Generation of oxyradicals (O₂^• and H₂O₂), mitochondrial activity and induction of apoptosis of PBMC of Cyprinus carpio carpio treated in vivo with halomethanes and with recombinant HSP60 kDa and with LPS of Klebsiella pneumoniae

Halomethanes (HM) can be immunotoxic in mammals; however, in the fish immune system HM effects are unknown. In the current study, we evaluated the mitochondrial activity (MA) by MTT, induction of apoptosis by SubG0 technique and quantified serum ROS concentration (O₂^• and H₂O₂) and ROS production in PBMC of Cyprinus carpio carpio treated i.p. with CH₂Cl₂, CHCl₃ and BrCHCl₂ (0.004-40.0 mg/kg) for 96 h. Positive controls were recombinant heat shock protein of 60 kDa (rHSP60 kDa) of Klebsiella pneumoniae and its LPS. In addition, for in vitro PBMC cultures, two culture media and two sources of sera were tested. Both positive controls increased the MA more than 4-fold as well as the production of O₂^•(26-fold) and H₂O₂ (5-fold) compared to their controls. HM induced different effects on MA, ROS production and an induction of apoptosis, depending on the chlorination patterns and the dose; however, a systemic damage prevails. To fish treated with CH₂Cl₂, the apoptosis was related with serum ROS concentration and with MA. In contrast, in fish dosed with CHCl₃ relationships were not found, deducing a systemic damage. However, in fish treated with BrCHCl₂, serum O₂^• concentration and in vitro ROS generation performed by PBMC were involved in the induction of apoptosis of these cells but not with MA suggesting also immunotoxic effects. The current study demonstrated that HMs are immunomodulators increasing an acute inflammatory response and that rHSP60kDA of K. pneumoniae and its LPS are appropriate antigens to assess the immune response of C. c. carpio.