Diclofenac-induced cytotoxicity in cultured carp leukocytes

Abstract

Diclofenac is a drug commonly used in human and veterinary medicine for the treatment of diseases associated with inflammation and pain. Medicinal products enter waste and surface waters on an everyday basis and contaminate the aquatic environment. Fish are therefore permanently exposed to these chemicals dissolved in their aquatic environment. To simulate variable environmental conditions, the aim of our study was to examine adverse effects of diclofenac under different temperatures of cell incubation (18, 21, 24, 27 and 30 °C). Cyto-toxic and -static effects of diclofenac in concentrations of 0.001 mcg/ml, 0.01 microg/ml, 0.1 mcg/ml, 1 mcg/ml, 10 mcg/ml and 100 mcg/ml for the carp (Cyprinuscarpio) cultured leukocytes were quantified using detection of lactate dehydrogenase released from damaged cells. Overall DCF cytotoxicity was relatively low and its impact was pronounced at higher temperature and DCF concentration. Cells growth inhibition is changing more rapidly but it is high mainly at the highest concentration from low temperature. DNA fragmentation was not detected in tested leukocyte cell line. CYP450 increased diclofenac cytotoxicity only at the highest concentration but at incubation temperatures 18 and 27 °C. Leukocyte viability is essential for immune functions and any change can lead to reduction of resistance against pathogens, mainly in cold year seasons, when the immune system is naturally suppressed.

Fig. 1

The dependence of calculated percentage of cell death on growth inhibition level. The correlation between two observed cytotoxic effects (cell death versus inhibition of growth) caused by diclofenac in carp (Cyprinus carpio) leukocyte cultivated in vitro in Experiment 1, after 48 incubation hours at different temperatures (18, 21, 24, 27 and 30 °C). Cells were treated with various diclofenac concentrations (0.001 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 10 μg/ml and 100 μg/ml).

Fig. 2

Diclofenac-induced cytotoxicity in cultured carp (Cyprinus carpio ) leuko-cytes. The percentage of killed carp leukocytes in Experiment 1, determined after 12 (blue line) and 24 (red line) hours of incubation at different temperatures (18, 21, 24, 27 and 30 °C). Cells were treated with various concentrations diclofenac (c1: 0.001 μg/ml, c2: 0.01 μg/ml, c3: 0.1 μg/ml, c4: 1 μg/ml, c5: 10 μg/ml and c6: 100 μg/ml).

Fig. 3

Diclofenac-induced cytostatic effect in cultured carp (Cyprinus carpio ) leukocytes. The percentage provides information about growth inhibition level in carp leukocyte cell culture in Experiment 1, determined after 12 (blue line) and 24 (red line) hours of incubation at different temperatures (18, 21, 24, 27 and 30 °C). Cells were treated with various concentrations diclofenac (c1: 0.001 μg/ml, c2: 0.01 μg/ml, c3: 0.1 μg/ml, c4: 1 μg/ml, c5: 10 μg/ml and c6: 100 μg/ml).

Fig. 4

Contour plots of the levels of overall response desirability for (a) diclofenac-induced cytotoxicity and (b) diclofenac-induced growth inhibition based on desirability profiling with the least squares fit. There is obvious difference in the response of analyzed parameters to the highest diclofenac concentration i.e. cytotoxic effect only at the highest temperatures but cytostatic effect at all temperatures.

Fig. 5

Cytotoxic effect caused by combination of diclofenac and cytochrome P450 in carp (Cyprinus carpio ) leukocyte cultivated in vitro. The percentage of killed carp leukocytes in Experiment 2, determined after 24 hours of incubation at different temperatures (18 and 27 °C). Cells were treated with 100 μg/ml of diclofenac (DCF) in combination with different cytochrome P450 concentrations (CYP low: 0.5 μg/ml or CYP high: 5 μg/ml).