Tissue uptake of paraquat and diquat

(;en f'harmac., 1977. Vol 8, pp. 173 to 176. I'erfamon Press. Printed in Great Britain

MINIREVIEW

TISSUE UPTAKE OF PARAQUAT AND DIQUAT M . S. ROSE AND L. L. SMITH

Biochemical Mechanisms Section, l.C.I. Central Toxicology Laboratory, Alderley Park Nr. Macclesficld, Cheshire SKI0 4TJ, U.K. (Received 25 February 1977)

INTRODUCTION

Paraquat (1,1 dimethyl 4,4' bipyridilium) and diquat (1,1' ethylene 2,2' bipyridilium) are closely related herbicides. They have similar chemical and physical properties and have a similar mode of action against green plants (Calderbank, 1968). Paraquat has been responsible for human deaths as the result of either the accidental or intentional ingestion of the concentrated product sold to agricultural users. In these cases the most commonly affected organs are the kidney (Oreopoulos et al., 1968) and the lung (Bronkhorst et al., 1968), with lung damage often described as the major factor in the death of the patient. The toxic effects of paraquat in the rat were first described by Clark et al. (1966). As in man, the lung is the organ most severely affected and the damage contributes largely to the death of the rat. Diquat, in contrast, does not produce lung damage in the rat (Clark & Hurst, 1970) although it has approximately the same LDso when administered systemically (Clark et al., 1966; Clark & Hurst, 1970). In the studies reviewed here, we have attempted to explain (1) the difference between the toxic effects of paraquat and diquat in the lung and (2) the selective toxic effect shown by paraquat for the lung compared with other organs.

high concentrations of both bipyridyls, greatly in excess of the concentrations in plasma (Rose et al., 1976) and these concentrations in the kidney remained high throughout the 30hr of the studies. The concentration of paraquat in the lung, however, started at about the level of the other tissues (including the plasma) and then increased slowly so that after 30 hr it was 6-7 times higher than the plasma (Smith et al., 1974). There was no similar increase observcd in the concentration of diquat in lung (Rose et al., 1976). Apart from kidney and lung, there was no evidence that any other organs accumulated either paraquat or diquat to any significant extent (Rose et al., 1976).

THE UPTAKE OF PARAQUATAND DIQUAT INTO RAT LUNG SLICES

In order to study the mechanism of the apparent selective uptake of paraquat and not diquat into rat lung, lung slices were incubated in media containing various concentrations of either ~4C-labelled paraquat or ~4C-labelled diquat. The amount of diquat found in the lung slices incubated in media containing 10-SM diquat remained constant (the slice/medium ratio being between 0.5 and 0.8) over the time of incubation (Fig. 1). In contrast there wag a linear increase in the slice/medium ratio with slices incubated in T H E IN V I V O DI.KrRIBUTION OF PARAQUAT 10-SM paraquat and by 2 hr the slices had accumuAND D I Q U A T lated paraquat to concentrations over 8 times that Sharp et al. (1972) first demonstrated that following of the medium (Fig. 1). This time-dependent increase intravenous dosing Of paraquat to rats, the lung had in the amount of paraquat in lung slices was inhibited the highest concentration compared with other by the addition of KCN and iodoacetate or when organs and appeared to retain the compound after the incubation was carried out under nitrogen (Fig. the plasma concentration had fallen to low levels. 1). We have concluded therefore that paraquat acThey also showed that following intravenous dosing cumulation by slices of rat lung is dependent upon with an equimolar amount of diquat, the concen- energy production. The rate of uptake of paraquat by slices of rat lung tration of diquat in the lung was much lower than that of paraquat despite the similarity of the plasma was shown to be dependent upon the paraquat conconcentrations. Retention of paraquat in the lungs of centration in the incubation medium and the process mice following intravenous administration was also obeys saturation kinetics (Rose et al., 1974). It is interdemonstrated by Litchfield et al. (1973) and in the esting to note that slices of lung in vitro accumulated rabbit by llett et al. (1974). A "transient" uptake of paraquat at the rate of 3 0 4 0 nmole/g wet weight tisparaquat by rat lung following oral dosing was sue/he when incubated with 10-SM paraquat (Rose et al., 1974), whereas in rivo, the rate of accumulation reported by Murray and Gibson (1974). We subsequently carried out detailed measure- was 4--6 nmole/g wet weight tissue from a plasma conments of the tissue distribution in rats following oral centration of approx 10-~M (Smith et al., 1974). We doses of 680/~moles of paraquat or diquat/kg body have suggested that this difference may in part be weight. In these studies, the kidney rapidly achieved due to the presence of endogenous inhibitors in 173 f;,I,. 83 C

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F i g I. f h c ell'cot of metabolic mhihitors and nitrogen atmosphere on the accumulation of paraqual. The medium

contained: (ll) paraquat Ill) SM); (O1 diquat (10 s M): (;_7) paraqual (lO 5 MI phis K C N (lO " M) and iodoacetale(10 ' 3 M i : ( i ) paraquat l l O S M l p l u s K C N Ill) )Nil and iodoacetate(10 ~ M i a d d e d after I h r m c u b a t i o n - ( A ) paraqual (tO s M t in a rlitrogcn atmosphere. The resulls arc expressed as mt2an t ..~.t{.ltv|. t~itll four deternlinations per point

plasma which compete with paraquat tor entry into the lung (Lock el al.. 1976). INVESFIG4I'IONS 01." "tile BINDING OF" PARAQL A t "10 I.t N(;

Since paraquat was shown to hc accumulated b) the hing {Rose el a[., 1976) but not to be covalently bound h) lung macromolccules (lieu et ,/.. 1974) we explored the possibility that thc accumulation and retenlion of p a r a q u a t might be d u e to a reversible binding phenomenon. In a se.ics of unpublished experiments with paraquat and lung homogcnales, we were unable to lind any evidence for reversible binding to macromolecules using the technique of equilibrium dialysis as described by Rose (19691. Furthermore. when lung slices which had accumulated p a r a qt, al for several hours v,e,'e genii 5 homogenised, the paraquat was found in the supernatant fraction. When this was passed through a column containing Sephadex G25 gel. the elution volume for the paraqual was not significantly different fiom that for paraquat dissolx, ed m buffer (Jaques & Smith, unpublished dalai, indicating that paraqual was nol bound to any small molecular weight material. Thus it appears that the accumulation and retention of paraquat by lung is not related to any binding phenomenon. TIlE I ' I " I ' A K F ) el." P.%RAQL AT ANt) D I Q I I A T B~' VARIOI;N r.r%l" "l'lS%I E SI.I('ES

Following the discovery of ene,g)-depcndent accunlulation of paraquat by sliced rat lung, slices c,f ',arious other rat organs were examined for their ability to accumulate paraquat and diquat. Only slices of rat brain cortex appeared able to accumulate p a r a qt, at in a lmear fashion although the rate of uptake was I0",, or less of that observed with lung slices (Rose c t a / . . 1976). This uptake was shown to be energ)-depcndent {Rose eta/.. 1976}. l)iquat was not accumulated significantly by any tissue slices with the

exception of tho~,e I'rom kMncv ~ith ~hich nlaxunal slice nledium r:.tlJOs o { 2 were obtained. I"hi, uplakc was rapid compared with lllal of lung ~lice~ and ~a,, complete by I hr (Rose cta/.. Ig76L Although slices of ral brahi corlc\ accumulalcd paraqtiat, the brain dM not accunlulaic i~al~lquai in lil'O (Rose ('l (ll.. 19761. This is pl'obab[} due I0 the rdlc of ihc blood brain harrier and dClllOllSlrillCS the limitations of tissue slices in predicting di',;llibkllh)ll m rite. [{quail}. ahhough the kidne\ COllCentralcd both paraquai and diqmiI m (.71.o. uptake b', slicc~ i#l I'itro %%.asrchttivel,, poor iRose ('t
l.ung slices from dog. monkey, rabbit and man have been showri to possess itle abilit\ to accumuhllc paraquat and this process has been shown to obey salutation kinetics. The K,,,'s h x the process ,ire all in the range 1() -a 10 5 M but the ll',,.,x'S range I'roln lO (for dog) to 3IX) {tor irlttn and rail nln(iics of p.:ilaquat accumulated.g lung per hr (Table IL l.ung slices fiom the rat. therefl)re, beha'.c nlost like those from man. ( ' I I A R A C I E R I Z A ' I I O N OF t I l E ~;Sy,II.:M PRESE'~,I IN I.L N(.; fOR -%('('t'ML t.ATIN(; P.kR,kQ! %1

The lung is known to play an important r61c in the regulation of the blood levels of several ~asoactive amines (Vane. 1968). Studies using isolated pcrfu,,cd hings have sh(mn that the endogenous amines 5-Mdroxytryptamine [Alabaster & Bahkle. 1970: Junod. 19721 and noradrenaline (Gillis & lwasav, a. 1972) are accumulated by the lung. We examined, ihcrctbrc, the effects of a range of endogenous ttmines on the :toc u m u l a t i o n el + paraquat b). slices of rat lung+ and sl-lowed that both 5-hydroxytr).ptammc (5lIT} and noradrenalirie v,ere able to inhibit (Lock ¢'t al.. 19761. Several drugs inchidirig propranalol, imipraminc. betazole, bt, rimimide and cll]orpheniramme were also shown to inhibit the accumuhttion of paraquai m vitro ( l . o c k et al., 1976}. Howe~er, the }I~ antagoifist chlorpheniraminc inhibited uptake ~hilst the l l~ antagonist mepyramine did not, and the tie antagonist burimimide inhibited whiNl file H_, anlagonist metias i d e did not. mdicatmg that inhibition is iltd rchltcd tO lhe pharmacological ;tctivilv of these C('qllpOtltldx.

Table I. Kinetic conslaillH ft)r the acCtllntllaIloll e l p;lraqnat b} hn].~ slices from dJffu'rcnl Sl'.:cic~, If,,.,. (nnlolcs o[ paraqu:atg h m b, per htg

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Dog Monkc.~ Rabbit Man Rat

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Tissue uptake of paraquat and diquat

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lablc 2. Uptake of paraquat or 5-hydroxytryptamine by slices of lung from rats killed at m,crvals after an intravctmus dose of paraquat l i m e after paraquat {hr) () 2 16

Paraquat uptake (nmolesg per hr) " 36.9 + 1.5 17) 35.5 ± 1.3 (10) 18.1 -~ 1.1 110)

Inhibition of paraquat uptake by compounds such as 5141 cannot he interpreted as indicating unequivocall} that the inhihitors are themselves accumulated by the same system, or that paraquat uptake is via that system which has been idcntiticd for the uptake of the vasoaclixe amines. We examincd, therefore, thc ability of lung slices to accumulate 5HT and shov, cd that as with the isolated pcrfused lung preparation. the c o m p o u n d is accumulated. The process in slices obeys saturation kinetics, is inhibited by metabolic inhibitors such as KCN. and is sodium dependent (Smith ct it/.. 1976). In this last properly, however. the uptake ploCCSs for paraquat dill'ors in that it is considerabl} stimulated m the absence of sodium ISmith el al.. 1976). This increase is not due to nonspecilic binding of paraquat it) the absence of sodium since ahnost all of the enhanced uptake can be prevented b.~ addition of K ( ' N qSmith et al.. 1976). 1-urthcr c~idencc that the uptake systems for paraquat and 5lIT are different lms been obtained by comparing the accumulation of these compounds into lung slices taken from rats which had previously been dosed with paraquat. It is known that following the administration of paraqt, at to rats. it is the type I and type II alveolar epithelial cells of the lung which arc tirst damaged (Kimbrough & Gaines. 19"70; Vileyaratnarn & Corrin. 1971; Smith & tteath, 1974). Sykes et al. (1977)haxc shown ultrastructural changes in the type I and t}pe 11 alveolar epithelial cells 4 hours after the intravenous administration of 65 innolcs of paraquat kg b o d y weight to rats which progressed ',~ith time. B} 16 hours there was significant dcstruclion of [hese cells, l.ung slices taken from rats ,,a,ious times after this dose of paraquat progrcssivcly lost their ability to accumulate paraquat whereas thc uptake of 5HT into these slices was unaffected ITable 2~. Since paraquat accumulation into lung slices taken two hours after intravenous dosing is simiktr to that in lung slices taken from control rats {Table 2) it is clear that it is not the presence of paraquat in the slicc per se which inhibits the accumt, lation process. Hence 16 hours after dosing, when the c o n c e n t r a t i o n o f paraquat in the lung is less than at 2 hours lSharp et al.. 1972). the inhibition of paraquat uptake is probably a consequence of the destruction of cells which have accumulated paraquat. Tht~s we have concluded that the system present in the lung for the accumulation of paraquat differs f i o m that ~hich accumulates 5 H T , and is probably present in the type I and type I1 alveolar epithelial cells. The cytoplasmic concentration of paraquat achie,,ed in these cells will be very high and the metabolic consequences that follow will include oxidation of N A D P I I . production of toxic oxygen radicals and subsequent cell death (Smith L. I.. & Rose M. S.. in preparation).

5-Hydroxytryptamine uptake (nmoles.:g per min) 0.9 -.- 0.1 (41 1.13_+ 0.1 (4)

SUMMARY It is now possible to conclude that the major difference between the toxic effects of paraquat and diquat in lung is a consequence of the selective accumulation of paraquat by lung. The lung has been shown to possess an energydependent uptake system fbr paraquat which other organs (with the possible exceptions of the brain and kidney) do not possess. The uptake system for paraquat has been shown to be different from that responsible for the accumulation of 5-hydroxytryptamine and is probably located in the type I and II alveolar epithelial ceils. The r61e of this system in the functioning of the lung is unknown. REFERENCES

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17+'+

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