Impact of solvents on the in vitro genotoxicity of TMPTA in human HepG2 cells

Highlights

• TMPTA induces γH2AX and Caspase-3 in HepG2 cells.

• PEG-400 does not prevent in vitro genotoxicity effect of TMPTA.

• The genotoxic effect of TMPTA is more pronounced when it is diluted in PEG400 than in DMSO.

Abstract

Small hydrophobic chemical compounds require solvents to produce suitable solutions for toxicological studies. However, some solvents can modify the biological properties of substances and therefore their toxicity. This specific issue has been raised for PEG-400 as an anti-inflammatory and anti-oxidative compound. Recently, in the context of the REACH Regulation, PEG-400 was used to test the in vivo genotoxicity of trimethylolpropane triacrylate (TMPTA) in the comet assay. TMPTA failed to increase DNA damage whereas it induces genotoxicity in vitro in DMSO. Therefore, we questioned whether PEG-400 could modify the genotoxicity of TMPTA. The aim of this study was to determine the potential impact of PEG-400 on the in vitro genotoxicity of TMPTA, compared to DMSO. TMPTA was dissolved in either PEG-400 or DMSO, and the induction of γH2AX and Caspase-3 was analyzed in HepG2 cells. TMPTA induced γH2AX and Caspase-3 with both PEG-400 and DMSO. However, TMPTA induced effects at 4-fold lower concentrations when PEG-400 is used as the solvent compared to DMSO. While genotoxic effects are observed at much lower concentrations with PEG-400, it does not modify the in vitro genotoxicity of TMPTA. However, further in vitro studies with small hydrophobic compounds should be done to clarify the effect of PEG-400. Moreover, in vivo studies should be performed to confirm that PEG-400 remains suitable for in vivo genotoxicity tests.

Introduction

Many chemicals, pesticides, drugs, environmental contaminants are insoluble in water and require the use of a solvent for in vitro and in vivo toxicology studies. Among them, dimethylsulfoxyde, DMSO, is the solvent the most commonly used, which solubilizes small hydrophobic molecules at high concentrations. Other hydrophobic solvents such as ethanol or acetone are also frequently used depending on the nature of chemicals and their condition of administration (Buggins et al., 2007). Polyethylene glycols (PEGs) are widely used in pharmaceutical and cosmetic formulations because they are not toxic, very well tolerated and easy to use (Fruijtier-Pölloth, 2005; Gullapalli and Mazzitelli, 2015). Although not commonly used as a solvent to assess the toxicity of chemicals, the use of PEG-400 as a solvent has been recently reported in several toxicity studies according to the registration dossier of TMPTA (ECHA, 2018).

However, it is important to ensure that the solvent chosen, in particular for in vitro and in vivo toxicity studies, does not interfere with the biological activity of the substance studied and also does not protect from its intrinsic toxicity. For example, the cytotoxicity of platinum salts differs in function of solvent used. Indeed, the in vitro cytotoxicity of cis‑platinum is drastically reduced when it is dissolved in DMSO compared to other solvents such as PEG-400 in different human cell lines (Hall et al., 2014; Marzano et al., 2009). Indeed, DMSO reacted with the platinum complex, and ligand displacement and changes in the structure of the complex inhibited its cytotoxicity and its ability to initiate cell death (Hall et al., 2014).

Similarly, in the context of in vivo studies, it is possible that the solvent not only modifies the chemical substance itself (e.g; ionization, structure, etc.) but also modifies the toxicokinetics of the substance and therefore its toxicity. The solvent can also have protective antioxidant and anti-inflammatory properties and interfere with the biological response induced by the substance. For example, polyethylene glycol 400 (PEG-400) is generally listed as an inactive ingredient in drug formulation but it can influence the toxicokinetics of the substances studied by affecting its gastrointestinal absorption (Ma et al., 2017) or the renal elimination of drugs (Hodoshima et al., 2004). In addition, PEG of low molecular weight has been associated with antioxidant and anti-inflammatory potential (Ackland et al., 2010; Ferrero-Andrés et al., 2020; Juarez-Moreno et al., 2015).

The solvents must not interfere with the biological activity of the compounds in toxicology studies, which the aim is to adequately identify and characterize the effects of chemical substances for human health. Recently, in the context of the REACH Regulation (CEC, 2006), trimethylolpropane triacrylate (TMPTA, CAS 15625–89-5) was tested in different solvents, including two studies performed with PEG-400. Whereas maternal mortality was observed in a prenatal toxicity study at a dose of 500 mg/kg/day of TMPTA diluted in corn oil, no mortality was reported in pregnant rats exposed up to 1000 mg/kg/d TMPTA in PEG-400 for 14 days. TMPTA was also prepared in PEG-400 before being injected intravenously in mice, in the in vivo Comet genotoxicity assay performed in liver and bone marrow. No statistical effect was noted in the liver. The statistically significant increase of DNA migration observed in the bone marrow was not dose-related. Moreover, in the negative (solvent) controls, the mean tail intensity in the bone marrow was lower with PEG 400 (0.18) than that reported with the historical controls with carboxymethylcelluloseas solvent (0.24–0.72). Based on the results, the authors considered the substance as devoid of genotoxicity potential in vivo, whereas in vitro studies indicated that TMPTA induced genotoxicity in lymphoma cells lines and primary human lymphocytes (ANSES, 2019; Kirkland and Fowler, 2018). DMSO was used as a solvent in the in vitro studies. However, it cannot be excluded that PEG-400 could mask the genotoxicity of TMPTA in vivo, in particular due to its antioxidant and anti-inflammatory potential (Ackland et al., 2010; Juarez-Moreno et al., 2015).

The aim of the present study was to investigate the impact of the solvents, DMSO or PEG-400, on the in vitro genotoxicity of TMPTA in human hepatocytes HepG2 cell lines by using the γH2AX test as a marker of DNA damage and the induction of Caspase-3 as a marker of apoptosis.