Commet

A High Throughput Nanogenotoxicity Assay

Research Area: Nanogenotoxicity

Funding Agencies: NIEHS Center at HSPH

Protocol for the high throughput Comet Assay. (A) Assembly of macrowell comet array. Agarose gel with microwells is sandwiched between a glass substrate and a bottomless 96-well plate and sealed with mechanical force. Approximately 300 arrayed microwells comprise the bottom of each macrowell. (B) Preparation of the nanoparticle suspension according to the protocol by Cohen at al. (C) Protocol for exposing the cells to the nanoparticles. (D) Loading of the exposed cell samples in the macrowells and running the microwell assay.

Protocol for the high throughput Comet Assay. (A) Assembly of macrowell comet array. Agarose gel with microwells is sandwiched between a glass substrate and a bottomless 96-well plate and sealed with mechanical force. Approximately 300 arrayed microwells comprise the bottom of each macrowell. (B) Preparation of the nanoparticle suspension according to the protocol by Cohen at al. (C) Protocol for exposing the cells to the nanoparticles. (D) Loading of the exposed cell samples in the macrowells and running the microwell assay.

There is mounting evidence that engineered nanoparticle (ENPs) exposure can lead to DNA damage that ultimately contributes to cytotoxicity and mutations that drive cancer. Engineered nanoparticle exposures are on the rise due to the inclusion of these novel materials into several consumer products such as sunscreens, cosmetics, and toner ink formulations. More specifically, metal oxide ENPs are widely used in consumer products due to their physicochemical properties. However, metal oxide ENPs have the ability to generate reactive oxygen species in target tissues, which can induce oxidative stress, the precursor of DNA damage and gene mutation. Currently, there is a lack of high throughput tools to assess the toxicity of the vast ENP libraries currently in the market or being developed. Thus, the development of novel high throughput tools to screen and identify possible hazardous ENPs is essential to protecting public health and also developing safer ENP formulations. In this effort, Dr. Christa Watson (lead investigator) in collaboration with Dr. Bevin Engelward of MIT, has developed a high throughput assay to assess the genotoxicity of engineered nanoparticles called the “NanoCometChip”. The NanoCometchip is a microfabricated platform that enables the simultaneous evaluation of multiple engineered nanoparticles with various physicochemical properties, which provides insight into structure-bioactivity relationships. Future efforts using this novel system aim to elucidate possible gene mutations associated with engineered nanoparticle exposures, thus shedding light to uncover possible ENP mediated carcinogenic mechanisms.