Bioluminescent enzyme inhibition-based assay to predict the potential toxicity of carbon nanomaterials

Highlights

• Bioluminescent enzymatic assay for CNM potential toxicity was proposed.

• The assay predicts toxicity decreasing in the following order: MWCNT > SWCNT > C₆₀HyFn.

• Soluble and immobilised enzymes differ qualitatively in CNM toxicity prediction.

Abstract

A bioluminescent enzyme inhibition-based assay was applied to predict the potential toxicity of carbon nanomaterials (CNM) presented by single- and multi-walled nanotubes (SWCNT and MWCNT) and aqueous solutions of hydrated fullerene С₆₀ (C₆₀HyFn). This assay specifically detects the influence of substances on parameters of the soluble or immobilised coupled enzyme system of luminescent bacteria: NAD(P)Н:FMN-oxidoreductase + luciferase (Red + Luc). A protocol based on the optical properties of CNM for correcting the results of the bioluminescent assay was also developed. It was shown that the inhibitory activity of CNM on Red + Luc decreased in the following order: MWCNT > SWCNT > C₆₀HyFn. The soluble enzyme system Red + Luc had high sensitivity to MWCNT and SWCNT, with values of the inhibition parameter IC₅₀ equal to 0.012 and 0.16 mg/L, respectively. The immobilised enzyme system was more vulnerable to C₆₀HyFn than its soluble form, with an IC₅₀ equal to 1.4 mg/L. Due to its technical simplicity, rapid response time and high sensitivity, this bioluminescent method has the potential to be developed as a general enzyme inhibition-based assay for a wide variety of nanomaterials.

Introduction

Due to the increasing scale of production and usage of a vast number of new materials in industrial and economic activities, society is faced with problems associated with a lack of materials safety assessment regarding humans, ecosystems and the biosphere as a whole. These materials include engineered nanomaterials, which are actively applied in medicine, perfumes, cosmetics and the food industry (Zhou, 2015). The lack of regulation over the biological safety of such nanomaterials has raised concerns about their toxicity in biology and medicine (Sahu and Casciano, 2009, Jain, 2012, Bottero, 2016). Evaluation of the potential risks of using nanomaterials is complicated due to their physical and chemical properties such as size, distribution, agglomeration state, shape, crystal structure, chemical composition, surface area, surface chemistry, surface charge and porosity (Oberdörster et al., 2005, Mu et al., 2014). Moreover, the connection between the size of nanomaterials and their toxicity, in terms of whether there is a specific type of nanotoxicity, remains unknown (Donaldson and Poland, 2013). Nowadays, numerous toxicological investigations using living organisms, cell lines, etc. are carried out in laboratories in order to assess the potential risks of using these materials and their biological effects on human health and the environment (Powers and Reineke, 2012, ENRHES (Engineered Nanoparticles: Review of Health and Environmental Safety), 2009, Maurer-Jones et al., 2013, Ema et al., 2015, Zhang et al., 2015). Good results in terms of rapidity and reproducibility have been obtained by using bioluminescent methods based on recombinant or natural strains of luminescent bacteria (Mortimer et al., 2008, Zheng et al., 2010, Deryabin et al., 2012).

The first reports on the toxic effect of carbon nanotubes (CNT) were published in 2003 (Service, 2003); since then, several research studies have proven that CNT are characterised by cyto- and genotoxicity (ENRHES (Engineered Nanoparticles: Review of Health and Environmental Safety), 2009, Lam et al., 2006, Lanone et al., 2013). Moreover, CNT can amplify the toxic effect of other contaminants (Sanchís et al., 2015). Many authors believe that the toxic effect of CNT is associated with their ability to generate reactive oxygen species (ROS) followed by the development of oxidative stress in cells (Johnston et al., 2010). However, this ROS generation is often explained by the fact that commercial preparations of CNT contain various contaminants, for example, iron, which enters nanotubes during the process of production (Kagan et al., 2006, Pulskamp et al., 2007). Templeton et al. (2006) have shown that the electrophoretic cleaning of commercial preparations leads to a significant reduction of their toxicity. However, it is critical to evaluate the toxicity of commercial preparations of CNT, because they can have a negative effect on biological objects during their manufacture and use.

The published data on the toxicity of the other group of CNM – fullerenes – at first glance seem contradictory. For example, Oberdörster (2004) showed that fullerene suspensions prepared by the method of Colvin et al. (2004) have toxic effects on the brain cells of fish at concentrations of 0.003–5 μМ. At the same time, many authors indicated no negative effects, even at much higher concentrations of fullerenes. For instance, Gharbi et al. (2005) showed that using water dispersions of fullerene С₆₀ in experiments with rats, even at very high doses (2.5 g/kg and higher), had no toxic effects on their liver. Importantly, Andrievsky et al. (2005) stated that the toxic effect of fullerenes depends on the characteristics of the preparations, primarily from the number and physical-chemical properties of impurities contained in these preparations. The authors of this review hypothesised that the main reason for the inhibitory (toxic) effect of Colvin's dispersions of fullerene on the biological systems is the presence of tetrahydrofuran (THF) molecules and products of their oxidative modification and subsequent polymerisation. This conclusion was further confirmed by Zhu et al. (2006) in a study showing that using THF as a solvent for making С₆₀ suspensions leads to a considerable increase in fullerene toxicity determined by the death rate of Daphnia magna.

Information about the ability of fullerenes to generate ROS is also contradictory. Some authors stated that С₆₀ stimulates ROS generation in solutions (Wang et al., 2011), while others evidenced the antioxidative properties of fullerenes (Gharbi et al., 2005, Andrievsky et al., 2009, Wang et al., 1999). Apparently, the latter statement refers only to aqueous solutions of pristine fullerene and can be explained by an ordered hydration shell surrounding the molecule of C₆₀ that prevents the formation of ROS (Andrievsky et al., 2005). Thus, it is clear that the toxic effects of fullerenes are determined by their structure and physical-chemical properties. Investigations based on human red blood cell haemolysis have also examined the cytotoxic effects of fullerenes (Tramer et al., 2012).

The molecular mechanism behind the effects of nanomaterials consists mainly of DNA degradation or enzyme inhibition (Wang et al., 2009, Wang et al., 2010, Zhang et al., 2012, Chang et al., 2014, Kӓkinen et al., 2013, Vale et al., 2015). In this work, the bacterial coupled enzyme system NAD(P)H:FMN-oxidoreductase and luciferase (Red + Luc), which catalyses the following reactions (1 and 2), was used as a test system in our attempt to replace luminescent bacteria:

$$\mathrm{NAD}\left(\mathrm{P}\right)\mathrm{H}+\mathrm{FMN}+{\mathrm{H}}^{+}\stackrel{\mathrm{Red}}{\to }\mathrm{NAD}{\left(\mathrm{P}\right)}^{+}+\mathrm{FMN}\cdot {\mathrm{H}}_2$$

$$\mathrm{FMN}\cdot {\mathrm{H}}_2+\text{RCHO}+{\mathrm{O}}_2\stackrel{\mathrm{Luc}}{\to }\mathrm{FMN}+\text{RCOOH}+{\mathrm{H}}_2\mathrm{O}+\mathrm{h\nu }$$

where FMN and FMN·H₂ are the oxidised and reduced forms of flavin mononucleotide, NAD(P)⁺ and NAD(P)H are oxidised and reduced forms of nicotinamide adenine dinucleotide (phosphate), RCHO is myristic aldehyde and RCOOH is the corresponding fatty acid.

The principle of bioluminescent enzymatic bioassay is to detect the toxic properties of substances and mixtures based on their influence on the parameters of these bioluminescent enzymatic reactions (Kratasyuk and Esimbekova, 2011, Esimbekova et al., 2013, Esimbekova et al., 2014). This and other similar assays were developed earlier for environmental monitoring and medical diagnostics (Esimbekova et al., 2014, Esimbekova et al., 1999, Kratasyuk and Sovtsov, 1992). In addition, this bioassay was used previously to determine the toxicity of biopolymers (Shishatskaya et al., 2002). Moreover, it has been shown that nanodiamonds are able to inhibit the coupled enzyme system Red + Luc (Kudryasheva et al., 1994).

The commercially available carbon-based nanomaterials, including single-walled carbon carboxylated nanotubes (SWCNT), multi-walled carbon nanotubes (MWCNT) and chemically un-modified С₆₀ fullerene water solution (C₆₀FWS), were tested in this study. The effect of these CNM on the activity of the soluble and immobilised coupled enzyme system Red + Luc was verified.