Assessment of the genotoxicity of the tyrosine kinase inhibitor imatinib mesylate in cultured fish and human cells.

The selective tyrosine kinase inhibitor imatinib mesylate (IM) is a widely used anticancer drug. Recent studies showing that IM can induce DNA and chromosomal damage in crustaceans and higher plants prompted us to re-examine its potential genotoxicity. IM was not mutagenic in the Ames assay (Salmonella typhimurium). Cytotoxicity and genotoxicity were evaluated in vitro in zebrafish (Danio rerio) liver (ZFL), human hepatoma (HepG2), and human peripheral blood lymphocyte (HPBL) cells. Genotoxicity was determined with the comet assay and with the cytokinesis-block micronucleus cytome assay. ZFL and HPBL cells showed comparable sensitivity to IM cytotoxicity, while HepG2 cells were less sensitive. At non-cytotoxic concentrations, IM induced DNA strand breaks in ZFL and HepG2 cells. An increase in the number of micronuclei was observed in ZFL and HPBL cells. In HPBLs, IM also induced an increase in the number of nucleoplasmic bridges and nuclear buds. Based on the data of the consumption of IM in European countries the predicted environmental concentrations (PEC) were calculated to be in the range between 3.3 and 5.0ng/L, which are several orders of magnitude lower from those that caused adverse effects in fish and human derived cells. However, based on the in vitro studies it is not possible to quantitatively predict the hazard for wildlife and humans, therefore further studies are warranted to explore the underlying molecular mechanisms of induced IM genotoxic effects as well as the studies of the occurrence of IM in the aquatic and occupational environment to establish the relevance of these observations for aquatic organisms and occupationally exposed personnel.

Setting Occupational Exposure Limits for Genotoxic Substances in the Pharmaceutical Industry.

In the pharmaceutical industry, genotoxic drug substances are developed for life-threatening indications such as cancer. Healthy employees handle these substances during research, development, and manufacturing; therefore, safe handling of genotoxic substances is essential. When an adequate preclinical dataset is available, a risk-based decision related to exposure controls for manufacturing is made following a determination of safe health-based limits, such as an occupational exposure limit (OEL). OELs are calculated for substances based on a threshold dose-response once a threshold is identified. In this review, we present examples of genotoxic mechanisms where thresholds can be demonstrated and OELs can be calculated, including a holistic toxicity assessment. We also propose a novel approach for inhalation Threshold of Toxicological Concern (TTC) limit for genotoxic substances in cases where the database is not adequate to determine a threshold.

Genotoxic potential of selected cytostatic drugs in human and zebrafish cells.

Due to their increasing use, the residues of anti-neoplastic drugs have become emerging pollutants in aquatic environments. Most of them directly or indirectly interfere with the cell's genome, which classifies them into a group of particularly dangerous compounds. The aim of the present study was to conduct a comparative in vitro toxicological characterisation of three commonly used cytostatics with different mechanisms of action (5-fluorouracil [5-FU], cisplatin [CDDP] and etoposide [ET]) towards zebrafish liver (ZFL) cell line, human hepatoma (HepG2) cells and human peripheral blood lymphocytes (HPBLs). Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and acridine orange/ethidium bromide staining. All three drugs induced time- and dose-dependent decreases in cell viability. The sensitivity of ZFL and HepG2 cells towards the cytotoxicity of 5-FU was comparable (half maximal inhibitory concentration (IC50) 5.3 to 10.4 µg/mL). ZFL cells were more sensitive towards ET- (IC50 0.4 µg/mL) and HepG2 towards CDDP- (IC50 1.4 µg/mL) induced cytotoxicity. Genotoxicity was determined by comet assay and cytokinesis block micronucleus (CBMN) assay. ZFL cells were the most sensitive, and HPBLs were the least sensitive. In ZFL cells, induction of DNA strand breaks was a more sensitive genotoxicity endpoint than micronuclei (MNi) induction; the lowest effective concentration (LOEC) for DNA strand break induction was 0.001 µg/mL for ET, 0.01 µg/mL for 5-FU and 0.1 µg/mL for CDDP. In HepG2 cells, MNi induction was a more sensitive genotoxicity endpoint. The LOEC values were 0.01 µg/mL for ET, 0.1 µg/mL for 5-FU and 1 µg/mL for CDDP. The higher sensitivity of ZFL cells to cytostatic drugs raises the question of the impact of such compounds in aquatic ecosystem. Since little is known on the effect of such drugs on aquatic organisms, our results demonstrate that ZFL cells provide a relevant and sensitive tool to screen genotoxic potential of environmental pollutant in the frame of hazard assessment.

A cell-based biosensor system HepG2CDKN1A-DsRed for rapid and simple detection of genotoxic agents.

The regulatory requirements for genotoxicity testing rely on a battery of genotoxicity tests, which generally consist of bacterial and mammalian cell assays for detection of gene mutations and chromosomal aberrations. However, for rapid screening, these methods are not appropriate. We have developed a new cell-based biosensor system that provides rapid and simple detection of genotoxic substances. This is based on stable transfection of human hepatoma HepG2 cells with a plasmid that encodes the red fluorescent protein DsRed2 under the control of the CDKN1A promoter (HepG2CDKN1A-DsRed cells). As the major downstream target gene of activated TP53, the tumour-suppressor gene CDKN1A is responsible for cell-cycle arrest following DNA damage, and it has been shown to be specifically up-regulated by genotoxic carcinogens. The assay is optimised for a 96-well microplate format and spectrofluorimetric quantification of induced DsRed expression. The assay was evaluated by testing direct-acting and indirect-acting genotoxic compounds with different mechanisms of action, along with non-genotoxic compounds. Out of 25 compounds that are known to be genotoxic in vitro and in vivo, 21 (84%) are detected as positive at non-cytotoxic doses, whereas of 12 compounds not considered genotoxic, 11 (92%) are negative. These data indicate the high sensitivity and specificity of our biosensor system. Based on its simplicity and sensitivity, this biosensor developed with HepG2CDKN1A-DsRed cells has the potential to become a valuable tool for genotoxicity screening for chemical safety evaluation, as well as for environmental and occupational monitoring of exposure to genotoxic agents and their complex mixtures.

Synthesis of poly(ɛ-caprolactone) nanospheres in the presence of the protective agent poly(glutamic acid) and their cytotoxicity, genotoxicity and ability to induce oxidative stress in HepG2 cells.

Nanospheres of poly(ɛ-caprolactone) (PCL) with sizes smaller than 200 nm were produced by combining the freeze drying method and the physicochemical solvent/non-solvent approach. The influence of various types of cryoprotectants (poly(glutamic acid) (PGA) or sacharose) and their concentrations on the outcome of freeze-dried poly(ɛ-caprolactone) particles was evaluated. The physiochemical properties, structural and morphological characteristics of thereby obtained PCL particles were determined by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The cytotoxicity of the samples was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay). The formation of intracellular reactive oxygen species was measured spectrophotometrically using a fluorescent probe (DCFH-DA assay). In addition, the genotoxic response of PCL particles obtained using PGA as a cryoprotectant was investigated by the Comet assay. This paper focuses on the role of PGA in the synthesis of PCL particles and demonstrates that PGA plays a dual role in the synthesis, i.e. it acts as a stabilizer but also as a cryoprotective agent. The sufficient and optimal concentration of PGA for producing uniform, spherical but also biocompatible PCL nanoparticles is established to be 0.05%.

Multifunctional PLGA particles containing poly(l-glutamic acid)-capped silver nanoparticles and ascorbic acid with simultaneous antioxidative and prolonged antimicrobial activity.

A water-soluble antioxidant (ascorbic acid, vitamin C) was encapsulated together with poly(l-glutamic acid)-capped silver nanoparticles (AgNpPGA) within a poly(lactide-co-glycolide) (PLGA) polymeric matrix and their synergistic effects were studied. The PLGA/AgNpPGA/ascorbic acid particles synthesized by a physicochemical method with solvent/non-solvent systems are spherical, have a mean diameter of 775 nm and a narrow size distribution with a polydispersity index of 0.158. The encapsulation efficiency of AgNpPGA/ascorbic acid within PLGA was determined to be >90%. The entire amount of encapsulated ascorbic acid was released in 68 days, and the entire amount of AgNpPGAs was released in 87 days of degradation. The influence of PLGA/AgNpPGA/ascorbic acid on cell viability, generation of reactive oxygen species (ROS) in HepG2 cells, as well as antimicrobial activity against seven different pathogens was investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated good biocompatibility of these PLGA/AgNpPGA/ascorbic acid particles. We measured the kinetics of ROS formation in HepG2 cells by a DCFH-DA assay, and found that PLGA/AgNpPGA/ascorbic acid caused a significant decrease in DCF fluorescence intensity, which was 2-fold lower than that in control cells after a 5h exposure. This indicates that the PLGA/AgNpPGA/ascorbic acid microspheres either act as scavengers of intracellular ROS and/or reduce their formation. Also, the results of antimicrobial activity of PLGA/AgNpPGA/ascorbic acid obtained by the broth microdilution method showed superior and extended activity of these particles. The samples were characterized using Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, transmission electron microscopy, zeta potential and particle size analysis. This paper presents a new approach to the treatment of infection that at the same time offers a very pronounced antioxidant effect.