Molecular Biotechnology Laboratory, Department of Biology, Faculty of Science, Shiraz University, P.O. Box: 71467-13565, Shiraz 71454, Iran; Department of Pharmaceutical Chemistry, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, CCB - Centrum for Chemistry and Biomedicine, Innrain 80/82, 6020 Innsbruck, Austria.
Department of Pharmaceutical Chemistry, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, CCB - Centrum for Chemistry and Biomedicine, Innrain 80/82, 6020 Innsbruck, Austria.
Toxicol In Vitro. 2021 Jun;73:105148. doi: 10.1016/j.tiv.2021.105148. Epub 2021 Mar 16.
Phthalates are widely used in polymer science and have potential toxicity related to their chemical structures. However, lots of evidence indicate that phthalate derivatives are undoubtedly produced as secondary metabolites by organisms, including plants, animals, and microorganisms. In the present study, Bacillus velezensis strain RP137 was cultured under optimized conditions. Its biomass was extracted with ethyl acetate with one fraction showing cytotoxic properties. A pure compound was isolated from the active fraction using combined silica gel and LH20 size exclusion column chromatography. Structural evaluation including FT-IR, H NMR, C NMR, HR-MS and CHN analysis identified the purified compound as di(2-ethylhexyl)phthalate (DEHP) with the formula CHO and the molecular weight of 389.29 Da. The microorganism-derived (stereospecific) DEHP was strongly reduced the proliferation and induced cytotoxic effects on various eukaryotic cell lines in compare to the synthetic racemic mixture of the compound when assessed by MTT assay. Furthermore, crystal violet assay and morphological changes confirmed the cytotoxic effect of DEHP. Interestingly, non-malignant SV40-immortalized fibroblast cells were less affected by the purified DEHP. Further evaluation on the antibacterial activity of DEHP documented no effect toward Gram-positive (S. aureus) and Gram-negative (E. coli and P. aeruginosa) pathogens even at a high concentration of 100 μM. In conclusion, existence of DEHP as byproduct of microorganism's metabolism can seriously be considered as a warning to human health.
邻苯二甲酸酯广泛应用于聚合物科学，并因其化学结构而具有潜在的毒性。然而，大量证据表明，邻苯二甲酸酯衍生物无疑是植物、动物和微生物等生物作为次生代谢产物产生的。在本研究中，优化了地衣芽孢杆菌 RP137 的培养条件，并用乙酸乙酯提取其生物量，其中一个馏分具有细胞毒性。用硅胶和 LH20 尺寸排阻柱色谱法从活性馏分中分离出一种纯化合物。通过傅里叶变换红外光谱（FT-IR）、核磁共振氢谱（H NMR）、核磁共振碳谱（C NMR）、高分辨率质谱（HR-MS）和元素分析（CHN）等结构评估，鉴定出该纯化合物为邻苯二甲酸二（2-乙基己基）酯（DEHP），其化学式为 CHO，分子量为 389.29 Da。与该化合物的合成外消旋混合物相比，微生物衍生的（立体特异性）DEHP 强烈抑制了各种真核细胞系的增殖，并通过 MTT 测定诱导了细胞毒性作用。此外，结晶紫测定和形态变化证实了 DEHP 的细胞毒性作用。有趣的是，非恶性 SV40 永生化成纤维细胞受纯化 DEHP 的影响较小。进一步评估 DEHP 的抗菌活性发现，即使在 100 μM 的高浓度下，它对革兰氏阳性（金黄色葡萄球菌）和革兰氏阴性（大肠杆菌和铜绿假单胞菌）病原体均无作用。总之，微生物代谢产物中存在 DEHP 可能会严重威胁人类健康，这应引起高度重视。
