Integrated metabolic models for xenobiotic induced mitochondrial toxicity in skeletal muscle (compound 177 experiments)

There is a need for robust in vitro models to sensitively capture skeletal muscle adverse toxicities early in the research and development of novel xenobiotics. To this end, an in vitro rat skeletal muscle model (L6) was used to study the translation of transcriptomics data generated from an in vivo rat model. Novel sulfonyl isoxazoline herbicides were associated with skeletal muscle toxicity in an in vivo rat model. Gene expression pathway analysis on skeletal muscle tissues taken from in vivo repeat dose studies identified enriched pathways associated with mitochondrial dysfunction, oxidative stress, energy metabolism, protein regulation and cell cycle. Mitochondrial dysfunction and oxidative stress were further explored in an in vitro L6 model. This model demonstrated that the sulfonyl isoxazoline compounds induced mitochondrial dysfunction, mitochondrial superoxide production and apoptosis. These in vitro findings accurately concurred with the in vivo transcriptomics data, thereby confirming the ability of the L6 skeletal muscle model to identify relevant in vivo mechanisms of xenobiotic-induced toxicity. Moreover, these results highlight the sensitivity of the L6 galactose media model to study mitochondrial perturbation associated with skeletal muscle toxicity; this model may be utilised to rank the potency of novel xenobiotics upon further validation. Total RNA obtained from isolated thigh skeletal muscle obtained from Han Wistar rats fed 150ppm sulfonyl isoxazoline compound 177 for 28 days (repeat dose), n=4, compared to n-4 control rats fed standard lab chow.

在新型外源性化合物（xenobiotics）的研发早期阶段，亟需能够灵敏捕捉骨骼肌不良毒性反应的稳健体外模型。为此，本研究采用大鼠骨骼肌体外模型（L6），对源自大鼠体内模型的转录组学（transcriptomics）数据进行转化验证研究。新型磺酰基异恶唑啉类除草剂（sulfonyl isoxazoline herbicides）在大鼠体内模型中被证实与骨骼肌毒性相关。对体内重复给药研究中获取的骨骼肌组织进行基因表达通路分析后，发现了与线粒体功能障碍、氧化应激、能量代谢、蛋白质调控及细胞周期相关的富集通路。研究团队进一步在体外L6模型中探究了线粒体功能障碍与氧化应激相关机制。该模型证实，磺酰基异恶唑啉类化合物可诱导线粒体功能障碍、线粒体超氧化物生成及细胞凋亡（apoptosis）。上述体外实验结果与体内转录组学数据高度吻合，从而验证了L6骨骼肌模型能够识别外源性化合物诱导毒性的相关体内机制。此外，本研究结果凸显了L6半乳糖培养基模型在研究与骨骼肌毒性相关的线粒体扰动方面的敏感性；经进一步验证后，该模型可用于对新型外源性化合物的毒性效力进行分级排序。本研究的总RNA样本来自两部分：一是连续28天喂食150ppm磺酰基异恶唑啉化合物177的Han Wistar大鼠（Han Wistar rats），分离其大腿骨骼肌后提取总RNA，样本量n=4；二是喂食标准实验室饲料的对照组大鼠，样本量同样为n=4，将两组样本进行对比分析。