‘Site of contact genotoxicity’ assessment for implants - Potential use of single cell gel electrophoresis in biocompatibility testing of medical devices

Toxicological risk assessment of medical devices requires genotoxicity assessment as per ISO 10993, Part 3, which is designed to address gene mutations, clastogenicity and/or aneugenicity endpoints. ‘Site of contact genotoxicity’ is a potential genotoxic risk especially for medical implants, that is currently not addressed in biocompatibility standards. We therefore performed initial validation study on the use of alkaline single cell gel electrophoresis (comet assay) for detecting ‘site of contact genotoxicity’ of medical devices, using test items made of acrylic implants impregnated with ethyl methanesulphonate (EMS). Comet assay detected increased DNA migration at the site of implantation, but not in the liver. The same implants also failed to show any genotoxicity potentials, when tested on the standard test battery using Salmonella/microsome and chromosome aberration assays. The study suggested that some medical implants can cause ‘site of contact genotoxicity’, without producing systemic genotoxicity. In conclusion, comet assay will add new dimension to safety assessment of medical devices, and this assay can be added to the battery of genetic toxicology tests for evaluating biocompatibility of medical implants

Emerging risks from ballast water treatment: The run-up to the International Ballast Water Management Convention

AbstractUptake and discharge of ballast water by ocean-going ships contribute to the worldwide spread of aquatic invasive species, with negative impacts on the environment, economies, and public health. The International Ballast Water Management Convention aims at a global answer. The agreed standards for ballast water discharge will require ballast water treatment. Systems based on various physical and/or chemical methods were developed for on-board installation and approved by the International Maritime Organization. Most common are combinations of high-performance filters with oxidizing chemicals or UV radiation. A well-known problem of oxidative water treatment is the formation of disinfection by-products, many of which show genotoxicity, carcinogenicity, or other long-term toxicity. In natural biota, genetic damages can affect reproductive success and ultimately impact biodiversity. The future exposure towards chemicals from ballast water treatment can only be estimated, based on land-based testing of treatment systems, mathematical models, and exposure scenarios. Systematic studies on the chemistry of oxidants in seawater are lacking, as are data about the background levels of disinfection by-products in the oceans and strategies for monitoring future developments. The international approval procedure of ballast water treatment systems compares the estimated exposure levels of individual substances with their experimental toxicity. While well established in many substance regulations, this approach is also criticised for its simplification, which may disregard critical aspects such as multiple exposures and long-term sub-lethal effects. Moreover, a truly holistic sustainability assessment would need to take into account factors beyond chemical hazards, e.g. energy consumption, air pollution or waste generation

An integrated approach to determine interactive genotoxic and global gene expression effects of multiwalled carbon nanotubes (MWCNTs) and benzo[a]pyrene (BaP) on marine mussels: evidence of reverse ‘Trojan Horse’ effects

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Application of the rainbow trout derived intestinal cell line (RTgutGC) for ecotoxicological studies: molecular and cellular responses following exposure to copper.

There is an acknowledged need for in vitro fish intestinal model to help understand dietary exposure to chemicals in the aquatic environment. The presence and use of such models is however largely restrictive due to technical difficulties in the culturing of enterocytes in general and the availability of appropriate established cell lines in particular. In this study, the rainbow trout (Oncorhynchus mykiss) intestinal derived cell line (RTgutGC) was used as a surrogate for the "gut sac" method. To facilitate comparison, RTgutGC cells were grown as monolayers (double-seeded) on permeable Transwell supports leading to a two-compartment intestinal model consisting of polarised epithelium. This two-compartment model divides the system into an upper apical (lumen) and a lower basolateral (portal blood) compartment. In our studies, these cells stained weakly for mucosubstances, expressed the tight junction protein ZO-1 in addition to E-cadherin and revealed the presence of polarised epithelium in addition to microvilli protrusions. The cells also revealed a comparable transepithelial electrical resistance (TEER) to the in vivo situation. Importantly, the cell line tolerated apical saline (1:1 ratio) thus mimicking the intact organ to allow assessment of uptake of compounds across the intestine. Following an exposure over 72 h, our study demonstrated that the RTgutGC cell line under sub-lethal concentrations of copper sulphate (Cu) and modified saline solutions demonstrated uptake of the metal with saturation levels comparable to short term ex situ gut sac preparations. Gene expression analysis revealed no significant influence of pH or time on mRNA expression levels of key stress related genes (i.e. CYP3A, GST, mtA, Pgp and SOD) in the Transwell model. However, significant positive correlations were found between all genes investigated suggesting a co-operative relationship amongst the genes studied. When the outlined characteristics of the cell line are combined with the division of compartments, the RTgutGC double seeded model represents a potential animal replacement model for ecotoxicological studies. Overall, this model could be used to study the effects and predict aquatic gastrointestinal permeability of metals and other environmentally relevant contaminants in a cost effective and high throughput manner

Pharmaceutical Metabolism in Fish: Using a 3-D Hepatic In Vitro Model to Assess Clearance

At high internal doses, pharmaceuticals have the potential for inducing biological/pharmacological effects in fish. One particular concern for the environment is their potential to bioaccumulate and reach pharmacological levels; the study of these implications for environmental risk assessment has therefore gained increasing attention. To avoid unnecessary testing on animals, in vitro methods for assessment of xenobiotic metabolism could aid in the ecotoxicological evaluation. Here we report the use of a 3-D in vitro liver organoid culture system (spheroids) derived from rainbow trout to measure the metabolism of seven pharmaceuticals using a substrate depletion assay. Of the pharmaceuticals tested, propranolol, diclofenac and phenylbutazone were metabolised by trout liver spheroids; atenolol, metoprolol, diazepam and carbamazepine were not. Substrate depletion kinetics data was used to estimate intrinsic hepatic clearance by this spheroid model, which was similar for diclofenac and approximately 5 fold higher for propranolol when compared to trout liver microsomal fraction (S9) data. These results suggest that liver spheroids could be used as a relevant and metabolically competent in vitro model with which to measure the biotransformation of pharmaceuticals in fish; and propranolol acts as a reproducible positive control

Assessing the impact of low level laser therapy (LLLT) on biological systems: a review

PURPOSE: Low level laser therapy (LLLT) in the visible to near infrared spectral band (390-1100 nm) is absorption of laser light at the electronic level, without generation of heat. It may be applied in a wide range of treatments including wound healing, inflammation and pain reduction. Despite its potential beneficial impacts, the use of lasers for therapeutic purposes still remains controversial in mainstream medicine. Whilst taking into account the physical characteristics of different qualities of lasers, this review aims to provide a comprehensive account of the current literature available in the field pertaining to their potential impact at cellular and molecular levels elucidating mechanistic interactions in different mammalian models. The review also aims to focus on the integral approach of the optimal characteristics of LLLT that suit a biological system target to produce the beneficial effect at the cellular and molecular levels. METHODS: Recent research articles were reviewed that explored the interaction of lasers (coherent sources) and LEDs (incoherent sources) at the molecular and cellular levels. RESULTS: It is envisaged that underlying mechanisms of beneficial impact of lasers to patients involves biological processes at the cellular and molecular levels. The biological impact or effects of LLLT at the cellular and molecular level could include cellular viability, proliferation rate, as well as DNA integrity and the repair of damaged DNA. This review summarizes the available information in the literature pertaining to cellular and molecular effects of lasers. CONCLUSIONS: It is suggested that a change in approach is required to understand how to exploit the potential therapeutic modality of lasers whilst minimizing its possible detrimental effects

Modified comet assay to determine oxidative damage to DNA in aquatic organisms: role of confounding factors influencing the results

In common with human health arena, oxidative damage to DNA could play an important role in the development of pathophysiological conditions and ultimately Darwinian fitness of the wild species. Despite the importance, not enough information is available in the literature pertaining to the determination of oxidative damage to DNA in wild species. Among the aquatic organisms, bivalve molluscs and fish species have been widely used for ecotoxicological studies and for the determination of DNA strand breaks using the standard comet assay. For these organisms, the widely used cell types are haemocytes for molluscs and erythrocytes for fish, although other cell types (e.g. gill cells) are also used. Whilst a large number of studies have been carried out using standard comet assay, applications of modified comet assay using bacterial enzymes to determine oxidative damage to DNA has been very limited. We have used these cell types from fish and bivalve species and have determined the oxidative damage to DNA following exposure to a range of environmental stressors (e.g. metals, nanoparticles and hypoxia). Overall these studies suggest that while in vitro optimisation studies using hydrogen peroxide provide a concentration-dependent response, the results do not always mirror the in vitro response in in vivo situations. This disparity could be attributed to different study designs, stressors used in addition to different confounding factors which influence the determination of different biological parameters. More research is therefore required to optimise the application of modified comet assay in different cell types of aquatic organisms before it is widely used for ecotoxicological studies