Body weight Raw Data

<p>The file shows the animal number information on the first row and the exposure group information in the second row. Then, the first column has the study day information, and each body weight recorded (in grams) is recorded in corresponding cells.</p

Lung Histopathology and Histomorphometry Raw Data

<p>The file contains histopathology scores and histomorphometry measurements . Briefly, it has the exposure group information on the first row, the time point information on the second row, and the animal number information in the third row. Then, the first column has the name of the finding evaluated, or the parameter in the histomorphometry measured and the second column the level at which this was done.</p

Smoke chemistry

<p>The file contains the concentrations of 58 harmful and potentially harmful constituents measured in aerosols from 3R4F and the pMRTP.</p

2D-PAGE data - normalized matrix

<p>Normalilzed matrix of protein measurements in lung by 2d-gel analysis.</p

Proteomics 2D gel images

<p>images of the 2d gels for lung proteomics analysis</p

Proteomics reverse phase protein array data

<p>For the correction of anti-species secondary antibody staining, arrays were assayed in the absence of primary antibodies. For the measurement of spotted protein, one blank chip (i.e., without antibody incubation) was stained with SYPROÒRuby. Scanned images were analyzed using ZeptoVIEW 3.1 software (Bayer Technology Services GmbH). Normalized fluorescence intensities (NFIs) for each sample and protein target were calculated as reference fluorescence intensities of primary antibody stained arrays (RFIprimary) corrected for secondary antibody staining (RFIsecondary), as well as relative spotted protein concentration (RFIprotein), determined by (RFIprimary − RFIsecondary) / RFIprotein, using ZeptoVIEW 3.1 software. NFI values were used for subsequent analysis.</p> <p>A series of bovine serum albumin references were spotted on each array. Each sample underwent serial dilution (100%, 75%, 50%, and 25%) to assess linearity. Prior to RPA analysis, all antibodies were validated by western blotting to assess their specificity.</p

Toxicity of aerosols of nicotine and pyruvic acid (separate and combined) in Sprague–Dawley rats in a 28-day OECD 412 inhalation study and assessment of systems toxicology

<div><p></p><p>Toxicity of nebulized nicotine (Nic) and nicotine/pyruvic acid mixtures (Nic/Pyr) was characterized in a 28-day Organization for Economic Co-operation and Development 412 inhalation study with additional transcriptomic and lipidomic analyses. Sprague–Dawley rats were nose-only exposed, 6 h/day, 5 days/week to filtered air, saline, nicotine (50 µg/l), sodium pyruvate (NaPyr, 33.9 µg/l) or equimolar Nic/Pyr mixtures (18, 25 and 50 µg nicotine/l). Saline and NaPyr caused no health effects, but rats exposed to nicotine-containing aerosols had decreased body weight gains and concentration-dependent increases in liver weight. Blood neutrophil counts were increased and lymphocyte counts decreased in rats exposed to nicotine; activities of alkaline phosphatase and alanine aminotransferase were increased, and levels of cholesterol and glucose decreased. The only histopathologic finding in non-respiratory tract organs was increased liver vacuolation and glycogen content. Respiratory tract findings upon nicotine exposure (but also some phosphate-buffered saline aerosol effects) were observed only in the larynx and were limited to adaptive changes. Gene expression changes in the lung and liver were very weak. Nic and Nic/Pyr caused few significant changes (including Cyp1a1 gene upregulation). Changes were predominantly related to energy metabolism and fatty acid metabolism but did not indicate an obvious toxicity-related response. Nicotine exposure lowered plasma lipids, including cholesteryl ester (CE) and free cholesterol and, in the liver, phospholipids and sphingolipids. Nic, NaPyr and Nic/Pyr decreased hepatic triacylglycerol and CE. In the lung, Nic and Nic/Pyr increased CE levels. These data suggest that only minor biologic effects related to inhalation of Nic or Nic/Pyr aerosols were observed in this 28-day study.</p></div

A framework for <i>in vitro</i> systems toxicology assessment of e-liquids

<p>Various electronic nicotine delivery systems (ENDS), of which electronic cigarettes (e-cigs) are the most recognized prototype, have been quickly gaining ground on conventional cigarettes because they are perceived as less harmful. Research assessing the potential effects of ENDS exposure in humans is currently limited and inconclusive. New products are emerging with numerous variations in designs and performance parameters within and across brands. Acknowledging these challenges, we present here a proposed framework for an <i>in vitro</i> systems toxicology assessment of e-liquids and their aerosols, intended to complement the battery of assays for standard toxicity assessments. The proposed framework utilizes high-throughput toxicity assessments of e-liquids and their aerosols, in which the device-to-device variability is minimized, and a systems-level investigation of the cellular mechanisms of toxicity is an integral part. An analytical chemistry investigation is also included as a part of the framework to provide accurate and reliable chemistry data solidifying the toxicological assessment. In its simplest form, the framework comprises of three main layers: (1) high-throughput toxicity screening of e-liquids using primary human cell culture systems; (2) toxicity-related mechanistic assessment of selected e-liquids, and (3) toxicity-related mechanistic assessment of their aerosols using organotypic air–liquid interface airway culture systems. A systems toxicology assessment approach is leveraged to enable in-depth analyses of the toxicity-related cellular mechanisms of e-liquids and their aerosols. We present example use cases to demonstrate the suitability of the framework for a robust <i>in vitro</i> assessment of e-liquids and their aerosols.</p

Effects of cigarette smoke, cessation and switching to a candidate modified risk tobacco product on the liver in <i>Apoe</i>^−/− mice – a systems toxicology analysis

<p>The liver is one of the most important organs involved in elimination of xenobiotic and potentially toxic substances. Cigarette smoke (CS) contains more than 7000 chemicals, including those that exert biological effects and cause smoking-related diseases. Though CS is not directly hepatotoxic, a growing body of evidence suggests that it may exacerbate pre-existing chronic liver disease. In this study, we integrated toxicological endpoints with molecular measurements and computational analyses to investigate effects of exposures on the livers of <i>Apoe^−/−</i>mice. Mice were exposed to 3R4F reference CS, to an aerosol from the Tobacco Heating System (THS) 2.2, a candidate modified risk tobacco product (MRTP) or to filtered air (Sham) for up to 8 months. THS2.2 takes advantage of a “heat-not-burn” technology that, by heating tobacco, avoids pyrogenesis and pyrosynthesis. After CS exposure for 2 months, some groups were either switched to the MRTP or filtered air. While no group showed clear signs of hepatotoxicity, integrative analysis of proteomics and transcriptomics data showed a CS-dependent impairment of specific biological networks. These networks included lipid and xenobiotic metabolism and iron homeostasis that likely contributed synergistically to exacerbating oxidative stress. In contrast, most proteomic and transcriptomic changes were lower in mice exposed to THS2.2 and in the cessation and switching groups compared to the CS group. Our findings elucidate the complex biological responses of the liver to CS exposure. Furthermore, they provide evidence that THS2.2 aerosol has reduced biological effects, as compared with CS, on the livers of <i>Apoe^−/−</i>mice.</p

Study overview file

<p>The file gives for each sample the animal<br>(with its coded animal number, CAN), the exposure group to which it belongs, the test or endpoint that was measured, and the name of the file where the data is given.</p