Ninety-Day Inhalation Study in Rats, Using Aged and Diluted Sidestream Smoke from a Reference Cigarette: DNA Adducts and Alveolar Macrophage Cytogenetics

To study the genotoxic effects of subchronic exposure to environmentaltobacco smoke, Sprague-Dawley rats were exposed to 0, 0.1, 1.0,and 10 mg total particulate matter (TPM)/m³ of aged and dilutedsidestream smoke (ADSS) from 1R4F reference cigarettes 6 hrper day, 5 days a week for 13 weeks. DNA from lung, heart, larynx,bladder, and liver was tested for adduct formation by the ³²P-postlabelingassay after 28 (except bladder) and 90 days of exposure and90 days after cessation of exposure. In addition, alveolar macrophagesfrom animals exposed for 28 or 90 days were examined for chromosomalaberrations. Exposure-related DNA adducts were not observedin any tissue in any of the animals exposed to 0.1 or 1.0 mgTPM/m³. However, increased levels of DNA adducts with diagonalradioactive zones were observed in lung, heart, and larynx DNAof animals exposed to the highest concentration of ADSS (10mg TPM/m³). Adduct analyses with varying amounts of DNA fromlungs of mid-and high-exposure animals clearly indicate thatthe dose-response for DNA adduct formation is nonlinear. Theadduct levels were highest after 90 days of exposure and weresignificantly reduced in all target tissues 90 days after cessationof exposure. Chromosomal aberrations in alveolar macrophageswere not elevated in any group after 28 or 90 days of exposure.These results indicate a no-observed-effect-level (NOEL) ofat least 1.0 mg/m³ for DNA adduct formation in lung, heart,and larynx, and a NOEL of at least 10 mg/m³ for the inductionof chromosome aberrations in alveolar macrophages, under theconditions of this study.     

Replicative dna synthesis in tissues of the rat exposed to aged and diluted sidestream smoke

Abstract

Male Sprague-Dawley rats were exposed to aged and diluted sidestream smoke (ADSS) from Kentucky 1R4F reference cigarettes for 6 h/day, 5 days/wk, for a 13-wk period. Exposure concentrations were 0, 0.1, 1, and 10 mg ADSS/m³. Exposures were conducted in whole-body inhalation chambers. Rats were held in nose-only exposure tubes for the 6-h exposures to minimize pelt deposition and subsequent ingestion of ADSS. Groups of 10 rats from each exposure group were killed after 5, 28, and 90 d of exposure to examine the rates of replicative DNA synthesis; 6 rats from each exposure group were kept for a 90-day recovery period after termination of exposures to examine replicative DNA synthesis rates. Three days prior to each scheduled necropsy, an osmotic pump containing 5-bromo-2′-deoxyuridine (BrdU) was implanted subcutaneously. After necropsy, tissues were processed for examination of BrdU-containing cells at several sites. Incorporation of BrdU was assessed either by counting the number of labeled cells along a length of an epithelial surface or by counting the number of labeled cells in an area of tissue. Tissues examined were from the nasal cavity, ventral larynx, and trachea, in addition to bronchial, bronchiolar, and alveolar regions of the lung. Endocardium, myocardium, epicardium, and aortic smooth muscle sites were also examined. Increased replicative DNA synthesis occurred in some sites of the respiratory tract at the 5-day timepoint at the mid or high exposure concentrations, although at 28 and 90 days, these effects had diminished in intensity or were not present, indicating adaptation to the ADSS exposure. The only tissues with elevated rates of replicative DNA synthesis at 90 days were the cuboidal and respiratory epithelium at the most rostra! portion of the nasal cavity at the highest exposure concentration. Increased rates of replicative DNA synthesis were not noted in heart tissues or lung alveolar epithelium at any concentration at any time point. Examination of rats killed after the end of the 90-day recovery period indicated that the increase in replicative DNA synthesis was not sustained after termination of exposures. The no observed effect level (NOEL) for increased replicative DNA synthesis after subchronic exposure to ADSS in the rat is greater than 1 mg ADSS/m³.     

Deposition of Cigarette Smoke Particles in the Rat

The fractional deposition of cigarette smoke particles in therespiratory tracts of rats was studied. Male and female ratswere conditioned in nose-only exposure tubes 25 min/day for2 days, exposed to cigarette smoke at mass concentrations of95 or 341 mg/m³ 25 min/day for 3 days, and then exposed to smokeat mass concentrations of 212 and 657 mg/m³, 25 min/day for5 days. Mainstream cigarette smoke was generated by a modifiedWalton smoking machine from two 1R3 research cigarettes burnedsequentially for each exposure. Deposition studies were conductedby placing the rats in plethysmograph tubes to allow respiratoryminute volume measurements during exposure, then exposing themto [¹⁴C] cigarette smoke at mass concentrations of 202 or 624mg/m³ for 25 min, using the same smoking machine. Size distribution,real-time concentration, and ¹⁴C activity of the smoke particleswere determined using a multijet Mercer impactor, a real-timeaerosol monitor, and filter samples, respectively. Immediatelyafter the exposure, the rats were terminated to determine thedistribution of the ¹⁴C. Individual lung lobes, trachea andlobar bronchi, head, larynx, kidneys, liver, gastrointestinal(GI) tract, blood, and depelted carcass of each rat were analyzedfor ¹⁴C content. Results showed that the GI tract contained16–31% of the total activity, indicating significant clearancefrom the large airways and nose to the GI tract during the exposureand during the 10–15 min between the cessation of theexposure and the removal ofthe organs. Total deposition of theinhaled ¹⁴C activity was 20.1 ? 1.6% for both exposure concentrations.The intrapulmonary deposition fractions (lung lobes plus airwaysbelow the lobar bronchi) were 12.4 ? 0.9 and 15.9 ? 1.4% forconcentrations of 202 and 624 mg/m³ respectively, suggestinga slight enhancement in upper airway deposition for animalsexposed to the higher smoke concentration.     

A 90-Day Inhalation Toxicity Study with Benomyl in Rats

A 90-Day Inhalation Toxiaty Study with Benomyl in Rats. WARHEIT,D. B., KELLY, D. P., CARAKOSTAS, M. C., AND SINGER, A. W. (1989).Fundam Appl Toxicol./ 12, 333-345. Benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate,CAS Registry No. 17804-35-2] is a fungicide and the possibilityfor inhalation exposure exists for field workers. To assessthe toxicity of benomyl, groups of 20 male and 20 female CDrats were exposed nose-only 6 hr a day, 5 days a week, to concentrationsof 0, 10, 50 or 200 mg/m³ of a benomyl atmosphere. At the midpoint(approximately 45 days on test) and at the end of the exposureperiod, blood and urine samples for clinical evaluation werecollected from 10 rats/group/sex, and these animals were sacrificedfor pathological examination. Similar evaluations were performadon all remaining rats at the end of the 90-day test period.After approximately 45 days on test, compoundrelated degenerationof the olfactory epithelium was observed in all males and in8 of 10 female rats exposed to 200 mg/m³ benomyl. Two male ratsexposed to 50 mg/m³ had similar, although less severe, areasof olfactory epithelial degeneration. After approximately 90days of exposure, the remaining 10 rats/group/sex were sacrificedand examined. Of these rats, all of the males and females exposedto 200 mg/m³ had olfactory degeneration, along with 3 malesexposed to 50 mg/m³ of benomyl. No other observed lesions wereinterpreted to have been caused by the benomyl exposure. Inaddition, male rats exposed to 200 mg/m³ benomyl had depressedmean body weights compared to controls and this finding correlatedwith a reduction in food consumption. Based on pathologicalobservations, 10 mg/m³ represents the no-observable-effect level(NOEL) for the male rats, and 50 mg/m³ is the NOEL for the femalerats.     

Four-Week Inhalation Toxicity Study with Ludox Colloidal Silica in Rats: Pulmonary Cellular Responses

Four-Week Inhalation Toxicity Study with Ludox Colloidal Silicain Rats: Pulmonary Cellular Responses. WARHEIT, D. B., CARAKOSTAS,M. C, KELLY, D. P., AND HARTSKY, M. A. (1991). Fundam. Appl.Toxicol. 16, 590–601. This study was designed to complementa traditional subchronic inhalation toxicity study with Ludoxcolloidal silica. CD rats were exposed nose-only for 2 or 4weeks at concentrations of 0, 10, 50, and 150 mg/m³ Ludox (driedSiO₂). Additional groups of rats exposed for 4 weeks were givena 3-month recovery period. Following exposure and/or recovery,fluids and cells were recovered from the lungs by bronchoalveolarlavage (BAL) and measured for cellular and biochemical parameters.Additional groups of animals were processed for cell labelingstudies or lung deposition studies. Inhaled doses of Ludox colloidalsilica were measured after 4-week exposures and were found tobe 489 µg/lung (10 mg/m³ group), 2418 µg/lung (50mg/m³), and 7378 µg/lung (150 mg/m³), respectively. Resultsshowed that exposures to 150 mg/m³ Ludox for 2 or 4 weeks producedpulmonary inflammation along with increases in BAL protein,LDH, and alkaline phosphatase values (p<0.05) and reducedmacrophage phagocytosis. Inflammatory responses, evidenced byincreased numbers of neutrophils, were also measured in thelungs of the 50 mg/m³ group following 2 and/or 4 weeks of exposure.Most biochemical parameters for all groups returned to controlvalues following a 3-month recovery period. Autoradiographicstudies demonstrated that the labeling indices of terminal bronchiolarand lung parenchymal cells were generally increased in the 50and 150 mg/m³ groups after 2 and 4 weeks of exposure but, withone exception, returned to normal levels following a 3-monthpostexposure period. No significant alterations in any measuredparameters were detected in rats exposed to 10 mg/m³ Ludox atany time postexposure. The determination of a no-observable-effectlevel (NOEL) of 10 mg/m³ was consistent with results obtainedby conventional toxicology methods and affirms the utility ofthese biochemical, cellular, and autoradiographic techniquesfor providing a predictive screen to assess the toxicity ofinhaled particles.     

Fourteen-Day Inhalation Study in Rats, Using Aged and Diluted Sidestream Smoke from a Reference Cigarette: I. Inhalation Toxicology and Histopathology

Sprague-Dawley rats were exposed 6 hr per day for 14 consecutivedays to aged and diluted sidestream smoke (ADSS), used as asurrogate for Environmental Tobacco Smoke (ETS), at concentrationsof 0.1 (typical), 1 (extreme), or 10 (exaggerated) mg of particulatesper cubic meter. Animals were exposed nose-only, inside whole-bodychambers, to ADSS from the 1R4F reference cigarette. End-pointsincluded histopathology, CO-ox-imetry plasma nicotine and cotinine,clinical pathology, and organ and body weights. The only pathologicalresponse observed was slight to mild epithelial hyperplasiaand inflammation in the most rostral part of the nasal cavity,in the high-exposure group only. No effects were noted at mediumor low exposures. The minimal changes noted were reversible,using a subgroup of animals kept without further treatment foran additional 14 days. Overall, the end-points used in the studydemonstrated that there was no detectable biological activityof ADSS at typical or even 10-fold ETS concentrations and thatthe activity was only minimal at very exaggerated concentrations(particle concentrations 100 times higher than typical real-worldconcentrations).     

Short-term exposure to aged and diluted sidestream cigarette smoke enhances ozone-induced lung injury in B6C3F1 mice.

To determine the effects of aged and diluted sidestream cigarette smoke (ADSS) as a surrogate of environmental tobacco smoke (ETS) on ozone-induced lung injury, male B6C3F1 mice were exposed to (1) filtered air (FA), (2) ADSS, (3) ozone, or (4) ADSS followed by ozone (ADSS/ozone). Exposure to ADSS at 30 mg/m3 of total suspended particulates (TSP) for 6 h/day for 3 days, followed by exposure to ozone at 0.5 ppm for 24 h was associated with a significant increase in the number of cells recovered by bronchoalveolar lavage (BAL) compared with exposure to ADSS alone or ozone alone. The proportion of neutrophils and lymphocytes, as well as total protein level in BAL, was also significantly elevated following ADSS/ozone exposure, when compared with all other groups. Within the centriacinar regions of the lungs, the percentage of proliferating cells identified by bromodeoxyuridine (BrdU) labeling was unchanged from control, following exposure to ADSS alone, but was significantly elevated following exposure to ozone (280% of control) and further augmented in a statistically significant manner in mice exposed to ADSS/ozone (402% of control). Following exposure to ozone or ADSS/ozone, the ability of alveolar macrophages (AM) to release interleukin (IL)-6 under lipopolysaccharide (LPS) stimulation was significantly decreased, while exposure to ADSS or ADSS/ozone caused a significantly increased release of tumor necrosis factor alpha from AM under LPS stimulation. We conclude that ADSS exposure enhances the sensitivity of animals to ozone-induced lung injury.     

Quantitative Histology and Cytochrome P-450 Immunocytochemistry of the Lung Parenchyma Following 6 Months of Exposure of Strain A/J Mice to Cigarette Sidestream Smoke

Abstract

Male strain A/J mice were exposed for 6 h/day, 5 days/wk to aged and diluted cigarette sidestream smoke (ADSS) at a chamber concentration of 4 mg/m³ of total suspended particulate matter (TSP). After 6 mo, the lungs were examined for altered expression of cytochrome P-450 isozymes and for differences in total alveolar tissue volume or surface area, as well as changes in the numbers of epithelial type II cells and alveolar macrophages. Morphologic measurements showed no statistically significant differences for the air, alveolar tissue, or capillary volumes of the lungs or changes in the total number of epithelial type II cells or alveolar macrophages. In contrast, cytochrome P-4501A1 was elevated in the lungs of ADSS-exposed animals and localized in capillary endothelial cells. CYP2B1 was present in airway epithelial cells as well as in epithelial cells throughout the lung parenchyma, but its distribution was not changed by ADSS exposure. Isozyme CYP2E1 was also found in airway epithelial cells, but not in the lung parenchyma, with no differences noted between ADSS exposed animals and controls. CYP2F2 was found in the bronchiolar Clara cells and in type II cells located within the alveolar parenchyma, but was also unchanged. It is concluded that chronic exposure to cigarette ADSS at 4 mg/m³ of TSP produces no changes in alveolar macrophages or epithelial type II cells in mouse lung but increases the expression of cytochrome P4501A1.     

Inhaled cigarette smoke induces the formation of DNA adducts in lungs of rats

Cigarette smoking causes a variety of adverse human health effects, including lung cancer. The molecular events associated with smoke-induced carcinogenesis are thought to be related in part to the genotoxic activities of the chemicals associated with smoke. The purpose of this investigation was to determine the molecular dosimetry of compounds in cigarette smoke in lungs of rats exposed by inhalation. These studies investigated the effects of exposure mode, sex, and time (adduct persistence) on the level of DNA adducts. Male and female F344/N rats were exposed 6 hr/day, 5 days/week for 22 days to cigarette smoke by nose-only intermittent (NOI), nose-only continuous (NOC), or whole-body continuous (WBC) exposures. Separate groups of rats were sham-exposed nose-only (NOS) or whole-body (WBS) to filtered air. All smoke exposure modes yielded daily smoke exposure concentration X time products of 600 mg particulate.hr/m3 for the first week and 1200 mg particulate.hour/m3 thereafter. Groups of rats were killed at 18 hr and 3 weeks after the 22-day exposure period and DNA adducts in lung tissues were quantified by the 32P-postlabeling method. There were significant (p less than 0.05) increases in levels of clearly resolved lung DNA adducts in male and female rats exposed to smoke compared to sham-exposed rats. There were no significant effects of exposure mode or sex on lung DNA adducts. Mean levels (+/- SE) of clearly resolved lung DNA adducts for both sexes combined in NOI, NOC, WBC, NOS, and WBS groups were 50 +/- 4, 52 +/- 6, 52 +/- 7, 21 +/- 6, and 22 +/- 4 adducts per 10(9) bases, respectively. Levels of clearly resolved DNA adducts were significantly less in lungs of rats killed 3 weeks after exposure and had declined to near control levels, suggesting that smoke-induced adducts are repaired by lung DNA repair enzymes. A single unidentified adduct accounted for about 20% of the total clearly resolved lung DNA adducts quantified in smoke-exposed rats and was increased 9- to 14-fold over control levels. Levels of this adduct in NOI, NOC, WBC, NOS, and WBS were 14 +/- 0.9, 9 +/- 0.7, 11 +/- 0.9, 1.4 +/- 0.2, and 1.1 +/- 0.2 adducts per 10(9) bases, respectively. The results from these experiments indicate that inhaled cigarette smoke induces lung DNA adducts which may play an important role in cigarette smoke-induced lung carcinogenesis.     

Chronic Inhalation Toxicity of Size-Separated Glass Fibers in Fischer 344 Rats

This study was initiated to determine the chronic biologicaleffects in Fisher 344 rats of inhaled size-separated respirablefractions of fibrous glass (FG) having compositions representativeof common building insulation wools. Rats were exposed usingnose-only inhalation chambers, 6 hr/day, 5 days/week, for 24months to three concentrations (3, 16, and 30 mg/m³) of twodifferent compositions of FG (designated MMVF 10 and MMVF 11),or to filtered air (negative control). Fibrous glass findingswere compared to those from a concurrent inhalation study ofchrysotile asbestos and refractory ceramic fiber (RCF). TheFGs used in this study were size selected to be largely respirablein the rat and the aerosol generation technique did not alterthe dimensions of the fibers. Interim euthanizations took placeat 3- to 6-month intervals to monitor progression of pulmonarychanges. Fibers were recovered from digested lung tissue fordetermination of changes in fiber number and morphology. Inanimals exposed to 30 mg/m³ of MMVF 10 or MMVF 11, 4.2±0.9x10⁵and 6.4±3.1x10⁵ fibers/mg dry lung tissue, respectively,were recovered after 24 months of exposure. Exposure to chrysotileasbestos (10 mg/m³) and to a lesser extent RCF (30 mg/m³) resultedin pulmonary fibrosis as well as mesothelioma and significantincreases in lung tumors. FG exposure was associated with anonspecific inflammatory response (macrophage response) in thelungs that did not appear to progress after 6–12 monthsof exposure. These cellular changes are reversible and are similarto the effects observed after inhalation of an inert dust. Nolung fibrosis was observed in the FG-exposed animals. Further,FG exposure resulted in no mesotheliomas and no statisticallysignificant increase in lung tumor incidence when compared tothat of the negative control group. These findings, along withprevious inhalation studies, suggest that respirable fibrousglass does not represent a significant hazard for fibrotic orneoplastic lung disease in humans.     

Alterations in DNA methylation and airway hyperreactivity in response to in utero exposure to environmental tobacco smoke

Abstract

Growing evidence indicates that prenatal exposure to maternal smoking is a risk factor for the development of asthma in children. However, the effects of prenatal environmental tobacco smoke (ETS) exposure on the genome and lung immune cells are unclear. This study aims to determine whether in utero ETS exposure alters DNA methylation patterns and increases airway hyperreactivity (AHR) and inflammation. Pregnant C57BL/6 mice were exposed daily to a concentration of 1.0?mg/m³ ETS. AHR was determined in the 6-week-old offspring by measurement of airway resistance. Global and gene promoter methylation levels in lung DNA from offspring were analyzed by luminometric methylation and pyrosequencing assays, respectively. Offspring exposed to ETS showed a marked increase in the number of alveolar macrophages in the bronchoalveolar lavage fluid and level of IL-13 in the airways compared with offspring of filtered-air exposed dams (controls). ETS exposure significantly augmented AHR compared with controls. In the methylation analysis, ETS-exposed offspring had a significantly lower level of global DNA methylation than the controls. We observed a significant increase in IFN-γ, and significant decrease in IL-13 methylation levels in the ETS group compared with controls. Collectively, these data suggest that in utero ETS exposure increases the risk of pulmonary inflammation and AHR through altered DNA methylation, but additional studies are needed to fully determine the causal link between changes in methylation and cytokines levels, as well as AHR.     

The Pulmonary Response and Clearance of Ludox Colloidal Silica after a 4-Week Inhalation Exposure in Rats

Rats were exposed to Ludox colloidal silica (CS) at concentrationsof 0, 10, 50, and 150 mg/m³ for 6 hr/day, 5 days/week for 4weeks. Rats were killed after 4 weeks of exposure and 10 daysor 3 months post exposure (PE). The exposure concentration of10 mg/m³ Ludox CS is considered to be the no-effect concentration.There were no exposure-related clinical signs in any group.After 4 weeks exposure, lung weights were increased significantlyin rats exposed to 50 and 150 mg/m³ Ludox CS, but lung weightswere similar to those of controls at 3 months PE. After 4 weeksexposure to 50 mg/m³ Ludox CS, a slight alveolar macrophageresponse, polymorphonuclear leukocytic infiltration, and TypeII pneumocyte hyperplasia in alveolar duct regions were present.After 3 months PE, these pulmonary lesions had almost disappearedwith removal of most dust-laden alveolar macrophages (AMs).The pulmonary response to 150 mg/m³ Ludox CS was similar incharacter but increased in magnitude from that seen at 50 mg/m³At 3 months PE, most particleladen AMs had disappeared and theremaining AMs were aggregated and sharply demarcated. A fewaggregates of particle-laden AMs appeared to transform intosilicotic nodules comprising macrophages, epithelioid cells,and lymphocytic infiltration in some animals. Some silicoticnodules showed reticular fiber networks with minute collagenfiber deposition. Tracheobronchial lymph nodes were enlargedwith aggregates of particle-laden AMs and hyperplastic histiocyticcells. Lung-deposited Ludox cleared rapidly from the lungs withhalf-times of approximately 40 and 50 days for the 50 and 150mg/m³ groups, respectively.     

Chronic Inhalation Toxicity and Carcinogenicity Study of Respirable Polymeric Methylene Diphenyl Diisocyanate (Polymeric MDI) Aerosol in Rats

Four groups of 60 Wistar rats of each sex were exposed by inhalationto 0, 0.2, 1.0, or 6.0 mg/m³ respirable polymeric methylenediphenyl diisocyanate (polymeric MDI) aerosol (93.5% < 4.2µm)for 6 hr a day, 5 days a week for up to 24 months. In addition,satellite groups of 10 rats/sex/group received the same treatmentfor 12 months. There was no adverse effect on general health,survival, body weight, or hematological or clinical chemistryparameters. Lung weights were increased in both males and femalesexposed to 6.0 mg polymeric MDI/m³ for 12 or 24 months. Grossexamination at autopsy of males exposed to 6.0 mg polymericMDI/m³ for 24 months revealed an increased incidence of spottedand discolored lungs. Increased incidences of degeneration andbasal cell hyperplasia of the nasal olfactory epithelium, oftenaccompanied by hyperplasia of Bowman's glands, were found inthe 1.0 and 6.0 mg/m³ groups. Light and electron microscopicstudies of the lungs revealed accumulations of alveolar macrophagescontaining polymeric MDI-associated refractile yellowish materialat the level of the alveolar duct in all exposed groups. Alveolarduct epithelialization as well as fibrosis of tissues surroundingthe macro phage accumulations occurred at the 1.0 and 6.0 mg/m³exposure levels. In addition, increased incidences of calcareousdeposits and localized alveolar bronchiolization were seen inthe 6.0 mg/m³ group. Moreover, eight pulmonary adenomas (sixin males and two in females) and one pulmonary adenocarcinoma(in a male) were observed in the 6.0 mg/m³ exposure group. Thetime sequence of the spectrum of pulmonary changes indicatesthat recurrent alveolar wall damage by polymeric MDI and/orpolymeric MDI-containing alveolar macrophages leads to alveolarbronchiolization and ultimately to bronchioloalveolar tumors.No lung tumors were found in the lower concentration groupsand in the control group. The incidence and distribution ofother types of tumors were not influenced by polymeric MDI.It was concluded that in the present study, the "no-observed-adverse-effectlevel" of polymeric MDI was 0.2 mg/m³ and that chronic exposureto polymeric MDI at a level of 6.0 mg/m³ was related to theoccurrence of pulmonary tumors. It was also concluded that exposureto polymeric MDI at concentrations not leading to recurrentlung tissue damage will not produce pulmonary tumors.     

Mucous Cell Metaplasia in the Airways of Rats Exposed to Machining Fluids

Occupational exposure to microbial-contaminated machining fluidsis associated with a variety of adverse pulmonary effects includingchronic bronchitis and increased sputum production. We havepreviously demonstrated in F344 rats that inhaled endotoxincan increase the amount of stored intraepithelial mucosubstances(Vs) in the respiratory tract. The purpose of the present studywas to examine the effect of endotoxin-contaminated machiningfluid aerosols on mucous production. Rats were exposed to aerosolsof pyrogen-free water, 1 or 10 mg/m³ used machining fluid, or10 mg/m³ unused machining fluid for 3 hr/day for 3 days. Twenty-fourhours after the final exposure, right lung lobes were lavagedand the nasal cavity and left lung were fixed in formalin. Theamount of Alcian blue/periodic acid-Schiff-stained mucosubstanceswas determined by morphometry. Exposure to 10 mg/m³ used machiningfluid (equivalent to 0.8 µg/m³ endotoxin) produced a significantincrease in Vs in the epithelial lining of both the nasal septumand intrapulmonary airways. These changes in Vs were accompaniedby a significant increase in total cells and neutrophils inthe lavage fluid. No changes in stored mucosubstances or lavageparameters were found in animals exposed to 1 mg/m³ used machiningfluid aerosols. A significant increase in Vs was observed inthe nasal septum but not in the intrapulmonary airways of animalsexposed to 10 mg/m³ unused machining fluids (no measurable endotoxin).These results suggest that in addition to endotoxin, nonendotoxincomponents of machining fluids may contribute to the increasein sputum and chronic bronchitis reported for workers exposedto machining fluid aerosols.     

Effect of Chronic Cigarette Smoke Exposure on Lung Clearance of Tracer Particles Inhaled by Rats

Cigarette smoking can influence the pulmonary disposition ofother inhaled materials in humans and laboratory animals. Thisstudy was undertaken to investigate the influence of cigarettesmoke exposures of rats on the pulmonary clearance of inhaled,relatively insoluble radioactive tracer particles. Following13 weeks of whole-body exposure to air or mainstream cigarettesmoke for 6 hr/day, 5 days/week at concentrations of 0, 100,or 250 mg total particulate matter (TPM)/m³, rats were acutelyexposed pernasally to ⁸⁵Sr-labeled fused aluminosilicate (⁸⁵Sr-FAP)tracer particles, then air or smoke exposures were resumed.A separate group of rats was exposed to the ⁸⁵Sr-FAP then seriallyeuthanized through 6 months after exposure to confirm the relativeinsolubility of the tracer particles. We observed decreasedtracer particle clearance from the lungs that was smoke concentration-dependent.By 180 days after exposure to the tracer aerosol, about 14,20, and 40% of the initial activity of tracer was present incontrol, 100 mg TPM/m³, and 250 mg TPM/m³ groups, respectively.Body weight gains were less in smoke-exposed rats than in controls.Smoke exposure produced lung lesions which included increasednumbers of pigmented alveolar macrophages distributed throughoutthe parenchyma and focal collections of enlarged alveolar macrophageswith concomitant alveolar epithelial hyperplasia and neutro-philicalveolitis. The severity of the lesions increased with smokeexposure duration and concentration to include interstitialaggregates of pigmented macrophages and interstitial fibrosis.Our data confirm previous findings that exposure to cigarettesmoke decreases the ability of the lungs to clear inhaled materials.We further demonstrate an exposure-concentration related magnitudeof effect, suggesting that the cigarette smoke-exposed rat constitutesa useful model for studies of the effects of cigarette smokeon the disposition of inhaled particles.     

Subchronic and Chronic Inhalation Toxicity of Antimony Trioxide in the Rat

Fischer 344 rats were exposed by inhalation to Sb₂O₃ (antimonytrioxide) dust at exposure levels of 0, 0.25, 1.08, 4.92, and23.46 mg/m³ for 6 hr/day, 5 days/week for 13 weeks followedby a 27-week observation period. Subsequently, an inhalationon-cogenicity study was conducted at exposure levels of 0, 0.06,0.51, and 4.50 mg/m³ for 12 months followed by a 12-month observationperiod. The Sb₂O₃ in the subchronic study had a mass medianaerodynamic diameter (MMAD) of 3.05 ± 0.21 microns (mean± SD) with a geometric standard deviation (GSD) of 1.57± 0.06. In the chronic study, the MMAD was 3.76 ±0.84 and the GSD was 1.79 ± 0.32. Except for the eyes,no adverse clinical observations were attributed to Sb₂O₃ ineither study. In the subchronic study, corneal irregularitieswere seen after about 2 weeks of exposure and did not abateduring the observation period. In the chronic study, ophthalmoscopicevaluation at 24 months revealed a dose-related increase incataracts of 11, 24, 28, and 32% (both sexes combined) for eachgroup, respectively. Body weights were significantly lower (6%)than the control group's weights in the 23.46 mg/m³ males inthe subchronic study. These rats did not recover this weightduring the 27-week observation period. Body weights of the femalesin both studies and males in the chronic study were unaffected.There were no Sb₂O₃ effects on clinical chemistry or he-matologyin either study. Mean absolute and relative lung weights weresignificantly increased in the 4.92 and 23.46 mg/m³ groups inthe subchronic study. The 23.46 mg/m³ group's lung weights didnot recover to control levels during the 27-week observationperiod. Lung weights for rats in the chronic study were unaffected.Microscopic changes in the lungs in the subchronic and chronicstudy were limited to subacute-chronic interstitial inflammation,increased numbers of alveolar-in-traalveolar macrophages, foreignmaterial in the alveolar-in-traalveolar macrophages in the peribronchialand perivascular (chronic study only) lymphoid aggregates andin the peribronchial lymph nodes, granulomatous inflammation/granulomas,and fibrosis. In the chronic study, any observed neoplasms occurredwith comparable incidence among all groups and were within thehistorical range for controls. Clearance of Sb₂O₃ from the lungwas burden dependent and was reduced by 80/ in the 4.50 mg/m³group in the chronic study. The previously reported studies,which found Sb₂O₃ to be a carcinogen, were run at higher lungburdens. Under the exposure conditions of the current study,Sb₂O₃ was not a carcinogen.     

Lack of Elevation of Stress-Inducible Heat-Shock Protein 70 in the Ferret Lung After Chronic Cigarette Smoke Inhalation

Abstract

Few investigations have examined in vivo expression of lung heat-shock proteins (HSPs) following environmental stress. This study was performed to ascertain if stress-inducible HSP 70 in the ferret lung is elevated following inhalant exposure to 2 high concentrations of a mixture of mainstream and sidestream cigarette smoke: (a) 381 ± 97 (SD) mg/m³ and (b) 38.2 ± 13.3 (SD) mg/m³. Ferrets (n = 5 for each concentration) starting at 5 wk of age were exposed head-only to cigarette smoke for 2 h/day, 5 days/wk for a total of 15 wk; control ferrets (n = 4) were exposed to filtered air. Samples of the peripheral lung from the right apical lobe were obtained for analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), protein immunoblotting, and subsequent chemiluminescent detection. Results revealed that stress-inducible HSP 70 is present constitutively in the control ferret lung and is not elevated significantly (p = .7758) in lungs from both groups of smoke-exposed ferrets.     

Benzene Hemoglobin Adducts in Mice and Rats: Characterization of Formation and Physiological Modeling

Benzene Hemoglobin Adducts in Mice and Rats: Characterizationof Formation and Physiological Modeling. SUN, J. D., MEDINSKY,M. A., BIRNBAUM, L. S., LUCIER, G., AND HENDERSON, R. F. (1990).Fundam. Appl. Toxicol 15, 468–475. Benzene is a myelotoxinand a human leukemogen. Humans are exposed to this compound,both occupationally and environmentally. This study was conductedto determine whether formation of benzene-derived adducts withblood hemoglobin (Hb) can be used as a biomarker of exposureto benzene. B6C3F₁ mice and F344/N rats were given 0.1 to 10,000µmol [¹⁴C]benzene/kg body wt, orally. Twenty-four hourslater, animals were euthanized, and globin was isolated fromblood samples. The globin was analyzed by liquid scintillationspectrometry for the presence of [¹⁴C]benzene-de-rived adducts.Hb adduct formation was linear with respect to dose for amountsof up to 500 µmol [¹⁴C]benzene/kg body wt, for both rodentspecies. Within this linear dose-response range, mice formedadducts from [¹⁴C]benzene approximately 3.5 times less efficiently[0.022 ± 0.010 (pmol adducts/mg globin)/(µmol/kgbody wt dose)] than did rats [0.076 ± 0.014 (pmol adducts)/µmol/kg body wt dose)]. Benzene-derived Hb adducts alsoaccumulated linearly when mice and rats were given up to threedaily doses of 500 µmol [¹⁴C]benzene/kg body wt. Thesedata were used to develop a physiological model for benzene-derivedHb adduct formation. Both first-order and saturable pathwaysfor adduct formation were incorporated. The results showed thatthe model simulated the levels of Hb adducts in both mice andrats after oral exposures to benzene and predicted the levelsof Hb adducts present after inhalation exposure. These studiessuggest that Hb adducts might be useful biomarkers for humanexposures to benzene.     

Short-Term Effects of Sidestream Smoke on Respiratory Epithelium in Mice: Cell Kinetics

Male strain A/J and C57BL/6 mice were exposed on five consecutivedays, for 6 hr a day, to sidestream smoke generated from Kentucky1R4F reference cigarettes. Chamber concentrations were 1 mg/m³of total suspended particulate matter and 528 to 549 µg/m³of nicotine. Cumulative labeling indices in the airways andin the pulmonary parenchyma were measured following 1, 3, or5 days exposure to unfiltered or filtered sides tream smoke.A significantly increased labeling index was found in A/J micein the epithelium lining large intrapulmonary airways and terminalbronchioles after 3 and 5 days exposure to unfiltered smoke,whereas following exposure to filtered smoke labeling indicesremained normal. The alveolar labeling index was not increasedfollowing smoke exposure. In C57BL/6 mice, sidestream smokedid not produce signs of increased cell proliferation in therespiratory tract. It is concluded that the response to sidestreamsmoke inhalation in mice may depend upon the strain of miceexamined.     

Alterations in Particle Accumulation and Clearance in Lungs of Rats Chronically Exposed to Diesel Exhaust

Alterations in Particle Accumulation and Clearance in Lungsof Rats Chronically Exposed to Diesel Exhaust. WOLFF, R. K.,HENDERSON, R. F., SNIPES, M. B., GRIFFITH, W. C., MAUDERLY,J. L., CUDDIHY, R. G., AND MCCLELLAN, R. 0. Fundam Appl. Toxicol9, 154–166. F344 rats were chronically exposed to dieselexhaust at target soot concentrations of 0 (control, C), 0.35(low, L), 3.5 (medium, M), and 7.0 (high, H) mg/m³ Accumulatedlung burdens of diesel soot were measured after 6, 12, 18, and24 months of exposure. Parallel measurements of particle depositionand clearance were made to provide insight into the mechanismsof particle accumulation in lungs. The fractional depositionof inhaled ⁶⁷Ga₂O₃ particles after 6, 12, 18, and 24 monthsof exposure and of inhaled ¹³⁴Cs-fused aluminosilicate particlesafter 24 months were similar for all groups. Progressive increasesin lung burdens of soot particles were observed in M and H exposedrats, reaching levels of 11.5 ± 0.5 and 20.5 ±0.8 mg/lung (SE), respectively, after 24 months. Rats in theL group had smaller relative increases in lung burden, reachinglevels of 0.60 ± 0.02 mg/lung after 24 months. Trachealmucociliary clearance measurements, using ^99mTc-macroaggitatedalbumin deposited in the trachea, showed no changes at anytime.There were statistically significant increases inclearance half-timesof inhaled radiolabeled particles of ⁶⁷Ga₂O₃ as early as 6 monthsat the H level and 18 months at the M level; no significantchanges were seen at the L level. Rats inhaled fused aluminosilicateparticles labeled with ¹³⁴Cs after 24 months of diesel exhaustexposure to measure long-term components of pulmonary clearance.The long-term clearance half-times were 79 ± 5, 81 ±5, 264 ± 50, and 240 ± 50 days (± SE) forthe C, L, M, and H groups, respectively. Differences were significantbetween the C and both the M and H exposure groups (p <0.01).Lung burdens of diesel soot were more than expected at the Hand M levels and were also associated with impaired particleclearance while smaller responses were observed in both burdensand clearance at the L level.