Inhibition of ornithine decarboxylase alters neurological responsiveness to a tremorigen

Difluoromethylornithine an of ornithine decarboxylase (ODC; 200-800 mg/kg, s.c.), to rats has no detectable behavioral effects using a battery of tests to assess sensorimotor function. In contrast, the induction of tremor by chlordecone, a neuro toxic agent that affects neuronal ionic processes, is significantly attenuated by pretreatment with DFMO. The effects of DFMO on chlordecone-induced tremor were reversed by pretreatment with putrescine. DFMO had no effects on p,p' -DDT, a tremorigen hav ing a mechanism of action different from chlordecone. These findings imply that polyamines may play a role in select neuronal processes.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the biosynthesis of the polyamines, putrescine, spermidine and spermine 13 · 20 • 23 . Polyamines appear to be essential for cell division and ODC is present in high concentrations in proliferating tissues 14 · 17 . The irreversible inhibitor of ODC, difluoromethylornithine (DFM0) 14 , is under trial as an experimental drug in cancer chemotherapy 4 .l 5 · 21 . Although basal levels of ODC are relatively low in the mature nervous system, they can be rapidly elevated in response to a variety of stimuli, including chemical or physical insults to the brain and electroconvulsive shock 2 · 16 . A role for ODC in the adult nervous system is further suggested by the finding that intraventricular injection of DFMO produces hyperexcitability, tremors, muscular rigidity, seizures and at lethal doses, histological damage to the hippocam-pus6. Systemic administration of polyamines such as spermidine and spermine to mice has been reported to depress spontaneous motor activity, enhance pentobarbital sleeping times, inhibit the writhing response induced by acetic acid and attenuate the effects of strychnine 18 . Similar results have been obtained following central administration of polya-mines 5 · 19 . In spite of these observations, there has been little published concerning the possible neurobiological consequences of ODC inhibition in mature animals. The present series of experiments were undertaken to determine the effects of systemically administered DFMO on sensorimotor function of intact, mature animals.
Male, adult rats of the Fischer-344 strain were used in these studies. Initial experiments were performed to determine the amount of inhibition produced by 200 mg/kg DFMO s.c., a dose suggested by pilot studies to cause marked inhibition of ODC. ODC was assayed by the measurement of evolved 14 C0 2 from DL-[1-14 CJornithine (55.9 mCi/mmol; New England Nuclear, Boston, MA) 17 . Tissues were homogenized in 19 vols. of 0.04 M Tris-HCI pH 7.4 and the homogenate centrifuged (26,000 g) for 20 min. An aliquot (0.9 ml) of the supernatant was added to 50 µI of pyridoxal phosphate solution (0.001 M) and 50 µI DL-[1-1 4 C)ornithine in 0.0445 M diethiothreitol. Incubation was for 30 min at 37 °C in a sealed tube and labeled C0 2 was trapped on a paper wick containing hyamine, suspended above the reaction mixture. The decarboxylation reaction was linear with time under these conditions. Background decarboxylation not attributable to ODC was determined by running a parallel incubation in the presence of 5 mM DFMO, a specific inhibitor of ODC. For the determination of DFMO content in the brains of treated animals, 30 µl of radioactive DL-a-[3 ,4-3H]DFMO (26.5 Ci/mmol; NEN, Boston, MA) was mixed with unlabeled DFMO to a specific activity of 132 µCilmmol. This mixture was injected s.c. into rats in a final concentration of 200 mg/kg. Five hours after injection, the rats were decapitated and the brains removed, weighed, and dissolved in tissue solubilizers (NCS, Nuclear, Chicago) and radioactivity measured. At 5 h after 200 mg/kg DFMO, brain ODC was inhibited by 87 ± 0.5% and the cerebral concentration of DFMO was 0.39 ± 0.12µM. Subsequent behavioral studies were performed in rats using water vehicle, 400 or 800 mg/kg of DFMO. Injections were given s.c. in a volume of 1 ml/kg b. wt. The rats were tested for alterations in sensorimotor function 5 h later. Vertical and horizontal components of motor activity were independently assessed during a 30-min test session in black Plexiglas test chambers (22 x 44 x 18 cm) located within a light-and sound-attenuated test chamber. Arrays of I 0 equally spaced infrared photodetector/emitter pairs were located 5.5 cm from the floor to measure horizontal activity and 13 cm from the floor to measure vertical activity. After testing motor activity. rats were assessed for their responsiveness to a novel acoustic stimulus using a commercially available reflex monitor. During testing each animal was placed into a small plastic cage enclosed within a light-and sound-attenuated chamber equipped with a ventilation fan. Test stimuli consisting of 10 consecutive 110 dB, 8 kHz tones superimposed on a continuous 75 dB white noise background were presented following a 2-min acclimation period in the test chamber. The stimulus duration was 200 ms and there was a fixed 15-s interstimulus interval. The startle response corresponded to the largest magnitude (from 0-1800 units) occurring within a 200-ms sampling period following onset of the tone. After the test for startle responsiveness, the rats were assessed for their responsiveness to a thermal stimulus using a commercially available hot-plate apparatus. During the test. each rat was picked up by the tail and gently placed onto the surface of the apparatus, which was maintained at 57.5 °C. The latency to lick one of the hindpaws was recorded. The animals were removed as soon as a response was made or after 45 s. whichever came first. Table I shows that DFMO had no effect on any of the behavioral measures taken.
The behavioral experiments indicated that DFMO had little or no effect on sensorimotor function of rats in the normal state. However, it was possible that these measures of neurological functioning may have been insensitive to more subtle effects of DFMO on nerve function. To assess this possibility, we studied the effects of DFMO on the neurological effects of a chemical known to produce its effects by interfering with ionic processes at the level of the nerve membrane. In these experiments, rati, were dosed with 200 mg/kg of DFMO 30 min prior to receiving a tremorigenic dose of chlordecone (75 mg/kg, i.p.) 22 . This neurotoxicant has been reported to interfere with mitochondrial uptake of calcium and has been postulated to increase unbound Ca-'+ intracellularly. which may lead to a destabilization of the membrane and increased transmitter release 3 . Five hours after exposure to chlordecone, rats were tested for tremorigenic activity by placing rats onto a platform attached to a load cell-transducer assembly 7 · 8 . The analog signal generated by the movement of the animal was processed by a Model 3582A Hewlett Packard spectral analyzer. A fast Fourier transformation of the signal was performed over a 1.3-min sampling period. Movement was quantified as power (-dBV) occurring from 0-25 Hz. In the present experiments, the average power of the curves was obtained for each rat and differences between groups were determined using analysis of variance (ANO VA) followed by Fisher's least significant difference test 24 .    group. If DFMO (200 mg/kg. s.c.) was given 30 min prior to chlordecone. significant attenuation of tremor was seen 24 (A) and 5 (B) h after chlordecone. If DFMO was given 19 h after exposure to chlordecone. no effect was seen (C). Data were analyzed for overall significance using ANOVA: asterisks indicate significant difference relative to chlordecone.
shows that chlordecone increased the average power of movement as determined by spectral analysis, while DFMO had no effect relative to controls. Rats treated 30 min previously with DFMO and given chlordecone had significantly less tremorigenic activity than rats given only chlordecone. In a subsequent experiment. rats were dosed with DFMO 30 min prior to receiving either corn oil or chlordecone and tremorigenic activity was measured 24 h after exposure to chlordecone. Fig. lB shows that under these conditions, DFMO still significantly attenuated chlordecone-induced alteration of motor movement. However. if rats were first exposed to chlordecone followed by DFMO 19 h later, then no attenuation of tremor was seen 24 h after exposure to chlordecone (Fig. lC). We reasoned that if the interaction between DFMO and chlordecone were related to the inhibition of ODC, then the effects of DFMO on chlordecone-induced tremor should be attenuated with putrescine, the primary decarboxylation product of ornithine. Other investigators have reported that putrescine pretreatment can attenuate other effects produced by DFM0 10 . Putrescine has also been reported to reverse DFMO-blockage of traumatically induced brain edema 11 • In the following experiment. some rats were given 120 mg/kg putrescine s.c .. in a 149 volume of 1 ml/kg immediately before and at a different injection site than the DFMO injection (200 mg/kg). Tremorigenic activity of chlordecone was determined 5 h after i.p. injection. Fig. 2A shows that putrescine alone had no effect on the spectral analysis of movement. while chlordecone significantly increased the average power of the spectrum. DFMO significantly attenuated the tremor produced by chlordecone and this effect was reversed by coadministration of putrescine. Earlier experiments found that a dose of 60 mg/kg of putrescine was insufficient to affect the DFMO-chlordecone interaction (unpublished observation).
Subsequent experiments were performed to determine the generality of the observation that DFMO affects tremorigenic activity produced by neuronally active chemicals. In these studies, the effects of DFMO pretreatment on the tremorigenic activity of another tremorigen. p,p'-DDT were studied. This agent is believed to act by holding the sodium channel open once it is open, causing repetitive firing of the nerve 25 . Rats were pretreated with 200 mg/kg of DFMO s.c., followed 30 min later by 75 mg/kg of p,p'-DDT dissolved in corn oil and given by gavage in a volume of 1 ml/kg. Fig. 28 shows that DFMO had no significant effect on spectral analysis of movement. while p,p' -DDT produced a significant increase in the average power. Pretreatment with DFMO had no significant effect on the tremorigenic activity produced by p,p'-DDT.
The data reported here are compatible with recent reports suggesting polyamines play a role in calcium mobilization in several tissues including the brain 12 and may be involved in modulating intracellular membrane fusion 9 . DFMO has been shown to inhibit calcium-stimulated release of D-aspartate from nerve endings while the concurrent presence of putrescine prevents this inhibition 1 . Thus, the induction of tremor by chlordecone and its apparent dependence on polyamines such as putrescine may be related to modulation of calcium fluxes within nerve tissue. This conclusion is in accord with the potential effects of chlordecone on intracellular calcium disposition 3 . The finding that DFMO had no effect on tremorigenic activity produced by p,p'-DDT while attenuating Bondy, S.C. and Walker, C.H., Polyamines contribute to calcium-stimulated release of aspartate from brain particulate fractions, Brain Research, in press. 2 Dienel, G.A. and Cruz. N.F., Induction of ornithine decarboxylase during brain recovery from metabolic, mechanical, thermal or chemical injury. that of chlordecone lends further support to the conclusion that these two chlorinated insecticides have different mechanisms of action 22 . Finally. our results suggest that, although ODC is very low in the mature nervous system, polyamines appearto play a role in neuronal processes. Furthermore. these results suggest that care be taken in the treatment of individuals with DFM0 15 · 21 , who may also be receiving pharmacological agents having effects on nerve function.
We thank Dr. P. McCann of the Merrell Research Center, Cincinnati, OH, for the DFMO used in this research. The technical assistance of Mr. Ronnie McLamb in performing some of these experiments and Mrs. Loretta Moore in typing this manuscript is gratefully acknowledged.