In Vitro Assessment of Cytotoxicity and Carcinogenic Potential of Chemicals: Evaluation of the Cytotoxicity Induced by 58 Metal Compounds in the Balb/3T3 Cell Line

Abstract

A new, mechanistically based, in vitro strategy involving Balb/c 3T3 clone A31-1-1 mouse embryo fibroblasts has been proposed for the determination of the carcinogenic potential of inorganic chemicals, in order to establish priority of metal compounds to be tested and, whenever possible, to compare the in vitro results with the corresponding in vivo data. As a first step in this research, this study reports on the cytotoxic effects of 58 metal compounds in the Balb/3T3 cell line. After harmonisation and standardisation of the Balb/3T3 protocol, cells were exposed for 72 hours to a fixed dose (100μM) of 58 individual compounds. The cytotoxicity induced by some metal compounds was found to be related to their chemical form (for example, Cr(NO3)3 and Na2CrO4), suggesting that the Balb/3T3 cell line is a valuable cellular model in relation to this aspect of metal speciation. The results of the systematic study on the metal-induced cytotoxic effects in the Balb/3T3 cell line could be arbitrarily classified into three groups according to the degree of cytotoxicity. Group I includes 26 species that induced no observable effect or only a slight cytotoxic effect; Group II includes 13 metal compounds that exhibited an obvious degree of cytotoxicity; and Group III includes 19 metal species that displayed a strong cytotoxic response. Metal compounds of Groups II and III are considered to be of the highest priority for setting of dose–effect relationships for a subsequent in vitro study on metal-induced concurrent cytotoxicity and morphological transformation in the Balb/3T3 cell line.

Mutagenicity, Carcinogenicity, and Teratogenicity

“…Recently, in-vitro transformation of SHE cells into a neoplastic state was shown to be associated with overexpression of c-myc and c-Ha-ras oncogenes . The Balb3T3 mouse cell line which has also been a classical model for cell transformation has recently been used for the assessment of cytotoxicity of a large series of metal compounds in order to predict their carcinogenic potential (Mazzotti et al 2001(Mazzotti et al , 2002.…”

Chromium(III) and iron(III) inhibits replication of DNA and RNA viruses

“…The concentrations of chromium chloride and iron chloride for these studies were chosen on the basis of other reports (Mazzotti et al 2001, 2002) and our earlier investigations. Our previous experiments have shown that both of them slightly stimulated cell proliferation (Terpiłowska and Siwicki 2009, 2010).…”

“…The aim of this study was to examine the effect of chromium(III) and iron(III) used separately and simultaneous treating of chromium(III) and iron(III) on HSV-1 an BVDV replication. The concentrations of chromium chloride and iron chloride for these studies were chosen on the basis of other reports (Mazzotti et al 2001, 2002) and our earlier investigations (Terpiłowska and Siwicki 2009, 2010, 2012). …”

In Vitro Setting of Dose–effect Relationships of 32 Metal Compounds in the Balb/3T3 Cell Line, as a Basis for Predicting their Carcinogenic Potential

“…The procedures for the maintenance and subculture of Balb/3T3 cells were as previously described (10). Whenever possible, metal salts were dissolved in sterile MilliQ water (sometimes at 30°C), at a concentration of 10 -2 M or 10 -3 M. Before incubation with the cells, aliquots of the concentrated metal solutions (stored at 4°C) were added to complete growth medium to give final metal concentrations in the required range of molarity.…”

“…The standardised experimental conditions used in this assay were as previously described (10). Cells were plated in 60mm cell culture dishes (FALCON; Cadorago, Como) in 4ml of Dulbecco's modified Eagle's medium (DMEM) low-glucose culture medium containing sodium pyruvate and sodium bicarbonate, without L-glutamine (DME/LOW; EuroClone, CELBIO, Pero, Milano, Italy), supplemented with 10% FCIII serum (200 cells/dish, six dishes per experimental point), and allowed to attach for 24 hours.…”