Adaptive changes caused by intermittent styrene inhalation on xenobiotic biotransformation

TOXICOL.OGY

AND

APPLIED

PHARMACOLOGY

49,

7-14 (1979)

Adaptive Changes Caused by Intermittent Styrene Inhalation on Xenobiotic Biotransformation HARRI Department

VAINIO,JORMAJ~RVISALO, AND EEROTASKINEN

of Industrial

Hygiene and Toxicology, Institute SF-00290 Helsinki 29, Finland

Received March

20,1978;

of Occupational

Health,

accepted January 14,1979

Adaptive Changes Caused by Intermittent Styrene Inhalation on Xenobiotic Biotransformation. VAINIO, H., JKRVISALO, J., AND TASKINEN, E. (1979). Toxicol. Appf. Pharrnacof. 49, 7-14. Intermittent 11-week exposure to styrene by inhalation (300 ppm 6 hr/day, 5 days/week) enhanced the activities of both drug hydroxylating (ethoxycoumarin Odeethylase, cytochrome P-450) and conjugating (epoxide hydratase, UDP glucuronosyltransferase) enzymes in the liver and kidneys of adult male rats. The increases in drug monooxygenation reached a peak within 2 weeks, whereas enhancement in UDP glucuronosyltransferase activity (p-nitrophenol as the aglycone) was observed only after 6 weeks. A dose-dependent depression of glutathione occurred after Cdays of exposure when measured 0.5 hr subsequent to the last exposure. The following morning (18 hr after exposure), the hepatic glutathione concentrations were slightly increased in styrene-exposed animals. Degenerative morphologic alterations were also observed in the parenchymal cells of the liver already after 2 weeks exposure to 300 ppm.

In the present work we have studied the effects of short-term (2-11 weeks) styrene exposure on the drug metabolizing enzymes of rat liver, kidney,’ and lung. In a shorter experiment (4 days) we also measured the glutathione content to determine the doseeffect relationship between styrene exposure and hepatic nonprotein sulfhydryl content.

Styrene (vinyl benzene) is widely used in the production of plastics and resins (polyor styrenestyrene resins, copolymers) butadiene rubber. Many workers in the polyester plastic industry are exposed to styrene, mainly via the lungs, due to the evaporation of styrene into the air of the workroom. The metabolic pathways of styrene are well known, and they are similar in many species (Ohtsuji and Ikeda, 1971; Leibman, 1975; Parkki et al., 1976). The first step is the formation of the epoxide, styrene oxide (Leibman and Ortiz, 1969), which is then hydrated by epoxide hydratase to styrene glycol (Oesch, 1973) or conjugated with glutathione (James and White, 1967; Marniemi and Parkki, 1975). Styrene glycol can be further conjugated with glucuronic acid (El Masri et al., 1958) or converted to’mandelic acid (Ohtsuji and Ikeda, 1970).

METHODS Animals and exposures. Forty adult male Wistar rats were exposed to 300 ppti (7.9 pmol/liter) of styrene (obtained from Koch-Light Laboratories Ltd., Colnbrook, England) 6 hr daily, 5 days/week in a dynamic exposure chamber of 1 ma. Styrene used in our experiments contained 0.60 ppm (v/v) ethylbenzene, 0.75 ppm (v/v) a-methylstyrene, and 0.5 ppm (v/v) styrene oxide according to gas chromatographic analysis. Forty littermate control rats were exposed to circulating air in an identical chamber. 7

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8

VAINIO,

JiiRVISALO,

The animals were deprived of food during exposure, but drinking water was given ad libirum. Saturated styrene vapor was diluted with room air and the styrene concentration in the chamber air was continuously monitored with infrared analysis (wavelength 11.0 pm, slit 0.5 mm, path length 12.75 m) (Miran 1, Wilks Scientific Corp.) and regulated by Eurotherm 090 controller. The calibration was done with the closed loop calibration system (volume = 5.64 dm3, 7.9 ~1 liquid styrene). Rats were killed by decapitation on Day 5 of Week 2, 4, 6, 8, and II of the experiment, immediately after the daily exposure. The livers, lungs, and kidneys were excised. A small slice of the anterior lobe of the liver was fixed in 10% formaldehyde and was later used for conventional histological evaluation. The rest of the livers and other tissues were stored for 3-5 days at -70°C until the biochemical analysis was performed. The liver, kidneys, and lungs were homogenized in 4 vol of the 0.25 M sucrose solution with a glassTeflon Potter-Elvehjem homogenizer driven by an electric drill at 500 rpm. The homogenate was spun in a Sorvall RC-5 refrigerated centrifuge at 12000x g,,, for 10 min. The microsomes were isolated by the Ca2+-aggregation method spinning at 27OOOg,,, for 15 min to sediment the microsomal fraction, as described by Aitio and Vainio (1976). The pellet was resuspended in 0.15 M KCI and recentrifuged in a similar way, and the new pellet resuspended finally in 0.25 M sucrose. Protein was measured with the method of Lowry et al. (1951). When the effect of various air concentrations of styrene on free GSH in the liver was studied, rats were exposed to the respective styrene concentrations 6 hr daily for 4 days (from 8 AM till 2 PM). Thirty minutes after exposure on Day 4, four control and four styrene-exposed animals were killed by decapitation, and their livers were removed, placed into an ice bath, and stored at -70°C till the analysis. Another set of exposed and control animals was killed the next morning at 8 AM so that the reversibility of the changes could be determined. Enzyme assays. The content of cytochrome P-450 was determined from the carbon monoxide difference spectrum of dithionite-reduced microsomes recorded according to the method of Omura and Sato (1964). Microsomal ‘I-ethoxycoumarin O-deethylase activity was measured by a modified fluorometric method of Ullrich and Weber (1972). One hundred microliters of 1.5 mM 7-ethoxycoumarin in acetone was added into the assay tube and then vehicle was evaporated. The incubation mixture in a final volume of 0.5 ml consisted of 0.3 mM 7-ethoxycoumarin, 125 mM TrisHCI buffer (pH 7.4), 20 mtvt KC], 5 mM MgCl,, 0.005 mM MnCI,, and a NADPH-regenerating system (0.5 mM NADP+ disodium salt, 4.5 mM isocitric acid

AND

TASKINEN

trisodium salt, 0.0125 unit isocitrate dehydrogenase, EC 1.1.1.42). The enzyme reaction was started by adding microsomes equivalent to about 0.1 mg of microsomal protein, and stopped after 10 min at 37°C with 0.5 ml of 5% trichloroacetic acid. After mixing 4 ml 1.6 M NaOH-glycine buffer, pH 10.3, was added. The protein precipitate was spun down, and the fluorescence of 7-hydroxycoumarin and appropriate standards was determined at 390 nm for excitation and 460 nm for emission with a Perkin-Elmer Double Beam 512 spectrofluorometer. The 7-ethoxycoumarin synthesized by us (Ullrich and Weber,l972) melted at 8%89°C and yielded a single spot after paper chromatography using benzene/ethanol/acetic acid (96.5/3/0.5). Epoxide hydratase (EC 4.2.1.63) activity was determined by the radiometric assay of Oesch et al. (1971) with the following modifications (Vainio and Parkki, 1974). [7JH]Styrene oxide (code No. TRQ. 1107, Radiochemical Centre, Amersham, England) was pipetted in 25 ~1 of acetone into 1.0 ml of incubation mixture at a specific activity of 140 &i/ mmol and at a radioactive concentration of I2 &i/ml to start the reaction. Radioactivity was measured from an aliquot of the aqueous phase in a LKB Wallac 81000 liquid scintillation counter with external standardization method for quench correlations. UDP glucuronosyltransferase (EC 2.4. I. 17) was assayed with 0.35 mM p-nitrophenol as aglycone and 4.5 mM UDP glucuronate (ammonium salt) as described by Hlnninen (1968). The liver microsomes were treated with digitonin prior to the UDPglucuronosyltransferase measurement (Vainio, 1973); 1.0 ml of 1% aqueous digitonin (Merck AG, Darmstadt, Germany) was added to 0.5 ml of the microsomal suspension incubated for 30 min at O”C, spun as washing the microsomes, and resuspended in 1 ml of sucrose. Determination of the free glutathione in the tissue was performed by the method of Cohn and Lyle (1966) with slight modifications. The proteins of 20% liver homogenate were sedimented with 5 % trichloroacetic acid (final concentration). After centrifugation at 3000g for IO min, IOO-~1 aliquots of the supernatants were taken for the free GSH determination, as described by Hissin and Hilf (1976). The statistical treatment of the data was done with the Student’s I test.

RESULTS The amount of styrene inhaled by the rats during 6-hr daily exposure to 300 ppm styrene was estimated to be between 60 and 120 mg (Table 1). About 70-90x of this dose will be taken up (Astrand et al., 1974). The rats tolerated the dose well; only after the

9

STYRENE AND DRUG METABOLISM TABLE DOSE OF

ESTIMATED RAT

EXPOSED

1

STYRENE TO

TABLE

INHALED

300 ppm

IN

6

HR

BY A

EFFECT DAILY,

OF STYRENE

OF STYRENE 5 DAYS/WEEK)

RAT

65/min 24 ml 130-260 ml

Rat breathing rhythm Rat breathing capacity Air inhaled in 1 min Ambient air concentration of styrene In 1 min the rat will inhale In 6 hr the rat will inhale

1.26 mg/liter 0.160.32 mg styrene 60-120 mg styrene

did the animals tend to be slightly somnolent. The styrene exposure did not change the liver weights of the rats. Intermittent styrene exposure during 2 weeks decreased the content of free glutain rat liver

significantly

(59%)

EFFECT

OF ST~RENE

MICROSOMAL

Exposure (weeks) 2 4 6 8 11

INHALATION

C~TOCHROME

(300 ppm, 6

P-450CONTENT,

Free GSH (pmol/g wet ,wt)

HR

AND

CONTENT

LUNG’

Free GSH (pmol/g lung wet wt) Controls

Exposed

2 4 6 11

1.89kO.23 2.82 + 0.27 2.27 + 0.40 2.15kO.40

1.36+0.23* 2.02 f 0.54* 2.49kO.18 2.16kO.46

weeks of exposure to 300 ppm, but no further change occurred (Table 2). The cytochrome P-450 concentration was also somewhat increased in the kidneys of the styrene-exposed animals (Table 5). The activity of ethoxycoumarin deethylase in both the liver and kidney resembled the behaviour of cytochrome P-450 (Tables 2 and

5). The epoxide hydratase activity in liver,, measured with styrene oxide as the substrate, was increased somewhat by the inhalation (Table 6), but the increment was less than that observed in the content of cytochrome P-450 or the activity of ethoxycoumarin deethylase (Table 2).

TABLE

2

DAILY,

5

DAYS/WEEK)

ETHOXYCOUMARIN

ON

FREE

DEETHYLASE

Cytochrome P-450 (nmol/mg mic. prot.)

GLUTATHIONE ACTIVITY

CONTENT,

IN RAT

LIVER’

Ethoxycoumarin deethylase (nmol x mg mic. prot.-r x min-I)

Controls

Exposed

Controls

Exposed

Controls

Exposed

3.48 kO.32 3.47 + 0.45 3.3OkO.18 4.19kO.14 4.16kO.27

1.44 + 0.24b 2.02 + 0.34’ 2.66+_0.18’ 3.53?0.17b 3.OOf.O.21*

0.47 f 0.04 0.45kO.16 0.59kO.08 0.53kO.13 0.56 + 0.08

0.92+0.17” 0.68kO.16 0.75+0.12 0.79 i- 0.03” 0.79 + 0.06’

0.34 + 0.06 0.54kO.12 ND“ 0.37+0.14 0.39kO.13

0.49 f 0.03’ 0.84 f 0.07’ ND 0.81+ 0.09’ 0.96kO.16’

a The values are means f SD (n = 4). b Statistical difference from control group: p
HR IN

11The values are means f SD (n = 4). b Statistical difference from control group: p < 0.05.

(Table

2). This depression was alleviated as the experiment continued but it was never totally abolished. Free GSH in rat lung was somewhat decreased after an exposure period of 2 weeks (Table 3). Later, however, no such change was observed. In a 4-day exposure experiment even lower styrene levels decreased free glutathione in rat liver (Table 4). This decrease was reversible, however; the next morning the free glutathione concentration was even slightly higher in the livers of exposed animals than those of controls. The cytochrome P-450 content of hepatic microsomes was doubled during the first 2

(300 ppm, 6

Exposure (weeks)

first few exposures

thione

3

INHALATION ON GLUTATHIONE

10

VAINIO, J;iRVISALO, AND TASKINEN TABLE EFFECT OF VARIOUS

4

4

ATMOSPHERIC CONCENTRATIONS OF STYRENE DAYS) ON FREE GLUTATHIONE IN RAT LIVER

(6 HR DAILY

FOR

Reduced glutathione (fimol/g liver wet wt) Styrene (ppm in the chamber air)

Time after exposure ON 0.5 18

Control

100

200

400

3.88&0.36(12) 5.89kO.49 (12)

3.56kO.29 (4) 5.82kO.71 (4)

2.91 kO.19 (4)b 6.21 f 0.45 (4)

1.71 f 0.23 (4)* 6.41+ 0.55 (4)

’ The values are means+ SD. The number of animals is given in parentheses. b Statistical differences from control group : p < 0.001.

EFFECT

P-450

OF STYRENE INHALATION CONTENT, ETHOXYCOUMARIN

TABLE 5 (300 ppm, 6 HR DAILY, 5 DAYS/WEEK)

ON MICRO~~MAL CVTOCHROME DEETHYLA~E AND UDP GLUCURONOSYLTRANSFERASE (p-NITROPHENOL AS AGLYCONE) ACTIVITY IN RAT KIDNEY’

Cytochrome P-450 (nmol/g wet wt)

Exposure (weeks)

Controls

Exposure

2 6 8 11

l.OOkO.36 1.19kO.27 1.3OkO.30 1.52kO.32

1.9410.98 1.72 & 0.44 1.74f0.45 1.94+0.08’

Ethoxycoumarin deethylase (nmol x g wet wt.? x min-*) Controls

Exposure

0.02 + 0.008 0.01 ir 0.009 0.07 ck0.92 0.06+ 0.01

0.09 + 0.02* 0.07+0.01* 0.24 f O.O@ 0.10~0.01’

UDP glucuronosyltransferase (nmol x g wet wt.-r x min-r) Controls

Exposure

7.90+ 1.03 5.66kO.82 5.03 + 1.43 6.39+ 1.66

9.09 + 0.47 8.08 * 0.90” 11.90_+ 1.21b 12.84+2.03’

a The values are means f SD (n = 4). * Statistical difference from control group: p < 0.001. c Statistical difference from control group: p < 0.01. d Statistical difference from control group : p < 0.05. TABLE

6

EFFECT OF STYRENE INHALATION (300 ppm, 6 HR DAILY, 5 DAYS/WEEK) ON MICRO~OMAL EPOXIDE HYDRATASE AND UDP GLUCURONOSYLTRANSFERASE (MEASURED FROM DIGITONIN-TREATED MICROSOMES) ACTIVITIES IN RAT LIVER’

Exposure (weeks) 2 4 6 8 11

Epoxide hydratase (nmol x mg micros. prot.-r x mm-l) ND* 7.76+ 1.81 ND 7.52kO.60 7.11 to.28

UDP glucuronosyltransferase (nmol x mg micros. prot.-r x mm-r)

ND 9.12* 1.66 ND 9.75+ 1.79 8.35kO.78’

n The values are means + SD (n = 4). bW’ Not determined. e Statistical difference from control group: p < 0.05. d Statistical difference from control group: p < 0.01.

Controls

Exposed

5.02kO.80 4.82kO.90 4.74* 0.59 4.62 310.71 5.44+ 1.25

5.82 f 0.77 5.4lkO.81 6.41 f 0.92’ 7.56f0.71’ 7.75 ~0.60’

STYRENE

AND

DRUG

METABOLISM

FIG. 1. Photomicrographs of Iivers from styrene-exposed rats showing parenchymal aheratio ns. (a) Hydropic degeneration. x 660. H & E staining; (b) Intracellular vacuolization (steatosis) of parenchymal cells. x 250. H & E staining. Both micrographs were taken from liver samples of anim lals killed after 11 weeks exposure to styrene (300 ppm).

12

VAINIO,

JliRVISALO,

Intermittent exposure to styrene (300 ppm) also enhanced the UDP glucuronosyltransferase activity in rat liver, the increase being detectable only after a 6-week period of exposure (Table 6). In rat kidney the enzyme activity was doubled in 11 weeks (Table 5). No significant increase in UDP glucuronosyltansferase activity was observed in the lungs. The exposed (300 ppm) animals exhibited histological liver alterations consisting of parenchymal hydropic degeneration, steatosis, and congestion. These were also observed in the first group killed after an exposure of 2 weeks (see Fig. 1). DISCUSSION The air concentrations of styrene exposure used in the present study are not rare in industry (Engstrbm et al., 1976), although they exceed the present threshold limit value of Finland and the United States (100 ppm). Intermittent inhalation exposure to styrene (300 ppm, 6 hr/day, 5 days/week) caused adaptive changes in various organs. Clear changes have been observed in the concentration of styrene in fat tissue, the 1 l-week perinephric fat content of styrene being only half of the peak value (1567 nmol/g) measured after 4 weeks of exposure (Savolainen and Pfaffli, 1977). The decrease in styrene concentration in fat may reflect biotransformation changes that took place during the exposure period. Both the mixed-function oxidase system (cytochrome P-450, ethoxycoumarin deethylase) and the conjugation phase of drug metabolism (epoxide hydratase, UDP glucuronosyltransferase) were enhanced in liver and kidney. The extent of enhancement was, however, fairly small and therefore agreed with data obtained with the intraperitoneal administration of styrene (Parkki et al., 1976). The increase in mixedfunction oxidase reached its maximum after 2 weeks of exposure, whereas the UDP glucuronosyltransferase activity was enhanced only after an exposure of 6 weeks. A

AND

TASKINEN

similar difference, an increase in drug hydroxylation preceding that in glucuronidation, has been found with many inducing agents, e.g., polycyclic aromatic hydrocarbons (Aitio et al., i972), phenobarbital (Vainio, 1973), and DDT (Vainio, 1974). Epoxide hydratase, which converts styrene oxide into phenylethylene glycol, is also induced by styrene inhalation. Styrene oxide readily reacts with glutathione or tissue macromolecules (Marniemi et al., 19771, and it is a mutagenic (Vainio et al., 1976; Milvy and Garro, 1976) and embryotoxic compound (Vainio et al., 1977). Consequently, the conversion of styrene oxide to its glycol can be regarded as a detoxifying step in the biotransformation of styrene. In agreement with earlier observations following intraperitoneal administration (Vainio and Makinen, 1977), styrene inhalation exposure caused a depression in the hepatic nonprotein sulfhydryl content. The changes that occurred in nonprotein sulfhydryl groups suggest that the reactive metabolite of styrene, styrene oxide, interacts with these endogenous nucleophiles. Epoxides are frequently formed from vinyl compounds, and they undergo an enzymatic as well as nonenzymatic interaction with reduced glutathione (Arias and Jakoby, 1976). Glutathione and, more generally, the nonprotein sulfhydryl groups prevent the cytotoxicity of many reactive compounds by acting as scavengers of toxic intermediates. There appears to be a clear dose-effect relationship in the free glutathione depression (Table 4). As reported earlier, various animal species differ in their sensitivity toward styreneinduced depletion of liver glutathione, mouse being the most sensitive and rat the most resistant species (Vainio and Makinen, 1977). The depletion of glutathione was observed immediately following exposure only. Eighteen hours later the liver glutathione concentrations of styrene-exposed rats were similar to, or even somewhat higher than, those of control animals. This rebound phenomenon has been observed after the

STYRENE AND DRUG METABOLISM administration of other chemicals as well (e.g., trichloroethylene, vinyl chloride) (Moslen et al., 1977; Watanabe et al., 1976). ACKNOWLEDGMENTS We wish to thank Ms. Anja Sarasjoki, Ms. Elvi Leskinen, and Mr. Paavo MIkell for their technical assistance and the Finnish Academy of Sciences for its financial aid. REFERENCES AITIO, A., AND VAINIO, H. (1976). UDP glucuronosyltransferase and mixed-function oxidase activity in microsomes prepared by differential centrifugation and calcium aggregation. Acta Pharmacol. Toxicol. 39, 555-561.

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