Document pmpMD1Og6JLrOQwdeGMKJr3L6
1 W. llimpfel anil li. Hahermann
Interestingly, Dio perecnlngc of motoneurons afflicted (not more than 1 in til) was
Tory (m ill. The nnl hors jmijiose a neuronal migration from tho injected muscle
into tlio repiesentalivo ports of the spinal cord.Thoir observations are concordant
with ours. c-
References
Bizzini, 0., Turpin, A., Raynaud, M.: Production et puriliention do la toxino
tftnniipic. Ami. Inst. Pasteur 11(5, 08(5--712 (1909)
Fedinoc, A. A.: Absorption and distribution of tetanus toxin experimental animals,
in; L, Kckmann, ed: Principles on tetanus, pp. 109 -- 170. l'roe. of the Internat.
Confereneo on tetanus. Bern und Stuttgart; Huber Vertag 10(10
Gcmi-mian, Yn. Yn,, Kryzb.-movskv, G. N., lairinn, V. N., Mnts, V. N., Folgnr,
A. The- nucleic arid content of spinal motoneurons and pcriucuronnl glia
with increased functional activity caused by tetanrd toxin. Scchennv physiol.
J. U.S.S.K. 57, 1422-1428 (1971) Hahermann, 13.: Phnnnakokinctischc Rcsonderhcilen des Tclanusloxins und iliro
ItczirluHigen znr PaDingeiirsc des lolialeu bziv. generniiaierten Tetanus. Naunyn-
Schmicdchergs Arch. I'hmmak. 207, 1 -- 19 (1970n)
Hahermann, E.: Bin ticucs Priuzip zur quantitativun Beatimiming hochmolc-
kiilarer Antigenc (VcrknupfunpMcst) und scino Amvendung auf Tctanustoxin,
SciuninUumim und Ovalbumin. Z. Klin. Cltcm. Klin. Biorhcm. 8, 51--58
(1970b)
Ifabermaim, K.: Hist ribut ion of ,S4I-tctanus toxin and ,BI-toxoid in rats with
local tetanus, as influenced by antitoxin. Namiyn-Schmicdebcrg's Arch. Phar
macol. 272, 75--88 (1972)
Habermaini, 13.: Interaction of labeled tetanus toxin and toxoid with substruc
tures of rat luain and spinal coul in vitro. Kaunyn-Schmicdchcrg's Arch. Phar
macol. 270, 311--359 (1973a)
llabeimaijii, K.: Discrimination between binding to CiYS, toxicity and innnun-
rt activity of derivatives of tetanus toxin, tied. Microbiol. Immunol. 159,
89-100 (1973h)
Habcrmnim, K., Bimpfcl, W.: Distribution of '"I-tctanus toxin and mI-toxoid in
lots with geiiriuliml tetanus, os inllucnrcd by antitoxin. Nnunyn-Schmicdc-
hcig's Arch. Pharmacol. 27ti, 327 -- 340 (1973)
Habi imami, K., Dimpfel, W., Ritker, K. O.: Interaction of labeled tetanus toxin
villi substructures of rat spinal cord in vivo. Kautiyu-Rebiniodebcrg'a Arch.
Pharmacol. 27li, 301--373 (1973)
Haberumnn, K., Wellhiiner, H. 11.: Studies on the jiaHiogrncsia of tetanus with
Ii;,J-toxin. Badioaetivc tracers in mierohial imrmmolngy. IAEA, Vienna 1972,
pp. C7 --70 Pal.iv, S. I-., Ptilade, C.K.: The fine structure of neurons. J. biophys. biochcm.
Cylol. 1, 09-88 (1955)
St-ili, U. C., Heiisel, U., Wiegand.il., Wellhoner, II. If.: SupjKirting evidence for
a neural . ent of toxin in Die jiallirigr-iieaw of general tetanus in eats. Nniiiiyn-
Rehmiedelrt rg'H Arch. Pharmacol. 2715, 403--411 (1973)
Wcllhriner, 11.11., Hcnsel, U., Seib, U. C.: I.oca) telamis in cats; Ketirophammco-
kinetics of ,,'I-tetanus toxin. A'annj ri fichinicdebeig's Arch. Pharmacol. 27(1,
375-3SC (1973)
K. llaherniaun
Phurinakoliigisclics Institut
de.r.lustua Liebig -.Uiiiversiliit
1) 9300 GicQcn, Si.liuhertalraflo 1
Federal Bepuhlie of Germany
teuei svu
III. Decrease in Liver Non-Protein Thiol after Intrngastric Application of tlic Drug
G. Noack and J. Portig
Institut ffir Toxikologio und Fhurmakolngic der riiilipjis-Universitat Marburg
Received June 20,1973
. Summary, 1. Alpha-hexachloroeyclohexanc (alpha-IICll) reduces total nonprotein thiol In the liver of tho rat through a reduction in glutathione (C.'ll) content. Brain and kidney non-protein thiol is not affected.
2. Maximum reduction was by about 50/# 18 h after 400 mg/kg, the highest doso tested. At this time, tho decrease in liver non-profein thiol was linearly related to doso and to alpha-llCH-concentration in liver as a whole and in thr liver cytosol fraction.
3. Tim effect is thought to bo causally related to GSH-depcndcnt degradation of alphs-IICH in tho liver, previously shown to take place in vitro.
A"*y words; Alplia-Bcxachlorocyclohexane -- Biodegradation -- Liver Chita thionc.
Studies in vitro have implicated glutathione (GSII) as a cofietnr in enzymic reactions by ivhieh alpha-hcxncli1orocyeh>hexane (nlpha-IK'(l) is converted to hydrophilic substance in Dip rat (Kraus t( a!., 1973). The liver cytosol fraction, in particular, was shown to contain enzymic activity that catalyses a reaction between GSII and alpha-liCIf re-ulting, presumably, in the formation of a GSR-conjugate (Portig etal., I'.'T.l) This paper reports on experiments the result of which is thought to .nt>i some weight to these observations in that injection of alphu-JiCII ini. the rat's stomach is shown to lie followed by a transitory dccrca-e in I'.o GSlI-content of the liver.
Methods
Alpha-1,2,3,4,5,6-hexachloroeyclohexanc (atpha-llCH) was fror.i the b.iirl used by Portig ct til. (1073). L-glutnthionc (GSII) was purchased fmm 13. Men-k AG Darmstadt, yeast glyoxnhiae I from llochringcr Biorhcmiea, Tulxing. I'll ` reagent was obtained through Servix Chemicals, Heidelberg, methyl glwiwil throu.;' Roth Chemicals, Karlsruhe.
Anitnn! Experiments
Female Wfatar rata of 120--ISO g body weight (7 to 8 works old) sv.pplWl i> Zontmlinstitut fur Versuchstierzuelit, Hannover, were used. In the l.iber.iloi;
* Aided by Deutsche Forseh11ngsgeineinschaft.
j
' 'i !
< *1 -i ' >i * . . , . ;
tv' "A!l i'-lllirl II Ilf* j* ", + , ,i M I lip wafer.
' t it, t t . I........... > ! I IV
Alpji.t-JU'll. ii ,i*-<lisa`lvril in filin' nil (Olriim Olivurtim 1)A 117) amt fldminwvtvl liy stomach InlJ Tlu> vehicle linn found to produco a tlrni'ano in liver lk'n-j'rnTVlH thin) unittilt when given alone (Table 1). Hccausc of this, tlw (lose of oil van kept constant at lOml/kg ami 11011 -concent ra t ion hi the oil wan varied.
The animal* were killed In-1 ween 9,00 nnd 10.01} In the morning, this time of vrefreatoieiil being net accordingly. In order for the assays of tissue thiol to lie completed in 2 h, the maximum inimlter of rats killed on any one day waB eight. N'.'lmg was 1y decapitation; after cessation of reflex movements, the organs to ho c\ iniiiied were dissected out ami weighed. 2 g of liver was assembled from small pieces nit offeaeh lobe and wan put into ice'cold |>crchloric acid for estimation of tieu-proletu thiol; the remninder of the organ was used for determination of HClfcoiicrntrutioti ns appropriate. Drain and kidney* were taken in totn for estimation of thiol content. 1.3 min elapsed hetween picking an animal up and immersion of
t issue tit acid.
Analytical Procedures
Tissue itoit-protein thiol was estimated on extracts prepared an follows: 1 part of tissue was homogenized in 4.5 parts of I N 11CI04 with an Ultva-Turrnx hlendor and centrifuged at 12000 X gfor 10 min. 2.5 ml of tho supernatant waB added to 0.9 ml of 1.75 M Kjl'O,, left in an ice bath for 15 min, nnd filtered (Itcrnt and Derguieyer, 1070). Aliquots of filtrate were then taken for estimation of total thiol content or of total thiol anil of GSH content. Every assay was run in triplicate. Total thiol and ('.''II content of the extracts was found to remain constant for 3 h
after which time it started to decline.
Tut a) Ihiol was estimated using the method of Elliuau (1U5!>). Aliquot volume of extract added to the reaction mixture was 50, 100, and 200 pi for liver, kidneys and brain, respectively. For liver extracts from untreated ruts readings taken at 412 lim were 10 to 15 times a water blank. 10 and/or 20 pg of GSM freshly dissolved were routinely tested for colour formation and consistently gave a mean e4(1 of I39UH (Kilman: 1300**). Absorption at 412 nm increased an expected when known amounts of GS1I were added to extracts of liver Iadore or after removal of perch'orate, hut loss of endogenous thiol was not checked on.
GSli-content of liver was estimated on lOOpl aliquots of extinct using tho method of Wk-Und cl til. (1955) as modified by Dcrnt nml Bcrgmcyer (1970), 0.1 M phosphate ItufTer being used instead of water, r,lt was 3400 -- 3600.
.4Iphn-HCli --content of lirer was estimated using (1LG. Liver was homogenized in 5 vo). of water with a Voltcr-Elvchjem ty|ie glass homugciinter. The homogenate was weighed and divided in [tortious hy wciglit. One portion was extracted with 2 voi of cyclohexane, tlie other was spun at lOOOOfl X g{av)--140000 X g (max) for 90 min, and weighed aliquots of the supematunt, wliieh was called tho cytosol friction, were also ext rut ted w ith 2 vu) of cyclohexane. Conditions of assay were
those described hy Dm tig el of. (1973). Values for /* refer to application of Student's t-lest.
Results
Table I shows how the non-pr(item thiol eon lent of nit liver was found to chnn^o with lime during the 2-1 It following oral application A 50, 200, and -100 mo/kg of alplurltCH. Olivo oil, thu vehicle used fur
till 11 tun , l.i pi Cull I .ml .
8UCt SSU
Table 1. Non-protein thiol content (pEq/g) of rat liver after three liff.-ii-nt d.j,e* of alpha-hexnehlonwyelohevHiir
Figures aro menu S.I>.; tiunilier of estimation* in brmk'.-i*
Time after appli cation
<>)
Olive oil only (10 nil/kg)
Phi* alpliu-lh.'II
0.5% (= 60 mg/kg)
2% (= 2u0 mg kg)
( - 0 -tm;.')-)
0 7.60 0.70 (63)
6 6.26 0.56*112) 5.40 0.46 (1) t.22 0.42 (4) 5.9s -- 0.5s (4)
12 6.78 0.92* (24) 5.77 l.ti-l** (8) 4.5u: . 0.7ti* * (10; 4.so 1
(s)
18 7.16 0.61 (27) 6.53-0.St**(l2) 5.55_ (l.xii** (12; 4.n7 . u.701* (2n)
24 7.48 0.50 (10) 7.51 0.32 (4) 0.19.^0.53** (x 6.i.i2 i.rr (9)
* Untreated rats. * J*<.025 fur difference to untreated rats. ** /'< 0.025 for difference to rats given the vehicle may.
Total non-protein thiol hr the livers of w/ttn-ah-d r.its v.iriid I > -mac extent
(range 6.89--S.Sti pEq'g in 6.1 animals), hut this variation .is well a- th.- u, m
(see Table 1} are iff the order riser,tly repmli*! front ot!e t 1,1.. ,i i /11 V.)
anil Chasseaud, 197<ij Jocelyn, 1972) for this spe.-ies. tSIl,
i.it'i
glyoxatasi' metliod, accounted for S'h4 -J 5.7% (.V t!*i of total r: -1T ri i! i,.I
in the livers of untreated, oil-treated, and nlph.i-IKTI tix uted lat-. i.!, t<t.:l
thiol content ranged froin 8.32 down to 3.21 ptxq g. Tin's, too, ei-nfii m- v i,!. : s
have found (see Jocelyn, 1972) and, in the context of tlii- study, ne .rt- tint de-
fall ill liver non-protein thiol brought about by alpli.i-IH'ft is due, pu-loTm'ii.'ntly,
to a full in liver GS1I.
At tent ton was paid to the influence that reduced intake of food >r. ty I: ou
liver GSH content (l.indan nnd Work, 1953). To monitor the iivtivi-b.it I it s
feeding behaviour between dosing and killing, body weight and relume liwr
weight were recorded and were i'omt>ared to body weight ion.-use or deer ease and
to relative liver weight in untreated rats that had or had not access to food for the
same period* of time.
Injection of olive oil alone into the stomach reduced ffvd intake som-wh it
for the next 0 h, hut the reduction was hy no means so large as to explain the fall
in liver GXH. When 1ICI1 was added lo the oil. feeding was impaired itv-ve, However,
this ouly became apparent alter the huge doses, and ii could not. In h-ctf, ;u n-m.i
for tho decrease in liver GSH. "E.g., the mean denes e Is It after -hsi me kg of
alplia lll-H (see Table If, the Inrgc.-I dose lesled, exceeded hy 1.S rtl\j s the iu-mii
decrease seen after 18 h starvation, xilien the niiinnls ln.l lost oil the ux.rage
I2g of their initial lavly weight, and their nirnii relative liver .wei'hi w.i-. 3. Id g1
l(Ki g us compared lo 4.3 p.'IOtl g in mm starn d tats of the sam,- age. lo eoutr i-',
18 h after 100 mg kg itlplia Ill'll the mean reduction in body weight v..n by only
3.6g, and mean relative liver weight wus 3.95g'loug.
G.Noack and J. Portig
^^7"Table S. Ding content (nmotrs/g) of rat liver after three different dose*
of nIpha-bcxachlorocyctohexnno
Figure* arc mean S.D.; number of estimations in brockets. I,iho a) gives drug content of wbolo liver, line b) drug eontent of cytosol fraction (equivalent of I g
of fresh liver)
Time aftrr application 0>)
6
12
18
24
ft
b
ft
b
ft
b
ft
CO mg/kg
133 10 (7) 8.0 3.0 (3) 115 14 (7) 6.1 0.5 (3) 64 16 (S) 8.0 0.5 (4) 70 5 (4)
200 nig/kg
232 79 (7) 21.3 6.3 (3) 304 08 (7) 10.5 1.3 (3) 341 05 (8) 21.2 4.6 (4) 290 22 (4)
400 mg/kg
668 88 (6) 49.7 11.0(3) 850 72 (4)
48.7 0.5 (3)
054 69 (8) 40.6 5.6 (7)
592 23 (3)
The decrease in liver non-protein thiol produced by alpha-llCII was significant at P< 0.025 or less (for difference to oil-treated animals) with ail three doses tested 12 and 18 h after application, and with the two large doses also at 24 h. Although impaired feeding behaviour con tributed to some extent, it was clear that the decrease was largely due to a different cause. A likely cause was thought to be consumption of GS1I in an enzymic reaction with alphn-lICH, shown previously to lake place in vitro (Portig ef/., 11173), and it was of interest, therefore, to determine the amount of alpha-llCII held hy the liver whilo the drug passes through it. This was done (Tabic 2) for liver as a whole and for its soluble fraction (cytosol) which, when tested hr vitro, appeared to contribute most to GSH-dei>c-mlcnt JJCJI-dcgradation in the liver cell
(Kraus etui., 1973).
Kol surprisingly in view of the hydrophobia character of alpha-IICH, moro
than 00*/(i of the total amount of drug contained in tho organ was always found
to be associated with the membranous particles. Also, whilo drug concentration in
the whole organ changed with time, particularly after the two largo duxes, drug concentration in the membralie-free fraction remained comparatively uonntnnt for the first 18 h.
Drug concentration in the whole organ is maximal about 12 h after application (Table 2), and it is after approximately this interval of time that, with increasing dose, the decrease in GS11 content becomes most marked. 18 h after application there was a si might-line relationship be tween drug effect and dose, and between drug rffeet and drug concen tration in the whole of (he liter as well as in the liver cytosol fraction (Fig. 1, lower curve). It may he worth noting Unit this curve intercepts
Alpha. JtexiicMorncyclohexnm) mid Liver Glulathsi::*
Cone, in liver cytosol
ij\ 6
2t2 nmoles/g CC E
Fig. 1. Alphn-IIClI-cffect on non-protein thiol content of r.it li\r i., r. l.v-'ii t<
dose and to drug concentration in the organ. Ordinate: l)if jem
_o j-, ]h. i
non-protein thiol content between rats given the drug mul ih r.'.-'ii t' > v.-!,i h
only. Abscissae: Doso of nlpha-IICIt injected into the stoic n h i'.-I. nlj i
IICll-concentration in whole liver (umolcs/g), and in livt-r i \ - ! f:
(nmolcs/g fresh liver equivalent). Upper curve: Sum of ePtviS nc-;..] ty |^
and 21 h after application in relation to dose. Lower cmve: L:Tcvi ,.,ii lx !i
application in relation to dose (X), to drug concentration in whole liver (j), not
to drug concent rut ion in liver cytosol (c)
the ordinate at a point > 0 which might represent an average x able up L which factors such as reduced intake of fond during the horns follow in. application of the drug contributed to the total drug effect on liver Gsi seen in nn individual animal. When (he drug effects seen 12, IS, and 21 l after application are added tip, their relation to do.-e is as shown by tin upper rurve in Fig. I. This relation may he expected to become moo nearly linear when n time interval greater-than the 21 h tested la te i studied, since (see Table 1) the return to normal of liver non-piohm thiol after the two larger doses of Ill'll is not yet complete after 21 h and it is slowest with the largc.-d dose.
Apart from liver, non-protein thiol eoidenl was deln initud iti hi -:i and kidneys 12 h alter oral application of 200 mg kg ami IS It afo : 400 mg/kg of alpha-] It'll. This was done because experiments with eigai
erne i s'su
homogenates had i mlionled Hint GSII-mediated degradation of n 1ICII may oeciir in these --and in other--organa (Kraus d al., loi.i;. However, brain content of non-protein thiol was found not to chnngo at all afler dosing with the drug, whilo in the kidneys total non-protein thiol appeared to even increase somewhat but this was not entirely convincing (J'2:0.05 for cither dose with N = 2x8); It may be added that ding concentration in the brain after oral application of 200 mg/kg was determined in se|iiiratc experiments and found to change with time exactly as it does in liver; it seems reasonable to suppose that the timecourse is not very different in the kidneys.
umsj.
Discussion
Dames cl of. (1050), in the course of their work on mcrcapturic acid formation in the mammal, were the first to show that administration to rats of hnlogcnnted organic compounds, which are mercapturic acid pre cursor's, lends to a fall in liver GSII content, and that the fall in liver GSII is roughly equivalent to the amount of mcrcapturic. acid produced. It was on the basis of this work, which has subsequently been extended (c.g. Borland and Chasseaud, 1069,1970) to include other types of mercapturic acid precursor, and in view of evidence (Fortig cl at,, 1973) that rat liver cytosol converts alpha-llClI to a hydrophilic metabolite in a GSII-dcpondont enzymic reaction, that the experiments described in this 'paper were undertaken. The finding that liver GSII decreases following injection of alpha-HCII info the rat's stomach, and the,good correlation that was found to exist between the extent of the decrease and the amount of drug administered as well as the amount of drug present in the liver, are consequently considered with preference for a causa) relationship with 11 Cl I-biodegradalion. More specifically, tho result of this investigation is taken to indicate that a reaction consuming GSII is of some quantitative imi>oitanco in tho biodegradation of nljihaJJCJ1 in the rat-though, presumably, less so than this apjxtars to he tho case in flies (Bradbury and Sianden, 1959; Ishida and Jhdim, 1965). Information on wliat proportion of the compound is, in.fact, excreted by a rat as pi odnet(s) of reaction with reduced sulfur is currently sought to be obtained.
In contrast to liver, brain and kidney content of non-protein thiol ap]aaied to be unaffected when high amounts of alplm-DCfl were present in these organs. This may be so either because GSII-mediated conversion of the drug in these tissues, which was found to occur under conditions in vitro (Kraus ef of., 1973), is of only minor importance, quantitatively, in the living animal or, alternatively, the role of GSU in HC11-degradation in these tissues might not be that of cosubslralo. This issue should be clarified through further investigation.
. I
Deferences
ifnrnea.M. M., James, S. I'., Wood, 1*. 11.; The furmatinn of ni*-r<--x|t urio arid-. I. Formation of mere-aptmic arid mid the b-v.-Is of gluintbiune in ti,.,ifi-s. J:iochem. J. 71, (ISO - Wm {I 'J.VJJ
Herat, K., Ik-rgimyer, It. L\: Melhodon der m/j mntbcli'-n Analy 2nd id.,
ed. by II. U. Ikignu-ycr, p. tWi.. Wcinln-hn '!Vr,;.tr.: Vr-rtig f'ln-i do J97u Boylaod, K., Cfiaasmiud, L. K.: Tim rote of glnUtliioiie mid g1nl,-ii|ii.,w.-S-ti-.iri..
fi-ruses in mercapturic acid biosynthesis. .Advime. Kn/ymol. !J2, i7.i-219 M'.'ipj
BoylamJ, E., Chasseainl, L. F: Tho effect of some c-m-honil compounds on r..' tivtr
glutathionelevels. Iliixhen). Pharmacol. J!l, I52t --132ii (19 "1
Bradbury, F. II., Staniltii.il.: Mctidmli^ni of ben/rne lew u ),!,,rid, |ty r.
bouseBits, Miisea domestics. Nature (bond.) lvf,
'j'l flO-Vij
Ellronn, O. L.: Tissue mdfliyiliyl groups. Arrh, liio' liem. liiophys. S2, 70 -- 77
(1059)
Isliid.i, M., Dahm, I*.: Metabolism of benzene lu-vur ldorid<- i-ouu-is uod i>-! ,i.-d
compounds in vitro. 1. Properties and dMi.'butiun of tie- unju,<-. J, 1\. Eiitomol. 55, 383-392 (ItHi.7) Jocelyn, 1*. C.: iiioelicinUlry of theSfl group, p. 291. London-Xew Yurk: .V- td- n,i l*rt>s 1972
Kraus, I*., Knack, G., Purlig, J.: Biodegradation of alpli.vlst-xiicldot-oeyrlikrxatr-.
II. Glutathione-mediated conversion to hydrophilii- fid,-', -me l,y p.n ti-il.Ue
fraclions of rat liver and by homogenates of variiv.i- rat wguit. N.nntyuSchnnVdel,erg's Arch. Pharmacol. 279, 199--2tt2 (1973) Lintlan, 0., Work, Elizabeth; Exjvrimental Imr neeru-is in rat--. 1. flnuo-- it;
liver, blood, and spleen glutathione and nscotbit arid tcxels in dietetic liver
necrosis, Eioebem. J. 55, 5.71--iiti2 (1953)
Porlig, J., Kraus, l*,, Brnlonmnn, S., Noafk, ti.: Hindi grad
of alpha-)i< \.i-
thlrtrocyi-lohrxnnc. I. C'dlitatliintieih-piiidriil nnivn-i.>ji to a )rid,oj,l,;'ie net ibolhe bv rat liver cytosol, Xmuiyn-.Schmiedt heiy's Aich. l`h iiJn.i-.-'l. 279, 155 -- 195 (1973) Wit-land, Tb., Dose, K., l'lleiderer, (!.: Fine eiizyiiintM lie lk-st immu tig dos Ghdithions. Biot-hcm. 7,. U2U, 412--445 (1953)
G. Xo.uk J. l'orlig
Jnatitut fiir Toxikologie und 1'ii irtn.iki-logic I'Jiifipps-l'nivri.'il.il
D-.M.uhtirg a.if. I.ahn I'ilgiiniileiii 2 Federal lleputdie of Gcnn.iny
*t