Document DMdo0963XBmkLbyxJXNXkK9OO

tyt1; K- iVII- Cioo) Hf*. ISSN 00*1-OOSX Volume 62, Number I January 1982 Toxicology and R&S 024284 Pharmacology Founding Editors Frederick Coulston Harry W. Hays Arnold J. Lehman Editors Emeritus C. Boyd Shaffer Gabriel L. Plaa Robert A. Neal Associate Editors Marion W. Anders James E. Gibson Frederick Guengerich Curtis D. Klaassen Tom S. Miya Donald J. Reed Jane F. Robens Hanspeter Witschi Official Journal of the Society of Toxicology Editor: A. WALLACE HAYES SSfcsiio-i ACADEMIC PRESS New York J.rtndnn Tnrontn ,Wn Son F'mortom aV> TOXICOLOGY AM) AWI.IH) PIIAKM AC'OI OGY 61, I - 10 (19X2) K- 1*711 - (toe) The Effect of Repeated Vinyl Chloride Exposure on Rat Hepatic Metabolizing Enzymes1 Julii; T. Du,: Mich Mil. T. T.nlncj, and Carlo II. Tamiiukko' Liver Research Center, Division of Digestive Diseases and Nutrition, Departmenis of Medicine and Anatomy, and Regional Cancer Center, University of Louisville School of Medicine, Louisville, Kentucky 40292 Received July 17. 1980; accepted September 12. 1981 The Effect of Repealed Vinyl Chloride Exposure on Rat Hepatic Metabolizing En/ymcs. Du, J. T,, Tseng, M. T., and Tamuukko, C. H. (19X2), Toxicol Appl Pharmacol 62. 1 10, Spraguc-Dawley rats were exposed to 2.8% vinyl chloride for 2 (70 hr), 4 (140 hr), and 6 (210 hr) weeks to determine the sequential biochemical changes related to the oxidation and detoxification ability of hepatic tissue. Glutathione^'*transferase(sl activity using 1.2epoxy-(/>*nitrophenoxy)propanc and /^nitrobcnzyl chloride as substrates was elevated 17 to 24, 28, and 35 to 42% after 2, 4, and 6 weeks of exposure, respectively, suggesting cn/yme(s) induction. Reduced glutathione, the major substrate required to conjugate the toxic metabolites of vinyl chloride, was also consistently elevated. Similarly, the activity of glutathione reductase, the enzyme necessary for the regeneration of reduced glutathione from its oxidized form, was also increased following vinyl chloride exposure. Cytochromes /*-450, the major protein in volved with vinyl chloride metabolism, was reduced after vinyl chloride exposure, confirming reports of others that vinyl chloride metabolites destroy /M50. No abnormalities of standard clinical biochemical blood tests of liver function were found during 6 weeks of vinyl chloride exposure. The only consistent ultrustructural modification was the dilation of endoplasmic reticulum. The biochemical and ultrastructural alterations could reflect early hepatocellular adaptation to vinyl chloride exposure. Vinyl chloride, al high conce'ntrations, has been shown to be carcinogenic in both lab oratory animals (Malloni and Lefcmine, 1975; Viola et al., 1971) and man (Creech and Johnson, 1974). Present data support the metabolism of. vinyl chloride by hepatic microsomal mixed-function oxidase system into toxic intermediates, chloroethylcne ox ide (Bolt el al., 1975; Hefner ei al., 1975; Kappuser al., 1976) and chloroacetaldehyde 1 ThU work was supported by a grant from the Man ufacturing Chemists Association, Washington, D C. Portions of this study have been presented (Fed. Pro:. 37, IJ45. 1978), 'Present address: Clement Associates, Inc., 1010 Wisconsin Avenue, N.W,, Suite 660, Washington, D.C. 20007. ' Address requests for reprints to: Carlo H. Tamburro. University of Louisville. (Gross and Freiberg, 1969), These two in termediates are considered to be the ultimate carcinogens (Barbin ei al., 1975; Jaeger et al., 1974b; Van Duuren, 1975), to be mu tagenic in bacterial systems (Elmore ei al., 1976; Greim ei al., 1975; Malaveille el al., 1975; McCann el at., 1975), to act as an alkylating agent by reacting with adenosine (Barbin et al., 1975) and cytidine (Laib and Bolt, 1978), and to bind with protein (Bolt et al., 1976; Kappus et al.. 1976; Watanabc et al., 1978), Detoxification of these metab olites occurs mainly by conjugation with glu tathione and is catalyzed by hepatic gluta thione transferases; the conjugates arc excreted in the urine as substituted cysteine derivatives (Watanabe et al., 1976b,c; Green and Hathway, 1975, 1977). Chloroacetal dehyde can be further oxidized to ch^ro- 0041 *008X /K2/010001 -10J02.00/0 Copyright C 1942 b> Academic preu. Inc. A|l p|llll of rcproiuciiM in any form ftMncd. f , r.^Tvw ECS *n DU. TSI.NG. AND TAMHURKO acetic acid (Hefners al., 1975). These data arc compiled in a metabolic scheme in Tig. J as an updated hypothesized metabolic fate of vinyl chloride in the adult rat. The me tabolism of chlorocthylcnc oxide via epoxide hydratase is not listed in the scheme because its product has not been identified. There have been a few in vivo studies con cerning the effects of vinyl chloride exposure on hepatic concentrations of glutathione (Hefner et al., 1975; Watanabe et al., 1976c; Du and Tamburro, 1978), cytochromes P~ 450 (Reynolds el al., 1975) and on mixedfunction oxidase activity (Drew et at., 1975; Reynolds el al., 1975). However, informa tion about the sequential alterations in he patic oxidation and detoxification of vinyl chloride, during prolonged exposure, simu lating the occurrence in workers, is still lack ing. It was reported previously that the en zymatic changes in rat liver following prolonged exposure to vinyl chloride were similar to those found in rat hepatoma (Du and Tamburro, 1976; Du et at., 1979). The sequential biochemical changes related to the hepatic oxidation and detoxification of' vinyl chloride following prolonged exposure , are reported here. METHODS Animals and experimental design, bight- lo Icftwcek-old Sprague-Dawlcy male rats (~300g), sup plied by Laboratory Supply of Indianapolis, Indiana, were randomized prior to the experiment into three groups: a vinyl chloride-exposed group and the air-ex- ( posed group housed in identical chambers and a second ) control group housed in the University's Central Animal ; Care Center. The exposure level was 2X.OOO ppm vinyl 1 chloride, 7 hr/day, 3 days/week for 2, 4, and 6 weeks. ) The exposure chambers were 4400-liter airtight vats. ' Vinyl chloride (--300 to 340 g) was added lo the vat ' to give a time-weighted average concentration of 211,000 ] - 1000 ppm. The chamber air was changed daily and i the vinyl chloride concentration was determined by gas chromatography. The air was constantly circulated by J a stirrer. The rats' respirations had negligible effect on the composition of the chamber's atmosphere because j of the chamber's large volume. Animals were fed on standard laboratory chow pellets ad libitum. All animals were anesthetized with ether, blood was drawn from the inferior vena cava, and the animals were R&s 024286 m,. fec: ' "'l "'r ^ ^ ` Fig. I. The proposed metabolic fate of vinyl chloride. (GSH. glutathione; MFO. mixed-function oxidase; VC, vinyl chloride; GEST. glutathione 5-cpoxidc transferase; and GaST, glutathione ^-al dehyde transferase). i? 4-..J r'Swjy, s4rV. \ IMI < III OKIUI- I I'H-.fl ON I.N/YMI N killed ,ippro\mi:iicl) 20 hr alter exposure at 1,00 pin each day. NADPIL glutathione. and glutathione disulfide were obtained from Sigma Chemical ( onipiny St Louis. Missouri, 1.2-cpoxy J-U>*nHf>pheno\y fpropanc was purchased from Kastman Kodak Company. Rochester, New York; /*nitfohcnz>l chloride was obtained from Malhoon, Coleman and Bell, La>t Rutherford. New Jersey; hen/pheumme was donated h> the Upjohn Company, Kalamazoo. Michigan. Double-distilled water was used throughout. Sample preparation and btuchcimcni dvierminatiun. Homogenates and subcellular fractions were prepared as described previously (Du ct ul, 11J7V) l*uch sample was prepared from a single organ and kepi at 4C during preparation. The remaining liver was frozen rapidly in liquid nitrogen and stored at -70C. Cytochromes /*450 concentrations were determined in the frozen mi* erosomal fractions the day following sacrifice. The glu tathione (GSH) concentration, as well as glutathioncV'transfcrusc. glutathione reductase, and mi.xed-functton oxidase activities were determined in the freshly fractionated frozen liver. l:or the assays using frozen tissue, the liver* from control and experimental rats were frozen in an identical manner for the same length ol time Cytochromes /*-450 concentration (Otnura and Sato. 1964), nonprotcin sulfhydryl content (Sedlak and Lindsay, 1968), and glutathione reductase activity (Curlberg and Murmcrvik, 1975) were determined in the microsomal or cytosol fractions by methods de scribed previously (Du ft u/.. 1979). Gluiathione^'-transferasc activity was determined using the 100,000 X g supernatant fraction. 1,2-ttpoxvM/>-niirophcnoxy)propane and />-nnrohenz.yl chloride were the substrates for glutathione-,S`-cpoxide transfer ase and gluiaihionc'.V-uralkyl transferase (GAST), re spectively. lin/ymc activity was determined as described by others (llabig ft al., 1974; Kaplowitz. et u/., 1975) All assays were linear functions of protein concentration and limed for at least 2 min. Solutions of 1.2-epoxy*3</>*nUrophenoxy)propanc and />-nitroben^yl chloride were prepared in absolute ethanol; the final ethanol con centration in the incubation mixture was 0.5%, Mixedfunction oxidase activity was estimated in the microwmial fraction by measuring NADPH disappearance m the NADIHi-dependent demethylution reaction of beniphetamine (Lu ft al., 1972), The protein content was determined by the method of Lowry ft al. (1951) The serum clinical liver tests including aspartate ami notransferase, alanine aminotransferase, alkaline phos phatase, bilirubin, cholesterol, and triglyceride were determined by Technicon sequential multiple analyzer computer (SMAC) system. Light and electron microscopy Small strips of liver cre removed under ether anesthesia, sliced into small wubes. placed immediately in ice-cold osmium icitoxide (pH 7.4), and fixed for 2 hr at 4*C. Subse quently, samples were washed mcrmght in phosphjlc buffer, dehydrated in ascending alcohol, and embedded in Upon. Tissue blocks were polymerized at 60*CJ for 2 days. Thin sections were t.ul with a diamond knife and stained with uranyl acetate and lead citrate before ex amination tin a Philip' 100 electron microscope l or ultrastructural analysis, three rats r.mdomiy selected from controls and groups exposed for 2. 4. and t* weeks to vinyl chloride were studied p'or light microscopy, a block of tissue wjs ti\ed in huffered formalin and processed routinely for paraftm embedding Sections < ^m thick were stained with he matoxylin and eosin Stattuical anal\`\t\ Analysis of variance was per formed for the various groups at the different tune pe riods and multiple comparisons were performed hased on the results of the analysis of variance KhSWITS The protein content (1114; protein/g liver) in the subcellular (ructions in both control and vinyl chloride-exposed groups was .the same throughout the exposure (data not shown); the enzymatic results, therefore, are expressed as mieromoles of substrate con verted per minute per milligram of protein. There were no statistical differences be tween the normal and air-exposed groups in the glutathione and cytochromes /'-450 con tents or in any of the en/ymo activities. The nonprotein sulfhydrxl content (Table I) was significantly elevated from 26 to 54ri at 2. 4, and 6 weeks in the vinyl chloride-exposed group compared to both control groups. Al though the nonprotein sulfhydryl content in creased with exposure, the increases were not statistically significant. Glutathione re ductase activity (Table 1 ) in the exposed group was increased by 53 to 77'v at all three time periods. The increase tr. glutathione reductase was the same at 2 and 4 weeks of exposure but showed a further significant increase after 6 weeks of exposure. Gluuthione-.V-epoxidc transferase (GliST, Table 1) and glutathione-.S'-aralkvl transferase (G.-\ST. Table I) activities were signifi cantly higher than controls after 6 weeks of exposure, 37 and 45ty. respectively. The cy tochromes /'-450 content, on the other hand. O IO IO 00 -J K" TABLE 1 StrjULNTIAL ClIANGLS IN J ll.PATIC NoNPKOTLJN Si PLHIYDKYL, CyTOCHROMHS P-450 CONTtNT AND ACTlVITItS Of GlUTATIIIONP: RltMJCTASI- AND Glutaihioni;-5-Thansh:kasij:{Epoxidii and Aralkyl) in Rats Exposto to Vinyl Chloridk' Time (weeks) Treatment 0 I 4 6 Nonprolein sulfhydr)! (*imol/g liver) Glutathione reductase (100 X ^mol/min/mg protein) (JEST (100 x (.mol/ rnin/mg protein) C!AST ( U) x pmol/ min/mg prolein) Cytochrome /M5U (nmol/g In nr) Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-ex posed Air control Normal control Vinyl chloride-exposed Air control 7.9 0.3 -- 5.0 0.' -- 9 1 i 1.6 -- -- 2.4 i 0.4 17.0 i 3.7 -- 7.8 4 0 4* <) 4 3 0 7,p 7.1 t 0 V 4.3 0 4' 0.7 0 6"' 4.5 * 0 7 7 8 0.4 9.1 * 1.7 8 1 i 1.7 ' i . o.: 7.6 0, > > *_ 0,2 17.1 4 1.7 13 7 l.l 1 5.5 4- 1.3 7.1 4 0.4" 10.2 + 0.6"' 6.9 0.4 4.2 4 0.4" 6.3 0.5*3.' 0.3 7.5 4 1.0 9.7 + 0 T 6.3 t 0.7' 1.9 4 0.7 2.4 + 0.3 1.9 4. 0.2 19.7 1.3* 15.3 + 1.3" 19.6 + 7.7 69 0.4* 1 1.4 1 0.6"' : 9 1 0.3 4 S * 0.3" S 9 i 0.7"' 5 3 i 0.4 7.7 * 1.0* 11.0 1 1 3*X 4 .* 0.9 2 4 i 0.3" J.7 i 0 I*' 2 \ t 0.7' 15.5 * 1.4* 10 b i.o*14.3 i 1.8' 7. 2 x C X JC O * Rais were exposed to 28,000 ppm of vinyl chloride; normal controls and iht air controls were exposed to air only, liach number represents the mean am) the SUM from a group of six rats. * Normal vs vinyl chloride-exposed, p < 0.05. f Air control vs vinyl chloride-exposed, p < 0.05, J Vinyl chloride (6 weeks) exposed vs vinyl chloride (2 and 4 weeks) exposed, p < 0.05. Kf:,, 882*20 S9U -s,Viw Sil VINVI CIII.ORIDP. l-FI-'hCT ON hNZYMIS was significantly lower ihan controls after 6 weeks of exposure to vinyl chloride (Table 1). No differences were found in the hepatic mixed-function oxidase activity or in the serum clinical liver tests. After 2 weeks of exposure, the two control groups had gained weight but the vinyl chloride-exposed group did not (Table 2). After 4 weeks of exposure, the normal control group housed at the an imal care center had gained significantly more weight than either the air-control or vinyl chloride-exposed group. After 6 weeks of exposure, however, the vinyl chloride-exposed group failed to gain weight; the nor mal control group gained more than the aircontrol group (Table 2). Morphological examination revealed poly hedral hepatocytes arranged in irregular plates interposed by vascular sinusoids in the livers of the control rats. This general cytoarchitecture was maintained after vinyl chloride exposure. Hepatocytes in control rats contained a prominent spherical nu cleus, numerous ovoid mitochondria, stacks of rough endoplasmic reticulum (RER), some aggregates of smooth endoplasmic re ticulum (SER), and varying amounts of ly- sosomes and glycogen particles (Fig. 2a).Few interstitial cells were scattered among the hepatocytes. These cells contained few cy toplasmic organelles and could be readily discerned at the light microscopic level by their hypcrchromatic nuclei. The sinusoids were linked by fenestrated endothelium and some of the lining cells displayed phagocytic activity. After 2 to 6 weeks of vinyl chloride exposure, the principal organelle affected appeared to be the endoplasmic reticulum. Cistcrnae of the RER became dilated in a relatively small population of the hepato cytes in the 2-week treatment group. Four weeks after exposure to vinyl chloride, patches of dilated endoplasmic reticulum were prominently displayed in some hepa tocytes (Fig. 2b). At this stage the SER was relatively unaffected. In the 6-weck treat ment group, vesieulalion of SER and dis tention of RER were easily discernible in a large number of hepatocytes (Fig. 2c). How ever, other cell organelles showed no de monstrable change. These changes, though, are still beyond the resolving limit of the light microscope. The nonhepatocyte com ponents showed minimum changes which !i TAHLU 2 Body Wunutrs or Rats bllokl and altlk vinyl Ciilokiul Lxposukl" Duration (week) Treatment Initial weight <g) Final weight (gi Percentage gain 2 Normal control 400 15 433 - 16 VC-exposcd 405 - 12 396 14 Air control 396 - 17 414 * 18 4 Normal control 398 - U 450 - 15 VC-cxposed 410 i 16 421 T 15 Air control 395 - 10 419 * 5 6 Normal control 402 * 14 486 * .14 VC-exposed 396 - 9 398 = 10 Air control 398 x 14 449 4 8* 5' 13" 3' 6" 21 <i*` 13" * Analysis of body weight was by regression analysis followed b) an analysis of variance on the residuals from [ the regression equation. (Residual e observed final weight -- predicted final weight from regression equation.) -Sid. * Norms) control vs vinyl chloride exposed, p < 0.05 , -hi-'l Iv ' Air control vs vinyl chloride exposed, p < 0.05. ' Air control vs normal control, p < 0.05. 11991 VINYL CHLORIDE EFFECT ON ENZYME.S 7 were characterized by an increased accu mulation of lysosomal-like substances in some of the sinusoidal lining cells as well as a greater tendency to accumulate lipids in the interstitial cells (Fig. 2d). DISCUSSION Glutathione conjugation is an important pathway for the metabolism of potentially harmful electrophilic metabolites of xenobiotics. Studies by Watanabe et al. (1976b,c) indicate this to be the major route Tor in activation of the vinyl chloride metabolites. Glutathione-S-transfcrases arc a group of cytosol enzymes catalyzing the reaction of glutathione and electrophilic compounds to form less toxic and more water-soluble con jugates. Their activity during chronic ex posure to xenobiotics, like vinyl chloride, could be a key determinate in the ultimate outcome of such exposures us illustrated by the longer arrow in Fig. 1. The increase in hepatic GEST activity at 4 weeks and the later increase in GAST activity at 6 weeks suggested that in the earlier stages of ex posures most of the chlorooxiranc interme diate is being adequately detoxified. During the later stages of chronic exposure, more chlorooxiranc may become rearranged to yield more chloroacetaldchyde and, in turn, react with other available glutathione trans ferases or become further metabolized to chloroacctic acid. Alternatively, the exces sive chlorooxiranc could rearrange sponta neously to form chloroethanol and be further oxidized to chloroacetaldchyde, which in turn may react with glutathione, or be oxi dized to monochloroacetic acid (Johnson, 1967). This would be consistent with the later increases in the aralkyl-transferases and the finding by Hefner et al. (1975) that monochloroacetic acid is found only in the urine of rats exposed for an extended time to higher levels (5000 ppm) of vinyl chloride. The increased use of alternate pathways, for chlorooxiranc and chloroacctaldehyde de toxification, may reflect increased concen tration of these active metabolites allowing greater opportunity for DNA injury. A single exposure to vinyl chloride de creased hepatic nonprotein sulfhydryl com pounds in rats (Watanabe, 1976a); similar decreases of glutathione concentrations were produced in rats by other xenobiotics such as 1,1-dichloroethylene (Jaeger et al., 1974a; Reichert et al., 1978) and acetaminophen (Mitchell et al., 1973), In the present study, repeated exposure to vinyl chloride caused a significant increase of nonprotcin sulfhy dryl concentrations (Table 1) analogous to the elevation of glutathione concentrations seen after the administration of carcinogens to rats (Fiala et al.. 1976). In addition, the results showed that repealed exposure to vi nyl chloride also caused an increase in he patic glutathione-S-transferase activity (Ta ble I) similar to that seen after the administration of phenobarbital and 3-melhylcholanthrcnc to rats (Mukhtar and Bresnick, 1976). These data suggest a mecha nism for compensatory synthesis of hepatic glutathione and glutathione-S-transferases after repeated exposure to vinyl chloride. The decreased concentration of cyto chromes P~450 found in rats after repeated exposure to vinyl chloride (Table 1) is con- FiC. 2. (a) Portion or a hepatocyte from control. Slacks of rough endoplasmic reticulum (RER) are separated by many ovoid mitochondria (M). Chromatin is finely dispersed in the nucleus (N). 9300X, (b) Hepatocyte after 2 weeks of vinyl chloride exposure. Dilation of RER appeared widespread in these two cells. Bile (B) canaliculus appeared unaltered in these rats. 5300X. (c) Four weeks after vinyl chloride exposure. Golgi complex (G) appeared unaffected while cisternal dilation continued. Distinction between SER and RER is complicated by the detachment or ribosomes. Lipid droplets (L) and glycogen (GL) often accumulated. 9500X, (d) A fat-storing interstitial cell is surrounded by several hepatocytcs in a vinyl chloride-treated animal. Unlike lipid stored in hepatocytcs. the shape of lipids (L) appeared irregular in these cells. gOOOx. X DU. TSENG, AND TaMBUKRO sistcni with work by Reynolds et al. (1975). This decrease in cytochromes P-450 content was also shown in vitro (Guengcrich and Strickland, 1977; Ivanetich et al., 1977) sug gesting that a metabolite of vinyl chloride destroys the cytochrome. Mixed-function oxidase activity, with benzphetamine as sub strate was unaltered. With regard to the structural alterations produced by vinyl chloride, the present find ings confirmed previous observations on the selective effect of this carcinogen in the en doplasmic reticulum (Du et al., 1979). A gradual increase in the number of hepatocytes affected and the involvement of both smooth and rough ER were shown in this study. The endoplasmic reticulum is the pri mary site of protein synthesis and its dilation suggested the presence of a vinyl chloriderelated effect. The lack of a concomitant Golgi hypertropy is noteworthy since this organelle serves as the site of glycosylation and packaging of many exportable proteins. It may be inferred that the vinyl chloride effect is mainly an intracellular phenome non, The relatively late involvement of SER could reflect a further attempt at detoxifi cation by the hcpatocytc. The tendency for the interstitial cells to accumulate lipid and the heightened phagocytic activity correlate with increases in collagen formation and may be evidence of low-grade cellular injury. This may prove to be the initial histological response to vinyl chloride exposure. Ultrastructural responses to chronic in halation of vinyl chloride were reported by Feron et at. (1979). Unlike our findings, the principal effects observed were swollen mi tochondria and some proliferation of SER. The mitochondrial swelling presumably re flects the extensive vacuolization observed by light microscopy. The discrepancy prob ably resulted from differences in the dose and duration in vinyl chloride exposure since Feron et al. (1979) exposed rats to 5000 ppm vinyl chloride for 52 weeks. Such an exten sive mitochondrial lesion could result in bio chemical modifications such as a change in ATPase or cytochrome oxidase levels. The only enzyme activity measured, glucosc-6phosphatase, was reduced. The authors believed that the altered glu tathione metabolism, as reflected by the in creased nonprotein sulfhydryl content, and the increased activities of glulathionc-Stransferascs and glutathione reductase, in rat liver after repeated exposure to high doses of vinyl chloride represent an early hepatocellular adaptation to vinyl chloride exposure. ACKNOWLEDGMENTS The authors wish to express iheir gratitude to the staffof the B. F. Goodrich Plant in Louisville, Kentucky, for their cooperation in the exposure studies, to Dr. Richard A. Greenberg for his review, to Mr. John P. Sandaz (or his help with the statistical analysis, and Mr. John Krcislc and Ms. Debra S. Eades for technical as sistance. REFERENCES Bakbin, A., BtttstL, H., Croisy, A., Jacouignon, P., Malaveille, C,, Montesano, R.. and Bartscii. H. (1975). Livcr-microsome-mcdiated formation of alkylating agents from vinyl bromide and vinyl chlo ride, Biochem. Biophys. firs. Commun. 67, 596-603. Bolt, H. M,, Kappus, H., Buciiter, A., and Bolt. W. (I97S). Metabolism of vinyl chloride. Lancet 1, 1425. Bolt, H, M.. Kappus. H., Kaufmann, R,, Appel, K. E.. Buciiter, A., and Bolt. W. (1976). Metabolism of "C-vinyl chloride in vitro and in vivo, laserm Sym posia Ser, 52, 151-164,1ARC Scientific Publications No. 13. Carlberg. 1.. and MaNnervjk, B. (1975). Purifica tion and characterization or the flavoenzyme gluta thione reductase from rat liver. J. Biol. 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