Investigation of photogenotoxicity of pesticides irradiated with near UV light or visible light

以大肠杆菌WP2uvrA/pKM101菌株为研究材料，在uva照射（320 ~ 400 nm）下进行Trp^+反向突变试验，发现进口柑橘常用的采后杀菌剂噻苯达唑（Thiabendazole， TBZ）具有光致突变性。S9mix是一种具有代谢激活辅助因子的大鼠肝脏匀浆微粒体组分，在S9mix的存在下没有观察到光致突变性。我们发现NADH和NADPH作为辅助因子在S9mix中有效地抑制了TBZ的光致突变性。这一证据有力地表明，S9mix存在时的非诱变性不是由于TBZ的代谢解毒或S9mix中的大分子清除uva活化的TBZ。相反，NADH和NADPH的猝灭作用可能是抑制uva活化TBZ的主要原因，因为氧化形式的NAD^+和NADP^+没有表现出抑制作用。研究了TBZ （50-400 μg/ml）和uva （320-400 nm, 250 μW/cm^2）短脉冲处理10 min对细菌和人细胞的dna损伤活性、致突变性和致裂性的影响。uva辐照TBZ对大肠杆菌和人淋巴母细胞WTK1细胞的DNA损伤分别采用umu试验和单细胞凝胶电泳（comet）试验。在鼠伤寒沙门菌和大肠杆菌的改良Ames试验中，检测到-1移码突变和碱基置换突变的强烈诱导。在体外细胞松弛素- b微核实验中，50-100 μg/ml的TBZ与uva辐照显著诱导WTK1细胞微核。单独用TBZ脉冲治疗10分钟无遗传毒性。虽然TBZ是一种诱导非整倍体的纺锤体毒性，但我们假设本研究中TBZ的光毒性是由不同的机制产生的，可能是DNA加合物的形成。我们得出结论，uva激活的TBZ在体外对人细胞具有遗传毒性。

Thiabendazole (TBZ), a post-harvest fungicide commonly used on imported citrus fruits, exhibited photo-mutagenicity following UVA-irradiation (320-400 nm) in Trp^+ reverse mutation assay using Escherichia coli WP2uvrA/pKM101 strain. The photo-mutagenicity was not observed in the presence of S9mix, a rat liver homogenate microsome fraction with co-factors for metabolic activation. We found that NADH and NADPH used as co-factor in the S9mix efficiently suppressed the photo-mutagenicity of TBZ. This evidence strongly suggested that non-mutagenicity in the presence of S9mix was not due to the metabolic detoxification of TBZ or the scavenging of UVA-activated TBZ by macromolecules in the S9mix. Rather quenching effect of NADH and NADPH may be more responsible for suppression of UVA-activation of TBZ, because oxidized forms of NAD^+ and NADP^+ did not show inhibitory effects.Potential of DNA-damaging activity, mutagenicity, and clastogenicity were investigated by short pulse treatment for 10 min with TBZ (50-400 μg/ml) and UVA-irradiation (320-400 nm, 250 μW/cm^2) in bacterial and human cells. UVA-irradiated TBZ caused DNA damage in Escherichia coli and human lymphoblastoid WTK1 cells assayed, respectively, by the umu-test and the single cell gel electrophoresis (comet) assay. In a modified Ames test using Salmonella typhimurium and E.coli, strong induction of -1 frameshift mutations as well as base-substitution mutations were detected. TBZ at 50-100 μg/ml with UVA-irradiation significantly induced micronuclei in WTK1 cells in the in vitro cytochalasin-B micronucleus assay. Pulse treatment for 10 min with TBZ alone did not show any genotoxicity. Although TBZ is a spindle poison that induces aneuploidy, we hypothesize that the photogenotoxicity of TBZ in the present study was produced by a different mechanism, probably by DNA adduct formation. We concluded that UVA-activated TBZ is genotoxic in human cells in vitro.