Comparative behavioral, biochemical and pigmentary effects of MPTP, MPP+ and paraquat in rana pipiens

Life Sciences, Vol. 37, pp. 1529-1538 Printed in the U.S.A.

Pergamon Press

COMPARATIVE BEHAVIORAL, BIOCHEMICAL AND PIGMENTARY EFFECTS OF MPTP, MPP+ AND PARAQUAT IN RANA PIPIENS A. Barbeau, L. Dallaire, N.T. Buu, J. Poirier 8 E. Rucinska Clinical Research Institute of Montreal, Montreal, Quebec, Can. H2W 1RJ (Received in final form August 12, 1985) Summary We demonstrate that injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenyl-pyridinium ion (MPP+) and Paraquat (PQ+> produce in Rana Piplens different behavioral, biochemical and skin pigmentation changes. MPTP causes in frogs the main symptoms of Parkinsonism (rigidity, akinesia and tremor) and it darkens the skin of animals. It also decreases brain and, less so, adrenal medulla dopamine. These effects are blocked by Pargylme. MPP+ causes the same symptoms but more rapidly. In contrast, skin pigmentation is clearly lightened. Brain and particularly adrenal dopamine reserves are nearly abolished. Pargyline increases these effects. Paraquat, in a cumulative fashion, eventually causes the same behavioral changes and a slight increase in pigmentation. It initially produces an increase in brain and adrenal dopamine concentrations, but later a significant dopamine concentration decrease. Pargyline potentiates these long term effects, blocks the dopamlne increase, but reverses the PQ+ effect upon melanin, producing the same depigmentation as MPP+ alone. 1-methyl-4-phenyl-1,2,3,6_tetrahydropyridine (MPTP) has been shown to produce a parkinsonian syndrome in man (1) and in various animal species, from monkeys (2) to amphibians (3). MPTP is metabolized to 1-methyl-4-phenylpyridinium ion (MPP+> (4,8), a compound structurally very similar to the herbicide Paraquat (PQ+). Since the frog model of MPTP action (3) has proved to be useful in producing clinical and biochemical changes similar to those seen in Parkinson's disease and even some pigmentary alterations, we compared the effects of MPTP, MPP+ or PQ+ upon these three parameters. Material and Methods The amphibians utilized for these studies were the frog Rana Pipiens, as opposed to Rana Clamitans Clamitans used in the previous report (3). The reason for the change was the short supply of the latter during the winter months. In preliminary investigations some differences were noted between the two species: R. Piplens are much smaller (mean 25g as opposed to 19Og); their base pigmentation is darker (melanophore index around 3.7 as compared to 1) and they have more numerous dark melanized patches on the back. However their response to MPTP is the same as that of R. Clamitans Clamitans. A total of 343 frogs were used (at a cost equivalent to that of one Rhesus monkey). Mean weight of the animals was 25g (range 20-32g). MPTP (free base, Aldrich Chemical Co.) was dissolved in water. Solutions were prepared fresh daily for lntraperitoneal injection. MPP+ was obtained through the generosity of Drs. I. Irwin and J.W. Langston. It was prepared daily in a manner identical to that for MPTP. Paraquat was purchased locally (as methyl viologene, 0024-3205185 $3.00 + -00 Copyright (c) 1985 Pergamon Press Ltd.

Life Sciences, Vol. 37, pp. 1529-1538 Printed in the U.S.A.

Pergamon Press

COMPARATIVE BEHAVIORAL, BIOCHEMICAL AND PIGMENTARY EFFECTS OF MPTP, MPP+ AND PARAQUAT IN RANA PIPIENS A. Barbeau, L. Dallaire, N.T. Buu, J. Poirier & E. Rucinska Clinical Research Institute of Montreal, Montreal,

Quebec, Can. H2W IR7

(Received in final form August 12, 1985) Summar 7 We demonstrate that injections of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP), l-methyl-4-phenyl-pyridinium ion (Mpp+) and Paraquat (PQ+) produce in Rana Piplens different behavioral, biochemical and skin pigmentation changes. MPTP causes in frogs the main symptoms of Parkinsonism (rigidity, aklnesia and tremor) and it darkens the skin of animals. It also decreases brain and, less so, adrenal medulla dopamine. These effects are blocked by Pargyline. MPP + causes the same symptoms but more rapidly. In contrast, skin pigmentation is clearly lightened. Brain and particularly adrenal dopamine reserves are nearly abolished. Pargyline increases these effects. Paraquat, in a cumulative fashion, eventually causes the same behavioral changes and a slight increase in pigmentation. It initially produces an increase in brain and adrenal dopamine concentrations, but later a significant dopamine concentration decrease. Pargyline potentiates these long term effects, blocks the dopamlne increase, but reverses the PQ+ effect upon melanin, producing the same depigmentatlon as MPP + alone. l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) has been shown to produce a parkinsonlan syndrome in man (i) and in various animal species, from monkeys (2) to amphibians (3). MPTP ms metabolized to l-methyl-4-phenylpyridinium ion (MPP +) (4,8), a compound structurally very similar to the herbicide Paraquat (PQ+). Since the frog model of MPTP action (3) has proved to be useful in producing clinical and biochemlcal changes similar to those seen in Parkinson's disease and even some pigmentary alterations, we compared the effects of MPTP, MPP + or PQ+ upon these three parameters. Material and Methods The amphibians utilized for these studies were the frog Rana P1piens, as opposed to Rana Clamitans Clamitans used in the previous report (3). The reason for the change was the short supply of the latter during the winter months. In preliminary investigations some differences were noted between the two species: R. Pipmens are much smaller (mean 25g as opposed to 190g); their base pigmentation is darker (melanophore index around 3.7 as compared to i) and they have more numerous dark melanized patches on the back. However their response to MPTP is the same as that of R. Clamitans Clamitans. A total of 343 frogs were used (at a cost equivalent to that of one Rhesus monkey). Mean weight of the animals was 25g (range 20-32g). MPTP (free base, Aldrich Chemical Co.) was dissolved in water. Solutions were prepared fresh daily for mntraperitoneal Injection. MPP + was obtained through the generosity of Drs. I. Irwin and J.W. Langston. It was prepared daily in a manner identlcal to that for MPTP. Paraquat was purchased locally (as methyl viologene, 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.

1530

MPTP, MPP + and Paraquat in E. p~p~ens

Vol. 37, No. 16, 1985

INHITION BY PARGYLINE (PARG) OF MPTP-INDUCED BEHAVORIAL CHANGES IN R. PIPIENS

(n:6frogs/group )

4

MPTP: PARG:

40mg/kg

2mg/kg

Qc

~o

2

MPTP ~/MPTP+PARG

'::)

| I- - O,-- -~'-- "'¢P- '-(~-- ~---O'--

C~

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--O-'-O--'-O--

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l

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DAYS OF INJECTION FIG. I-A

POTENTATION OF PARAQUAT (PQ+)AND MPP + EFFECTS BY PARGYLINE (PARG) IN R. PIPIENS (n=6 frogs/group) _ MPP +. lOmglkg PQ+ 40mglkg PARG 5 mg/kg

Q::

3

I1,~ MPP++PARG

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11 12 13 14 15 16 17 f8

DAYS OF INJECTION FIG. I-B

Vol. 37, No. 16, 1985

MPTP, MPP + and Paraquat in R. pipiens

1531

Sigma-) and used similarly. Control animals recezved water. All liquids were passed through a 0.22 pm f11ter to assure sterility. The injection volume was always less than 1.5 ml. The frogs were acclimatized for 2-3 days in a temperature-controlled room with fixed lighting (12 hours on, 12 hours off). The basins were always kept at a fixed distance from the source of light. The water was refrigerated by daily addition of crushed ice over the cage, which filtered down to the basins as it melted. The water level in the basins was kept uniform at 3 cm. Rest plateaus were provided in each basin. Before the dazly injection the animals underwent an observatlon and test sesszon. The frogs were placed on a table with constant illuminatlon and their displacements observed, tlmed and calibrated. This was followed by a series of test during which they were placed on their backs and held supine wlth one hand. Upon release, the time (in seconds) requlred to turn over (righting reflex or "flip-over") was recorded.

CUMULATIVE EFFECT OF MPTP, PARAOUATAND MPP+ UPON BRAIN DOPAMINE CONCENTRATION IN R PIPIENS

500 F

( n= 4~group-4 da,ly rejections)

~ 400I

F • 200I ¢~

z

PARAQUAT

100 ~

MPP~

MPTP

0

1 2 3 CUMULATIVE INJECTED DOSE

4

5

(mg)

FIG. 2 The Rua!it~ of movements was calibrated on a i to 6 scale for the flip-over test and a I to 4 scale for the jump. For the fllp-over test a score of i is normal (flip-over rapid, usually under 1.5 seconds, does not require external stimulus); 2: rapid, but rigid in the movements; 3: slow (> 2 secs) movement, also rigid; ~: rigidity, tremor but normal time; 5: same as 4 but wlth slow time; 6: unable to turn-over (maximum time of observation 5 mlns). For the jump test: ~: normal Jump without stimulus; 2: requires important stimulus to Jump; 3: crawling instead of Jumping, requires external stimulus to move; 4: no movement despite stimulus. Inter-observer concordance error for these tests has been found to be less than 5 %. There was a constant correlation between the results of the fllp-over test and those of the Jump test (r - 0.90).

1532

MPTP, MPP + and Paraquat in

R. pipiens

Vol. 37, No. 16, 1985

To determine the melanophore index, the method of Hogben and Slome (5) was used. Flve stages are recognized, stage 1 being the most aggregated state and stage 5 being the most dispersed (darkest). Verbal designation of these stages would be: i: punctuate; 2: punctuate-stellate; 3: stellate; 4: stellate-reticulate; 5: reticulate. As stated above R. P~piens, in winter, have a mean melanophore index of 3.7, higher than that of R. Clamitans Clamltans.

EFFECT OF CHRONIC DAILY INJECTION OF MPTP, MPP + AND PARAQUAT UPON BRAIN DOPAMINE CONCENTRATIONS IN R. PIPIENS ( n = 2 to 6 ~ g r o u p ) 600

r

5oo I Q=

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The monoamine oxldase inhibitor Pargyline (Sigma) was also given intraperitoneally. In R. Clamitans Clamitans, we had reported that doses of 80 mg/kg completely abolished the neurobehavioral effects of MPTP (3). We found that this dose uniformally killed the R. Pipiens frogs after I0 to 15 days. The anlmals then became dyspneic and "ballooned-out". A preliminary study showed that this effects was also present (after 15-18 days) at a dose as low as 5 mg/kg and that it increased, with earller onset, in a dose-dependent manner. However at 2 mg/kg, Pargyline was well tolerated by all anlmals for up to 30 days (maximum duration of our experiments). For the studies to be reported here, groups of 6 frogs were always used, for each test substance and each end point, unless otherwise specified. Statistical evaluations, when approprlate, used the student T test for paired data, or an analysis of variance (ANOVA). Brain and adrenal catecholamines were also determined. The animals were decapitated after a blow to the head, brains and adrenals immediately dissected and placed at -70°C. Ad3acent renal areas were also dissected as control tissue for the adrenal which, in the frog, are embedded in a groove of the kidney. The tissue ~Tas homogenized and catecho!amlnes determined simultaneously by hlgh perfor~'ance liquid chromatography (HPLC) with electroLhe~ical detection

Vol. 37, No. 16, 1985

MPTP, MPP + and Paraquat in R. p~p~en8

1533

after extraction by alumina (6). Because of inherent variation in the sensltivity of the HPLC method, approprlate controls were run with each experiment and on each working day. Results Behavioral studies In R. P1piens hlgher doses of MPTP than in R. Clamltans Clamitans were required to produce the components of Parkinsonlsm, but at 40 mg/kg the full plcture of rlgidity, slowness in initlatlon of movements and, in 30 % of anlmals, tremor was observed. This is best illustrated by the results of the fllp-over test (Fi$. l-A). As previously reported by us (3), MPP + is much more toxic and at lower doses (Fig. I-B) produces severe rigidity and a decrease in movements, before killing the animals. The herbicide Paraquat, at 40 mg/ kg, became toxic in a cumulative fashion. After 10-12 days the anlmals started to become rlgld and slow (Fl$. l-B)and most died by day 26. The quallty of rigldity and akinesla was the same for all three compounds. They differed only in the intensity and in the rapidlty of the reaction. On the other hand when Pargyline was injected simultaneously with the test substances, the reactlons were totally divergent. As previously noted in R. Clamitans Clamltans (3), Pargyllne also modified the neurologlcal and behavloral effects of MPTP In R. Pipiens (Fi$. l-A). However, it potentlated the toxic effects of both MPP + and Para]uat (Fig. IFB). This observation had not been prevlously reported. It has been repeated at various doses of MPP +, PQ+ and Pargyllne (including 2 mg/kg). The higher concentrations of the latter, the higher the resulting comblned toxicity (data not shown). Respiratory dlstress also became evldent with combined treatment after 14-15 days of injectlons. Biochemical studles in the brain The effect upon brain dopamlne concentratlon of cumulative doses of each of the three test compounds follows essentially the same pattern, but with differences in absolute values (Fi$. 2). In these experiments 4 injections daily of i0 mg/kg (40 mg/kg/day) were glven. At low doses, there is an increase in the braln concentratlon of dopamlne and to a lesser degree of other catecholamlnes (latter data not shown). With the three compounds, as soon as a cumulatlve dose of 1 mg is reached, brain dopamine concentrations start to fall, precipltously wlth MPP + and MPTP, but very slowly with Paraquat. These results, based on cumulatlve dose, were obtained over a maxlmum perlod of 5 days. The dlfferences in pattern are even more recognizable over long perlods of time. It is seen in Fig. 3 that MPTP causes a 90 % loss in brain dopamine concentration withln 2 days as reported by others (7), Pargyline alone (2 mg/kg) produced a i0 % increase in whole braln dopamine. With MPTP it prevented the decrease by 70 %. Desplte continued use of MPTP, there is no further reductlon in concentration. MPP + produces similar catastrophlc brain dopamine loss at any dose between 5 and 20 mg/kg with or without Pargyllne (data not shown). Other mechanisms of general toxicity are probably also in actlon when MPP + is used. Long term Paraquat utilisation is shown, for the first tlme, to produce a decrease in braln dopamlne concentration but not quite to threshold levels for extrapyramidal symptoms (3). Pargyline did not influence the latter results. The neurological syndrome observed after 12 days in the PQ+-injected anlmals must result also from other toxic events, in addition to the brain dopamine decrease.

1534

MPTP, MPP + and Paraquat

B1ochemlcal

in R. pzpiens

Vol.

37, No. 16, 1985

studles in the adrenal medulla

The adrenal medulla gland is known to be very rich in catecholamlnes, partlcularly noradrenaline and adrenaline. It also contains signlficant amounts of dopamlne. In fact the concentratlon of dopamine in the adrenal gland of the frog is i0 tlmes that in the whole braxn per gram/tissue. It has also be shown by Langston and collaborators (9) that the adrenal gland is the organ with the highest concentration of MPP + after in3ectlon of MPTP. It can be seen in Fx~. 4, that the three test compounds have different effects, after 5 days of injection, upon the concentrations of the varlous catecholamines with or without concomitant use of Pargyline. Thus MPTP slgnlficantly decreases dopamine and noradrenallne, but not adrenaline. Pargyline given simultaneously does not affect these changes. Paraquat, on the other hand, lncreases the concentration of dopamine and noradrenaline, but this effect is prevented by the simultaneous in3ectlon of Pargyllne. Finally, MPP + lowers the concentratlon of adrenal dopamlne to near zero whereas It only moderately reduces that of noradrenallne (by 50 %) and of adrenallne (by 48 %).

EFFECT )? 6F ~

MPP + AND PARAQUAT(+-PARGYLINE) UPON ADRENAL CATECHOLAMINES IN R PIPIENS DOPAMINE I C" CONTROLS (H20)

OF MPTP,

P PARGYLINE = 80mglKg MPTP : 40mglKg M +. MPP + =20mg/Kg Q PARAQUAT= 40mg/Kg

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M

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FIG. 4 Pargyllne, which itself has different effects upon dopamine concentration on the one side (increase) and upon noradrenaline on the other (decrease), does not prevent the complete depletion of dopamine after MPP +. It does however partially block the effect upon the other catecholamines. Because the other amines are not reduced in a similar manner, we can conclude that the dose related decrease in dopamlne after MPP + is a specific local toxic effect and not the result of general toxicity.

Vol. 37, No. 16, 1985

MPTP, MPP + and Paraquat in

R. p~p~ens

1535

Studles on the melanophore effect As seen in Fig. 5, the three test compounds have dlvergent effects upon the skin pigmentation of the frog as measured by the melanophore index, MPTP, as prevlously shown (3), markedly increases the dlsperslon and amount of melanln in the skin, including the palms of the feet where it is studied under the mlcroscope. This darkening of the frog skln perslsts untll at least day 18. Pargyllne (2 mg/kg) given slmultaneously partlally reduces thls darkening and eventually nearly abollshes it. When glven before MPTP, Pargyllne prevents the darkenlng reactlon (data not shown). On the other hand, MPP +, with or wlthout slmultaneous Pargyline, clearly decreases the skln pigmentatlon much below the normal state in R. P1plens. Melanln, which in that specles is usually in the stellate (stage 3) or stellate-retlculate (stage 4) state, is reduced to punctuate. Melanin bearlng cells are also decreased in number. Thls latter point is belng investlgated further in a hlstologlcal study to be reported elsewhere. Paraquat, alone, produces a slight increase in plgmentatlon (by 0.5 to i stage over the controls). When Pargyllne is glven slmultaneously wlth Paraquat, a clear-cut decrease in plgmentatlon (slmllar to that wlth MPP+) is then observed. Thls is a totally new flndlng.

EFFECT OF MPTP, MPP" AND PARAOUAT (PO)+_PARGYLINE (PARG) UPON THE MELANOPHORE INDEX IN R. PIPIENS ( n=6 frogs ~grOup) MPTP

MPTP+PARG

IJj LIJ

CONTROLS

3

Q= 2= Q..

2

Mpp÷

Z~

CONTROLS PARG OR H20 MPTP 40rag~k@ MPP + PQ PARG

10m@/kg 40mq/kg 2mg/kg

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DAYS OF INJECTION

FIG. 5

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1536

MPTP, MPP + and Paraquat zn R. pzpzens

Vol. 37, No. 16, 1985

Dlscusslon As is obvious from perusal of the summary of neurotoxlc manzfestatlons in Fig. 6, the behavzoral, blochemzcal and skzn pigmentation effects of MPTP, its metabolzte MPP +, and the related herbzclde Paraquat (PQ+) are not zdentzcal. However, study of thls dzverszty of effects helps zn understandzng the underlying mechanzsms of actlon, and possibly the pathophysiology of Parklnson's dzsease.

SUMMARY

A.

OF CLINICAL, EFFECTS

BIOCHEMICAL IN R. PIPIENS

+PARG

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FIG. 6 Although Pargylzne completely blocked the behavzoral effects of MPTP in frogs by znhibztlng the fzrst step zn the metabolzsm of the substance (4,10) and preventlng the eventual formatzon of MPP +, zt clearly zncreased the behavzoral manzfestzons produced by MPP + ztself and by Paraquat, a herbzczde wzth a szmzlar structure. Our studies are compatzble wzth the fact that the first step in the process of MPTP actzon zs blockzng of the dopamine reuptake system, as shown by others (2,11). Early effects zndeed indzcate an increase in dopamzne concentratzon (Fz$. 2) followed later by a marked decrease, particularly wzth MPTP and MPP + (Fig. 3). A multl-stage process is znvolved zn the transformatzon of MPTP znto MPP + (4,12), only the fzrst step bezng monoamine-oxzdase (MAO) sensitzve. Pargylzne glven for many days before MPTP wzll totally block both the behavzoral and blochemzcal effects, whzle Pargylzne gzven concurrently wlth MPTP, but not before, blocks the behavzoral effect but only partially reduces the dopamzne decrease (3). Because of this, we thlnk that the dopamine-cell toxzczty (in acute conditions such as those we used in frogs) zs most likely due to MPP + whzch zs zmmedzately formed and accumulated despzte

Vol. 37, No. 16, 1985

MPTP, MPP + and Paraquat in

R. pipien8

]537

the partial blocking of MAO. This toxicity occurs very rapidly and can be zndependent of the intermediates, as our results in the adrenal medulla demonstrate clearly (Fig. 4). In that gland MPTP only slightly decreases dopamine, whzle MPP + totally abolishes the dopamlne reserve, despite concomittent Pargyizne. This observation implies that even in the presence of MAO (such as zs the case in the adrenal medulla and zn many parts of the brain), MPTP is only slowly metabolized, in a controlled manner, to MPP +, the actual cytotoxzc subtance, whereas zn the substantia nzgra this transformatzon is almost instantaneous, within 120 seconds in vitro (13). Studies from our laboratory (12) have recently shown that the speclfic catalyst zn this reactzon is the presence of free iron. Our observations on skin pzgmentatzon (Fm~. 5) further confirm that the acute toxicity of MPTP injections is due to immediate MPP + formation and not to MPTP itself or not even to the generation of amznochromes and melanin. MPTP does cause a marked mncrease In skln pzgmentation (increased dzspersion plus increased synthesis), a process inhibzted by Pargyline and involving dopamine and melanocyte stimulatory hormone (3), but the cytotoxiclty through thzs mechanism is slow (8). More to the point, MPP + markedly decreases the skzn plgmentatzon, probably by interfering with the electron transfer chaln. We have indeed shown (POlrzer & Barbeau, Neurosczence Letters, 1985, in press) that MPP +, but not MPTP, inhzblts NADH cytochrome c reductase. Skin melanocytes could thus become a model for the study of what happens in the brain pzgmented cells, despite the fact that the bzochemzcal orlgin of the melanzn therein is different. In this respect, it should always be remembered that the "depigmentatlon of the substantia nigra" reported in Parkinson's disease zs not due to impairment of melanin syntheszs, but to death of the pigment bearing cells, with clearing of the remnants by glial cells (14). Since MPP + or MPTP are unlikely to be largely present in nature, the toxzc process of the herbzclde PQ+, partzcularly when associated wlth Pargylzne,may thus be a new model for the study of Parkznson's dzsease. Acknowledgements The studies reported in thzs paper were supported by grants from the Medlcal Research Councll of Canada (MT-4938), the W.G. Weston Charztable Foundatzon and La Fondation Parkznson du Quebec. The authors would like to thank Drs. J.W. Langston and I. Irwzn for gift of MPP+; Miss L. Lafrance, veterinarian, for znvaluable help wzth the upkeep and handling of the frogs; Mr. O. Taliano and Miss I. Morin for the illustrations and Mrs. N. Guay-Pozrier for typing the manuscript. References i. 2. 3.

4.

5. 6.

J.W. LANGSTON, P. BALLARD, J.W. TETRUD, and I. IRWIN, Science 219, 979-980, (1983). R.S. BURNS, C.C. CHIUEH, S.P. MARKEY, M.H. EBERT, D.M. JACOBOWITS, and I.J. KOPIN, Proc. Nat. Acad. Sci. USA 80, 4546-4550 (1983). A. BARBEAU, L. DALLAIRE, N.T. BUU, F. VEILLEUX, H. BOYER, L.E. DE LANNEY, I. IRWIN, E.B. LANGSTON, and J.W. LANGSTON, Life Sczences 36, 1125-1134 (1985). K. CHIBA, A. TREVOR, a n d N. CASTAGNOLI, B i o c h e m . B i o p h y s . R e s . Comm. 1 2 0 , 574-578 (1984). L.T. HOGBEN, and D. SLOME, Proc. Roy. Soc. (London) B-108, 10-53 (1931). P.T. KISSINGER, R.M. RIGGIN, R.L. ALCORN, and L.D. RAY, Biochem. Med. 13, 299-306 (1975).