Suzuki Nobuo, Honda Masato, Sato Masayuki, Yoshitake Shuhei, Kawabe Kimi, Tabuchi Yoshiaki, Omote Toshiki, Sekiguchi Toshio, Furusawa Yukihiro, Toriba Akira, Tang Ning, Shimasaki Yohei, Nagato Edward G, Zhang Lulu, Srivastav Ajai K, Amornsakun Thumronk, Kitani Yoichiro, Matsubara Hajime, Yazawa Takashi, Hirayama Jun, Hattori Atsuhiko, Oshima Yuji, Hayakawa Kazuichi
Noto Marine Laboratory, Institute of Nature and Environmental Technology, Kanazawa University, Noto-cho, Ishikawa 927-0553, Japan.
Botanical Garden, Institute of Nature and Environmental Technology, Kanazawa University, Kakuma-machi, Ishikawa 920-1192, Japan.
Ecotoxicol Environ Saf. 2022 Apr 1;234:113401. doi: 10.1016/j.ecoenv.2022.113401. Epub 2022 Mar 14.
To study the toxicity of 3-hydroxybenzo[c]phenanthrene (3-OHBcP), a metabolite of benzo[c]phenanthrene (BcP), first we compared it with its parent compound, BcP, using an in ovo-nanoinjection method in Japanese medaka. Second, we examined the influence of 3-OHBcP on bone metabolism using goldfish. Third, the detailed mechanism of 3-OHBcP on bone metabolism was investigated using zebrafish and goldfish. The LCs of BcP and 3-OHBcP in Japanese medaka were 5.7 nM and 0.003 nM, respectively, indicating that the metabolite was more than 1900 times as toxic as the parent compound. In addition, nanoinjected 3-OHBcP (0.001 nM) induced skeletal abnormalities. Therefore, fish scales with both osteoblasts and osteoclasts on the calcified bone matrix were examined to investigate the mechanisms of 3-OHBcP toxicity on bone metabolism. We found that scale regeneration in the BcP-injected goldfish was significantly inhibited as compared with that in control goldfish. Furthermore, 3-OHBcP was detected in the bile of BcP-injected goldfish, indicating that 3-OHBcP metabolized from BcP inhibited scale regeneration. Subsequently, the toxicity of BcP and 3-OHBcP to osteoblasts was examined using an in vitro assay with regenerating scales. The osteoblastic activity in the 3-OHBcP (10 to 10 M)-treated scales was significantly suppressed, while BcP (10 to 10 M)-treated scales did not affect osteoblastic activity. Osteoclastic activity was unchanged by either BcP or 3-OHBcP treatment at each concentration (10 to 10 M). The detailed toxicity of 3-OHBcP (10 M) in osteoblasts was then examined using gene expression analysis on a global scale with fish scales. Eight genes, including APAF1, CHEK2, and FOS, which are associated with apoptosis, were identified from the upregulated genes. This indicated that 3-OHBcP treatment induced apoptosis in fish scales. In situ detection of cell death by TUNEL methods was supported by gene expression analysis. This study is the first to demonstrate that 3-OHBcP, a metabolite of BcP, has greater toxicity than the parent compound, BcP.
为了研究苯并[c]菲（BcP）的代谢产物3-羟基苯并[c]菲（3-OHBcP）的毒性，首先我们在日本青鳉中采用卵内纳米注射法将其与其母体化合物BcP进行比较。其次，我们利用金鱼研究了3-OHBcP对骨代谢的影响。第三，利用斑马鱼和金鱼研究了3-OHBcP影响骨代谢的详细机制。BcP和3-OHBcP在日本青鳉中的半数致死浓度分别为5.7 nM和0.003 nM，这表明该代谢产物的毒性是母体化合物的1900倍以上。此外，纳米注射的3-OHBcP（0.001 nM）可诱导骨骼异常。因此，我们检查了钙化骨基质上同时具有成骨细胞和破骨细胞的鱼鳞，以研究3-OHBcP对骨代谢的毒性机制。我们发现，与对照金鱼相比，注射BcP的金鱼的鳞片再生受到显著抑制。此外，在注射BcP的金鱼胆汁中检测到了3-OHBcP，这表明从BcP代谢而来的3-OHBcP抑制了鳞片再生。随后，利用再生鳞片的体外试验研究了BcP和3-OHBcP对成骨细胞的毒性。在3-OHBcP（10至10 M）处理的鳞片中，成骨细胞活性受到显著抑制，而BcP（10至10 M）处理的鳞片对成骨细胞活性没有影响。在每个浓度（10至10 M）下，BcP或3-OHBcP处理均未改变破骨细胞活性。然后，利用鱼鳞进行全基因组表达分析，研究了3-OHBcP（10 M）对成骨细胞的详细毒性。从上调基因中鉴定出8个与细胞凋亡相关的基因，包括APAF1、CHEK2和FOS。这表明3-OHBcP处理可诱导鱼鳞细胞凋亡。基因表达分析支持了通过TUNEL方法对细胞死亡进行的原位检测。本研究首次证明，BcP的代谢产物3-OHBcP比母体化合物BcP具有更大的毒性。
