• dopamine;
  • methamphetamine;
  • neurotoxicity;
  • protein carbonyls;
  • serotonin;
  • TBARs


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The neurotoxic actions of methamphetamine (METH) may be mediated in part by reactive oxygen species (ROS). Methamphetamine administration leads to increases in ROS formation and lipid peroxidation in rodent brain; however, the extent to which proteins may be modified or whether affected brain regions exhibit similar elevations of lipid and protein oxidative markers have not been investigated. In this study we measured concentrations of TBARs, protein carbonyls and monoamines in various mouse brain regions at 4 h and 24 h after the last of four injections of METH (10 mg/kg/injection q 2 h). Substantial increases in TBARs and protein carbonyls were observed in the striatum and hippocampus but not the frontal cortex nor the cerebellum of METH-treated mice. Furthermore, lipid and protein oxidative markers were highly correlated within each brain region. In the hippocampus and striatum elevations in oxidative markers were significantly greater at 24 h than at 4 h. Monoamine levels were maximally reduced within 4 h (striatal dopamine [DA] by 95% and serotonin [5-HT] in striatum, cortex and hippocampus by 60–90%). These decrements persisted for 7 days after METH, indicating effects reflective of nerve terminal damage. Interestingly, NE was only transiently depleted in the brain regions investigated (hippocampus and cortex), suggesting a pharmacological and non-toxic action of METH on the noradrenergic nerve terminals. This study provides the first evidence for concurrent formation of lipid and protein markers of oxidative stress in several brain regions of mice that are severely affected by large neurotoxic doses of METH. Moreover, the differential time course for monoamine depletion and the elevations in oxidative markers indicate that the source of oxidative stress is not derived directly from DA or 5HT oxidation.

Abbreviations used



dopamine transporter


3,4 dihydroxyphenylacetic acid




5-hydroxyindolacetic acid




1-methyl-4-phenylpyridinium ion






thiobarbituric acid reactive substances


reactive oxygen species.

d-Methamphetamine (METH) is a widely abused substance that has the potential of causing neurotoxicity in humans. Recent findings in humans using brain imaging of dopamine transporters (DAT) by PET techniques in abstinent METH abusers implicate long-lasting alterations or neurodegenerative effects on striatal dopamine (DA) nerve terminals (McCann et al. 1998; Volkow et al. 2001). Additionally, postmortem studies on the brains of METH abusers also demonstrate decreases in dopaminergic measures after acute exposure to this stimulant although these studies did not provide conclusive evidence of damage (Wilson et al. 1996). In animal models, METH administration leads to damage of dopaminergic and serotonergic nerve terminals within several brain regions including the striatum, hippocampus, and cortex, as well as the potential for some cell body degeneration in these regions (for a review see Seiden et al. 1993 and Sonsalla et al. 1989, 1991, 1996; O'Callaghan and Miller 1994; Albers and Sonsalla 1995; Schmued and Bowyer 1997; Eisch and Marshall 1998; Deng et al. 1999).

Several studies link the formation of reactive oxygen species (ROS) to the CNS neurotoxicity following METH administration. Increases in oxidized and reduced glutathione levels immediately following METH administration have been reported (Harold et al. 2000) suggesting a role for antioxidant mechanisms in reducing METH-induced toxicity. In support of this, METH-induced neurotoxicity is attenuated by concurrent treatment with antioxidants or free radical spin-trapping agents (De Vito and Wagner 1989; Cappon et al. 1996; Hirata et al. 1998; Yamamoto and Zhu 1998). Furthermore, METH toxicity is also reduced in mice that over-express the antioxidant enzyme CuZn superoxide dismutase (Cadet et al. 1994; Hirata et al. 1996). Additionally, salicylate trapping techniques demonstrate early elevations in striatal 2,3-dihydroxybenzoic acid (DHBA) indicative of acute increases in ROS formation following METH treatment (Giovanni et al. 1995; Fleckenstein et al. 1997; Yamamoto and Zhu 1998; Kita et al. 1999). These studies support the hypothesis that increased ROS formation may be a contributing factor to toxicity produced by METH.

Toxic doses of METH lead to brain lipid peroxidation and the formation of various oxidation products including malondialdehyde (MDA; Yamamoto and Zhu 1998). Most METH studies have reported elevations of lipid oxidation products using a coupled thiobarbituric acid spectrophotometric assay (thiobarbituric acid reactive substances or TBARs) which measures a variety of substances including MDA as well as contaminating carbonyls from proteins, nucleic acids and other lipid metabolites of non-ROS origin. However, despite its relatively lower assay sensitivity, these studies have reported elevated TBARs in striatum and hippocampus following METH treatment in rodents (Acikgoz et al. 1998; Kita et al. 2000; Wan et al. 2000), and these elevations persist for as long as seven days after the last dose (Wan et al. 2000). Furthermore, salicylate trapping studies performed at 7 days after METH demonstrate the persistence of ROS production for days following the initial toxic insult (Wan et al. 2000). These findings suggest that there might also be delayed mechanisms of ROS production, the latter being independent of acute METH-associated ROS formation.

Free radical formation, possibly resulting from the oxidation of DA or 5-HT (Ohmori et al. 1993; Cadet and Brannock 1998; Hirata et al. 1998; Wrona and Dryhurst 1998; Yamamoto and Zhu 1998; Berman and Hastings 1999; LaVoie and Hastings 1999) has been implicated in the neurotoxicity produced by METH. However, the extent to which these oxidation products contribute to neuronal damage in the monoaminergic innervated regions is unclear. If early onset ROS formation (i.e. which occurs within hours after METH treatment) derives from the direct oxidation of DA or 5-HT then it might be expected that elevations in oxidative markers would occur within the same time frame as the decreases in monoamine levels. Little is known about the time course for the formation of oxidative markers after METH treatment. DA and 5HT levels are rapidly reduced within only a few hours after METH exposure. Thus, one of the primary objectives of the study was to determine if increased formation of oxidative markers paralleled depletions in monoamine content in METH-treated mice.

It is also unclear to what extent ROS formation leads to oxidative damage to other macromolecules including proteins. Evidence that proteins are modified following METH exposure includes the formation of cysteinyl-DA adducts, formed by the nucleophilic attack of cysteinyl sulfur with DA-quinone intermediates (LaVoie and Hastings 1999). However, direct oxidation of protein side groups are not detected by this latter method. One reliable index of oxidative stress to proteins involves the measurement of protein carbonyl formation, resulting from the reaction of ROS with various protein side groups including lysine and arginine (Rice-Evans et al. 1991). Protein carbonyl measurements offer certain advantages over TBARs because of its lack of interference with other non-protein substances and are a more dependable measure of protein oxidative stress (Levine et al. 1990). Whether METH treatment affects protein carbonyls in the striatum or other brain regions is not known. Therefore, we have simultaneously measured TBARs and protein carbonyls to provide a more complete picture of the damage to proteins and lipids produced by ROS following METH treatment. We recently demonstrated a good correlation between elevations in protein carbonyls and TBARs in the hippocampus and striatum following exposure to an excitotoxin (Gluck et al. 2000). Thus, the second objective of the study was to determine whether protein carbonyls were elevated in various brain regions of mice after METH treatment and to assess how these increases compare with the formation of TBARs as a measure of lipid peroxidation within the same brain regions.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References


[3H]-Sodium borohydride (specific activity 90 Ci/mmole) was obtained from American Radiolabeled Chemicals (St Louis, MO, USA). d-Methamphetamine hydrochloride was purchased from Sigma (St Louis, MO, USA). All reagent grade chemicals used in the study were of the highest-grade purity available.


Experiments were conducted in male Swiss–Webster mice (30–40 g; Taconic Farms, Germantown, NY, USA) in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care Committee. Mice were housed in groups of 4–5 per cage in a room maintained at 22°C on a 12-h light–dark cycle with food and water available ad libitum, and habituated to the animal facilities for a period no less than 1 week before use.


Mice received i.p. injections of saline or METH (four 10 mg/kg/injection q 2 h). The METH was dissolved in water and administered in doses equivalent to its free base form. Mice were killed at 4 h and 24 h and 7 days after the last injection of METH. The striatum, hippocampus, cortex and cerebellum were dissected, rapidly frozen, and stored at − 80°C until assayed. Tissues from four mice were pooled for the lipid and protein oxidative marker assays.

MDA measurements by TBARs

The MDA was measured using a modified method (Esterbauer et al. 1991). Brain tissue (30–40 mg wet weight) was homogenized in 1.15% KCl/0.4 mm sodium azide (3–4 mg protein/mL) and incubated at 37°C for 15 min. Proteins were precipitated by the addition of 20% (w/v) trichloroacetic acid. The samples were centrifuged at 14 000 g for 10 min and the supernatant was added to an equal volume of 0.75% (w/v) 2-thiobarbituric acid. These samples were incubated at 50°C for 20 min. Bismalondialdehyde tetraethyl acetal was used as a standard. Protein concentrations were assayed according to previous methods (Lowry et al. 1951).

Protein carbonyl measurements

Protein carbonyl content was measured by tritiated sodium borohydride reduction as described originally (Levine et al. 1990) but modified in the following ways: 50–75 mg of wet weight brain tissue was homogenized in 0.75 mL of buffer (0.1% w/v digitonin/0.1 m potassium dihydrogen phosphate/pH 7.4) and incubated at room temperature (22°C) for 20 min. The sample was centrifuged at 3000 g for 10 min. The supernatant was applied to a Kontes chromatography column (0.7 × 20cm) packed with 6.5 mL Sephadex-DEAE (A50-120) equilibrated in 0.075 m Tris buffer/pH 7.0. The protein was eluted using 0.15 m Tris HCl/pH 7.8 at room temperature. Fractions (500 µL) were collected and A280/A260 ratios (indicating protein purity) measured. Fractions having a ratio greater than 1.50 were pooled and freeze-dried. The dried samples were reconstituted in 100 µL of distilled water containing 3 µL of 2 mm EDTA. Ten µL of [3H]NaBH4 (5 mCi/0.10 mmoles/mL NaBH4) in 0.1 N Na0H was added to the protein solutions. Samples were incubated for 30 min at 37°C, precipitated with 1 mL of 10% TCA, incubated for 5 min in a vented hood and centrifuged for 5 min at 11 000 g. The supernatent was discarded and the pellet was washed three times using 10% trichloroacetic acid. After the final wash, the precipitate was dissolved in 100 µL of 1.0 N NaOH and incubated at 37°C for 15 min The radiolabeled protein solution was transferred to 20 mL glass scintillation vials, 15 mL of Formula 989 (Packard) scintillation fluid was added and the sample counted using a LKB Rackbeta scintillation counter. Typically, one nanomole of carbonyl corresponded to approximately 72 000 cpm.

HPLC measurements

For the 4 h and 24 h measurements of the monoamines and their metabolites, an aliquot of the MDA homogenate was added to 0.4 N perchloric acid in a 1 : 1 dilution and centrifuged. Dopamine, 5-HT and their metabolites in the supernatants were measured by HPLC as previously described (Sonsalla et al. 1991). Norepinephrine was quantified by HPLC using a modified buffer without THF. For the 7 day measurements, brain tissue was dissected and frozen until assayed. The tissue was directly homogenized in 0.2 m perchloric acid. The supernatant was used for measurements.


TBARs and protein carbonyls were compared by a three-way anova using SAS software (V.8; SAS Institute, Cary, NC, USA). Highly significant interactions found between treatment, brain region and time were further analyzed for treatment and time by two way anova with region as a repeated measure factor. Any significant interactions between time and group were further analyzed by one-way anova followed by Bonferroni’s multiple comparison tests. Neurochemical data were analyzed by one-way anova followed by Bonferroni multiple comparison tests. Correlation coefficients and the significance of the linear regressions were determined using GraphPad Prism (V. 3.02, GraphPad, San Diego, CA, USA). Differences were considered significant at p < 0.05.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

METH treatment produces rapid depletion of monoamines

To assess the acute and long-term effects of METH administration on tissue content of monoamines and metabolites, mice were euthanized at 4 h, 24 h and 7 days after the last of four injections of METH. In the striatum, both DA and its metabolite DOPAC were maximally reduced (by > 95%) within 4 h after the last injection of METH with no evidence of any recovery by 7 days (Fig. 1). These dramatic reductions in striatal DA and DOPAC values at 7 days following drug administration are indicative of damage to the striatal dopaminergic system (Sonsalla et al. 1989, 1991, 1996; Hogan et al. 2000). Furthermore, 5-HT in the striatum was decreased by 58%, 50% and 70% at 4 h, 24 h and 7 days, respectively (Fig. 2). Reductions in 5-hydroxyindole acetic acid (5-HIAA) were similar in magnitude.


Figure 1.  METH treatment produces persistent striatal DA depletion. Mice were administered METH (4 × 10 mg/kg i.p. q 2 h) and killed at 4 h, 24 h or 7 days after the last injection. Each sample determination represents pooled striata from four different animals for the 4 h and 24 h time points and from individual animals for the 7 day data. Data are the mean percentage of control ± SD. The dotted line represents control values. Control values (µg/g tissue) are DA (9.3 ± 0.9) and DOPAC (1.0 ± 0.2). Two-way anova indicated a significant effect of treatment on levels of DA [F1,25 = 571; p < 0.0001] and DOPAC [F1,21 = 173; p < 0.0001]. No significant effect of time was observed. aStatistically different versus respective control group as determined by subsequent one-way anova.

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Figure 2.  METH treatment produces persistent depletion of 5-HT in several brain regions. Mice and tissue samples were treated as described in Fig. 1. Data are the mean percentage of control ± SD. The dotted line represents control values. Control values (µg/g tissue) for hippocampus, striatum and cortex are 5-HT (0.56 ± 0.08, 0.70 ± 0.10, and 0.47 ± 0.03, respectively) and 5-HIAA (0.42 ± 0.07, 0.26 ± 0.06, and 0.17 ± 0.04, respectively). Two-way anova indicated significant effects of treatment on levels of 5HT and 5-HIAA in all three brain regions (F-values ranged from 41.5 to 117; p < 0.0001). A significant effect of time was observed only for 5HT in the cortex [F1,24 = 4.8; p = 0.018). aStatistically different vs. respective control group; bstatistically different versus 4 h METH group as determined by subsequent one-way anova.

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Significant reductions in 5-HT and 5-HIAA were also observed in the hippocampus and the cortex of METH-treated mice (Fig. 2). In the hippocampus, 5-HT was reduced by 89%, 78% and 38% at 4 h, 24 h and 7 days, respectively. Similar reductions in 5-HIAA were also observed at these time points. There was no significant evidence of recovery in the hippocampus, although a non-significant trend towards higher levels of 5-HT were observed at 7 days. In the cortex, 5-HT content was maximally reduced at 4 h (87%) with slight but significantly higher levels at 24 h (56%) and 7 days (52%). However, 5-HT and 5HIAA content at 24 h and 7 days were nearly identical indicating no recovery between 1 and 7 days. Although the 7 day decrements in 5-HT and 5-HIAA in the various brain regions were not as dramatic as the losses sustained by the dopaminergic system, the persistent decreases suggest that the serotonergic pathways in these brain regions were damaged. The METH treatment did not alter 5-HT or 5-HIAA in the cerebellum (data not shown).

Unlike the severe and persistent decrements in DA and/or 5-HT observed in METH-treated mice, changes in NE content in the hippocampus and cortex exhibited a different profile. Hippocampal and cortical NE content at 4 h was dramatically reduced to less than 10% of the respective control values (Fig. 3). However, substantial recovery of NE concentrations occurred by 24 h and complete recovery was seen at 7 days. These results indicate that METH produces rapid and reversible changes in NE, reflective of a pharmacological action and not of neurotoxicity.


Figure 3.  METH administration transiently reduces NE content in several brain regions. Mice and tissue samples were treated as described in Fig. 1. Data are the mean percentage of control ± SD. The dotted line represents control values. Control values for NE in hippocampus and cortex were 0.82 ± 0.11 and 0.70 ± 0.06 µg/g tissue, respectively. Two-way anova revealed significant effects of both treatment and time in the two brain regions (F-values ranged from 23.7 to 64.9; p < 0.0001. aStatistically different versus respective control group; bstatistically different versus 4 h METH group as determined by subsequent one-way anova.

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Lipid markers of oxidative stress are elevated after METH administration

Methamphetamine treatment resulted in regionally selective and time-dependent elevations of lipid markers of oxidative stress. Statistically significant elevations of TBARs were observed in the striatum and hippocampus following METH administration (Fig. 4). In the striatum, TBARs were significantly elevated at 4 h (33%) and persisted at 24 h (37%) after METH treatment. In the hippocampus, TBARs were significantly elevated following METH administration only after 24 h (94%). In the cortex, a two-way anova revealed a significant main drug effect of METH treatment on TBARs (F1,16 = 9.39, p < 0.007); however, no significant effects were observed as a function of time [F1,16 = 1.4 (NS)]. In the cerebellum, there were no significant effects of METH treatment on TBARs.


Figure 4.  TBARs are elevated following METH treatment in mice. Mice were treated as described in Fig. 1 and methods. Each sample determination represents pooled brain regions from four mice. Data are the mean ± SD. aStatistically different vs. respective control group; bstatistically different versus 4 h METH; cstatistically different versus 4 h control group.

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METH administration increases protein markers of oxidative stress

As observed with the TBARs measurements, METH treatment resulted in regionally selective and time-dependent elevations of protein carbonyls. Significant elevations in protein carbonyls were observed in the hippocampus and striatum after METH administration (Fig. 5). At 4 h after METH treatment, protein carbonyls were significantly elevated in the striatum but not the hippocampus. At 24 h, protein carbonyls were significantly elevated in both the striatum (47%) and the hippocampus (63%). Furthermore, levels of striatal protein carbonyls were significantly greater at 24 h than 4 h. In the cortex there was a marginally significant main drug effect (F1,16 = 4.62, p = 0.047) but there were no significant effects with respect to time [F1,16 = 2.6 (NS)]. No significant effect of METH-treatment was observed in the cerebellum.


Figure 5.  Protein carbonyls are increased in METH-treated mice. Mice were treated as described in Fig. 1 and methods. Each sample determination represents pooled brain regions from 4 mice. Data are the mean ± SD. aStatistically different vs. respective control groups; bstatistically different versus 4 h METH.

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Correlation between protein and lipid markers of oxidative stress

To examine if there was a correlation between the protein and lipid oxidative markers within each METH affected brain region, the data from controls and both of the treated groups (4 h and 24 h) were plotted using linear regression analysis. Concentrations of protein carbonyls correlated significantly with TBARs in the hippocampus (r = 0.771, p = 0.0001) and the striatum (r = 0.914, p < 0.0001; Figs 6a and b).


Figure 6.  Lipid and protein oxidative markers strongly correlate with one another in a given brain region. Correlation between protein carbonyls and TBARs in hippocampus (a) and striatum (b). Pooled data points from saline, 4 h and 24 h treated groups were used for analysis.

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Various lines of evidence implicate ROS as mediators of neuronal damage following METH exposure. In this study we show that the administration of toxic doses of METH to mice produces brain region-selective and time-dependent elevations of protein carbonyls and TBARs, providing the first evidence that METH administration leads to ROS-mediated oxidative stress to proteins. Concentrations of TBARs and protein carbonyls were significantly increased in the hippocampus and striatum, marginally increased in the frontal cortex and unchanged in the cerebellum following METH administration. These results indicate that protein carbonyl measurements provide an additional reliable marker of ROS mediated oxidative stress, and in conjunction with TBARs measurements offers a complementary technique for detecting regional differences in oxidative markers following METH treatment. In affected brain regions, the magnitude and time course for protein carbonyl formation closely paralleled that of TBARs generation (i.e. greater concentrations were observed at 24 h than at 4 h after METH treatment). Furthermore, the highly significant correlation between levels of protein carbonyls and TBARs taken from the same sample in a given brain region demonstrates that protein and lipid oxidation is probably arising from similar mechanisms. This might be expected given the highly reactive and non-discriminatory nature of hydroxyl and superoxide radicals.

A cause and effect relationship between ROS production following METH administration (evidenced by increased elevations in oxidative markers) and subsequent nerve terminal damage (as measured by neurotransmitter content) remains unresolved. In order to examine whether early elevations in protein carbonyls and TBARs could accurately predict the extent of toxicity, we determined monoamine levels in a parallel group of euthanized mice 7 days after METH treatment. These measures indicated a profound toxicity to the dopaminergic and serotonergic terminals in the striatum as well as the serotonergic terminals in the hippocampus and frontal cortex. Unexpectedly, neurotransmitter loss following METH administration did not coincide with the elevations of oxidative markers measured at 4 h and 24 h post-METH treatment in the brain regions. For example, the striatum appeared to be the most severely damaged brain region based on the neurochemical deficits (70% 5-HT loss and > 95% DA loss at 7 days), but this region did not have the greatest relative increase in TBARs or protein carbonyls. Depletions in hippocampal 5-HT (38% loss) at 7 days were less than that observed in the striatum. Although hippocampal DA loss was not determined due to technical limitations, we have previously observed that DA content in the hippocampus of mice is more than 20-fold lower than in the striatum (unpublished observations). Thus, if catecholamine-associated oxidation is a primary source of ROS formation and considering the extensive amount of DA in the striatum, DA oxidation would be expected to contribute a much greater oxidative burden in the striatum than in the hippocampus. We would therefore have predicted that the relative increase in oxidative markers would have been higher in the striatum than other brain regions. However, levels of oxidative markers were similar in the striatum and the hippocampus.

A further example of the incongruity between oxidative markers and nerve terminal damage is illustrated by data obtained in the frontal cortex. TBARs and protein carbonyls were both substantially elevated in the striatum and hippocampus, and were significantly higher at 24 h than at 4 h post METH treatment. However, in the frontal cortex there were no substantial elevations in oxidative markers, although a small but statistically significant effect was observed with respect to treatment but not time. Neurochemically, however, it appears that all three regions appeared to have sustained substantial nerve terminal injury. The lack of substantial elevations in oxidative markers in the frontal cortex was unexpected because the 7 day measures of 5-HT and 5-HIAA indicated damage to serotonergic nerve terminals. Although it might be argued that the decrements in 5-HT and 5-HIAA in the frontal cortex observed at the 7 day time point are not indicative of toxicity but rather of pharmacological actions, this seems unlikely for several reasons. First, 5-HT and 5-HIAA concentrations in the frontal cortex at 7 days after METH treatment were significantly reduced and were similar to the reductions observed in the hippocampus, a brain region that showed substantial elevations in TBARs and protein carbonyls. Second, concentrations of NE in frontal cortex as well as the hippocampus were as profoundly depleted as 5-HT at 4 h (∼ 90% reductions), but unlike 5-HT, they had nearly recovered by 24 h and were completely recovered by 7 days. These findings are indicative of a pharmacological effect of METH on NE neurons and a toxic effect on 5-HT neurons. Furthermore, O'Callaghan and Miller (1994) have shown that decreases in 5-HT of the magnitude observed in our studies are associated with increases in glial acidic fibrillary protein and reflect neurotoxic damage in the cortex.

In summary, differential elevations of oxidative markers can occur in brain regions damaged by METH. However, our findings also suggest that a neurotoxic insult following METH treatment may not always be associated with measurable elevations in oxidative markers. Therefore, an important conclusion to draw from our studies is that increases in oxidative markers are not necessarily reflective or predictive of the degree of neurotransmitter loss and nerve terminal damage. The reasons for a mismatch between the magnitude of the oxidative stress for lipids and proteins produced by METH and the extent of neurotoxic damage that ensues in brain regions are unclear and warrant further investigation, but may arise from differences in antioxidant buffering capacities or differential susceptibilities to oxidative stress. Furthermore, our finding of serotonergic nerve terminal damage without significantly elevated oxidative markers in the cortex provides further support that METH may initiate additional pathophysiological cell injury pathways involving apoptosis or nitric oxide (Stumm et al. 1999; Deng et al. 1999; Imam et al. 1999, 2001; Kuhn and Geddes 2000).

The cellular source of ROS production in the METH-treated mice is not known. It is proposed that oxidation products derived from DA or 5-HT (for a review see Graham et al. 1978; Wrona and Dryhurst 1998; LaVoie and Hastings 1999) factor heavily into the toxicity associated with METH administration. A recent report by Wrona and Dryhurst (1998) summarized cumulative data and highlighted the potential neurotoxic role for a group of serotonin metabolites arising from superoxide-mediated oxidation. Dopamine quinones have also been implicated in METH toxicity (LaVoie and Hastings 1999). Exposure of neurons to METH leads to disruption of vesicular monoamine stores, increased amounts of neurotransmitter in the cytosol and enhanced release into the extracellular space (Sulzer et al. 1995). These adverse events are thought to initiate the oxidative stress that leads to oxidative damage. However, monoamines are rapidly depleted following initiation of METH administration within only a few hours after the last METH injection; see (LaVoie and Hastings 1999) and the present findings. In the striatum, DA and 5HT are depleted by over 75% as early as 1 h after the last injection of METH (unpublished observations). Oxidative markers were not significantly or only modestly elevated in the hippocampus and striatum, respectively, at 4 h after METH whereas substantially greater amounts were observed at 24 h. These data show that monoamine depletion clearly precedes evidence of ROS-induced damage to cellular proteins or lipids. In contrast, protein cysteinyl-DA levels peak as early as 2 h after the last injection of METH, suggesting a rapid but short-lived formation of DA-quinone attack on proteins (LaVoie and Hastings 1999).

Based on these observations, it appears that the formation of ROS may depend initially on oxidation products derived from the monoamines but that continued ROS formation is independent of monoamine oxidation. If ongoing release of monoaminergic neurotransmitters does not account for the increase in oxidative markers at 24 h then additional pathophysiologic mechanisms must be operative. Continued formation of ROS independent of catechol or indole monoamine oxidation could occur with compromised mitochondrial function and aberrant electron transport function leading to increased production of superoxide and decreased ATP production (Burrows et al. 2000a; Lotharius and O'Malley 2000; Nakamura et al. 2000). There are several findings to suggest that mitochondrial function and energy production may be significantly decreased following METH treatment (Chan et al. 1994; Li and Dryhurst 1997; Berman and Hastings 1999). Additionally, coadministration of METH with mitochondrial inhibitors exacerbates toxicity in mice and rats (Albers and Sonsalla 1995; Bowyer et al. 1996; Burrows et al. 2000b). In a recent study, Yamamoto and colleagues reported a rapid but transient decrease in cytochrome oxidase staining in the striatum of METH treated rats (Burrows et al. 2000a), which supports related studies demonstrating decreases in striatal ATP in METH-treated mice (Chan et al. 1994). These studies are consistent with a METH-induced impairment of mitochondrial function. In agreement with these findings, our laboratory has recently observed that methamphetamine is equipotent as 1-methyl-4-phenylpyridinium ion (MPP+) in inhibiting rotenone sensitive NADH oxidase and NADH-ubiquinone reductase activities in submitochondrial particles isolated from pig heart (unpublished data), although its relevance to effects on intact mitochondria remain unclear. Nonetheless, it is possible that the delayed increase in oxidative stress seen in the hippocampus and striatum in our study may reflect direct METH-induced mitochondrial dysfunction. Future studies will need to be performed to obtain a more thorough analysis of the time-course formation of oxidative markers following METH administration as well as further clarifying any inhibitory effect of METH on mitochondrial function and the electron transport chain.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors would like to acknowledge statistical expertise from Ron Cody with SAS software. This study was supported by the Department of Veterans Affairs Career Development Award and the Presidential Early Career Award for Scientists and Engineers (MRG), NIH grants DA 06236 and AG 08479 (PKS), and National Institute of Health grant MH 12390 (LYM).


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • Acikgoz O., Gonenc S., Kayatekin B. M., Uysal N., Pekcetin C., Semin I., Gure A. (1998) Methamphetamine causes lipid peroxidation and an increase in superoxide dismutase activity in the rat striatum. Brain Res. 813, 200202.
  • Albers D. S. & Sonsalla P. K. (1995) Methamphetamine-induced hyperthermia and dopaminergic neurotoxicity in mice: pharmacological profile of protective and nonprotective agents. J. Pharmacol. Exp. Ther. 275, 11041114.
  • Berman S. B. & Hastings T. G. (1999) Dopamine oxidation alters mitochondrial respiration and induces permeability transition in brain mitochondria: implications for Parkinson's disease. J. Neurochem. 73, 11271137.
  • Bowyer J. F., Clausing P., Schmued L., Davies D. L., Binienda Z., Newport G. D., Scallet A. C., Slikker W. Jr (1996) Parenterally administered 3-nitropropionic acid and amphetamine can combine to produce damage to terminals and cell bodies in the striatum. Brain Res. 712, 221229.
  • Burrows K. B., Gudelsky G., Yamamoto B. K. (2000a) Rapid and transient inhibition of mitochondrial function following methamphetamine or 3,4-methylenedioxymethamphetamine administration. Eur J. Pharmacol. 398, 1118.
  • Burrows K. B., Nixdorf W. L., Yamamoto B. K. (2000b) Central administration of methamphetamine synergizes with metabolic inhibition to deplete striatal monoamines. J. Pharmacol. Exp. Ther. 292, 853860.
  • Cadet J. L. & Brannock C. (1998) Free radicals and the pathobiology of brain dopamine systems. Neurochem. Int. 32, 117131.
  • Cadet J. L., Sheng P., Ali S., Rothman R., Carlson E., Epstein C. (1994) Attenuation of methamphetamine-induced neurotoxicity in copper/zinc superoxide dismutase transgenic mice. J. Neurochem. 62, 380383.
  • Cappon G. D., Broening H. W., Pu C., Morford L., Vorhees C. V. (1996) alpha-Phenyl-N-tert-butyl nitrone attenuates methamphetamine-induced depletion of striatal dopamine without altering hyperthermia. Synapse 24, 173181.
  • Chan P., Di Monte D. A., Luo J. J., DeLanney L. E., Irwin I., Langston J. W. (1994) Rapid ATP loss caused by methamphetamine in the mouse striatum: relationship between energy impairment and dopaminergic neurotoxicity. J. Neurochem. 62, 24842487.
  • De Vito M. J. & Wagner G. C. (1989) Methamphetamine-induced neuronal damage: a possible role for free radicals. Neuropharmacology 28, 11451150.
  • Deng X., Ladenheim B., Tsao L. I., Cadet J. L. (1999) Null mutation of c-fos causes exacerbation of methamphetamine-induced neurotoxicity. J. Neurosci. 19, 1010710115.
  • Eisch A. J. & Marshall J. F. (1998) Methamphetamine neurotoxicity: dissociation of striatal dopamine terminal damage from parietal cortical cell body injury. Synapse 30, 433445.
  • Esterbauer H., Schaur R. J., Zollner H. (1991) Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med. 11, 81128.
  • Fleckenstein A. E., Wilkins D. G., Gibb J. W., Hanson G. R. (1997) Interaction between hyperthermia and oxygen radical formation in the 5-hydroxytryptaminergic response to a single methamphetamine administration. J. Pharmacol. Exp. Ther. 283, 281285.
  • Giovanni A., Liang L. P., Hastings T. G., Zigmond M. J. (1995) Estimating hydroxyl radical content in rat brain using systemic and intraventricular salicylate: impact of methamphetamine. J. Neurochem. 64, 18191825.
  • Gluck M. R., Jayatilleke E., Shaw S., Rowan A. J., Haroutunian V. (2000) CNS oxidative stress associated with the kainic acid rodent model of experimental epilepsy. Epilepsy Res. 39, 6371.
  • Graham D. G., Tiffany S. M., Bell W. R. J., Gutknecht W. F. (1978) Autoxidation versus covalent binding of quinones as the mechanism of toxicity of dopamine, 6-hydroxydopamine, and related compounds toward C1300 neuroblastoma cells in vitro. Mol. Pharmacol. 14, 644653.
  • Harold C., Wallace T., Friedman R., Gudelsky G., Yamamoto B. (2000) Methamphetamine selectively alters brain glutathione. Eur. J. Pharmacol. 400, 99102.
  • Hirata H., Ladenheim B., Carlson E., Epstein C., Cadet J. L. (1996) Autoradiographic evidence for methamphetamine-induced striatal dopaminergic loss in mouse brain: attenuation in CuZn-superoxide dismutase transgenic mice. Brain Res. 714, 95103.
  • Hirata H., Asanuma M., Cadet J. L. (1998) Melatonin attenuates methamphetamine-induced toxic effects on dopamine and serotonin terminals in mouse brain. Synapse 30, 150155.
  • Hogan K. A., Staal R. G. W., Sonsalla P. K. (2000) Analysis of VMAT2 binding after methamphetamine or MPTP treatment: Disparity between homogenates and vesicle preparations. J. Neurochem. 74, 22172220.
  • Imam S. Z., Crow J. P., Newport G. D., Islam F., Slikker W. J., Ali S. F. (1999) Methamphetamine generates peroxynitrite and produces dopaminergic neurotoxicity in mice: protective effects of peroxynitrite decomposition catalyst. Brain Res. 837, 1521.
  • Imam S. Z., Newport G. D., Itzhak Y., Cadet J. L., Islam F., Slikker W. J., Ali S. F. (2001) Peroxynitrite plays a role in methamphetamine-induced dopaminergic neurotoxicity: evidence from mice lacking neuronal nitric oxide synthase gene or overexpressing copper-zinc superoxide dismutase. J. Neurochem. 76, 745749.
  • Kita T., Takahashi M., Kubo K., Wagner G. C., Nakashima T. (1999) Hydroxyl radical formation following methamphetamine administration to rats. Pharmacol. Toxicol. 85, 133137.
  • Kita T., Shimada K., Mastunari Y., Wagner G. C., Kubo K., Nakashima T. (2000) Methamphetamine-induced striatal dopamine neurotoxicity and cyclooxygenase-2 protein expression in BALB/c mice. Neuropharmacology 39, 399406.
  • Kuhn D. M. & Geddes T. J. (2000) Molecular footprints of neurotoxic amphetamine action. Ann. NY Acad. Sci. 914, 92103.
  • LaVoie M. J. & Hastings T. G. (1999) Dopamine quinone formation and protein modification associated with the striatal neurotoxicity of methamphetamine: evidence against a role for extracellular dopamine. J. Neurosci. 19, 14841491.
  • Levine R. L., Garland D., Oliver C. N., Amici A., Climent I., Lenz A. G., Ahn B. W., Shaltiel S., Stadtman E. R. (1990) Determination of carbonyl content in oxidatively modified proteins. Meth. Enzymol. 186, 464478.
  • Li H. & Dryhurst G. (1997) Irreversible inhibition of mitochondrial complex I by 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1): a putative nigral endotoxin of relevance to Parkinson's disease. J. Neurochem. 69, 15301541.
  • Lotharius J. & O'Malley K. L. (2000) The parkinsonism-inducing drug 1-methyl-4-phenylpyridinium triggers intracellular dopamine oxidation. A novel mechanism of toxicity. J. Biol. Chem. 275, 3858138588.
  • Lowry O. H., Rosenbrough N. J., Farr A. L., Randall R. J. (1951) Protein measurement with folin reagent. J. Biol. Chem. 193, 265272.
  • McCann U. D., Wong D. F., Yokoi F., Villemagne V., Dannals R. F., Ricaurte G. A. (1998) Reduced striatal dopamine transporter density in abstinent methamphetamine and methcathinone users: evidence from positron emission tomography studies with [11C]WIN-35,428. J. Neurosci. 18, 84178422.
  • Nakamura K., Bindokas V. P., Marks J. D., Wright D. A., Frim D. M., Miller R. J., Kang U. J. (2000) The selective toxicity of 1-methyl-4-phenylpyridinium to dopaminergic neurons: the role of mitochondrial complex I and reactive oxygen species revisited. Mol. Pharmacol. 58, 271278.
  • O'Callaghan J. P. & Miller D. B. (1994) Neurotoxicity profiles of substituted amphetamines in the C57BL/6J mouse. J. Pharmacol. Exp. Ther. 270, 741751.
  • Ohmori T., Koyama T., Muraki A., Yamashita I. (1993) Competitive and noncompetitive N-methyl-d-aspartate antagonists protect dopaminergic and serotonergic neurotoxicity produced by methamphetamine in various brain regions. J. Neural Transmission – General Section 92, 97106.
  • Rice-Evans C. A., Diplock A. T., Symons M. C. R. (1991) Mechanisms of Radical Production. In: Laboratory Techniques in Biochemistry and Molecular Biology: Techniques in Free Radical Research (Burdon, R. H. and Van Knippenberg, P. H., eds), pp. 1950. Elsevier, New York.
  • Schmued L. C. & Bowyer J. F. (1997) Methamphetamine exposure can produce neuronal degeneration in mouse hippocampal remnants. Brain Res. 759, 135140.
  • Seiden L. S., Sabol K. E., Ricaurte G. A. (1993) Amphetamine: effects on catecholamine systems and behavior. Annu. Rev. Pharmacol. Toxicol. 33, 639677.
  • Sonsalla P. K., Nicklas W. J., Heikkila R. E. (1989) Role for excitatory amino acids in methamphetamine-induced nigrostriatal dopaminergic toxicity. Science 243, 398400.
  • Sonsalla P. K., Riordan D. E., Heikkila R. E. (1991) Competitive and noncompetitive antagonists at N-methyl-d-aspartate receptors protect against methamphetamine-induced dopaminergic damage in mice. J. Pharmacol. Exp. Ther. 256, 506512.
  • Sonsalla P. K., Jochnowitz N. D., Zeevalk G. D., Oostveen J. A., Hall E. D. (1996) Treatment of mice with methamphetamine produces cell loss in the substantia nigra. Brain Res. 738, 172175.
  • Stumm G., Schlegel J., Schafer T., Wurz C., Mennel H. D., Krieg J. C., Vedder H. (1999) Amphetamines induce apoptosis and regulation of bcl-x splice variants in neocortical neurons. FASEB J. 13, 10651072.
  • Sulzer D., Chen T. K., Lau Y. Y., Kristensen H., Rayport S., Ewing A. (1995) Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport. J. Neurosci. 15, 41024108.
  • Volkow N. D., Chang L., Wang G. J., Fowler J. S., Leonido-Yee M., Franceschi D., Sedler M. J., Gatley S. J., Hitzemann R., Ding Y. S., Logan J., Wong C., Miller E. N. (2001) Association of dopamine transporter reduction with psychomotor impairment in methamphetamine abusers. Am. J. Psychiatry 158, 377382.
  • Wan F. J., Lin H. C., Huang K. L., Tseng C. J., Wong C. S. (2000) Systemic administration of d-amphetamine induces long-lasting oxidative stress in the rat striatum. Life Sci. 66, L205L212.
  • Wilson J. M., Kalasinsky K. S., Levey A. I., Bergeron C., Reiber G., Anthony R. M., Schmunk G. A., Shannak K., Haycock J. W., Kish S. J. (1996) Striatal dopamine nerve terminal markers in human, chronic methamphetamine users. Nat. Med. 2, 699703.
  • Wrona M. Z. & Dryhurst G. (1998) Oxidation of serotonin by superoxide radical: implications to neurodegenerative brain disorders. Chem. Res. Toxicol. 11, 639650.
  • Yamamoto B. K. & Zhu W. (1998) The effects of methamphetamine on the production of free radicals and oxidative stress. J. Pharmacol. Exp. Ther. 287, 107114.