Sabrina Lin

Metal and silicate particles including nanoparticles are present in electronic cigarette cartomizer fluid and aerosol.

Background Electronic cigarettes (EC) deliver aerosol by heating fluid containing nicotine. Cartomizer EC combine the fluid chamber and heating element in a single unit. Because EC do not burn tobacco, they may be safer than conventional cigarettes. Their use is rapidly increasing worldwide with little prior testing of their aerosol. Objectives We tested the hypothesis that EC aerosol contains metals derived from various components in EC. Methods Cartomizer contents and aerosols were analyzed using light and electron microscopy, cytotoxicity testing, x-ray microanalysis, particle counting, and inductively coupled plasma optical emission spectrometry. Results The filament, a nickel-chromium wire, was coupled to a thicker copper wire coated with silver. The silver coating was sometimes missing. Four tin solder joints attached the wires to each other and coupled the copper/silver wire to the air tube and mouthpiece. All cartomizers had evidence of use before packaging (burn spots on the fibers and electrophoretic movement of fluid in the fibers). Fibers in two cartomizers had green deposits that contained copper. Centrifugation of the fibers produced large pellets containing tin. Tin particles and tin whiskers were identified in cartridge fluid and outer fibers. Cartomizer fluid with tin particles was cytotoxic in assays using human pulmonary fibroblasts. The aerosol contained particles >1 µm comprised of tin, silver, iron, nickel, aluminum, and silicate and nanoparticles (<100 nm) of tin, chromium and nickel. The concentrations of nine of eleven elements in EC aerosol were higher than or equal to the corresponding concentrations in conventional cigarette smoke. Many of the elements identified in EC aerosol are known to cause respiratory distress and disease. Conclusions The presence of metal and silicate particles in cartomizer aerosol demonstrates the need for improved quality control in EC design and manufacture and studies on how EC aerosol impacts the health of users and bystanders.

Comparison of electronic cigarette refill fluid cytotoxicity using embryonic and adult models

Electronic cigarettes (EC) and refill fluids are distributed with little information on their pre- and postnatal health effects. This study compares the cytotoxicity of EC refill fluids using embryonic and adult cells and examines the chemical characteristics of refill fluids using HPLC. Refill solutions were tested on human embryonic stem cells (hESC), mouse neural stem cells (mNSC), and human pulmonary fibroblasts (hPF) using the MTT assay, and NOAELs and IC(50)s were determined from dose-response curves. Spectral analysis was performed when products of the same flavor had different MTT outcomes. hESC and mNSC were generally more sensitive to refill solutions than hPF. All products from one company were cytotoxic to hESC and mNSC, but non-cytotoxic to hPF. Cytotoxicity was not due to nicotine, but was correlated with the number and concentration of chemicals used to flavor fluids. Additional studies are needed to fully assess the prenatal effect of refill fluids.

Identification of toxicants in cinnamon-flavored electronic cigarette refill fluids

In a prior study on electronic cigarette (EC) refill fluids, Cinnamon Ceylon was the most cytotoxic of 36 products tested. The purpose of the current study was to determine if high cytotoxicity is a general feature of cinnamon-flavored EC refill fluids and to identify the toxicant(s) in Cinnamon Ceylon. Eight cinnamon-flavored refill fluids, which were screened using the MTT assay, varied in their cytotoxicity with most being cytotoxic. Human embryonic stem cells were generally more sensitive than human adult pulmonary fibroblasts. Most products were highly volatile and produced vapors that impaired survival of cells in adjacent wells. Cinnamaldehyde (CAD), 2-methoxycinnamaldehyde (2MOCA), dipropylene glycol, and vanillin were identified in the cinnamon-flavored refill fluids using gas chromatography-mass spectrometry and high-pressure liquid chromatography (HPLC). When authentic standards of each chemical were tested using the MTT assay, only CAD and 2MOCA were highly cytotoxic. The amount of each chemical in the refill fluids was quantified using HPLC, and cytotoxicity correlated with the amount of CAD/product. Duplicate bottles of the same product were similar, but varied in their concentrations of 2MOCA. These data show that the cinnamon flavorings in refill fluids are linked to cytotoxicity, which could adversely affect EC users.

Methods for Culturing Mouse and Human Embryonic Stem Cells

Mouse embryonic stem cells (mESCs) were first derived and cultured almost 30 years ago and ever since have been valuable tools for creating knockout mice and for studying early mammalian development. More recently (1998), human embryonic stem cells (hESCs) have been derived from blastocysts, and numerous methods have evolved to culture hESCs in vitro in both complex and defined media. hESCs are especially important at this time as they could potentially be used to treat degenerative diseases and to access the toxicity of new drugs and environmental chemicals. For both human and mouse ESCs, fibroblast feeder layers are often used at some phase in the culturing protocol. The feeders - often mouse embryonic fibroblasts (mEFs) - provide a substrate that increases plating efficiency, helps maintain pluripotency, and facilitates survival and growth of the stem cells. Various protocols for culturing embryonic stem cells from both species are available with newer trends moving toward feeder-free and serum-free culture. The purpose of this chapter is to provide basic protocol information on the isolation of mouse embryonic fibroblasts and establishment of feeder layers, the culture of mESCs on both mEFs and on gelatin in serum-containing medium, and the culture of hESCs in defined media on both mEFs (hESC culture medium) and Matrigel (mTeSR). These basic protocols are intended for researchers wanting to develop stem cell research in their labs. These protocols have been tested in our laboratory and work well. They can be modified and adapted for any relevant users particular purpose.

Comparison of the Toxicity of Smoke from Conventional and Harm Reduction Cigarettes Using Human Embryonic Stem Cells

This study evaluated the hypothesis that smoke from harm reduction cigarettes impedes attachment and proliferation of H9 human embryonic stem cells (hESCs). Smoke from three harm reduction brands was compared with smoke from a conventional brand. Doses of smoke were measured in puff equivalents (PE) (1 PE = the amount of smoke in one puff that dissolves in 1 ml of medium). Cytotoxic doses were determined using morphological criteria and trypan blue staining, and apoptosis was confirmed using Magic Red staining. Attachment and proliferation of hESC were followed at a noncytotoxic dose in time-lapse videos collected using BioStation technology. Data were mined from videos either manually or using video bioinformatics subroutines developed with CL-Quant software. Mainstream (MS) and sidestream (SS) smoke from conventional and harm reduction cigarettes induced apoptosis in hESC colonies at 1 PE. At 0.1 PE (noncytotoxic), SS smoke from all brands inhibited attachment of hESC colonies to Matrigel with the strongest inhibition occurring in harm reduction brands. At 0.1 PE, SS smoke, but not MS smoke, from all brands inhibited hESC growth, and two harm reduction brands were more potent than the conventional brand. In general, hESC appeared more sensitive to smoke than their mouse ESC counterparts. Although harm reduction cigarettes are often marketed as safer than conventional brands, our assays show that SS smoke from harm reduction cigarettes was at least as potent or in some cases more potent than smoke from a conventional brand and that SS smoke was more inhibitory than MS smoke in all assays.

Comparison of toxicity of smoke from traditional and harm-reduction cigarettes using mouse embryonic stem cells as a novel model for preimplantation development

BACKGROUND Embryonic stem cells (ESC), which originate from the inner cell mass of blastocysts, are valuable models for testing the effects of toxicants on preimplantation development. In this study, mouse ESC (mESC) were used to compare the toxicity of mainstream (MS) and sidestream (SS) cigarette smoke on cell attachment, survival and proliferation. In addition, smoke from a traditional commercial cigarette was compared with smoke from three harm-reduction brands. METHODS MS and SS smoke solutions were made using an analytical smoking machine and tested at three doses using D3 mESC plated on 0.2% gelatin. At 6 and 24 h, images were taken and the number of attached cells was evaluated. RESULTS Both MS and SS smoke from traditional and harm-reduction cigarettes inhibited cell attachment, survival and proliferation dose dependently. For all brands, SS smoke was more potent than MS smoke. However, removal of the cigarette filter increased the toxicity of MS smoke to that of SS smoke. Both MS and SS smoke from harm-reduction cigarettes were as inhibitory, or more inhibitory, than their counterparts from the traditional brand. When preimplantation mouse embryos were cultured for 1 h in MS or SS smoke solutions from a harm-reduction brand, blastomeres became apoptotic, in agreement with the data obtained using mESC. CONCLUSIONS mESC provide a valuable model for toxicological studies on the preimplantation stage of development and were used to show that MS and SS smoke from traditional and harm-reduction cigarettes are detrimental to embryonic cells prior to implantation.

Mouse and human embryonic stem cells: can they improve human health by preventing disease?

Given the vast number of chemicals that are released into the environment each year, it is imperative that we develop new predictive models to identify toxicants before unavoidable exposure harms the health of humans and other organisms. In vitro models are especially attractive in predictive toxicology as they can greatly reduce assay costs and animal usage while identifying those chemicals that may require further in vivo evaluation. With the derivation of both mouse and human embryonic stem cells, new opportunities have developed that could revolutionize the field of predictive toxicology. Stem cells themselves can be used to model earliest stages of development, or they can be differentiated to study later aspects of development and thereby model post-implantation. Because embryos and fetuses are usually more sensitive to environmental toxicants than adults, stem cells provide an unique tool for studying the prenatal phase in our life cycle. The embryonic stem cell test (EST), which has been validated for use with mouse ESC (mESC), is an accurate predictor of embryotoxic compounds, particularly those that are highly embryotoxic. Human embryonic stem cells (hESCs), although not yet incorporated into a validated test, are a particularly attractive platform for toxicological testing as they can give us direct information on humans and avoid concerns about species variation in response. This review discusses toxicological studies and strategies that have been used with embryonic stem cells during the past five years and possible directions that could lead to improvements in the development of predictive assays in the future.

The effect of cigarette smoke on fertilization and pre-implantation development: assessment using animal models, clinical data, and stem cells

Numerous studies have repeatedly shown that women who smoke experience problems establishing and maintaining pregnancies, and recent work has further demonstrated that the in utero effects of smoke may not be manifested until months or even years after birth. The purpose of this review is to examine the recent literature dealing with the effects of cigarette smoke on the earliest stages of human prenatal development. Studies in this area have included the use of animal models, patients undergoing in vitro fertilization, and embryonic stem cell models. Events leading to fertilization, such as cumulus expansion, hyperactivation of sperm motility, and oocyte pick-up by the oviduct are all impaired by smoke exposure in animal models. Steps crucial to fertilization such as the acrosome reaction and sperm binding to the zona pellucida are likewise inhibited by cigarette smoke. Preimplantation embryos and stem cells that model embryos show a number of adverse responses to smoke exposure, including poor adhesion to extracellular matrices, diminished survival and proliferation, and increased apoptosis. The current literature demonstrates that the earliest stages of prenatal development are sensitive to smoke exposure and indicates that pregnant women should be advised not to smoke during this time.

A method for rapid dose-response screening of environmental chemicals using human embryonic stem cells

INTRODUCTION Human embryonic stem cells (hESC) provide an invaluable model for assessing the effects of environmental chemicals and drugs on human prenatal development. However, hESC are difficult to adapt to 96-well plate screening assays, because they survive best when plated as colonies, which are difficult to count and plate accurately. The purpose of this study is to present an experimental method and analysis procedure to accomplish reliable screening of toxicants using hESC. METHODS We present a method developed to rapidly and easily determine the number of cells in small colonies of hESC spectrophotometerically and then accurately dispense equivalent numbers of cells in 96-well plates. The MTT assay was used to evaluate plating accuracy, and the method was tested using known toxicants. RESULTS The quality of the plate set-up and analysis procedure was evaluated with NIH plate validation and assessment software. All statistical parameters measured by the software were acceptable, and no drift or edge effects were observed. The 96-well plate MTT assay with hESC was tested by performing a dose-response screen of commercial products, which contain a variety of chemicals. The screen was done using single wells/dose, and the reliability of this method was demonstrated in a subsequent screen of the same products repeated three times. The single and triple screens were in good agreement, and NOAELs and IC(50)s could be determined from the single screen. The effects of vapor from volatile chemicals were studied, and methods to monitor and avoid vapor effects were incorporated into the assay. DISCUSSION Our method overcomes the difficulty of using hESC for reliable quantitative 96-well plate assays. It enables rapid dose-response screening using equipment that is commonly available in laboratories that culture hESC. This method could have a broad application in studies of environmental chemicals and drugs using hESC as models of prenatal development.

Video bioinformatics analysis of human embryonic stem cell colony growth.

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software. Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT. To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images. To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time. The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.