The effect of a 2-h exposure to cigarette smoke on the metabolic activation of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mice.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, induces lung adenomas in A/J mice following a single intraperitoneal (i.p.) injection. However, inhalation of mainstream cigarette smoke does not induce or promote NNK-induced lung tumors in this mouse strain purported to be sensitive to chemically-induced lung tumorigenesis. The critical events for NNK-induced lung tumorigenesis in A/J mice is thought to involve O(6)-methylguanine (O(6)MeG) adduct formation, GC-->AT transitional mispairing, and activation of the K-ras proto-oncogene. The objective of this study was to test the hypothesis that a smoke-induced shift in NNK metabolism led to the observed decrease in O(6)MeG adducts in the lung and liver of A/J mice co-administered NNK with a concomitant 2-h exposure to cigarette smoke as observed in previous studies. Following 2 h nose-only exposure to mainstream cigarette smoke (600 mg total suspended particulates/m(3) of air), mice (n=12) were administered 7.5 micromol NNK (10 microCi [5-3H]NNK) by i.p. injection. A control group of 12 mice was sham-exposed to HEPA-filtered air for 2 h prior to i.p. administration of 7.5 micromol NNK (10 microCi [5-3H]NNK). Exposure to mainstream cigarette smoke had no effect on total excretion of NNK metabolites in 24 h urine; however, the metabolite pattern was significantly changed. Mice exposed to mainstream cigarette smoke excreted 25% more 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) than control mice, a statistically significant increase (P<0.0001). Cigarette smoke exposure significantly reduced alpha-hydroxylation of NNK to potential methylating species; this is based on the 15% reduction in excretion of the 4-(3-pyridyl)-4-hydroxybutanoic acid and 42% reduction in excretion of 4-(3-pyridyl)-4-oxobutanoic acid versus control. Detoxication of NNK and NNAL by pyridine-N-oxidation, and glucuronidation of NNAL were not significantly different in the two groups of mice. The observed reduction in alpha-hydroxylation of NNK to potential methylating species in mainstream cigarette smoke-exposed A/J mice provides further mechanistic support for earlier studies demonstrating that concurrent inhalation of mainstream cigarette smoke results in a significant reduction of NNK-induced O(6)MeG adduct formation in lung and liver of A/J mice compared to mice treated only with NNK.

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.

Quantitative 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.

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.

The effect of cotinine or cigarette smoke co-administration on the formation of O6-methylguanine adducts in the lung and liver of A/J mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, induces lung adenomas in A/J mice, following a single intraperitoneal (i.p.) injection. However, inhalation of tobacco smoke has not induced or promoted tumors in these mice. NNK-induced lung tumorigenesis is thought to involve O6-methylguanine (O6MeG) formation, leading to GC-->AT transitional mispairing and an activation of the K-ras proto-oncogene in the A/J mouse. NNK can be metabolized by several different cytochromes P450, resulting in a number of metabolites. Formation of the promutagenic DNA adduct O6MeG is believed to require metabolic activation of NNK by cytochrome P450-mediated alpha-hydroxylation of the methylene group adjacent to the N-nitroso nitrogen to yield the unstable intermediate, methanediazohydroxide. Nicotine, cotinine (the major metabolite of nicotine), and aqueous cigarette tar extract (ACTE) have all been shown to effectively inhibit metabolic activation of NNK to its mutagenic form, most likely due to competitive inhibition of the cytochrome P450 enzymes involved in alpha-hydroxylation of NNK. The objective of the current study was to monitor the effects of cotinine and cigarette smoke (CS) on the formation of O6MeG in target tissues of mice during the acute phase of NNK treatment. To test the effect of cotinine, mature female A/J mice received a single intraperitoneal injection of NNK (0, 2.5, 5, 7.5, or 10 mumole/mouse) with cotinine administered at a total dose of 50 mumole/mouse in 3 separate i.p. injections, administered 30 min before, immediately after, and 30 min after NNK treatment. To test the effect of whole smoke exposure on NNK-related O6MeG formation, mice were exposed to smoke generated from Kentucky 1R4F reference cigarettes at 0, 0.4, 0.6, or 0.8 mg wet total particulate matter/liter (WTPM/L) for 2 h, with a single i.p. injection of NNK (0, 3.75, or 7.5 mumole/mouse) midway through the exposure. Cigarette smoke alone failed to yield detectable levels of O6MeG. The number of O6MeG adducts following i.p. injection of NNK was significantly (p < 0.05) reduced in both lung and liver by cotinine and by cigarette smoke exposure. Our results demonstrate that NNK-induced O6MeG DNA adducts in A/J mice are significantly reduced when NNK is administered together with either cotinine, the major metabolite of nicotine, or the parental complex mixture, cigarette smoke.

Chemical and biological studies of a new cigarette that primarily heats tobacco. Part 3. In vitro toxicity of whole smoke.

Mainstream smoke from Kentucky reference low "tar" (1R4F) and ultra-low "tar" (1R5F) cigarettes and a test cigarette (TOB-HT), that primarily heats tobacco, was compared for cytotoxic and genotoxic potential using cellular smoke exposure technology (CSET). CSET includes a computer controlled 30-port AMESA/Battelle-Geneva smoke generator which exposes cultured mammalian Chinese hamster ovary cells (CHO) to whole smoke. Cytotoxicity was assessed using the neutral red assay and genotoxicity was assessed using the sister chromatid exchange (SCE) assay. Compared on a per cigarette basis, mainstream smoke from 1R5F and the TOB-HT cigarette was significantly less cytotoxic and genotoxic than the smoke from the 1R4F cigarette. The cytotoxic and genotoxic activity of smoke from the TOB-HT cigarettes was slightly greater than the smoke from the ultra-low "tar" Kentucky 1R5F reference cigarettes. In conclusion, in these assays mainstream whole smoke of the TOB-HT cigarette had slightly greater cytotoxic and genotoxic potential compared with an ultra-low "tar" 1R5F Kentucky reference cigarette and significantly less activity compared with the whole mainstream smoke from a low "tar" 1R4F Kentucky reference cigarette, representative of the US market average cigarette for FTC yields of "tar", CO and nicotine.

Evaluation of the genotoxic and cytotoxic potential of mainstream whole smoke and smoke condensate from a cigarette containing a novel carbon filter.

A novel carbon filter has been developed which primarily reduces the amount of certain vapor phase constituents of tobacco smoke with greater efficiency than the charcoal filters of cigarettes currently in the market. In vitro indicators of genotoxic and cytotoxic potential were used to compare the cigarette smoke condensate (particulate phase) or whole cigarette smoke (vapor phase and particulate phase) from cigarettes containing the novel carbon filter with smoke condensate or whole smoke from commercial or prototype cigarettes not containing the novel carbon filter. Ames bacterial mutagenicity, sister chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells, and neutral red cytotoxicity assays in CHO cells were utilized to assess the genotoxic and cytotoxic potential of the cigarette smoke condensates. SCE and neutral red cytotoxicity assays were utilized to assess the genotoxic and cytotoxic potential of the whole smoke. As expected, the novel carbon filter did not significantly affect the genotoxic or cytotoxic activity of the smoke condensate, although we did observe that the use of low-nitrogen tobacco reduced the mutagenicity of the condensate in Salmonella typhimurium strain TA98. However, the whole smoke from cigarettes containing the novel carbon filter demonstrated significant reductions in genotoxic and cytotoxic potential compared to cigarettes without the novel carbon filter. The toxicity of the smoke was correlated (r = 0.7662 for cytotoxicity and r = 0.7562 for SCE induction) to the aggregate mass of several vapor phase components (acetone, acetaldehyde, acrolein, acrylonitrile, 1,3-butadiene, ammonia, NOx, HCN, benzene, isoprene, and formaldehyde) in the smoke of the cigarettes utilized in this study. In conclusion, this novel carbon filter, which significantly reduced the amount of carbonyls and other volatiles in mainstream cigarette smoke, resulted in significant reductions in the genotoxic and cytotoxic activity of the smoke as measured by these assays.

Relevant exposure to environmental tobacco smoke surrogate does not produce or modify secretory otitis media in the rat.

Parental smoking is a possible risk factor in the development of secretory otitis media (SOM) in children. This experiment was designed to determine, using rats as an experimental model, whether exposures to environmental tobacco smoke (ETS) produce SOM and whether ETS exposure affects the rate of clearance of an experimentally induced effusion. Male Sprague-Dawley rats were exposed to 3 different concentrations of aged and diluted sidestream smoke, a surrogate for ETS, from IR4F research cigarettes for 6 hr per day for 5 days. Experimental SOM was induced bilaterally in subgroups of animals from each group, by cold air exposure to the external auditory canals. Ears of rats were examined during the in-life portion of the study. Histopathologic examination of the middle ear was conducted at the termination of the 5-day period. The production of SOM was not induced by ETS exposure, nor were there differences noted between the groups in the rates of clearance of the experimentally induced SOM. Short-term exposure to ETS did not affect the acquisition or clearance of SOM in the rat.

Comparative study of DNA adduct formation in mice following inhalation of smoke from cigarettes that burn or primarily heat tobacco.

The genotoxic potential of mainstream smoke from a test cigarette (TOB-HT) that primarily heats tobacco and a representative tobacco-burning cigarette (Kentucky reference 1R4F) was compared in male B6C3/F1 mice after nose-only inhalation exposure. Mice were exposed 1 hr per day, 5 days/ week for a 4 week period to mainstream smoke at concentrations of 0, 0.16, 0.32, and 0.64 mg total particulate matter/liter of air. Micronuclei formation in bone marrow and peripheral blood polychromatic erythrocytes (PCE) of animals exposed to either the TOB-HT or 1R4F cigarette was not significantly different compared with control animals exposed nose-only to filtered and humidified air (sham controls). DNA adduct measurement by the 32P-post-labeling method indicated an exposure-dependent increase in lung adducts of animals exposed to 1R4F cigarette smoke at all three concentrations with the mid and high exposure groups exhibiting statistically significant increases (P < 0.05) in adduct formation compared to sham-exposed animals. The concentration of DNA adducts in the lungs of animals exposed to the TOB-HT cigarette was not significantly increased (P < 0.05) at any concentration compared to sham-exposed controls. A statistically significant (P < 0.05) concentration-dependent formation of DNA adducts was also observed in the heart tissues of animals exposed to smoke from the 1R4F cigarette at all three concentrations, but no significant increase in adduct formation was observed in heart DNA of the animals exposed to the TOB-HT cigarette (P < 0.05). Under the conditions of this experiment, the mainstream smoke from the TOB-HT cigarette was demonstrated to be less genotoxic in mice than mainstream smoke from the 1R4F cigarette, which is representative of cigarettes in the current U.S. market.

Molecular toxicology endpoints in rodent inhalation studies.

Although histopathology will continue to be essential for assessing the results of rodent inhalation studies, molecular toxicology endpoints are of increasing importance, as these techniques often complement and extend histopathological examinations. One of the primary uses of molecular toxicology is determining the delivered dose of the inhaled material to macromolecules in target tissues. During inhalation studies this is most often done by measuring DNA adducts in the respiratory tract. DNA adducts may be measured specifically (e.g. using monoclonal antibodies or mass spectrometry) or non-specifically (e.g. by using the 32P-post-labeling assay). Another major use of molecular toxicology techniques is the assessment of cellular and molecular changes in target tissues which may precede or be more sensitive than histopathologic alterations. For example, rates of cellular DNA synthesis occurring in target tissues may be quantified at any time during the study by administering the animals either radiolabelled thymidine or the non-radiolabelled thymidine analog bromodeoxyuridine (BrdU). Pulmonary changes may be assessed in bronchoalveolar lavage fluid using either cellular (e.g. macrophage number, granulocyte number) or biochemical (e.g. alkaline phosphatase, lactate dehydrogenase) techniques. The potential of the inhaled material to produce genetic alterations may be evaluated by examining the chromosomes of pulmonary alveolar macrophages for cytogenetic changes. To illustrate the use of these endpoints, an experiment was conducted to determine the molecular toxicology of aged and diluted sidestream smoke (a surrogate for environmental tobacco smoke) in rodent inhalation studies. The endpoints measured were DNA adducts in target and non-target tissue, chromosome aberrations in pulmonary alveolar macrophages, and DNA synthesis in the epithelial lining of the nasal turbinates.

Effects of low humidity on the rat middle ear.

Secretory otitis media is common in the winter, and the possible risk factors are numerous. This study examines the effect of low humidity on the middle ear using a Sprague-Dawley rat model: 23 test rats housed for 5 days in a low-humidity environment (10% to 12% relative humidity) and 23 control rats housed at 50% to 55% relative humidity. Microscopic ear examinations were graded for otitis media with effusion (OME) before testing and on test days 3 and 5. The mucosa of the middle ears and eustachian tubes was examined histopathologically. Significantly more effusions were observed in the low-humidity group on test days 3 (P = .003) and 5 (P = .01), but no intergroup histopathologic differences were noted. We conclude that a low-humidity environment contributed to the development of OME in the test animals, and that low-humidity warrants further investigation as a contributing factor in childhood middle ear disease.

Design, construction, and evaluation of an inhalation system for exposing experimental animals to environmental tobacco smoke.

An inhalation system was designed to expose experimental animals to aged and diluted sidestream smoke (ADSS), used as a surrogate for environmental tobacco smoke (ETS). The construction of the smoke generator and of the smoke dilution systems is described. Target ADSS concentrations in a 90-day inhalation study were 0.1, 1, and 10 mg/m3 of respirable suspended particulates (RSP). Data is presented on the physical and chemical composition of the smoke presented to animals at or near these target RSP concentrations. The design of the inhalation laboratory was shown to result in highly reproducible respirable aerosols that were effective surrogates of ETS.

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 consecutive days to aged and diluted sidestream smoke (ADSS), used as a surrogate for Environmental Tobacco Smoke (ETS), at concentrations of 0.1 (typical), 1 (extreme), or 10 (exaggerated) mg of particulates per cubic meter. Animals were exposed nose-only, inside whole-body chambers, to ADSS from the 1R4F reference cigarette. End-points included histopathology, CO-oximetry, plasma nicotine and cotinine, clinical pathology, and organ and body weights. The only pathological response observed was slight to mild epithelial hyperplasia and inflammation in the most rostral part of the nasal cavity, in the high-exposure group only. No effects were noted at medium or low exposures. The minimal changes noted were reversible, using a subgroup of animals kept without further treatment for an additional 14 days. Overall, the end-points used in the study demonstrated that there was no detectable biological activity of ADSS at typical or even 10-fold ETS concentrations and that the activity was only minimal at very exaggerated concentrations (particle concentrations 100 times higher than typical real-world concentrations).

Histological sectioning of the rodent larynx for inhalation toxicity testing.

In rodents, the larynx is a major site of histopathologic alteration following inhalation exposure to particulates, vapors, and aerosols. Specifically, the epithelial lining of a narrowly delineated region on the ventral floor of the larynges of rats and mice appears to be especially vulnerable to inhaled materials, and is recognized as a preferred site for histopathological evaluation in inhalation studies. This site is located at the base of the epiglottis, cranial to the ventral laryngeal diverticulum (ventral pouch). The presence of underlying seromucinous glands is critical for histologic identification of this site. We report a histologic sectioning technique, using the ventral laryngeal diverticulum as the anatomical landmark, to obtain tissue sections from this area of predilection in rats and in mice.

Analysis of cytogenetic effects in bone-marrow cells of rats subchronically exposed to smoke from cigarettes which burn or only heat tobacco.

The genotoxic effects of 90-day nose-only exposures to smoke from new cigarettes, which heat but do not burn tobacco (New), or from reference cigarettes, which burn tobacco, were evaluated in Sprague-Dawley rats by examining the cytogenetic endpoints of sister-chromatid exchanges (SCE), chromosome aberrations, and micronuclei in bone-marrow cells. The concentrations of wet total particulate matter (WTPM) and carbon monoxide in the smoke from both cigarette types were similar. The mainstream smoke from both New and reference cigarettes was adjusted to WTPM concentrations of approx. 200 and 400 micrograms/l for low and high smoke exposure. Rats were exposed to smoke 1 h per day, 5 days per week for 13 consecutive weeks. Inhalation of smoke by the exposed animals was confirmed by analysis of blood carboxyhemoglobin and plasma nicotine. Examination of bone-marrow cells following the final day of exposure showed that smoke from neither the New nor reference cigarette induced a positive response in the SCE, chromosome aberration, or micronucleus assays in rats.

Histologic changes in the respiratory tract induced by inhalation of xenobiotics: physiologic adaptation or toxicity?

Toxicologists and pathologists are often faced with the dilemma of categorizing changes observed in the respiratory tract of laboratory animals as either "adaptive" or "toxic." However, it is often difficult to interpret the nature of a given change as either "adaptive" or "toxic." Certain lesions or changes in the respiratory tract are to be expected from the concentration of materials given or the experimental design of a study. Careful analysis suggests that some of these changes may be more properly described as adaptive rather than toxic within the context of a given study or situation. Tissue changes discussed in this paper include squamous metaplasia of laryngeal epithelium, goblet cell change in respiratory epithelium, macrophage accumulation within alveoli, and bronchiolization of alveolar epithelium. Examples provided show that some of these changes observed in inhalation studies are similar in severity but slightly increased in frequency over sham control animals. The introduction of exogenous material into the respiratory tract of laboratory animals in an experimental setting should be expected to result in certain changes. The challenge scientists must accept is to interpret these changes so that toxic events may be separated from adaptive changes. In order to meet this challenge, studies incorporating several species and novel technologies may have to be utilized.

Ninety-day inhalation study in rats, comparing smoke from cigarettes that heat tobacco with those that burn tobacco.

Eight groups of 30 male and 30 female rats were exposed 1 hr per day, 5 days per week for 13 weeks, to smoke from reference (tobacco burned) or test (tobacco only heated) cigarettes, at nicotine concentrations of 5, 15, or 30 micrograms/liter of air. Similar smoke concentrations of wet total particulate matter and carbon monoxide were produced in each of the test/reference comparisons. There was a pronounced depression of minute ventilation of animals in the reference groups, but not in the test animals. Blood carboxyhemoglobin concentrations were similar in animals exposed to smoke from test and reference cigarettes. Plasma concentrations of nicotine and cotinine in the test groups were higher than in the reference groups. There were no differences between the smoke-exposed groups in terms of body weight or feed consumption. At necropsy, an increase in heart weight was noted in both high exposure groups. There were notable differences in histopathology, with fewer and less-pronounced changes in the test groups than in the reference groups. Many of the histopathological responses induced in the reference groups were absent in the test groups. Overall, the study demonstrated a substantial reduction in the biological activity of smoke from the test cigarette when compared with the reference.

The fate of inhaled azodicarbonamide in rats.

Azodicarbonamide (ADA) is widely used as a blowing agent in the manufacture of expanded foam plastics, as an aging and bleaching agent in flour, and as a bread dough conditioner. Human exposures have been reported during manufacture as well as during use. Groups of male F344/N rats were administered ADA by gavage, by intratracheal instillation, and by inhalation exposure to determine the disposition and modes of excretion of ADA and its metabolites. At 72 hr following gavage, 30% of the administered ADA was absorbed whereas following intratracheal instillation, absorption was 90%. Comparison between groups of rats exposed by inhalation to ADA to achieve body burdens of 24 or 1230 micrograms showed no significant differences in modes or rates of excretion of [14C]ADA equivalents. ADA was readily converted to biurea under physiological conditions and biurea was the only 14C-labeled compound present in excreta. [14C]ADA equivalents were present in all examined tissues immediately after inhalation exposure, and clearance half-times on the order of 1 day were evident for all tissues investigated. Storage depots for [14C]ADA equivalents were not observed. The rate of buildup of [14C]ADA equivalents in blood was linearly related to the lung content as measured from rats withdrawn at selected times during a 6-hr inhalation exposure at an aerosol concentration of 25 micrograms ADA/liter. In a study extending 102 days after exposure, retention of [14C]ADA equivalents in tissues was described by a two-component negative exponential function. The results from this study indicate that upon inhalation, ADA is rapidly converted to biurea and that biurea is then eliminated rapidly from all tissues with the majority of the elimination via the urine.

Disposition and metabolism of [14C]dibenzo[c,g]carbazole aerosols in rats after inhalation.

Dibenzo[c,g]carbazole (DBC) is a nitrogen-containing polycyclic aromatic hydrocarbon that has been detected in tobacco tars, industrial oils, and diesel engine exhaust fumes. DBC is carcinogenic in respiratory tract tissue of hamsters and in lungs, kidneys, and livers of mice. The purpose of this research was to determine the respiratory tract deposition, distribution in tissues, metabolism, and excretion of DBC in rats after inhalation. Rats were exposed nose-only to 1.1 or 13 micrograms [14C]DBC/liter air for 60 min. Activity median aerodynamic diameters for the two concentrations of DBC ranged from 0.7 to 0.8 micron. Urine, feces, and selected tissues were collected for various times after exposure. The fractional deposition for the 1.1 and 13 micrograms/liter exposure concentrations was similar, 13 and 16%, respectively. The dominant route of excretion of 14C following exposure to either concentration of DBC was the feces, accounting for approximately 95% of the total 14C eliminated. Half-time for fecal excretion was 20 +/- 6 hr (means +/- SE). Gastrointestinal absorption of [14C]DBC was 43%. Radioactivity was widely distributed to all tissues examined, with the respiratory tract (lung, trachea, larynx, and nasal turbinates), upper gastrointestinal tract (stomach and small intestine), the liver, and the adrenals containing the highest concentrations of [14C]DBC equivalents within 1 hr after exposure. At both concentrations of DBC tested, clearance of 14C from tissues was rapid, with approximately 60 to 98% of the initial tissue burden being cleared with half-times ranging from 1 to 16 hr. The remaining 2 to 40% in the tissues was cleared with half-times that ranged from 1.5 to 14 days. Several metabolites were detected in the urine and feces, none of which appeared to be either glucuronide or sulfate conjugates. Small quantities of [14C]DBC were detected in the urine, although quantities were less than 1% of the initial respiratory tract burden of [14C]DBC. The results from this research indicate that DBC was rapidly absorbed from the lungs and translocated to many tissues. Prior to elimination, primarily in the feces, DBC was extensively metabolized. There appeared to be no effect of exposure concentration on the toxicokinetics of inhaled DBC.

Scanning and transmission electron microscopic observations of the acute morphological response of the mouse urinary bladder to 4-ethylsulfonylnaphthalene-1-sulfonamide.

A total of 75 BALB/cStCrlfC3H/Nctr male weanling mice were administered either 0 or 250 ppm of 4 ethylsulfonylnaphthalene-1-sulfonamide (ENS) in the diet for periods up to 14 days to evaluate the early morphological changes of the transitional epithelium of the urinary bladder with scanning (SEM) and transmission (TEM) electron microscopy. Primary TEM changes included hyperplasia of the epithelium, loosening of the intercellular junctions, autophagic vacuoles and electron dense granules in the mitochondria. Primary SEM changes included sloughing of epithelial cells, irregularity in the size and shape of the transitional epithelial cells and the presence of microvilli. Although pleomorphic microvilli were present after only three days of treatment with ENS, it appears that they are a transient observation in a series of morphological changes. The reversibility or transient nature of the pleomorphic microvilli may indicate that they are an acute toxic response and may not necessarily indicate a preneoplastic change.