Cellular responses of Prochilodus lineatus hepatocytes after cylindrospermopsin exposure

Abstract

Cylindrospermopsin is a potent toxicant for eukaryotic cells produced by several cyanobacteria. Recently, primary hepatocyte cultures of Neotropical fish have been established, demonstrating to be a quite efficient in vitro model for cellular toxicology studies. In the current study, a protocol for culture of Prochilodus lineatus hepatocytes was established and utilized to investigate the cellular responses to purified cylindrospermopsin exposure. Hepatocytes were successfully dissociated with dispase, resulting in a cell yield of 6.36 × 10(7)cells g(-1) of liver, viability of 97% and attachment on uncoated culture flasks. For investigation of cylindrospermopsin effects, hepatocytes were dissociated, cultured during 96 h and exposed to three concentrations of the toxin (0.1, 1.0 or 10 μgl(-1)) for 72 h. Cylindrospermopsin exposure significantly decreased cell viability (0.1 and 1 μgl(-1)) and multixenobiotic resistance mechanism, MXR (all exposed groups), but increased reactive oxygen/nitrogen species levels (all exposed groups) and lipid peroxidation (10 μgl(-1)). On the other hand no significant alterations were observed for other biochemical biomarkers as 2GSH/GSSG ratio, protein carbonyl levels and DNA strand breaks or glutathione S-transferase and glucose 6-phosphate dehydrogenase activities. In conclusion, hepatocytes might be made sensitive to cylindrospermopsin, at least in part, due to reduction of xenobiotics and endobiotics efflux capacity by MXR. Additionally, the toxin exposure suggests important issues regarding hepatocytes survival at the lowest cylindrospermopsin concentrations.

Low concentrations of cylindrospermopsin induce increases of reactive oxygen species levels, metabolism and proliferation in human hepatoma cells (HepG2)

“…Differently than expected and reported for other models (Kinnear, 2010;Lankoff et al, 2007;Liebel et al, 2011) HepG2 cells were not deleteriously affected by CYN exposure at concentrations up to 10-fold the safe limit of 1 lg l À1 as these concentrations were not cytotoxic and, surprisingly, promoted metabolism increase and cell proliferation. These controversy data are then discussed in order to highlight that CYN at low concentrations promote a proliferation of human hepatoma cells (HepG2) and CYP-induction slightly increase CYN toxicity.…”

“…However, values lower than 1 lg l À1 has been suggested by Humpage (2005, 2006). In a previous study a decreases of cell viability and multidrug resistance transporters activity on primary cultured fish hepatocytes exposed to CYN (1-10 lg l À1 ) were described (Liebel et al, 2011). Additionally, the same authors showed an increase of reactive oxygen and nitrogen species concentration related with significant lipid peroxidation in the same cells.…”

“…In the latter, GST activity has increased at 10 lg l À1 of CYN and 12 h, but then decreased later at 24 h. These results may be due to the variability of cell number. We have previously reported efflux transporters activity decrease in primary cultured fish hepatocytes, which may express levels of biotransformation enzymes very close to the in vivo levels (Liebel et al, 2011). These cells appear to be more sensitive to CYN than HepG2, since efflux transporters activity was generally not affected by CYN exposure, except for 10 lg l À1 in 12 h-exposed CYPs-induced cells.…”

Experimental immunology Assessment of the potential genotoxic and proapoptotic impact of selected cyanotoxins on fish leukocytes

“…In the present study, a similar increase in DNA fragments in comet tails was observed. On the other hand, Liebel et al [37] did not detect any DNA damage in hepatocytes from Prochilodus lineatus exposed to the toxin at concentrations up to 10 µg/ml for 72 h. In that study, however, increased reactive …”

Evaluation of pollution of Ganga River water using fish as bioindicator

“…Hence, accumulation of metals in the fish tissues affects their physiology. Several studies have reported alteration in level of protein, lipid, carbohydrate, and activities of different enzymes in the fish in response to different xenobiotics (Palanisamy et al 2011;Yousafzai and Shakoori 2011;Yacoub and Gad 2012;Liebel et al 2011;Nogueira et al 2011). Since they act as biomarker of the health of fish, their assessment is very important to evaluate the effect of pollution on the fish health.…”