红霉素、环丙沙星和磺胺甲噁唑对羊角月牙藻的毒性效应及其作用机理
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摘要
抗生素的广泛使用和不当处置所引起的环境效应已经引起了国际环境科学界乃至公众的广泛关注。为了研究抗生素对水生态系统中初级生产者—藻类的毒性效应及其作用机理并准确评估其生态风险,本研究选择乳糖酸红霉素、盐酸环丙沙星和磺胺甲嗯唑三种广泛使用的抗生素作为研究对象,以羊角月牙藻模式生物作为代表,通过其生长速率、叶绿素合成、光合系统、次生代谢、抗氧化系统以及细胞色素P450系统等生理、生化方面的不同参数,综合评价三种抗生素对羊角月牙藻生长及生理代谢的影响。结果表明：(1)红霉素对羊角月牙藻的毒性最大,环丙沙星次之,磺胺甲嗯唑最小,三者96 h IC50分别为0.20、3.07、4.40 mg L-1,安全浓度分别为0.019、0.297、0.312 mg L-1。(2)三种抗生素均可显著导致羊角月牙藻叶绿素含量的下降,红霉素在处理浓度为0.06 mg L-1时即可引发叶绿素含量的显著下降,而环丙沙星与磺胺甲(?)唑则在处理浓度高于1.5 mg L-1才会产生同样的抑制效应；另一方面,只有红霉素可以显著抑制叶绿素生物合成过程,并对叶绿素生物合成第一步反应(6-氨基乙酰丙酸合成)产生直接的抑制效应。(3)三种抗生素均可显著抑制羊角月牙藻原初光化学反应、电子传递、光合磷酸化以及碳同化等光合生理过程,红霉素在处理浓度为0.06 mg L-1时即可对光合系统产生明显的急性毒性效应,而环丙沙星与磺胺甲嗯唑则在处理浓度高于1.0 mg L-1时产生同样的抑制效应。(4)三种抗生素均对羊角月牙藻次生代谢产生强烈影响。红霉素可造成苯丙氨酸解氨酶活性下降,并使各类次生代谢产物含量降低,其中对类黄酮的影响最大；而环丙沙星和磺胺甲嗯唑则会使苯丙氨酸解氨酶活性以及各类代谢产物含量上升,其中环丙沙星对于缩酚酸、黄酮及黄烷醇的影响要强于磺胺甲(?)唑,而花青素则对磺胺甲(?)唑更为敏感。(5)三种抗生素均可对羊角月牙藻抗氧化系统产生显著影响,红霉素在处理浓度为0.06 mg L-1时即可引发强烈的氧化胁迫,并显著抑制抗坏血酸与谷胱甘肽的生物合成,进而导致抗氧化系统整体功能下降,而环丙沙星与磺胺甲嗯唑则分别在处理浓度高于1.0和1.5 mg L-1时才会引起明显的氧化胁迫。对于环丙沙星暴露,羊角月牙藻主要通过抗坏血酸-谷胱甘肽循环以及其它抗氧化酶,包括过氧化氢酶和超氧化物歧化酶等活性的上升来抵抗氧化损伤,而对于磺胺甲(?)唑暴露,则可能主要通过叶黄素循环以及谷胱甘肽-S-转移酶活性的上升来抵御氧化损伤。(6)三种抗生素对羊角月牙藻细胞色素P450系统的影响差异较大,红霉素在处理浓度高于0.12 mg L-1时对细胞色素P450系统产生强烈的抑制效应；CPF仅对细胞色素P450系统中与外源物质代谢相关的酶活性有较强的抑制效应,对内源物质代谢酶以及总细胞色素P450含量影响不大；SMZ对细胞色素P450系统总体表现为促进效应,包括总细胞色素P450含量以及相关酶活性的上升等。综上所述,与其它两种抗生素相比,红霉素对羊角月牙藻表现出较强的毒性,表明红霉素在水生态系统中具有更大的生态风险,此外,藻类一些生理指标如叶绿素荧光、核酮糖-1,5-二磷酸羧化酶、苯丙氨酸解氨酶、反式肉桂酸-4-羟化酶等,对红霉素暴露表现出较高的特异性,这些指标均可作为大环内酯类抗生素污染的潜在生物标志物。
The ecological effects of antibiotics have attracted increasing attentions from both public and environmental fields due to its widespread usage and the improper disposal. In order to investigate the toxic mechanism and evaluate the ecological risk of antibiotics to photosynthetic organisms, three widely used antibiotics, erythromycin, ciprofloxacin and sulfamethoxazole were chosen to be target compounds and Selenastrum capricornutum, a model algae, was used as tested object to evaluate the effects of those antibiotics on growth and physiological metabolism of algae through the following aspects:growth rate, chlorophyll biosynthesis, photosynthetic apparatus, secondary metabolism, antioxidation system and cytochrom P450 system. The results indicated:(1) The toxicity of erythromycin was higher than ciprofloxacin and sulfamethoxazole, their 96 h IC50 were 0.20,3.07 and 4.40 mg L-1, respectively; and the safe concentration were 0.019,0.297 and 0.312 mg L-1, respectively. (2) Three types of antibiotics could significantly decrease chlorophyll content. Erythromycin could induce decreasing of chlorophyll content at the concentration of 0.06 mg L-1, while ciprofloxacin and sulfamethoxazole achieved the same results at higher than 1.5 mg L-1. Otherwise, only erythromycin could significantly inhibit the chlorophyll biosynthesis at the first step (synthesis ofδ-aminolevulinic acid). (3) Three antibiotics could significantly inhibit the physiological metabolism process including primary photochemistry, electron transport, photophosphorylation and carbon assimilation. Erythromycin could bring about acute toxic effects at the concentration of 0.06 mg L-1, while the same results were exhibited for ciprofloxacin and sulfamethoxazole at higer than 1.0 mg L-1. (4) The secondary metabolism of S. capricornutum could be affected strikingly by three antibiotics. The phenylalanine ammonialyase activity and the content of each secondary metabolic product were decreased after erythromycin treatment, but increased after ciprofloxacin and sulfamethoxazole treatments. The flavonoids content was decreased significantly after erythromycin treatment. The phenylpropanoids, flavones and flavanols were more sensitive to ciprofloxacin than sulfamethoxazole, however, the anthocyanins was more sensitive to sulfamethoxazole than ciprofloxacin. (5) All three types of antibiotics could significantly affect the antioxidant system in S. capricornutum. Erythromycin could significantly induce oxidative stress and lead to the increase of the lipid peroxidation at the concentration of 0.06 mg L-1, while ciprofloxacin and sulfamethoxazole induced significant oxidative stress at concentrations higher than 1.0 and 1.5 mg L-1, respectively. Erythromycin was the most toxic to S. capricornutum mainly due to its inhibited effects on ascorbic acid and glutathione biosynthesis. In comparison, S. carpricornutum was more tolerant to ciprofloxacin and sulfamethoxazole. The tolerant of S. carpricornutum to ciprofloxacin were probably due to the induction of ascorbate-glutathione cycle and some other antioxidants, such as catalase and superoxide dismutase and sulfamethoxazole, while the tolerant of S. carpricornutum to sulfamethoxazole might be attributed to the induction of xanthophylls cycle and glutathione-S-transferase activity. (6) The effects of three antibiotics on cytochrome P450 system exhibited great diversity. Erythromycin could induce great inhibited effects on cytochrome P450 system while its treatment concentration was higher than 0.12 mg L-1; ciprofloxacin could inhibit the activities of enzymes related to the metabolism of xenobiotics, but no significant effects on the enzymes related to endogenuous substances metabolism and total cytochrome P450 contents; Sulfamethoxazole could induce activation effects on cytochrome P450 system including total cytochrome P450 contents and related enzymes activities. Overall, erythromycin was considerably more toxic than ciprofloxacin and sulfamethoxazole to tested algae and may pose a higher potential risks to the aquatic ecosystem. Some indices like chlorophyll fluorescence, activities of ribulose-1,5-bisphosphatecarboxylase, phenylalanin ammonialyase and cinnamic acid 4- hydroxylase showed a high sensitivity to the exposure of erythromycin, and may be potentially used as candidate biomarkers for macrolide antibiotics pollutions.
The ecological effects of antibiotics have attracted increasing attentions from both public and environmental fields due to its widespread usage and the improper disposal. In order to investigate the toxic mechanism and evaluate the ecological risk of antibiotics to photosynthetic organisms, three widely used antibiotics, erythromycin, ciprofloxacin and sulfamethoxazole were chosen to be target compounds and Selenastrum capricornutum, a model algae, was used as tested object to evaluate the effects of those antibiotics on growth and physiological metabolism of algae through the following aspects:growth rate, chlorophyll biosynthesis, photosynthetic apparatus, secondary metabolism, antioxidation system and cytochrom P450 system. The results indicated:(1) The toxicity of erythromycin was higher than ciprofloxacin and sulfamethoxazole, their 96 h IC50 were 0.20,3.07 and 4.40 mg L-1, respectively; and the safe concentration were 0.019,0.297 and 0.312 mg L-1, respectively. (2) Three types of antibiotics could significantly decrease chlorophyll content. Erythromycin could induce decreasing of chlorophyll content at the concentration of 0.06 mg L-1, while ciprofloxacin and sulfamethoxazole achieved the same results at higher than 1.5 mg L-1. Otherwise, only erythromycin could significantly inhibit the chlorophyll biosynthesis at the first step (synthesis ofδ-aminolevulinic acid). (3) Three antibiotics could significantly inhibit the physiological metabolism process including primary photochemistry, electron transport, photophosphorylation and carbon assimilation. Erythromycin could bring about acute toxic effects at the concentration of 0.06 mg L-1, while the same results were exhibited for ciprofloxacin and sulfamethoxazole at higer than 1.0 mg L-1. (4) The secondary metabolism of S. capricornutum could be affected strikingly by three antibiotics. The phenylalanine ammonialyase activity and the content of each secondary metabolic product were decreased after erythromycin treatment, but increased after ciprofloxacin and sulfamethoxazole treatments. The flavonoids content was decreased significantly after erythromycin treatment. The phenylpropanoids, flavones and flavanols were more sensitive to ciprofloxacin than sulfamethoxazole, however, the anthocyanins was more sensitive to sulfamethoxazole than ciprofloxacin. (5) All three types of antibiotics could significantly affect the antioxidant system in S. capricornutum. Erythromycin could significantly induce oxidative stress and lead to the increase of the lipid peroxidation at the concentration of 0.06 mg L-1, while ciprofloxacin and sulfamethoxazole induced significant oxidative stress at concentrations higher than 1.0 and 1.5 mg L-1, respectively. Erythromycin was the most toxic to S. capricornutum mainly due to its inhibited effects on ascorbic acid and glutathione biosynthesis. In comparison, S. carpricornutum was more tolerant to ciprofloxacin and sulfamethoxazole. The tolerant of S. carpricornutum to ciprofloxacin were probably due to the induction of ascorbate-glutathione cycle and some other antioxidants, such as catalase and superoxide dismutase and sulfamethoxazole, while the tolerant of S. carpricornutum to sulfamethoxazole might be attributed to the induction of xanthophylls cycle and glutathione-S-transferase activity. (6) The effects of three antibiotics on cytochrome P450 system exhibited great diversity. Erythromycin could induce great inhibited effects on cytochrome P450 system while its treatment concentration was higher than 0.12 mg L-1; ciprofloxacin could inhibit the activities of enzymes related to the metabolism of xenobiotics, but no significant effects on the enzymes related to endogenuous substances metabolism and total cytochrome P450 contents; Sulfamethoxazole could induce activation effects on cytochrome P450 system including total cytochrome P450 contents and related enzymes activities. Overall, erythromycin was considerably more toxic than ciprofloxacin and sulfamethoxazole to tested algae and may pose a higher potential risks to the aquatic ecosystem. Some indices like chlorophyll fluorescence, activities of ribulose-1,5-bisphosphatecarboxylase, phenylalanin ammonialyase and cinnamic acid 4- hydroxylase showed a high sensitivity to the exposure of erythromycin, and may be potentially used as candidate biomarkers for macrolide antibiotics pollutions.
引文
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