The Lysosomal Pathway Of DNA Damage Induced By Patulin In HEK-293Cells

Objective:Patulin(PAT) is a mycotoxin belonging to a class of toxic compounds,mainly Penicillium, Aspergillus and other fungi genus bare capsule. Patulinwas first isolated in1940, but until recent years, people know that patulin is aglobal presence mycotoxin in apples and apple products.PAT by International Agency for Research on Cancer (IARC) as carcinogenic tohumans of the third category. Namely, the existing evidence is not yet on its humancarcinogenicity classification. World Health Organization (WHO) provides patulin thehighest limit of in food is50μg/L, the EU has more stringent requirements of the trendof infant food within patulin content limit of10μg/kg.Toxicology tests showed that patulin has teratogenic, carcinogenic and mutageniceffects. The study also shows that there are genetic toxicity and immune toxicity of PAT.Some studies have shown that the genetic toxicity of PAT has a relationship withoxidative stress on cells. Some recent studies have shown that DNA damage has arelationship with changes in lysosomal membrane stability. Therefore, we studied therelationship between patulin induced DNA damage and intracellular ROS levels, alsoand the lysosomes, to investigate the PAT genotoxic mechanism.In this study we choose hunan embryonic kidney HEK-293cells to explore the roleof patulin in possible mechanism of DNA damage. It may provide experimental data onthe risk of PAT for human health.Methods:HEK-293cells were selected as test system. We evaluate the genetictoxicity of PAT by MTT assay and single cell gel electrophoresis (SCGE) test fordetection of DNA damage.To investigate the possible genotoxic mechanism, we usedthe2’,7’-dihydro-dichlorofluorescein (DCFH) to detect intracellular ROS levels; we used acridine orange (AO) to measure the changes of lysosomal membrane stability;the use of N-acetylcysteine (NAC) and ammonium chloride(NH4CL) protection of thelysosomal intervention; we used NAC, NH4CL, gastric inhibitory peptide (pepstatina A)interfere with PAT induced DNA damage. The results with SPSS v11.5statisticalpackage for statistical analysis.Results: After being treated with PAT (5-20μM) in HEK-293cells for1h, DNAstrand has breaks. In the comet assay, a significant dose-dependent increase of strandbreaks was found; With PAT (2.5-40μM) in HEK-293cells1h, it is caused the changeof lysosomal membrane stability. And PAT (80μM) caused an increase of ROS levelsafter being treated for1h. With10mM of NH4CL,500mM of NAC pretreatment ofHEK-293cells after1h, it was significantly protected cell lysosomal membrane stability.With10mM of NH4CL,500mM of NAC and100μM of pepstatin A pretreatment ofHEK-293cells after1h, PAT induced DNA strand breaks was almost completelyblocked. But with30μM of desipramine pretreatment, PAT induced DNA strand breakshave not been significantly improved, it may be separately desipramine causedHEK-293cell DNA damage.Conclusion:Patulin induced DNA strand breaks in HEK-293cells, and itsmechanism may be related to lysosomal signaling pathway, through the release oflysosomal membrane stability of the destruction of some lysosomal enzymes, leading toDNA strand breaks. NAC is an effective antioxidants, while protecting the lysosomalmembrane stability with NH4CL, it may indicated that generation of ROS because ofPAT itself is toxic lead, and cause DNA strand breaks through the lysosomal pathway.Lysosome may be one of the biological targets in cytotoxicity of PAT, and membranestability caused by ROS damage.