Effects of tri butyl tin acetate on dopamine biosynthesis and l-dopa-lnduced cytotoxicity in pc12 cells

The effects of tributyltin acetate (TBTA) on dopamine biosynthesis and L-3,4-dihydroxyphenylalanine (L-DOPA)-induced cytotoxicity in PC12 cells were examined. TBTA at concentrations of 0.1-0.2 microM inhibited dopamine biosynthesis by reducing tyrosine hydroxylase (TH) activity and TH gene expression in PC12 cells. TBTA at 0.1-0.4 microM also reduced L-DOPA (20-50 microM)-induced increases in dopamine content for 24 h in PC12 cells. TBTA at concentrations up to 0.3 microM did not affect cell viability. However, TBTA at concentrations higher than 0.4 microM caused apoptotic cytotoxicity. Exposure of PC12 cells to non-cytotoxic (0.1 and 0.2 microM) or cytotoxic (0.4 microM) concentrations of TBTA with L-DOPA (20, 50 and 100 microM) significantly increased the cell loss and the percentage of apoptotic cells after 24 or 48 h compared with TBTA or L-DOPA alone. These data suggest that TBTA inhibits dopamine biosynthesis and enhances L-DOPA-induced cytotoxicity in PC12 cells.     

Aggravation of L-DOPA-induced neurotoxicity by tetrahydropapaveroline in PC12 cells

Tetrahydropapaveroline (THP) is formed in Parkinsonian patients receiving L-DOPA therapy and is detected in the plasma and urine of these patients. In this study, we have investigated the effects of THP on L-DOPA-induced neurotoxicity in cultured rat adrenal pheochromocytoma, PC12 cells. Exposure of PC12 cells up to 10 microM THP or 20 microM L-DOPA after 24 or 48 hr, neither affected the cell viability determined by MTT assay, nor induced apoptosis by flow cytometry and TUNEL staining. However, at concentrations higher than 15 microM, THP showed cytotoxicity through an apoptotic process. In addition, THP at 5-15 microM for both incubation time points significantly enhanced L-DOPA-induced neurotoxicity (L-DOPA concentration, 50 microM). Exposure of PC12 cells to THP, L-DOPA and THP plus L-DOPA for 48 hr resulted in a marked increase in the cell loss and percentage of apoptotic cells compared with exposure for 24hr. The enhancing effects of THP on L-DOPA-induced neurotoxicity were concentration- and treated-time-dependent. THP, L-DOPA and THP plus L-DOPA produced a significant increase in intracellular reactive oxygen species generation and decrease in ATP levels, supporting the involvement of oxidative stress in THP- and L-DOPA-induced apoptosis. The antioxidant N-acetyl-L-cysteine strongly inhibited changes in apoptosis, decreases in cell viability and ROS generation induced by THP associated with L-DOPA. These results suggest that THP aggravates L-DOPA-induced oxidative neurotoxic and apoptotic effects in PC12 cells. Therefore, Parkinsonian patients treated with L-DOPA for long-term need to be monitored for the relationship between plasma concentration of THP and the symptoms of neurotoxicity.     

Effects of harman and norharman on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells

The effects of harman and norharman on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Harman and norharman at a concentration of 20 microM and 100 microM showed 49.4% and 49.5% inhibition of dopamine content for 48 h, respectively. The IC50 values of harman and norharman were 21.2 microM and 103.3 microM. Dopamine content, tyrosine hydroxylase (TH) activity and TH mRNA levels were decreased during the first 6 h, maintained for up to 48 h and then gradually recovered at 72 h after exposure to 20 microM harman and 100 microM norharman. Under the same conditions, the intracellular cyclic AMP levels and Ca2+ concentrations were also decreased by harman and norharman. In addition, harman and norharman at concentrations higher than 80 microM and 150 microM caused cytotoxicity at 48 h in PC12 cells. Non-cytotoxic ranges of 10-30 microM harman and 50-150 microM norharman inhibited L-DOPA (20-50 microM)-induced increases in dopamine content at 48 h. Harman at 20-150 microM and norharman at 100-300 microM also enhanced L-DOPA (20-100 microM)-induced cytotoxicity at 48 h with an apoptotic process. These results suggest that harman and norharman inhibit dopamine biosynthesis by reducing TH activity and enhance L-DOPA-induced cytotoxicity in PC12 cells.     

Effects of (1R,9S)-beta-hydrastine on l-DOPA-induced cytotoxicity in PC12 cells

(1R,9S)-beta-Hydrastine in lower concentrations of 10-50 microM inhibits dopamine biosynthesis in PC12 cells. In this study, the effects of (1R,9S)-beta-hydrastine on L-DOPA (L-3,4-dihydroxyphenylalanine)-induced cytotoxicity in PC12 cells were investigated. (1R,9S)-Hydrastine at concentrations up to 250 microM did not reduce cell viability. However, at concentrations higher than 500 microM it caused cytotoxicity in PC12 cells, as determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, TUNEL (terminal deoxynucleotidyltransferase dUTP nick-end labeling) method and flow cytometry. Exposure of PC12 cells to cytotoxic concentrations of (1R,9S)-beta-hydrastine (500 and 750 microM) in combination with L-DOPA (20, 50 and 100 microM) after 24 or 48 h resulted in a significant decrease in cell viability compared with the effects of (1R,9S)-beta-hydrastine or L-DOPA alone, and apoptotic cell death was observed. However, the decrease in cell viability induced by (1R,9S)-beta-hydrastine was not prevented by the antioxidant N-acetyl-L-cysteine, indicating that it is not mediated by membrane-based oxygen free radical damage. These data suggest that (1R,9S)-beta-hydrastine has a mild cytotoxic effect and at higher concentration ranges aggravates L-DOPA-induced cytotoxicity in PC12 cells.     

Effects of asimilobine on dopamine biosynthesis and l-DOPA-induced cytotoxicity in PC12 cells

The effects of asimilobine, an aporphine isoquinoline alkaloid, on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Asimilobine at concentration ranges of 0.05-0.2 microM showed a significant inhibition of intracellular dopamine levels for 24 h in a concentration-dependent manner with an IC50 value of 0.13 microM. Asimilobine at 0.15 microM inhibited tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) activities at 24 h (73.2% inhibition of TH activity): the inhibition of TH activity was stronger and longer than that of AADC activity. Asimilobine also decreased TH mRNA levels and intracellular cyclic AMP levels, but not the basal Ca2+ concentrations. In addition, asimilobine at 0.05-5.0 microM, but not 10 microM, did not alter cell viability toward PC12 cells. A non-cytotoxic asimilobine (0.15 microM) associated with l-DOPA (20, 50, and 100 microM) for 24 h inhibited L-DOPA-induced increases in dopamine levels and enhanced L-DOPA-induced cell death when compared with L-DOPA alone. These results suggest that asimilobine inhibits dopamine biosynthesis by mainly reducing the TH activity and TH mRNA expression, and enhances L-DOPA-induced cytotoxicity in PC12 cells.     

Liriodenine inhibits dopamine biosynthesis and L-DOPA-induced dopamine content in PC12 cells

The inhibitory effects of liriodenine, an aporphine isoquinoline alkaloid, on dopamine biosynthesis and L-DOPA-induced dopamine content increases in PC12 cells were investigated. Treatment of PC12 cells with 5-10 microM liriodenine significantly decreased the intracellular dopamine content in a concentration-dependent manner (IC50 value, 8.4 microM). Liriodenine was not cytotoxic toward PC12 cells at concentrations up to 20 microM. Tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) activities were inhibited by 10 microM liriodenine to 20-70% and 10-14% of control levels at 3-12 h, respectively; TH activity was more influenced than AADC activity. The levels of TH mRNA, intracellular cyclic AMP and basal Ca2+ concentration were also decreased by 10 microM liriodenine. In addition, 10 microM liriodenine reduced L-DOPA (20-100 microM)-induced increases in dopamine content. However, 10 microM liriodenine resulted in a protective effect against L-DOPA (50-100 microM)-induced cytotoxicity. These results suggest that liriodenine regulates dopamine biosynthesis by partially reducing TH activity and TH gene expression and has protective effects against L-DOPA-induced cytotoxicity in PC12 cells.     

Modulation of PC12 cell viability by forskolin-induced cyclic AMP levels through ERK and JNK pathways: an implication for L-DOPA-induced cytotoxicity in nigrostriatal dopamine neurons

The intracellular levels of cyclic AMP (cAMP) increase in response to cytotoxic concentrations of L-DOPA in PC12 cells, and forskolin that induces intracellular cAMP levels either protects PC12 cells from L-DOPA-induced cytotoxicity or enhances cytotoxicity in a concentration-dependent manner. This study investigated the effects of cAMP induced by forskolin on cell viability of PC12 cells, relevant to L-DOPA-induced cytotoxicity in Parkinson's disease therapy. The low levels of forskolin (0.01 and 0.1 μM)-induced cAMP increased dopamine biosynthesis and tyrosine hydroxylase (TH) phosphorylation, and induced transient phosphorylation of ERK1/2 within 1 h. However, at the high levels of forskolin (1.0 and 10 μM)-induced cAMP, dopamine biosynthesis and TH phosphorylation did not increase, but rapid differentiation in neurite-like formation was observed with a steady state. The high levels of forskolin-induced cAMP also induced sustained increase in ERK1/2 phosphorylation within 0.25-6 h and then led to apoptosis, which was apparently mediated by JNK1/2 and caspase-3 activation. Multiple treatment of PC12 cells with nontoxic L-DOPA (20 μM) for 4-6 days induced neurite-like formation and decreased intracellular dopamine levels by reducing TH phosphorylation. These results suggest that the low levels of forskolin-induced cAMP increased dopamine biosynthesis in cell survival via transient ERK1/2 phosphorylation. In contrast, the high levels of forskolin-induced cAMP induced differentiation via sustained ERK1/2 phosphorylation and then led to apoptosis. Taken together, the intracellular levels of cAMP play a dual role in cell survival and death through the ERK1/2 and JNK1/2 pathways in PC12 cells.     

Effects of catalpalactone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells

The effects of catalpalactone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Catalpalactone at 5–30 μM decreased intracellular dopamine content with the IC₅₀ value of 22.1 μM. Catalpalactone at 5–20 μM, but not 30 μM, did not alter cell viability. Catalpalactone at 20 μM inhibited tyrosine hydroxylase (TH) and aromatic-l-amino acid decarboxylase (AADC) activities. Catalpalactone also decreased cyclic AMP levels and inhibited TH phosphorylation. In addition, catalpalactone at 20 μM reduced the increases in dopamine levels induced by L-DOPA (20–50 μM). Catalpalactone (5–30 μM) associated with L-DOPA (50–100 μM) enhanced L-DOPA-induced cytotoxicity at 48 h, which was prevented by N-acetyl-l-cysteine. These results suggest that catalpalactone inhibited dopamine biosynthesis by reducing TH and AADC activities and enhanced L-DOPA-induced cytotoxiciy in PC12 cells.     

Enantio-selective inhibition of (1R,9S)- and (1S,9R)-beta-hydrastines on dopamine biosynthesis in PC12 cells

The inhibitory effects of (1R,9S)- and (1S,9R)-enantiomers of beta-hydrastine (BHS) on dopamine biosynthesis in PC12 cells were investigated. (1R,9S)-BHS decreased the intracellular dopamine content with the IC50 value of 14.3 microM at 24 h, but (1S,9R)-BHS did not. (1R,9S)-BHS was not cytotoxic at concentrations up to 250 microM towards PC12 cells. In these conditions, (1R,9S)-BHS inhibited tyrosine hydroxylase (TH) activity mainly in a concentration-dependent manner (33% inhibition at 20 microM) and decreased TH mRNA level in PC12 cells. The inhibitory patterns of dopamine content and TH activity by (1R,9S)-BHS showed similar behavioral curves. (1R,9S)-BHS at 10-50 microM also reduced the intracellular cyclic AMP level and Ca2+ concentration. In addition, treatment of L-DOPA at 20-50 microM for 24 h increased the intracellular dopamine content to 198-251% compared with the control in PC12 cells. However, the increase in dopamine levels induced by L-DOPA (20-50 microM) was reduced when L-DOPA was combined with (1R,9S)-BHS (10-50 microM). These results indicate that (1R,9S)-BHS, but not (1S,9R)-BHS, reduced dopamine content and L-DOPA-induced increase in dopamine content, in part, through the inhibition of TH activity and TH gene expression in PC12 cells: thus, (1R,9S)-BHS proved to have a function to regulate dopamine biosynthesis.     

Modulatory effects of sesamin on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells

The effects of sesamin on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Sesamin at concentration ranges of 20-75 μM exhibited a significant increase in intracellular dopamine levels at 24 h: 50 μM sesamin increased dopamine levels to 133% and tyrosine hydroxylase (TH) activity to 128.2% of control levels. Sesamin at 20-100 μM rapidly increased the intracellular levels of cyclic AMP (cAMP) to 158.3%-270.3% of control levels at 30 min. At 50 μM, sesamin combined with L-DOPA (50, 100 and 200 μM) further increased the intracellular dopamine levels for 24 h compared to L-DOPA alone. In the absence or presence of L-DOPA (100 and 200 μM), sesamin (50 μM) increased the phosphorylation of TH, cAMP-dependent protein kinase (PKA), and cAMP-response element-binding protein (CREB), as well as the mRNA levels of TH and CREB for 24 h, an effect which was reduced by L-DOPA (100 and 200 μM). In addition, 50 μM sesamin exhibited a protective effect against L-DOPA (100 and 200 μM)-induced cytotoxicity via the inhibition of reactive oxygen species (ROS) production and superoxide dismutase reduction, induction of extracellular signal-regulated kinase (ERK)1/2 and BadSer112 phosphorylation and Bcl-2 expression, and inhibition of cleaved-caspase-3 formation. These results suggested that sesamin enhanced dopamine biosynthesis and L-DOPA-induced increase in dopamine levels by inducing TH activity and TH gene expression, which was mediated by cAMP-PKA-CREB systems. Sesamin also protected against L-DOPA (100-200 μM)-induced cytotoxicity through the suppression of ROS activity via the modulation of ERK1/2, BadSer112, Bcl-2, and caspase-3 pathways in PC12 cells. Therefore, sesamin might serve as an adjuvant phytonutrient for neurodegenerative diseases.     

Inhibitory effects of ethaverine, a homologue of papaverine, on dopamine content in PC12 cells

The inhibitory effects of ethaverine on dopamine content in PC12 cells were investigated. Ethaverine decreased dopamine content in a concentration-dependent manner in PC12 cells and showed 33.6% inhibition of dopamine content at a concentration of 1.0 microM for 24-48 h. The IC50 value of ethaverine was 1.4 microM. Dopamine content was lowered at 6h and reached a minimal level at 12h after exposure to ethaverine at 2.0 microM. The decreased dopamine level was maintained up to 48 h and then recovered to the control level at about 72 h. Tyrosine hydroxylase (TH) was inhibited at 6 h following treatment with ethaverine in PC12 cells and the activity was maintained at a reduced level up to 36 h (12-22% inhibition at 2.0 microM). These results indicate that ethaverine leads to a decrease in dopamine content by inhibition of TH activity.     

Induction of dopamine biosynthesis by l-DOPA in PC12 cells: implications of L-DOPA influx and cyclic AMP

The effects of 3,4-dihydroxyphenylalanine (l-DOPA) on dopamine biosynthesis and cytotoxicity were investigated in PC12 cells. l-DOPA treatment (20-200 microM) increased the levels of dopamine by 226%-504% after 3-6 h of treatment and enhanced the activities of tyrosine hydroxylase (TH) and aromatic l-amino acid decarboxylase (AADC). l-DOPA (20-200 muM) treatment led to a 562%-937% increase in l-DOPA influx at 1 h, which inhibited the activity of TH, but not AADC, during the same period. The extracellular releases of dopamine were also increased by 231%-570% after treatment with 20 and 200 microM l-DOPA for 0.5-3 h. l-DOPA at a concentration of 100-200 microM, but not 20 microM, exerted apoptotic cytotoxicity towards PC12 cells for 24-48 h. l-DOPA (20-200 microM) increased the intracellular cyclic AMP levels by 318%-557% after 0.5-1 h in a concentration-dependent manner. However, the elevated cyclic AMP levels by l-DOPA could not protect against l-DOPA (100-200 microM)-induced cytotoxicity after 24-48 h. In addition, l-DOPA (20-200 microM)-induced increases in cyclic AMP and dopamine were significantly reduced by treatment with SCH23390 (dopamine D(1) receptor antagonist). The increased levels of dopamine by l-DOPA were also reduced by H89 (protein kinase A, PKA, inhibitor) and GF109203X (protein kinase C inhibitor); however, the reduction by GF109203X was not significant. l-DOPA at 20-200 microM stimulated the phosphorylation of PKA and cyclic AMP-response element binding protein and induced the biosynthesis of the TH protein. These results indicate that 20-200 microM l-DOPA induces dopamine biosynthesis by two pathways. One pathway involves l-DOPA directly entering the cells to convert dopamine through AADC activity (l-DOPA decarboxylation). The other pathway involves l-DOPA and/or released dopamine activating TH to enhance dopamine biosynthesis by the dopamine D(1) receptor-cyclic AMP-PKA signaling system (dopamine biosynthesis by TH).     

Oxidative and non-oxidative mechanisms of neuronal cell death and apoptosis by L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine

1. The present study was designed to evaluate the nature of intervening agents in L-DOPA- and dopamine-induced neurotoxicity in Neuro-2A cells. 2. In the absence of cells and in conditions of light protection, at 37 degrees C, L-DOPA or dopamine (1 mM) in culture medium degraded spontaneously in a time-dependent manner, this being prevented by ascorbic acid (200 microM) and other antioxidants, namely glutathione (1 mM), N-acetyl-L-cysteine (1 mM), sodium metabisulphite (200 microM), but not N-ter-butyl-alpha-phenylnitrone (1 mM) and deferoxamine (100 microM). 3. The viability of Neuro-2A cells declined following treatment with L-DOPA or dopamine in a concentration- and time-dependent manner. The decrease in cell viability by L-DOPA (10+/-4% of control) or dopamine (15+/-4% of control) was markedly attenuated by antioxidants (ascorbic acid, glutathione, N-acetyl-L-cysteine and sodium metabisulphite). Autoxidation of L-DOPA or dopamine was accompanied by the formation of H(2)O(2) in a time-dependent manner, this being completely prevented by ascorbic acid at 24 h or markedly reduced at 48 h. 4. Protective effects of 100 U ml(-1) catalase (40+/-1% of control) against L-DOPA-induced cell death were lower than those conferred by 200 microM ascorbic acid (70+/-3% of control). Catalase-induced protection (59+/-5% of control) against dopamine-induced cell death was similar to that conferred by 200 microM ascorbic acid (57+/-4% of control). L-DOPA-induced neuronal cell death was also accompanied by increases in caspase-3 activity, this being insensitive to ascorbic acid. Dopamine-induced increase in caspase-3 activity occurred only when autoxidation of the amine was prevented by ascorbic acid. 5. It is suggested that in addition to generation of H(2)O(2) and quinone formation, L-DOPA- and dopamine-induced cell death may result from induction of apoptosis, as evidenced by increases in caspase-3 activity. Dopamine per se induces apoptosis by a mechanism independent of oxidative stress, as evidenced by the fact that increases in caspase-3 activity occurred only when autoxidation of the amine was prevented.     

Triorganotin-induced cytotoxicity to rat thymus, bone marrow and red blood cells as determined by several in vitro assays

To further investigate the immunotoxic effects of tri-n-propyltin chloride (TPTC), tri-n-butyltin chloride (TBTC) and triphenyltin chloride (TPhTC) several cytotoxicity tests with a series of trialkyltin chlorides and TPhTC were carried out, using isolated rat thymocytes as target cells. Thymocytes, cultured in a serum-supplemented medium, were exposed to organotin concentrations ranging from 0.01 to 10 microM for periods up to 30 h. Parameters such as cell count, trypan blue exclusion, chromium release, thymidine incorporation and cyclic AMP production were used to evaluate the cytotoxicity of these compounds. The more lipophilic compounds TPTC, TBTC, tri-n-hexyltin chloride (THTC) and TPhTC appeared most cytotoxic, reducing thymidine incorporation at concentrations as low as 0.05-1 microM. Membrane damage as determined by trypan blue exclusion and chromium release occurred at higher levels (1-10 microM). The water soluble homologue trimethyltin chloride (TMTC) was least effective in all test models. When phosphate-buffered saline supplemented with glucose was used as incubation medium, TBTC appeared more cytotoxic to thymocytes. Using this medium in 5-h incubations the cytotoxicity of TBTC to thymus, bone marrow and red blood cells was compared. Bone marrow cells were slightly less sensitive than thymocytes, while red cells were relatively resistant. In conclusion, of the triorganotin compounds especially the lipophilic homologues are cytotoxic in vitro.     

Alteration of catecholamines in pheochromocytoma (PC12) cells in vitro by the metabolites of chlorotriazine herbicide.

The effects of four major chlorotriazine metabolites on the constitutive synthesis of the catecholamines dopamine (DA) and norepinephrine (NE) were examined, using undifferentiated PC12 cells. NE release and intracellular DA and NE concentrations were quantified, for up to 24 h after initiation of treatment with different concentrations, ranging from 0 to 400 microM, of the metabolites hydroxyatrazine (HA), 2-amino-4-chloro-6-isopropylamino-s-triazine (deethylchlorotriazine), 2-amino-4-chloro-6-ethylamino-s-triazine (deisopropylchlorotriazine), and diaminochlorotriazine (DACT). Hydroxyatrazine significantly decreased intracellular DA and NE concentrations in a dose- and time-dependent manner. This metabolite also caused a significant inhibition of NE release from the cells. In contrast, deethylchlorotriazine and deisopropylchlorotriazine significantly increased intracellular DA concentration following exposure to 50-200 microM from 12 to 24 h. Intracellular NE was significantly reduced at these same concentrations of deethylchlorotriazine at 24 h while the concentration of NE in PC12 cells exposed to deisopropylchlorotriazine was not altered at any dosage or time point measured. NE release was decreased at 18 (200 microM) and 24 h (100 and 200 microM) following exposure to deethylchlorotriazine and at 24 h (100 and 200 microM) following deisopropylchlorotriazine. DACT, at the highest concentration (160 microM), significantly increased intracellular DA and NE concentrations at 18 and 24 h. NE release was also increased at 40-160 microM at 24 h. The viability of the PC12 cells was tested using the trypan blue exclusion method. Following 18 to 24 h of treatments with HA, cell viability was reduced 10-12% at the two higher concentrations (200 and 400 microM), but, with other metabolites, the viability was reduced by only 2 to 5% at the highest concentrations. These data indicate that HA affects catecholamine synthesis and release in PC12 cells in a manner that is similar to synthesis of atrazine and simazine. On the other hand, deethylchlorotriazine and deisopropylchlorotriazine altered catecholamine synthesis in a manner similar to that observed in the rat brain following in vivo exposure (i.e., increased DA and decreased NE concentration), whereas DACT appeared to produce an increase in NE release as well as in the intracellular DA and NE concentrations. Overall, these findings suggest that the catecholamine neurons may be a target for the chlorotriazines and/or their metabolites, that the metabolites produce a unique pattern of catecholamine response, and that all of the changes were seen within the same range of doses.     

Differential modulation of catecholamines by chlorotriazine herbicides in pheochromocytoma (PC12) cells in vitro.

Epidemiological, wildlife, and laboratory studies have pointed to the possible adverse health effects of chlorotriazine herbicide (i.e. , atrazine, simazine, and cyanazine) exposure. However, the cellular mechanism(s) of action of these compounds remains unknown. Recently, it was reported by Cooper et al. (2000, Toxicol. Sci. 53, 297-307) that atrazine disrupts ovarian function by altering hypothalamic catecholamine concentrations and subsequently the regulation of luteinizing hormone (LH) and prolactin (PRL) secretion by the pituitary. In this study, we examined the effect of three chlorotriazines on catecholamine metabolism in vitro using PC12 cells. Intracellular norepinephrine (NE) and dopamine (DA) concentrations and spontaneous NE release were measured following treatment with different concentrations of atrazine, simazine (0, 12. 5, 25, 50, 100, and 200 microM) and cyanazine (0, 25, 50, 100, and 400 microM) for 6, 12, 18, 24, and 48 h. Atrazine and simazine significantly decreased intracellular DA concentration in a concentration-dependent manner. Intracellular NE concentration was also significantly decreased by 100 and 200 microM atrazine and 200 microM simazine. Similarly, there was a dose-dependent inhibition of NE release with 100 and 200 microM concentrations of both compounds. Although 100 and 400 microM cyanazine increased intracellular NE concentration, 50, 100, and 400 microM cyanazine significantly increased NE release at 24 and 36 h. In contrast, intracellular DA concentration was decreased by cyanazine, but only at 400 microM. The GABA(A)-receptor agonist, muscimol (0, 0.01, 0.1, and 1.0 microM) had no effect on either the release or on intracellular catecholamine concentrations from 6 through 24 h of treatment. Cell viability was somewhat lower in the groups exposed to 100 and 200 microM atrazine and simazine. However, the reduction in viability was significant only in the highest dose of atrazine used (200 microM) at 24 h. Cyanazine did not have an effect on the viability at any of the doses tested, and the cells were functional, even up to 48 h of exposure. These data indicate that both atrazine and simazine inhibit the cellular synthesis of DA mediated by the tyrosine hydroxylase (TH), and NE mediated by dopamine beta-hydroxylase (DbetaH), and, as a result, there is a partial or significant inhibition of NE release. Cyanazine, on the other hand, stimulated the synthesis of intracellular NE, and not DA. Thus, chlorotriazine compounds presumably act at the enzymatic steps or sites of CA biosynthesis to modulate monoaminergic activity in PC12 cells.     

维生素C对左旋多巴致自由基系统动态平衡损害的保护作用

目的通过体外实验探讨维生素C对左旋多巴(L-DOPA)致胶质细胞培养液中自由基系统动态平衡损害的保护作用,防治L-DOPA加速帕金森病进展。方法在含有胶质细胞的中脑培养液中,加入L-DOPA(100μmol/L)、维生素C(200μmol/L)+L-DOPA(100μmol/L),分别在24、48和72h时间点取标本,设空白对照组,测定谷胱甘肽(GSH)的含量、谷胱甘肽过氧化物酶(GSH-Px)的活性、超氧化物歧化酶(SOD)的活力及丙二醛(MDA)的水平。结果L-DOPA100μmol/L各个时间点GSH的含量、GSH-Px的活性、SOD的活力较维生素C+L-DOPA、对照组显著的下降(P<0.05～0.01),MDA的水平显著的升高(P<0.05～0.01);维生素C+L-DOPA组各个时间点GSH的含量、GSH-Px的活性、SOD的活力、MDA的水平与对照组比较差异无统计学意义(P>0.05～0.01)。结论维生素C可增强GSH、GSH-Px、SOD水平或活性,以增强其清除自由基的能力,降低MDA的水平,维持自由基系统的动态平衡,降低L-DOPA对神经元的毒性作用。     

Effects of methylmercury and mercuric chloride on differentiation and cell viability in PC12 cells.

The effects of methylmercury (CH3Hg) or mercuric chloride (HgCl(2)) on neurite outgrowth and cell viability were quantified using undifferentiated (unprimed) and differentiated (primed) pheochromocytoma (PC12) cells. In unprimed cells, following 24-h exposure, CH3Hg significantly decreased NGF-stimulated neurite outgrowth at concentrations of 0.3-3 microM. However, HgCl(2) significantly increased both neurite outgrowth and the number of branch points, a component of neurite outgrowth. In primed PC12 cells, following 24-h exposure, both CH3Hg and HgCl(2) inhibited NGF-stimulated neurite outgrowth with an EC(50) of approximately 0.03 microM; however, there was a difference between CH3Hg and HgCl(2) effects on the subcomponents of total neurite outgrowth. CH3Hg significantly decreased both the number of branch points (0.3 microM) and fragment length (0.01 microM), while HgCl(2) only decreased fragment length (0.03 microM). Cell viability was assessed in the same cultures by trypan-blue exclusion. In unprimed cells, the EC(50) for cytotoxicity of CH3Hg in the presence and absence of NGF was 0.21 +/- 0.04 and 0.87 +/- 0.12 microM, respectively, and for HgCl(2) in the presence and absence of NGF was 8.18 +/- 1.52 and 5.02 +/- 0.74 microM, respectively. In primed cells, the EC(50) for cytotoxicity of CH3Hg in the presence or absence of NGF was 1.17 +/- 0.38 and 0.73 +/- 0.14 microM, respectively, and for HgCl(2) in the presence or absence of NGF was 3.96 +/- 0.82 and 3.81 +/- 0.91 microM, respectively. In the primed PC12 model, cytotoxicity occurred at concentrations that were at least 30-fold higher than the EC(50) for neurite outgrowth, suggesting that the mercurial compounds can act selectively on the process of differentiation.     

The role of oxidative stress in hormesis induced by sodium arsenite in human embryo lung fibroblast (HELF) cellular proliferation model

Hormetic dose-response relationships induced by environmental agents are often characterized by a low-dose stimulation and a high-dose inhibition. The mechanisms underlying hormesis induced by environmental agents still remain an enigma; however, hormetic consequences may have significant implications for health risk assessments. To investigate the role of oxidative stress in hormetic phenomena associated with cell proliferation induced by sodium arsenite, the levels of reactive oxygen species (ROS), lipid peroxidation (LPO), and heat-shock proteins (HSP) and the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were measured in human embryo lung fibroblast (HELF) cells after treatment with sodium arsenite at various concentrations for differing times. Results showed that sodium arsenite induced significant cell proliferation at low concentrations (0.5 microM for 12, 24, and 48 h), but inhibited cell growth at high amounts (5 and 10 microM for 24 and 48 h), reflected as a beta concentration-response curve. Data indicated that the relationship between ROS levels and sodium arsenite exposure concentration displayed a positive correlation. It was found out that sodium arsenite at high concentrations induced LPO damage. The activities of SOD were enhanced at low metal concentrations but inhibited with high amounts in a concentration-dependent manner. Similarly, heat-shock protein 27 (HSP27) levels were increased by sodium arsenite of low concentrations with early exposure time (3, 6, and 12 h), but decreased with high metal concentrations with greater exposure time (24 and 48 h).Sodium arsenite decreased HSP70 expression at lower concentrations, but increased HSP70 expression at higher concentration. The results indicated that this cellular hormetic model of cell proliferation induced by sodium arsenite occurred in HELF cells, which may explain contradictory effects seen with this metal. Sodium arsenite at low concentrations induced enhanced ROS generation without cytotoxicity and a cellular protective effect. In contrast, sodium arsenite at high concentrations produced marked ROS formation, marked oxidative stress, and cellular damage, as evidenced by LPO.