William Slikker

Methamphetamine-Induced Dopaminergic Neurotoxicity: Role of Peroxynitrite and Neuroprotective Role of Antioxidants and Peroxynitrite Decomposition Catalysts

Abstract: Oxidative stress, reactive oxygen (ROS), and nitrogen (RNS) species have been known to be involved in a multitude of neurodegenerative disorders such as Parkinsons disease (PD), Alzheimers disease (AD), and amyotrophic lateral sclerosis (ALS). Both ROS and RNS have very short half‐lives, thereby making their identification very difficult as a specific cause of neurodegeneration. Recently, we have developed a high performance liquid chromatography/electrochemical detection (HPLC/EC) method to identify 3‐nitrotyrosine (3‐NT), an in vitro and in vivo biomarker of peroxynitrite production, in cell cultures and brain to evaluate if an agent‐driven neurotoxicity is produced by the generation of peroxynitrite. We show that a single or multiple injections of methamphetamine (METH) produced a significant increase in the formation of 3‐NT in the striatum. This formation of 3‐NT correlated with the striatal dopamine depletion caused by METH administration. We also show that PC12 cells treated with METH has significantly increased formation of 3‐NT and dopamine depletion. Furthermore, we report that pretreatment with antioxidants such as selenium and melatonin can completely protect against the formation of 3‐NT and depletion of striatal dopamine. We also report that pretreatment with peroxynitrite decomposition catalysts such as 5, 10,15,20‐tetrakis(N‐methyl‐4′‐pyridyl)porphyrinato iron III (FeTMPyP) and 5, 10, 15, 20‐tetrakis (2,4,6‐trimethyl‐3,5‐sulfonatophenyl) porphinato iron III (FETPPS) significantly protect against METH‐induced 3‐NT formation and striatal dopamine depletion. We used two different approaches, pharmacological manipulation and transgenic animal models, in order to further investigate the role of peroxynitrite. We show that a selective neuronal nitric oxide synthase (nNOS) inhibitor, 7‐nitroindazole (7‐NI), significantly protect against the formation of 3‐NT as well as striatal dopamine depletion. Similar results were observed with nNOS knockout and copper zinc superoxide dismutase (CuZnSOD)‐overexpressed transgenic mice models. Finally, using the protein data bank crystal structure of tyrosine hydroxylase, we postulate the possible nitration of specific tyrosine moiety in the enzyme that can be responsible for dopaminergic neurotoxicity. Together, these data clearly support the hypothesis that the reactive nitrogen species, peroxynitrite, plays a major role in METH‐induced dopaminergic neurotoxicity and that selective antioxidants and peroxynitrite decomposition catalysts can protect against METH‐induced neurotoxicity. These antioxidants and decomposition catalysts may have therapeutic potential in the treatment of psychostimulant addictions.

Prevention of Dopaminergic Neurotoxicity by Targeting Nitric Oxide and Peroxynitrite: Implications for the Prevention of Methamphetamine-induced Neurotoxic Damage

Methamphetamine (METH) is a neurotoxic psychostimulant that produces catecholaminergic brain damage by producing oxidative stress and free radical generation. The role of oxygen and nitrogen radicals is well documented as a cause of METH‐induced neurotoxic damage. In this study, we have obtained evidence that METH‐induced neurotoxicity is the resultant of interaction between oxygen and nitrogen radicals, and it is mediated by the production of peroxynitrite. We have also assessed the effects of inhibitors of neuronal nitric oxide synthase (nNOS) as well as scavenger of nitric oxide and a peroxynitrite decomposition catalyst. Significant protective effects were observed with the inhibitor of nNOS, 7‐nitroindazole (7‐NI), as well as by the selective peroxynitrite scavenger or decomposition catalyst, 5,10,15,20‐tetrakis(2,4,6‐trimethyl‐3,5‐sulfonatophenyl)porphyrinato iron III (FeTPPS). However, the use of a nitric oxide scavenger, 2‐phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (c‐PTIO), did not provide any significant protection against METH‐induced hyperthermia or peroxynitrite generation and the resulting dopaminergic neurotoxicity. In particular, treatment with FeTPPS completely prevented METH‐induced hyperthermia, peroxynitrite production, and METH‐induced dopaminergic depletion. Together, these data demonstrate that METH‐induced dopaminergic neurotoxicity is mediated by the generation of peroxynitrite, which can be selectively protected by nNOS inhibitors or peroxynitrite scavenger or decomposition catalysts.

Acute effects of dexfenfluramine (d-FEN) and methylenedioxymethamphetamine (MDMA) before and after short-course, high-dose treatment.

ABSTRACT: The acute behavioral effects of methylenedioxymethamphetamine (MDMA) and dexfenfluramine (d‐FEN) were assessed in six rhesus monkeys using performance in the National Center for Toxicological Research (NCTR) Operant Test Battery (OTB); three additional animals served as controls for neurochemical endpoints. The OTB consists of five food‐reinforced tasks designed to model aspects of learning, short‐term memory and attention, time estimation, motivation, and color and position discrimination. Shortly after the acute effects of each drug were determined, three of the monkeys received a short‐course, high‐dose exposure (2×/day × 4 days, intramuscular (i.m.) injections) of MDMA (10 mg/kg), while three monkeys were exposed to an identical regimen of d‐FEN (5 mg/kg). Approximately one month later, the acute effects of each drug were again determined. In monkeys exposed to high‐dose d‐FEN, the sensitivities of the OTB tasks to acute disruption by either MDMA or d‐FEN were essentially unchanged. Conversely, monkeys treated with high‐dose MDMA were less sensitive to the acute behavioral effects of both drugs, although such an effect was seen more frequently for d‐FEN and was OTB task specific. Thus a residual behavioral tolerance to the acute behavioral effects of MDMA and d‐FEN was noted after high‐dose MDMA exposure, but not after high‐dose d‐FEN exposure. These findings are surprising, as similar neurochemical effects (i.e., significant decreases of ca. 50% in serotonin in frontal cortex and hippocampus) were observed in all monkeys approximately six months after short‐course, high‐dose MDMA or d‐FEN treatment.

Methamphetamine‐Induced Dopaminergic Neurotoxicity and Production of Peroxynitrite Are Potentiated in Nerve Growth Factor Differentiated Pheochromocytoma 12 Cells

Abstract: Methamphetamine (METH) is a widely abused psychomotor stimulant known to cause dopaminergic neurotoxicity in rodents, nonhuman primates, and humans. METH administration selectively damages the dopaminergic nerve terminals, which is hypothesized to be due to release of dopamine from synaptic vesicles within the terminals. This process is believed to be mediated by the production of free radicals. The current study evaluates METH‐induced dopaminergic toxicity in pheochromocytoma 12 (PC12) cells cultured in the presence or absence of nerve growth factor (NGF). Dopaminergic changes and the formation of 3‐nitrotyrosine (3‐NT), a marker for peroxynitrite production, were studied in PC12 cell cultures grown in the presence or absence of NGF after different doses of METH (100‐1,000 μM). METH exposure did not cause significant alterations in cell viability and did not produce significant dopaminergic changes or 3‐NT production in PC12 cells grown in NGF‐negative media after 24 hours. However, cell viability of PC12 cells grown in NGF‐positive media was decreased by 45%, and significant dose‐dependent dopaminergic alteration and 3‐NT production were observed 24 hours after exposure to METH. The current study supports the hypothesis that METH acts at the dopaminergic nerve terminals and produces dopaminergic damage by the production of free radical peroxynitrite.

Adaptation to Repeated Cocaine Administration in Rats

Abstract: Quantitative electroencephalogram (EEG) studies in cocaine‐dependent human patients show deficits in slow‐wave brain activity, reflected in diminished EEG power in the delta and theta frequency bands. In the present study, electrophysiological measures were monitored in 10 nonanesthetized, adult male Sprague‐Dawley rats via bipolar, epidural electrodes implanted over the somatosensory cortex. Control electrocorticograms (ECoG) were recorded twice within a two‐week interval to establish a baseline. Rats were subsequently injected daily with cocaine HCl at 15 mg/kg, i.p., for two weeks. The ECoG was recorded during a 1‐h session one day after the last injection. Total concentrations of dopamine (DA) and its metabolites were assayed in caudate nucleus (CN) and frontal cortex (FC) using HPLC/EC. Compared with controls, marked increases in DA concentrations were observed in both regions. The DA turnover decreased significantly. The power spectra, obtained by use of a fast Fourier transformation, revealed a significant decrease in slow‐wave delta frequency bands following repeated exposure to cocaine. These data are consistent with reported findings in humans that repeated exposures to cocaine result in a decrease in slow‐wave brain activity. Further studies are necessary to establish whether regional alterations in blood flow and metabolic activity may underlie such observations.

Alteration in Electroencephalogram and Monoamine Concentrations in Rat Brain following Ibogaine Treatment

ABSTRACT: Ibogaine (IBO) is a psychoactive indole alkaloid that has antiaddictive properties. However, treatment with IBO may lead to neurotoxicity, since IBO and its metabolites interact persistently with many neurotransmitter systems. Here, we recorded cortical electroencephalogram (EEG) signals from rats anesthetized with isoflurane. The heart rate (HR) was monitored via electrocardiogram (EKG) electrodes. After the baseline EEG was recorded, rats received one intraperitoneal (i.p.) dose of 50 mg/kg IBO. EEG signals were recorded for 2 hr. Rats were then sacrificed and brains dissected into frontal cortex (FC), caudate nucleus (CN), hippocampus (HIP), and brain stem (BS). The level of dopamine (DA), serotonin (5‐HT), and their metabolites were determined by high‐performance liquid chromatography with electrochemical detection (HPLC‐ECD). Compared with baseline, a decrease in HR immediately after IBO injection and a decrease in δ, θ, α, and β power spectra frequency bands (1–4, 4–8, 8–13, 13–32Hz) during the first 30 min after IBO administration was observed. EEG recovered within the next 15 min. In CN, the level of DA decreased and DA turnover rate increased significantly. The levels of 5‐HT increased in FC. The pattern of EKG and EEG response to IBO may be due to multiple receptor interactions of IBO.

Application of Electrophysiological Method to Study Interactions between Ibogaine and Cocaine

The psychoactive indole alkaloid, ibogaine (IBO), has been investigated for over a decade concerning its reported anti‐addictive properties for opioids as well as psychomotor stimulants. The mechanism for the anti‐addictive action of IBO is still unclear. IBO interactions with opioid, NMDA, nicotinic, adrenergic, and serotonergic receptor sites have been suggested. The involvement of the dopaminergic system in IBO action is well documented. Increased or decreased levels of dopamine (DA) in specific brain regions following IBO pretreatment have been seen concomitantly with increased or decreased motor activity after subsequent amphetamine or cocaine administration. In this report, in vivo electrophysiological measures were monitored in awake adult male rats in order to investigate alterations of the electrocorticogram (ECoG) resulting from interactions between IBO and cocaine (COC). Rats were implanted bilaterally with bipolar ECoG electrodes. They were either injected with saline, COC alone (20 mg/kg, i.p.) or IBO (50 mg/kg, i.p.) and COC 1 hr later. The concentrations of DA, 5‐HT, and their metabolites DOPAC, HVA, and 5‐HIAA were assessed in the caudate nucleus in separate groups of saline‐, COC‐, and IBO/COC‐treated rats. An alpha1 power increase was observed within 10 min after COC injection, which lasted for less than 20 min. A desynchronization over alpha2 and both beta power bands was observed throughout the recording. In IBO/COC‐treated rats, a significant increase in delta, theta, and alpha1 power occurred within 20 min after COC injection (p <0.05). This effect lasted for up to an hour. DA levels significantly increased after COC only and decreased after IBO administration. A further decrease in levels of DA was observed in IBO/COC‐treated rats. DA turnover increased significantly after IBO alone but was not observed after IBO/COC treatment. The alterations in ECoG and neurotransmitter levels suggest a decreased response to COC following IBO pretreatment.

Current and Future Approaches to Neurotoxicity Risk Assessmenta

The U.S. Food and Drug Administration (FDA) has the responsibility for the safety and efficacy of drugs, biologics and medical devices as well as the task of ensuring the safety of food and food-related chemicals, cosmetics, electronic products that emit radiation and veterinary drugs. Under the food, drug and cosmetics laws, the FDA is charged with the responsibility for products that account for 25 cents out of every dollar spent by American consumers. Although the FDA generally follows a rather structured review and approval process in making its regulatory decisions about these products, there are few formally written guidelines. The most detailed account of the Agency’s approach for assessing risk of adverse health effects is described in the FDA’s Redbook I. This publication released in 1982 described the nature and extent of toxicological testing that was considered necessary for the agency to assure the safety of foods and color additives intended for use in human foods. Recently, a proposed set of guidelines, referred to as Redbook 11, has been drafted and disseminated for public comment and revision. These proposed guidelines include specific attention to neurotoxicity and describe the nature and extent of information deemed necessary for the assessment of neurotoxic potential. The Redbook I1 guidelines, which suggest a strategy for obtaining this information as part of the safety evaluation process, will be discussed and compared to other approaches for neurotoxicity risk assessment published in the open literature.