‘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

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

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies

DNA damage in B and T lymphocytes of farmers during one pesticide spraying season

Purpose The effect of one pesticide spraying seasonon DNA damage was measured on B and T lymphocytesamong open-field farmers and controls.Methods At least two peripheral blood samples were collectedfrom each individual: one in a period without anypesticide application, several weeks after the last use (January,at period P0), and another in the intensive pesticidespraying period (May or June, at period P4). DNA damagewas studied by alkaline comet assay on isolated B or Tlymphocytes.Results Longitudinal comparison of DNA damageobserved at both P0 and P4 periods revealed a statisticallysignificant genotoxic effect of the pesticide spraying seasonin both B (P = 0.02) and T lymphocytes (P = 0.02) in exposed farmers. In contrast, non-farmers did not showany significant modifications. DNA damage levels in Band T lymphocytes were significantly higher in farmersthan in non-farmers during the P4 period (P = 0.003 andP = 0.001 for B and T lymphocytes, respectively) but notduring the P0 period. The seasonal effect observed amongfarmers was not correlated with either total farm area, farmarea devoted to crops or recent solar exposure. On average,farmers used pesticides for 21 days between P0 and P4.Between the two time points studied, there was a tendencyfor a potential effect of the number of days of fungicidetreatments (r2 = 0.43; P = 0.11) on T lymphocyte DNAdamage.Conclusions A genotoxic effect was found in lymphocytesof farmers exposed to pesticides, suggesting in particularthe possible implication of fungicides

Assignment of phosphorus-31 chemical shifts to isomers of 2,3-dialkoxy-λ3\lambda 3-diazadiphosphetidines. Crystal and molecular structure of trans−[PhNP(OCH2CF3)]2-[PhNP(OCH_2CF_3)]2

Geometrical isomers of $\lambda$3-diazadiphosphetidines show large differences in their 31P chem. shifts. Trifluoroethoxylation of cis-$(PhNPCl)_2$ gives only the low-field isomer initially, for which the crystal structure is detd. The alkoxy groups are trans to each other. On standing in soln., trans-$[PhNP(OCH_2CF_3)]2$transforms slowly and almost completely into its cis analog with a high-field 31P chem. shift

NMR Spectroscopic and X-Ray Structural Studies of Two Diazadiphosphetidines

The crystal structures of the two diazadiphosphetidines, [PhNP(OCH2CF3)]2 (1) and [MeNP(NMe2)(O2C6H4)]2 (2) have been determined. The trifluoroethoxy groups in (1) have a trans orientation. The phosphorus chemical shift for (1) is at 189.8 δ. On standing in solution, (1) transforms slowly (∼ 10 days) and almost completely into its 'high-field' (cis) isomer (142.2δ)