Postnatal development of parvalbumin and calbindin D-28k immunoreactivities in the canine hippocampus.

The calcium-binding proteins, parvalbumin and calbindin D-28k, are markers of different classes of GABAergic interneurons and display different functions. The present study was attempted to determine immunoreactivities and colocalization of the parvalbumin and calbindin D-28k in the developing canine hippocampus by immunohistochemistry. The calcium-binding protein-containing neurons showed different developmental patterns. The first appearance of parvalbumin immunoreactive nonpyramidal cells was observed at P7. Parvalbumin immunoreactivity was elicited by the sequence from CA3 to CA1 to reach an adult-like distribution pattern, which was reached at P60, while calbindin D-28k immunoreactivity appeared from P0, including pyramidal and nonpyramidal cells. The characteristic distribution of calbindin D-28k immunoreactive pyramidal cells was clarified by P28, and an adult-like distribution pattern was reached by the end of the second postnatal month. Double-labeled nonpyramidal cells were frequently seen in the subareas, CA3 of P14/CA1-CA2 of P28, where parvalbumin immunoreactive nonpyramidal cells were emerging. These data suggest that the colocalization of the two calcium-binding proteins during development is related closely to the area-specific maturation of parvalbumin expression, although either prenatal expression of calbindin D-28k or parvalbumin was not determined.

Expression of transforming growth factor alpha in plutonium-239-induced lung neoplasms in dogs: investigations of autocrine mechanisms of growth.

We have previously shown that 47% of radiation-induced lung neoplasms in dogs exhibit increased expression of epidermal growth factor receptor (EGFR). In this study, we investigated the expression of transforming growth factor alpha (TGF-alpha), a ligand for EGFR, to determine if an autocrine mechanism for growth stimulation was present in these tumors. As determined by immunohistochemistry, 59% (26/44) of the lung neoplasms examined had increased expression of TGF-alpha. Expression of TGF-alpha was not related to the etiology of the tumor, e.g., spontaneous or plutonium-induced; however, it was related to the phenotype of the tumor. Statistical analysis of the correlation of EGFR and TGF-alpha expression within the same tumor did not show a positive association; however, specific phenotypes did have statistically significant expression of EGFR or TGF-alpha, suggesting that overexpression of either the ligand or its receptor conferred a growth advantage to the neoplasm. Twenty-seven percent (32/117) of radiation-induced proliferative epithelial foci expressed TGF-alpha, and a portion of those foci (8/32) expressed both EGFR and TGF-alpha. This supports the hypothesis that these foci represent preneoplastic lesions, and suggests that those foci exhibiting increased expression of the growth factor or its receptor are at greater risk for progressing to neoplasia.

Influence of dose rate on survival time for 239PuO2-induced radiation pneumonitis or pulmonary fibrosis in dogs.

Beagle dogs were exposed once or repeatedly to 0.75-microns-diameter monodisperse aerosols of 239PuO2 by pernasal inhalation. The dogs that were exposed once received alveolar depositions (+/- standard deviation) of 3.9 +/- 1.9 kBq/kg body mass and accumulated doses of 23 +/- 8 Gy to the lung before death at 5.4 +/- 1.7 years after exposure. Dogs exposed repeatedly received a total alveolar deposition of 5.3 +/- 0.9 kBq/kg body mass during 7 to 10 semiannual exposures and accumulated doses of 22 +/- 5 Gy to the lung before death at 4.9 +/- 0.7 years after first exposure. Clearance of the plutonium from the lung in the dogs exposed repeatedly was slower than in the dogs exposed once. All dogs in the repeated-exposure study and all but one dog in the single-exposure study died from radiation effects. Pulmonary fibrosis accounted for 72% of the radiation-related deaths in the single-exposure study and 87% in the repeated-exposure study. The remaining dogs died with pulmonary cancer. Based on total cumulative radiation dose, the times after exposure to death from radiation pneumonitis and pulmonary fibrosis were not significantly different for single and repeated exposures. Thus dose rate does not appear to be an important factor in predicting death from radiation pneumonitis or pulmonary fibrosis for dogs inhaling 239PuO2.

Biological effects of 137CsCl injected in beagle dogs.

The toxicity of intravenously administered 137CsCl in the beagle dog was investigated as part of a program to evaluate the biological effects of internally deposited fission-product radionuclides. The intravenous route of exposure was chosen for simplicity and accuracy because it was known that after intravenous injection, inhalation or ingestion, internally deposited 137CsCl is rapidly absorbed and distributed throughout the body, exposing the whole body to beta-particle and gamma radiations. Fifty-four dogs were injected intravenously with 137Cs to provide one group of six dogs with mean initial body burdens of 141 MBq 137Cs/kg body mass and four groups of 12 dogs each with mean initial body burdens of 104, 72, 52 and 36 MBq 137Cs/kg. Twelve dogs were injected with isotonic saline as study controls. Because the number of study control dogs was small, data from an additional 49 control dogs from other studies at the Inhalation Toxicology Research Institute that were performed over a similar span of years were also used. There was a significant, dose-dependent decrease in survival of the 137Cs-injected dogs. Eleven 137Cs-injected dogs, including all six in the highest initial body burden group, died within 81 days after injection, primarily due to hematopoietic cell damage resulting in severe pancytopenia. An additional 25 dogs had transient hematological dyscrasia but survived for long times. All 137Cs-injected male dogs had marked damage to the germinal epithelium of the testicular seminiferous tubules with azoospermia in the long-term survivors. Benign and malignant neoplasms occurred in a variety of organs in 137Cs-injected dogs, rather than in a single target organ. When individual organs were considered, the incidence of malignant neoplasms was increased in the liver and in the nasal cavity and paranasal sinuses of the 137Cs-injected dogs. There was a 137Cs treatment effect in the incidence of malignant neoplasms (P < 0.001) in male dogs but no 137Cs-related treatment effect in female dogs. However, when malignant mammary neoplasms were excluded from the analysis, there was no gender difference, and there was a dose-related response (P < 0.001) in both males and females for the incidence of malignant neoplasms.

Sequential analysis of the pathogenesis of plutonium-induced pulmonary neoplasms in the rat: morphology, morphometry, and cytokinetics.

Light microscopy, morphometry, and cytokinetic techniques were used to examine the dynamics of plutonium-induced pulmonary proliferative lesions and neoplasms in rats at several intervals to 450 days after inhalation exposure to aerosols of 239PuO2. Maximal increases in alveolar and bronchiolar epithelial cell labeling were seen at 30 days; decreasing subsequently, the levels remained elevated above control indices. Focal proliferative epithelial lesions developed in the lung by 180 days and before the onset of pulmonary neoplasms. Pulmonary neoplasms, predominantly adenocarcinomas and squamous cell carcinomas, were initially observed at 308 days. The proliferative lesions progressed through a succession of morphological changes leading to the development of neoplasms. The volume density (fraction) and epithelial surface area of foci of alveolar epithelial hyperplasia increased progressively between 180 and 450 days after exposure, in contrast to the other proliferative lesions. We conclude that plutonium-induced pulmonary neoplasms develop through a succession of focal proliferative lesions that represent developmental preneoplastic lesions. Progressive increases in volume and epithelial surface area of the alveolar epithelial hyperplasias suggest that they may be more at risk for neoplastic transformation than the other histological types of proliferative foci.

ATP-sensitive K+ current and its modulation by substance P in gastric myocytes isolated from guinea pig.

To investigate whether ATP-sensitive K+ channels exist in gastric smooth muscle of the guinea pig and whether they are modulated by substance P, we recorded lemakalim-activated K+ currents from freshly isolated cells using the standard whole-cell configuration. With 0.1 mM ATP and 140 mM K+ in the pipette and 90 mM K+ in the bath solution and a holding potential of -80 mV, lemakalim (10 microM) activated a glibenclamide-sensitive inward current with a mean amplitude of -224+/-34 pA. These currents were voltage-independent from -90 to 0 mV and K+-selective. Increasing the intracellular ATP concentrations from 0.1 to 3 mM reduced the lemakalim-activated currents by about five-fold. External barium and cesium inhibited the lemakalim-activated currents in a dose-dependent manner. External tetraethylammonium (10 mM) inhibited the lemakalim-activated currents by 66+/-15%. Bath application of substance P (5 microM) inhibited the lemakalim-activated currents by 53+/-13% and this inhibition was absent when 0.5 mM guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) was in the pipette. Phorbol 12,13-dibutyrate (PDB) inhibited the lemakalim-activated currents by 71+/-3%. Chelerythrine (1 microM) reduced the substance P-induced inhibition of lemakalim-activated currents by 22.2+/-11.3%. These results suggest the presence of ATP-sensitive K+ channels in gastric smooth muscle and that substance P inhibits ATP-sensitive K+ channels via G-protein through protein kinase C activation.

Successful treatment of all-trans retinoic acid resistant and chemotherapy naïve acute promyelocytic patients with arsenic trioxide--two case reports.

Arsenic trioxide(As2O3) has proved highly effective in treating both refractory or primary cases of acute promyelocytic leukemia (APL). The role of arsenic trioxide in APL treatment has been confirmed by study groups in China and in the USA. However, what is the role of As2O3 in treating APL? Should it be used as first line therapy, or should it be used as a second line drug. This still remains to be defined. Here, we report two cases of APL, who were treated successfully with As2O3 when they relapsed. Initially, both received all-trans retinoic acid (ATRA) for primary remission induction therapy, and obtained a complete remission. For ethical or personal reasons, they did not receive chemotherapy as consolidation therapy and when they relapsed at 23 months and 12 months later respectively, they both received As2O3 therapy after being resistant to ATRA treatment. Two courses of As2O3 were given and both reached complete remission. There were very few adverse reactions to the drug, only mild abdominal cramps, mild fluid retention, and transient elevation of transaminases. They both had rather good quality of life throughout the treatment and both remain in remission for 32 months and 10 months since therapy, respectively.

Retinoic acid syndrome induced by arsenic trioxide in treating recurrent all-trans retinoic acid resistant acute promyelocytic leukemia.

Arsenic Trioxide (As2O3) is an effective agent for treating acute promyelocytic leukemia achieving a complete remission rate of about 60% to 90%. It is similar to all-trans retinoic acid (ATRA) when treating acute promyelocytic leukemia (APL), because both agents have limited side effects compared to conventional chemotherapy, although the treatment period is more prolonged. During treatment, both agents may induce leukocytosis, and in patients taking ATRA, leukocytosis appears to be related to the development of retinoic acid syndrome (RAS). We report here a case of APL treated with ATRA in combination with chemotherapy 3 years earlier. During treatment, an episode of RAS with fever, edema, pericardiac effusion etc. was encountered. Recently, she had a relapse of leukemia, and As2O3 therapy was used. Leukocytosis developed again, and symptoms of fever, skin rash, edema resembling a RAS also developed, which was quickly relieved by steroid administration in a manner resembling response to RAS.

Biological markers of exposure to SO2: S-sulfonates in nasal lavage.

S-sulfonate levels were measured in the nasal lavage (NAL) fluid of humans exposed to sulfur dioxide as a potential biological marker of exposure. These levels were determined by treating NAL fluid protein with cyanide to cleave the S-S linkage and release the sulfite. The cyanolytically released sulfite was measured by ion chromatography. In two experiments, humans were exposed to air or 1 ppm SO2 for 10 minute, and to air or 7 ppm SO2 for 20 minutes and lavaged immediately after exposure. Releasable sulfite levels in NAL fluid were 1.06 +/- 0.24 and 2.61 +/- 0.55 micrograms SO=3/mg protein, respectively (mean +/- SE, n = 5), for the first experiment, and 1.16 +/- 0.37 and 4.91 +/- 0.76 micrograms SO=3/mg protein, respectively (mean +/- SE, n = 8), for the second. The subjects in the former study were persons with asthma. In both experiments, S-sulfonate levels were statistically elevated in the exposed group compared with the control groups (p < 0.05, paired t-test). The same individuals in the second experiment received five additional 20-minute exposures to 7 ppm SO2 every other day, for a total of six exposures. NAL fluid taken at the conclusion of the final exposure had releasable sulfite levels of 4.99 +/- 1.36 micrograms SO=3/mg protein; these levels were statistically elevated relative to controls but were not elevated relative to the 1-day exposure (mean +/- SE, n = 8). The lack of accumulation of S-sulfonates after 6 days of short-term exposure suggests clearance of these compounds from the nasal passages within 24 hours. The levels of S-sulfonates observed in NAL fluid in this study are almost three orders of magnitude higher than those measured in plasma following similar SO2 exposures. Measurement of S-sulfonates in the nasal passage may be an effective short-term biomarker of exposure to SO2.

Pulmonary toxicity of inhaled diesel exhaust and carbon black in chronically exposed rats. Part I: Neoplastic and nonneoplastic lung lesions.

This study compared the pulmonary carcinogenicities and selected noncancer effects produced by chronic exposure of rats at high rates to diesel exhaust and carbon black. The comparison was intended to provide insight into the likely importance of the mutagenic organic compounds associated with the soot portion of diesel exhaust in inducing pulmonary carcinogenicity in diesel exhaust-exposed rats. The role of the organic fraction has become important in judging the usefulness of the substantial data base on carcinogenicity in rats for predicting lung cancer risk for humans, and for determining the most appropriate method of extrapolating results across species and exposure concentrations. Rats were exposed chronically to either diesel exhaust or carbon black, which served as a surrogate for diesel exhaust soot with much reduced mutagenic activity associated with its organic fraction. The sequestration of particles in the lung and the induction of pulmonary neoplasia and non-neoplastic changes in the lung were compared in detail. Samples also were provided to collaborators to examine adduct formation in lung DNA and hemoglobin. Approximately 140 female and 140 male F344/N rats were exposed for 16 hours per day, 5 days per week for up to 24 months, beginning at eight weeks of age, to diesel exhaust or carbon black at 2.5 mg or at 6.5 mg particles/m3 of air, or to clean air as controls. The diesel exhaust was generated by light-duty engines burning certification fuel and operating on an urban-duty cycle. The carbon black was selected because it had particle size and surface area characteristics similar to those of diesel exhaust soot, but markedly less mutagenic activity associated with its organic fraction when analyzed using procedures typically used in studies of diesel soot. Rats were killed after 3, 6, 12, 18, or 23 months of exposure to measure lung and lung-associated lymph node burdens of particles, lung weight, bronchoalveolar lavage indicators of inflammation, DNA adducts in whole lung and alveolar type II cells, and chromosome injury in circulating lymphocytes, and to perform histopathologic assessment. In addition, after 3 and 18 months of chronic exposure, one group of rats was acutely exposed to radiolabeled carbon black particles or to fluorescent microspheres. These exposures were conducted to examine the clearance of radiolabeled particles and the sequestration of the fluorescent microspheres in the lungs. These experiments provided information on clearance overload and particle dosimetry. The growth characteristics of lung neoplasms also were examined by transplanting neoplastic cells into athymic mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Detection of serum alpha-fetoprotein in dogs with hepatic tumors.

Serum alpha-fetoprotein (AFP) concentration was detected by use of 2 commercially available kits containing antibodies to human AFP--a radioimmunoassay and an enzymetric test. Using neonatal canine serum (a source high in AFP), it was determined that reagents from both kits were able to bind to canine AFP, but a significant difference was detected in AFP concentration. The enzymetric test was superior in detecting canine AFP. Sera from dogs were classified into 6 groups: from dogs with primary hepatic tumors only (group 1); from dogs with primary hepatic tumors and other tumors (group 2); from dogs with normal liver but with other types of neoplasia (group 3); from dogs with nonneoplastic hepatic disease and tumors originating in other organs (group 4); from dogs with nonneoplastic hepatic disease only (group 5); and from clinically normal dogs (group 6). Serum biochemical determinations (alkaline phosphatase, alanine transaminase, albumin, total protein, total bilirubin, and serum bile acids) and values from the 2 AFP assays were obtained for all dogs. Serum AFP concentration detected by the enzymetric test was significantly higher in dogs with hepatocellular carcinoma and cholangiocarcinoma. Values greater than 250 ng/ml were detected in 5 of 9 dogs with cholangiocarcinoma and in 3 of 4 dogs with hepatocellular carcinoma. High serum AFP concentration also was indicative of liver involvement in 2 of 3 dogs with primary hepatic lymphosarcoma; 2 dogs had values greater than 225 ng/ml. Serum AFP concentration in dogs with other types of hepatic tumors was less than 250 ng/ml, and serum AFP concentration could not be correlated with such tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

The critical role of catalase in prooxidant and antioxidant function of p53.

The tumor suppressor p53 is an important regulator of intracellular reactive oxygen species (ROS) levels, although downstream mediators of p53 remain to be elucidated. Here, we show that p53 and its downstream targets, p53-inducible ribonucleotide reductase (p53R2) and p53-inducible gene 3 (PIG3), physically and functionally interact with catalase for efficient regulation of intracellular ROS, depending on stress intensity. Under physiological conditions, the antioxidant functions of p53 are mediated by p53R2, which maintains increased catalase activity and thereby protects against endogenous ROS. After genotoxic stress, high levels of p53 and PIG3 cooperate to inhibit catalase activity, leading to a shift in the oxidant/antioxidant balance toward an oxidative status, which could augment apoptotic cell death. These results highlight the essential role of catalase in p53-mediated ROS regulation and suggest that the p53/p53R2-catalase and p53/PIG3-catalase pathways are critically involved in intracellular ROS regulation under physiological conditions and during the response to DNA damage, respectively.

The p53-inducible gene 3 (PIG3) contributes to early cellular response to DNA damage.

The p53-inducible gene 3 (PIG3) is originally isolated as a p53 downstream target gene, but its function remains unknown. Here, we report a role of PIG3 in the activation of DNA damage checkpoints, after UV irradiation or radiomimetic drug neocarzinostatin (NCS). We show that depletion of endogenous PIG3 sensitizes cells to DNA damage agents, and impaired DNA repair. PIG3 depletion also allows for UV- and NCS-resistant DNA synthesis and permits cells to progress into mitosis, indicating that PIG3 knockdown can suppress intra-S phase and G2/M checkpoints. PIG3-depleted cells show reduced Chk1 and Chk2 phosphorylation after DNA damage, which may directly contribute to checkpoint bypass. PIG3 exhibited diffuse nuclear staining in the majority of untreated cells and forms discrete nuclear foci in response to DNA damage. PIG3 colocalizes with gamma-H2AX and 53BP1 to sites of DNA damage after DNA damage, and binds to a gamma-H2AX. Notably, PIG3 depletion decreases the efficient induction and maintenance of H2AX phosphorylation after DNA damage. Moreover, PIG3 contributes to the recruitment of 53BP1, Mre11, Rad50 and Nbs1 to the sites of DNA break lesions in response to DNA damage. Our combined results suggest that PIG3 is a critical component of the DNA damage response pathway and has a direct role in the transmission of the DNA damage signal from damaged DNA to the intra-S and G2/M checkpoint machinery in human cells.

Ribonucleotide reductase small subunit p53R2 suppresses MEK-ERK activity by binding to ERK kinase 2.

The p53-dependent RR small subunit (p53R2) protein, a newly identified member of the ribonucleotide reductase family, plays a key role in the p53-dependent cellular response to DNA. Several recent studies have suggested that p53R2 also plays an important role in suppressing the invasive potential of human cancer cells. However, the cellular mechanism that regulates invasiveness remains largely unknown. In this study, we show that p53R2 interacts with MEK2 (extracellular signal-regulated kinase (ERK) kinase 2-mitogen-activated protein kinase (MAPK) kinase 2), the molecule immediately upstream of ERK in the Ras-Raf-MAPK signaling cascade. In co-immunoprecipitation and immunofluorescence analyses, we found that p53R2 and MEK2 interact physically in cultured mammalian cells, and that the p53R2 segment comprising amino acids 161-206 is critical for this interaction. Moreover, serum-induced phosphorylation of MEK1/2 and ERK1/2 was greatly augmented in human cancer cells expressing small-interfering RNA against p53R2. On the other hand, phosphorylation of MEK1/2 and ERK1/2 in human cancer cells was markedly attenuated by overexpression of p53R2. Furthermore, MEK2 was required for p53R2 knockdown-induced enhancement of the invasive ability and anchorage-independent growth of human lung cancer H1299 cells. Taken together, these findings show that p53R2 negatively modulates serum-induced MEK-ERK activity and inhibits the MEK-ERK-mediated malignancy potential of human cancer cells.

Calbindin D-28k, parvalbumin and calcitonin gene-related peptide immunoreactivity in the canine spinal cord.

This study was conducted as a comparative analysis of the immunohistochemical localization of calbindin D-28k, parvalbumin and the calcitonin gene-related peptide (CGRP) in the cervical through the sacral spinal cord of mongrel dogs, to reveal any distinct patterns of distribution and possible involvement in spinal processing. In laminae I and II of the substantia gelatinosa, both calbindin D-28k and CGRP showed strong immunoreactivity, with calbindin D-28k being positive in both cells and fibres, while CGRP was positive in fibres only. Parvalbumin and CGRP immunoreactive cells were widely distributed in various nuclei and lower motor neurones in the ventromedial horn. In addition, the lower motor neurones expressed CGRP as well as parvalbumin, but not calbindin D-28k. These results are generally consistent with previous reports, and the co-localization of parvalbumin and CGRP may explain the functional improvement of lower motor neuron disease.

Disposition of inhaled isobutene in F344/N rats.

Isobutene (2-methylpropene) (CAS No. 115-11-7) is a gas widely used in the chemical manufacturing industry. As an aid to planning long-term toxicity studies, research was conducted to determine the effect of exposure concentrations on the absorption and metabolism of isobutene in F344/N rats. Male F344/N rats (11-15 weeks of age) were exposed for 2 hr to 0, 40, 400, or 4000 ppm isobutene, and a time-course evaluation of blood levels of isobutene was performed using headspace analysis methods. Blood levels of isobutene were linearly related to exposure concentrations between 40 and 400 ppm but increased in a supralinear fashion at the highest concentration, suggesting that the capacity of the rats to metabolize isobutene had been exceeded. Total uptake, excretion patterns, and metabolic conversions were studied in rats exposed for up to 6 hr to 0, 2, 40, 400, or 4000 ppm [14C]isobutene. Absorption of the inhaled isobutene was approximately 8% up to 40 ppm isobutene, but decreased at the higher concentrations. The amount of isobutene metabolized per ppm.hr of exposure was also linear up to 40 ppm but decreased at higher concentrations. Over 90% of the absorbed isobutene was metabolized at exposure concentrations up to 400 ppm, but the exposure to approximately 4000 ppm isobutene resulted in approximately 20% of the absorbed dose exhaled as the unmetabolized isobutene. Two urinary metabolites were identified as isobutenediol and 2-hydroxyisobutyric acid. Two other urinary metabolites were tentatively identified as sulfate conjugates of isobutenediol. Based on these studies, linear dose-response relationships would be expected in chronic toxicity studies for exposures up to 40 ppm isobutene. Additional studies would be required to determine if repeated exposures would induce higher metabolic capacities in the exposed rats.

Effect of dose on the disposition of 2-ethoxyethanol after inhalation by F344/N rats.

Glycol ethers such as 2-ethoxyethanol (EE) are widely used as solvents because they are miscible in aqueous and organic solutions. Toxic effects of EE in rodents include teratogenicity, fetotoxicity, hematotoxicity, and testicular atrophy. The purpose of this study was to determine the effect of dose on the absorption, metabolism, and excretion of 2-ethoxy [U-14C]ethanol by F344/N rats after inhalation exposure. Rats were exposed to either 5 ppm EE for 5 hr 40 min or 46 ppm EE for 6 hr. The uptake and metabolism of EE were linear in the concentration range studied. Significant percentages of the retained doses were exhaled during (22%) and after exposure (16%) as 14CO2. Forty-six percent of the retained dose was excreted in the urine. Approximately 10% of the retained dose was detected in the carcass 66 hr after exposure. The major urinary metabolite was ethoxyacetic acid (EAA), the toxic metabolite of EE. The amount of EAA excreted was linearly related to exposure concentration. Ethylene glycol and N-ethoxyacetyl glycinate were identified as minor metabolites excreted in the urine. The results of this study suggest that the toxicity of inhaled EE should be directly proportional to the exposure concentration up to 46 ppm if the toxicity of EE is due to EAA.

Effect of chronic cigarette smoke exposure on lung clearance of tracer particles inhaled by rats.

Cigarette smoking can influence the pulmonary disposition of other inhaled materials in humans and laboratory animals. This study was undertaken to investigate the influence of cigarette smoke exposures of rats on the pulmonary clearance of inhaled, relatively insoluble radioactive tracer particles. Following 13 weeks of whole-body exposure to air or mainstream cigarette smoke for 6 hr/day, 5 days/week at concentrations of 0, 100, or 250 mg total particulate matter (TPM)/m3, rats were acutely exposed 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 serially euthanized through 6 months after exposure to confirm the relative insolubility of the tracer particles. We observed decreased tracer 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 in control, 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 increased numbers of pigmented alveolar macrophages distributed throughout the parenchyma and focal collections of enlarged alveolar macrophages with concomitant alveolar epithelial hyperplasia and neutrophilic alveolitis. The severity of the lesions increased with smoke exposure duration and concentration to include interstitial aggregates of pigmented macrophages and interstitial fibrosis. Our data confirm previous findings that exposure to cigarette smoke decreases the ability of the lungs to clear inhaled materials. We further demonstrate an exposure-concentration related magnitude of effect, suggesting that the cigarette smoke-exposed rat constitutes a useful model for studies of the effects of cigarette smoke on the disposition of inhaled particles.

Species differences in urinary butadiene metabolites: comparisons of metabolite ratios between mice, rats, and humans.

We have previously identified two metabolites, 1,2-dihydroxy-4-(N-acetylcysteinyl-S-)-butane (M-I) and 1-hydroxy-2-(N-acetylcysteinyl-S-)-3-butene (M-II) in the urine of mice, rats, hamsters, and monkeys exposed by inhalation to 8000 ppm [14C]butadiene. The sum of these two metabolites constituted between 50 and 90% of the total urinary [14C]butadiene equivalents. When comparing species, the ratios of excreted M-I relative to the total of M-I + M-II were linearly related to hepatic epoxide hydrolase activities, with mice displaying the lowest ratios and monkeys displaying the highest ratios. Because humans are known to have epoxide hydrolase activities more similar to those of monkeys than mice, we postulated that after inhalation of butadiene, humans would excrete predominantly M-I and little M-II. To address this hypothesis, we measured the two metabolites in the urine of workers occupationally exposed to butadiene. We initially developed an assay to measure the two metabolites in urine using techniques not dependent on radiolabeled compounds. The assay is based on isotope-dilution gas chromatography/mass spectroscopy. After addition of deuterated internal standards, the metabolites were isolated from urine samples by solid-phase extraction and selective precipitation. The metabolites were converted to volatile derivatives by trimethylsilylation prior to analysis. The assay is sensitive down to at least 100 ng/ml of both metabolites in urine. The assay was applied to urine samples of humans occupationally exposed to butadiene in a production plant. M-I, but not M-II, could be readily identified and quantitated in the urine samples at levels frequently greater than 1 microgram/ml, thus supporting our hypothesis. Employees who worked in production areas with historical atmospheric concentrations of 3-4 ppm butadiene could be distinguished as a group from those outside controls. Finally, mice and rats were exposed to 11.7 ppm butadiene for 4 hr, and the ratio of the two metabolites was measured. For mice, the ratios of M-I to M-I + M-II were similar to those reported previously following exposure to 8000 ppm. In contrast, for rats, M-I represented a higher proportion of the excreted metabolites at the lower exposure level. These results confirm earlier in vitro studies that suggested the predominant pathway for clearance of BDO in humans is by hydrolysis rather than direct conjugation with glutathione.

Effect of cumulative exposure on nasal response to ozone.

To assess the potential health risks associated with exposure to low levels of ozone, it is essential to know if the ozone-induced responses are dependent on cumulative exposures or on the peak concentrations. To answer this question female F344/N rats, 11-13 weeks of age, were exposed to a matrix of equal concentration x time values that included exposures to 0, 0.12, 0.24, and 0.48 ppm ozone for 3, 6, 12, or 24 hr. the response of the nasal epithelium was measured as induced DNA synthesis determined by the uptake of bromodeoxyuridine (a thymidine analog) into epithelial cells lining the nasal anterior maxilloturbinates. No increased DNA synthesis was observed in rats exposed to 0.12 ppm ozone for any of the time periods. For exposures higher than 0.12 ppm ozone, the response of the nasal epithelium was similar for equal cumulative exposures. The responses, however, were not linearly related to the cumulative (concentration x time) exposures. It appeared that some mitigating factor was present which decreased the responses at the higher cumulative exposures. No frank toxicity or cellular necrosis was observed, indicating that sublethal cell damage was sufficient to induce DNA synthesis. A simple mathematical model was developed to describe the relationship between ozone exposure and the induction of DNA synthesis in the nasal epithelium. The model predicted that the threshold concentration of ozone for inducing DNA synthesis in the nasal epithelium was 0.1 +/- 0.1 ppm. For one measure of ozone toxicity (induced DNA synthesis) at a sensitive site in the respiratory tract (maxilloturbinates), the effects of ozone were dependent on cumulative exposures at concentrations > 0.12 ppm and within the time and concentration ranges used in this study.