Psychopharmacology Psychopharmacology (1989) 97:32(~330 9 Springer-Verlag 1989 Evidence for monoaminergic involvement in triadimefon-induced hyperactivity* K.M. Crofton 1, V.M. Boncek 2, and R.C. MacPhail 1 1 Neurotoxicology Division, Health Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 2771 i, USA 2 Northrop Services, Inc., Environmental Sciences, Research Triangle Park, NC 27709, USA Abstract. Triadimefon is a triazole fungicide that produces hyperactivity in both mice and rats similar to that seen following administration of compounds with catecholaminergic activity (e.g., d-amphetamine). To determine whether the triadimefon-induced hyperactivity is due to an action on CNS catecholaminergic systems, we evaluated the effects of combined treatment of triadimefon with either the tyrosine hydroxlase inhibitor d,l-~-methyl-p-tyrosine methyl ester HC1 (c~MPT) or the amine depletor reserpine. Adult male Long-Evans hooded rats, approximately 70 days of age were used. Dosage-effect functions were determined for c~MPT (0 200 mg/kg IP), reserpine (0-2.5 mg/kg IP), d-amphetamine (0-3 mg/kg IP), and methylphenidate (0-40 rag/ kg IP). Motor activity was measured as photocell interruptions in figure-eight mazes. The interaction between triadimefon and ~MPT was determined with the following groups: 1) vehicle control; 2) 200 mg/kg triadimefon PO; 3) 100 mg/kg ~MPT; and 4) both c~MPT and triadimefon. A similar design was used to determine the interaction between triadimefon and reserpine (0.62 mg/kg), ~MPT and d-amphetamine (1.5 mg/kg), and reserpine and methylphenidate (5.0 mg/kg). In the first experiment e M P T did not block the increased motor activity produced by triadimefon (i.e., both triadimefon alone and c~MPT in combination with triadimefon produced significant increases in motor activity), c~MPT did, however, block d-amphetamine-induced hyperactivity. Since e M P T did not antagonize the effect of triadimefon, these data suggest that increased motor activity produced by triadimefon is not mediated through release of newly synthesized catecholamines. In contrast, pretreatment with reserpine blocked the hyperactivity induced by both triadimefon and methylphenidate, which suggests that triadimefon-induced hyperactivity may be due to an interaction with CNS catecholamines stored in reserpine-sensitive pools. Key words: Triadimefon Hyperactivity - Reserpine - ctMethyl-p-tyrosine - d-Amphetamine - Methylphenidate * The research described in this article has been reviewed by the Health Effects Research Laboratory, US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Presented in part at the Annual Meeting of the Society for Neuroscience, New Orleans, LA, November, 1987 Q{'fprint requests to." K.M. Crofton Triadimefon, [1- (4- chlorophenoxy)- 3,4- dimethyl- 1- ( I H 1,2,4-triazole-l-yl)-2-butanone], is a substituted triazole fungicide. Although the mechanism of fungicidal action has been well documented (Buchenauer 1977, 1978; Brent and Skidmore 1979) little information is available on the effects of triadimefon in mammals. Triadimefon is readily absorbed following oral exposure in rats. Maximal tissue and plasma levels in rats occur 1 2 h following oral administration of [14C]-triadimefon (FAO 1981). Triadimefon exposure in humans has been associated with contact dermatitis (Winter and Kurtz 1985). Knaak and coworkers (1984) demonstrated greater than 50% percutaneous absorption of triadimefon in rats, with a tl/2 of elimination of 29 54 h. Recent work has demonstrated triadimefon-induced hyperactivity following oral administration in both mice (MacPhail 1986) and rats (Crofton et al. 1988) with a similar time-course to the tissue and plasma kinetics (FAO 1981). This hyperactivity is accompanied, at higher dosage levels, by stereotyped behaviors (Moser 1987; Walker et al. 1988) similar to those seen following administration of apomorphine, d-amphetamine, and other psychomotor stimulants (Randrup and Munkvad 1967; Scheel-Krfiger 1971; Wallach 1974). Due to the similarity in the behavioral effects of triadimefon and CNS stimulants such as apomorphine and damphetamine, it was hypothesized that triadimefon-induced hyperactivity may involve dopaminergic systems in the CNS. Evidence from various investigators has led to the hypothesis of two distinct classes of indirect acting CNS stimulants, those antagonized by reserpine (methylphenidate-like compounds), and those antagonized by a-methylp-tyrosine (amphetamine-like compounds) (see McMillen et al. 1980; McMillen 1983). The purpose of this work was to determine whether the CNS stimulant properties of triadimefon are similar to amphetamine-like or methylphenidate-like compounds. We evaluated the interaction of prior treatment with either d,l-~-methyl-p-tyrosine methyl ester HC1 (~MPT) or reserpine. ~-MPT pretreatment depletes the newly-synthesized pool of catecholamines (Widerlov and Lewander 1978a), whereas reserpine depletes the stored-vesicular pool of CNS amines (Carlsson et al. 1963; Anden 1967). These two compounds can be used, both in vivo and in vitro, to differentiate "amphetamine-like" and "methylphenidate-like" compounds (Weissman et al. 1966 ; Aceto et al. 1967; Stolk and Rech 1967; Frey and Magnussen 1968; Scheel-Kriiger 1971 ; Thornberg and Moore 1973; Hollister et al. 1974; Braestrup 1977; Chiueh and Moore 1974, 1975; Widerlov and Lewander 1978b; Fessler et al. 327 t980; McMilten et al. 1980; McMillen 1983; Niddam eL al. 1985; Butcher et al. 1988). d-Amphetamine and methylphenidate were used for comparative purposes as positive controls for indirect agonist activity resulting from release of newly-synthesized, or stored catecholamine pools, respectively. Methods Animals. Male Long-Evans hooded rats (Blue Spruce Farms, Altamont, NY), were obtained at approximately 60 days of age, and were housed two per cage in standard plastic hanging cages (24 x 20 x 45 cm). All animals were given a 10-day acclimation period and were maintained on a 12:~2 h photoperiod, L : D (0600:1800). Food (Purina Lab Chow) and water were provided ad lib. Temperature was maintained at 21.0_+2.0~ C and relative humidity at 40 + 20%. Motor activity. Motor activity was measured in 16 figureeight mazes, each consisting of a series of interconnected alleys (10 x 10 cm) converging on a central arena and covered with transparent acrylic plastic (Reiter et al. 1975). Motor activity was detected by eight phototransistor/photodiode pairs; each time a photobeam was interrupted, an activity count was registered. Dosage-effect data were collected and stored in 5-min intervals to allow analysis of the temporal pattern of activity. Animals were tested only once for I h, except in experiments with methylphenidate where animals were tested for 2 h. Animal exposures. Acute dosage-effect functions were determined as follows: 0 200 mg/kg (d,l)-ct-methyl-p-tyrosine methyl ester HC1 (Sigma Chemical Co., St. Louis, M e ) was administered IP in 1.0 ml/kg saline (Abbott Laboratories, North Chicago, IL) 3 h prior to testing; 0-2.5 mg/kg reserpine (Sigma ChemicaI Co.) was administered IP in 2.0 ml/kg deionized water (dissolved first in a few drops of glacial acetic acid) 18 h prior to testing; and both methylphenidate HC! (0-40 mg/kg, Sigma Chemical Co.) and damphetamine sulfate (0-3.0 mg/kg, Sigma Chemical Co.) were administered IP 20 min prior to testing in 1.0 ml/kg saline. All dosages are expressed as the salt form, except reserpine which is the free base. These dosages and treatment times were based on published values. For the interaction studies, non-effective dosages of c~-methyl-p-tyrosine (100 mg/kg) and reserpine (0.62 mg/kg), and maximally effective dosages of methylphenidate (5.0 mg/kg) and d-amphetamine (1.5 mg/kg) were selected. Triadimefon (0 or 200 mg/kg, Chem Service, Inc., West Chester, PA) was administered PO in 2.0 ml/kg corn oil (Fisher Scientific, Inc., Pittsburgh, PA) I h prior to testing. This dosage of triadimefon was based on previously published data (Crofton et al. 1988). Following treatment, rats were returned to their home cages until testing. All rats were randomly assigned to treatment groups and to individual test chambers. Separate groups of rats were used for each experiment (n = 7-16/ group). The interaction between triadimefon and reserpine was determined using the following design: 1) vehicle controls; 2) 200 mg/kg triadimefon; 3) 0.62 mg/kg reserpine; and 4) both triadimefon and reserpine. A similar design was used to determine the interaction of triadimefon and c~-MPT (100 mg/kg), reserpine and methylphenidate (5.0 mg/kg), and c,-MPT and d-amphetamine (1.5 mg/kg). All treatment times were as above. I 400 I I I I A c~-MethyJ-p-Tyrosine 350 300 250 200 150 !~< 100 v , 8 VEH 400 l 50 t 100 I 350 o 150 I 200 l f3 Reserpine 3oo ~.= 250 2OO 15(} 100 50 0 VEH 0.31 0.62 1.25 Dosage 2.5 (mg/kg) Fig. ~A, B. The effects of c~-methyl-p-tyross (A) on figure-eight maze activity during a l-h test session 3 h post-administration. VEH= saline vehicle, (n ~ 9-10/group). The effects of reserpine (B) on figure-eight maze activity during a t-h test session 18 h postadministration. VEH = vehicle, (n = 6-10/group). * significantly different from vehicle control group (P<0.05, Tukey's). AlI data are presented as group means (+ SE) Analysis of variance (ANOVA) procedures were used for all main-effects tests (SAS 1986). An c~ level of 0.05 was selected to determine significance. Mean contrast comparisons were made using Tukey's studentized range test (SAS 1985). Results Exposure to ~MPT had no effect on motor activity at dosages up to 200 mg/kg (Fig. 1 a [F(4,42)= 3.28, P > 0.05]. Reserpine, however, produced large decreases in motor activity (Fig. 1 b). There was a significant overall effect of reserpine [F(5,37)= 16.00, P < 0.0001], due to significant decreases in the groups receiving 1.25 and 2.5 mg/kg ( P < 0.05). Both d-amphetamine and methylphenidate produced non-monotonic alterations in motor activity (Fig. 2a and b). For d-amphetamine there was a significant effect of treatment [F(4,45)= 5.01, P<0.002], Although mean contrast tests indicated significant increases in activity in the 0.375, 0.75 and ~.5 mg/kg dosages (P<0.05), the highest dosage of 3.0 mg/kg was not different when compared to contro!s ( P > 0.05). For methylphenidate there was a significant effect of treatment [F(5,34)= 13.4I, P < 0.0001i. Activity in the 5.0 and 10.0 mg/kg groups was significantly increased relative to vehicle controls (both P<0.05). A 2-h test session was used for the methylphenidate-treated animals because pilot data indicated non-significant effects with l-h test times. Behavioral stereotypies including licking, gnawing, rearing and head weaving were observed in the groups receiving the largest dosages of d-amphetamine and methylphenidate. Reserpine pretreatment blocked the hyperactivity induced by both triadimefon and methylphenidate (Fig. 3a and b). There were overall effects of treatment for both 328 450 I I I I the triadimefon/reserpine experiment [F(3,51)=6.12, P < I 0.0012] and the methylphenidate/reserpine experiment [F(3,26) = 5.55, P < 0.0044]. The reserpine alone dosage was not significantly different from control in either experiment (P>0.05). Both the 200 mg/kg dosage of triadimefon and the 5.0 mg/kg dosage of methylphenidate produced significant increases in motor activity, 58% and 64% when compared to controls, respectively. These increases were blocked by pretreatment with reserpine; groups that received both reserpine and either triadimefon or methylphenidate were not significantly different from controls ( P > 400 § IX 350 "l- 300 '~ 250 -.t O 200 O ---- 150 O 100 ~ 50 0 I V I I 0.375 0.75 I 1.5 I 3.0 0.05). Pretreatment with c~MPT blocked the d-amphetamine- ~ " 700 U) +1 Ix 0oo "r" induced hyperactivity but not triadimefon-induced hyperactivity (Fig. 4a and b). There were significant overall effects of treatment for the triadimefon/eMPT [F(3,28)=30.69, P < 0.0001] and the d-amphetamine/c~MPT [F(3,28) = 7.40, P < 0.0008] experiments. Triadimefon alone significantly increased motor activity 83% compared to the control group (P<0.05), but pretreatment with ~MPT failed to significantly attentuate this hyperactivity. The group that received both triadimefon and c~MPT had significantly increased motor activity compared to both the control and the ~ M P T alone gronps (P<0.05), and did not differ from the group that received triadimefon alone ( P > 0.05). d-Amphetamine significantly increased motor activity 82% compared to the vehicle control group (P<0.05). However, in contrast to the lack of effect of e M P T on triadimefon hyperactivity, 500 400 ,~ 300 "~ 200 O a, 100 0 I I I V 2.5 5.0 I 10.0 I 40.0 I 20.0 Dosage (mg/kg) Fig. 2A, B. Hyperactivity during a 1-h figure-eight maze test session following amphetamine administration (A) or during a 2-h test session following methylphenidate administration (B). Data are presented as group means (+ SE). VEH= saline vehicle, (n = 910/group) * significantly different from vehicle control group (P < 0.05, Tukey's) I ~" I I I 700 500 F T "F "] VEH MPD RSP RSP/MPD A § IX v "I" 400 c 300 o 0 -- I1:~ 600 v 500 400 I 200 100 g. VEH TDF A RSP 8 300 "~ 200 .~ 100 0 RSP/TDF Fig. 3A, B. Antagonism of triadimefon-induced hyperactivity (A) and methylphenidate-induced hyperactivity (B) by reserpine. Data are presented as group means (_SE). VEH-vehicle controls, TDF=200 mg/kg triadimefon 1 h prior to testing, RSP=O.62mg/kg reserpine 18 h prior to testing, RSP/TDF-reserpine pretreatment plus triadimefon. MPD=5.0 mg/kg methylphenidate 20 rain prior to testing, and RSP/MPD=reserpine pretreatment plus methylphenidate (n= 13 16/group and n=7-8/group for the RSP/MPD and the RSP/TDF experiments, respectively). * significantly different from vehicle control group (P< 0.05, Tukey's) - - T F ~ - - T T ~ " 500 r +1 IX v 400 "r" 500 ~- 300 300 F T "f 400 O - - 200 200 100 100 0 o g. 0 VEH TDF ~MPT o~MPT/TDF VEH AMP o~MPT ~MPT/AMP Fig. 4A, B. Lack of antagonism of triadimefon-induced hyperactivity by c~-methyl-p-tyrosine (A) and antagonism of d-amphetamineinduced hyperactivity by c~-methyl-p-tyrosine (B). Data are presented as group means (_+SE). VEH= vehicle controls, TDF= 200 mg/kg triadimefon I h prior to testing, ~MPT= 100 mg/kg ~-methyl-p-tyrosine 3 h prior to testing, c~MPT/TDF= c~-methyl-p-tyrosine pretreatment plus triadimefon, A M P - 1 . 5 mg/kg d-amphetamine 20 min prior to testing, and c~MPT/AMP=~-methyl-p-tyrosine pretreatment plus d-amphetamine (n = 8/group). * significantly different from vehicle control group (P < 0.05, Tukey's) 329 c~MPT pretreatment blocked d-amphetamine-induced hyperactivity. Discussion The results of these experiments implicate involvement of reserpine-sensitive catecholamine pools in the hyperactivity induced by triadimefon. In rats this hyperactivity was antagonized by prior treatment with reserpine. The hyperactivity produced by methylphenidate was also blocked by reserpine pretreatment, c~MPT pretreatment faited to block the hyperactivity produced by triadimefon. However, prior treatment with c~MPT did antagonize the activity increases following exposure to d-amphetamine. These data provide preliminary evidence that triadimefon may act in a manner similar to the "methylphenidate-Iike" CNS stimulants. c~MPT alone had no effect on figure-eight maze activity at dosages up to 200 mg/kg, Aithough these dosages are associated with reductions in brain catecholamine concentrations (Widerlov and Lewander 1978a; Butcher et ai. 1988), others have also reported a lack of effect on motor activity (Menon et al. 1967; Rech etal. 1968; Widerlov and Lewander 1978b). The 100 mg/kg dosage of c~MPT used in the present study was reported to dep!ete both dopamine and norepinephrine to 50% of control !evels (Frey and Magnussen 1968; Widerlov and Lewander 1978a). In contrast to the lack of effect of ~MPT, reserpine produced striking reductions in motor activity. This is in agreement with numerous previous reports using similar dosages and treatment times (e.g., Aceto et ai. ~967; Stolk and Rech 1967; Sansone 1978). Depletion of CNS amine concentrations of approximately 50% has been demonstrated using dosages (0.5 1,0 mg/kg) and treatment times (24 h) similar to those used in the present study (Anden i967; Rech etaI. 1968). As expected, both methylphenidate and d-amphetamine produced dosage-dependent, non-monotonic increases in motor activity. Hyperactivity produced by d-amphetamine at these dosages has been previously demonstrated numerous times (e.g., Aceto et al. 1967; Norton eta!. 1975; Bush~ nell 1986). Methylphenidate has aiso been previously shown to increase motor activity (Ward et al, !981; Brenneman et at. 1982; Menon et al. 1984). In the present study hyperactivity following methylphenidate exposure was significant only when rats were tested for 2 h, but not for 1 h, This was most likely due to an interaction of the tack of effect on initial activity (i.e., no change in activity in the first 5 rain) and the level of hyperactivity sustained throughout she rest of the experiment. The centre1 animals habituated to a lower level than the methylphenidate-treated animals. Thus testing for a longer interval increased the difference between controi and treated animals. For instance there was a 140% increase in activity of the 5.0 mg/kg group during the last hour of the 2-h test session, while there was only a 44% increase during the 1st hour of the test session. Pilot data (not shown) indicated that this effect was not related the to the treatment-[o-test interval; the same effect was seen with treatment-to-test intervals from 20 to 120 rain. Methylphenidate is different from d-amphetamine in this respect, since the latter produced significant increases in activity the Jst hour of testing. This effect of methylphenidate was observed in pilot experiments as well as in the dosage-effect and interaction experiments. The differences between these two drugs points out the necessity in future work to test compounds for longer test sessions when using this device. Test sessions ~hat are too short may not reliably detect hyperactivity produced by compounds like methylphenidate. Pretreatment with c~MPT failed to block the triadimefen-induced hyperactivity, although c~MPT pretreatment did block d-amphetamine-induced hyperactivity. This is in agreement with previous reports on antagonism of d-amphetamine-induced behaviors (Aceto etal. 1967; Weissman et al. 1967; Frey and Magnussen 1968; Scheel-Krfiger 1971; Braestrup t977; Widerlov and Lewander ~978b). These resuits indicate that triadimefon may not increase activity by the same mechanism as d-amphetamine. In contrast, pretreatment with reserpine blocked the hyperactivity produced by both triadimefon and methylphenidate. Antagonism by reserpine has been taken as evidence for an aminergic involvement in me~hylphenidate-induced behaviors (Scheei-Kr/iger 1971; Braestrup 1977; Fessler et al. 1980). The fact that pretreatment with reserpine also blocks the hyperactivity produced by triadimefon administration indicates that triadimefon may act through a mechanism similar to the "methylphenidate-like" CNS stimulants. Reserpine is known to deplete other CNS aminergic transmitters (e.g., serotonin) (Anden 1967; Shore and Giachetti 1978). Taken alone, this fact makes it difficult to attribute the triadimefen-induced hyperactivi:y to an interaction with CNS dopaminergic systems. However, recent evidence sheds interesting light on this issue. Walker et ai. (1988) and Moser (1987) have reported triadimefon-induced stereotyped behaviors that are remarkably similar to those seen following dopaminergic stimuiation (e.g.~ sniffing, licking, gnawing, head weaving, and circling). Although no systematic attempt was made in the present study to quantify stereotypies, the 200 mg/kg PO dosage did not produce any overtly observabl~e stereotypies. Clearly further work is needed to investigate the potential interactions of triadimefon with CNS aminergic systems. In summary, these results demonstrate that triadimefoninduced hyperactivity can be antagonized by pretreatment with reserpine. Pretreatment with c~MPT, however, does not block the hyperactivity produced by triadimefon administration. The dosages of triadimefon used in the present study (200 mg/kg PO), along with the tack of published data on pharmacokinetics and absorption in rats or humans, precludes determination at this time of the hazard potential for human health. 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