Shareen H. Doak

NanoGenotoxicology : The DNA damaging potential of engineered nanomaterials

With the rapid expansion in the nanotechnology industry, it is essential that the safety of engineered nanomaterials and the factors that influence their associated hazards are understood. A vital area governing regulatory health risk assessment is genotoxicology (the study of genetic aberrations following exposure to test agents), as DNA damage may initiate and promote carcinogenesis, or impact fertility. Of late, considerable attention has been given to the toxicity of engineered nanomaterials, but the importance of their genotoxic potential on human health has been largely overlooked. This comprehensive review focuses on the reported abilities of metal nanoparticles, metal-oxide nanoparticles, quantum dots, fullerenes, and fibrous nanomaterials, to damage or interact with DNA, and their ecogenotoxicity is also considered. Many of the engineered nanomaterials assessed were found to cause genotoxic responses, such as chromosomal fragmentation, DNA strand breakages, point mutations, oxidative DNA adducts and alterations in gene expression profiles. However, there are clear inconsistencies in the literature and it is difficult to draw conclusions on the physico-chemical features of nanomaterials that promote genotoxicity, largely due to study design. Hence, areas that require that further attention are highlighted and recommendations to improve our understanding of the genotoxic potential of engineered nanomaterials are addressed.

Potential toxicity of superparamagnetic iron oxide nanoparticles (SPION)

Superparamagnetic iron oxide nanoparticles (SPION) are being widely used for various biomedical applications, for example, magnetic resonance imaging, targeted delivery of drugs or genes, and in hyperthermia. Although, the potential benefits of SPION are considerable, there is a distinct need to identify any potential cellular damage associated with these nanoparticles. Besides focussing on cytotoxicity, the most commonly used determinant of toxicity as a result of exposure to SPION, this review also mentions the importance of studying the subtle cellular alterations in the form of DNA damage and oxidative stress. We review current studies and discuss how SPION, with or without different surface coating, may cause cellular perturbations including modulation of actin cytoskeleton, alteration in gene expression profiles, disturbance in iron homeostasis and altered cellular responses such as activation of signalling pathways and impairment of cell cycle regulation. The importance of protein–SPION interaction and various safety considerations relating to SPION exposure are also addressed.

Mechanistic Influences for Mutation Induction Curves after Exposure to DNA-Reactive Carcinogens

A mechanistic understanding of carcinogenic genotoxicity is necessary to determine consequences of chemical exposure on human populations and improve health risk assessments. Currently, linear dose-responses are assumed for DNA reactive compounds, ignoring cytoprotective processes that may limit permanent damage. To investigate the biological significance of low-dose exposures, human lymphoblastoid cells were treated with alkylating agents that have different mechanisms of action and DNA targets: methylmethane sulfonate (MMS), methylnitrosourea (MNU), ethylmethane sulfonate (EMS), and ethylnitrosourea (ENU). Chromosomal damage and point mutations were quantified with the micronucleus and hypoxanthine phosphoribosyltransferase forward mutation assays. MNU and ENU showed linear dose-responses, whereas MMS and EMS had nonlinear curves containing a range of nonmutagenic low doses. The lowest observed effect level for induction of chromosomal aberrations was 0.85 microg/mL MMS and 1.40 microg/mL EMS; point mutations required 1.25 microg/mL MMS and 1.40 microg/mL EMS before a mutagenic effect was detected. This nonlinearity could be due to homeostatic maintenance by DNA repair, which is efficient at low doses of compounds that primarily alkylate N(7)-G and rarely attack O atoms. A pragmatic threshold for carcinogenicity may therefore exist for such genotoxins.

Confounding experimental considerations in nanogenotoxicology

The development of novel nanomaterials with unique physico-chemical properties is increasing at a rapid rate, with potential applications across a broad range of manufacturing industries and consumer products. Nanomaterial safety is therefore becoming an increasingly contentious issue that has intensified over the past 4 years, and in response, a steady stream of studies focusing on nanotoxicology are emerging. However, it is becoming increasingly evident that nanomaterials cannot be treated in the same manner as chemical compounds with regards to their safety assessment, as their unique physico-chemical properties are also responsible for unexpected interactions with experimental components that generate misleading data-sets. In this report, we focus on nanomaterial interactions with colorimetric and fluorometric dyes, components of cell culture growth medium and genotoxicity assay components, and the resultant consequences on test systems are demonstrated. Thus, highlighting some of the potential confounding factors that need to be considered in order to ensure that in vitro genotoxicity assays report true biological impacts in response to nanomaterial exposure.

Cytotoxic effects of gold nanoparticles: a multiparametric study.

The in vitro labeling of therapeutic cells with nanoparticles (NPs) is becoming more and more common, but concerns about the possible effects of the NPs on the cultured cells are also increasing. In the present work, we evaluate the effects of poly(methacrylic acid)-coated 4 nm diameter Au NPs on a variety of sensitive and therapeutically interesting cell types (C17.2 neural progenitor cells, human umbilical vein endothelial cells, and PC12 rat pheochromocytoma cells) using a multiparametric approach. Using various NP concentrations and incubation times, we performed a stepwise analysis of the NP effects on cell viability, reactive oxygen species, cell morphology, cytoskeleton architecture, and cell functionality. The data show that higher NP concentrations (200 nM) reduce cell viability mostly through induction of reactive oxygen species, which was significantly induced at concentrations of 50 nM Au NPs or higher. At these concentrations, both actin and tubulin cytoskeleton were deformed and resulted in reduced cell proliferation and cellular differentiation. In terms of cell functionality, the NPs significantly impeded neurite outgrowth of PC12 cells up to 20 nM concentrations. At 10 nM, no significant effects on any cellular parameter could be observed. These data highlight the importance of using multiple assays to cover the broad spectrum of cell-NP interactions and to determine safe NP concentrations and put forward the described protocol as a possible template for future cell-NP interaction studies under comparable and standardized conditions.

In vitro genotoxicity testing strategy for nanomaterials and the adaptation of current OECD guidelines

Highlights ► We consider current in vitro OECD genotoxicity tests for nanomaterials. ► Ames test does not appear to be suitable for nanomaterial assessment. ► In vitro HPRT and micronucleus assays require nanomaterial specific protocols. ► We recommend a strategic in vitro genotoxicity testing strategy for nanomaterials.

The role of iron redox state in the genotoxicity of ultrafine superparamagnetic iron oxide nanoparticles.

Ultrafine superparamagnetic iron oxide nanoparticles (USPION) hold great potential for revolutionising biomedical applications such as MRI, localised hyperthermia, and targeted drug delivery. Though evidence is increasing regarding the influence of nanoparticle physico-chemical features on toxicity, data however, is lacking that assesses a range of such characteristics in parallel. We show that iron redox state, a subtle though important physico-chemical feature of USPION, dramatically modifies the cellular uptake of these nanoparticles and influences their induction of DNA damage. Surface chemistry was also found to have an impact and evidence to support a potential mechanism of oxidative DNA damage behind the observed responses has been demonstrated. As human exposure to ferrofluids is predicted to increase through nanomedicine based therapeutics, these findings are important in guiding the fabrication of USPION to ensure they have characteristics that support biocompatibility.

Chromosome 4 hyperploidy represents an early genetic aberration in premalignant Barrett’s oesophagus

Background and aims: Characterisation of the underlying molecular mechanisms that promote Barrett’s progression may ultimately lead to identification of potential predictive genetic markers that classify patients’ malignant risk. In an attempt to understand these causative pathways, fluorescence in situ hybridisation (FISH) was used in this study to determine when specific genetic alterations arise during Barrett’s associated neoplastic progression. Methods: Endoscopic cytology brushings were obtained from 28 patients with Barrett’s metaplasia, 28 with dysplasia (20 low grade dysplasia (LGD) and eight with high grade dysplasia (HGD)), and seven with adenocarcinoma, together with paired control brushings from regions of normal proximal squamous cell epithelium. The exfoliated epithelial cells were washed and deposited onto slides. Probes specific for the centromeres of chromosomes 4, 8, 20, and Y, and locus specific probes for the tumour suppressor genes p16, p53, and Rb were subsequently hybridised. Results: Aneuploidy was found early in progression, with metaplastic tissues displaying increased copy numbers of chromosomes 4 and 8. Chromosome 4 hyperploidy was found in 89%, 90%, 88%, and 100% of metaplasias, LGD, HGD, adenocarcinomas, respectively, while chromosome 8 hyperploidy occurred in 71%, 75%, 100%, and 100% of patients with the respective staging. Loss of the p16 tumour suppressor gene also presented in metaplastic epithelium (7%) but most other genetic aberrations were only seen in HGD. Conclusions: Genetic instability arises well before dysplasia in Barrett’s oesophagus, with chromosome 4 and 8 hyperploidy representing the earliest and most common alterations identified. As these aberrations are widespread at all the premalignant stages, there may be genes on chromosomes 4 and 8 that are involved in both the initiation and progression of Barrett’s oesophagus.

Generic epitaxial graphene biosensors for ultrasensitive detection of cancer risk biomarker

A generic electrochemical method of ?bioreceptor? antibody attachment to phenyl amine functionalized graphitic surfaces is demonstrated. Micro-channels of chemically modified multi-layer epitaxial graphene (MLEG) have been used to provide a repeatable and reliable response to nano-molar (nM) concentrations of the cancer risk (oxidative stress) biomarker 8-hydroxydeoxyguanosine (8-OHdG). X-ray photoelectron spectroscopy, Raman spectroscopy are used to characterize the functionalized MLEG. Confocal fluorescence microscopy using fluorescent-labelled antibodies indicates that the anti-8-OHdG antibody selectively binds to the phenyl amine-functionalized MLEG?s channel. Current?voltage measurements on functionalized channels showed repeatable current responses from antibody?biomarker binding events. This technique is scalable, reliable, and capable of providing a rapid, quantitative, label-free assessment of biomarkers at nano-molar (<20 nM) concentrations in analyte solutions. The sensitivity of the sensor device was investigated using varying concentrations of 8-OHdG, with changes in the sensor?s channel resistance observed upon exposure to 8-OHdG. Detection of 8-OHdG concentrations as low as 0.1 ng ml?1 (0.35 nM) has been demonstrated. This is five times more sensitive than reported enzyme linked immunosorbent assay tests (0.5 ng ml?1).

Non-linear dose-response of DNA-reactive genotoxins: Recommendations for data analysis

Until recently, there has only been a limited amount of data available on the kinetics of mutation induction in the low dose region of exposure. In our publication Doak et al. [S.H. Doak, G.J. Jenkins, G.E. Johnson, E. Quick, E.M. Parry, J.M. Parry, Mechanistic influences for mutation induction curves after exposure to DNA-reactive carcinogens, Cancer Res. 67 (2007) 3904-3911] we showed that the two alkylating agents methyl-methanesulfonate (MMS) and ethyl-methanesulfonate (EMS) possess non-linear dose-response curves with no observed effect levels (NOEL) for mutation or chromosomal damage in vitro. These experiments were carried out in the AHH-1 human lymphoblastoid cell line, using the hypoxanthine phosphoribosyl transferase (HPRT) assay and the cytokinesis-block micronucleus (CBMN) assay, respectively. We have now carried out more advanced statistical analyses to define threshold values, which is critical as it has a dramatic impact on hazard and risk assessment. To do this, we re-analysed the data to see if the linear model or a more complex model (hockey stick or quadratic) gave a significant better fit of the data. For both EMS and MMS cytokinesis-block micronucleus data sets, the hockey stick model gave the most significant fit. The same was true for EMS, MMS and surprisingly ethylnitrosourea (ENU) in the HPRT assay in human AHH-1 cells. However, methylnitrosourea (MNU) was linear in both assays. These further analyses have shown that EMS and MMS have clear thresholds for both gene mutation and chromosome damage, as does ENU for gene mutation in AHH-1 cells. MNU was linear for gene and chromosome mutation and so was ENU for chromosome mutations at the concentrations tested. These findings correlate closely with those in vivo findings of Gocke et al. [E. Gocke, L. Müller, In vivo studies in the mouse to define a threshold for the genotoxicity of EMS and ENU, Mutat. Res. (this issue)] and together these data show a true threshold for EMS both in vitro and in vivo. In this report, we will discuss the approaches that were taken to investigate potential threshold dose-response curves for DNA-reactive genotoxic compounds, with recommendations for further studies.