crease in GSH in lung and only a 16% decrease in liverone hour following exposure.

4-Vinylphenol is a minor metabolite of styrene andyet it is an order of magnitude more potent than styreneor styrene oxide in producing pneumotoxic and hep-atotoxic effects in mice (Carlson et al., 2002; Vogieet al., 2004). However, while 4-vinylphenol did causesignificant decreases in GSH in both liver and lung,these changes were small in comparison to styreneand styrene oxide. The dose could not be increaseddue to lethality at higher doses. The metabolic path-way for 4-vinylphenol is unknown although it has beensuggested that it may be via ring opening (Boogaardet al., 2000). As such it may not involve conjugationwith GSH leading to depletion and thus may act viaanother mechanism.

The role of the decrease in GSH in causing the toxic-ity to both the liver and lung is as yet unclear. However,the data suggest that a very profound but transient ef-fect occurs. Now that the current studies have identifiedtime and dose dependence, further studies are neededto characterize this oxidative stress in both tissues in-cluding an evaluation of the enzymes, which controlGSH levels in these tissues.

A

theS

ependent decrease in GSH in both tissues. The eas observed as early as one hour after administrith a peak effect observed at 3 h. This effect was s

ived with a return to control levels by 12 h. Styrenede also gave a dose-dependent response in both tith a return to control by 12 h. As might be expectffects due to styrene oxide were noted at lower d

cknowledgment

This work was supported in part by a gift fromtyrene Information and Research Center.

388 M. Turner et al. / Toxicology 206 (2005) 383–388

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