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The effects of 4-nonylphenol (NP) on functional responses of haemocytes from the clam Tapes philippinarum were investigated after 7 d exposure to sublethal NP concentrations (0, 0 + ace- tone, 0.025, 0.05, 0.1 and 0.2 mg l -1 NP). Haemocytes from both controls and exposed clams were col- lected, and the effects of NP on uptake of the vital dye Neutral Red (NR), superoxide dismutase (SOD) and lysozyme activities, total haemocyte count (THC) and volume of circulating cells were evaluated. The capability of NP to induce apoptosis was also investigated. Exposure of clams to 0.2 mg l -1 NP significantly increased (p < 0.05) NR uptake when compared with controls, suggesting that NP caused alterations in cell membrane stability. Significant decreases in both SOD and lysozyme activ- ity were observed from 0.05 mg l -1 NP (p < 0.01 and p < 0.05, respectively) with respect to controls, indicating that NP causes oxidative stress and reduces the immunocompetence of the exposed clams. From 0.05 mg l -1 NP, the apoptotic index (percentage of haemocytes showing positivity to the TUNEL reaction) significantly increased (p < 0.001). Apoptotic haemocytes showed shrinkage and lost their amoeboid shape. Moreover, NP exposure significantly increased THC (p < 0.05 at 0.2 mg l -1 NP) and caused a different distribution in size frequency. On the basis of the threshold concentration values, the most sensitive endpoints for NP were SOD, lysozyme and apoptosis assays. Our results highlight a relationship between NP exposure and changes in the functional responses of haemocytes, sug- gesting that the contaminant induces immunomodulation in T. philippinarum, mainly by altering cell morphology, cell membrane stability and enzymatic activities, and by promoting oxidative stress and apoptotic events.

4-Nonylphenol induces immunomodulation and apoptotic events in the clam Tapes philippinarum