Many pesticides are used increasingly in combinations during crop protection and their stability ensures the presence of such combinations in foodstuffs. The effects of three fungicides, pyrimethanil, cyprodinil and fludioxonil, were investigated together and separately on U251 and SH-SY5Y cells, which can be representative of human CNS glial and neuronal cells respectively. Over 48h, all three agents showed significant reductions in cellular ATP, at concentrations that were more than tenfold lower than those which significantly impaired cellular viability. The effects on energy metabolism were reflected in their marked toxic effects on mitochondrial membrane potential. In addition, evidence of oxidative stress was seen in terms of a fall in cellular thiols coupled with increases in the expression of enzymes associated with reactive species formation, such as GSH peroxidase and superoxide dismutase. The glial cell line showed significant responsiveness to the toxin challenge in terms of changes in antioxidant gene expression, although the neuronal SH-SY5Y line exhibited greater vulnerability to toxicity, which was reflected in significant increases in caspase-3 expression, which is indicative of the initiation of apoptosis. Cyprodinil was the most toxic agent individually, although oxidative stress-related enzyme gene expression increases appeared to demonstrate some degree of synergy in the presence of the combination of agents. This report suggests that the impact of some pesticides, both individually and in combinations, merits further study in terms of their impact on human cellular health.
Reliable, high throughput, in vitro preliminary screening batteries have the potential to greatly accelerate the rate at which regulatory neurotoxicity data is generated. This study evaluated the importance of astrocytes when predicting acute toxic potential using a neuronal screening battery of pure neuronal (NT2.N) and astrocytic (NT2.A) and integrated neuronal/astrocytic (NT2.N/A) cell systems derived from the human NT2.D1 cell line, using biochemical endpoints (mitochondrial membrane potential (MMP) depolarisation and ATP and GSH depletion). Following exposure for 72 h, the known acute human neurotoxicants trimethyltin-chloride, chloroquine and 6-hydroxydopamine were frequently capable of disrupting biochemical processes in all of the cell systems at non-cytotoxic concentrations. Astrocytes provide key metabolic and protective support to neurons during toxic challenge in vivo and generally the astrocyte containing cell systems showed increased tolerance to toxicant insult compared with the NT2.N mono-culture in vitro. Whilst there was no consistent relationship between MMP, ATP and GSH log IC(50) values for the NT2.N/A and NT2.A cell systems, these data did provide preliminary evidence of modulation of the acute neuronal toxic response by astrocytes. In conclusion, the suitability of NT2 neurons and astrocytes as cell systems for acute toxicity screening deserves further investigation.
Abstract:The effects of the α-diketone derivatives 2,3-and 3,4-hexanediones were investigated in three non-neuronal cell lines (MCF7, HepG 2 and CaCo-2) as well as in the neuroblastoma line, SH-SY5Y. The MTT reduction assay was employed to determine the necrotic effects of the α-diketones and the neurotoxin 2,5-hexanedione over 4, 24 and 48 hr exposures. Flow cytometry was also used to study the effects of the three isomers on the cell cycle of the SH-SY5Y line only. With 2,5-hexanedione, the mean MTT IC 50 decreased more than 10-fold from 4 to 48 hr. The toxicities of both α-diketones were similar, with a more than 18-fold increase in sensitivity of the SH-SY5Y at 24 hr compared to that of 4 hr. With flow cytometry at 48 hr, SH-SY5Y apoptosis with 2,5-hexanedione rose throughout the concentration range evaluated (0-30 mM) while 2,3-and 3,4-hexanediones showed apoptosis over the concentration range 1-1.6 mM, with 3,4-hexanedione being the more potent compared to the 2,3-isomer. At 1.6 mM nearly all the cells had entered apoptosis in the presence of the 3,4-isomer, (94.9 ± 1.4%) but only 57.5 ± 4.1% of the 2,3-isomer-treated cells had reached that stage. The 2,3-and 3,4-isomers in diets alone may not pose a serious threat to human health. Further studies may be necessary to evaluate the effects of other dietary components on their toxicity. These α-diketones also display a degree of toxic selectivity towards neuroblastoma cells, which may have therapeutic implications.Hexanediones are formed biologically (2,5-hexanedione) as a result of exposure to the organic solvent hexane [1] and they also are present in the diet as food colourings and flavouring agents. 2,3-Hexanedione occurs naturally in beer, coffee and fermented soya beans and is used commercially to yield a buttery-cheesy taste in various foodstuffs and fragrances. 3,4-Hexanedione also occurs naturally in coffee, as well as cauliflower and it yields a sweet caramel-buttery odour and taste in butter, margarine and fruit flavourings [2]. The 2,5-isomer is an established human neurotoxin, which causes the cross-linking of neurofilaments; this process is linked to the onset of a polyneuropathy that is associated with occupational exposure to hexane [3,4]. In contrast, the 2,3-and 3,4-isomers have been generally viewed as non-toxic [2,5] although the 2,3-derivative induced mitotic chromosomal loss in the presence of an inducer of chromosomal malsegregation [6]. More recent studies have revealed the 2,3-and 3,4-isomers to be several fold more acutely toxic over 4 and 24 hr compared to the 2,5-isomer in a number of neural and astrocytic cell lines [7] and they are also capable of disrupting the cell cycle and causing apoptosis in the human SK-N-SH neuroblastoma cell line [8].To date, there have been no comparative studies aimed at determining if the 2,3-and 3,4-hexanediones illustrate any degree of specific toxicity towards neuroblastoma lines or are cytotoxic to human cell lines in general. Hence, the cytotoxicity of the three hexanedione derivatives was evalua...
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