The Effect of Modifiers of Microsomal Electron Transport On Carbon Tetrachloride Hepatoxicity

ACKNOWLEro EMEi"JTS TABIE OF COHTENTS LIST OF TABT,F:S • • • • LIST OF FIGURES • • • llJTRODUCTION • • • • LITERATURE SURVEY • The P'11ospholipid Hyy:othesis 1'he Nitochondrial Hypothe0is The Catecholcir.ri.rw H:,rpothesis • • • • • • • • • • • • • • • • • 0 • • • • • • • • • • • • • • • • . -. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 'l'he Lipid Pc:ro::;d_dation Hyp'.)thesis Alteration of c arbon tct1·achloride Hicrosor11;U Electron TTansport S-yste!'ll hep::>.totox.-i_c:i.ty by drugs • EXPERIHE:ITAL ~ • • • • • • • • • • Haterials • • • • • Caroon Tctr·achlodc.e Ex:pos-0.re • Collection of Biolo;$iCDJ. fo;;2p10s Serur.i • • • • L:i.vcr P-re_p<).r~.tio~1 of Ll vcr ViicrocoEtos • In Vi tr·o L·i cub~tion P-.c0e;e:c1JX'.'e • .Ar.rlJ101-~~v·1·5~r1~.: cl t:;.:·;~etb.yJ_o._·(. ~. on P-Yli t,1,)t1 lJisoJ.c cJ.c! :ie:·~·.! ~:.,.-l ~"t· .... io11 • • • •

LITERA'i'UHE SUR VE:i The toxicity of carbon tetr8.chlodde was recognized shortly after its introduction as an anesthetic by Simpson, the discov8rer of chloroform anesthesia (Robinson, 1946). The inhalation of cI'J.oroforn or carton tetrachloride frequently produced. an acute yellow atrophy of the liver that ofte.."1 proved. fatal. While both age."1ts produce si.!li.la.r pathologicct.l changes, car'con tetrachloride is cons:id8rably r.!ore toxic (Drill, 1952). Chloroform has bee.."rl used for ane1:;thesia but is aJ;;10st nGver used for that p·J.rp.:>se today. Although c arbon tetrachloride ¥Tas used for so:n~ tin'3 as an f..,1thell:U.nthic (Hall , 1921), it h2..S no~-w be0n rcplacBd by more effec·U ve and less t?.>xic compounds .  Drill, 1952;and Recknagel, 1967). The peripheral cells are the first exposed to car"bo!1 tetrachloride and yet are not usually involved in the overall pathology (Drill , 1952 An ea.rly approach to the study of ca-r'txm tetrachloride hepatotorlci ty involved the use of ciecnts effective in preventing or decreasing the severity of the lesions in an effort to clucid.:>.te the mechanism of tox::i.ci ty. }iuch of the evidence acctunula.ted f:rori such investigations suggested that antioxida.'1ts were particularly effective in reducing the pathological consequences of carbon tetr2.chloricle poisoning (Recknagel, 196?). Hove (1948) reported that rats on vi tarn.in E deficient diets were more susceptible to ca:cbon tetrach_1oride poisoning than rats fed diets &1.l.pplemcnted with alphar.:tocopherol. F..ove (1953)  th2.t CEtl'oon tetra chlo1 1 icle :i.s metaoolize<l to the trichloromethyl fi'Ce p:>lysomes isolated from rat liver after car"bon tetrachloride pQisoning were disaggregated and had depressed protein synthesizing capacity 2...s ascertained by incorporation of mm.no acids into protein 5.n vit£Q.. How-cYer, ca.r"bon tetrachloride in vitro produced no such effects . The metabolism of carbon tetrachloride appeared. to be a prerequisite for the ef .• feet.
Using diene conjugation absorption as an indication of lipid pero:rld.ation, Ghoshal and Recknagel (1965) showe:d that the loss of glucose-6-phosphatase activity fro:.71 a rtlcl .. osomal preparaticn in vitro was paral-  The ability of this enzyme to reduce cytochroT'le £ in vi..tro provides a convenient method for the quc:>riti tation of enzyme D.cti vity , T"no in viy_q_ electron acceptor for NADPH cytochrome £ reductase is not known .
Liver microsomes 2~lso contain a hemoprotoin which was first described. by Klingcnburg (19.58) and is now generally referred to as CO-  . !   x.idation was reported as the cliffe:renc.;l betwee:n the rneai1 absorbmce at 240 nm for mj_crosornal lipids from car"oon t etr.:i.ch1oride exposed rats cind their respective controls . The mean optical densi ty obtained by difference is the diem' conjugation absorption .for peroxidized lipid at a final conc€:ntration of O.l per cent , which when multipl:i.ed by 10 , is reported as delta J2-:t (Rao and Recknc..gel , 1968 ) are presented in figure 1. In the phenobarbitcil (PB) pretreat ed anirnals which were exposed, SGOT and EGPT values were elevated two: fold when compared to salJne treated controls that were simi.la:l:'J.y exposed. However, the J-methylcholanth:cene (JHC) pretreated group had significantly J.ower (p(.0,5) value s of SJOT and SJP'f than the corn oil tre £~-Ced controls.
Hmmve:!'.', the SGP' r ;:md SJOT values for the J..:'1ctbylchol a11t.h."t'c:.r1c pretr.::e..ted groups were onJ.y 28 p3J.' cent and 57 per c ent , resp.~ct.i vely , of the cor n oil g1·oup Hhen l:ot.h p•oups w~"re eKposed to c a::.~hon t et.rachlol'-lc1e .  to centrolo'bular necrosis and hydropic degeneration (in aoout one hill of the liver lobules) in other rats. In the rats showing the most marked lesions, the injury was confined to only a few cells in the centrolobular areas and appeared to be much less intense than the morphological alterations .found in cininaJ.s receiving corn oil and carton tet-rach1oride~ The histological find:L-ig of less liver d.2mage due to caroon tet ..
re.chloride inhaJ.ation in the ~methylcholar.tlu·e.Yle pretreated animals than in the corn oil controls is of special interest since Reuber r.:t £.l· (1968 , 1970)      Whether carbon tetrachloride produces an incre~o e in lipid peroxidation tr.r ar:!tivating a lipid peroxidation shunt or by formation of toxic free radicals from interaction with intermediates in microsomal electron traJ1sport remains undetermined at this time. In either event , an increase in UADPH cytochrome .9. rod\.lctasc and CO-bindi."lg pigment bJ phenobarbital pret:i:·eatment wou1d account for the increase in the hcpatotoxic effect of carbon tetrachlo1~ide a.11d enhanced lipid po:cox:i.da.tion as reported b-.r Rao et a~. (1970). However , a_f'ter J-methylcholc:inthrcne induction an increase in CO-binding pigment without a concormnitant increase in NA.DPH cyl;ochro1:ie £ :a.·eductase was 2.ssocia:t.ed with decreased car'ron tet-   (1971) have r eported that acute exposure to car'OOn monoxide resulted in the inhibition in vivo of drug metabolism in rats as measure:l by prolonged response t o he:;r..obarbital and zoxazol2.1iune . Ho·rever, these authors did not conclude whether the effect ·nas clue to direct inhibition of CO::binding pigment or to induced tissue hypoxia. T'nesc .findings were the founda tion for ad.ditional experir11a'l'lts to deternl.i.ne whether or not caroon monoxide could mm.nee ill vivo lipid peroriclation and hepatotoxici ty due to carl::vn tetra..: chloride by virtue oi' its af.fini ty for CO-binding pig.-::en t . Ha ts were exposed to carton monoxide (approxinately 1000 pf."1 ) for 60 minutes t o reach EX 1 ttllibrium saturation. Carbon tct.rachlo1'ide l ms thD..   N.s. a.Anir.l~.ls were exposed to 9.58 ppm c.'U'bon Illono.xidc or hypoxi<.. for 60 minutes followed by c...xposuro for an additional 30 minutes to carbon totrD..c!°'..lor-ld.c vapors combined with c_,.rbon monoxide or hypoxia. (.05 N.s. e