Cytotoxicity of three graphene-related materials in rainbow trout primary hepatocytes is not associated to cellular internalization

Abstract

As a consequence of increasing production and use of graphene-related materials (GRM), their release into the aquatic environment is likely to be expected. Development of appropriate model systems to assess their potential toxicity toward aquatic organisms is undoubtedly needed. Of particular relevance are primary cultures of fish hepatocytes, since they maintain similar functionalities as those of the original tissue. Isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) were exposed to ranges of concentrations of different forms of GRM, two graphene oxides (GO) of sheet-like structure and one tubular-shaped carbon nanofiber (CNF) in the presence or absence of fetal bovine serum (FBS) for 24 and 72 h. Metabolic activity, cell membrane integrity, lysosomal function, reactive oxygen species (ROS) formation and interaction with cytochrome P450 1 A enzyme were assessed by using AlamarBlue, 5-carboxyfluorescein diacetate-acetoxymethyl ester, neutral red uptake, dichlorofluorescein and 7-ethoxyresorufin-O-deethylase (EROD) assays, respectively. In the presence of FBS, GO affected metabolic activity and cell membrane integrity more than CNF, whilst absence of serum further reduced cell viability in GRM-exposed cells. GRM did not alter lysosomal function nor did it induce ROS formation or EROD activity. Intracellular uptake was observed only in the case of CNF when incubated without FBS. Primary hepatocytes from rainbow trout appear to be a suitable model to screen for cytotoxicity and to reveal any interaction with GRM. Results emphasize the role of serum proteins in the toxicological responses following exposure to GRM with important implications for the environmental risk assessment of these nanomaterials.