Patulin: Mechanism of genotoxicity

Patulin is a frequently found food contaminant mainly produced by the fungi Aspergillus and Penicillium. Patulin is suspected to be clastogenic, mutagenic, teratogenic and in higher concentrations cytotoxic. Here, we investigate the mechanism of the patulin-induced genotoxicity. Chromosomal damage was detected as micronucleus and nucleoplasmic bridge formation. Due to the activity of patulin on SH-groups, glutathione is a major compound in the cellular defense against patulin and the depletion of glutathione level with buthionine sulfoximine led to a strong increase in the genoxicity of patulin. A modified version of the alkaline comet assay was carried out to show the cross-linking properties of patulin. As a mechanistic hypothesis, we suspect patulin to cause structural DNA damage by cross-linking, yielding nucleoplasmic bridges and as a later consequence, micronucleus formation. The structural DNA damage may also lead to cell cycle delays, the consequence of which may be the observed centrosome amplification and formation of multipolar mitotic spindles.     

HPLC-MS/MS法测定中药材枳壳中展青霉素

目的:建立中药材枳壳中展青霉素的HPLC-MS/MS测定方法。方法:样品经果胶酶酶解,用水提取、经过展青霉素免疫亲和柱净化后,用高效液相色谱-三重四级杆串联质谱进行分析测定。结果:展青霉素在5.08 ng/ml～254.0 ng/ml范围内与峰面积呈良好的线性关系,线形方程为Y=2.17×103X-222,r=0.9977,回收率在80.0%～101.1%之间。在枳壳中最低检测限为0.02μg/kg。结论:本法灵敏、快速、准确、专属性强,可用于中药枳壳中展青霉素的测定。     

Mutagenicity of the mycotoxin patulin in cultured Chinese hamster V79 cells, and its modulation by intracellular glutathione

Because the ability of the mycotoxin patulin (PAT) to cause gene mutations in mammalian cells is still ambiguous, we have studied the mutagenicity of PAT at the hypoxanthine–guanine phosphoribosyltransferase (HPRT) gene locus in cultured Chinese hamster V79 cells with normal, depleted, and elevated glutathione (GSH) levels. PAT was more toxic to GSH-depleted cells than to normal cells and caused an increase of the intracellular GSH level in normal and GSH-depleted cells. It also caused synchronization of the cell cycle due to a temporary accumulation of cells in the G2/M phase; this G2/M arrest was more persistent in GSH-depleted than in normal cells. PAT gave rise to a clear and concentration-dependent induction of HPRT mutations at non-cytotoxic concentrations in V79 cells with normal GSH level; the lowest PAT concentration causing a significant number of mutant cells was 0.3 micromolar, and the mutagenic potency of PAT equaled that of the established mutagen 4-nitroquinoline-N-oxide. The mutagenicity of PAT was again more pronounced, by a factor of about three, in GSH-depleted V79 cells. Elevated GSH levels abolished all observed effects of PAT. These data support the notion that PAT is a mutagenic mycotoxin, in particular in cells with low GSH concentration. The ability of PAT to cause gene mutations in mammalian cells might have a bearing on its carcinogenicity.     

Correlation between the destruction of tight junction by patulin treatment and increase of phosphorylation of ZO-1 in Caco-2 human colon cancer cells

Patulin is a mycotoxin and its contamination of food has been reported to cause gastrointestinal inflammation, ulcers, and bleeding. The toxicity of patulin is thought to be due to the destruction of tight junctions (TJs) in gastrointestinal tissues. However, the precise mechanism has not been clarified. Here, we investigated the phosphorylation of TJ components. The transepithelial electrical resistance (TER) of Caco-2 human colon cancer cells decreased gradually during the first 24 h of treatment with 50 μM patulin. Immunofluorescence microscopy showed that the TJ proteins ZO-1 and claudin-4, but not occludin, had decreased after 24 h and decreased from the cell-cell contact regions of TJs after 48 h of patulin treatment. Western blotting showed that the level of ZO-1 decreased after 48 h of patulin treatment, but the levels of claudin-4 and occludin remained at the initial level until 72 h. Phosphorylation of ZO-1 was detected by 24 h and increased markedly after 72 h of patulin treatment. However, phosphorylation of claudin-4 and occludin was not detected by probing with anti-phosphotyrosine antibody. Immunoprecipitation showed that interaction of ZO-1 with claudin-4 had decreased after 48 h and was completely absent after 72 h. These results suggest that phosphorylation caused the degradation of ZO-1 protein and the decrease in TER induced by patulin treatment of Caco-2 cells.     

HPLC法测定含山楂类保健食品中展青霉素的含量

目的：建立含山楂类保健食品中展青霉素的含量测定方法,完善相关保健食品中展青霉素的质量控制。方法：色谱柱：Agilent TC-C18（2）柱（250 mm×4.6 mm,5μm）;流动相：0.8%四氢呋喃溶液;流速：1.0 ml·min^-1;柱温：30℃;检测波长：276 nm。结果：展青霉素在1-20 ng范围内线性关系良好（r=1.000 0）,平均加样回收率为92.1%,RSD=2.2%（n=6）。结论：本法简便、可靠、准确,可用于含山楂类保健食品中展青霉素的含量测定。     

Evaluation of genotoxic risk and oxidative DNA damage in mammalian cells exposed to mycotoxins,patulin and citrinin

Mycotoxins are fungal secondary metabolites with very diversified toxic effects in humans and animals. In the present study, patulin (PAT) and citrinin (CTN), two prevalent mycotoxins, were evaluated for their genotoxic effects and oxidative damage to mammalian cells, including Chinese hamster ovary cells (CHO-K1), human peripheral blood lymphocytes, and human embryonic kidney cells (HEK293). PAT, but not CTN, caused a significant dose-dependent increase in sister chromatid exchange (SCE) frequency in both CHO-K1 and human lymphocytes. PAT also elevated the levels of DNA gap and break in treated CHO-K1. In the single cell gel electrophoresis (SCGE) assay, exposure of HEK293 to concentrations above 15 microM of PAT induced DNA strand breaks; the tail moment values also greatly increased after posttreatment with formamidopyrimidine-DNA glycosylase (Fpg). This suggests that in human cells PAT is a potent clastogen with the ability to cause oxidative damage to DNA. However, no significant change in the tail moment values in CTN-treated cultures was found, suggesting that CTN is not genotoxic to HEK293. Incubation of HEK293 with CTN increased the mRNA level of heat shock protein 70 (HSP70), but not that of human 8-hydroxyguanine DNA glycosylase 1 (hOGG1). PAT treatment did not modulate the expression of either HSP70 or hOGG1 mRNA.     

高效液相色谱-质谱联用法检测果汁中的展青霉素

目的:应用高效液相色谱-质谱联用技术测定果汁中的展青霉素。方法:用振荡器和样品浓缩仪优化样品提取方法,以液质联用仪分析展青霉素。结果:该方法相对标准偏差在1.05%～3.41%之间,回收率在86.3%～97.5%之间,线性相关系数大于0.9995。结论:本方法准确可靠,可应用于果汁中展青霉素的测定。     

Evaluation of the reproductive toxicity of patulin in growing male rats

Patulin is a mycotoxin produced by several Penicillium, Aspergillus and Byssachlamys species. Patulin can be produced on different food products including fruits, grains, cheese, cured meats, but in natural situations patulin is exclusively found in apple and apple products. Patulin, at dose of 0.1 mg/kg bw/day, was administered by gavage to the growing male rats aged 5–6 week for 60 or 90 days. At the end of the experiment, sperm counts and morphology were investigated. Also, effects of patulin on the epididymis, seminal vesicle and prostate tissues were examined histopathologically and morphologically.

While sperm counts increased in patulin-treated rats for 60 days, sperm counts in patulin-treated rats for 90 days decreased compared to the corresponding control group. Patulin affected sperm morphology of growing male rats. Tail abnormalities like bent and/or coiled tails, and sticking of sperm tails were observed. A significant change was not determined in absolute and relative weights of the seminal vesicle and prostate of patulin-treated rats. While absolute cauda epididymal weights increased in rats treated with patulin for 60 days, absolute and relative cauda epididymal weights reduced in rats treated with patulin for 90 days. In histologic examination, some histopathological changes were observed in the epididymis and prostate tissues of rats in patulin treatment groups.