Cytotoxic, genotoxic, and neurotoxic effects of Mg, Pb, and Fe on pheochromocytoma (PC-12) cells.

Metals such as lead (Pb), magnesium (Mg), and iron (Fe) are ubiquitous in the environment as a result of natural occurrence and anthropogenic activities. Although Mg, Fe, and others are considered essential elements, high level of exposure has been associated with severe adverse health effects including cardiovascular, hematological, nephrotoxic, hepatotoxic, and neurologic abnormalities in humans. In the present study we hypothesized that Mg, Pb, and Fe are cytotoxic, genotoxic and neurotoxic, and their toxicity is mediated through oxidative stress and alteration in protein expression. To test the hypothesis, we used the pheochromocytoma (PC-12) cell line as a neuro cell model and performed the LDH assay for cell viability, Comet assay for DNA damage, Western blot for oxidative stress, and HPLC-MS to assess the concentration levels of neurological biomarkers such as glutamate, dopamine (DA), and 3-methoxytyramine (3-MT). The results of this study clearly show that Mg, Pb, and Fe, respectively in the form of MgSO4 , Pb(NO3 )2 , FeCl2 , and FeCl3 induce cytotoxicity, oxidative stress, and genotoxicity in PC-12 cells. In addition, exposure to these metallic compounds caused significant changes in the concentration levels of glutamate, dopamine, and 3-MT in PC-12 cells. Taken together the findings suggest that MgSO4 , Pb(NO3 )2 , FeCl2 , and FeCl3 have the potential to induce substantial toxicity to PC-12 cells.

Fast quantification of amino acids by microchip electrophoresis-mass spectrometry.

A fast microchip electrophoresis-nano-electrospray ionization-mass spectrometric method (MCE-nanoESI-MS) was developed for analysis of amino acids in biological samples. A glass/poly(dimethylsiloxane) hybrid microchip with a monolithic nanoESI emitter was used in the platform. The proposed MCE-nanoESI-MS analytical method showed high separation efficiency for amino acids. Baseline separation of an amino acid mixture containing Lys, Arg, Val, Tyr, and Glu was completed within 120 s with theoretical plate numbers of >7,500. The method was applied to study cellular release of excitatory amino acids (i.e., aspartic acid (Asp) and glutamic acid (Glu)) under chemical stimulations. Linear calibration curves were obtained for both Asp and Glu in a concentration range from 1.00 to 150.0 µM. Limits of detection were found to be 0.37 µM for Asp and 0.33 µM for Glu (S/N = 3). Assay repeatability (relative standard deviation, n = 6) was 4.2 and 4.5%, for Asp and Glu at 5.0 µM, respectively. In the study of cellular release, PC-12 nerve cells were incubated with alcohol at various concentrations for 1 h. Both extra- and intracellular levels of Asp and Glu were measured by the proposed method. The results clearly indicated that ethanol promoted the release of both Asp and Glu from the cells.

Chiral capillary electrophoresis-mass spectrometry of 3,4-dihydroxyphenylalanine: evidence for its enantioselective metabolism in PC-12 nerve cells.

A fully automated chiral capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) method was developed for enantiomeric quantification of 3,4-dihydroxyphenylalanine (DOPA) and its precursors, phenylalanine (Phe) and tyrosine (Tyr). To avoid MS source contamination, a negatively charged chiral selector, sulfated ß-cyclodextrin (sulfated ß-CD), that migrated away from the detector was used in combination with the partial filling technique. The six stereoisomers were simultaneously quantified in less than 12 min. Detection limits were 0.48 and 0.51 µM for l- and d-DOPA enantiomers, respectively. Assay reproducibility values (relative standard deviations [RSDs], n=6) were 4.43, 3.15, 4.91, 5.16, 3.96, and 3.25% for l- and d-DOPA, l- and d-Tyr, and l- and d-Phe at 10 µM, respectively. Thanks to the high enantioseparation efficiency, detection of trace d-DOPA in l-/d-DOPA mixtures could be achieved. The assay was employed to study the metabolism of DOPA, a well-known therapeutic drug for treating Parkinson's disease. It was found that l-DOPA was metabolized effectively in PC-12 cells. Approximately 88% of l-DOPA disappeared after incubation at a cell density of 2×10(6)cells/ml for 3 h. However, d-DOPA coexisting with l-DOPA in the incubation solution remained intact. The enantiospecific metabolism of DOPA in this neuronal model was demonstrated.

A comparison of corneal cellular responses after 213-nm compared with 193-nm laser photorefractive keratectomy in rabbits.

PURPOSE: Corneal refractive surgery is typically performed using a 193-nm excimer laser. However, a recently developed 213-nm solid-state (5th harmonic) Nd:YAG laser presents some practical and user safety advantages, although the biological impact of using this wavelength remains poorly characterized. Here, we provide in vivo and in vitro comparisons of the corneal cellular outcomes after irradiation with 213 and 193 nm wavelengths. METHODS: New Zealand White rabbits underwent photorefractive keratectomy with -5 diopters and a 6.5-mm optical zone and studied at time points up to 1 year. The development of haze was examined ophthalmologically and by detecting myofibroblasts immunohistochemically. Cell death was quantified using a terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay, and the number of stromal keratocytes undergoing apoptosis estimated histologically. Superoxide dismutase activity was estimated in vitro by enzyme-linked immunosorbent assay in irradiated rabbit corneal keratocytes. RESULTS: Our results demonstrate subtle differences in the cellular outcomes after irradiation with 213- and 193-nm lasers, despite similar degrees of corneal haze developing in both treatment groups. In vivo, the 213-nm laser results in more stable stromal cell numbers, implying a more predictable ablation outcome. In vitro, higher levels of superoxide dismutase in corneal keratocytes irradiated with 213 nm compared with 193 nm wavelengths suggest a better endogenous protection against free radicals induced by laser surgery. CONCLUSIONS: The more favorable cellular responses after irradiation with 213 nm compared with 193 nm wavelengths are consistent with good clinical outcomes previously reported. Ablation with a 213 nm wavelength may result in better wound healing, leading to a more reliable correction of refractive errors.