Apoptotic and necrotic effects of hexanedione derivatives on the human neuroblastoma line SK-N-SH.
The potential cytotoxicity of two hexanedione food additives (2,3 and 3,4 isomers) was evaluated in comparison with the neurotoxic hexane metabolite 2,5-hexanedione in the human SK-N-SH neuroblastoma line using the MTT assay to indicate mitochondrial dehydrogenase activity and flow cytometry to monitor the cell cycle over 48 h. The IC(50)s of the 2,3-hexanedione (3.3+/-0.1 mM) and 3,4-hexanedione (3.5+/-0.1 mM), indicated that the sensitivity of the cells was approximately seven-fold greater to these toxins compared with the 2,5 derivative (IC(50) of 22.4+/-0.2 mM). Comparison between the respective IC(50)s of the 2,3-hexanedione and 3,4-hexanedione revealed no difference between the two isomers in terms of their effects on MTT turnover. With flow cytometry analysis, all three hexanediones showed increases in apoptosis within their respective concentration ranges of toxicity shown previously by MTT. In the presence of 2,5-hexanedione, between 8.5 and 17 mM concentrations, there was a significant increase in apoptotic nucleoids which was accompanied by a significant fall in the percentage of nucleoids in the G0/G1 phase (72.4+/-0.3-45.3+/-0.6%,), and a rise in the numbers of cells in the G2/M phase. This is likely to indicate growth arrest at cell cycle G2/M checkpoint in response to toxin damage. G2/M accumulation was also shown with 3,4 and 2,3 HD, which was maximal at much lower concentrations (approximately 4 and 3mM, respectively). Arrest at G1 and G2/M phase is indicative of inhibition of the cell cycle at the stages of DNA replication and chromosome segregation, respectively. It was also apparent that flow cytometry, rather than the MTT assay, did distinguish between the effects of the alpha-diketones 2,3-hexanedione and 3,4-hexanedione on the cell cycle. At a concentration of 5.8mM 3,4-hexanedione, the percentage of apoptotic nucleoids was 10.9+/-0.8% whilst apoptosis induced by 3,4-hexanedione had already reached a maximal level of 60.4+/-0.5%. In summary, flow cytometry indicated that the 3,4-hexanedione derivative was more toxic than its 2,3 isomer and that both food additives caused interruption in the neuroblastoma cell cycle and further investigation may be required to assess if these alpha-diketones present in diets pose any possible risks to human health.
Zilz
Thomas R
TR
School of Life and Health Sciences, Aston University, Birmingham, UK.
Griffiths
Helen R
HR
Coleman
Michael D
MD
D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005503:Food Additives / Q000633:toxicity*; D005562:Formazans / Q000378:metabolism; D006588:Hexanones / Q000633:toxicity*; D006801:Humans; D007536:Isomerism; D009336:Necrosis / Q000139:chemically induced*; D009447:Neuroblastoma; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009498:Neurotoxins / Q000633:toxicity; D013778:Tetrazolium Salts / Q000378:metabolism
D005503:Food Additives; D005562:Formazans; D006588:Hexanones; D009498:Neurotoxins; D013778:Tetrazolium Salts; C079782:MTT formazan; C043231:2,3-hexanedione; C011269:2,5-hexanedione