Tissue concentrations of MPTP and MPP+ after administration of lethal and sublethal doses of MPTP to mice

Toxicology Letters, 54 (1990) 253-262

253

Elsevier

TOXLET

02472

Tissue concentrations of MPTP and MPP+ after administration of lethal and sublethal doses of MPTP to mice Ray W. Fuller and Susan K. Hemrick-Luecke Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis. IN (U.S.A.) (Received

27 April 1990)

(Accepted

27 July 1990)

Key words: MPTP; Deprenyl;

I-Methyl-4-phenyl-1,2,3,6_tetrahydropyridine;

I-Methyl-4-phenylpyridinium;

Mouse lethality

SUMMARY Tissue concentrations mice after MPP+

of MPP+

administration

given subcutaneously

lethality

resulting

of MPP+

doses

or orally,

and somewhat

MPTP

caused

an LDSo of 54 mgjkg.

were measured

the relationships

concentrations

between

of MPP+

antagonized

whereas

the lethality

in brain and in other tissues after MPTP injection. of MPTP

although

both compounds

of deprenyl

against

after MPTP

injection.

levels of MPTP Lethality

MPTP

could be measured

lethality

The reduced

or MPP+

after MPP+

are thought

than of phenylethylamine

to be oxidized by monoamine

implicates lethality

MPP+ (or possibly of MPTP

after oral MPTP implicate

administration

almost

tissues than did MPTP subcutaneous

of subcutaneous

and longer-lasting

oxidation

Deprenyl

oxidation,

MPTP

MPTP

oxidase type B. The protective some other metabolite)

when given orally

and the relative

in brain after oral or subcutaneous

does not involve the brain,

had

and re-

caused a greater especially

in liver, effect

in the lethality lack of brain

the brain as a target organ in the lethality

certainly

or and

led to much higher concentrations

duced MPP+ concentrations inhibition

River CFW

tissue concentrations

in peripheral

at oral doses up to 160 mg/kg

pretreatment

in Charles

(I-methyl-4-phenyl-1,2,3,6_tetrahydropyridine)

MPTP given subcutaneously

higher

no lethality

Deprenyl

of MPTP

to investigate

from these compounds.

in brain

given orally.

(1-methyl-4-phenylpyridinium)

of high

of MPTP.

since little or no MPP+

dosing of MPP+

INTRODUCTION

1-Methyl-4-phenyl-1,2,3,6_tetrahydropyridine (MPTP) has been studied intensively during the past several years because of its ability to cause movement disorders resembling Parkinson’s disease in humans [l] and in non-human primates [2]. MPTP is neurotoxic to nigrostriatal dopamine neurons in several species [see Ref. 31, due Address for correspondence:

R.W. Fuller, Lilly Research

porate

IN 46285, U.S.A.

Center,

0378-4274/90/S

Indianapolis,

3.50 @ 1990 Elsevier Science Publishers

Laboratories,

B.V. (Biomedical

Eli Lilly and Company,

Division)

Lilly Cor-

254

to its metabolism by monoamine oxidase type B (MAO-B) to I-methyl-4-phenylpyridinium (MPP+) and the subsequent accumulation of MPP+ into dopamine neurons via the dopamine uptake carrier [4,5]. The neurotoxicity toward dopamine neurons is prevented by inhibitors of MAO-B [6,7] and by inhibitors of the dopamine uptake carrier [5,8]. Recently, we studied the acute lethality of subcutaneously administered MPTP in mice and found that the lethality was prevented by MAO-B inhibitors but not by dopamine uptake inhibitors [9]. The protection against lethality by MAO-B inhibitors seemed to be associated with inhibition of MPTP conversion to MPP+ in brain. MPTP is relatively non-lethal when given orally [lo]. The present experiments were undertaken to measure tissue concentrations of MPTP and MPP+ at lethal and non-lethal doses of MPTP given by oral or subcutaneous routes to mice in an attempt to provide additional insight into mechanisms and sites of lethality associated with MPTP. METHODS

Male CRLjCFW mice weighing 20-30 g (Charles River Breeding Laboratories, Portage, MI) were given MPTP hydrochloride (synthesized in the Lilly Research Laboratories by Dr. David W. Robertson and associates) by subcutaneous injection or by oral gavage. Tissues were removed from decapitated mice, frozen on dry ice, and stored at - 70°C prior to analysis. MPTP and MPP+ concentrations were determined by liquid chromatography with detection by ultraviolet absorbance [9]. Tissues were homogenized in 10 volumes of 0.1 N trichloroacetic acid and centrifuged. An aliquot (100 ~1) of the supernatant fluid was adjusted to a pH of 5-7 with 1 M sodium acetate and mixed with 1 ml of water. Aliquots of the samples were adsorbed onto a CBA (carboxylic acid) cassette and eluted directly onto a Zorbax ODS Cl8 column. The mobile phase was 1 M sulfuric acid + 0.05 M triethylamine + 10% acetonitrile (pH 2.3); flow rate was 1.5 ml/min, and MPTP and MPP+ peak heights were detected by ultraviolet absorbance at 260 nm. Standards were added to tissue homogenates from untreated mice. MAO activity in whole homogenates of mouse tissues was measured in vitro using 12.5 PM 1-[i4C]-phenylethylamine hydrochloride (New England Nuclear, Boston, MA) as substrate. The oxidation of MPTP by mitochondrial preparations in vitro was measured by a modification of the method of Szutowicz et al. [l l]. Hydrogen peroxide formation was measured calorimetrically 1 h after the addition of 5 mM MPTP as substrate. All results are shown as mean values + standard errors for 5 mice per group. Statistical comparisons were made by analysis of variance using Tukey’s test. RESULTS

Figure 1 shows the tissue concentrations

of MPTP and of MPP+ 1 h after graded

255

Fig. 1. Tissue concentrations of MPTP (open bars) and MPP+ (cross-hatched bars) 1 h after administration of MPTP, at 30,60 or 90 mg/kg S.C.or at 90, 120 or 180 mg/kg p.o., to mice.

doses of MPTP were given to mice. The subcutaneous doses were 30,60 and 90 mg/ kg, and the oral doses were 90, 120 and 180 mg/kg. In brain, MPTP and MPP+ concentrations were nearly equal to each other at all doses after subcutaneous administration, and both increased proportionately to dose. After oral administration, very low amounts of MPTP and MPP+ were present in brain. In the heart, MPP+ concentrations were much higher than those of MPTP after all doses by either route of administration. MPPf concentrations after the 90-180 mg/kg oral doses were nearly as high in heart as those after the 3&90 mg/kg subcutaneous doses, although MPTP concentrations in heart were lower after the oral doses than after the subcutaneous doses. The MPP+ concentrations in heart were higher than those in any of the other tissues examined. In the liver, limited dose proportionality was found after oral ad-

256 TABLE I LETHALITY OF MPTP AND MPPf AFTER SUBCUTANEOUS IN MICE

AND ORAL ADMINISTRATION

Compound p.0.

S.C.

> 160

MPTP (48ZO)

49 (41-59) -

MPP+ (9Z4)

LDSa values are shown with confidence intervals as determined by least-squares analysis of data from 5--6 doses with each compound, 10 treated mice per dose group.

ministration of MPTP, a little clearer dose proportionality after subcutaneous administration. The concentrations of MPTP and MPP+ were approximately equal in liver after oral doses of MPTP, whereas MPP+ concentrations were somewhat higher than those of MPTP after subcutaneous administration of MPTP. In the lung, MPP+ concentrations were similar to MPTP concentrations after subcutaneous administration of MPTP, but after oral administration of MPTP the concentrations of MPTP were much lower than those of MPP+. In neither group was the relationship between dose and tissue concentration as distinct as in brain and heart. In the kidney, dose proportionality was poor after either route of administration, and approximate-

TABLE II DEPRENYL PRETREATMENT AFTER MPTP ADMINIST~TION Tissue

REDUCED IN MICE

CONCENTRATIONS

f&g)

OF MPP+

Pretreatment None

Brain Heart Liver Lung Kidney

TISSUE

19.2& 1.8 49.5k3.1 28.lk2.7 18.5i: 1.2 14.0& 1.5

Deprenyl (10 trig/kg, 3 d)

Deprenyl (10 r&kg,

6.6&0.4* 32.1+1.5* L%+o.4* 10.9f0.7* 6.2kO.l’

2.9&0.2* 14.5& 1.0* 5.3rto.3* 3.7+0.3* 8.8f 1.7*

Deprenyl

1h)

(1 m&z, 1 h) 3.4+0.1* I3.4f3.4* 8.0* 1.0, 10.0+3.0* 5.2+0.8*

MPTP was injected at 90 mg/kg S.C. 1 h before mice were sacrificed. Deprenyl was injected at 1 mg/kg i.p. 1 h before MPTP, at 10 mg/kg i.p. 1 h before MPTP, or at 10 mg/kg 3 days before MPTP. Mean values + standard errors for 5 mice per group are shown. Asterisks indicate significant difference from group without pretreatment (‘&key’s test, PcO.05).

251

ly equal amounts of MPTP and MPP+ were present, less after oral than after subcutaneous administration of MPTP. Although the doses of MPTP injected subcutaneously in this experiment are high enough to produce lethality, deaths do not begin occurring before 2 h, so in this experiment all mice survived to the time of sacrifice (1 h). Table I compares the lethality of MPTP and of MPP+ given by subcutaneous or oral routes to mice. MPTP was not lethal at any dose tested orally, although its subcutaneous LDso was 54 mg/kg. MPPf was more lethal than MPTP by the subcutaneous route and its lethality shifted by only about 2-fold when given orally. Table II shows that pretreatment with deprenyl at a single dose, either 1 or 10 mg/ kg, given 1 h or 3 days before MPTP injection, markedly reduced MPP+ concentrations in all tissues after MPTP administration. These dosage regimens have previously been shown to protect against MPTP lethality [9]. In some but not all cases, MPTP concentrations were increased by deprenyl pretreatment (data not shown). The percentage reduction in MPP+ concentration was generally highest after the 1 h pretreatment with the higher dose of deprenyl. Figure 2 shows the recovery in several tissues of MAO-B activity measured with 14C-phenylethylamine as substrate after a single 10 mg/kg dose of deprenyl in mice. As reported earlier [9], MAO-B activity recovered more rapidly in liver than in brain. In this experiment, only the l-day time point showed marked (53%) inhibition of MAO-B by deprenyl in liver. At 3 and 5 days there was only slight (13 and 17% respectively) but statistically significant inhibition of MAO-B in liver. The activity of MAO-B in other tissues (heart, lung and kidney) recovered at very similar rates to those of MAO-B in brain. The basal activity of MAO-B measured with t4C-phenyl-

Days

Fig. 2. Time course (10 mg/kg

of inhibition

i.p.) treatment

mean value for untreated and lung (P
of MAO-B

Deprenyl

after

activity

(W-phenylethylamine

in mice. Rate of phenylethylamine

oxidation

mice. All data points differed significantly

In liver, only the l-, 3- and 5-day (PC 0.05).

data

points

as substrate) is expressed

from control differed

after deprenyl

as percentage

of the

for brain, heart, kidney

significantly

from control

258 TABLE III MAO-B ACTIVITY IN MOUSE TISSUES -_ Tissue

Liver Heart Lung Brain Kidney

Substrate oxidized (nmol/min/g tissue) ~henylethylamine

MPTP

355.8 + IS.0 94.3F3.6 77.4+ 1.8 34.1kO.7 12.6IfO.4

256.4i23.2 17.4* 1.6 4.9io.4 8.9+0.5 l.O&O.l

_-

ethylamine or MPTP as substrate was very high in liver and much lower in the other tissues studied (Table III}. Figure 3 shows the recovery of MPTP oxidative capacity in the same tissues after the single 10 mg/kg dose of deprenyl in mice. With most tissues (except for kidney), MPTP oxidation was inhibited more at all times than was phenylethylamine oxidation, and MPTP oxidative capacity recovered more slowly. Enzyme activity in brain was inhibited more than enzyme activity in liver, as had been true with phenylethylamine. But MPTP oxidation in kidney recovered faster than in liver, opposite to the case with phenylethylamine. Figure 4 compares tissue concentration of MPP+ after the administration of MPP+ subcutaneously or orally. No MPP+ was detected in brain after either route of administration. MPP+ was absorbed orally, with relatively high concentrations

Days

stter

Deprenyl

Fig. 3. Time course of inhibition of MTPP oxidation after deprenyl (10 mg/kg i.p.) treatment in mice. Rate of MPTP oxidation is expressed as percentage of the mean value for untreated mice. All data points differed significantly from control for brain, heart and lung (PC 0.05). In liver, all data points except at 14 days differed significantly from control (P
259

being found in all other tissues. After subcutaneous administration of the 30 mg/kg dose of MPP+, tissue concentrations varied in the order kidney> liver = lung> heart, but after oral administration the order of tissue concentrations was liver > lung > heart > kidney. Reasonable proportionality between tissue concentrations and dose was found in all cases. Some of the doses of MPP+ used in this experiment cause lethality, and deaths begin occurring with MPP+ before 1 h. More than 5 mice per group were treated with MPP+, and only those mice that survived to 1 h (time of sacrifice) were used for analysis of MPP+ levels.

Fig. 4. Tissue concentrations

of MPP+

1 h after administration

of MPP+,

at 30.40 or 60 mg/kg p.o., to mice.

at 10, 20 or 30 mg/kg

S.C. or

260 DISCUSSION

Previous data have implicated MPP+ (or possibly some other metabolite of MPTP) in the acute lethality of MPTP at high doses in mice [9], based on protection by MAO inhibitors against MPTP lethality. An objective of the present study was to compare MPP+ levels in various tissues after lethal doses of MPTP given subcutaneously and after higher but non-lethal doses of MPTP given orally. The most striking difference between MPP’ concentrations attained after oral administration of MPTP versus those attained after subcutaneous administration of MPTP was in brain (Fig. 2). In the brain, MPP+ concentrations after the non-lethal 180 mg/kg oral dose of MPTP were much lower than those after the lethal 60 and 90 mg/kg subcutaneous doses of MPTP. In the other tissues, MPP+ concentrations after the 180 mg/ kg oral dose of MPTP were only marginally lower than those after the 60 and 90 mg/kg subcutaneous doses of MPTP. The findings are consistent with an important role of the brain as a site of MPTP-induced lethality but do not rule out an involvement of other tissues as well. One of the most clear findings in the tissue distribution experiment (Fig. 1) was the predominance of MPP+ concentrations over MPTP concentrations in heart after either subcutaneous or oral administration of MPTP. In no other tissues were such high concentration ratios of MPP’./MPTP found. Because the heart has a much lower capacity to oxidize MPTP than does liver (Table III), the high concentrations of MPP+ in heart may represent accumulation by that tissue, not local formation. Deprenyl, at doses and treatment regimens that prevented MPTP-induced lethality in mice [9], significantly reduced MPP+ concentrations in all tissues studied (Table II). We had earlier found that MAO-B activity in liver recovered rapidly after deprenyl injection whereas protection against MPTP-induced lethality persisted for at least 14 days, leading to the idea that MPTP conversion to MPP+ in brain might be a key step in MPTP-induced lethality [9]. The liver is the richest source of MAO-B in the mouse (Table III), suggesting that liver would be the major source of MPP+ formation in the periphery. In the current experiment (Fig. 2) MAO-B in liver recovered more rapidly after deprenyl treatment than did MAO-B in brain, as reported previously [9]. MAO-B was depressed significantly (PC 0.05) at 3 days after deprenyl pretreatment even though the change was small (13%), whereas in the earlier study there was no statistically significant effect at 3 days [9]. The current studies also show that MAO-B activity in other peripheral tissues does not recover as fast as in liver. Further, MPTP oxidation (Fig. 3) does not recover as fast as phenylethylamine oxidation (Fig. 2) in peripheral tissues, including liver. Therefore, deprenyl pretreatment (Table II) reduces MPP+ levels in various tissues, not just brain, even at 3 days when liver MAO-B activity has largely recovered. Although MPTP is oxidized by MAO-B [4], inhibition of MPTP oxidation did not follow an identical time course to that of phenylethylamine oxidation. MPTP oxidation was reduced to a greater extent and recovered more slowly than phenylethyla-

261

mine oxidation after deprenyl administration. Thus MPP+ concentrations were decreased in all tissues, not just in brain, when MPTP was administered 3 days after deprenyl injection. Since deprenyl pretreatment in 3 different dosage regimens that earlier had been shown to protect against MPTP lethality [9] reduced MPP+ concentrations not just in brain but in other tissues as well, it was not possible to link MPTP-induced lethality with a particular target tissue on the basis of these experiments. Administration of MPP+ causes greater lethality than administration of MPTP by the subcutaneous and especially by the oral route (Table I). Lethality produced by MPP+ injection is not likely to involve the brain, since very little MPP+ could be detected in brain. If the lethality involved the heart or the kidney, MPP+ would have been more than twice as lethal when given subcutaneously than when given orally, based on tissue concentrations of the drug. MPP+ was only about twice as lethal when given subcutaneously as compared to orally, but tissue concentrations in heart and kidney were greater than 6-fold higher after subcutaneous compared to oral administration. The relative tissue concentrations after MPPf administration via the two routes would be compatible with lung or liver as target organs producing lethality. Johannessen et al. [12] had reported that systemically administered MPP+ produced its major pathology in the lung. Adams et al. [13] considered that MPP+ formed from administered MPTP in mice might also be responsible for histopathological changes in the lung. Referring to Figure 1, one sees that MPP+ concentrations in lung at non-lethal oral doses are slightly lower than after lethal subcutaneous doses (60 and 90 mg/kg), although those differences in lung and in other peripheral tissues are not nearly as great as in the brain. The present findings do not definitively indicate which tissue is the site of the lethal effects after MPTP administration. REFERENCES 1 Langston,

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