In vitro effect of cadmium and copper on separated blood leukocytes of Dicentrarchus labrax

Abstract

a b s t r a c tThe immunotoxic effects of heavy metals on blood leukocytes of sea bass (Dicentrarchus labrax) were examined. The cells, separated by a discontinuous Percoll-gradients, were exposed in vitro to various sublethal concentrations of cadmium and copper (10 À 7 M, 10 À 5 M, and 10 À 3 M) and their immunotoxic effect was then evaluated by measuring neutral red uptake, MTT assay, DNA fragmentation and Hsp70 gene expression. First of all, we demonstrated that the cells treated in vitro could incorporate Cd and Cu. A relationship between heavy metal exposure and dose-time-dependent alterations in responses of leukocytes from blood was found for both metals, but copper was more immunotoxic than cadmium in all assays performed. A significant reduction in the cells' ability to uptake neutral red and viability by MTT assay was recorded, indicating that both cadmium and copper could change the membrane permeability, inducing cellular apoptosis when the concentration of metals reached 10 À 3 M. The apoptotic effect may also explain the high level of cytotoxicity found when the leukocytes were exposed to higher concentration of metals. These results demonstrated that toxic effect of copper and cadmium affect on the mechanisms of cellmediated immunity reducing the immune defences of the organism.

Short-term cadmium exposure induces stress responses in frog (Pelophylax bergeri) skin organ culture

“…The time-course experiments performed in skin explants treated with 500 mMCd during the 24-h period, revealed that P. bergeri Hsp70 mRNA levels increased in a time-dependent manner. Transcriptional activation of Hsp70 gene observed upon Cd exposure is a common phenomenon that has been demonstrated in many organisms and suggests a protective and adaptive response of the cell (Vazzana et al, 2014;Kim et al, 2014;. With regard to the constitutive member Hsc70, the results showed that Cd did not significantly affect P. bergeri Hsc70 transcript expression, in agreement with previous studies on other organisms (Deane and Woo, 2006;Planelló et al, 2010).…”

Toxicological in vitro effects of heavy metals on gilthead seabream (Sparus aurata L.) head–kidney leucocytes

“…We have chosen a very short time for leucocyte exposure to heavy metals (30 min and 2 h) since it is known that fish primary cultures of leucocytes show their viability rapidly reduced, mainly in the case of lymphocytes, which in turn are more affected than phagocytes by heavy metals (Witeska and Wakulska, 2007). Thus, previous assays done in fish leucocytes exposed to heavy metals ranged from 10 min to 6 days of exposure (Bennani et al, 1996;Ghanmi et al, 1989;MacDougal et al, 1996;Sarmento et al, 2004;Steinhagen et al, 2004;Teles et al, 2011;Vazzana et al, 2014;Witeska and Wakulska, 2007). This short time was already enough to show some immunotoxicological signs in fish leucocytes and in our Fig.…”

“…Then, 20 μl/ well of water (controls) or heavy metal solutions, to make final concentrations of 0.05-5 mM of Cd, 0.005-0.1 mM Hg forms or 2-10 mM As, were added and exposed for short time, 30 min or 2 h, at 25°C. Selected concentrations were higher and lower than the effective concentration producing the 50% cell death (EC 50 ) obtained after seabream HKL exposure to the same heavy metals for 24 h whilst exposure times were previously tested in fish (Bennani et al, 1996;Vazzana et al, 2014;Witeska and Wakulska, 2007). In all the assays, we used HKLs from 6 independent specimens (not pooled HKLs) and assayed separately (tested in different days) except in the gene expression studies that we used 4 independent fish specimens.…”

“…Thus, common carp macrophages exposed to Cu upregulated the expression of immune-relevant genes [interleukin-1β (il1b), il6, tumour necrosis factor-α (tnfa), serum amyloid A (saa) and trout C-polysaccharide binding protein (tcpbp)] (Teles et al, 2011). In European sea bass PBLs, the transcription of heat-shock protein 70 (hsp70) was also up-regulated by Cd, but mainly by Cu (Vazzana et al, 2014). Therefore, more studies are needed to clarify the immunotoxicological effects and mechanisms of heavy metals in fish leucocytes.…”

Metals content in otoliths of Dicentrarchus labrax from two fish farms of Sicily

“…The European sea bass (D. labrax) is a demersal specie with a high commercial value, and due to some of its ecological features (e.g., large distribution, eurythermal and euryahaline specie), it is considered a good model organism for environmental monitoring study (Kerambrun et al 2012). Actually, many researchers have performed on D. labrax for ecotoxicological investigations by monitoring its physiological status under different rearing systems (Maricchiolo et al 2011;Kalantzi et al 2013), exposure to heavy metals (Vazzana et al 2009(Vazzana et al , 2014, or stress conditions (Celi et al 2012). The aim of this study was to evaluate the impact of different environmental systems on otoliths elemental signature of D. Labrax and thus investigate the use of this biogenic carbonate as a valuable indicator of environmental condition.…”