Cancer Letters, 2 (1976) 109--114
© El.qevier/North-Holland Biomedical Press
CYTOCHROME P-450 AND THE METABOLISM O F VINYL CHLORIDE
A N D R E W G. SALMON
Central Toxicology Laboratory, Imperia~ Chemiea! Industries Limited, Aldcrley Par~=, Nr. Macclesfield, Ct~¢shireSK10 4TJ ( U.K. )
(Received 18 June 1976) (Revised version receive~ 2 August :[976)
SUMMARY The oxidation of vinyl ehlol~de to non-volatile products is dependen~ on NADPH and microsomal enzymes. The addition of vinyl chloride to microsomes causes a Type 1 spectral shift, similar to that seen for p h e n o b a r b i ~ l [ 1 1 ] , which indicate:, the direct involvement o f a cytochrome P-450 species; this difference spectrum is characteristic of substrate binding to this ~Tpe of enzyme. A glutathione conjugate is probably formed, perhaps via a reactive intermediate.
INTRODUCTION The carcinogenic [12] and mutagenic [1] effects o f vinyl chloride are thought to depend on its conversion t o reactive metabolites. The products formed on exposure of the intact animal ;~o vinyl chloride [5,6] are apparently the result of oxidation and conjugation with glutathione. Similar products have been observed with other xenobiotics, including carcinogens [2]. It is not clear which enzymes are involved; some experiments on vinyl chloride metabolism in vitro [1,4] suggest microsomal enzymes, while others implicate soluble enzymes, such as alcohol dehydrogenase E6]. The following experiments were undertaken to clarify a possible role of microsomal enzymes in this reaction. MATERIALS AND METHODS Rat liver microsomes were obtained from male Alder!y Park strain SPF albino rats weighhng 150--200 g. The livers were homogenized in a W~ing blender with 3 volumes of 50 mM Tris buffer, pH 7.5, containh~g 0.25 M sucrose. Microsomes were prepared from the homogenate by the s~and-,,rd me'thod k[7]. After one ~ h , they were resuspended in Tris/sucrose buffer at 2,5 mg protein m1-1 and stored ~.nder argon a t - 20°C. [U-~"C,] vinyl chloride ::(specific activity 1 m C i - m m o r ' ) was prepared by Dr A. Hestop (ICI PeSo-
110
chemicals Division); its pt~rity was better than 99% by gas--Iiquid chromatography. Tris buffe~ (Trizma reagent grade) and soybean lecithin were obt~zined from Sigma Chemical Co., St. Louis, Mo,, U.S.A. Other ,chemicals were obtained from BDH Chemicals Ltd., Poole, U.K. Difference spectra (Perkin-Elmer 356 spectrophotometer, integrating scattered light over 180 °) were recorded at 25°C. Microsome suspensions were diluted with Tris/suc]tose buffer to an optical density of 1.0 at 400 nm. Vinyl chloride was passed thro--gh one sample until saturated (about 5 min). Enzymic oxidation of vinyl chloride was assayed by the formation of non-volatile product~. Vinyl chloride was added ei~l~er in ethanol (finat concentration less than 0,5%) or as a solution it, cor,~ off containing 10% lecithin, emulsified in a buffer containing 1% Twecn 60 (1 ml emulsion per sample). Vinyl chloride was added to 2 ml of oxygen saturated Tris/sucrose buffer, containing 400 pM NADPH, 3 mM MgCl: and the microsomes. The sealed tube was ~ncubated at 30°C, after which 1 ml of ace:tone was added to stop the reaction, Excess vinyl chlortde and other volatile materials were removed by freeze-drying for 12 h, and soluble materials extracted with 2 ml water. Solids and solution were separated by centrifug,~'~ion, and the radioactivity of each fraction was determined by scintillatiorJ counting (tntertechnique SL30). Efficiencies were compared with a [~'*C]hexadecane internal star, dural. Protein concen'~rations were determined by the method o:~ ]Lowry et al. [9] adapted to a Technicon Auto Analyser.
6000"-
4000"
~ 0
,JJ~ /// 2OCt"
o -
~..............................
I ..........................
,5
I0
1- ' ~
15 ,,,,,, ,1,,,
F~g. !, Produetio~ of non-volatile clericaL[yesfrom vinyl cifloride in the pre'Jeneeof m]erosomes and N A D P ' - L MethodE~as Ueseribed in the text. Mic,-osom~lprotein ¢,r,n c e n ~ r a t i o n 2.46 rng ral-', vlny~ch~ori2,.~concentration 300 ~M, 3,5 × 10" dpm, added in coen oi~ emulsion.
iii
RESULTS
A t i m e - d e p e n d e n t increase in t h e l a b e l i n c o r p o r a t e d i n t o non=volatile w a t e r soluble m a t e r i a l is s h o w n in Fig. 1. T h e r e a c t i o n r a t e r e m a i n e d c o n s t a n t f o r 1 5 rain, b u t d e c l i n e d later, p o s s i b l y d u e t o e x h a u s t i o n o f s u b s t r a t e s a n d f u r t h e r m e t a b o l i s m o f p r o d u c t s . I n c o r p o r a t i o n , o f label i n t o m i c r o s o m a l p r o t e i n was also observed. O n t h e o t h e r h a n d , n o labelling o f lipids was o b s e r v e d w h e n t h e n o n - v o l a t i l e m a t e r i a l w e r e e x t r a c t e d i n t o e t h y l acel~ate a n d e x a m i n e d for r a d i o a c t i v i t y . V i n y l c h l o r i d e was a d d e d in a c o r n oil e m u l s i o n in t h e e x p e r i m e n t s h o w n in Fig. 1. S i m i l a r result,,; were o b t a i n e d w h e n vinyl c h l o r i d e was a d d e d in e t h a n o l i c s o l u t i o n , suggesting t h a t n e i t h e r s o l v e n t i n h i b i t s t h e e n z y m i c r e a c t i o n . N o i n c o r p o r a t i o n o f label r o t e e i t h e r soluble o r solid f r a c t i o n was seen in t h e a b s e n c e o f m i c r o s o m e s of of N A D P H , or in t h e p r e s e n c e of m i c r o s o m e s p r e v i o u s l y h e a t e d t o X00°C fo~ 5 rain. TABLE 1 EFFECT OF REDUCED GLUTATHIONE ON MICROSOMAL OXIDATION OF VINYL CHLORIDE Fzaction
Aqueous Aqueous Solid Solid
GSH Concentration -lmM -lmM
Percentage incorporation of label in 20 rain Mean Mean
Standard deviation (n = 4)
2.70 4.55 7.30 6.61
0.75 0.14 4.2 2.i2
Vinyl chloride (100 ~M, 1 × 10 ~ dpm) was added as a solution in ethanol to Tris/sucrose buffer, pH 7.5, containing oxygen (saturated), NADPH (400 ,=M), MgCI= (3 raM) and microsomes (2.45 mg ml-:). Incubation was for 20 rain at 30°C, after which the sample~ were fractionated and radioactivity determined as described in the text. A d d i t i o n o f 1 mM redu,~:ed g l u t a t h i o n e t o t h e r e a c t i o n m i x t u r e c a u s e d a n increase in t h e micros~,me-catalysed f o r m a t i o n o f non-volatile, w a t e r soluble p r o d u c t s (Table 1). A statistically i n s i g n i f i c a n t d e c i ~ a s e in label i n c o r p o r a t e d i n t o t h e solid f r a c t i o n was also o b s e r v e d , t n t h e a b s e n c e of active e n z y m e s , t h e r e was n o r e a c t i o n of vinyl c h l o r i d e w i t h glutathi,one. T h e c o n c e n t r a t i o n e f c y t o c h r o m e P - 4 5 0 , est':mated as t h e r e d u c e d c a r b o n m o n o x i d e c o m p l e x with a m o l a r e x t i n c t i o n i n c r e m e n t of 9 ! mM -~ c m -i a t 4 5 0 n m ~ i 0 ] , was f o u n d t o b e 0 . 6 n M p e r mg p r o t e i n . T h e a m o u n t o f P - 4 2 0 d e t e c t e d was very small. A d d i t i o n of 1 0 0 ~M p h e n o b a r b i t a l t o a m i c r o s o m e s u s p e n s i o n c a u s e d an increase in a b s o r b a n c e at 3 9 0 n m a n d a d e c r e a s e a t 4 2 0 a m , i.e. a t y p i c a l T y p e 1 difference s p e c t r u m . A similar d i f f e r e n c e s p e c t r u m was o b s e r v e d a f t e r s a t u r a t i o n o f a m i c r o s o m a l s u s p e n s i o n w i t h v~nyl c h l o r i d e (Fig. 2).
112
c,d, unJt~ ~10 ~
Rb~l',ubulbllel -.o.=tlW9[ tbloFpdW
d..- ~ 2.-
/;ro
-6.--
~o o
....
//
-8.-Fig. 2. Difference spectra induced by phenobarbital and by vinyl chloride. I ern path length celia containing microsomes (2.23 mg/rrd"~ protein, A,o,, n m = 0.97). Phenobarbital concentration 100 ~M, vinyl chloride saturated solution. Buffer ~s described in the text. DISCUSSION The o x i d a t i o n o f vinyl c h l o r i d e t o non-volatile p r o d u c t s in vitro is depen-. d e n t i n m i c r o s o m a l e n z y m e s a n d o n available NADPH. T h e p r o d u c t s f o r m e d p r e s u m a b l y i n c l u d e c o n j u g a t e s w i t h a d d e d glutathion,e. T h e m i c r o s o m a l m a c r o m o l e c u l e s w e r e ~ s o labelled. T h e s e p r o d u c t s m a y r e s u l t f r o m t h e f o r m a t i o n of r e a c t i v e i n t e r m e d i a t e m e t a b o l i t e s , w h i c h are partially d e a c g v a t e d b y c o n j u g a t i o n , while s o m e f o r m c o v a l e n t p r o d u c t s w i t h p r o t e i n s a n d n u c l e i c acids [ 8 ] . T h e r e q u i r e m e n t o f N A D P H is c h a r a c t e r i s t i c of a r e a c t i o n eatalysed b y m i x e d f u n c t i o n oxidases, s u c h as t h e c y t o c h r o m e P , 4 5 0 c o n t a i n i n g mic~csoma] enzymes. T h e o b s e r v a t i o n of a T y p e 1 d i f f e r e n c e s p e c t r u m suggests an i n t e r a c t i o n with a c y t o c h r o m e P-450 p r e s e n t in liver m i c r o s o m e s w i t h o u t i n d u c t i o n . This is in c o n s t r a s t t o t h e polycyclic a r o m a t i c h y d r o e m : b o n carcinogens, w h i c h causes a T y p e 1 d i f f e r e n c e s p e c t r u m w i t h c y t e , c h r o m e P-448, w h i c h is i n d u c i b l e , b u t n o t d e t e c t e d in n o r m a l Iiver [ 3 ] . REFERENCES 1 Bartsch, H., Malavielle, G. and Montezano, R. (1975) Human, rat and mouse livermediated mutagenieity of ~inyl chloride. Int. J. Cancer, 7.5, 429--437. 2 Boyland, E. and Sims, P. (1965) The metabolism of benz[a]antiar'~cene a~d dibenz[a,h]anthracene and their 5,6-epoxy-5,6 dihydro derivativez by rat liver homog.cnates. Bioehem. J., 97, 7~16.
113 3 Fujita, T., Shoeman, D.W. and Mannerlng, C.J. (1973) Differences in P-450 cytochrome~ from liversof rats treated with phenobarbital and with 3-methylcholanthrene. J. Bio]~ Chem., 248, 2192--2201. 4 GSthv, R., Calleman, C.J., Ehrenberg, L. and Waehtmelster, C,A, (1974) Trapping wlth 3,4-diehlorobenzenethiol of reactive metabolites formed in vitro from vinyl chloride. Ambio, 3, 234--236. 5 Green, T. and Hathway, D.E. (1975) The biological fate in rats of vinyl chloride in relation to its oneogenieity. Chem. Biol. Interactions, 11,545--562. 6 He£ner, R.E., Wamnabe, P.G. and Gehring, P.J. (1975) Preliminary studies of the fa~.e of iwhaled vinyl choride monomer in rats.Ann. N.Y. Acad. SeL, 246, 135--148. 7 Hogeboom, G.H. (1955} Fractionation of cell components of animal tL~sues~ In: Methods in Enzymology, Vol. 1, pp. 16--19. Editors: Colewick, S.P. and Kaplan, N.O. Academic Press, N e w York. S Kappus, H., Bolt, H.M., Buehter, A. and Bolt, W. (1975) Rat livermierosomcs catalyse covalent binding of [14C]vinyl chloride to macromolecules. Nature, 257, 134r135. 9 Lowry, O.H., Rosebrough, H.J., Farr, A.L, and Randall, R.J. (1951) Protein me,surement with the Folin phenol reagent. J. Biol. Chem., 193, 245--275. 10 Omura, T. and Sato, R. (1964) The carbon monoxide-binding pigment of liver microsomes. 1. Evidence for its hemopz'otien nature. J. Biol. Chem., 239, 2370--2378. 11 Returner, H., Schenk.man, L., Estabrook, B..W., Sasame, H., Gillette, J., Narasimhuiu, S., Cooper, D.T. and Rosenthal, O. (1966) Drug in.~eractionwith hepatic microsomal Cy~oehrome. Mol. Pharmacol., 2, 187--!90. 12 Viola, P.L., Bigotti, A. and Caputto, A. (1971) Oncogenic response of rat skin, lungs and bones to vinyl chloride. Cancer Res., 31, 516--519.
© El.qevier/North-Holland Biomedical Press
CYTOCHROME P-450 AND THE METABOLISM O F VINYL CHLORIDE
A N D R E W G. SALMON
Central Toxicology Laboratory, Imperia~ Chemiea! Industries Limited, Aldcrley Par~=, Nr. Macclesfield, Ct~¢shireSK10 4TJ ( U.K. )
(Received 18 June 1976) (Revised version receive~ 2 August :[976)
SUMMARY The oxidation of vinyl ehlol~de to non-volatile products is dependen~ on NADPH and microsomal enzymes. The addition of vinyl chloride to microsomes causes a Type 1 spectral shift, similar to that seen for p h e n o b a r b i ~ l [ 1 1 ] , which indicate:, the direct involvement o f a cytochrome P-450 species; this difference spectrum is characteristic of substrate binding to this ~Tpe of enzyme. A glutathione conjugate is probably formed, perhaps via a reactive intermediate.
INTRODUCTION The carcinogenic [12] and mutagenic [1] effects o f vinyl chloride are thought to depend on its conversion t o reactive metabolites. The products formed on exposure of the intact animal ;~o vinyl chloride [5,6] are apparently the result of oxidation and conjugation with glutathione. Similar products have been observed with other xenobiotics, including carcinogens [2]. It is not clear which enzymes are involved; some experiments on vinyl chloride metabolism in vitro [1,4] suggest microsomal enzymes, while others implicate soluble enzymes, such as alcohol dehydrogenase E6]. The following experiments were undertaken to clarify a possible role of microsomal enzymes in this reaction. MATERIALS AND METHODS Rat liver microsomes were obtained from male Alder!y Park strain SPF albino rats weighhng 150--200 g. The livers were homogenized in a W~ing blender with 3 volumes of 50 mM Tris buffer, pH 7.5, containh~g 0.25 M sucrose. Microsomes were prepared from the homogenate by the s~and-,,rd me'thod k[7]. After one ~ h , they were resuspended in Tris/sucrose buffer at 2,5 mg protein m1-1 and stored ~.nder argon a t - 20°C. [U-~"C,] vinyl chloride ::(specific activity 1 m C i - m m o r ' ) was prepared by Dr A. Hestop (ICI PeSo-
110
chemicals Division); its pt~rity was better than 99% by gas--Iiquid chromatography. Tris buffe~ (Trizma reagent grade) and soybean lecithin were obt~zined from Sigma Chemical Co., St. Louis, Mo,, U.S.A. Other ,chemicals were obtained from BDH Chemicals Ltd., Poole, U.K. Difference spectra (Perkin-Elmer 356 spectrophotometer, integrating scattered light over 180 °) were recorded at 25°C. Microsome suspensions were diluted with Tris/suc]tose buffer to an optical density of 1.0 at 400 nm. Vinyl chloride was passed thro--gh one sample until saturated (about 5 min). Enzymic oxidation of vinyl chloride was assayed by the formation of non-volatile product~. Vinyl chloride was added ei~l~er in ethanol (finat concentration less than 0,5%) or as a solution it, cor,~ off containing 10% lecithin, emulsified in a buffer containing 1% Twecn 60 (1 ml emulsion per sample). Vinyl chloride was added to 2 ml of oxygen saturated Tris/sucrose buffer, containing 400 pM NADPH, 3 mM MgCl: and the microsomes. The sealed tube was ~ncubated at 30°C, after which 1 ml of ace:tone was added to stop the reaction, Excess vinyl chlortde and other volatile materials were removed by freeze-drying for 12 h, and soluble materials extracted with 2 ml water. Solids and solution were separated by centrifug,~'~ion, and the radioactivity of each fraction was determined by scintillatiorJ counting (tntertechnique SL30). Efficiencies were compared with a [~'*C]hexadecane internal star, dural. Protein concen'~rations were determined by the method o:~ ]Lowry et al. [9] adapted to a Technicon Auto Analyser.
6000"-
4000"
~ 0
,JJ~ /// 2OCt"
o -
~..............................
I ..........................
,5
I0
1- ' ~
15 ,,,,,, ,1,,,
F~g. !, Produetio~ of non-volatile clericaL[yesfrom vinyl cifloride in the pre'Jeneeof m]erosomes and N A D P ' - L MethodE~as Ueseribed in the text. Mic,-osom~lprotein ¢,r,n c e n ~ r a t i o n 2.46 rng ral-', vlny~ch~ori2,.~concentration 300 ~M, 3,5 × 10" dpm, added in coen oi~ emulsion.
iii
RESULTS
A t i m e - d e p e n d e n t increase in t h e l a b e l i n c o r p o r a t e d i n t o non=volatile w a t e r soluble m a t e r i a l is s h o w n in Fig. 1. T h e r e a c t i o n r a t e r e m a i n e d c o n s t a n t f o r 1 5 rain, b u t d e c l i n e d later, p o s s i b l y d u e t o e x h a u s t i o n o f s u b s t r a t e s a n d f u r t h e r m e t a b o l i s m o f p r o d u c t s . I n c o r p o r a t i o n , o f label i n t o m i c r o s o m a l p r o t e i n was also observed. O n t h e o t h e r h a n d , n o labelling o f lipids was o b s e r v e d w h e n t h e n o n - v o l a t i l e m a t e r i a l w e r e e x t r a c t e d i n t o e t h y l acel~ate a n d e x a m i n e d for r a d i o a c t i v i t y . V i n y l c h l o r i d e was a d d e d in a c o r n oil e m u l s i o n in t h e e x p e r i m e n t s h o w n in Fig. 1. S i m i l a r result,,; were o b t a i n e d w h e n vinyl c h l o r i d e was a d d e d in e t h a n o l i c s o l u t i o n , suggesting t h a t n e i t h e r s o l v e n t i n h i b i t s t h e e n z y m i c r e a c t i o n . N o i n c o r p o r a t i o n o f label r o t e e i t h e r soluble o r solid f r a c t i o n was seen in t h e a b s e n c e o f m i c r o s o m e s of of N A D P H , or in t h e p r e s e n c e of m i c r o s o m e s p r e v i o u s l y h e a t e d t o X00°C fo~ 5 rain. TABLE 1 EFFECT OF REDUCED GLUTATHIONE ON MICROSOMAL OXIDATION OF VINYL CHLORIDE Fzaction
Aqueous Aqueous Solid Solid
GSH Concentration -lmM -lmM
Percentage incorporation of label in 20 rain Mean Mean
Standard deviation (n = 4)
2.70 4.55 7.30 6.61
0.75 0.14 4.2 2.i2
Vinyl chloride (100 ~M, 1 × 10 ~ dpm) was added as a solution in ethanol to Tris/sucrose buffer, pH 7.5, containing oxygen (saturated), NADPH (400 ,=M), MgCI= (3 raM) and microsomes (2.45 mg ml-:). Incubation was for 20 rain at 30°C, after which the sample~ were fractionated and radioactivity determined as described in the text. A d d i t i o n o f 1 mM redu,~:ed g l u t a t h i o n e t o t h e r e a c t i o n m i x t u r e c a u s e d a n increase in t h e micros~,me-catalysed f o r m a t i o n o f non-volatile, w a t e r soluble p r o d u c t s (Table 1). A statistically i n s i g n i f i c a n t d e c i ~ a s e in label i n c o r p o r a t e d i n t o t h e solid f r a c t i o n was also o b s e r v e d , t n t h e a b s e n c e of active e n z y m e s , t h e r e was n o r e a c t i o n of vinyl c h l o r i d e w i t h glutathi,one. T h e c o n c e n t r a t i o n e f c y t o c h r o m e P - 4 5 0 , est':mated as t h e r e d u c e d c a r b o n m o n o x i d e c o m p l e x with a m o l a r e x t i n c t i o n i n c r e m e n t of 9 ! mM -~ c m -i a t 4 5 0 n m ~ i 0 ] , was f o u n d t o b e 0 . 6 n M p e r mg p r o t e i n . T h e a m o u n t o f P - 4 2 0 d e t e c t e d was very small. A d d i t i o n of 1 0 0 ~M p h e n o b a r b i t a l t o a m i c r o s o m e s u s p e n s i o n c a u s e d an increase in a b s o r b a n c e at 3 9 0 n m a n d a d e c r e a s e a t 4 2 0 a m , i.e. a t y p i c a l T y p e 1 difference s p e c t r u m . A similar d i f f e r e n c e s p e c t r u m was o b s e r v e d a f t e r s a t u r a t i o n o f a m i c r o s o m a l s u s p e n s i o n w i t h v~nyl c h l o r i d e (Fig. 2).
112
c,d, unJt~ ~10 ~
Rb~l',ubulbllel -.o.=tlW9[ tbloFpdW
d..- ~ 2.-
/;ro
-6.--
~o o
....
//
-8.-Fig. 2. Difference spectra induced by phenobarbital and by vinyl chloride. I ern path length celia containing microsomes (2.23 mg/rrd"~ protein, A,o,, n m = 0.97). Phenobarbital concentration 100 ~M, vinyl chloride saturated solution. Buffer ~s described in the text. DISCUSSION The o x i d a t i o n o f vinyl c h l o r i d e t o non-volatile p r o d u c t s in vitro is depen-. d e n t i n m i c r o s o m a l e n z y m e s a n d o n available NADPH. T h e p r o d u c t s f o r m e d p r e s u m a b l y i n c l u d e c o n j u g a t e s w i t h a d d e d glutathion,e. T h e m i c r o s o m a l m a c r o m o l e c u l e s w e r e ~ s o labelled. T h e s e p r o d u c t s m a y r e s u l t f r o m t h e f o r m a t i o n of r e a c t i v e i n t e r m e d i a t e m e t a b o l i t e s , w h i c h are partially d e a c g v a t e d b y c o n j u g a t i o n , while s o m e f o r m c o v a l e n t p r o d u c t s w i t h p r o t e i n s a n d n u c l e i c acids [ 8 ] . T h e r e q u i r e m e n t o f N A D P H is c h a r a c t e r i s t i c of a r e a c t i o n eatalysed b y m i x e d f u n c t i o n oxidases, s u c h as t h e c y t o c h r o m e P , 4 5 0 c o n t a i n i n g mic~csoma] enzymes. T h e o b s e r v a t i o n of a T y p e 1 d i f f e r e n c e s p e c t r u m suggests an i n t e r a c t i o n with a c y t o c h r o m e P-450 p r e s e n t in liver m i c r o s o m e s w i t h o u t i n d u c t i o n . This is in c o n s t r a s t t o t h e polycyclic a r o m a t i c h y d r o e m : b o n carcinogens, w h i c h causes a T y p e 1 d i f f e r e n c e s p e c t r u m w i t h c y t e , c h r o m e P-448, w h i c h is i n d u c i b l e , b u t n o t d e t e c t e d in n o r m a l Iiver [ 3 ] . REFERENCES 1 Bartsch, H., Malavielle, G. and Montezano, R. (1975) Human, rat and mouse livermediated mutagenieity of ~inyl chloride. Int. J. Cancer, 7.5, 429--437. 2 Boyland, E. and Sims, P. (1965) The metabolism of benz[a]antiar'~cene a~d dibenz[a,h]anthracene and their 5,6-epoxy-5,6 dihydro derivativez by rat liver homog.cnates. Bioehem. J., 97, 7~16.
113 3 Fujita, T., Shoeman, D.W. and Mannerlng, C.J. (1973) Differences in P-450 cytochrome~ from liversof rats treated with phenobarbital and with 3-methylcholanthrene. J. Bio]~ Chem., 248, 2192--2201. 4 GSthv, R., Calleman, C.J., Ehrenberg, L. and Waehtmelster, C,A, (1974) Trapping wlth 3,4-diehlorobenzenethiol of reactive metabolites formed in vitro from vinyl chloride. Ambio, 3, 234--236. 5 Green, T. and Hathway, D.E. (1975) The biological fate in rats of vinyl chloride in relation to its oneogenieity. Chem. Biol. Interactions, 11,545--562. 6 He£ner, R.E., Wamnabe, P.G. and Gehring, P.J. (1975) Preliminary studies of the fa~.e of iwhaled vinyl choride monomer in rats.Ann. N.Y. Acad. SeL, 246, 135--148. 7 Hogeboom, G.H. (1955} Fractionation of cell components of animal tL~sues~ In: Methods in Enzymology, Vol. 1, pp. 16--19. Editors: Colewick, S.P. and Kaplan, N.O. Academic Press, N e w York. S Kappus, H., Bolt, H.M., Buehter, A. and Bolt, W. (1975) Rat livermierosomcs catalyse covalent binding of [14C]vinyl chloride to macromolecules. Nature, 257, 134r135. 9 Lowry, O.H., Rosebrough, H.J., Farr, A.L, and Randall, R.J. (1951) Protein me,surement with the Folin phenol reagent. J. Biol. Chem., 193, 245--275. 10 Omura, T. and Sato, R. (1964) The carbon monoxide-binding pigment of liver microsomes. 1. Evidence for its hemopz'otien nature. J. Biol. Chem., 239, 2370--2378. 11 Returner, H., Schenk.man, L., Estabrook, B..W., Sasame, H., Gillette, J., Narasimhuiu, S., Cooper, D.T. and Rosenthal, O. (1966) Drug in.~eractionwith hepatic microsomal Cy~oehrome. Mol. Pharmacol., 2, 187--!90. 12 Viola, P.L., Bigotti, A. and Caputto, A. (1971) Oncogenic response of rat skin, lungs and bones to vinyl chloride. Cancer Res., 31, 516--519.
