The protocol describes the method of quantification of two tobacco specific nitrosamines, N-Nitrosonornicotine (NNN), and 4-(methylnitrosamino)-1-(3-pyridil)-1-butanone (NNK) in air samples collected in a dedicated Indoor Air Quality room (IAQ-room) by LC-MS/MS analysis.
The protocol describes the quantification of gas-phase tobacco specific markers, nicotine and 3-ethenylpyridine (3-EP).
In order to expose in a reproducible manner, the aerosols were generated under specific conditions and well characterized.
The protocol describes the quantification of Respirable Suspended Particles (RSP), ultraviolet particulate matter (UVPM), fluorescent particulate matter (FPM), and solanesol that are marker constituents in Environmental Tobacco Smoke (ETS).
Analytical procedure to measure TPM, Nicotine and Acrolein in aerosol fractions prepared for in vitro assays (PMI)Lydia Gautier
Total particulate matter (TPM) and gas and vapor phase (GVP) are trapped from aerosol generated by THS 2.2 or cigarette. Nicotine is determined from TPM and acrolein from GVP fractions, respectively. These measurements are used as quality checks to evaluate the repeatability between the different batches of aerosol fractions generated for in vitro assays.
This protocol describes the assessment of cell viability and apoptosis in response to aqueous aerosol extracts.
Point mutations are the cause of many human genetic diseases and there is substantial evidence that point mutations in oncogenes and tumour suppressor genes of somatic cells are involved in tumour formation in humans and experimental animals. The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria...
This protocol describes an high-throughput method to measure the concentrations of pro-inflammatory cytokines secreted into the basolateral medium of 3D organotypic tissue cultures before and after the exposure to test substances.
Measurement of body weight in mice at regular intervals during exposure.
The bronchoalveolar lavage fluid (BALF) collected from mice can be analyzed in order to assess the inflammation status of the lungs after the exposure to toxicants. This protocol describes the methodology required to measure the concentrations of several inflammatory mediators (cytokines and chemokines) in cell-free BALF supernatants.
The protocol lists analytical methods from Health Canada and Labstat International to detect and quantify major analytes in aerosol and/or smoke. The list of these analytes was defined by PMI as the 58 Harmful or Potentially Harmful Constituents (58 HPHCs), and is divided into 14 analyte groups, plus glycerol.
This protocol describes a method to measure the ciliary beating frequency in organotypic nasal or bronchial tissue cultures exposed at the air-liquid interface. The cilia are recorded with a digital high-speed camera connected to an inverted microscope system, and a software calculates the beating frequency.
This protocol describes measurement of ciliary beating functionality of the ciliated pseudostratified epithelium exposed at the air liquid interface.
Assessment of clinical chemistry parameters in blood.
This protocol describes the indirect quantification of DNA double-strand breaks in response to whole aerosol exposure by assessment of H2AX histone phosphorylation in cell nuclei.
Estimation of the oxidative stress response to aqueous aerosol extracts with the ARE reporter assay (BAT)Damien Breheny, Jason Adamson, David Azzopardi, Andrew Baxter, Emma Bishop, Tony Carr, Ian Crooks, Katherine Hewitt, Tomasz Jaunky, Sophie Larard, Frazer Lowe, Oluwatobiloba Oke, Mark Taylor, Simone Santopietro, David David, Benjamin Zainuddin, Marianna Gaça, Chuan Liu, James Murphy & Christopher Proctor
This protocol describes the luciferase-based antioxidant response element (ARE) reporter assay for the detection and quantification of Nrf2 transcriptional activity, as a marker of oxidative stress response
The protocol describes how the food and water consumed by rodents during a defined period of time is measured, as part of the health monitoring program during whole body aerosol/smoke exposure studies.
Characterization of blood cell populations in in vivo samples
This protocol describes how to use an HCS-based platform for the toxicological assessment in vitro of different test items. The platform runs in parallel cellular assays with definite endpoints such as cytotoxicity, mitochondrial fitness, oxidative stress, apoptosis and necrosis, DNA damage, epithelial to mesenchymal cell transition, and NF-kB pathway activation. Depending on the study design, not all endpoints may be evaluated.
This protocol describes a method to analyze the microRNAs of rodent tissues. The miRNA labelling is performed with the (ThermoFisher) FlashTag™ Biotin HSR RNA kit and hybridized in ThermoFisher microarrays.
Summary of transcriptomics analysis for various tissues collected in an inhalation study in mice, from sample collection to biological interpretation and network perturbation analysis.
The principle of the NRU in vitro cytotoxicity assay is based on the ability of viable cells to incorporate and bind neutral red. Exposure of cells to chemicals can alter this ability due to death or inhibition of growth. In that case, the uptake of neutral R^red by the cells decreases. Consequently, the concentration of neutral red dye desorbed from the cultured cells is directly proportional to the number of living cells. In the Neutral...
The protocol describes the impactor technique used to characterize the particle/droplet size distribution of the aerosol. The physical properties of an aerosol determine the fraction of aerosol that is able to pass through the upper respiratory tract to reach the lungs and the fraction that is retained on the respiratory system.
Transepithelial Electrical Resistance (TEER) Measurement with Chopstick Electrode (in vitro organotypic, PMI)Stefan Frentzel, Stephanie Johne, Anita Iskandar & Laura Ortega Torres
This protocol describes a measurement of the trans-epithelial electrical resistance (TEER) of 3D organotypic tissues. TEER values indicate tissue integrity and can be measured after cultures are exposed to test substances using the STX2 Chopstick electrode and the EVOM2 Epithelial Voltohmmeter. Using the STX2 electrode, a current is passed through the 3D organotypic tissue via two electrodes. A second pair of electrodes measures the voltage and a resistance value is calculated and displayed by EVOM2...
The protocol describes the determination of the sum of volatile organic compounds (TVOC) concentrations, expressed as toluene equivalent in air samples collected in an indoor air quality (IAQ) room.