利用多種生物檢測法評估二苯甲酮類防曬物質的生態毒性與潛在效益

Abstract

二苯甲酮類化合物（Benzophenones, BPs）為化學性防曬劑與保養品中常見之原料，其利用過濾或吸收紫外線的方式，達到保護皮膚的效果。近十年來隨著防曬霜與美白產品市場擴大，其使用量也隨之增加，特別在亞洲國家更是明顯。雖然二苯甲酮類化合物在化妝品和個人護理產品中的用量仍處於規定範圍內，但有多篇研究報告顯示，二苯甲酮類化合物中有幾種衍生物，像是BP-3會對哺乳類動物引起過敏反應和非目標生物的毒性反應。然而目前針對二苯甲酮類防曬物質，同時分析其防曬能力與環境生態毒性的研究仍非常有限。因此，本研究的兩個目的如下：一、分析 BP 類防曬物質對青鱂魚的急毒性和次致死毒性；二、利用in vitro蘑菇酪胺酸酶測定法和in vivo斑馬魚評估 BP 防曬物質的酪氨酸酶抑制效力 (tyrosinase inhibition potential)。經由96小時急毒性測驗測得了7種BP化合物的半致死濃度，其中BP-2 (LC50 = 19.16 µM) 毒性最低且BP-8 (LC50 = 1.42 µM) 毒性最高。本研究評估魚苗泳動行為 (locomotor activity) 作為BP防曬物質的次致死毒性的指標。 研究結果顯示在BP-2中暴露24小時後，並無改變魚苗泳動行為。然而暴露48小時後，魚苗的活動時間百分比 (percent time active) 有了顯著降低，而BP-3影響了魚苗的絕對旋轉角度 (absolute turn angle); 最後，BP-8為最高急性毒性的BP防曬物質，影響三個泳動行為參數，分別降低魚苗平均游泳速度 (mean velocity)、最大游泳速度及活動時間百分比。同時，蘑菇酪氨酸酶測定法結果顯示只有BP-2能抑制酪氨酸酶活性 (IC50 = 18.2 ± 3.7 μM)。本研究更使用斑馬魚作為模式生物進一步評估BP-2對黑色素合成的抑制效力，結果顯示BP-2明顯的降低黑色素含量 (melanin content) 和酪氨酸酶活性。相比對照組，BP-2分別使斑馬魚體內的黑色素含量降低20% 及酪氨酸活性降低15%。綜合而論，本研究顯示在7個商業用二苯甲酮中，BP-2的毒性最低且具有酪氨酸酶抑制特性的額外功能。

Benzophenones (BPs) are commonly used in the formulation of chemical sunscreen and other personal care products as UV filters to protect our skin from UV overexposure. In the recent decades, there has been a rise in the markets for sunscreen and skin-whitening products to meet the demand from consumers, especially in the Asian countries. Although BP usage in cosmetics and personal care products is within regulation, some studies have reported allergic reactions of several BPs (e.g. BP-3) in mammals and toxicity to non-target organisms. However, there has been no research studying both the ecotoxicity as well as the benefits of these BP-type UV filters. Hence, the objectives of this study include (1) to analyze the acute and sublethal toxicities of BPs in medaka (Oryzias latipes) larvae and (2) to evaluate potential tyrosinase inhibition of less toxic BPs using in vitro mushroom tyrosinase assay and zebrafish (Danio rerio) as an in vivo model organism. The LC50 value from 96 h acute mortality of 7-day post hatching (dph) medaka larvae showed that BP-2 was the least toxic compound (LC50 = 19.16 µM) and BP-8 (LC50 =1.42 µM) was the most toxic compound. Swimming behavior in medaka larvae was evaluated from 24 h to 7 days as an indicator for sublethal toxicities of BP-type UV filters. The result showed that BP-2 at environmentally-relevant concentrations (5 – 50 nM) did not significantly alter larval locomotion after 24 h of exposure while BP-3 (50 nM) had significantly altered the maximum velocity of larvae. At 48-h exposure, BP-2 and BP-3 at 50 nM significantly altered one parameter each: the percent time active and absolute turn angle of medaka larvae, respectively. In contrast, BP-8 significantly altered 3 parameters of swimming behavior after 48 h. Meanwhile, the result from screening of whitening potential using in vitro mushroom tyrosinase assay showed that only BP-2 was able to inhibit tyrosinase activity, with EC50 value of 18.2 ± 3.7 µM. Further evaluation indicated that BP-2 was able to dose-dependently decrease melanin content and tyrosinase activity of zebrafish embryos after 40-h exposure. BP-2 at 20 µM was able to decrease melanin content and tyrosinase activity of zebrafish significantly by 20% and 15%, respectively, relative to the control group. Overall, among the seven tested BPs, this study showed that BP-2 was the least toxic UV filter at both acute and sublethal concentrations with extra benefit of tyrosinase inhibition property.