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Effects of leptophos on rat brain levels and turnover rates of biogenic amines and their metabolites
ECOTOXICOLOGY
AND
ENVIRONMENTAL
SAFETY
6. 570-576
(1982)
Effects of Leptophos on Rat Brain Levels and Turnover Rates of Biogenic Amines and Their Metabolites’ C. N. ALDOUS,~ C. H. FARR,~ AND R. P. SHARMA~ To.uico1og.vProgram, Utah State University. Logan, Utah 84322 Received May 7. 1982 Leptophos is a potent acetylcholinesterase inhibitor which causes delayed central-peripheral neuropathy. Rats were administered multiple doses of leptophos until motor deficits were observed in rotorod performance in the highest dosage group. Doses lower than the median effective dose were then administered to other rats and alterations of brain catecholamine and serotonin levels and turnover rates were determined. Turnover rates of brain norepinephrine and dopamine were elevated in rats administered cumulative doses of 75 mg/kg leptophos over a 15-day period. Levels of the major dopamine metabolite. 3,4-dihydroxyphenylacetic acid, appeared to be slightly elevated at this dose level and levels of dopamine were also higher than controls. These observations suggest that leptophos increases brain adrenergic activity. Rats administered the same dose levels had significantly reduced scrotonin turnover rates. This observation was possibly artifactual. because rats administered a cumulative dose of 225 mg/ kg leptophos showed no difference from controls.
INTRODUCTION Certain classes of organophosphates, including a number of effective pesticides, have been shown to elicit delayed-onset neuropathy in experimental animals (Johnson, 1975). Among these, O-methyl-O-4-bromo-2,5-dichlorophenyl phenylphosphonothioate (leptophos) has received much attention since major losses of water buffalo in the Nile delta were attributed to this pesticide (Abou-Donia and Preissig, 1976). The pattern of delayed-onset central-peripheral distal axonopathy is similar to that elicited by other compounds such as acrylamide and hexanedione (Cavanagh, 1973; Bouldin and Cavanagh, 1979). Among delayed-neuropathy agents, certain organophosphates and a few closely related groups of chemicals appear to be unique in their capacity to bind irreversibly to a specific “neurotoxic esterase” (Johnson, 1975). This capacity is closely correlated to the neurotoxic potential of organophosphates. and may represent a mechanism of toxicity fundamentally different from that elicited by other delayed neurotoxic compounds. Leptophos is among the potent inhibitors of “neurotoxic esterase” (Hussain and Oloffs, 1979). Pathological lesions are similar to those of other delayed-neurotoxic compounds. Abou-Donia and Preissig ( 1976) reported axonal damage in the sciatic nerve and the spinal cord, but not in the brain anterior to the medulla. Lowndes ef ul. (1974) inferred from experiments with delayed-neurotoxic diisopropyl fluo’ Utah State University Agricultural Experiment Station Journal Paper No. 2690. This work was supported in part by NIH Grant ES 07097. ’ Current address: Department of Neurology, The University of Mississippi Medical Center, 2500 N State Street, Jackson. Miss. 39216. ’ Current address: Phillips Petroleum Co., Medical Division, Bartlesville, Okra. 74004. ’ To whom reprints requests should be sent: Utah State University, UMC 56, Logan, Utah 84322
0147-6513/82/060570-07$0200/O Copyright IQ 1982 by Academic Press. Inc. All rights of reproduction m any form reserved.
570
LEPTOPHOS
AND
BRAIN
AMINES
571
rophosphate (DFP) that peripheral axon injury occurred in distal parts of motor axons and that it was a trophic disturbance. One manifestation of axonal impairment, which appears to be characteristic of the organophosphates which cause delayed distal neuropathy, is inhibition of fast axoplasmic transport, as demonstrated by Reichlert and Abou-Donia ( 1980). Similar findings were reported in response to another (delayed-neurotoxic compound, acrylamide (Weir et al., 1978). The possibility that delayed-neurotoxic organophosphates might have marked effects on axons of higher brain centers, which might potentiate peripheral and spinal cord damage, has not yet been adequately investigated. The recent findings that acrylamide apparently damages nigrostriatal fibers (Agrawal et al., 1981) raises questions as to whether delayed-neurotoxic organophosphates might do likewise. METHODS
Animals Male Sprague-Dawley-derived rats (Simonson Laboratories, Gilroy, Calif.) weighing 2 15 :t 8 and 232 & 4 g were used for catecholamine and for serotonin studies, respectively. All rats were acclimated to the animal facility surrounding for at least 1 week before testing. They were fed a commercial chow and given free accessto feed and water during experiments. The animals were exposed to 12 hr of light per day.
Chemicak
and Chemical Assay Equipment
The following standards were purchased from Sigma Chemical Company (St. Louis, MO): 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA ), dopamine HCl, norepinephrine HCl, serotonin creatinine sulfate, L-tryptophan, and L-tyrosine. t,-[ri?zg-2,6-3H]tyrosine, 38 Ci/mmol, and L[G-3H]tryptophan, 7.88 Ci/rnmol, were purchased from New England Nuclear (Boston, Mass.). Leptophos (98% analytical standard, Chem. Service Co., Westchester, Pa.) was dissolved in propylene glycol in concentrations such that each animal received 1 ml of the vehicle per 100 g body wt. Atropine sulfate purchased from Sigma Chemical Company was administered ip at 50 mg/kg to leptophos-treated animals and controls prior to leptophos administration. All gels. resins, and reagents used in sample assays were of a quality suitable for fluorescence studies. Fluoro.metric assayswere performed on an Aminco-Bowman spectrophotofluorometer (Aminco, Silver Spring, Md.). The Packard Tri-Carb 2660 Liquid Scintillation System (Packard Instrument Co., Downers Grove, Ill.) was used for all scintillation counting.
Rotorod Studies The rotorod apparatus used was similar to that described by Kaplan and Murphy (1972). A wooden dowel (7.6-cm diameter) was placed 33 cm above the floor, which was formed of stainless-steelrods supplied with a scrambled shock device. Rats were trained for seven days before treatment, mild shocks on the floor grid providing the incentive for learning to walk the dowel. Final testing speed was 20 rpm. Tests proceeded1with groups of five randomly selected rats. Leptophos dissolved in propylene glycol was administered at 3-day intervals by oral gavage at initial dosesof
512
ALDOUS.
FARR.
AND
SHARMA
90, 30, and 9 mg/kg. The criterion for normal performance in rotorod tests was the ability to walk on the dowel for 1 min during any of three attempts. Rotorod performance tests were conducted on the days of leptophos administration, prior to gavage. Based on the results of this experiment, the dose levels were selected for subsequent trials to measure brain amines.
Assays of Tyrosine, Norepinephrine, Estimations for Norepinephrine
Dopamine, and DOPAC; and Dopamine
Turnover Rate
Rats were administered 0, 5, or 15 mg/kg/feeding of leptophos at 3-day intervals for a total of five feedings. Two days after the last administration the animals were injected iv with 62 &i L-[ring-2,6-3H]tyrosine, which was dissolved in 0.5 ml saline. Rats were sacrificed by decapitation during the fifth and sixth hours of the daylight cycle at 2.0 or 3.5 hr after injection of the labeled precursor amino acid. Their brains were quickly excised, frozen on dry ice, and subsequently stored at -90°C until assay. Perchloric acid ( 0.4 &I) was used to extract norepinephrine, dopamine, tyrosine, and DOPAC from rats preinjected with labeled tyrosine. Fractions were neutralized with potassium formate and subjected to Sephadex G-10 chromatography, and DOPAC was assayed fluorometrically by the method of Westerink and Korf (1977). The balance of the perchloric acid extracts were neutralized with 5 A4 KOH. Supernatants were separated into fractions containing norepinephrine, dopamine, and tyrosine on Dowex 50 columns by the method of Neff et al. (1977). Portions of the isolated fractions of the three amines were added to the scintillation cocktail and counted. Fluorometric assays of norepinephrine and dopamine were performed by the manner of Karasawa et al. (1975). Tyrosine was assayed fluorometrically by the method of Smith et al. (1975).
Tryptophan, Serotonin, and 5-HIAA Estimates
Assays and Serotonin
Turnover Rate
Rats were gavaged with leptophos every third day for five feedings. Groups received 4.5, 15, or 45 mg/kg/feeding. Two days after the last dosing, rats were administered 50 PCi L-[G-3H]tryptophan iv. Half of each dosage group were sacrificed at 40 min, the balance at 120 min, after injection. Sacrifices occurred during the third and fourth hours of the daylight cycle. Removal, freezing, and storage of brains followed, as described for catecholamines. On the day of assay, the brains were homogenized in 0.4 M perchloric acid. Neutralized extracts were fractionated on Dowex 50 columns by the method of Marini et al. (1979). The fluorometric method of Atack and Lindquist (1973) was used for 5-HIAA assay. Tryptophan was assayed by the fluorometric method of Marini et af. (1979), and serotonin was determined fluorometrically according to Karasawa et al. (1975). Tryptophan and serotonin fractions were also subjected to liquid scintillation counting, and the method of Neff et al. ( 197 1) was used to estimate turnover rates.
Data Analysis External standards were used to obtain values for biogenic amine and metabolite levels. These values were corrected for fractional recovery and adjusted to represent nanomoles of compound per gram wet weight of brain. A one-way analysis of
LEPTOPHOS
AND
BRAIN
AMINES
573
variance was employed to assess dose effects on animal weight gain and on levels of biogenic amines and their metabolites. Significant differences among treatment means (I’ < 0.05) were calculated by Tukey’s HSD multiple mean comparison test (Neter and Wasserman, 1974). Turnover rates were calculated on the basis of averaged specific activities of biogenic amines and their precursors for groups treated alike. Standard errors for turnover rate estimates were obtained by applying specific activities of pairs of rats, which had been given the same dose but were preinjected at different times, to the formula of Neff et al. (197 1). RESULTS Rotorod Studies and Clinical
Signs of Toxicity
Leptophos-treated animals showed signs of tremors after the first dose of 90 mg/ kg. Two animals in that group died after the third atropine dose but prior to oral administration of the leptophos. Dosing was then discontinued because the animals were very lethargic and emaciated. Animals dosed with either 30 or 9 mg/kg had little difficulty performing the rotorod test after six doses over 16 days (Fig. 1). Animals in the 45 mg/kg dose group showed a significantly reduced weight gain in 16 days compared to control animals. Leptophos had no effect on weight gain at lower dose levels. Ej2ct.s OH Catecholamines
and DOPAC
Treatrnent of rats with leptophos had no effect on the total level of brain tyrosine or the specific activity of labeled tyrosine. Since the specific activity differed at the two testing periods after injection of labeled amine, only total values are indicated in Table 1. Similarly, no effect was noticed on the brain levels of norepinephrine but the dopamine level was significantly elevated in animals treated with a cumulative dose of 75 mg leptophos/kg body wt. The change in the whole brain dopamine concentration in this group was nearly 14% over controls. No change in dopamine level wa:s observed in the group treated with a cumulative leptophos dose of 25 mg/kg.
Days
of Leptophos
Dosing
FIG. 1. Percentage of rats unable to remain on the rotorod for 60 set at 20 rpm 3 days after the most recent leptophos treatment (n = 4 in each group). The animals were treated every 3 days by oral gavage. Dosing was discontinued in the group given repeated doses of 90 mg/kg after two treatment periods.
514
ALDOUS, FARR, AND SHARMA TABLE 1
WHOLE BRAIN LEVELSOF TYROSINE,NOREPINEPHRINE, DOPAMINE,ANDDOPAC: ALSO CATECHOLAM~NETURNOVERRATESAFTERLEPTOPHOSTREATMENTOFRATS~
Cumulative doseof leptophos(mg/kg) Control
(n = Tyrosine (nmol/g) Norepinephrine(nmol/g) Dopamine (nmol/g) 3,4-Dihydroxyphenylacetic acid (nmol/g) Norepinephrineturnover rate (nmol/g/hr) Dopamineturnover rate (nmd/g/hr)
14)
116.3 + 3.3 2.60 k 0.05 4.93 -t 0.09
109.7 f 3.5 2.57 + 0.08 4.95 * 0.20
112.9 * 2.2 2.58 t 0.04 5.61 + 0.19'
0.88
f
0.02
0.90
*
0.03
0.99
f
0.46
f
0.08
0.49
2 0.10
0.77
+ 0.10’
1.92 i
0.20
1.68
f
2.74 + 0.39
0.35
0.05
’ Mean f SEM. Group size indicated in parentheses. b Three rats died in the 75 mg/kg group. ‘Significant, P < 0.05.
Synthesis rates of both norepinephrine and dopamine were increased in the group treated with 75 mg leptophos/kg (Table 1). Increases in the synthesis rate constants for these two biogenic amines were 67 and 43%, respectively. These values indicate a considerable rise in adrenergic activity in leptophos-treated rats. The levels of the dopamine metabolite, DOPAC, showed a 13% increase, which was not statistically significant at this dose level. Eflects on Serotonin and its Metabolite In the groups of animals treated with labeled tryptophan, neither the total levels nor the specific activities of tryptophan were influenced by 15 days of leptophos treatment. Again, only the total levels are depicted in Table 2, since the specific activities varied with the time of sacrifice relative to injection of labeled precursor amino acid. The levels of serotonin and its metabolite, 5-HIAA, in the animals given leptophos are also given in Table 2. Levels of these compounds were not altered by leptophos treatments. The turnover rate for serotonin was significantly decreased at the cumulative dose of 75 mg leptophos/kg, but no variation from the control value was observed when the dose of leptophos was increased to 225 mg/kg over the same period. DISCUSSION Most of the economically important organophosphates that cause delayed distal neuropathy are potent acetylcholinesterase inhibitors. For this reason, it is difficult to determine whether alterations in neurotransmitter levels and functions represent primary sequelae of altered choline& function, alterations in neuronal function associated with delayed neuropathy, or other unexplained interactions. Alterations in catecholamine levels or turnover rates have been reported after the administration
LEPTOPHOS
AND BRAIN AMINES
575
TABLE 2 WHOLE
BRAIN
LEVELS TURNOVER
OF ENDOGENOUS RATES AFTER
INDOLE COMPOUNDS AND LEPTOPHOS TREATMENT’
SEROTONIN
Cumulative doseof leptophos(mg/kg) Control Tryptophan (nmol/g) Serotonin (nmol/g) 5-HIAA
(mmol/g)
Serotonin turnover rate (nmol/g/hr)
23.07
2
0.97
2.72 fc 0.07 1.52
3~0.08
1.88 f 0.09
0 14 animals in each treatment group, ’ Significant, P < 0.05.
15
22.5
values
24.87 2.63 1.67
f i +
0.82 0.09 0.15
1.96 f 0.05 are means
225
f 1.04 + 0.05 t 0.07
23.23 2.78 1.43
1.58 + 0.05’
2.07
22.92 2.69 1.59
k
0.86
k 0.11 +- 0.10
+ 0.10
+ SEM.
of potent cholinesterase inhibitors that do not cause delayed neuropathy (Holt and Hawkins,, 1978; Fiscus and Van Meter, 1977). Freed et al. (1976) found reductions in dopamine levels after administering mipafox and leptophos, both of which cause delayed distal neuropathy. Fenitrothion, however, which lacks delayed neuropathy effects, did not alter steady state dopamine levels. In the present studies, levels of dopamine were slightly elevated in rats receiving a moderate dose of leptophos ( 15 mg/kg every third day for a cumulative dose of only 75 mg/kg). The present study involved animals receiving a dose of Ieptophos not sufficient to cause signsofdelayed neuropathy, whereas rats in the Freed et al. (1976) study had received small daily dosesof (delayed-neurotoxic compounds over enough time to elicit motor deficits. In control rats, the whole brain levels of norepinephrine, dopamine, serotonin, the acid metabolites, and the turnover rates of biogenic amines studied here were comparable to those reported previously (Marini et al. 1979, Neff et al. 197 1~Smith et al. 1975). Neurotransmitter turnover rate alterations are generally more sensitive indicators of altered brain function than are changesin steady-state levels of neurotransmitters (Neff et al., 1971). The reduction in the serotonin turnover rate that followed a repeated dose of 15 mg/kg leptophos was not observed after 45 mg/kg, and may represent.an artifact. The increasesin norepinephrine and dopamine turnover rates and the apparent increase in DOPAC levels after five repeated dosesof 15 mg/kg leptophos indicate an increase in activity of catecholaminergic neurons. Inhibition of catecholamine re-uptake by the presynaptic membrane may permit an increased extracellular destruction of the neurotransmitters, and thus necessitatean increased synthesis rate to maintain normal steady-state levels. It is also possible that altered postsynaptic receptor affinities for, or responsiveness toward, neurotransmitters might cause reflex alterations in the quantities of neurotransmitters discharged per action potential. One compound that causesdelayed central-peripheral distal neuropathy, acrylamide. increases the number of dopamine receptors in rat striatum and decreasesthe responsivenessof such receptors to dopamine agonists (Agrawal et al., 1981). The possibility that chemicals which cause delayed neuropathy characteristically alter neurotransmitter receptor function, with possible subsequent alterations in neurotransmitter synthesis rates. has apparently not been investigated.
576
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REFERENCES ABOU-DONIA, M. B., AND PREISSIG. S. H. (I 976). Delayed neurotoxicity of leptophos: Toxic effects on the nervous system of hens. Toxicol. Appl. Pharmacol. 35, 269-282. AGRAWAL. A. K. SETH, P. K. SQUIBB, R. E., TILSON. H. A., UPHOUSE, L. L.. AND BONDY, S. C. (198 1). Neurotransmitter receptors in brain regions of a&amide-treated rats. I: Effects of a single exposure to acrylamide. Pharmacol. Biochern. Behav. 14, 527-531. ATACK, C., AND LINDQUIST, M. (1973). Conjoint native and orthopthaldialdehyde-condesate assaysfor the fluorimetric determination of 5hydroxyindoles in brain. Naunyn-Schmiedeberg’s.4rch. Pharmacol. 279, 267-284.
BOULDIN, T. W., AND CAVANAGH. J. B. (1979). Organophosphorus neuropathy. II. A fine-structural study of the early stages of axonal degeneration. Amer. J. Pathol. 94, 253-270. CAVANAGH. J. B. (I 973). Peripheral neuropathy caused by chemical agents. CRC Crit. Rev. Tosicol. 2, 365-417.
Rscus. R. R.. AND VAN METER, W. G. (1977). Effects of parathion on turnover and endogenous levels of norepinephrine (NE) and dopamine (DA) in rat brain. Fed Proc. 36, 951. FREED, V. H., MATIN, M. A., FANG, S. C.. AND KAR, P. P. (I 976). Role of striatal dopamine in delayed neurotoxic effects of organophosphorus compounds. Eur. J. Pharmacol. 35, 229-232. HOLT, T. M., AND HAWKINS, R. K. (1978). Rat hippocampal norepinephrine response to cholinesterase inhibition. Res. Commun. Chem. Pathol. Pharmacol. 20, 239-251. HUSSAIN, M. A.. AND OLOFFS, P. C. ( 1979). Neurotoxic effects of Leptophos (phosvel) in chickens and rats following chronic low-level feeding. J. Environ. Sci. Health B 14, 367-386. JOHNSON, M. K. (1975). The delayed neuropathy caused by some organophosphorus ester: Mechanism and challenge. CRC Crit. Rev. Toxicol. 3, 289-3 16. KAPLAN, M. L., AND MURPHY. S. D. (1972). Effect of acrylamide on rotorod performance and sciatic nerve &glucuronidase activity of rats. To+xicol. Appl. Pharmacol. 22, 259-268. KARASAWA. T., FURUKAWA, K.. YOSHIDA, K., AND SHIMIZU, M. (1975). A double column procedure for the simultaneous estimation of norepinephrine, normetanephrine, dopamine. 3-methoxytyramine and 5-hydroxytryptamine in brain tissue. Japan. J. Pharmacol. 25, 727-736. LOWNDES, H. E., BAKER, T., AND RIKER, W. F. (1974). Motor nerve dysfunction in delayed DFP neuropathy. Eur. J. Pharmacol. 29, 66-73. MARINI, J. L.. WILLIAMS, S. P. AND SHEARD, M. H. (1979). Simultaneous assay for L-tryptophan, serotonin, 5-hydroxyindoleacetic acid. norepinephrine and dopamine in brain. Pharmacol. Biochem. Behav. 11, 183-187. NEFF, N. H.. SPANO. P. F.. GROPPETTI. A.. WANG, C. T.. AND COSTA, E. (1971). A simple procedure for calculating the synthesis rate of norepinephrine, dopamine and serotonin in rat brain. J. Pharmacol. Esp. Ther. 176, 701-710. NETER. J., AND WASSERMAN, W. (1974). Applied Linear Statistical Models. pp. 419-450, 473-477. Richard D. Irwin, Inc.. Homewood. IL. REICHERT, B. L.. AND ABOU-DONIA, M. B. (1980). Inhibition of fast axoplasmic transport by delayed neurotoxic organophosphorus esters: A possible mode of action. Mol. Pharmacol. 17, 56-60. SMITH, J. E., LANE, J. D., SHEA, P. A.. MCBRIDE, W. J., AND APRISON, M. H. (1975). A method for concurrent measurement of picomole quantities of acetylcholine, choline, dopamine, norepinephrine, serotonin, 5-hydroxytryptophan. 5-hydroxyindoleacetic acid. tryptophan. tyrosine. glycine, aspartate. glutamate. alanine, and gamma-aminobutyric acid in single tissue samples from different areas of rat central nervous system. Anal. Biochem. 64, 149-169. WEIR, R. L., GLAUBIGER. G., AND CHASE, T. N. (1978). Inhibition of fast axoplasmic transport by acrylamide. Environ. Res. 17, 25 l-255. WESTERINK, B. H. C.. AND KORF, J. (1977). Rapid concurrent automated fluorimetric assay of noradrenaline, dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 3-methoxytyramine in milligram amounts of nervous tissue after isolation on Sephadex GIO. J. Neurochem. 29, 697-706.
AND
ENVIRONMENTAL
SAFETY
6. 570-576
(1982)
Effects of Leptophos on Rat Brain Levels and Turnover Rates of Biogenic Amines and Their Metabolites’ C. N. ALDOUS,~ C. H. FARR,~ AND R. P. SHARMA~ To.uico1og.vProgram, Utah State University. Logan, Utah 84322 Received May 7. 1982 Leptophos is a potent acetylcholinesterase inhibitor which causes delayed central-peripheral neuropathy. Rats were administered multiple doses of leptophos until motor deficits were observed in rotorod performance in the highest dosage group. Doses lower than the median effective dose were then administered to other rats and alterations of brain catecholamine and serotonin levels and turnover rates were determined. Turnover rates of brain norepinephrine and dopamine were elevated in rats administered cumulative doses of 75 mg/kg leptophos over a 15-day period. Levels of the major dopamine metabolite. 3,4-dihydroxyphenylacetic acid, appeared to be slightly elevated at this dose level and levels of dopamine were also higher than controls. These observations suggest that leptophos increases brain adrenergic activity. Rats administered the same dose levels had significantly reduced scrotonin turnover rates. This observation was possibly artifactual. because rats administered a cumulative dose of 225 mg/ kg leptophos showed no difference from controls.
INTRODUCTION Certain classes of organophosphates, including a number of effective pesticides, have been shown to elicit delayed-onset neuropathy in experimental animals (Johnson, 1975). Among these, O-methyl-O-4-bromo-2,5-dichlorophenyl phenylphosphonothioate (leptophos) has received much attention since major losses of water buffalo in the Nile delta were attributed to this pesticide (Abou-Donia and Preissig, 1976). The pattern of delayed-onset central-peripheral distal axonopathy is similar to that elicited by other compounds such as acrylamide and hexanedione (Cavanagh, 1973; Bouldin and Cavanagh, 1979). Among delayed-neuropathy agents, certain organophosphates and a few closely related groups of chemicals appear to be unique in their capacity to bind irreversibly to a specific “neurotoxic esterase” (Johnson, 1975). This capacity is closely correlated to the neurotoxic potential of organophosphates. and may represent a mechanism of toxicity fundamentally different from that elicited by other delayed neurotoxic compounds. Leptophos is among the potent inhibitors of “neurotoxic esterase” (Hussain and Oloffs, 1979). Pathological lesions are similar to those of other delayed-neurotoxic compounds. Abou-Donia and Preissig ( 1976) reported axonal damage in the sciatic nerve and the spinal cord, but not in the brain anterior to the medulla. Lowndes ef ul. (1974) inferred from experiments with delayed-neurotoxic diisopropyl fluo’ Utah State University Agricultural Experiment Station Journal Paper No. 2690. This work was supported in part by NIH Grant ES 07097. ’ Current address: Department of Neurology, The University of Mississippi Medical Center, 2500 N State Street, Jackson. Miss. 39216. ’ Current address: Phillips Petroleum Co., Medical Division, Bartlesville, Okra. 74004. ’ To whom reprints requests should be sent: Utah State University, UMC 56, Logan, Utah 84322
0147-6513/82/060570-07$0200/O Copyright IQ 1982 by Academic Press. Inc. All rights of reproduction m any form reserved.
570
LEPTOPHOS
AND
BRAIN
AMINES
571
rophosphate (DFP) that peripheral axon injury occurred in distal parts of motor axons and that it was a trophic disturbance. One manifestation of axonal impairment, which appears to be characteristic of the organophosphates which cause delayed distal neuropathy, is inhibition of fast axoplasmic transport, as demonstrated by Reichlert and Abou-Donia ( 1980). Similar findings were reported in response to another (delayed-neurotoxic compound, acrylamide (Weir et al., 1978). The possibility that delayed-neurotoxic organophosphates might have marked effects on axons of higher brain centers, which might potentiate peripheral and spinal cord damage, has not yet been adequately investigated. The recent findings that acrylamide apparently damages nigrostriatal fibers (Agrawal et al., 1981) raises questions as to whether delayed-neurotoxic organophosphates might do likewise. METHODS
Animals Male Sprague-Dawley-derived rats (Simonson Laboratories, Gilroy, Calif.) weighing 2 15 :t 8 and 232 & 4 g were used for catecholamine and for serotonin studies, respectively. All rats were acclimated to the animal facility surrounding for at least 1 week before testing. They were fed a commercial chow and given free accessto feed and water during experiments. The animals were exposed to 12 hr of light per day.
Chemicak
and Chemical Assay Equipment
The following standards were purchased from Sigma Chemical Company (St. Louis, MO): 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA ), dopamine HCl, norepinephrine HCl, serotonin creatinine sulfate, L-tryptophan, and L-tyrosine. t,-[ri?zg-2,6-3H]tyrosine, 38 Ci/mmol, and L[G-3H]tryptophan, 7.88 Ci/rnmol, were purchased from New England Nuclear (Boston, Mass.). Leptophos (98% analytical standard, Chem. Service Co., Westchester, Pa.) was dissolved in propylene glycol in concentrations such that each animal received 1 ml of the vehicle per 100 g body wt. Atropine sulfate purchased from Sigma Chemical Company was administered ip at 50 mg/kg to leptophos-treated animals and controls prior to leptophos administration. All gels. resins, and reagents used in sample assays were of a quality suitable for fluorescence studies. Fluoro.metric assayswere performed on an Aminco-Bowman spectrophotofluorometer (Aminco, Silver Spring, Md.). The Packard Tri-Carb 2660 Liquid Scintillation System (Packard Instrument Co., Downers Grove, Ill.) was used for all scintillation counting.
Rotorod Studies The rotorod apparatus used was similar to that described by Kaplan and Murphy (1972). A wooden dowel (7.6-cm diameter) was placed 33 cm above the floor, which was formed of stainless-steelrods supplied with a scrambled shock device. Rats were trained for seven days before treatment, mild shocks on the floor grid providing the incentive for learning to walk the dowel. Final testing speed was 20 rpm. Tests proceeded1with groups of five randomly selected rats. Leptophos dissolved in propylene glycol was administered at 3-day intervals by oral gavage at initial dosesof
512
ALDOUS.
FARR.
AND
SHARMA
90, 30, and 9 mg/kg. The criterion for normal performance in rotorod tests was the ability to walk on the dowel for 1 min during any of three attempts. Rotorod performance tests were conducted on the days of leptophos administration, prior to gavage. Based on the results of this experiment, the dose levels were selected for subsequent trials to measure brain amines.
Assays of Tyrosine, Norepinephrine, Estimations for Norepinephrine
Dopamine, and DOPAC; and Dopamine
Turnover Rate
Rats were administered 0, 5, or 15 mg/kg/feeding of leptophos at 3-day intervals for a total of five feedings. Two days after the last administration the animals were injected iv with 62 &i L-[ring-2,6-3H]tyrosine, which was dissolved in 0.5 ml saline. Rats were sacrificed by decapitation during the fifth and sixth hours of the daylight cycle at 2.0 or 3.5 hr after injection of the labeled precursor amino acid. Their brains were quickly excised, frozen on dry ice, and subsequently stored at -90°C until assay. Perchloric acid ( 0.4 &I) was used to extract norepinephrine, dopamine, tyrosine, and DOPAC from rats preinjected with labeled tyrosine. Fractions were neutralized with potassium formate and subjected to Sephadex G-10 chromatography, and DOPAC was assayed fluorometrically by the method of Westerink and Korf (1977). The balance of the perchloric acid extracts were neutralized with 5 A4 KOH. Supernatants were separated into fractions containing norepinephrine, dopamine, and tyrosine on Dowex 50 columns by the method of Neff et al. (1977). Portions of the isolated fractions of the three amines were added to the scintillation cocktail and counted. Fluorometric assays of norepinephrine and dopamine were performed by the manner of Karasawa et al. (1975). Tyrosine was assayed fluorometrically by the method of Smith et al. (1975).
Tryptophan, Serotonin, and 5-HIAA Estimates
Assays and Serotonin
Turnover Rate
Rats were gavaged with leptophos every third day for five feedings. Groups received 4.5, 15, or 45 mg/kg/feeding. Two days after the last dosing, rats were administered 50 PCi L-[G-3H]tryptophan iv. Half of each dosage group were sacrificed at 40 min, the balance at 120 min, after injection. Sacrifices occurred during the third and fourth hours of the daylight cycle. Removal, freezing, and storage of brains followed, as described for catecholamines. On the day of assay, the brains were homogenized in 0.4 M perchloric acid. Neutralized extracts were fractionated on Dowex 50 columns by the method of Marini et al. (1979). The fluorometric method of Atack and Lindquist (1973) was used for 5-HIAA assay. Tryptophan was assayed by the fluorometric method of Marini et af. (1979), and serotonin was determined fluorometrically according to Karasawa et al. (1975). Tryptophan and serotonin fractions were also subjected to liquid scintillation counting, and the method of Neff et al. ( 197 1) was used to estimate turnover rates.
Data Analysis External standards were used to obtain values for biogenic amine and metabolite levels. These values were corrected for fractional recovery and adjusted to represent nanomoles of compound per gram wet weight of brain. A one-way analysis of
LEPTOPHOS
AND
BRAIN
AMINES
573
variance was employed to assess dose effects on animal weight gain and on levels of biogenic amines and their metabolites. Significant differences among treatment means (I’ < 0.05) were calculated by Tukey’s HSD multiple mean comparison test (Neter and Wasserman, 1974). Turnover rates were calculated on the basis of averaged specific activities of biogenic amines and their precursors for groups treated alike. Standard errors for turnover rate estimates were obtained by applying specific activities of pairs of rats, which had been given the same dose but were preinjected at different times, to the formula of Neff et al. (197 1). RESULTS Rotorod Studies and Clinical
Signs of Toxicity
Leptophos-treated animals showed signs of tremors after the first dose of 90 mg/ kg. Two animals in that group died after the third atropine dose but prior to oral administration of the leptophos. Dosing was then discontinued because the animals were very lethargic and emaciated. Animals dosed with either 30 or 9 mg/kg had little difficulty performing the rotorod test after six doses over 16 days (Fig. 1). Animals in the 45 mg/kg dose group showed a significantly reduced weight gain in 16 days compared to control animals. Leptophos had no effect on weight gain at lower dose levels. Ej2ct.s OH Catecholamines
and DOPAC
Treatrnent of rats with leptophos had no effect on the total level of brain tyrosine or the specific activity of labeled tyrosine. Since the specific activity differed at the two testing periods after injection of labeled amine, only total values are indicated in Table 1. Similarly, no effect was noticed on the brain levels of norepinephrine but the dopamine level was significantly elevated in animals treated with a cumulative dose of 75 mg leptophos/kg body wt. The change in the whole brain dopamine concentration in this group was nearly 14% over controls. No change in dopamine level wa:s observed in the group treated with a cumulative leptophos dose of 25 mg/kg.
Days
of Leptophos
Dosing
FIG. 1. Percentage of rats unable to remain on the rotorod for 60 set at 20 rpm 3 days after the most recent leptophos treatment (n = 4 in each group). The animals were treated every 3 days by oral gavage. Dosing was discontinued in the group given repeated doses of 90 mg/kg after two treatment periods.
514
ALDOUS, FARR, AND SHARMA TABLE 1
WHOLE BRAIN LEVELSOF TYROSINE,NOREPINEPHRINE, DOPAMINE,ANDDOPAC: ALSO CATECHOLAM~NETURNOVERRATESAFTERLEPTOPHOSTREATMENTOFRATS~
Cumulative doseof leptophos(mg/kg) Control
(n = Tyrosine (nmol/g) Norepinephrine(nmol/g) Dopamine (nmol/g) 3,4-Dihydroxyphenylacetic acid (nmol/g) Norepinephrineturnover rate (nmol/g/hr) Dopamineturnover rate (nmd/g/hr)
14)
116.3 + 3.3 2.60 k 0.05 4.93 -t 0.09
109.7 f 3.5 2.57 + 0.08 4.95 * 0.20
112.9 * 2.2 2.58 t 0.04 5.61 + 0.19'
0.88
f
0.02
0.90
*
0.03
0.99
f
0.46
f
0.08
0.49
2 0.10
0.77
+ 0.10’
1.92 i
0.20
1.68
f
2.74 + 0.39
0.35
0.05
’ Mean f SEM. Group size indicated in parentheses. b Three rats died in the 75 mg/kg group. ‘Significant, P < 0.05.
Synthesis rates of both norepinephrine and dopamine were increased in the group treated with 75 mg leptophos/kg (Table 1). Increases in the synthesis rate constants for these two biogenic amines were 67 and 43%, respectively. These values indicate a considerable rise in adrenergic activity in leptophos-treated rats. The levels of the dopamine metabolite, DOPAC, showed a 13% increase, which was not statistically significant at this dose level. Eflects on Serotonin and its Metabolite In the groups of animals treated with labeled tryptophan, neither the total levels nor the specific activities of tryptophan were influenced by 15 days of leptophos treatment. Again, only the total levels are depicted in Table 2, since the specific activities varied with the time of sacrifice relative to injection of labeled precursor amino acid. The levels of serotonin and its metabolite, 5-HIAA, in the animals given leptophos are also given in Table 2. Levels of these compounds were not altered by leptophos treatments. The turnover rate for serotonin was significantly decreased at the cumulative dose of 75 mg leptophos/kg, but no variation from the control value was observed when the dose of leptophos was increased to 225 mg/kg over the same period. DISCUSSION Most of the economically important organophosphates that cause delayed distal neuropathy are potent acetylcholinesterase inhibitors. For this reason, it is difficult to determine whether alterations in neurotransmitter levels and functions represent primary sequelae of altered choline& function, alterations in neuronal function associated with delayed neuropathy, or other unexplained interactions. Alterations in catecholamine levels or turnover rates have been reported after the administration
LEPTOPHOS
AND BRAIN AMINES
575
TABLE 2 WHOLE
BRAIN
LEVELS TURNOVER
OF ENDOGENOUS RATES AFTER
INDOLE COMPOUNDS AND LEPTOPHOS TREATMENT’
SEROTONIN
Cumulative doseof leptophos(mg/kg) Control Tryptophan (nmol/g) Serotonin (nmol/g) 5-HIAA
(mmol/g)
Serotonin turnover rate (nmol/g/hr)
23.07
2
0.97
2.72 fc 0.07 1.52
3~0.08
1.88 f 0.09
0 14 animals in each treatment group, ’ Significant, P < 0.05.
15
22.5
values
24.87 2.63 1.67
f i +
0.82 0.09 0.15
1.96 f 0.05 are means
225
f 1.04 + 0.05 t 0.07
23.23 2.78 1.43
1.58 + 0.05’
2.07
22.92 2.69 1.59
k
0.86
k 0.11 +- 0.10
+ 0.10
+ SEM.
of potent cholinesterase inhibitors that do not cause delayed neuropathy (Holt and Hawkins,, 1978; Fiscus and Van Meter, 1977). Freed et al. (1976) found reductions in dopamine levels after administering mipafox and leptophos, both of which cause delayed distal neuropathy. Fenitrothion, however, which lacks delayed neuropathy effects, did not alter steady state dopamine levels. In the present studies, levels of dopamine were slightly elevated in rats receiving a moderate dose of leptophos ( 15 mg/kg every third day for a cumulative dose of only 75 mg/kg). The present study involved animals receiving a dose of Ieptophos not sufficient to cause signsofdelayed neuropathy, whereas rats in the Freed et al. (1976) study had received small daily dosesof (delayed-neurotoxic compounds over enough time to elicit motor deficits. In control rats, the whole brain levels of norepinephrine, dopamine, serotonin, the acid metabolites, and the turnover rates of biogenic amines studied here were comparable to those reported previously (Marini et al. 1979, Neff et al. 197 1~Smith et al. 1975). Neurotransmitter turnover rate alterations are generally more sensitive indicators of altered brain function than are changesin steady-state levels of neurotransmitters (Neff et al., 1971). The reduction in the serotonin turnover rate that followed a repeated dose of 15 mg/kg leptophos was not observed after 45 mg/kg, and may represent.an artifact. The increasesin norepinephrine and dopamine turnover rates and the apparent increase in DOPAC levels after five repeated dosesof 15 mg/kg leptophos indicate an increase in activity of catecholaminergic neurons. Inhibition of catecholamine re-uptake by the presynaptic membrane may permit an increased extracellular destruction of the neurotransmitters, and thus necessitatean increased synthesis rate to maintain normal steady-state levels. It is also possible that altered postsynaptic receptor affinities for, or responsiveness toward, neurotransmitters might cause reflex alterations in the quantities of neurotransmitters discharged per action potential. One compound that causesdelayed central-peripheral distal neuropathy, acrylamide. increases the number of dopamine receptors in rat striatum and decreasesthe responsivenessof such receptors to dopamine agonists (Agrawal et al., 1981). The possibility that chemicals which cause delayed neuropathy characteristically alter neurotransmitter receptor function, with possible subsequent alterations in neurotransmitter synthesis rates. has apparently not been investigated.
576
ALDOUS.
FARR.
AND
SHARMA
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