Mark A. Higuchi

Comparative subchronic inhalation study of smoke from the 1R4F and 2R4F reference cigarettes

A subchronic, nose-only inhalation study compared the effects of mainstream smoke from a 1R4F research cigarette to that of a 2R4F research cigarette. Male and female rats were exposed for 1 h/day, 5 days/wk, for 13 wk to mainstream smoke at 0, 0.06, 0.20, or 0.80 mg wet total particulate matter per liter of air. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin, serum nicotine, pulmonary plethysmography, gross pathology, and histopathology were determined. When histological changes resulting from exposure to smoke from the two types of cigarettes were compared, no biologically significant differences were observed. At the end of the exposure period, subsets of rats from each group were maintained without smoke exposures for an additional 13 wk (recovery period). At the end of the recovery period, there were no statistically significant differences in histopathological findings observed between the 1R4F and the 2R4F cigarettes. The complete toxicological assessment in this comparative inhalation study of 1R4F and 2R4F cigarettes suggests no overall biologically significant differences between the rats exposed to the two cigarettes.

Subchronic inhalation by rats of mainstream smoke from a cigarette that primarily heats tobacco compared to a cigarette that burns tobacco.

A subchronic, nose-only inhalation study comparing the potential biological activity of mainstream smoke from a cigarette that primarily heats tobacco (Eclipse) to mainstream smoke from a 1R4F reference cigarette was conducted using Sprague-Dawley rats of each gender. Smoke exposures were for 1 h/day, 5 days/wk for 13 wk, at concentrations of 0, 0.16, 0.32, or 0.64 mg wet total particulate matter (WTPM)/L air. Smoke was generated at the Federal Trade Commission standard of a 2-s puff of 35 ml, taken once per minute. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin, serum nicotine, plethysmography, gross pathology, and histopathology were determined. Plethysmography indicated that respiratory rate was decreased at all concentrations of 1R4F smoke, but only at the high concentration of Eclipse smoke. Tidal volume was depressed and minute volume was lower for all smoke-exposed rats. Rats exposed to Eclipse smoke inhaled more smoke at the low and mid-concentration exposures than rats exposed to equivalent concentrations 1R4F smoke. Carboxyhemoglobin and serum nicotine were directly related to the exposure concentrations of carbon monoxide (CO) and nicotine in an exposure-dependent manner. Body weights were slightly lower in smoke-exposed rats, while no treatment-related effects were seen in clinical signs, clinical chemistry, hematology, or gross changes at necropsy. The only treatment-related effect seen in organ weights was an increase in heart weight in females in the Eclipse high-concentration exposure group, attributed to higher CO in the Eclipse exposure atmosphere. Higher CO resulted from the lower dilution of Eclipse smoke required to maintain WTPM concentrations equal to those of the 1R4F smoke, and not from a higher CO yield from Eclipse cigarettes. Nasal epithelial hyperplasia and ventral laryngeal squamous metaplasia were noted after exposure to either the 1R4F or Eclipse smoke. The degree of change was less in Eclipse smoke-exposed rats. Lung macrophages were increased to a similar extent in the Eclipse and 1R4F smoke-exposed groups. Brown/gold pigmented macrophages were detected in the lungs of rats exposed to 1R4F smoke, but not those exposed to Eclipse smoke. Subsets of rats from each group were maintained for an additional 13 wk without smoke exposures. Most of the changes noted at the end of the smoke exposures had disappeared, while those that remained were regressing toward normal. Evaluation of these findings indicated the overall biological activity of Eclipse smoke was less than 1R4F smoke at comparable exposure concentrations.A subchronic, nose-only inhalation study comparing the potential biological activity of mainstream smoke from a cigarette that primarily heats tobacco (Eclipse) to mainstream smoke from a 1R4F reference cigarette was conducted using Sprague-Dawley rats of each gender. Smoke exposures were for 1 h/day, 5 days/wk for 13 wk, at concentrations of 0, 0.16, 0.32, or 0.64 mg wet total particulate matter (WTPM)/L air. Smoke was generated at the Federal Trade Commission standard of a 2-s puff of 35 ml, taken once per minute. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin, serum nicotine, plethysmography, gross pathology, and histopathology were determined. Plethysmography indicated that respiratory rate was decreased at all concentrations of 1R4F smoke, but only at the high concentration of Eclipse smoke. Tidal volume was depressed and minute volume was lower for all smoke-exposed rats. Rats exposed to Eclipse smoke inhaled more smoke at the low and mid-concentration exposures than rats exposed to equivalent concentrations 1R4F smoke. Carboxyhemoglobin and serum nicotine were directly related to the exposure concentrations of carbon monoxide (CO) and nicotine in an exposure-dependent manner. Body weights were slightly lower in smokeexposed rats, while no treatment-related effects were seen in clinical signs, clinical chemistry, hematology, or gross changes at necropsy. The only treatment-related effect seen in organ weights was an increase in heart weight in females in the Eclipse high-concentration exposure group, attributed to higher CO in the Eclipse exposure atmosphere. Higher CO resulted from the lower dilution of Eclipse smoke required to maintain WTPM concentrations equal to those of the 1R4F smoke, and not from a higher CO yield from Eclipse cigarettes. Nasal epithelial hyperplasia and ventral laryngeal squamous metaplasia were noted after exposure to either the 1R4F or Eclipse smoke. The degree of change was less in Eclipse smoke-exposed rats. Lung macrophages were increased to a similar extent in the Eclipse and 1R4F smoke-exposed groups. Brown/gold pigmented macrophages were detected in the lungs of rats exposed to 1R4F smoke, but not those exposed to Eclipse smoke. Subsets of rats from each group were maintained for an additional 13 wk without smoke exposures. Most of the changes noted at the end of the smoke exposures had disappeared, while those that remained were regressing toward normal. Evaluation of these findings indicated the overall biological activity of Eclipse smoke was less than 1R4F smoke at comparable exposure concentrations.

Generation and characterization of diesel engine combustion emissions from petroleum diesel and soybean biodiesel fuels and application for inhalation exposure studies

Abstract Biodiesel made from the transesterification of plant- and animal-derived oils is an important alternative fuel source for diesel engines. Although numerous studies have reported health effects associated with petroleum diesel emissions, information on biodiesel emissions are more limited. To this end, a program at the U.S. EPA assessed health effects of biodiesel emissions in rodent inhalation models. Commercially obtained soybean biodiesel (B100) and a 20% blend with petroleum diesel (B20) were compared to pure petroleum diesel (B0). Rats and mice were exposed independently for 4 h/day, 5 days/week for up to 6 weeks. Exposures were controlled by dilution air to obtain low (50 µg/m3), medium (150 µg/m3) and high (500 µg/m3) diesel particulate mass (PM) concentrations, and compared to filtered air. This article provides details on facilities, fuels, operating conditions, emission factors and physico-chemical characteristics of the emissions used for inhalation exposures and in vitro studies. Initial engine exhaust PM concentrations for the B100 fuel (19.7 ± 0.7 mg/m3) were 30% lower than those of the B0 fuel (28.0 ± 1.5 mg/m3). When emissions were diluted with air to control equivalent PM mass concentrations, B0 exposures had higher CO and slightly lower NO concentrations than B100. Organic/elemental carbon ratios and oxygenated methyl esters and organic acids were higher for the B100 than B0. Both the B0 and B100 fuels produced unimodal-accumulation mode particle-size distributions, with B0 producing lower concentrations of slightly larger particles. Subsequent papers in this series will describe the effects of these atmospheres on cardiopulmonary responses and in vitro genotoxicity studies.

Mutagenicity and pollutant emission factors of solid-fuel cookstoves: Comparison with other combustion sources

Background: Emissions from solid fuels used for cooking cause ~4 million premature deaths per year. Advanced solid-fuel cookstoves are a potential solution, but they should be assessed by appropriate performance indicators, including biological effects. Objective: We evaluated two categories of solid-fuel cookstoves for eight pollutant and four mutagenicity emission factors, correlated the mutagenicity emission factors, and compared them to those of other combustion emissions. Methods: We burned red oak in a 3-stone fire (TSF), a natural-draft stove (NDS), and a forced-draft stove (FDS), and we combusted propane as a liquified petroleum gas control fuel. We determined emission factors based on useful energy (megajoules delivered, MJd) for carbon monoxide, nitrogen oxides (NOx), black carbon, methane, total hydrocarbons, 32 polycyclic aromatic hydrocarbons, PM2.5, levoglucosan (a wood-smoke marker), and mutagenicity in Salmonella. Results: With the exception of NOx, the emission factors per MJd were highly correlated (r ≥ 0.97); the correlation for NOx with the other emission factors was 0.58–0.76. Excluding NOx, the NDS and FDS reduced the emission factors an average of 68 and 92%, respectively, relative to the TSF. Nevertheless, the mutagenicity emission factor based on fuel energy used (MJthermal) for the most efficient stove (FDS) was between those of a large diesel bus engine and a small diesel generator. Conclusions: Both mutagenicity and pollutant emission factors may be informative for characterizing cookstove performance. However, mutagenicity emission factors may be especially useful for characterizing potential health effects and should be evaluated in relation to health outcomes in future research. An FDS operated as intended by the manufacturer is safer than a TSF, but without adequate ventilation, it will still result in poor indoor air quality. Citation: Mutlu E, Warren SH, Ebersviller SM, Kooter IM, Schmid JE, Dye JA, Linak WP, Gilmour MI, Jetter JJ, Higuchi M, DeMarini DM. 2016. Mutagenicity and pollutant emission factors of solid-fuel cookstoves: comparison with other combustion sources. Environ Health Perspect 124:974–982; http://dx.doi.org/10.1289/ehp.1509852

ANALYSIS OF POTENTIAL TRANSFER OF CONTINUOUS GLASS FILAMENT FROM ECLIPSE CIGARETTES

This study was designed to determine if a prototype of the Eclipse cigarettes, which uses a special form of continuous glass filament (CGF) as an insulator around the carbon heat source, yielded CGFs via mainstream smoke. A method was developed that used electrostatic precipitation with a greater than 99% collection efficiency of mass to capture CGFs transferred to mainstream smoke. The cigarettes were smoked using an exaggerated puffing condition that was more than twice the Federal Trade Commission (FTC) standard. The cigarettes were subjected to handling procedures that simulated commercial shipping conditions prior to smoking. CGFs were intentionally added to a series of smoke condensate samples to determine CGF recovery efficiency. The recovery efficiency was determined for a series of four internal standards added to smoke condensate. The recovery efficiency was 86% for the Eclipse 5-014 prototype. The number of CGFs in smoke condensate collected from the Eclipse 5-014 prototype was approximately 0.06 ± 0.02 CGFs per cigarette ( ± standard deviation), including the background counts of CGFs and 0.03 CGFs per cigarette, when corrected for background contributions. The number of CGFs found in smoke condensates for this prototype was not statistically distinguishable from zero or background in these experiments, which were capable of detecting transfer rates of greater than 0.2 CGFs per cigarette.This study was designed to determine if a prototype of the Eclipse cigarettes, which uses a special form of continuous glass filament (CGF) as an insulator around the carbon heat source, yielded CGFs via mainstream smoke. A method was developed that used electrostatic precipitation with a greater than 99% collection efficiency of mass to capture CGFs transferred to mainstream smoke. The cigarettes were smoked using an exaggerated puffing condition that was more than twice the Federal Trade Commission (FTC) standard. The cigarettes were subjected to handling procedures that simulated commercial shipping conditions prior to smoking. CGFs were intentionally added to a series of smoke condensate samples to determine CGF recovery efficiency. The recovery efficiency was determined for a series of four internal standards added to smoke condensate. The recovery efficiency was 86% for the Eclipse 5-014 prototype. The number of CGFs in smoke condensate collected from the Eclipse 5-014 prototype was approximately 0.06 +/- 0.02 CGFs per cigarette (+/- standard deviation), including the background counts of CGFs and 0.03 CGFs per cigarette, when corrected for background contributions. The number of CGFs found in smoke condensates for this prototype was not statistically distinguishable from zero or background in these experiments, which were capable of detecting transfer rates of greater than 0.2 CGFs per cigarette.

Quantative Analysis of Potential Transfer of Continuous Glass Filament From Eclipse Prototype 9-014 Cigarettes

This study was designed to determine if the Eclipse prototype 9-014 cigarettes, which use a special form of continuous glass filament (CGF) as an insulator around the carbon heat source, yield CGFs via mainstream smoke. A previously developed method (Higuchi et al., 2000) that employed electrostatic precipitation—with a greater than 99% collection efficiency of mass—was used to capture CGFs transferred to mainstream smoke. The cigarettes were smoked using an exaggerated puffing condition more than twice the Federal Trade Commission (FTC) standard. Prior to smoking, cigarettes were subjected to handling procedures that simulated commercial shipping conditions. Using a modified standard addition method, and utilizing a mixture of water and glycerol as a mock condensate, CGFs were intentionally added to a series of (mock condensate) samples to develop knowledge of CGF recovery efficiency. The linear regression model of the recovered CGFs demonstrated a recovery efficiency of 86%. This efficiency rate was applied to the number of CGFs recovered from samples of smoke condensate and associated background samples. The number of CGFs in smoke condensate collected from the Eclipse 9-014 prototype was approximately 0.32 ± 0.17 CGFs per cigarette (± standard deviation), including the background counts of CGFs, and 0.16 CGFs per cigarette when corrected for background contributions. The number of CGFs found in the smoke condensates for this prototype was statistically (p = .00031) distinguishable from zero and background in these experiments. The low number of CGFs seen in the transfer data from this prototype studied, the unique physical characteristics of the filaments ( e.g., controlled physical dimensions), and the absence of biological activity of similar glass filaments/fibers indicate that biologically significant exposure to the Eclipse smoker does not occur.This study was designed to determine if the Eclipse prototype 9-014 cigarettes, which use a special form of continuous glass filament (CGF) as an insulator around the carbon heat source, yield CGFs via mainstream smoke. A previously developed method (Higuchi et al., 2000) that employed electrostatic precipitation-with a greater than 99% collection efficiency of mass-was used to capture CGFs transferred to mainstream smoke. The cigarettes were smoked using an exaggerated puffing condition more than twice the Federal Trade Commission (FTC) standard. Prior to smoking, cigarettes were subjected to handling procedures that simulated commercial shipping conditions. Using a modified standard addition method, and utilizing a mixture of water and glycerol as a mock condensate, CGFs were intentionally added to a series of (mock condensate) samples to develop knowledge of CGF recovery efficiency. The linear regression model of the recovered CGFs demonstrated a recovery efficiency of 86%. This efficiency rate was applied to the number of CGFs recovered from samples of smoke condensate and associated background samples. The number of CGFs in smoke condensate collected from the Eclipse 9-014 prototype was approximately 0.32 +/- 0.17 CGFs per cigarette (+/- standard deviation), including the background counts of CGFs, and 0.16 CGFs per cigarette when corrected for background contributions. The number of CGFs found in the smoke condensates for this prototype was statistically (p =.00031) distinguishable from zero and background in these experiments. The low number of CGFs seen in the transfer data from this prototype studied, the unique physical characteristics of the filaments (e.g., controlled physical dimensions), and the absence of biological activity of similar glass filaments/fibers indicate that biologically significant exposure to the Eclipse smoker does not occur.

Ambient Particulate Matter and Acrolein Co-Exposure Increases Myocardial Dyssynchrony in Mice via TRPA1

Air pollution is a complex mixture of particulate matter and gases linked to adverse clinical outcomes. As such, studying responses to individual pollutants does not account for the potential biological responses resulting from the interaction of various constituents within an ambient air shed. We previously reported that exposure to high levels of the gaseous pollutant acrolein perturbs myocardial synchrony. Here, we examined the effects of repeated, intermittent co-exposure to low levels of concentrated ambient particulates (CAPs) and acrolein on myocardial synchrony and the role of transient receptor potential cation channel A1 (TRPA1), which we previously linked to air pollution-induced sensitization to triggered cardiac arrhythmia. Female B6129 and Trpa1-/- mice (n = 6/group) were exposed to filtered air (FA), CAPs (46 µg/m3 of PM2.5), Acrolein (0.42 ppm), or CAPs+Acrolein for 3 h/day, 2 days/week for 4 weeks. Cardiac ultrasound was conducted to assess cardiac synchronicity and function before and after the first exposure and after the final exposure. Heart rate variability (HRV), an indicator of autonomic tone, was assessed after the final exposure. Strain delay (time between peak strain in adjacent cardiac wall segments), an index of myocardial dyssynchrony, increased by 5-fold after the final CAPs+Acrolein exposure in B6129 mice compared with FA, CAPs, or Acrolein-exposed B6129 mice, and CAPs+Acrolein-exposed Trpa1-/- mice. Only exposure to acrolein alone increased the HRV high frequency domain (5-fold) in B6129 mice, but not in Trpa1-/- mice. Thus, repeated inhalation of pollutant mixtures may increase risk for cardiac responses compared with single or multiple exposures to individual pollutants through TRPA1 activation.

A new cell culture exposure system for studying the toxicity of volatile chemicals at the air-liquid interface.

Abstract A cell culture exposure system (CCES) was developed to expose cells established at an air–liquid interface (ALI) to volatile chemicals. We characterized the CCES by exposing indigo dye-impregnated filter inserts inside culture wells to 125 ppb ozone (O3) for 1 h at flow rates of 5 and 25 mL/min/well; the reaction of O3 with an indigo dye produces a fluorescent product. A 5-fold increase in fluorescence at 25 mL/min/well versus 5 mL/min/well was observed, suggesting higher flows were more effective. We then exposed primary human bronchial epithelial cells (HBECs) to 0.3 ppm acrolein for 2 h at 3, 5, and 25 mL/min/well and compared our results against well-established in vitro exposure chambers at the U.S. EPA’s Human Studies Facility (HSF Chambers). We measured transcript changes of heme oxygenase-1 (HMOX1) and interleukin-8 (IL-8), as well as lactate dehydrogenase (LDH) release, at 0, 1, and 24 h post-exposure. Comparing responses from HSF Chambers to the CCES, differences were only observed at 1 h post-exposure for HMOX1. Here, the HSF Chamber produced a ∼6-fold increase while the CCES at 3 and 5 mL/min/well produced a ∼1.7-fold increase. Operating the CCES at 25 mL/min/well produced a ∼4.5-fold increase; slightly lower than the HSF Chamber. Our biological results, supported by our comparison against the HSF Chambers, agree with our fluorescence results, suggesting that higher flows through the CCES are more effective at delivering volatile chemicals to cells. This new CCES will be deployed to screen the toxicity of volatile chemicals in EPA’s chemical inventories.

Differential exposure and acute health impacts of inhaled solid-fuel emissions from rudimentary and advanced cookstoves in female CD-1 mice

Background: There is an urgent need to provide access to cleaner end user energy technologies for the nearly 40% of the worlds population who currently depend on rudimentary cooking and heating systems. Advanced cookstoves (CS) are designed to cut emissions and solid‐fuel consumption, thus reducing adverse human health and environmental impacts. Study premise: We hypothesized that, compared to a traditional (Tier 0) three‐stone (3‐S) fire, acute inhalation of solid‐fuel emissions from advanced natural‐draft (ND; Tier 2) or forced‐draft (FD; Tier 3) stoves would reduce exposure biomarkers and lessen pulmonary and innate immune system health effects in exposed mice. Results: Across two simulated cooking cycles (duration ˜ 3 h), emitted particulate mass concentrations were reduced 80% and 62% by FD and ND stoves, respectively, compared to the 3‐S fire; with corresponding decreases in particles visible within murine alveolar macrophages. Emitted carbon monoxide was reduced ˜ 90% and ˜ 60%, respectively. Only 3‐S‐fire‐exposed mice had increased carboxyhemoglobin levels. Emitted volatile organic compounds were FD ≪ 3‐S‐fire ≤ ND stove; increased expression of genes involved in xenobiotic metabolism (COX‐2, NQO1, CYP1a1) was detected only in ND‐ and 3‐S‐fire‐exposed mice. Diminished macrophage phagocytosis was observed in the ND group. Lung glutathione was significantly depleted across all CS groups, however the FD group had the most severe, ongoing oxidative stress. Conclusions: These results are consistent with reports associating exposure to solid fuel stove emissions with modulation of the innate immune system and increased susceptibility to infection. Lower respiratory infections continue to be a leading cause of death in low‐income economies. Notably, 3‐S‐fire‐exposed mice were the only group to develop acute lung injury, possibly because they inhaled the highest concentrations of hazardous air toxicants (e.g., 1,3‐butadiene, toluene, benzene, acrolein) in association with the greatest number of particles, and particles with the highest % organic carbon. However, no Tier 0–3 ranked CS group was without some untoward health effect indicating that access to still cleaner, ideally renewable, energy technologies for cooking and heating is warranted. HIGHLIGHTSMice inhaled emissions from advanced cookstoves (CS) and a 3‐stone (3‐S)‐fire.Tier 2 and 3 CS emitted significantly less particulate matter and carbon monoxide.3‐S fire (Tier 0)‐exposed mice were the only group to develop acute lung injury.Diminished macrophage phagocytosis was observed in Tier 2 CS‐exposed mice.Lung glutathione was significantly depleted across all Tier 0–3 CS‐exposed mice.