Histopathological and electrophysiological indices of rotenone-evoked dopaminergic toxicity: Neuroprotective effects of acetyl-L-carnitine.

Exposure to the natural pesticide, rotenone, a potent mitochondrial toxin, leads to degeneration in striatal nerve terminals and nigral neurons. Rotenone-induced behavioral, neurochemical and neuropathological changes in rats mimic those observed in Parkinson's disease (PD). Here, protective effects of acetyl-L-carnitine (ALC) in the brain dopaminergic toxicity after a prolonged exposure to rotenone were evaluated using electrophysiological and immunolabeling methods. Adult, male Sprague-Dawley rats were injected i.p. with rotenone alone (1 mg/kg) or rotenone with ALC (either 10 or 100 mg/kg; ALC10 or ALC100, respectively) once daily on days 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26, 29, 31, 33 and 37. Control rats received either 100mg/kg ALC or vehicle (30% Solutol HS 15 in 0.9% saline) injections. Animals were weighed on injection days and monitored daily. Motor nerve conduction velocity (MCV) was assessed within two days after treatment using compound muscle action potentials (CMAP) detected from the tail muscle through surface receiver electrodes installed around the distal part of the tail. Rats were perfused immediately after testing with 4% paraformaldehyde and immunohistochemical analysis of dopamine transporter (DAT), tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), and microglial CD11b marker was performed in the caudate-putamen (CPu) and the substantia nigra pars compacta (SNc) in order to estimate dopaminergic neuronal and transporter damage. Additionally, effects of ALC on preventing microglial or astrocytic hypertrophy were also evaluated. In rats exposed to rotenone and rotenone/ACL10, a significant increases in both proximal (S1) and distal (S2) motor latency and a decrease in MCV were detected in tail nerves (p<0.05). The conduction parameters in rats co-treated with rotenone/ACL100 were not different from control. It was found that 100 mg/kg ALC prevented loss of TH and a decline of DAT level in the midbrain and also prevented the activation of both microglia and astroglia after rotenone treatment. Data indicate neuroprotective effects of ALC in rotenone-evoked dopaminergic neurotoxicity.

Propentophylline increases striatal dopamine release but dampens methamphetamine-induced dopamine dynamics: A microdialysis study.

While there are currently no medications approved for methamphetamine (METH) addiction, it has been shown that propentofylline (PPF), an atypical methylxanthine, can suppress the rewarding effects of methamphetamine (METH) in mice. This experiment studied the interactions of PPF with METH in striatal dopaminergic transmission. Herein, the impact of PPF (10-40mM, intrastriatally perfused (80min) on the effect of METH (5mg/kg, i.p.) on striatal dopamine (DA) release was evaluated using brain microdialysis in Sprague-Dawley adult rats. METH was injected at the 60min time point of the 80min PPF perfusion. The extracellular levels of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were determined using high performance liquid chromatography with electrochemical detection (HPLC-ED). PPF induced a concentration-dependent increase in DA release beginning 30min after the onset of PPF perfusion. DA peak levels evoked by 40mM PPF were similar to those induced by 5mg/kg METH i.p. Only the highest concentration of PPF decreased the METH-induced DA peak (circa 70%). The significant decreases in extracellular levels of DOPAC and HVA evoked by METH were partially blocked by 10 and 20mM PPF. Although 40mM of PPF also partially blocked the METH-induced DOPAC decrease, it completely blocked HVA depletion after a transient increase in HVA levels in METH-treated rats. Data indicates for the first time that while PPF increases presynaptic striatal DA dynamics it attenuates METH-induced striatal DA release and metabolism.

Acetyl-L-Carnitine Modulates TP53 and IL10 Gene Expression Induced by 3-NPA Evoked Toxicity in PC12 Cells.

The neurotoxicity induced by the mitochondrial inhibitor 3-nitropropionic acid (3-NPA) is associated with a decrease of ATP synthesis and an increase of free radical production which can lead to apoptosis or necrosis. We have used the PC12, neuron-like rat pheochromocytoma cell line, to study further the mechanism of 3-NPA-evoked neurotoxicity and the effects of acetyl-L-carnitine (ALC) which has neuroprotective actions against various types of mitochondrial inhibitors.Cultured PC 12 cells were exposed to a low dose of 3-NPA 50 (microM) in the presence or absence of 5 mM ALC. The dose of 3-NPA was sub toxic and no changes in pro-apoptotic Bax or anti-apoptotic Bcl-2 gene expression were observed. We followed specific genetic markers to look for changes evoked by 3-NPA toxicity and also changes associated with neuroprotection exerted by the ALC treatment, using RT-PCR arrays (delta-delta method). 3-NPA exposure evoked a decrease in expression of the Tp53 gene. This down regulation was prevented by pretreatment of the cells with ALC. The Tp53 gene responds to cellular stresses and the effects seen here are possibly associated with the 3-NPA evoked changes in mitochondrial metabolism. Other genes associated with stress and apoptosis, Parp-1, Bcl-2, and Bax were not affected by 3-NPA or ALC. The decrease of inflammatory response Il-10 gene expression due to 3-NPA was further lowered by presence of ALC. Other inflammation related genes, Il1rn, Nr3c1 and Cxcr4 were not affected. Interestingly, the glutamate transporter slc17a7, carnitine-acylcarnitine translocase Slc25a20 and heat shock proteins genes, Hsp27, Hmox1 (Hsp32, HO1) as well as Hspa 1a (Hsp 70) increased only when both ALC and small dose of 3-NPA were present. The alterations in gene expression detected in this study suggest role of several intracellular pathways in the neurotoxicity of 3-NPA and the neuroprotection against 3-NPA-induced neurotoxicity by ALC.

Analysis of electrical brain waves in neurotoxicology: γ-hydroxybutyrate.

Advances in computer technology have allowed quantification of the electroencephalogram (EEG) and expansion of quantitative EEG (qEEG) analysis in neurophysiology, as well as clinical neurology, with great success. Among the variety of techniques in this field, frequency (spectral) analysis using Fast Fourier Transforms (FFT) provides a sensitive tool for time-course studies of different compounds acting on particular neurotransmitter systems. Studies presented here include Electrocorticogram (ECoG) analysis following exposure to a glutamic acid analogue - domoic acid (DOM), psychoactive indole alkaloid - ibogaine, as well as cocaine and gamma-hydroxybutyrate (GHB). The ECoG was recorded in conscious rats via a tether and swivel system. The EEG signal frequency analysis revealed an association between slow-wave EEG activity delta and theta and the type of behavioral seizures following DOM administration. Analyses of power spectra obtained in rats exposed to cocaine alone or after pretreatment with ibogaine indicated the contribution of the serotonergic system in ibogaine mediated response to cocaine (increased power in alpha(1) band). Ibogaine also lowered the threshold for cocaine-induced electrographic seizures (increased power in the low-frequency bands, delta and theta). Daily intraperitoneal administration of cocaine for two weeks was associated with a reduction in slow-wave ECoG activity 24 hrs following the last injection when compared with controls. Similar decreased cortical activity in low-frequency bands observed in chronic cocaine users has been associated with reduced metabolic activity in the frontal cortex. The FFT analyses of power spectra relative to baseline indicated a significant energy increase over all except beta(2) frequency bands following exposure to 400 and 800 mg/kg GHB. The EEG alterations detected in rats following exposure to GHB resemble absence seizures observed in human petit mal epilepsy. Spectral analysis of the EEG signals combined with behavioral observations may prove to be a useful approach in studying chronic exposure to drugs of abuse and treatment of drug dependence.

Striatal dopamine in the response of brain and liver unsaturated and saturated free fatty acids following administration of C75 in CD-1 mice.

In order to clarify the mechanism of action of cerulenin analog, C75, known to suppress feeding behavior, food intake was measured in adult CD-1 male mice n=5 per group, treated i.p. with 10 and 20mg/kg of C75. Animals in both treatment groups had significantly lower 24h food consumption rate relative to the control group injected with vehicle. Striatal monoamine neurotransmitters and striatal as well as liver long chain free fatty acids concentrations were subsequently evaluated in another group treated i.p. with 20mg/kg C75. Acute exposure to C75 at 20mg/kg led to approximately 50% increase in the striatal dopamine levels and a decrease in dopamine turnover for up to 24h following the injection. The concentration of serotonin remained unchanged. Concentration of saturated fatty acids in the liver and striatum did not change, while striatal unsaturated myristoleic acid (cis-9-tetradecenoic acid) levels were significantly higher as early as 2h post-injection and remained elevated at 24h post-injection. These preliminary data suggest a central regulatory role of unsaturated fatty acids under dopaminergic control in the C75-induced anorexia. Pharmacological alterations in fatty acid metabolism may prove beneficial in the treatment of obesity.

L-carnitine protects neurons from 1-methyl-4-phenylpyridinium-induced neuronal apoptosis in rat forebrain culture.

1-Methyl-4-phenylpyridinium ion (MPP+), an inhibitor of mitochondrial complex I, has been widely used as a neurotoxin because it elicits a severe Parkinson's disease-like syndrome with an elevation of intracellular reactive oxygen species (ROS) and apoptosis. L-carnitine plays an integral role in attenuating the brain injury associated with mitochondrial neurodegenerative disorders. The present study investigates the effects of L-carnitine against the toxicity of MPP+ in rat forebrain primary cultures. Cells in culture were treated for 24 h with 100, 250, 500 and 1000 microM MPP+ alone or co-incubated with L-carnitine. MPP+ produced a dose-related increase in DNA fragmentation as measured by cell death ELISA (enzyme-linked immunosorbent assay), an increase in the number of TUNEL (terminal dUTP nick-end labeling)-positive cells and a reduction in the mitochondrial metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). No significant effect was observed with the release of lactate dehydrogenase (LDH), indicating that cell death presumably occurred via apoptotic mechanisms. Co-incubation of MPP+ with L-carnitine significantly reduced MPP+-induced apoptosis. Western blot analyses showed that neurotoxic concentrations of MPP+ decreased the ratio of BCL-X(L) to Bax and decreased the protein levels of polysialic acid neural cell adhesion molecules (PSA-NCAM), a neuron specific marker. L-carnitine blocked these effects of MPP+ suggesting its potential therapeutic utility in degenerative disorders such as Parkinson's disease, Alzheimer's disease, ornithine transcarbamylase deficiency and other mitochondrial diseases.

Neuroprotective role of L-carnitine in the 3-nitropropionic acid induced neurotoxicity.

L-carnitine (LC) plays an important regulatory role in the mitochondrial transport of long-chain free fatty acids (FFA). 3-Nitropropionic acid (3-NPA) is known to induce cellular energy deficit and oxidative stress related neurotoxicity via an irreversible inhibition of the mitochondrial enzyme succinate dehydrogenase (SDH). Protective effects of L-carnitine on the neurotoxicity induced by 3-NPA have been shown in vitro. Here, the activities of SDH as well as the activity of the antioxidant enzymes, catalase (CAT), and superoxide dismutase (SOD) were measured in order to evaluate the protective action of LC against 3-NPA-induced neurotoxicity. Male, CD Sprague-Dawley rats, 3-month old, were injected with either 50 or 100 mg/kg of LC, i.p., 30-60 min prior to 3-NPA (30 mg/kg, s.c.) or with 3-NPA alone. Enzyme activities were assayed in caudate nucleus (CN), frontal cortex (FC), and hippocampus (HIP) post sacrifice. Increased activities of CAT and SOD were observed after treatment with 3-NPA alone. Pretreatment with low or high doses of LC was associated with attenuation of these increases equivalent to, or below, the control levels. In rats treated with 3-NPA alone, SDH activity was inhibited by 62% (CN), 50% (FC), and 65% (HIP) of controls. Pretreatment with LC prior to 3-NPA attenuated decreases of SDH activity in a dose-dependent manner. However, compared with control, the activity of SDH remained significantly lower in brain regions of treated rats despite the attenuation of inhibition by LC pretreatment (P<0.05). These data suggest protective effect of LC against 3-NPA-induced oxidative stress. It appears that the protective effect of LC against 3-NPA-induced oxidative stress is not mediated by the direct action of LC preventing the SDH inhibition but rather is achieved due to the actions of LC downstream of the SDH inhibition.

Effect of L-carnitine pretreatment on 3-nitropropionic acid-induced inhibition of rat brain succinate dehydrogenase activity.

L-Carnitine (LC) plays an important regulatory role in the mitochondrial transport of long chain free fatty acids (FFA). 3-Nitropropionic acid (3-NPA) is known to induce cellular energy deficit and oxidative stress-related neurotoxicity via an irreversible inhibition of mitochondrial succinate dehydrogenase (SDH). In the present study, activity of SDH was measured in order to evaluate neuroprotective effects of LC against the 3-NPA-induced neurotoxicity. Male, CD Sprague-Dawley rats, three months old, were injected with either 50 or 100 mg/kg of LC, i.p., 30 min prior to 3-NPA (30 mg/kg, s.c.) or with 3-NPA alone. The activity of brain SDH was quantified spectrophotometrically in caudate nucleus (CN), frontal cortex (FC), and hippocampus (HIP) 60 min after the 3-NPA injection. The SDH activity in the animals treated with 3-NPA alone was 38% (CN), 50% (FC), and 36% (HIP) that of saline controls. Pretreatment with LC prior to 3-NPA injection attenuated decreases of SDH activity by approximately 15 and 29% (LC low and high dose, respectively). Despite the attenuation of SDH inhibition, the activity of SDH in these regions remained significantly lower in treated than in control rats (p < 0.05). It appears that the protective effect of LC against 3-NPA-induced oxidative stress cannot be explained by the direct action of LC to interfere with the SDH inhibition but are rather achieved by LC actions downstream of the SDH inhibition.

Biomarkers of 3-nitropropionic acid (3-NPA)-induced mitochondrial dysfunction as indicators of neuroprotection.

In humans or animals, symptoms of mitochondrial energy dysfunction may be produced by mutations or inborn errors of the necessary enzymes, as well as by enzyme inhibitors or uncouplers of the oxidative phosphorylation process. 3-Nitropropionic acid (3-NPA) is a toxin that is sometimes produced on moldy crops (sugarcane, peanuts, etc.) in amounts sufficient to cause severe neuromuscular disorders when consumed by humans. In vitro, 3-NPA irreversibly inactivates SDH, a Complex II respiratory enzyme important for mitochondrial energy production. We have been studying biomarkers of 3-NPA exposure in the expectation that such markers may be useful in the screening process to identify neuroprotective agents against neurotoxicity produced by mitochondrial energy dysfunction. Animals were sacrificed at various times after 3-NPA exposure for histochemical visualization of SDH activity and measurement of immediate postmortem rectal temperature. 3-NPA-treated rats experienced progressive hypothermia that reached a loss of 3 degrees C or more in core body temperature by three hours after dosing. The optical density of the SDH stain in brain was reduced, following a similar time course, most prominently in the cerebellum and least sharply in the thalamus. Some rats were given injections of L-carnitine (an enhancer of fatty acid transport) either alone, or as a pretreatment prior to a dose of 3-NPA. Although L-carnitine deficiency by itself can produce mitochondrial dysfunction, pretreatment with L-carnitine was of limited efficacy at overcoming the effects of 3-NPA on either body temperature or quantitative SDH histochemistry. Body temperature and SDH histochemistry may be useful biomarkers for evaluating the efficacy of neuroprotective agents against lower doses of 3-NPA, against other pharmacological models of mitochondrial dysfunction, or even against genetic mitochondrial diseases.

Transplacental pharmacokinetics and fetal distribution of 2', 3'-didehydro-3'-deoxythymidine (d4T) and its metabolites in late-term rhesus macaques.

BACKGROUND: The overall goal of human immunodeficiency virus (HIV) therapy during pregnancy is to maintain maternal health and reduce the probability of vertical transmission during gestation and delivery, while keeping toxicity risks low. Azidothymidine (AZT) is currently recommended for pregnant women infected with HIV; however, many pregnant women are unable to tolerate AZT because of toxicity. In the present study, the placental transfer and fetal accumulation of the anti-HIV compound 2',3'-didehydro-3'-deoxythymidine (d4T) and its active (triphosphorylated) and inactive (thymine and beta-aminoisobutyric acid) metabolites were examined at steady state in late-term rhesus macaques. METHODS: On the day of the hysterotomy, the mother was administered an intravenous loading dose of d4T, followed by a 3-hr steady-state intravenous infusion that also included [(3)H]d4T as a tracer. After 3 hr of infusion, the fetus was delivered by cesarean section under halothane/N(2)O anesthesia. Plasma, amniotic fluid, and tissues were analyzed for d4T and its inactive metabolites by HPLC; tissue samples were analyzed for d4T and active (phosphorylated) metabolites by strong anion-exchange HPLC. RESULTS: Maternal steady-state plasma concentrations of d4T were 1-2 microg/ml, with a fetal-to-maternal plasma ratio of 0.85 +/- 0.09. The fetal tissue distribution of radioactivity was highest in the kidney and lowest in the brain. D4T, thymine, and beta-aminoisobutyric acid were detected in all fetal tissues examined. CONCLUSIONS: Our data indicate that d4T readily crosses the placenta and is present in the fetus as parent compound or its inactive metabolites after maternal infusion. Although fetal plasma concentrations of d4T were similar to clinical d4T concentrations, no phosphorylated metabolites were detected. Teratology 62:93-99, 2000. Published 2000 Wiley-Liss, Inc.

Transplacental pharmacokinetics and fetal distribution of azidothymidine, its glucuronide, and phosphorylated metabolites in late-term rhesus macaques after maternal infusion.

3'-Azido-3'-deoxythymidine (AZT) is currently prescribed to pregnant women infected with human immunodeficiency virus to reduce the risk of vertical transmission of the virus to the fetus. Consequently, more information is needed concerning the placental transfer and tissue distribution of AZT and its metabolites. In the present study, the placental transfer and fetal accumulation of AZT, its glucuronide metabolite [3'-azido-3'-deoxythymidine-beta-D-glucuronide (AZTG)], and phosphorylated metabolites were examined at steady-state in near-term rhesus macaques. One to 2 weeks before a chronic infusion, an intravenous bolus of 8 mg/kg AZT was administered to pregnant animals to determine the dose of AZT needed to reach steady-state plasma concentrations. On the day of hysterotomy, the mother was administered an intravenous loading dose of AZT, followed by a 3-hr steady-state intravenous infusion that also included a trace of [3H]AZT. After 3 hr of infusion, the mother was anesthetized, and the fetus was delivered. Plasma and amniotic fluid were analyzed for AZT and AZTG by HPLC, and tissue samples were analyzed for AZT, AZTG, and phosphorylated metabolites by strong anion exchange HPLC. Maternal steady-state plasma concentrations were 1.3-2.2 micrograms/ml for AZT and 2.3-8.0 micrograms/ml for AZTG. Fetal AZT and AZTG plasma concentrations were both lower (0.98-2.3 micrograms/ml and 1.3-5.4 micrograms/ml, respectively) than maternal concentrations, with fetal-to-maternal plasma ratios of 0.63-1.0 for AZT. Fetal tissue distribution of tritium was highest in the kidney and lowest in the brain. Although the active triphosphorylated metabolite was not detected in the fetus, the AZT-monophosphate was detected in almost all fetal tissues examined. Our data indicate that AZT is rapidly converted to the glucuronide and monophosphate metabolites in the fetus after maternal infusion.