Marchi B, Burlando B, Moore M N, Viarengo A
Dipartimento di Biologia Sperimentale Ambientale e Applicata, Università di Genova, Viale Benedetto XV 5, 16132 Genova, Italy.
Aquat Toxicol. 2004 Feb 10;66(2):197-204. doi: 10.1016/j.aquatox.2003.09.003.
Heavy metals are environmental pollutants able to produce different cellular effects, such as an alteration of Ca2+ homeostasis and lysosomal membrane destabilisation. The latter is one of the most used stress indices in biomonitoring programs. Recently, it has been demonstrated that cytosolic calcium increase can modulate lysosomal membrane destabilisation via activation of Ca(2+)-dependent phospholipase A2 (cPLA2). The aim of this work was to investigate the possible involvement of Ca(2+)-activated PLA2 in lysosomal membrane destabilisation induced by heavy metals in mussel haemolymph cells. We have studied the effects of Hg2+ and Cu2+ on free cytosolic calcium using Fura2/AM-loaded cells and lysosomal membrane destabilisation using neutral red (NR) staining. Hg2+ induced a [Ca2+]i rise from 100 to 780 nM in 30 min, and a lysosome destaining of 70% after 60 min that indicates destabilisation of lysosomal membranes. Both effects were reduced in a Ca(2+)-free medium, suggesting a cause-effect relationship. Exposure to Cu2+ produced the same effects, but with an intensity of about 50% respect to Hg2+. Metal-induced lysosomal destabilisation was also reduced in cells pre-exposed to a specific Ca(2+)-dependent cPLA2 inhibitor (AACOCF3). Conversely, haemocyte pretreatment with a Ca(2+)-independent PLA2 inhibitor (bromoenol-lactone (BEL)) did not prevent the destabilizing effect of heavy metals on lysosomes. Exposure to heavy metals also produced an increase in lysosomal volume of 1.8-2-folds, that was prevented by pre-incubation with AACOCF3 but not with BEL. These data indicate an involvement of cPLA2 in lysosomal membrane destabilisation induced by heavy metals.
重金属是能够产生不同细胞效应的环境污染物，例如改变钙离子稳态和破坏溶酶体膜稳定性。后者是生物监测项目中最常用的应激指标之一。最近，有研究表明胞质钙增加可通过激活钙依赖性磷脂酶A2（cPLA2）来调节溶酶体膜稳定性。本研究的目的是探讨钙激活的磷脂酶A2是否可能参与重金属诱导的贻贝血细胞溶酶体膜稳定性破坏。我们使用负载Fura2/AM的细胞研究了Hg2+和Cu2+对游离胞质钙的影响，并使用中性红（NR）染色研究了溶酶体膜稳定性破坏情况。Hg2+在30分钟内使细胞内钙离子浓度从100 nM升高到780 nM，并在60分钟后导致70%的溶酶体褪色，这表明溶酶体膜稳定性遭到破坏。在无钙培养基中，这两种效应均减弱，提示存在因果关系。暴露于Cu2+也产生了相同的效应，但强度约为Hg2+的50%。预先暴露于特异性钙依赖性cPLA2抑制剂（AACOCF3）的细胞中，金属诱导的溶酶体稳定性破坏也有所降低。相反，用非钙依赖性磷脂酶A2抑制剂（溴代烯醇内酯（BEL））预处理血细胞并不能阻止重金属对溶酶体的破坏作用。暴露于重金属还使溶酶体体积增加了1.8至2倍，预先与AACOCF3孵育可阻止这种增加，但BEL则不能。这些数据表明cPLA2参与了重金属诱导的溶酶体膜稳定性破坏。
