Alleviation of specific responses in the combined exposure of freshwater mussel Unio tumidus to psychoactive substances and microplastics

The environmentally relevant aquatic pollution is associated with the mixtures of xenobiotics, each in the low, picomolar to micromolar concentrations. Among these substances, the combinations of pharmaceuticals and microplastics (MP) have become an increasingly serious threat. The objective of this study was to track the specific and multi-stress responses of swollen river mussels (Unio tumidus) to the psychoactive substances caffeine (Caff) and chlorpromazine (Cpz) in the combined exposure with MP. The MP (1 mg•L-1, size 35–50 μm), Caff (20 µg•L-1), Cpz (12 ng•L-1) or their mixture (Mix) were administered to mussels for 14 days. The redox state, enzymes of biotransformation and apoptosis were analysed in the digestive gland. All exposures except Mix caused oxidative injury to lipids and proteins, accompanied by increased GSH and metallothionein levels, suppressed NAD+ and activation of GST (except Mix), and GTPase. MP had the lower particular impact. Specific responses to Caff were activation of Cyp450 (EROD) and cathepsin D, decreased GSH/GSSG ratio and prominent demetallation of metallothionein. The Cpz caused an increase in NADH/NAD+ ratio and caspase-3 inhibition. In the combined exposure, the specific responses to single xenobiotics were alleviated which was confirmed by discriminant analysis. The Mix-group was distinguished by the highest NADH/NAD+ and GSH/GSSG ratios, markedly increased caspase-3 activity accompanied by the decrease of protein carbonyl level and the highest IBR index, attesting to the negative cumulative effect of multi-stress exposure. The vulnerability of mussels to pM concentration of neuroleptic Cpz needs particular attention.

与环境相关的水生污染与多种外源性化合物（xenobiotics）的混合暴露密切相关，各化合物浓度普遍较低，处于皮摩尔至微摩尔范围。其中，药物与微塑料（microplastics, MP）的复合污染已成为愈发严峻的水生环境威胁。本研究旨在探究膨肚河蚌（Unio tumidus）在微塑料联合精神活性物质暴露下的特异性与多胁迫响应，所涉精神活性物质为咖啡因（caffeine, Caff）与氯丙嗪（chlorpromazine, Cpz）。实验设置了4种暴露体系：1 mg•L-1、粒径35–50 μm的微塑料，20 µg•L-1的咖啡因，12 ng•L-1的氯丙嗪，以及上述三者的混合体系（Mix），暴露时长为14天。随后对其消化腺的氧化还原状态、生物转化酶及细胞凋亡相关指标进行分析。除混合暴露组外，其余各单一暴露组均引发了脂质与蛋白质的氧化损伤，伴随谷胱甘肽（GSH）与金属硫蛋白水平上调、烟酰胺腺嘌呤二核苷酸（NAD+）水平下调，以及谷胱甘肽S-转移酶（GST，混合暴露组除外）与GTP酶的激活。微塑料单独暴露的影响相对较弱。咖啡因单独暴露的特异性响应包括：细胞色素P450（Cyp450，即EROD活性）与组织蛋白酶D激活、谷胱甘肽/氧化型谷胱甘肽（GSH/GSSG）比值降低，以及金属硫蛋白的显著去金属化。氯丙嗪单独暴露则导致还原型烟酰胺腺嘌呤二核苷酸/烟酰胺腺嘌呤二核苷酸（NADH/NAD+）比值升高，以及半胱氨酸天冬氨酸蛋白酶-3（caspase-3）活性受抑制。在复合暴露体系中，单一外源性化合物的特异性响应被削弱，该结论经判别分析得以验证。混合暴露组的特征为：最高的NADH/NAD+与GSH/GSSG比值、显著升高的caspase-3活性，伴随蛋白质羰基水平下降，以及最高的整合生物标志物响应（IBR）指数，证实了多胁迫暴露的负面累积效应。河蚌对皮摩尔级别的抗精神病药物氯丙嗪的易感性需引起特别关注。