Applying new approach methodologies to assess next-generation tobacco and nicotine products.

In vitro toxicology research has accelerated with the use of in silico, computational approaches and human in vitro tissue systems, facilitating major improvements evaluating the safety and health risks of novel consumer products. Innovation in molecular and cellular biology has shifted testing paradigms, with less reliance on low-throughput animal data and greater use of medium- and high-throughput in vitro cellular screening approaches. These new approach methodologies (NAMs) are being implemented in other industry sectors for chemical testing, screening candidate drugs and prototype consumer products, driven by the need for reliable, human-relevant approaches. Routine toxicological methods are largely unchanged since development over 50 years ago, using high-doses and often employing in vivo testing. Several disadvantages are encountered conducting or extrapolating data from animal studies due to differences in metabolism or exposure. The last decade saw considerable advancement in the development of in vitro tools and capabilities, and the challenges of the next decade will be integrating these platforms into applied product testing and acceptance by regulatory bodies. Governmental and validation agencies have launched and applied frameworks and "roadmaps" to support agile validation and acceptance of NAMs. Next-generation tobacco and nicotine products (NGPs) have the potential to offer reduced risks to smokers compared to cigarettes. These include heated tobacco products (HTPs) that heat but do not burn tobacco; vapor products also termed electronic nicotine delivery systems (ENDS), that heat an e-liquid to produce an inhalable aerosol; oral smokeless tobacco products (e.g., Swedish-style snus) and tobacco-free oral nicotine pouches. With the increased availability of NGPs and the requirement of scientific studies to support regulatory approval, NAMs approaches can supplement the assessment of NGPs. This review explores how NAMs can be applied to assess NGPs, highlighting key considerations, including the use of appropriate in vitro model systems, deploying screening approaches for hazard identification, and the importance of test article characterization. The importance and opportunity for fit-for-purpose testing and method standardization are discussed, highlighting the value of industry and cross-industry collaborations. Supporting the development of methods that are accepted by regulatory bodies could lead to the implementation of NAMs for tobacco and nicotine NGP testing.

Exploring in vitro to in vivo extrapolation for exposure and health impacts of e-cigarette flavor mixtures.

In vitro to in vivo extrapolation (IVIVE) leverages in vitro biological activities to predict corresponding in vivo exposures, therefore potentially reducing the need for animal safety testing that are traditionally performed to support the hazard and risk assessment. Interpretation of IVIVE predictions are affected by various factors including the model type, exposure route and kinetic assumptions for the test article, and choice of in vitro assay(s) that are relevant to clinical outcomes. Exposure scenarios are further complicated for mixtures where the in vitro activity may stem from one or more components in the mixture. In this study, we used electronic cigarette (EC) aerosols, a complex mixture, to explore impacts of these factors on the use of IVIVE in hazard identification, using open-source pharmacokinetic models of varying complexity and publicly available data. Results suggest in vitro assay selection has a greater impact on exposure estimates than modeling approaches. Using cytotoxicity assays, high exposure estimates (>1000 EC cartridges (pods) or > 700 mL EC liquid per day) would be needed to obtain the in vivo plasma levels that are corresponding to in vitro assay data, suggesting acute toxicity would be unlikely in typical usage scenarios. When mechanistic (Tox21) assays were used, the exposure estimates were much lower for the low end, but the range of exposure estimate became wider across modeling approaches. These proof-of-concept results highlight challenges and complexities in IVIVE for mixtures.

Evaluation of Inhalation Exposures and Potential Health Impacts of Ingredient Mixtures Using in vitro to in vivo Extrapolation.

In vitro methods offer opportunities to provide mechanistic insight into bioactivity as well as human-relevant toxicological assessments compared to animal testing. One of the challenges for this task is putting in vitro bioactivity data in an in vivo exposure context, for which in vitro to in vivo extrapolation (IVIVE) translates in vitro bioactivity to clinically relevant exposure metrics using reverse dosimetry. This study applies an IVIVE approach to the toxicity assessment of ingredients and their mixtures in e-cigarette (EC) aerosols as a case study. Reported in vitro cytotoxicity data of EC aerosols, as well as in vitro high-throughput screening (HTS) data for individual ingredients in EC liquids (e-liquids) are used. Open-source physiologically based pharmacokinetic (PBPK) models are used to calculate the plasma concentrations of individual ingredients, followed by reverse dosimetry to estimate the human equivalent administered doses (EADs) needed to obtain these plasma concentrations for the total e-liquids. Three approaches (single actor approach, additive effect approach, and outcome-oriented ingredient integration approach) are used to predict EADs of e-liquids considering differential contributions to the bioactivity from the ingredients (humectant carriers [propylene glycol and glycerol], flavors, benzoic acid, and nicotine). The results identified critical factors for the EAD estimation, including the ingredients of the mixture considered to be bioactive, in vitro assay selection, and the data integration approach for mixtures. Further, we introduced the outcome-oriented ingredient integration approach to consider e-liquid ingredients that may lead to a common toxicity outcome (e.g., cytotoxicity), facilitating a quantitative evaluation of in vitro toxicity data in support of human risk assessment.

E-vapor aerosols do not compromise bone integrity relative to cigarette smoke after 6-month inhalation in an ApoE-/- mouse model.

Cigarette smoke (CS) exposure is one of the leading risk factors for human health. Nicotine-containing inhalable products, such as e-cigarettes, can effectively support tobacco harm reduction approaches. However, there are limited comparative data on the effects of the aerosols generated from electronic vapor products (e-vapor) and CS on bone. Here, we report the effects of e-vapor aerosols and CS on bone morphology, structure, and strength in a 6-month inhalation study. Eight-week-old ApoE-/- mice were exposed to aerosols from three different e-vapor formulations-CARRIER (propylene glycol and vegetable glycerol), BASE (CARRIER and nicotine), TEST (BASE and flavor)-to CS from 3R4F reference cigarettes at matched nicotine concentrations (35 µg/L) or to fresh air (Sham) (N = 10 per group). Tibiae were analyzed for bone morphology by µCT imaging, biomechanics by three-point bending, and by histological analysis. CS inhalation caused a significant decrease in cortical and total bone volume fraction and bone density relative to e-vapor aerosols. Additionally, CS exposure caused a decrease in ultimate load and stiffness. In contrast, bone structural and biomechanical parameters were not significantly affected by e-vapor aerosol or Sham exposure. At the dissection time point, there was no significant difference in body weight or tibia bone weight or length among the groups. Histological findings revealed microcracks in cortical bone areas among all exposed groups compared to Sham control. In conclusion, because of the bone-preserving effect of e-vapor aerosols relative to CS exposure, e-vapor products could potentially constitute less harmful alternatives to cigarettes in situations in which bone health is of importance.

Comparison of experimentally measured and computational fluid dynamic predicted deposition and deposition uniformity of monodisperse solid particles in the Vitrocell® AMES 48 air-liquid-interface in-vitro exposure system.

Accurately determining the delivered dose is critical to understanding biological response due to cell exposure to chemical constituents in aerosols. Deposition efficiency and uniformity of deposition was measured experimentally using monodisperse solid fluorescent particles with mass median aerodynamic diameters (MMAD) of 0.51, 1.1, 2.2 and 3.3 µm in the Vitrocell® AMES 48 air-liquid-interface (ALI) in vitro exposure system. Experimental results were compared with computational fluid dynamic, (CFD; using both Lagrangian and Eulerian approaches) predicted deposition efficiency and uniformity for a single row (N = 6) of petri dishes in the Vitrocell® AMES 48 system. The average experimentally measured deposition efficiency ranged from 0.007% to 0.43% for 0.51-3.3 µm MMAD particles, respectively. There was good agreement between average experimentally measured and the CFD predicted particle deposition efficiency, regardless of approach. Experimentally measured and CFD predicted average uniformity of deposition was greater than 45% of the mean for all particle diameters. During this work a new design was introduced by the manufacturer and evaluated using Lagragian CFD. Lagragian CFD predictions showed better uniformity of deposition, but reduced deposition efficiency with the new design. Deposition efficiency and variability in particle deposition across petri dishes for solid particles should be considered when designing exposure regimens using the Vitrocell® AMES 48 ALI in vitro exposure system.

Biological changes in C57BL/6 mice following 3 weeks of inhalation exposure to cigarette smoke or e-vapor aerosols.

We compared early biological changes in mice after inhalation exposures to cigarette smoke or e-vapor aerosols (MarkTen® cartridge with Carrier, Test-1, or Test-2 formulations; 4% nicotine). Female C57BL/6 mice were exposed to 3R4F cigarette smoke or e-vapor aerosols by nose-only inhalation for up to 4 hours/day, 5 days/week, for 3 weeks. The 3R4F and e-vapor exposures were set to match the target nose port aerosol nicotine concentration (∼41 µg/L). Only the 3R4F group showed postexposure clinical signs such as tremors and lethargy. At necropsy, the 3R4F group had significant increases in lung weight and changes in bronchoalveolar lavage parameters, as well as microscopic findings in the respiratory tract. The e-vapor groups had minimal microscopic changes, including squamous metaplasia in laryngeal epiglottis, and histiocytic infiltrates in the lung (Test-2 group only). The 3R4F group had a higher incidence and severity of microscopic findings compared to any e-vapor group. Transcriptomic analysis also showed that the 3R4F group had the highest number of differentially expressed genes compared to Sham Control. Among e-vapor groups, Test-2 group had more differentially expressed genes but the magnitude of gene expression-based network perturbations in all e-vapor groups was ∼94% less than the 3R4F group. On proteome analysis in the lung, differentially regulated proteins were detected in the 3R4F group only. In conclusion, 3-weeks of 3R4F exposure induced molecular and microscopic changes associated with smoking-related diseases in the respiratory tract, while e-vapor exposures showed substantially reduced biological activities.

Biological responses in rats exposed to mainstream smoke from a heated cigarette compared to a conventional reference cigarette.

The heated cigarette (HC) generates mainstream smoke by vaporizing the components of the tobacco rod using a carbon heat source at the cigarette tip. Mainstream smoke of HC contains markedly less chemical constituents compared to combusted cigarettes. Mainstream smoke from HC was generated under Health Canada Intense regimen and its biological effects were compared to those of Reference (3R4F) cigarettes, using nose-only 5-week and 13-week inhalation studies. In the 13-week study, SD rats were necropsied following exposure to mainstream smoke from each cigarette at 200, 600 or 1000 µg wet total particulate matter/L for 1 h/day, 7 days/week or following a 13-week recovery period. Histopathological changes in the respiratory tract were significantly lesser in HC groups; e.g. respiratory epithelial hyperplasia in the nasal cavity and accumulation of pigmented macrophages in alveoli. After a 13-week recovery, the lesions were completely or partially regressed, except for accumulation of pigmented macrophages in alveoli, in both HC and 3R4F groups. In the 5-week study, SD rats were necropsied following exposure to mainstream smoke of either cigarette at 600 or 1000 µg/L for 1 h, two times/day (with 30 min interval), 7 days/week or following a 4-week recovery period. Bronchoalveolar lavage fluid (BALF) analysis of neutrophil percentages and enzyme levels like γ-GT, ALP and LDH indicated that pulmonary inflammation was significantly less in HC groups compared to 3R4F groups. In conclusion, HC demonstrated significantly lower biological effects compared to 3R4F, based on the BALF parameters and histopathology.

Comparison of dermal tumor promotion activity of cigarette smoke condensate from prototype (heated) cigarette and reference (combusted) cigarette in SENCAR mice.

Test cigarette (prototype "heated" cigarette) was evaluated on its dermal tumor promotion activity in SENCAR mice relative to conventional 3R4F cigarette. Mainstream cigarette smoke was generated under the modified Health Canada Intensive Regimen, and smoke condensate (CSCs) were collected using cold traps and extracted with acetone. Female mice received a topical application of 7,12-dimehtylbenz(a)anthracene (DMBA) as the tumor initiator on the back skin during Week 1. Subsequently, CSC was repeatedly applied as the tumor promoter at 5 doses, up to 30 mg tar/application, three times per week for 30 weeks. Test groups showed a clearly longer latency at lower doses (⩽15 mg), but the difference was less clear at higher doses (⩾22.5 mg), while mortalities were not affected throughout the study. Test groups also had consistently lower incidence and multiplicity of neoplasms, as well as lower incidences of non-neoplastic changes (e.g., inflammations and squamous epithelial hyperplasia on the site of application). The group without DMBA initiation did not induce any neoplasm but the respective Reference group showed an increase in tumorigenicity. In conclusion, the study demonstrated significant reduction in dermal irritancy and tumorigenicity of Test CSC compared to Reference CSC.

Comparison of biological responses in rats under various cigarette smoke exposure conditions.

A variety of exposure regimens of cigarette smoke have been used in animal models of lung diseases. In this study, we compared biological responses of smoke exposure in rats, using different smoke concentrations (wet total particulate matter [WTPM]), daily exposure durations, and total days of exposure. As a range-finding acute study, we first compared pulmonary responses between SD and F344 strains after a single nose-only exposure to mainstream cigarette smoke or LPS. Secondly, F344 rats were exposed to cigarette smoke for 2 or 13 weeks under the comparable daily exposure dose (WTPM concentration x daily exposure duration; according to Haber's rule) but at a different WTPM concentration or daily exposure duration. Blood carboxylhemoglobin was increased linearly to the WTPM concentration, while urinary nicotine plus cotinine value was higher for the longer daily exposure than the corresponding shorter exposure groups. Gamma glutamyl transferase activity in bronchoalveolar lavage fluid (BALF) was increased dose dependently after 2 and 13 weeks of cigarette smoke exposure, while the neutrophil content in BALF was not increased notably. Smoke-exposed groups showed reduced body weight gain and increased relative lung and heart weights. While BALF parameters and the relative lung weights suggest pulmonary responses, histopathological examination showed epithelial lesions mainly in the upper respiratory organs (nose and larynx). Collectively, the results indicate that, under the employed study design, the equivalent daily exposure dose (exposure concentration x duration) induces equivalent pulmonary responses in rats.

Impaired transcriptional response of the murine heart to cigarette smoke in the setting of high fat diet and obesity.

Smoking and obesity are each well-established risk factors for cardiovascular heart disease, which together impose earlier onset and greater severity of disease. To identify early signaling events in the response of the heart to cigarette smoke exposure within the setting of obesity, we exposed normal weight and high fat diet-induced obese (DIO) C57BL/6 mice to repeated inhaled doses of mainstream (MS) or sidestream (SS) cigarette smoke administered over a two week period, monitoring effects on both cardiac and pulmonary transcriptomes. MS smoke (250 µg wet total particulate matter (WTPM)/L, 5 h/day) exposures elicited robust cellular and molecular inflammatory responses in the lung with 1466 differentially expressed pulmonary genes (p < 0.01) in normal weight animals and a much-attenuated response (463 genes) in the hearts of the same animals. In contrast, exposures to SS smoke (85 µg WTPM/L) with a CO concentration equivalent to that of MS smoke (~250 CO ppm) induced a weak pulmonary response (328 genes) but an extensive cardiac response (1590 genes). SS smoke and to a lesser extent MS smoke preferentially elicited hypoxia- and stress-responsive genes as well as genes predicting early changes of vascular smooth muscle and endothelium, precursors of cardiovascular disease. The most sensitive smoke-induced cardiac transcriptional changes of normal weight mice were largely absent in DIO mice after smoke exposure, while genes involved in fatty acid utilization were unaffected. At the same time, smoke exposure suppressed multiple proteome maintenance genes induced in the hearts of DIO mice. Together, these results underscore the sensitivity of the heart to SS smoke and reveal adaptive responses in healthy individuals that are absent in the setting of high fat diet and obesity.

Diet-induced obesity reprograms the inflammatory response of the murine lung to inhaled endotoxin.

The co-occurrence of environmental factors is common in complex human diseases and, as such, understanding the molecular responses involved is essential to determine risk and susceptibility to disease. We have investigated the key biological pathways that define susceptibility for pulmonary infection during obesity in diet-induced obese (DIO) and regular weight (RW) C57BL/6 mice exposed to inhaled lipopolysaccharide (LPS). LPS induced a strong inflammatory response in all mice as indicated by elevated cell counts of macrophages and neutrophils and levels of proinflammatory cytokines (MDC, MIP-1γ, IL-12, RANTES) in the bronchoalveolar lavage fluid. Additionally, DIO mice exhibited 50% greater macrophage cell counts, but decreased levels of the cytokines, IL-6, TARC, TNF-α, and VEGF relative to RW mice. Microarray analysis of lung tissue showed over half of the LPS-induced expression in DIO mice consisted of genes unique for obese mice, suggesting that obesity reprograms how the lung responds to subsequent insult. In particular, we found that obese animals exposed to LPS have gene signatures showing increased inflammatory and oxidative stress response and decreased antioxidant capacity compared with RW. Because signaling pathways for these responses can be common to various sources of environmentally induced lung damage, we further identified biomarkers that are indicative of specific toxicant exposure by comparing gene signatures after LPS exposure to those from a parallel study with cigarette smoke. These data show obesity may increase sensitivity to further insult and that co-occurrence of environmental stressors result in complex biosignatures that are not predicted from analysis of individual exposures.

Comparison of mouse strains and exposure conditions in acute cigarette smoke inhalation studies.

Cigarette smoke exposures in mice have been conducted under various exposure conditions using different strains as animal models of smoke-related diseases. We exposed cigarette smoke to two strains of mice [C57BL/6J (C57) and AKR/J (AKR)] under two different exposure regimens (1 h or 4 h/day) at equivalent daily exposure amount (concentration × time). After 2 weeks exposure, mice were evaluated using exposure markers and biological responses. Smoke exposure suppressed respiratory parameters dependent on exposure concentration. The 1-h regimen groups generally showed a greater degree of respiratory suppression and relatively lower exposure markers of urinary nicotine metabolites than the corresponding 4-h regimen groups. Tidal volume was more suppressed in AKR compared to C57, while respiratory rate was more suppressed in C57. Plasma exposure markers and respiratory parameters suggested that C57 inhaled more volume of smoke than AKR. Changes in bronchoalveolar lavage fluid (BALF) cytology and enzyme parameters were most noticeable in the 1 h AKR groups. In BALF cytokine concentration, TARC concentration in C57 was higher than AKR, while KC and MCP-1 in AKR were higher than C57. Relative lung/body weight ratio in smoke-exposed C57 was generally higher, as well as the incidence and severity of lesions in respiratory organs compared to AKR. In summary, C57 appeared to inhale relatively more smoke and displayed greater inflammatory changes in respiratory tract than AKR. Comparison of exposure regimens suggests that a longer exposure duration at lower WTPM concentration might deliver a larger dose of smoke than a shorter exposure duration at higher WTPM concentration.

Comparison of the physiological and morphological effects of cigarette smoke exposure at comparable weekly doses on Sprague-Dawley rats.

A variety of dose x duration exposure regimens have been used in inhalation toxicity studies using rodents. We evaluated the effects of differences in smoke concentration and daily exposure duration under similar weekly cumulative exposures in rats to determine potential variation in type and severity of adverse effects in 13-week exposure studies. The weekly cumulative dosages were 2100 and 4200 µg wet total particle matter (WTPM)/L, and the daily exposure durations were 1 and 6 h. Weekly exposure duration was 5 or 7 days/week for groups exposed 1 h/day and 7 days/week for groups exposed 6 h/day. Recovery duration was 6 and 13 weeks. Mainstream smoke exposure suppressed body weight (BW) gain in both regimens. Lower dose groups exposed 1 h/day had a consistently greater of BW gain compared with corresponding 6 h/day groups. Respiratory rate, tidal volume, and minute volume (MV) were suppressed in a dose-dependent manner in both regimens. Higher MV in rats exposed for 6 h/day compared with rats exposed 1 h/day suggested that a lower concentration for longer duration resulted in a greater total inhaled mass (TIM) in rats exposed 6 h/day. Groups exposed for 6 h/day had lower blood carboxyhemoglobin and plasma nicotine levels than groups exposed 1 h/day, reflecting the lower carbon monoxide (CO) and WTPM concentrations in the 6 h/day groups. Data from examination of bronchoalveolar lavage fluids (BALF) and respiratory tract tissues indicated comparable effects between both regimens. Exposure-induced histopathological changes regressed similarly for both regimens after the recovery periods.

Comparison of two quantitative methods of discerning airspace enlargement in smoke-exposed mice.

In this work, we compare two methods for evaluating and quantifying pulmonary airspace enlargement in a mouse model of chronic cigarette smoke exposure. Standard stereological sample preparation, sectioning, and imaging of mouse lung tissues were performed for semi-automated acquisition of mean linear intercept (L(m)) data. After completion of the L(m) measurements, D(2), a metric of airspace enlargement, was measured in a blinded manner on the same lung images using a fully automated technique developed in-house. An analysis of variance (ANOVA) shows that although L(m) was able to separate the smoke-exposed and control groups with statistical significance (p = 0.034), D(2) was better able to differentiate the groups (p<0.001) and did so without any overlap between the control and smoke-exposed individual animal data. In addition, the fully automated implementation of D(2) represented a time savings of at least 24x over semi-automated L(m) measurements. Although D(2) does not provide 3D stereological metrics of airspace dimensions as L(m) does, results show that it has higher sensitivity and specificity for detecting the subtle airspace enlargement one would expect to find in mild or early stage emphysema. Therefore, D(2) may serve as a more accurate screening measure for detecting early lung disease than L(m).

Evaluation of a novel inhalation exposure system to determine acute respiratory responses to tobacco and polymer pyrolysate mixtures in Swiss-Webster mice.

Modern cigarette production processes are highly automated and yield millions of cigarettes per day. The forming cigarette and its components contact many different materials in the production process, some of which may leave minute residues. The potential for small inclusions of non-cigarette materials such as wood, plastic, cardboard and other materials exists from the bulk handling and processing of tobacco, in spite of vigilant workers and numerous online systems designed to keep the tobacco stream clean. Currently, there are no published methods that describe an approach to evaluate the potential toxicological impact of these non-tobacco residues and inclusions on the biological activity from exposure to the complex mixture of tobacco smoke. There are, however, many methods which describe toxicological evaluation approaches for pure materials, particularly synthetic polymers. We used the Deutsche Institute fur Normung (DIN) 53-436 tube furnace and nose-only exposure chamber in combination to conduct pilot studies in Swiss-Webster mice in order to develop a standardized methodology for the evaluation of sensory irritation and other potentially useful biological endpoints for predicting any potential hazards. Sensory and/or pulmonary irritation was assessed based on respiratory function parameters using the ASTM E981-84 method described by Alarie (1966) in mice, exposed to test atmospheres of 100% tobacco pyrolysate or tobacco/polymer pyrolysate mixtures. Other biological evaluations included respiratory function parameters, clinical signs, body weights, bronchoalveolar lavage fluid analysis, carboxyhemoglobin, blood cyanide concentrations and histopathology of the respiratory tract. These pilot studies have demonstrated that such an approach can detect biological changes resulting from exposure to unique tobacco/polymer pyrolysates. Small differences were detected in the sensory irritation responses (respiratory function), activation state of pulmonary macrophages, and histopathological findings in the nose of mice exposed to 100% tobacco or tobacco/polymer pyrolysates. Analytical measurements were also performed in order to characterize the test atmospheric changes that could occur from inclusion of the polymer into the tobacco. These included DIN-generated wet total particulate matter (TPM) DIN-Generated wet TPM (DWTPM), nicotine, cyanide, formaldehyde, acetaldehyde, acrolein, carbon monoxide, carbon dioxide, and nitrogen oxides. We attempted to correlate analyte differences in the test atmospheres with the resulting biological findings in the mice. The results demonstrated that this approach could detect minimal toxicological effects in mice exposed to test atmospheres of 100% tobacco or 70%/30% tobacco/polymer pyrolysates.

Characterization of the mouse bronchoalveolar lavage proteome by micro-capillary LC-FTICR mass spectrometry.

Bronchoalveolar lavage fluid (BALF) contains proteins derived from various pulmonary cell types, secretions and blood. As the characterization of the BALF proteome will be instrumental in establishing potential biomarkers of pathophysiology in the lungs, the objective of this study was to contribute to the comprehensive collection of Mus musculus BALF proteins using high resolution and highly sensitive micro-capillary liquid chromatography (microLC) combined with state-of-the-art high resolution mass spectrometry (MS). BALF was collected from ICR and C57BL/6 male mice exposed to nose-only inhalation to either air or cigarette smoke. The tandem mass spectra were analyzed by SEQUEST for peptide identifications with the subsequent application of accurate mass and time tags resulting in the identification of 1797 peptides with high confidence by high resolution MS. These peptides covered 959 individual proteins constituting the largest collection of BALF proteins to date. High throughput monitoring profiles of this extensive collection of BALF proteins will facilitate the discovery and validation of biomarkers that would elucidate pathogenic or adaptive responses of the lungs upon toxic insults.

Pulmonary inflammation in mice exposed to mainstream cigarette smoke.

Male C57Bl/6 (C57) and ICR mice were exposed by nose-only inhalation to mainstream cigarette smoke (MS) from 2R4F reference cigarettes, at concentrations of 75, 250, and 600 microg of total particulate matter (TPM) per liter, for up to 6 mo. Respiratory-tract tissue (nose, larynx, and lung), blood, and bronchoalveolar lavage fluid (BALF) samples were collected and analyzed at several time points. Blood samples were analyzed for biomarkers of exposure (COHb and nicotine). BALF was analyzed for biomarkers of cell injury, inflammation, oxidative stress, enzyme activity, and cytokines. Blood COHb and plasma nicotine concentrations increased in a dose-dependent manner, confirming smoke exposure. Mild emphysema was observed following 28 wk of exposure. Macrophage accumulation and inflammatory infiltrates were observed around the alveolar ducts and adjacent vasculature. There was an approximately 13% increase in mean linear intercept (Lm) only in ICR mice exposed to 600 microg/L TPM. There were no significant changes in biomarkers of oxidative stress secondary to smoke exposures; however, 8-isoprostane significantly increased following the 13-wk post-inhalation period. BALF macrophage and neutrophil counts were rapidly and consistently elevated, while lymphocyte counts gradually increased over time. MS-induced inflammatory responses observed in this study are comparable to changes reported in chronic smokers, supporting the role of chronic inflammation in the pathogenesis of emphysema. However, mild emphysema in minimal numbers of mice suggests that MS exposure concentration and/or duration in the current study were not sufficient to induce a definitive emphysema phenotype.

3-week inhalation exposure to cigarette smoke and/or lipopolysaccharide in AKR/J mice.

AKR/J mice were exposed to cigarette smoke (CS) and/or lipopolysaccharide (LPS) via inhalation for 3 wk and pulmonary responses were evaluated. The objective was to explore the feasibility of coexposing LPS with cigarette smoke under a subacute exposure, as a surrogate for viral or bacterial insults, that would mimic the pathogenesis of infection-related chronic obstructive pulmonary disease (COPD) exacerbations. The study was the first step in an effort to develop a rodent COPD model in which morphologic lesions of COPD develop in a shorter period of exposure and more closely simulate human COPD. Mice were exposed 6 h/day, 5 days/wk for 3 wk to one of the following: (1) sham control: filtered air; (2) CS: 250 microg/L wet total particulate matter (WTPM) for 5 h/day followed by 1 h/day air; (3) LPS: 0.5 microg/L LPS (055:B5 Escherichia coli; 3,000,000 EU/mg) for the last 1 h/day 2 day/wk (following 5 h/day of filtered air); and (4) CS/LPS: CS 5 h/day followed by air or LPS (2 days/wk) for 1 h/day. After the last exposure, animals were necropsied and subjected to bronchoalveolar lavage (BAL) or histopathology. The BAL neutrophil counts were highest in the LPS group, while macrophage counts were higher in the CS/LPS group than other exposed groups. The LPS group displayed the greatest increases in BAL cytokines, while KC (keratinocyte-derived chemokine) and TARC (thymus and activation-regulated chemokine) were highest in the CS group. The CS/LPS group had generally lower cytokine levels relative to the LPS or CS groups, except for the levels of RANTES and G-CSF (granulocyte-colony stimulating factor) comparable to the LPS group. At microscopic examination of lung sections, cellular inflammatory infiltrates were most notable in the CS/LPS group, which had a diffuse, predominantly macrophage infiltrate with fewer neutrophils. The LPS group had predominantly neutrophils in the pulmonary infiltrate and the CS group had a predominantly macrophage infiltrate in alveolar ducts and adjacent alveoli. Apoptotic labeling of lung cells was highest with the CS/LPS group. In summary, the CS/LPS group displayed greater cellular infiltration and apoptotic responses in the lung with an indication of immunosuppressive effects (lower BAL cytokines) than the CS or LPS group, suggesting that the CS/LPS model shows promise to be further explored as an animal model for studying pathogenesis of COPD exacerbations. A longer term study with interim assessments is needed to confirm that the subacute responses observed in the CS/LPS group will result in greater severity of COPD-related pulmonary lesions following prolonged exposures.

A study of spectral integration and normalization in NMR-based metabonomic analyses.

Metabonomics involves the quantitation of the dynamic multivariate metabolic response of an organism to a pathological event or genetic modification [J.K. Nicholson, J.C. Lindon, E. Holmes, Xenobiotica 29 (1999) 1181-1189]. The analysis of these data involves the use of appropriate multivariate statistical methods; Principal Component Analysis (PCA) has been documented as a valuable pattern recognition technique for 1H NMR spectral data [J.T. Brindle, H. Antti, E. Holmes, G. Tranter, J.K. Nicholson, H.W. Bethell, S. Clarke, P.M. Schofield, E. McKilligin, D.E. Mosedale, D.J. Grainger, Nat. Med. 8 (2002) 1439-1444; B.C. Potts, A.J. Deese, G.J. Stevens, M.D. Reily, D.G. Robertson, J. Theiss, J. Pharm. Biomed. Anal. 26 (2001) 463-476; D.G. Robertson, M.D. Reily, R.E. Sigler, D.F. Wells, D.A. Paterson, T.K. Braden, Toxicol. Sci. 57 (2000) 326-337; L.C. Robosky, D.G. Robertson, J.D. Baker, S. Rane, M.D. Reily, Comb. Chem. High Throughput Screen. 5 (2002) 651-662]. Prior to PCA the raw data is typically processed through four steps; (1) baseline correction, (2) endogenous peak removal, (3) integration over spectral regions to reduce the number of variables, and (4) normalization. The effect of the size of spectral integration regions and normalization has not been well studied. The variability structure and classification accuracy on two distinctly different datasets are assessed via PCA and a leave-one-out cross-validation approach under two normalization approaches and an array of spectral integration regions. The first dataset consists of urine from 15 male Wistar-Hannover rats dosed with ANIT measured at five time points, mimicking drug-induced cholangiolitic hepatitis [D.G. Robertson, M.D. Reily, R.E. Sigler, D.F. Wells, D.A. Paterson, T.K. Braden, Toxicol. Sci. 57 (2000) 326-337; J.P. Shockcor, E. Holmes, Curr. Top. Med. Chem. 2 (2002) 35-51; N.J. Waters, E. Holmes, A. Williams, C.J. Waterfield, R.D. Farrant, J.K. Nicholson, Chem. Res. Toxicol. 14 (2001) 1401-1412]. The second data is serum samples from young male C57BL/6 mice subjected to instillation of pancreatic elastase producing emphysema type symptoms [C. Kuhn, S.Y. Yu, M. Chraplyvy, H.E. Linder, R.M. Senior, Lab. Invest. 34 (1976) 372-380; C. Kuhn, R.M. Senior, Lung 155 (1978) 185-197]. This study indicates that independent of the normalization method the classification accuracy achieved from metabonomic studies is not highly sensitive to the size of the spectral integration region. Additionally, both datasets scaled to mean zero and unity variance (auto-scaled) have higher variability within classification accuracy over spectral integration window widths than data scaled to the total intensity of the spectrum. Of the top 10 latent variables for the ANIT dataset the auto-scale normalization has standard deviations larger than the total-scale in seven cases. In the case of the elastase all standard deviations are larger for the auto-scaling.

Mechanisms of circadian rhythmicity of carbon tetrachloride hepatotoxicity.

The toxicity of carbon tetrachloride (CCl(4)) and certain other chemicals varies over a 24-h period. Because the metabolism of some drugs follows a diurnal rhythm, it was decided to investigate whether the hepatic metabolic activation of CCl(4) was rhythmic and coincided in time with maximum susceptibility to CCl(4) hepatotoxicity. A related objective was to test the hypothesis that abstinence from food during the sleep cycle results in lipolysis and formation of acetone, which participates in induction of liver microsomal cytochrome P450IIE1 (CYP2E1), resulting in a diurnal increase in CCl(4) metabolic activation and acute liver injury. Groups of fed and fasted male Sprague-Dawley rats were given a single oral dose of 800 mg of CCl(4)/kg at 2- to 4-h intervals over a 24-h period. Serum enzyme activities, measured 24 h post dosing as indices of acute liver injury, exhibited distinct maxima in both fed and fasted animals dosed with CCl(4) near the beginning of their dark/active cycle. Blood acetone, hepatic CYP2E1 activity, and covalent binding of (14)CCl(4)/metabolites to hepatic microsomal proteins in untreated rats fed ad libitum followed circadian rhythms similar to that of susceptibility to CCl(4). Parallel fluctuations of greater amplitude were seen in rats fasted for 24 h. Hepatic glutathione levels were lowest at the time of greatest susceptibility to CCl(4). Acetone dose-response experiments showed high correlations between blood acetone levels, CYP2E1 induction, and CCl(4)-induced liver injury. Pretreatment with diallyl sulfide suppressed CYP2E1 and abolished the circadian rhythmicity of susceptibility to CCl(4). These findings provide additional support for acetone's physiological role in CYP2E1 induction and for CYP2E1's role in modulating CCl(4) chronotoxicity in rats.