To improve assessment of risks associated with pharmaceutical contamination of the environment, it is crucial to understand effects and mode of action of drugs in non-target species. The evidence is accumulating that species with well-conserved drug targets are prone to be at risk when exposed to pharmaceuticals. An interesting group of pharmaceuticals released into the environment is imidazoles, antifungal agents with inhibition of ergosterol synthesis as a primary mode of action in fungi. However, imidazoles have also been identified as competitive antagonists of calmodulin (CaM), a calcium-binding protein with phylogenetically conserved structure and function. Therefore, imidazoles would act as CaM inhibitors in various organisms, including those with limited capacity to synthesize sterols, such as arthropods. We hypothesized that effects observed in crustaceans exposed to imidazoles are related to the CaM inhibition and CaM-dependent nitric oxide (NO) synthesis. To test this hypothesis, we measured (i) CaM levels and its gene expression, (ii) NO accumulation and (iii) gene expression of NO synthase (NOS1 and NOS2), in the cladoceran Daphnia magna exposed to miconazole, a model imidazole drug. Whereas significantly increased CaM gene expression and its cellular allocation were observed, supporting the hypothesized mode of action, no changes occurred in either NO synthase expression or NO levels in the exposed animals. These findings suggest that CaM inhibition by miconazole leads to protein overexpression that compensates for the loss in the protein activity, with no measurable downstream effects on NO pathways. The inhibition of CaM in D. magna may have implications for effect assessment of exposure to mixtures of imidazoles in aquatic non-target species.

中文翻译：

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钙调蛋白抑制作为抗真菌咪唑药物在非靶标生物中的作用方式。

为了改进与环境药物污染相关的风险评估，了解药物在非目标物种中的作用和作用方式至关重要。越来越多的证据表明，具有良好药物靶标的物种在接触药物时容易面临危险。释放到环境中的一组有趣的药物是咪唑，这是一种抗真菌药，具有抑制麦角固醇合成的作用，是真菌的主要作用方式。但是，咪唑也已被确定为钙调蛋白（CaM）的竞争性拮抗剂，钙调蛋白是一种具有系统发育上保守的结构和功能的钙结合蛋白。因此，咪唑将在各种生物中充当CaM抑制剂，包括那些合成固醇（如节肢动物）的能力有限的生物。我们假设，在暴露于咪唑的甲壳类动物中观察到的影响与CaM抑制和CaM依赖的一氧化氮（NO）合成有关。为了检验这个假设，我们在锁骨中测量了（i）CaM水平及其基因表达，（ii）NO积累和（iii）NO合酶（NOS1和NOS2）的基因表达。大型蚤（Daphnia magna）暴露于咪唑类药物，一种咪唑类药物。尽管观察到CaM基因表达及其细胞分配显着增加，支持了假设的作用方式，但暴露动物中NO合酶表达或NO水平均未发生变化。这些发现表明，咪康唑对CaM的抑制作用会导致蛋白质过表达，从而补偿蛋白质活性的损失，并且对NO途径没有可测量的下游影响。Ca.M. magna的抑制作用可能对暴露于水生非目标物种中的咪唑混合物的效果评估有影响。