Changes in rat liver microsomal cytochrome P-450 and enzymatic activities after the inhalation of n-hexane, xylene, methyl ethyl ketone and methylchloroform for four weeks.

Changes in rat liver microsomal cyto chrome P-450 and enzymatic activities afber the inhalation of n-hexane, xylene, methyl ethyl ketone and methylchloroform for :flour weeks. Scand j work environ health 7 (1981) 31-37. Groups of Sprague-Dawley rats were exposed, by inhalation, to n-hexane (900 ppm, 3,240 mg/m3), xylene (600 ppm, 2,625 mg/m:!), methy11 ethyl ketone (800 ppm, 2,345 mg/m 3) and methylchlorofolm (800 ppm, 4,345 mg/m 3) :£or fuur weeks. Increased liver weights and liver to body weight ratios were observed ~or all the ,solvents except n-hexane. An ,increased in Vlitro formati'on of certain metabolites of all the investigated substrates was found only illl the raits exposed to xylene. The in vitro microsomal metabolism of biphenyl, benzo(a)'PY'rene, 4-androstene-3,17-d~one and 5a-androstane 3a, 17ji-diol in combination wHh sodium dodecyl sullfate-polyacrylamide gel electro phoresis showed that n-hexane was wi1!hout effect on rat liver microsomal cytochrome P-450 and that methyl ethyl ketone and mebhylc'hloroform depressed the formation of two metabolites of androstenedione but did not alter the concentration of cytochrome P-450 under the experimental e,onditions Xylene was shown to be a phenobar bital-like inducer of rat liver microsomal cytochrome P-450.

The enzyme system responsible for such activation 1!hrough the formation of epoxides or other r€acllive meta'bolites is, in most cases, the one dependent on liver mi,crosomal cytochrome P-450. The activity of 1!his enzyme system, which consists of separate isoenzymes with different substrate specificities (12,31), can be modulated 'by exogenous factors, including exposure to Qrganic solvents. This modula-0355-3140/81/010031-7 tion may lead to an altered susceptibility to the toxic effects of the solvent i1self or to other environmental contaminants. An inducing effect on liver microsomal cytochrome P-450 in rats has been shown after the inhalation of methylchloroform, benzene, carbon tetrachloride, and trichloroethylene (10,20). On the other hand, both methylchloroform and caT'bon tetrachloride have been reported to decrease the liver microsomal content of cytochrome P-450 When administered intragastrlically (28,32), and th'is finding underlines the importance of using relevant adm~nistra tion routes in the evaluation of the hiological effects of hydrocarbon solvents.
The present study was undertaken to investigate the effect of four commonly used hydrocarbon solvents with widely different chemical structures, n...:hexane, xylene, methyl ethyl ketone and methylchloroform, on the liver microsomal cytochrome P-450 enzyme system in the rat after inhalation. Changes in the different forms of cytodhrome P-450 4 and in the in vitro microsomal metabolism of biphenyl, benzo(a)pyrene, and the steroids 4-androstene-3,17-dione and 5a-androstane-3a, 17fi-diol were investigated.

Animals and experimental design
Male Sprague-Dawley rats weighing about 300 g were obtained from Anticimex (Sweden). The rats were kept in cages 5 d prior to treatment. They had free access to water and food and were kept in a room with controlled temperature and light (14 h light -10 h dark).
Groups of four rats were exposed during the light period of the day to solvent vapors [n-:hexane 910 ppm ± 240 (± SD) (3,276 ± 864 mg/m 3 ) and xylene 630 ppm ± 170 (± SD) (2,756 ± 744 mg/m 3 ) in one experiment and methyl ethyl ketone 760 4 The different cytochromes are referred to on the basis of the species, tissue, subcellular fraction, and molecular weight of the subunit as characterized by sodium dodecyl sulfatepolyacrylamide g,el electrophoresis. Thus, RLvMc P-450so denotes rat liver micI"osomal cytochrome P-450 with a subunit molecular weight of about 50,000. 32 ppm ± 200 (± SD) (2,229 ± 587 mg/m 3 ) and methylchloroform 820 ppm ± 130 (± SD) (4,451 ± 706 mg/m 3 ) in another experiment] 6 h each day, 5 d/week, during four weeks. Control groups were exposed to circulating air only. The an'imals were killed by decapitation on the morning of the day after the last exposure. No food or drinking water was offered during the exposures. Only water was allowed during the 24 h preceding sacrifice.

Inhalation exposure
The rats were exposed in a glass des'icca tor fitted with inlet and outlet tubing. The volume of the desiccator was 21 1, and an air How of 8 1 . mlin-1 wa's mainlta1ined during the exposure. The desired composition of the exposure atmosphere was obtained by the mixing of measured portions of air and saturated solvent vapor. Every 2 h the exposure level was monitored from 0.2-ml air samples taken in the animal's brea'thing zone with a prewarmed gastight syringe. These samples were injec!ted into a gas chromatograph (Varian Aerograph Series 1400). A calibra'tion curve was constructed with the use of gas standards containing known concentrations of the solvent.

Preparation of liver microsomes
Rat liver microsomes were prepared as previously described (31). The microsomes were suspended and diluted in a 0.05 M potassium buffer, pH 7.4, contalining 10-4 M ED'TA (etfuy1enediam'inetetraace-'tate) to a final ,concentration of about 30 mg of mi'crosomal protein per milli'lliter. The protein concentration was determined by the method of Lowry (16), bovine serum albumin be!ing used as the standard.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis
The liver microsomes were diluted to a concentration of 1 mg of protein per milliliter in 0.05 M sodium phosphate buffer, pH 7.4, contain'ing 10-4 M EDTA. The microsomal suspensions were treated with sodium dodecy'l. sulfate (SDS) [150 mg/mg of prote'in (0.5 mmoIlmg of protein)] and fi-mercaptoethanol [75 mg/mg of protein (1.0 mmo'l/mg of protein)]. Electrophoresis was perfoI1IIled on slalb g,els (model 220 Dual Veihical S~a'b Ge'l Electrophoresis Cel]'!, BIO-RAD Laboratories, USA) with the use of the diJSconitinuous buffer system of Laemmli (14). 'I1he slalb gels contained 7.5 % acryIramli'de in 1!he separating gel and had the dimensions 140 mm (m[grafion dlstance) X 100 mm X 0.75 mm (thikkness). Twenty sample wells were loaded Wlitih 5 fl,g of protein each; 5 mA was appHedper gel during s'taokling and 10 rnA per gel during separation. As judged from the mobilities of the standard proteins phosphorylase B (mol wt 94,000), bovine serum a:lbumin (mol wt 68,000), ovalbumin (mol wt 45,000), and carbonic anhydrase (mol wt 30,000), the resolution obtained during electrophoresis was sufficient to distinguish differences of 500 in the apparent molecular weights of the different types of liver microsomal cyltochrome P-450. The gels were stained for protein at 60°C wirth 0.2,5 Ofo Coomassie brilliant blue R-250 in water:ethanol:acetic acid (5:5 :1) for 20 min and desta,ined overnight at tihe same temperature in a'cetic acid :ethanol:water (1.5:1:17.5).
The qualitative identification, based on heme staining, of four different protein bands, induced by either phenobaribital or 3-methylcholanthrene, as cytochrome P-450s (RLvMc P-450 50 , RLvMc P-450 iYh RLvMc P-450 55 and RLvMc P-450 58 ) has 'been described e'1sewhere (31). Relative quantitation of the amount of protein in the different bands was performed by densiitometric scann'ing on a Beckman scanning densitometer model R-112 at 500 nm after the gels had been stained Wlith Coomassie brilliant blue R-250. A linear relationsh'ip between peak area and the amount of protein applied has been reported earlier (19).

Statistics
Student's t-test was used, and p-values of less than 0.05 were considered significant.

Results
Of the organic solvents investigated, only xylene sign'ificarrtly impaired the growth of the rats during the four weeks' exposure [35 ± 3 g (± SD) increase ,against 61 ± 2 g for the con'tr'Ol group].
The effects 'On liver weight and the total concentration of liver microsomal cyto-chrome P-450 are summarized in table 1. Significantly increased liver weights and liver to body weight ratios were observed after exposure to all the solvents studied except n~hexane. Xylene exposure caused a 20 Ofo increase (not statistically significant) lin the total concentration of cyto-chrome P-4'50, while there was no increase for the other solvents.
The SDS-polyacrylamide gel electrophoresis of liver m:icrosomes revealed no significant changes in the different forms of cytochrome P-450 after exposure to n-hexane, methyl ethyl ketone or methyl- " The experimental rats were exposed to 900 ppm of n-hexane (3,240 mg/m'), 600 ppm of xylene (2,675 mg/m'), 800 ppm of methyl ethyl ketone (2,345 mg/m') or 800 pp m of methyl chloroform (4,345 mg/m') lor 6 h a day, 5 d a week, during four weeks. The controls were exposed to circulating air only.
The effects of solvent exposure on the liver microsomal metabolism of 4-androstene-3,17-dione and 5a-androstane-3a 17,8-diol are shown in table 3. The format ion of 16-hydroxylated androstenedione metabolites increased 60 % fonowing exposure to xylene. ThIs phenomenon was mainly due to an increased formation of 16-ketotestosterone, as demonstrated by gas chromatography/mass spectrometry. Exposure to methyl ethyl ketone increased the formation of 7a-hydroxyandrostenedione but decreased the formation of both 6,8...Jhydroxyandrostenedione and 16-hydroxyandrostenedione. Exposure to meth-y1chloroform also decreased the f,ormation of ,the two latter metabolites. No effects were observed following the exposure to n-hexane.
No effects were observed follOWing the exposure to n-hexane.

Discussion
The results of this study and another recent study by Savolainen et al (26) imply that aromatic hydrocaflbon solvents such as xylene are potential inducing agents for cytochrome P-450 and cytochrome P-450dependent reactions in the liver. This assumption is also supported by the Similar effects of toluene on cytochrome P-450 (results to be published). Methyl ethyl ketone and me'thylchloroform do not seem to influence the total amount of liver microsomal 'cytochrome P-450 significantly, but they tend to depress some cytochrome P-450 dependent reactions. Although nhexane did not influence either the liver microsomal cytochrome P-450 or any of the ,cytochrome P-450-mediated reactions investigated, an induction of cytochrome P-450 has been reported after the exposure of mice to a higher dose during a short period of time (13). Similar results have also been reported with respect to the effects of me'thylchloroform on rats (10). These findings may suggest a dose-dependent influence of n-hexane and methylchloroform on cytochrome P-450, and they indicate the importance of the length of exposure. The inducing capacity of xylene may partly be due to the large uptake of xylene in comparison to the uptake of methylchloroform and aliphatic solvents (1,29). SDS-polya'Crylamide gel electrophoresis revealed that xylene induced RLvMc P-450 50 and RLvMc P-45 0 54 , which are the same cytochrome P-450 forms that are induced to the greatest extent by phenobarbital (31). Furthermore, both xylene and phenobarbital increased the formation of 2-and 4-hydroxyb'iphenyl. Phenobarbital increased the formation of 4,5-dihydroxy-4,5-d~hydrobenzo{a)pyrene more than tenfold (31), while xylene increased the formation of this metabolite f'ivefo'ld. The metaboiism of androstenedione and androstanediol was also affected in a similar manner following exposure to xy-en~or phenobarbital. These data strongly mdlcate that xylene is a phenobarbitallike inducer of liver microsomal cyto-chrome P-450 in the rat. Methyl ethyl ketone, methylchloroform, and n-hexane did not cause any detectable induction either in the total amount of liver microsomal cytochrome P-450 or in the multiple forms, as demonstrated by SDS-polyacrylamide gel electrophoresis. No increases were detected in the formation of the different metabolites of biphenyl or benzo{a)pyrene. However, methyl ethyl ketone and met'hylchloroform reduced the metabolism of the two mentioned substrates to some extent.
Since cytochrome P-450 metabolizes endogenous substrates such as steroids, an induction of liver microsomal cytochrome P-4'50 may cause endocrine disturbances (22,24). Whether the increased in vitro formation of 16-hydroxy-androstenedione after xylene exposure or the decreased formation of 1!he same metabol'ite after methyl ethyl ketone and methylchloroform exposure are relevant for the in vivo situation remains to be established. It may be noted that reproduction disturbances have been reported in men exposed to organic solvents (8).

36
The hepatotoxicity of trichloroethylene and the covalent binding of trichloroethylene metabolites to DNA (deoxyribo-nucleVc add) increase after pretreatment with phenobarbital (2). Since xYilene is shown to be a phenobarbital-like inducer, it is reasonable to assume that synergistic toxic effects may occur upon simultaneous exposure to xylene and other solvents.