Phenoloxidase and cytotoxicity in the compound ascidian Botryllus schlosseri

The vacuoles of morula cells (MC) of the colonial ascidian Botryllus schlos seri contain phenoloxidase (PO). As the release of their vacuolar content a t the border of incompatible contacting colonies is associated with the for mation of necrotic masses which characterize the rejection reaction, the ro le of PO in Botryllus cytotoxicity was investigated. When hemocytes are inc ubated with blood plasma from incompatible (heterologous) colonies, MC degr anulate and, after 60 min, the cytotoxicity index becomes significantly gre ater than that observed in controls incubated with autologous plasma. The r ise in cell mortality is completely inhibited by the addition of PO inhibit ors sodium benzoate, tropolone and phenylthiourea, and serine protease inhi bitors phenylmethylsulfonyl fluoride, benzamidine, N-tosyl-L-phenylalanine chloromethyl ketone and N-tosyl-L-lysine chloromethyl ketone. The addition of either reducing agents L-cysteine and ascorbic acid or reactive oxygen s pecies scavenger enzymes superoxide dismutase and catalase has a similar ef fect. Significant inhibition of cytotoxicity is also observed with the quin one scavenger, 3-methyl-2-benzothiazolinone hydrazone. In the presence of s odium benzoate and phenylthiourea, there is a significant reduction in the number, size and color intensity of necrotic masses along the contact borde r of incompatible colonies. A significant increase in superoxide anion prod uction, completely inhibited by sodium benzoate, is observed when hemocytes are incubated with heterologous blood plasma. These results indicate that: (i) PO is the enzyme responsible for the cytotoxicity observed in both hem ocyte cultures and rejection reactions; (ii) PO is present inside MC vacuol es as a proenzyme which is activated, upon release, by humoral proteases; ( iii) cytotoxicity appears to be mainly due to oxidative stress generated by PO during oxidation of polyphenols to quinones without the involvement of other oxidases such as NADPH oxidase and peroxidase. (C) 1998 Elsevier Scie nce Ltd. All rights reserved.