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化学物質の分子サイズや代謝分解性に着目した生物濃縮性評価手法に関する研究

宮田, 知代子 京都大学 DOI:10.14989/doctor.k23951

2022.03.23

概要

生物濃縮性をもつ化学物質は、環境中の生物に悪影響を及ぼす懸念があるため様々な規制を受け、新規な合成化学品の製造・販売にあたってはその生物濃縮性の評価が求められる。生物濃縮性の試験には魚が用いられ、化合物を水に溶解させる水曝露法、あるいは餌に混ぜて与える餌曝露法により魚体に取り込ませた後、体中の化合物濃度の水中あるいは餌中濃度に対する比を濃縮倍率として算出し、その値を評価する。しかしこの試験は、時間を要する上、多数の実験動物(魚)を必要とするため、開発コストの削減のみならず動物愛護の観点からも改良が求められている。本論文は動物実験を行うことなく化合物の生物濃縮性を正確に予測・評価するための手法開発を検討したもので、内容は以下のとおりである。

第1章序論に続く第2章では、化合物が鰓経由で取り込まれる際の濃縮性と分子サイズの関係が調べられている。従来から、立体的にかさ高い分子は生体膜を透過しにくいと考えられており、欧州では真空中の分子を内包する球の直径Dmax averを評価指標として、その値が17.4 Å以上であれば、生物濃縮の可能性は低い(濃縮倍率<5000)とみなしてよいことになっている。Dmax averは、分子が取り得るすべての安定コンフォメーションのそれぞれについてつくられる内包球の直径(Dmax)の平均値であるが、その算出には特定のソフトウエアが必要で、かつ使用には量子化学等の専門知識が求められるため汎用性に問題がある。そこで一般に利用されている分子モデリング・計算化学ソフトと自作のプログラムを組み合わせ、より簡便にDmax averを算出する方法を考案した。さらに水棲生物における濃縮性を評価するには水中でのDmaxを指標とする方が適切と考え、濃縮倍率が既知の1665 化合物について分子動力学シミュレーションを実施し、水中で取り得る安定コンフォメーションから求められるDmaxと濃縮倍率の関係を解析した。その結果、分子の水中Dmaxの平均値(Dmax aver)が17.1 Åあるいは最小値 (Dmax min)が 15.6 Åを上回る化合物であれば、鰓から取り込まれにくく低濃縮性と判断できることを明らかにし、膜透過性への寄与が大きいと考えられるコンパクトなコンフォメーションの分子構造を反映する後者の方が、より安全に配慮した評価基準であるとした。

第3章では魚の腸管膜経由の濃縮性と化合物の分子サイズの関係を検討している。
水中の化合物は鰓経由のみでなく口からも魚に取り込まれることが考えられる。経口摂取による濃縮ポテンシャルを評価する試験法では、化合物を餌に混ぜて与えることがガイドラインにより定められているが、魚が取り込む餌の量にばらつきが大きく暴露量の制御が困難であることが問題となっている。そこで、魚の腸管を用いたin vitro 反転腸管法によって分子量や分子サイズの異なる公知の9化合物の膜透過性を評価した。コイから採取した腸管を表裏反転させ、内部を緩衝液で満たした状態で、被験化合物の溶液に浸し、一定時間経過後に内部に移行した化合物の濃度を測定した。その結果、分子量 345 以下かつDmax min 13.9 Å 以下の4化合物は腸管内への透過が認められたのに対し、分子量が 548 以上かつDmax min 15.8 Å 以上の5化合物は腸管膜をほとんど透過しなかった。これにより、腸管膜経由の濃縮性も鰓経由と同様に分子サイズで判定できる可能性が示された。

第4章では魚による化合物の代謝速度と濃縮性の関係が調べられている。化学物質の濃縮性には物理化学的性質に加え、生物体内での代謝分解性も大きく影響する。魚による代謝については、現在、分子構造から代謝速度を予測する定量的構造活性相関( QSAR) 式が考案されているが、その予測力は不十分で、代謝されやすく濃縮性は低いと予測した化合物が実際には高い濃縮性を示す例が認められている。そこで現行法の予測力の低さを改善すべくOECD が 2018 年にガイドラインとして示した、魚の肝 S9 分画を用いるin vitro試験の有用性を検討した。生物試験でみられた代謝速度と QSAR による予測値に乖離が認められる一連のアルキルフェノール類を被験化合物として選定し、ニジマスの肝S9に補酵素を加え一定時間インキュベートしたところ、立体的にかさ高い置換基をもつ化合物の分解が他に比べ遅かった。この分解速度の違いは、化合物が代謝を受ける部位付近の立体的な環境の違いによると考えられ、現行 QSAR予測式はかさ高い置換基による立体的な反応阻害効果をうまく考慮できていないことが示された。さらにこのin vitro系での代謝分解が遅い化合物は、生物試験で求められた濃縮倍率も大きく、この試験を取り入れることで従来の予測が改善されると結論している。

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