Carbon black and soot: two different substances.

Carbon blacks are manufactured under controlled conditions for commercial use primarily in the rubber, painting, and printing industries. In contrast, soots are unwanted byproducts from the combustion of carbon-based materials for the generation of energy or heat, or for the disposal of waste. Unfortunately, the terms carbon black and soot often have been used interchangeably; however, carbon black is physically and chemically distinct from soot. Greater than 97% of carbon black consists of elemental carbon arranged as aciniform particulate. Depending on the type of soot, the relative amount of carbon (< 60% of the total particle mass), the type of particulate carbon, and particle characteristics (size, shape, and heterogeneity) can vary considerably. For both carbon black and soot, other elements and chemical compounds are associated with the particulate carbon. Total inorganics (ash) represent < 1% of the carbon black particle mass. Organic compounds can be extracted from particle surfaces (solvent extractable fraction [SOF]), and for carbon black, also are < 1% of the particle mass. Soots have much greater percentages of ash, SOF, or both, than carbon black. There has been concern about the adsorbed organic compounds because of potential biologic activity. For carbon black, the SOF is strongly adherent to carbon surfaces and is not released by biological fluids. The types of organic compounds consist primarily of unsubstituted polycyclic aromatic hydrocarbons and are not as biologically potent as those compounds present in soot. Thus, carbon black is distinctly different from soot, and when discussing potential health effects, care must be taken to differentiate between the two types of carbon-based particles.

Comparative mutagenic dose of ambient diesel engine exhaust.

Diesel engine exhaust contains carbon-based particles that can be inhaled and deposited on lung surfaces. Concern about the carcinogenic potential of diesel engine exhaust derives in part from the mutagenic activity of organics that can be extracted from exhaust particles. However, the lung cancer risk is controversial, and diesel exhaust is a candidate for further evaluation. A comparative potency approach can be used to rank the mutagenic risk of diesel exhaust with other combustion products. We compared the specific mutagenic activities of cigarette smoke condensate (CSC) and diesel exhaust particle extract (DEPE) and estimated "mutagenic dose" to the lungs. Although the specific mutagenic activities of CSC and DEPE are similar in magnitude, it is the dose reaching the lungs that is more relevant for comparing mutagenic potential. We calculated that, depending on the source of CSC and DEPE, a person would have to inhale approximately 63 to 181 mg of particulate from diesel engine exhaust to match the mutagenic dose of 1 cigarette. We also calculated that a person would have to breathe diesel exhaust (1.5 microg/m(3), estimated total personal exposure) for 6 to 16 yr to equal the mutagenic dose of 1 cigarette. Although instructive, comparative potency results should be used cautiously due to the need for simplifying assumptions. For example, the type of mutagenic assay and the source of cigarettes and diesel engine exhaust could affect dose estimates to some degree; however, the extent to which diesel particle mutagens are bioavailable would have an even greater effect on estimates of relative risk. For both cigarette smoke and diesel exhaust particles, we assumed that the organic mutagens are 100% bioavailable. In summary, our analysis showed a larger mutagenic dose-to-target-tissues in the smoke of one cigarette as compared to a year of exposure to diesel exhaust particulate at ambient levels.

Lung cancer rates in carbon-black workers are discordant with predictions from rat bioassay data.

The recent demonstration of lung tumors in rats chronically exposed to airborne carbon-black particles has prompted reevaluation of the carcinogenicity of carbon black. However, accumulating evidence suggests that rat responses to inhaled particles are unique and are mediated by the sequelae to lung overload conditions. To test the predictive value of the rat inhalation bioassay for human lung cancer risk, we examined epidemiologic studies of workers exposed to carbon black during its manufacture or use. Industries in which significant airborne carbon-black exposure has occurred were not associated with increased lung cancer risk. We also predicted the number of occupational lung cancer cases that one would expect in exposed workers, based on the cancer potency of carbon black derived from rat studies. Our quantitative comparison of the tumorigenicity of carbon black predicted from rat studies to the lung cancer rate in carbon-black workers showed a marked discrepancy between the lung cancers predicted and those actually observed. We found that far more lung cancers are predicted from the rat bioassay than can be demonstrated in workers. We determined that it is highly unlikely that this discrepancy is due to chance. We conclude that extrapolating the incidence of lung tumors in rats inhaling inert, insoluble particles, such as carbon black, to humans must be seriously questioned. Using rat inhalation bioassay data for carbon black to estimate lung tumor risk in humans must be seriously questioned unless the mechanisms of the rat's unique response are shown to be relevant to humans.

Analysis of diesel-exhaust unit-risk estimates derived from animal bioassays.

Chronic inhalation of diesel exhaust (DE) causes lung tumors in rats; epidemiologic studies suggest that DE may be a potential human carcinogen. We compared the DE unit risks for human lung cancer as derived by Smith and Stayner (1991), Hattis and Silver (1992), Pepelko and Chen (1993), California Environmental Protection Agency, Office of Environmental Health Hazard Assessment (1994), and the U.S. Environmental Protection Agency (USEPA) (1994). All five sets of authors used identical rat bioassay data (Mauderly et al., 1987). Although different in detail, the dose-response models were uniformly linear and nonthreshold. However, each set of authors chose a different approach in relating the rat data to DE unit risk in humans. The predicted unit risks for continuous lifetime exposure to 1 microgram/m3 DE differed by an 80-fold factor between the highest [8 x 10(-4), Hattis and Silver] and the lowest [0.1 x 10(-4), Pepelko and Chen]. The choice of dose-input parameters and how each group treated particle overload were the major factors affecting the different risk estimates. Several unanswered questions undermine the current use of the rat bioassay for DE risk assessment: (1) Differences in emission products and exposure scenarios between laboratory studies and human exposure, (2) occurrence of an apparent threshold in the rat-lung-tumor response, (3) uncertainty as to the appropriate lung-dose metric and its low-dose extrapolation, (4) the role of lung overload, and (5) the cause of species-specific biological susceptibility.

Cellular and subcellular colocalization of nerve growth factor and epidermal growth factor in mouse submandibular glands.

Immunocytochemical methods have been used to compare the cellular and subcellular distribution of nerve growth factor (NGF) and epidermal growth factor (EGF) in mouse submandibular glands. Rabbit antisera raised against purified proteins were characterized by immunoblot methods and were used to stain sections of salivary glands embedded in plastic. For light microscopy, antibodies were visualized by indirect immunofluorescence. For electron microscopy, thin sections were treated simultaneously with IgG against NGF and EGF coupled to colloidal gold particles of different size. Data indicate that NGF and EGF are present in all granular convoluted tubule cells and in no other cell type within the salivary gland. Ultrastructural analyses indicate that NGF and EGF are evenly distributed together within mature secretory granules, although a population of small granules was identified that is not immunoreactive for either protein. Taken together, the data suggest that granular convoluted tubule cells are homogeneous in the production and storage of NGF and EGF.

Alterations in the pituitary-thyroid axis and 5'-deiodinase activity in mice with muscular dystrophy.

Mice with hereditary muscular dystrophy have reduced levels of serum T3. To determine possible causes of T3 deficits, we evaluated pituitary thyrotroph ultrastructure by electron microscopy, thyroid gland morphology by light microscopy, and T4 to T3 conversion by measuring iodothyronine 5'-deiodinase activity. Differences were not evident between dystrophic and normal littermates in either the structure of pituitary thyrotrophs or thyroid tissues. Dystrophic mice, however, had only 50% the normal hepatic 5'-deiodinase activity. Cerebral 5'-deiodinase, which does not appear to contribute significantly to serum T3, was similar in normal and dystrophic mice. Submandibular gland concentrations of nerve growth factor and epidermal growth factor are reduced in dystrophic mice but can be increased by T4 treatment. To distinguish whether growth factor deficits are due to reductions in serum T3 or to deficiencies in T4 5'-deiodinase activity and subsequent T3 utilization within the salivary gland, we measured submandibular deiodinase activity. Gland homogenates were active in the deiodinase assay, but no differences were detected between normal and dystrophic mice. In order to evaluate tissue responses to reductions in circulating T4, we treated mice with methimazole. Structural analyses revealed that thyrotrophs in dystrophic mice were less stimulated than thyrotrophs in similarly treated normal littermates. Likewise, thyroid follicular cells appeared less active, and thyroid weights increased only 40-50% as much as in normals. Liver 5'-deiodinase activity decreased in both normal and dystrophic mice. Cerebral 5'-deiodinase activity increased more than 4-fold in normal females but only 2-fold in dystrophic females; 2- to 3-fold increases occurred in both normal and dystrophic males. In summary, the structure of pituitary and thyroid glands in dystrophic mice is similar to that of tissues from normal littermates, but hepatic conversion of T4 to T3 is reduced. When challenged by methimazole-induced reductions in serum T4, pituitary and cerebral tissues in dystrophic mice respond abnormally.

Enrichment of rat anterior pituitary cell types by metrizamide density gradient centrifugation.

Anterior pituitary cells derived from hypothyroid rats were enzymatically dispersed and then fractionated by equilibrium sedimentation through a metrizamide density gradient. Populations relatively enriched in thyrotrophs, somatotrophs, mammotrophs, or gonadotrophs were obtained. The thyrotroph-enriched fraction was put into short term culture and tested for hormone responsiveness. TRH stimulated TSH release with an ED50 of 8 X 10(-10) M, and this TRH effect was inhibited by T3 with an ED50 of 1.6 X 10(-11) M free T3. Metrizamide density gradient centrifugation is a useful technique for obtaining relatively enriched populations of viable anterior pituitary cell types.

Regulation of thyroxine 5'-deiodinase activity by 3,5,3'-triiodothyronine in cultured rat anterior pituitary cells.

The influence of the medium T3 concentration on iodothyronine 5'-deiodinase activity was studied in cultured anterior pituitary cells derived from chronically hypothyroid rats. Type II (propylthiouracil-insensitive) enzyme activity, measured with T4 as substrate, was reduced by T3 in a dose-dependent manner, with an ED50 of approximately 1.4 X 10(-10) M free T3. Density gradient centrifugation was used to obtain populations of pituitary cells relatively enriched in thyrotrophs, somatotrophs, mammotrophs, or gonadotrophs, and the effect of T3 on type II 5'-deiodinase activity was evaluated in each of these four populations. In the absence of T3, the enzyme activity was 1.5- to 2-fold greater in the somatotroph- and mammotroph-enriched cell pools than in the thyrotroph- and gonadotroph-enriched pools. In contrast, when the cells were cultured in the presence of T3, enzyme activity was reduced to the same low level in all four enriched pools. The results suggest that the increase in whole pituitary type II 5'-deiodinase activity associated with hypothyroidism is due largely or totally to increases occurring within somatotrophs and mammotrophs. The data also suggest that the intrinsic responsiveness of the deiodinase to hypothyroidism is greater in somatotrophs and mammotrophs than in other anterior pituitary cells.

Submandibular glands in mice with muscular dystrophy: studies with nerve growth factor.

Experiments have been carried out to examine the submandibular glands in mice with hereditary muscular dystrophy. Radioimmunoassay data confirm biological studies which show that submandibular glands in mice with muscular dystrophy contain less nerve growth factor (NGF) than glands of normal animals. Male dystrophics have half as much submandibular NGF as unafflicted mice, while females have only 10% of control levels. Gel filtration and electrophoretic studies detect no differences in the molecular properties of NGF in gland extracts from normal and dystrophic mice. Furthermore, NGF from both sources show equal activity in the sensory ganglion bioassay. Together, these results suggest that NGF deficits in submandibular glands of dystrophic mice are not due to measurement artifacts arising from alterations in the structure of the molecule. Morphological studies have uncovered a cytological basis for chemical deficits within submandibular glands of dystrophic mice. Stereological analysis of light and electron microscopic sections revealed that growth factor containing granular tubule cells (GTC) take up a smaller portion of the total gland volume, are smaller in size, and contain fewer secretory granules than comparable cells in glands from controls. Furthermore, the ultrastructure of GTC in dystrophic animals suggests that the cells are less active in producing secretory protein than GTC in glands from normal animals. These results are consistent with the idea that growth factor deficits arise from cellular abnormalities in the granular tubule segment of the gland.

The effect of SO2 on the uptake of particles by mouse bronchial epithelium.

In three experiments, we have explored the uptake and transport of colloidal gold (Au) and iron oxide (Fe2O3) by normal and SO2-injured bronchial epithelium. In the first experiment, mice were exposed to a 2-hr aerosol of Au; in the second experiment, mice were exposed to SO2 (250 ppm) for 3 hr, then to Au; and in the third experiment, mice inhaled SO2 and then were exposed to Fe2O3. In each experiment, animals were killed at 0 hr, 1 day, and 4 days postexposure. From each animal, samples of large airways were examined by electron microscopy, and the cell type and particle location were recorded. Au was not readily taken up by the bronchial epithelium in that only two cells out of 1162 contained particles. No Au was seen in the submucosa. Exposure to SO2 produced a mild, but nonnecrotic response characterized by an increase in mucus, vacuolated cells, detached cilia, and an influx of polymorphonuclear leukocytes. SO2 did not affect the uptake of Au; 2 out of 1177 epithelial cells contained Au. However, gold did reach the submucosa. Results from the SO2 plus Fe2O3 experiment were compared to a previous study of similar design in which mice were exposed only to Fe2O3. SO2 increased the uptake of iron and affected the distribution of intracellular iron among the different cell types. It is possible that increased iron uptake is due to either injury or to changes in particle solubility produced by the SO2.

Uptake of iron aerosols by mouse airway epithelium.

Spontaneously breathing mice were exposed to an aerosol of iron oxide for 3 hours. Participation of the tracheal and bronchial epithelium in the uptake of iron oxide was noted immediately following the exposure and at 1 day, 4 days, and 7 days postexposure. Observations with the electron microscope revealed that iron oxide was pinocytosed and converted to ferritin and hemosiderin in all epithelial cell types except mucous cells. Iron content increased over time and approximately 50% of the nonmucous cells contained hemosiderin by 4 days postexposure. Ferritin and hemosiderin, but not iron oxide, were noted in connective tissue cells in the submucosa beneath the airway epithelium. Soluble iron and/or ferritin produced in the airway epithelial layer was transported to the submucosa, but normal epithelium prevented the penetration of deposited iron oxide particles to the connective tissue compartment.