Brain Research 768 Ž1997. 345–348
Methamphetamine-induced serotonin neurotoxicity is attenuated in p53-knockout mice Hiroshi Hirata, Jean Lud Cadet
Molecular Neuropsychiatry Section, NIH r NIDA, DiÕision of Intramural Research, P.O. Box 5180, Baltimore, MD 21224, USA Accepted 17 June 1997
Abstract Methamphetamine ŽMETH. is a drug of abuse that causes deleterious effects to brain monoaminergic systems. The tumor suppressor gene, p53, is thought to play an important role in cell death. In the present study, we have assessed the participation of p53 in METH-induced serotonergic neurotoxicity, by using the mice lacking the gene for p53 protein. Three dosages Ž2.5, 5.0 and 10.0 mgrkg= 4. of METH were administered to wild-type Žp53qrq., heterozygous Žp53qry. and homozygous Žp53yry. p53-knockout mice. The two lower doses caused no significant changes in serotonin Ž5-HT. transporters in any of the groups. The highest dose Ž10.0 mgrkg. caused significant decreases in striatal 5-HT transporters in wild-type Žy31%. and heterozygous Žy18%. mice. In contrast, 5-HT transporters were not significantly decreased in homozygous mice. These results suggest that the tumor suppressor, p53, plays an important role in METH-induced serotonergic neurotoxicity in mice brain. These data provide further evidence for a role of p53 in the neurotoxic effects of METH. q 1997 Elsevier Science B.V. Keywords: Knockout mouse; Methamphetamine; Neurotoxicity; p53; Serotonin transporter
p53 protein plays an important role in both apoptosis and cell-cycle arrest w14,15,19x. DNA damage caused by a number of agents including free radicals causes accumulation of p53 protein w7,12,22x with subsequent induction of apoptosis in the damaged cells w20x. In addition to its neurotoxic effects on dopaminergic systems w3–5x, methamphetamine ŽMETH. can also damage serotonergic neuronal systems. This is evidenced by long-lasting changes in several neuronal markers, including serotonin Ž5-HT. synthesis w1x, concentration of 5-HT w11,16,18,21x and 5-HT transporters w13,17,21x. Previous studies from our lab have shown that free radicals might be involved in the toxic effects of METH on the 5-HT system w9x. Because free radicals can cause the p53 accumulation w7,22x, it was reasonable to think that p53 might play a role in METH-induced toxicity. We postulated that if this were indeed the case, then animals lacking the gene for p53 protein should show protection against the toxic effects of METH.
Corresponding author. Fax: q1 Ž410. 550-2745.
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 7 9 8 - 1
Male homozygous p53-knockout transgenic mice Žp53yry ., heterozygous p53-knockout transgenic mice Žp53qry . and wild-type Žp53qrq . mice ŽGenPharm, Mountain View, CA. aged 8 weeks were used in these experiments. These animals were produced by Donehower et al. w6x. All animal care and use procedures were according to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care and Use Committee of the NIDA. On the day of experiments, the animals were injected with saline or 2.5, 5.0 or 10.0 mgrkg of METH intraperitoneally. These were given at 2 h intervals for a total of 4 injections in one day according to a previously published protocol w10x. Two weeks later, the animals were sacrificed and their brains were rapidly removed, frozen in isopentane on dry ice and stored frozen at y708C. Sections Ž20 m m thick. were cut at y208C and thaw-mounted on gelatin-coated glass slides. The slides were kept at y708C until used in the autoradiographic studies using w 125 IxRTI-55 Žspec. act.: 2200 Cirmmol.. Binding assays were performed according to a published protocol w10x. Briefly, slide-mounted sections were incubated for 90 min at 48C with w 125 IxRTI-55 Ž150 000 cpmrml. in a binding buffer
H. Hirata, J.L. Cadetr Brain Research 768 (1997) 345–348
ŽBB. consisting of 55.2 mM sodium phosphate buffer, pH 7.4. The radioligand was made in a protease inhibitor cocktail containing BB, 1 mgrml BSA, chymostatin Ž25 m grml., leupeptin Ž25 m grml., EDTA Ž100 m M., and EGTD Ž100 m M.. LR1111 Ž10 m M. was used to block binding of w 125 IxRTI-55 to dopamine ŽDA. transporters. Specific binding was determined in the presence of 10 m M paroxetine and represented greater than 90% of total bind-
ing. At the end of the incubation period, the slides were washed in fresh ice-cold buffer, dipped in ice-cold distilled water and dried under a stream of cool air. The slides were then apposed to radiosensitive films ŽHyperfilm, Amersham, Buckinghamshire, UK. with plastic standards Žw 125 Ixmicroscales, Amersham. for 5 days at 48C. The films were then developed according to routine procedures. w 125 IxRTI-55 binding was quantified on both sides of the
Fig. 1. Autoradiographic representation of the dose-dependent effects of METH on w 125 IxRTI-55-labeled 5-HT transporters in p53qrq ŽA, D, G, J., p53qry ŽB, E, H, K. and p53yry ŽC, F, I, L. mice. Mice were treated with saline ŽA, B, C., or four injections of 2.5 mgrkg ŽD, E, F., 5.0 mgrkg ŽG, H, I. and 10.0 mgrkg ŽJ, K, L. of METH given 2 h apart. Mice were sacrificed 14 days later. Autoradiographic studies were carried out as described. The lighter images represent greater binding density. Note the obvious dose-dependent decreases in 5-HT transporters caused by METH in the p53qrq mice ŽG, J.. The terminal loss caused by METH was attenuated in both p53qry ŽH, K. and p53yry ŽI, L. mice. The quantitative data are given in Fig. 2.
H. Hirata, J.L. Cadetr Brain Research 768 (1997) 345–348
of METH on the serotonergic system. This result indicates that p53 protein may be an important mediator of the long-term effects of METH in the serotonergic system. It is thus possible that METH-induced toxicity in the serotonergic system might be through a process that resembles apoptosis because p53 accumulation is associated with this process of cell death w14,15,19x. The present results are compatible with our demonstration that METH-induced dopaminergic toxicity is also attenuated in p53-knockout mice w8x. Because we and others have previously shown that the toxic effects of this drug are secondary to oxidative stress w2,4,5,10x, the present data and those obtained previously on dopaminergic neuronal systems indicate that similar mechanisms might be involved in the toxicity of METH on both striatal 5-HT and DA systems of mice.
Acknowledgements The authors thank the staff of the Animal Care Facility at the Division of Intramural Research of NIHrNIDA for the impeccable care of the animals.
References Fig. 2. Dose-dependent effects of METH on 5-HT transporters in the striatum ŽA., the septal nucleus ŽB. and the nucleus accumbens ŽC.. The mice were injected with either saline or METH Ž2.5, 5.0 or 10.0 mgrkg =4 in one day. and sacrificed 14 days later. The values represent means"S.E.M. ŽnCirmg tissue. of 5–6 animals per group. Statistical analyses were done using analysis of variance ŽANOVA. followed by posthoc Fisher’s PLSD test. Criteria for significance were set at the 0.05 level. Key to statistics: ) p- 0.05, ) ) p- 0.005 in comparison to salinetreated mice of the same strain; ap- 0.05, aap- 0.005 in comparison to the p53qrq mice treated with the same dose of METH.
mice brains using a Macintosh computer-based image analysis system ŽImage, NIH. using standard curves generated from the w 125 Ixmicroscales. Non-specific binding was at the level of the film background. Statistical analyses were done using analysis of variance ŽANOVA. followed by posthoc Fisher’s PLSD test. Figs. 1 and 2 show the effects of METH on w 125 IxRTI55-labeled 5-HT transporters in the striatum, septal nucleus and nucleus accumbens of mice that were sacrificed 2 weeks after the administration of METH. The low dose Ž2.5 mgrkg= 4. caused no significant changes in striatal 5-HT transporters in any of the three strains. The high dose Ž10.0 mgrkg= 4. caused significant decreases Žy31%. in striatal 5-HT transporters of p53qrq mice. 5-HT transporters were also significantly decreased Žy18%. in p53qry mice. In contrast, no significant decreases were observed in the p53yry mice. This is the first demonstration that mice lacking the gene for p53 protein are protected against the toxic effects
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