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ラッ ト肝 の Steroid 5Β-Reductase 活性 に 対す る酢酸第 二水銀

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ラッ ト肝 の Steroid 5Β-Reductase 活性 に 対す る酢酸第 二水銀 〔614〕 ラ ッ ト肝 の steroid 5β-reductase 活 性 に 対 す る酢 酸 第 二 水 銀 の 影 響 The Effect of Mercuric Acetate on the Activity of Steroid 5β-reductase in the Rat Liver 北海道大学医学部衛生学教室 富 田 勤 ・佐 藤 敏 雄 ・高 桑 栄 松 北海道大学大学院環境科学研究科環境医学教室 斎 藤 和 雄 Tsutomu Tomita, Toshio Sato and Eimatsu Takakuwa Department of Hygiene and Preventive Medicine, Hokkaido University School of Medicine, Sapporo Kazuo Saito Department of Environmental Medicine, Graduate School of Environmental Science, Hokkaido University, Sapporo The increase of urinary δ-aminolevulinic acid in man and animals exposed to high concentration of mercury is studied, but the mechanism of the increase has not been clearly explained. In this paper, the content of 5β type steroid, inducing δ-aminolevulinic acid synthetase, was studied by noticing the activities of steroid 5β-reductase, which is the rate limiting enzyme in metabolism of steroid hormones, 3α- and 3β-hydroxysteroid dehydrogenase. For measurement of these activities, 105,000×g supernatant of rat liver was used. The testosterone and ⊿4-androstene-3, 17-dione 5β-reductase activities decreased on either the fourth or eleventh day after either intraperitoneal or subcutaneous administration of mercuric acetate, respe- ctively, and the adrenosterone 5β-reductase activity increased on the eleventh day after the subcutaneous administration of mercuric acetate. These changes mentioned above, however, were not significant. The progesterone and 17α-hydroxy progesterone 5β-reductase activities decreased on either the fourth or eleventh day after either intraperitioneal or subcutaneous administration of mercuric acetate, but there were no significant changes. The ⊿4-pregnen-20β-ol-3-one 5β-reductase activity decreased sig- nificantly on the fourth day after the intraperitoneal administration of mercuric acetate. The 3α-hydroxysteroid dehydrogenase activity on etiocholanolone decreased significantly on the fourth day after the intraperitoneal administration of mercuric acetate, but that on androsterone and epipregnanolone did not show any significant change after administration of mercuric acetate. No significant change was observed in the 3β-hydrogysteroid dehydrogenase activity on epiandrosterone with the administration of mercuric acetate. From these results, it was said that in mercury poisoning the increase of 5β type steroids was not verified and δ-aminolevulinic acid synthetase activity in heme synthesis was little affected by 5β type steroids. る ヘ ム 合 成 の 障 害 に つ い て 多 く の 報 告 が 見 ら れ る。 鉛 中 I. 序 毒 に よ り δ-aminolevulinic acid (ALAと 略 す) お よ び わが国においては, 目ざましい工業の発達により, 重 coproporphyrin が 増 加 し1～3), ま た, 血 中 の δ-amino- 金属に起因する中毒問題が注目されており, 特に鉛によ levulinic acid dehydrase (ALA-Dと 略 す) の 活 性 低 富 田 ・佐 藤 ・斎 藤 ・高 桑: ラ ッ ト肝 の steroid 5β-reductase 活 性 に 対 す る酢 酸 第 二 水 銀 の 影 響 〔615〕 下4), heme synthetase の 活 性上 昇5)が 認 め られ て い る。 た。 一方, 水 銀 に よ る ヘ ム合 成 障害 に 関す る報 告 で は, 鈴 木6), 反 応 条 件 は Tomkins ら13)の 方 法 に 準 じ, 0.2M tris- 土 屋 ら7)は coproporphyrin 量 の 増加 を認 め て い る。 ま HCl buffer, pH7.2, 5.5mlに 後 述 す る 基 質 (8.4×10-7 た, 低 濃度 水 銀 曝 露 で は Goldwater ら8)はALA量 が 変 moles) の エ タ ノ ー ル 溶 液0.1mlを 加 え, さ ら に 還 元 型 動 を 示 さな い と して い るが, 村居 ら9)は ラ ッ トの腹 腔 内 nicotinamide adenine dinucleotide phosphate (NAD- 水 銀 投 与 に よ り, 鉛 投 与 の場 合 程 著 明 とは 言 え ない が, PH), 3.3×10-6moles を 加 え, こ れ を2等 分 し, 各 々 約4倍 のALA量 増 加 を認 めて い る。 す な わ ち, 高 濃 度 に 酵 素 溶 液0.5mlを 加 え た。 こ の 一 方 を 対 照 溶 液 と し 水 銀 曝 露 で はALA量 に 変 動 を引 き起 こす こ とが認 め ら て 直 ち に dichloromethane (CH2Cl2) 4.0mlを 加 え 充 分 れ て い る。 この 水 銀 投与 に よ るALA量 の 増 加 の 原 因を 攪 拌 し, 他 方 を37℃に て20分 間 振 盪 後, CH2Cl2, 4.0ml ALA-Dの 活 性 低下 に求 め る こ とは可 能 で あ るが, 実 際 を 加 え 充 分 に 攪 拌 して 反 応 を 終 結 させ た。 こ の 溶 液 に 精 の in vivo で はALA-D活 性 が 鉛 の場 合 に 見 られ る程 顕 製 水4.0mlお よ びCH2Cl2, 4.0mlを 加 え て 充 分 抽 出 操 著 な低下 を示 して い な い10)。故 に, ALA量 の 増加 は δ- 作 を 行 い, 3,000rpmで 上 層 (水 層) と下 層 (CH2Cl2 aminolevulinic acid synthetase (ALA-Sと 略 す) 活 性 層) に 遠 心 分 離 し た。 こ の 水 層 を さ ら にCH2Cl2で 抽 出 の変 動 の 面か らも 考 察 を加 え る 必 要 が あ る と 考 え ら れ し, CH2Cl2層 と 先 の 下 層 と を 合 わ せ, 10mlのCH2Cl2 る。ALA-S活 性 が 上 昇 す るた め に は, ヘ ム生 成 量 の低 溶 液 と し た。 こ の 溶 液 を 分 光 光 度 計230～250mμの 波 下 に よ って フ ィー ドバ ックが 働 く場 合, お よびALA-S 長 に お け る 各 々 の 基 質 の 最 大 吸 光 度 で 測 定 し, 対 照 溶 液 を誘 導 生 成 す る と され て い る5β型 ス テ ロイ ドが過 剰 に と反 応 溶 液 と の 吸 光 度 の 差 を そ の 酵 素 の 活 性 と した｡活 増 加 す る場 合 が 考 え られ る11)(Fig. 1)。 性 値 は す べ て 蛋 白 量mgに 対 して 反 応 し た ス テ ロ イ ド量 著 者 ら12)は, 既 に 鉛 中毒 に お い て5β型 ス テ ロイ ドが で 示 し た。 ALA量 の 増加 に影 響 を 与 え る 一 因 子 で あ る 可 能 性 に つ 基 質 に は, androgen 関 連 化 合 物 と して testosterone い て 報 告 したが, 水 銀 中毒 に よ るヘ ム合 成 障 害 に 関 す る (Ia), ⊿4-androstene-3, 17-dione (IＩa), adrenosterone 研 究 で は, そ の機 構 解 明 に5β型 ス テ ロイ ドの変 動 の 面 (IIIa) を 用 い, こ の 基 質 のCH2Cl2溶 液 の 測 定 波 長 は そ か ら検 討 した報 告 は見 られ な い。 れ ぞ れ240mμ, 238mμ, 237mμと した｡ま た, pre- 本 研 究 は, 5β型 ス テ ロイ ド生 成 に及 ぼ す 水 銀 の 影 響 gnane 関 連 化 合 物 と し て progesterone (IVa), 17α-hy- を 見 るた め, そ の rate limiting enzyme で あ る生体 内の droxy progesterone (Va), ⊿4-pregnen-20β-ol-3-one 各 種 steroid 5β-reductase 活 性 を 測 定 し, 同時 に5β型 ス (VIa) を 用 い, こ れ ら の 測 定 波 長 は そ れ ぞ れ238mμ, テ ロイ ドの 代 謝 を も 検 討 す る こと に よ り, 5β型 ス テ ロ 242mμ, 242mμと した。 イ ド生 成 量 を推 測 し, ヘ ム合 成 系 へ の 関与 に つ い て 追求 3. 3α-hydroxysteroid dehydrogenase 活 性 の 測 定 しよ う と した。 3α-hydroxysteroid dehydrogenase 活 性 の 測 定 は Ko- ide14)の 方 法 に 準 じた。 II. 実 験 方 法 (1) etiocholanolone (IIc) を 基 質 に 用 い た 場 合 に は, 1. 実 験 動 物 お よ び水 銀 投与 方 法 nicotinamide adenine dinucleotide (NAD), 1.0×10-6 実 験 動 物 と して は ウ イス ター系 雄 性 ラ ッ ト (体重200～ moles を 含 む0.1M phosphate buffer (K salt), pH8.0, 300g) 28匹 を 用 い, 水 銀 投 与 群 は酢 酸 第二 水 銀 溶 液 (1 2.0mlに 精 製 水0.7mlを 加 え, さ ら に 酵 素 溶 液0.02ml ml/体 重100g) を 腹腔 内 に24時 間 毎 に3回 また は背 部皮 を 加 え て15分 間 プ レ イ ン キ ュ ベ ー シ ョ ン す る。 そ の 後, 下 に11日 間 に8回 投 与 した2群 で あ る。対 照 群 に は 精 製 基 質 (2.0×10-7moles) の メ タ ノ ー ル 溶 液0.01mlを 加 水 (1ml/体 重100g) を 投与 した。 酢 酸 第 二水 銀 溶 液 は え, 340mμの 波 長 で 反 応 速 度 を 測 定 す る こ と に よ り 求 酢 酸 第 二 水 銀 (Hg(OAc)2) を0.65μmole/mlの 割 合 で め た。 精 製 水 に 溶 か して 調 製 した。 動 物 は それ ぞれ4日 お よび (2) epipregnanolone (IVc) を 基 質 に 用 い た 場 合 に は 11日 後 に と殺 し, 取 り出 した 肝 臓 を4℃の0.25M庶 糖 溶 NAD (1.0×10-6moles) を 含 む0.1M phosphate buff- 液 で洗 浄 した。 er (K salt), pH8.0, 2.0mlに 精 製 水0.7mlを 加 え, 2.
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