Learning the hard way: the importance of accurate data.

AIM: The outcome of surgery for colorectal cancer in each unit in the UK is collated by the National Bowel Cancer Audit Project (NBOCAP). In 2008-2009 our unit had a raw 30-day postoperative mortality close to the national average, but when it was nationally adjusted it appeared to be an outlier. The purpose of this study was to identify reasons for this disparity. METHOD: All records were obtained for patients undergoing surgery for colorectal cancer over the 2 years. Data submitted to NBOCAP to determine adjusted rates were compared with actual data. RESULTS: There were major discordances between submitted and actual data for American Society of Anesthesiology grades and timing of surgery. This explained why the unit appeared to be an outlier. CONCLUSION: There is increasing emphasis on outcome of health service delivery, which has important implications. Submission of correct data is essential if objective comparison is to be made on which to base decisions on service delivery among units and within health regions.

Thinking outside the box: Aboriginal people's suggestions for conducting health studies with Aboriginal communities.

OBJECTIVES: Aboriginal people are under-represented in epidemiological research, largely due to past failures to engage and recruit Aboriginal communities, research fatigue and the use of culturally inappropriate methods. A qualitative study was undertaken in rural and urban Aboriginal communities in north-eastern and south-western Ontario to identify culturally congruent public health research methodologies. STUDY DESIGN: A qualitative participatory research study using focus group discussions. METHODS: This study employed a participatory research framework to elicit methodological suggestions for conducting public health research with Aboriginal communities during focus groups with healthcare providers from six diverse Aboriginal health organizations in Ontario, Canada. RESULTS: Continuing requests for participation in health research studies have led to community exhaustion. Discussions explored appropriate methods to obtain community approval and support for a study, the need for cultural sensitivity training for researchers, the value of conducting studies of interest and benefit to the community, advantages and disadvantages of qualitative and quantitative studies, the benefit of both Aboriginal and non-Aboriginal ethics reviews, the importance of safeguarding trusted information, types of incentives that may enhance study participation, suggestions to improve the collection of questionnaire information and biological specimens, how to resolve contentious issues and dissemination of study results. CONCLUSION: In order to successfully engage Aboriginal people in health studies, researchers need to build rapport with communities, have a community presence, be respectful and collaborative, utilize incentives, and employ flexible and adaptive methodologies of reasonable length. Oral interviews are preferred to self-completed information. The use of more mixed methods methodologies was suggested when quantitative data collection is necessary. Communities expect presentations about research findings.

Mitigation of patient harm from testing errors in family medicine offices: a report from the American Academy of Family Physicians National Research Network.

OBJECTIVES: Little research has focused on preventing harm from errors that occur in primary care. We studied mitigation of patient harm by analysing error reports from family physicians' offices. METHODS: The data for this analysis come from reports of testing process errors identified by family physicians and their office staff in eight practices in the American Academy of Family Physicians National Research Network. We determined how often reported error events were mitigated, described factors related to mitigation and assessed the effect of mitigation on the outcome of error events. RESULTS: We identified mitigation in 123 (21%) of 597 testing process event reports. Of the identified mitigators, 79% were persons from inside the practice, and 7% were patients or patient's family. Older age was the only patient demographic attribute associated with increased likelihood of mitigation occurring (unadjusted OR 18-44 years compared with 65 years of age or older = 0.27; p = 0.007). Events that included testing implementation errors (11% of the events) had lower odds of mitigation (unadjusted OR = 0.40; p = 0.001), and events containing reporting errors (26% of the events) had higher odds of mitigation (unadjusted OR = 1.63; p = 0.021). As the number of errors reported in an event increased, the odds of that event being mitigated decreased (unadjusted OR = 0.58; p = 0.001). Multivariate logistic regression showed that an event had higher odds of being mitigated if it included an ordering error or if the patient was 65 years of age or older, and lower odds of being mitigated if the patient was between age 18 and 44, or if the event included an implementation error or involved more than one error. Mitigated events had lower odds of patient harm (unadjusted OR = 0.16; p<0.0001) and negative consequences (unadjusted OR = 0.28; p<0.0001). Mitigated events resulted in less severe and fewer detrimental outcomes compared with non-mitigated events. CONCLUSION: Nearly a quarter of testing process errors reported by family physicians and their staff had evidence of mitigation, and mitigated errors resulted in less frequent and less serious harm to patients. Vigilance throughout the testing process is likely to detect and correct errors, thereby preventing or reducing harm.

Testing process errors and their harms and consequences reported from family medicine practices: a study of the American Academy of Family Physicians National Research Network.

CONTEXT: Little is known about the types and outcomes of testing process errors that occur in primary care. OBJECTIVE: To describe types, predictors and outcomes of testing errors reported by family physicians and office staff. DESIGN: Events were reported anonymously. Each office completed a survey describing their testing processes prior to event reporting. SETTING AND PARTICIPANTS: 243 clinicians and office staff of eight family medicine offices. MAIN OUTCOME MEASURES: Distribution of error types, associations with potential predictors; predictors of harm and consequences of the errors. RESULTS: Participants submitted 590 event reports with 966 testing process errors. Errors occurred in ordering tests (12.9%), implementing tests (17.9%), reporting results to clinicians (24.6%), clinicians responding to results (6.6%), notifying patient of results (6.8%), general administration (17.6%), communication (5.7%) and other categories (7.8%). Charting or filing errors accounted for 14.5% of errors. Significant associations (p<0.05) existed between error types and type of reporter (clinician or staff), number of labs used by the practice, absence of a results follow-up system and patients' race/ethnicity. Adverse consequences included time lost and financial consequences (22%), delays in care (24%), pain/suffering (11%) and adverse clinical consequence (2%). Patients were unharmed in 54% of events; 18% resulted in some harm, and harm status was unknown for 28%. Using multilevel logistic regression analyses, adverse consequences or harm were more common in events that were clinician-reported, involved patients aged 45-64 years and involved test implementation errors. Minority patients were more likely than white, non-Hispanic patients to suffer adverse consequences or harm. CONCLUSIONS: Errors occur throughout the testing process, most commonly involving test implementation and reporting results to clinicians. While significant physical harm was rare, adverse consequences for patients were common. The higher prevalence of harm and adverse consequences for minority patients is a troubling disparity needing further investigation.

Identification of a new urinary metabolite of carbon disulfide using an improved method for the determination of 2-thioxothiazolidine-4-carboxylic acid.

A new method is reported for the analysis of 2-thioxothiazolidine-4-carboxylic acid (TTCA) in urine that is amenable to automation and provides greatly simplified chromatograms. The method comprises the addition of tetrahydro-2-thioxo-2H-1,3-thiazine-4-carboxylic acid, which is chemically similar to TTCA, as internal standard, purification on an Oasis HLB solid-phase extraction column, and analysis by HPLC with UV detection. The limit of detection for TTCA was 40 pmol/mL of urine, recovery was 79.3 +/- 1.0%, and detection was linear over at least 3 orders of magnitude. In addition, during the analysis of urine samples from workers exposed to CS(2), a novel urinary metabolite of CS(2) was recognized. The new metabolite demonstrated a dose response, was present at approximately 30% the level of TTCA, and was charaterized to be 2-thioxothiazolidin-4-ylcarbonylglycine (TTCG). Administration of TTCG to rats resulted in excretion of TTCA suggesting that TTCG is a likely precursor of TTCA. Although urinary excretion of both TTCA and TTCG resulted from administration of captan, only TTCA was detected following administration of methyl isothiocyanate. The greater selectivity of TTCG suggests that co-analysis of TTCA and TTCG in urine may aid in differentiating exposures to CS(2), captan and isothiocyanates.

Enhanced N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice deficient in CuZn-superoxide dismutase or glutathione peroxidase.

Administration of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mammals causes damage to the nigrostriatal dopaminergic pathway similar to that observed in Parkinson disease (PD). Reactive oxygen species (ROS) are thought to be involved in the pathogenesis of MPTP-mediated dopaminergic neurodegeneration. To further clarify the role of superoxide anion radical (*O2-) and to study the possible involvement of hydroperoxides in MPTP-mediated neurodegeneration, MPTP neurotoxicity was induced in mice deficient in either CuZn superoxide dismutase (SOD), a scavenger enzyme for *O2-, or cellular glutathione peroxidase (GSHPx-1), a scavenger enzyme for hydroperoxides. Littermate control and homozygous deficient mice were injected intraperitoneally with a total cumulative dose of 0, 75, or 150 mg/kg of MPTP delivered over 5 d. All mice were killed 5 d after the last injection and the brains were processed for immunohistological analysis for tyrosine hydroxylase (TH) in the striatum and the substantia nigra pars compacta (SNc), as well as for direct measurements of dopamine concentrations in the striatum. The intensity of TH immunoreactivity in the striatum was evaluated by measuring the relative optical density (OD) with NIH IMAGE, and expressed as Log (OD of striatum)/Log (OD of white matter). Degeneration of TH-containing neurons was assessed by counting TH-positive neurons in the SNc. We found that this MPTP exposure protocol produced dose-dependent depletion of TH immunoreactivity and dopamine in the striatum in littermate control mice and both strains of knockout mice; however. reduction in TH immunoreactivity and dopamine content were significantly greater in CuZn-SOD or GSHPx-1 deficient mice compared with littermate controls. MPTP exposure did not significantly alter the number of TH-positive neurons in the SNc in littermate control or knockout mice. These data suggest that some of the deleterious effects of MPTP on striatal dopaminergic nerve terminals are mediated by both *O2- and hydroperoxides, and that they occur prior to dopaminergic neurodegeneration in the SNc. The similarity between the MPTP model and PD raises the possibility that both types of ROS may play a significant role in the early pathogenesis of dopaminergic neurodegeneration in PD.

Enhancement of dopaminergic neurotoxicity by the mercapturate of dopamine: relevance to Parkinson's disease.

The mechanisms that underlie dopaminergic neurodegeneration in Parkinson's disease (PD) are not known but have been proposed to involve oxidation of dopamine and related catechols. In other organ systems, cytotoxicity from catechol oxidation is profoundly influenced by mercapturate metabolism. Here we have tested the hypothesis that catechol thioethers produced in the mercapturic acid pathway may act as dopaminergic neurotoxins. A rat mesencephalic/neuroblastoma hybrid (MES) cell line was exposed to dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), or eight different catechol thioethers for up to 24 h, and the extent of apoptosis was quantified by a microculture kinetic assay. Apoptosis also was confirmed morphologically with Giemsa-stained cultures and by demonstration of internucleosomal DNA fragmentation. The results showed that dopamine at 5-50 microM produced concentration-dependent increases in the percentage of apoptotic MES cells. At 25 and 50 microM dopamine, the maximal proportions of apoptotic cells were detected at approximately 19 (20.7 +/- 2.0%) and 14 h (30.3 +/- 3.5%), respectively. None of the catechol thioethers (up to 5 microM) alone induced significant apoptosis in MES cells. However, when MES cells were incubated with dopamine (25 microM) and catechol thioethers (5 microM) to mimic pathological conditions, 5-S-N-acetylcysteinyldopamine, 5-S-homocysteinyldopamine, and 5-S-homocysteinyl-DOPAC significantly increased the percentage of apoptotic cells compared with dopamine alone. These results suggest that mercapturate metabolism of endogenous catechols may yield products that facilitate dopaminergic neurodegeneration.

Characterization of carbon disulfide neurotoxicity in C57BL6 mice: behavioral, morphologic, and molecular effects.

Female C57BL6 mice were exposed to 0 or 800 ppm carbon disulfide (CS2), 6 h/d, 5 d/wk for 20 weeks. The neurologic function of all mice was assessed once at the end of exposures using a functional observational battery. General health effects included a decrease in body weight gain, piloerection, hunched body posture, and ptosis. Treatment-related effects included altered gait (uncoordinated placement of hind limbs and ataxia) and impaired function on an inverted screen test. In addition, rearing and locomotor movement were decreased in treated mice. Focal to multifocal axonal swelling was seen predominantly in the muscular branch of the posterior tibial nerve, and occasionally giant axonal swelling was detected in the lumbar segment of the spinal cord. Electron microscopic examination revealed swollen axons with massive accumulation of neurofilament proteins within the axoplasm. Covalent cross-linking of erythrocyte spectrin (surrogate protein to neurofilament protein) was demonstrated in mice exposed to CS2 but not in mice receiving filtered air. These data provide supportive evidence that covalent cross-linking of neurofilament proteins is a significant feature of the axonal swellings in mice produced by inhalation exposure to CS2.

Dopamine mercapturate can augment dopaminergic neurodegeneration.

Pathological and biochemical studies have consistently associated endogenous catechol oxidation with dopaminergic neurodegeneration in Parkinson's disease (PD). Recently, it has been proposed that products of catechol oxidation, the catechol thioethers, may contribute to dopaminergic neurodegeneration. In other organ systems, thioether cytotoxicity is influenced profoundly by the mercapturic acid pathway. We have pursued the hypothesis that endogenous catechol thioethers produced in the mercapturic acid pathway contribute to dopaminergic neurodegeneration. Our results showed that the extent of in vitro metal-catalyzed oxidative damage by catechol thioethers varied with the structures of the parent catechol and thioether adduct. Catechol mercapturates uniquely produced more oxidative damage than their parent catechols. In dopaminergic cell cultures, dopamine induced apoptosis in a concentration-dependent manner from 5 to 50 microM. The apoptotic effect of dopamine was greatly enhanced by subcytotoxic concentrations of the mitochondrial inhibitor, N-methyl-4-phenylpyridinium (MPP+). Similarly, subcytotoxic levels of the mercapturate or homocysteine conjugate of dopamine significantly augmented dopamine-induced apoptosis. Finally, microsomal fractions of substantia nigra from PD patients or age-matched controls had comparable cysteine-S-conjugate N-acetyltransferase activity. These data indicate that the mercapturate conjugate of dopamine may augment dopaminergic neurodegeneration and that the mercapturate pathway exists in human substantia nigra.

High-pressure liquid chromatography quantitation of cytochrome c using 393 nm detection.

The release of cytochrome c from the mitochondrial intermembrane space can induce apoptotic cell death. Previous methods to detect cytochrome c release from mitochondria have relied upon immunoblotting, a procedure that can be limited by nonlinearity of signal, epitope masking, and impracticality for large numbers of samples. In order to circumvent these limitations, we have developed a reverse-phase high-pressure liquid chromatography method for cytochrome c detection and quantitation by taking advantage of a novel acid-induced absorbance maximum at 393 nm for cytochrome c in buffer containing 0.1% trifluoroacetic acid. Using a C4 reverse-phase analytical column, this assay had a quantitation limit of 10 ng (0.8 pmol) of cytochrome c. We demonstrated the detection and quantitation of cytochrome c from isolated mitochondria. This method of cytochrome c analysis may be useful for the study of agents that cause mitochondrial dysfunction and apoptotic cell death.

Endogenous catechol thioethers may be pro-oxidant or antioxidant.

Increased catechol thioether formation is associated with Parkinson's disease. In this study, we examined whether catechol thioethers, having a lower oxidation potential than their parent catechols, would cause greater oxidative damage than their parent catechols. We synthesized 5'-S-glutathionyl, cysteinyl, and N-acetylcysteinyl derivatives of dopamine and dopac, encompassing the known catechol thioethers of the mercapturate pathway. Cyclic voltametry studies showed that catechol thioethers had higher reduction potentials than their parent catechols. A higher reduction potential did not correlate with an increase in oxidative damage, measured by metal-catalyzed DNA strand breakage. 5'-S-Glutathionyldopamine and the cysteinyl adducts of dopamine and dopac mediated less oxidative damage than their parent catechols. In contrast, both N-acetylcysteinyl analogs were equipotent to dopamine. Oxygen consumption corresponded to DNA damage except for 5'-S-glutathionyldopamine. The glutathionyl and cysteinyl adducts of dopamine inhibited dopamine-mediated DNA damage indicating that these adducts may have antioxidant properties. 5'-S-Glutathionyldopamine potentiated H2O2-mediated damage whereas 5-S-cysteinyldopamine was inhibitory. Our results show that the ability of catechol thioethers to cause oxidative damage in vitro is not based simply upon the reduction potential but rather, reflects a complex relationship among structures of the parent catechol and thiol adduct, metal catalyst, and oxidant.

Increased cerebral cortical lipid peroxidation and abnormal phospholipids in aged homozygous apoE-deficient C57BL/6J mice.

Aged homozygous apolipoprotein E gene-deficient (apoE -/-) mice have been proposed as an experimental model for the role of human apoE isoforms in Alzheimer's disease (AD). However, results from different laboratories have been in conflict regarding the presence or absence of neurodegeneration in these mice. Moreover, despite apoE being the major lipid trafficking molecule in the central nervous system, there has been no investigation of brain lipid levels in apoE -/- mice. Here we have examined male and female apoE -/- and control mice aged 10 to 12 months, testing the hypothesis that lack of apoE leads to some of the neuropathological changes seen in AD. Our results failed to demonstrate significant neurodegeneration, histopathological changes, or reduction in cerebral cortical synaptophysin in apoE -/- mice. However, we did observe a significant reduction in cerebral cortical phospholipids and their constituent fatty acids, as well as elevated lipid peroxidation products, in apoE -/- mice compared to apoE +/+ mice with the same genetic background. Our results suggest that the brains of aged apoE -/- mice display some of the lipid abnormalities associated with AD; however, these changes alone, at the magnitudes achieved in the apoE -/- mice, do not directly lead to the major neurodegenerative changes of AD.

Exposure of C57BL/6 mice to carbon disulfide induces early lesions of atherosclerosis and enhances arterial fatty deposits induced by a high fat diet.

Even though atherosclerotic cardiovascular disease (ACVD) is the number one cause of death in the United States, the effects of environmental toxicants on this process are less well studied than the effects of chemicals on the second leading cause of death, cancer. There is considerable epidemiological evidence that workers exposed to carbon disulfide (CS2) have increased rates of ACVD, and there is conflicting evidence of the atherogenic potential of CS2 from animal studies. Chemical modification, such as oxidation of low-density lipoproteins (LDL), is tightly associated with increased LDL uptake by macrophages and the development of arterial fatty streaks. CS2 has been previously demonstrated to modify several proteins in vitro including LDL, and others in vivo through derivatization and covalent cross-linking. To investigate both the capacity of CS2 to induce arterial fatty deposits by itself, and its ability to enhance the rate of fatty deposit formation induced by a western style, high fat diet, groups of 20 female C57BL/6 mice were exposed to 0, 50, 500, or 800 ppm CS2 by inhalation. Half the animals in each group were placed on an atherogenic high fat diet and half on a control diet (NIH-07). Animals were sacrificed after 1, 4, 8, 12, 16, or 20 weeks of exposure, and the rates of fatty deposit formation under the aortic valve leaflets were evaluated. Exposure of mice on the control diet to 500 and 800 ppm CS2 induced a small but significant increase in the rate of fatty deposit formation over non-exposed controls. A more striking result was observed in the animals on the high fat diet. There was marked enhancement of the rate of fatty deposit formation in mice exposed to 500 and 800 ppm over the animals on the high fat diet alone. In addition, there was a small but significant enhancement in mice exposed to 50 ppm over the rate of fatty deposit formation induced by the high fat diet alone. Analysis of erythrocyte spectrin for protein cross-linking revealed a dose-dependent formation of alpha- and beta-heterodimers in animals on both diets. These data demonstrate that CS2 is atherogenic at high concentrations, but more importantly, suggest that, in conjunction with other risk factors, CS2 at relatively low concentrations can enhance atherogenesis.

The lipid peroxidation product 4-hydroxynonenal inhibits neurite outgrowth, disrupts neuronal microtubules, and modifies cellular tubulin.

Oxidative stress is believed to be an important factor in the development of age-related neurodegenerative diseases such as Alzheimer's disease (AD). The CNS is enriched in polyunsaturated fatty acids and is therefore particularly vulnerable to lipid peroxidation. Indeed, accumulation of lipid peroxidation products has been demonstrated in affected regions in brains of AD patients. Another feature of AD is a change in neuronal microtubule organization. A possible causal relationship between lipid peroxidation products and changes in neuronal cell motility and cytoskeleton has not been investigated. We show here that 4-hydroxy-2(E)-nonenal (HNE), a major product of lipid peroxidation, inhibits neurite outgrowth and disrupts microtubules in Neuro 2A cells. The effect of HNE on microtubules was rapid, being observed after incubation times as short as 15 min. HNE can react with target proteins by forming either Michael adducts or pyrrole adducts. 4-Oxononanal, an HNE analogue that can form only pyrrole adducts but not Michael adducts, had no effect on the microtubules. This suggests that the HNE-induced disruption of microtubules occurs via Michael addition. We also show that cellular tubulin is one of the major proteins modified by HNE and that the HNE adduction to tubulin occurs via Michael addition. Inhibition of neurite outgrowth, disruption of microtubules, and tubulin modification were observed at pathologically relevant HNE concentrations and were not accompanied by cytotoxicity. Our results show that these are proximal effects of HNE that may contribute to cytoskeletal alterations that occur in AD.

Parkinson's disease is associated with oxidative damage to cytoplasmic DNA and RNA in substantia nigra neurons.

Oxidative damage, including modification of nucleic acids, may contribute to dopaminergic neurodegeneration in the substantia nigra (SN) of patients with Parkinson's disease (PD). To investigate the extent and distribution of nucleic acid oxidative damage in these vulnerable dopaminergic neurons, we immunohistochemically characterized a common product of nucleic acid oxidation, 8-hydroxyguanosine (8OHG). In PD patients, cytoplasmic 8OHG immunoreactivity was intense in neurons of the SN, and present to a lesser extent in neurons of the nucleus raphe dorsalis and oculomotor nucleus, and occasionally in glia. The proportion of 8OHG immunoreactive SN neurons was significantly greater in PD patients compared to age-matched controls. Midbrain sections from patients with multiple system atrophy-Parkinsonian type (MSA-P) and dementia with Lewy bodies (DLB) also were examined. These showed increased cytoplasmic 8OHG immunoreactivity in SN neurons in both MSA-P and DLB compared to controls; however, the proportion of positive neurons was significantly less than in PD patients. The regional distribution of 8OHG immunoreactive neurons within the SN corresponded to the distribution of neurodegeneration for these three diseases. Nuclear 8OHG immunoreactivity was not observed in any individual. The type of cytoplasmic nucleic acid responsible for 8OHG immunoreactivity was analyzed by preincubating midbrain sections from PD patients with RNase, DNase, or both enzymes. 8OHG immunoreactivity was substantially diminished by either RNase or DNase, and completely ablated by both enzymes. These results suggest that oxidative damage to cytoplasmic nucleic acid is selectively increased in midbrain, especially the SN, of PD patients and much less so in MSA-P and DLB patients. Moreover, oxidative damage to nucleic acid is largely restricted to cytoplasm with both RNA and mitochondrial DNA as targets.

4-Hydroxy-2(E)-nonenal inhibits CNS mitochondrial respiration at multiple sites.

A destructive cycle of oxidative stress and mitochondrial dysfunction is proposed in neurodegenerative disease. Lipid peroxidation, one outcome of oxidative challenge, can lead to the formation of 4-hydroxy-2(E)-nonenal (HNE), a lipophilic alkenal that forms stable adducts on mitochondrial proteins. In this study, we characterized the effects of HNE on brain mitochondrial respiration. We used whole rat brain mitochondria and concentrations of HNE comparable to those measured in patients with Alzheimer's disease. Our results showed that HNE inhibited respiration at multiple sites. Complex I-linked and complex II-linked state 3 respirations were inhibited by HNE with IC50 values of approximately 200 microM HNE. Respiration was apparently diminished owing to the inhibition of complex III activity. In addition, complex II activity was reduced slightly. The lipophilicity and adduction characteristics of HNE were responsible for the effects of HNE on respiration. The inhibition of respiration was not prevented by N-acetylcysteine or aminoguanidine. Studies using mitochondria isolated from porcine cerebral cortex also demonstrated an inhibition of complex I- and complex II-linked respiration. Thus, in neurodegenerative disease, oxidative stress may impair mitochondrial respiration through the production of HNE.

Neurotoxicants and the cytoskeleton.

Exposure to occupational and environmental toxicants can result in distal axonopathies through reaction with various components of the axonal cytoskeleton. The solvents n-hexane and methyl n-butyl ketone are metabolized to the beta-diketone, 2,5-hexanedione, which covalently cross-links neurofilaments, resulting in large paranodal axonal swellings filled with neurofilaments. Carbon disulfide exposure leads to an identical axonopathy, achieving neurofilament cross-linking through a parallel series of reactions. Acrylamide and ethylene oxide, on the other hand, adduct proteins but do not lead to cross-linking. These toxicants appear to affect the function of microtubule-associated proteins, such as kinesin, and result in the impaired transport of synaptic vesicles.

Secondary excitotoxicity contributes to dopamine-induced apoptosis of dopaminergic neuronal cultures.

Dopamine (DA) and related catechols may contribute to selective degeneration of dopaminergic neurons in the substantia nigra in Parkinson's disease. To investigate whether DA induces apoptosis of dopaminergic neurons, we characterized the effects of various concentrations of exogenous DA on a substantia nigra/neuroblastoma hybrid cell line (MES 23.5 or MES). The hybrid MES cells were maintained in the presence of 50 microM glutamate in logarithmic growth on poly-D-lysine-precoated T-75 flasks and plated either onto petri dishes with glass coverslips for morphological studies or onto 6-well plates for quantification of apoptosis by flow cytometry. The results showed that DA exposure (0.5-20 microM) induced time- and dose-dependent apoptotic cell death of MES cells. To further analyze the mechanism responsible for DA-mediated apoptosis, we repeated the experiments at 20 microM DA in the presence or absence of 40 microM nomifensine, a DA re-uptake inhibitor, and 25 microM 2-amino-5-phosphonopentanoic acid (AP5), an N-methyl-D-aspartate (NMDA) receptor antagonist. The data indicate that both compounds significantly prevented DA-induced apoptosis of MES cells and that combination of AP5 and nomifensine provided greater protection against DA toxicity than AP5 alone. These results suggest for the first time that DA-induced apoptosis in dopaminergic neurons is partially attributable to increased vulnerability of these cells to non-toxic levels of excitatory amino acids, i.e., secondary excitotoxicity.