DataSheet1_Experimental exposure assessment of designed chemical mixtures in cell-based in vitro bioassays.PDF

Cell-based bioassays are useful tools for the effect assessment of complex mixtures, but so far exposure assessment has not been performed for mixtures of chemicals. In the present study, cytotoxicity and activation of oxidative stress response were measured for three designed chemical mixtures with up to twelve components. The measurements of biological responses were complemented by concentration measurements using solid-phase microextraction to derive the freely dissolved concentrations of the mixtures (Cfree,mix). The tested mixtures showed slightly higher cytotoxic effects than predicted by the concentration addition model. Nominal and freely dissolved effect concentrations of the mixtures were very similar (within a factor of 1.5), but nominal concentrations (Cnom) and Cfree of the individual mixture components were only similar for the hydrophilic chemicals (e.g., caffeine, coumarin, lamotrigine). For hydrophobic (e.g., fluoranthene) and acidic chemicals (e.g., diclofenac, naproxen) Cfree was up to 648 times lower than Cnom. Chemicals were dosed in equipotent nominal concentration ratios and therefore contributed equally to the detected effects. Hydrophilic chemicals with low potency dominated Cnom,mix (up to 95%) and Cfree,mix (up to 99%). Several mixture components (e.g., diclofenac, ibuprofen, naproxen and warfarin) showed increasing free fractions with increasing Cnom,mix and therefore also a concentration-dependent contribution to Cfree,mix. Based on the findings of this study, we concluded that Cnom,mix will be sufficient for evaluating the toxicity of mixtures that contain chemicals with diverse physicochemical properties at low concentration levels. In contrast, for risk assessment purposes and quantitative in vitro to in vivo extrapolations, Cfree,mix is a better parameter because the in vitro responses can be related to freely dissolved concentrations in human plasma.

基于细胞的生物测定法（cell-based bioassays）是评估复杂混合物效应的有效工具，但截至目前，尚未针对化学品混合物开展暴露评估工作。本研究针对三种设计制备的化学品混合物（每种最多包含12种组分），测定了其细胞毒性与氧化应激反应激活情况。研究通过固相微萃取（solid-phase microextraction）开展浓度测定，以获取混合物的自由溶解总浓度（Cfree,mix），以此补充生物学响应的检测数据。受试混合物表现出略高于浓度加和模型（concentration addition model）预测值的细胞毒性效应。混合物的标称效应浓度与自由溶解效应浓度十分接近（偏差在1.5倍以内），但仅针对亲水性化学品（如咖啡因、香豆素、拉莫三嗪），混合物各组分的标称浓度（Cnom）与Cfree才较为相近。对于疏水性化学品（如荧蒽）与酸性化学品（如双氯芬酸、萘普生），其Cfree较Cnom最高可低至1/648。本研究中各化学品以等效标称浓度比进行投加，因此各组分对检测到的毒性效应贡献均等。低活性的亲水性化学品在混合物标称总浓度（Cnom,mix，最高占比达95%）与自由溶解总浓度（Cfree,mix，最高占比达99%）中均占据主导地位。部分混合物组分（如双氯芬酸、布洛芬、萘普生与华法林）的自由分数随Cnom,mix升高而增加，因此其对Cfree,mix的贡献也呈现浓度依赖性。基于本研究结果，我们得出结论：当混合物中包含低浓度水平、理化性质各异的化学品时，采用Cnom,mix即可满足混合物毒性评估的需求。与之相对，若用于风险评估以及定量体外-体内外推（in vitro to in vivo extrapolations），则Cfree,mix是更优参数，因为体外检测得到的响应可与人体血浆中的自由溶解浓度建立关联。