Hydroxylated benzo[c]phenanthrene metabolites cause osteoblast apoptosis and skeletal abnormalities in fish

Nobuo Suzuki; Masato Honda; Masayuki Sato; Shuhei Yoshitake; Kimi Kawabe; Yoshiaki Tabuchi; Toshiki Omote; Toshio Sekiguchi; Yukihiro Furusawa; Akira Toriba; Ning Tang; Yohei Shimasaki; Edward G Nagato; Lulu Zhang; Ajai K Srivastav; Thumronk Amornsakun; Yoichiro Kitani; Hajime Matsubara; Takashi Yazawa; Jun Hirayama; Atsuhiko Hattori; Yuji Oshima; Kazuichi Hayakawa

doi:10.1016/j.ecoenv.2022.113401

Hydroxylated benzo[c]phenanthrene metabolites cause osteoblast apoptosis and skeletal abnormalities in fish

Ecotoxicol Environ Saf. 2022 Apr 1:234:113401. doi: 10.1016/j.ecoenv.2022.113401. Epub 2022 Mar 14.

Authors

Nobuo Suzuki¹, Masato Honda², Masayuki Sato³, Shuhei Yoshitake⁴, Kimi Kawabe⁵, Yoshiaki Tabuchi⁶, Toshiki Omote³, Toshio Sekiguchi³, Yukihiro Furusawa⁷, Akira Toriba⁸, Ning Tang⁹, Yohei Shimasaki⁴, Edward G Nagato¹⁰, Lulu Zhang⁹, Ajai K Srivastav¹¹, Thumronk Amornsakun¹², Yoichiro Kitani³, Hajime Matsubara¹³, Takashi Yazawa¹⁴, Jun Hirayama¹⁵, Atsuhiko Hattori¹⁶, Yuji Oshima⁴, Kazuichi Hayakawa¹⁷

Affiliations

¹ Noto Marine Laboratory, Institute of Nature and Environmental Technology, Kanazawa University, Noto-cho, Ishikawa 927-0553, Japan. Electronic address: nobuos@staff.kanazawa-u.ac.jp.
² Botanical Garden, Institute of Nature and Environmental Technology, Kanazawa University, Kakuma-machi, Ishikawa 920-1192, Japan.
³ Noto Marine Laboratory, Institute of Nature and Environmental Technology, Kanazawa University, Noto-cho, Ishikawa 927-0553, Japan.
⁴ Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University, Fukuoka 819-0395, Japan.
⁵ Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma, Ishikawa 920-1192, Japan.
⁶ Life Science Research Center, University of Toyama, Sugitani, Toyama 930-0194, Japan.
⁷ Department of Pharmaceutical Engineering, Faculty of Engineering, Toyama Prefectural University, Kurokawa, Toyama 939-0398, Japan.
⁸ Graduate School of Biomedical Sciences, Nagasaki University, Bunkyo-machi, Nagasaki 852-8521, Japan.
⁹ Institute of Nature and Environmental Technology, Kanazawa University, Kakuma-machi, Ishikawa 920-1192, Japan.
¹⁰ Graduate School of Faculty of Life and Environmental Sciences, Shimane University, Matsue, Shimane 690-8504, Japan.
¹¹ Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur 273-009, India.
¹² Fisheries Technology Program, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand.
¹³ Noto Center for Fisheries Science and Technology, Kanazawa University, Osaka, Noto-cho, Ishikawa 927-0552, Japan.
¹⁴ Department of Biochemistry, Asahikawa Medical University, Hokkaido 078-8510, Japan.
¹⁵ Department of Clinical Engineering, Faculty of Health Sciences, Komatsu University, Komatsu, Ishikawa 923-0961, Japan.
¹⁶ Department of Biology, College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Ichikawa, Chiba 272-0827, Japan.
¹⁷ Low Level Radioactivity Laboratory, Institute of Nature and Environmental Technology, Kanazawa University, Nomi city, Ishikawa 923-1224, Japan.

PMID: 35298967
DOI: 10.1016/j.ecoenv.2022.113401

Abstract

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 LC₅₀s 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^-10 to 10^-7 M)-treated scales was significantly suppressed, while BcP (10^-11 to 10^-7 M)-treated scales did not affect osteoblastic activity. Osteoclastic activity was unchanged by either BcP or 3-OHBcP treatment at each concentration (10^-11 to 10^-7 M). The detailed toxicity of 3-OHBcP (10^-9 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.

Keywords: Apoptosis; Fish scales; Monohydroxylated polycyclic aromatic hydrocarbons; Osteoblasts; Osteoclasts; Skeletal abnormalities.