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首页> 外文期刊>The Journal of toxicological sciences >Multiphoton spectral analysis of benzo(a)pyrene uptake and metabolism in breast epithelial cell lines
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Multiphoton spectral analysis of benzo(a)pyrene uptake and metabolism in breast epithelial cell lines

机译：乳腺癌上皮细胞系中苯并（a）py吸收和代谢的多光子光谱分析

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Quantification of polycyclic aromatic hydrocarbons (PAH) and their metabolites within living cells and tissues in real time using fluorescence methods is complicated due to overlaping excitation and/or emission spectra of metabolites. In this study, simultaneous analysis of several metabolites of a prototype carcinogenic PAH, benzo[a]pyrene (BaP) in undifferentiated (MCF10A) and differentiated (MCFlOCAlh) breast cancer cells was performed using single-cell multiphoton spectral analysis. The two cell types were selected for this study because they are known to have differences in BaP uptake and metabolism and induction of aryl hydrocarbon receptor-dependent ethoxyresorafin-O-deethylase (EROD) activity. Multiphoton microscopy spectral analysis performed in cells exposed to BaP for 24 hr identified 5 major peaks of fluorescence that were monitored within spectral bands. A comparison of the fluorescence peaks within these bands to those of BaP metabolite standards indicated that a peak in the spectral range of 393-415 nm matched benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide(+-),(anti) (BPDE), the ultimate carcinogenic BaP metabolite. In addition, the 426-447 nm band matched the major metabolites 3-hydroxybenzo[a]pyrene (3-OH BaP) and 9-hydroxybenzo[a]pyrene (9-OH BaP); the 458-479 nm band corresponded to the secondary metabolite benzo[a]pyrene-3,6-dione (3,6 BPQ); and a peak at 490-530 nm matched the parent compound, BaP. Multiphoton spectral analysis also revealed differences in fluorescence intensities between MCF10A and MCF10CAlh cells within three spectral bands: 393-415 nm, 426-447 nm and 458-479 nm which were partially reversed with cyclosporine A suggesting differences in efflux mechanisms between cell lines. These results demonstrate the feasibility of analyzing BaP metabolism in situ by multiphoton spectral analysis and also identifying cell-type differences in BaP accumulation and metabolism.
机译：由于代谢物的激发和/或发射光谱重叠，使用荧光方法实时定量活细胞和组织内多环芳烃（PAH）及其代谢物非常复杂。在这项研究中，使用单细胞多光子光谱分析技术同时分析了未分化（MCF10A）和分化（MCF10CAlh）乳腺癌细胞中原型致癌PAH，苯并[a]（（BaP）的几种代谢物。选择这两种细胞进行此项研究是因为已知它们在BaP摄取和代谢以及诱导芳基烃受体依赖性乙氧基间苯二酚-O-脱乙基酶（EROD）活性方面存在差异。在暴露于BaP 24小时的细胞中进行的多光子显微镜光谱分析确定了在光谱带内监控的5个主要荧光峰。将这些谱带中的荧光峰与BaP代谢物标准品的荧光峰进行比较表明，在393-415 nm光谱范围内的峰与苯并[a] py-r-7，t-8-二氢二醇-t-9,10相匹配-环氧（+-），（抗）（BPDE），最终致癌的BaP代谢产物。此外，426-447 nm谱带与主要代谢物3-羟基苯并[a] py（3-OH BaP）和9-羟基苯并[a] a（9-OH BaP）相匹配。 458-479 nm谱带对应于次生代谢产物苯并[a] py-3,6-二酮（3,6 BPQ）; 490-530 nm处的峰与母体化合物BaP相匹配。多光子光谱分析还揭示了在三个光谱带内的MCF10A和MCF10CAlh细胞之间的荧光强度差异：393-415 nm，426-447 nm和458-479 nm，部分被环孢霉素A逆转，表明细胞系之间的外排机理不同。这些结果证明了通过多光子光谱分析原位分析BaP代谢的可行性，以及鉴定BaP积累和代谢中的细胞类型差异。

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