Gary E. R. Hook

Nature of the enhancement of hepatic uridine diphosphate glucuronyltransferase activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats

Abstract After single low-level oral doses of 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) to rats, hepatic microsomal p -nitrophenol (PNP) glucuronyltransferase activity was elevated approximately 6-fold, whereas the hepatic glucuronyltransferase conjugating testosterone or estrone was unaffected. Solubilized and purified PNP glucuronyltransferase and steroid glucuronyltransferases from control and TCDD-treated rats exhibited the same relative activities (TCDD:control) as when the enzymes were bound to the endoplasmic reticulum. Elevation of PNP glucuronyltransferase was still evident 73 days after a single oral dose of 25 μg TCDD/kg. Female rats were more susceptible to TCDD actions on liver microsomal PNP glucuronyltransferase than males. The effects of TCDD treatment on PNP glucuronyltransferase appeared to be related to increased amounts of liver enzyme for the following reasons: (1) K m values for PNP and UDPGA were unchanged by TCDD treatments; (2) the magnitude of the TCDD-induced increase of PNP glucuronyltransferase activity was the same whether enzyme activity was measured in the presence or absence of Mg 2+ or Triton X-100; (3) TCDD, when added in in vitro , had no detectable effect on enzyme activity; (4) TCDD treatment of rats did not change total hepatic microsomal phospholipid or cholesterol contents: (5) pH optima were unaffected by TCDD treatment; (6) solubilization of enzyme was not accompanied by a change in the TCDD induction effect: and (7) actinomycin D appeared to block the initial phase of induction.

Postnatal stimulation of hepatic microsomal enzymes following administration of TCDD to pregnant rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) administered to pregnant rats at 3 mu-g/kg as a single oral dose during early, middle, or late gestation caused marked elevations of some maternal hepatic microsomal enzymes for at least 10 weeks after treatment. This dose was not teratogenic and fetal rates of glucuronidation of testosterone and p-nitrophenol (PNP) were unaffected. Increases in fetal liver benzpyrene hydroxylase (BPH) activities were evident during late gestation although cytochrome P-450 and cytochrome b-5 contents were unchanged. The offspring of pregnant rats administered TCDD had markedly elevated hepatic PNP UDP-glucuronyltransferase (UDPGT) BPH, and microsomal cytochrome contents whereas the perinatal development of testosterone UDPGT was unchanged. PNP glucuronidation attained a maximal 8-fold increase above controls by 3 weeks after birth and activities were twice that of controls 8 weeks after birth (adults). Maximal increases in benzpyrene hydroxylation rates occurred one day after birth when in the prenatally exposed group activities were approximately 20 times higher than controls. Foster mother experiments demonstrated that the postnatal inductive effect resulted both from exposure of newborns to TCDD via maternal milk and the activation of an inducing mechanism occurring after birth. Tese data demonstrate that multiple factors are responsible for the induction of hepatic microsomal enzymes in the newborn following administration of TCDD to pregnant rats.

Hypertrophy and hyperplasia of alveolar type II cells in response to silica and other pulmonary toxicants.

Alveolar Type II cells serve two major functions in the lung, both of which are essential for the preservation of normal lung function. First, Type II cells synthesize and secrete pulmonary surfactant, and second, they function as progenitor cells for maintaining the alveolar epithelium. The Type II cell population of the lung is quite sensitive to the deposition of toxicants in the distal lung, responding in two principal ways. Damage to the Type I epithelium stimulates Type II cells to proliferate and subsequently differentiate to replace the injured Type I cells. Second, a portion of the Type II cell population may become hypertrophic. Both of these events are frequent findings in the diseased or damaged lung. The Type II cell changes are often associated with increases in surfactant pools. In those cases where ultrastructural characteristics of hypertrophic Type II cells were examined, the appearance of these cells was consistent with that of an activated cell type. Alterations in the lamellar body compartment are a common finding in hypertrophic Type II cells, with increases in both lamellar body size and number. It is likely that the hypertrophic, or activated, Type II cells account for the increased levels of surfactant found in the lungs after exposure to a variety of toxic agents. We examined, in detail, Type II cell hyperplasia and hypertrophy induced by silica deposition. Both Type II cell hyperplasia and hypertrophy were prominent responses. The proliferative response led to an approximate doubling of the number of Type II cells in the lung.(ABSTRACT TRUNCATED AT 250 WORDS) ImagesFIGURE 1.FIGURE 2.FIGURE 3.FIGURE 5.FIGURE 6.FIGURE 7. aFIGURE 7. b

Mixed-function oxidases and the alveolar macrophage

Abstract Biphenyl 4-hydroxylase, benzpyrene hydroxylase and D-(+)-benzphetamine N -demethylase activities in subcellular fractions of the alveolar macrophage were investigated and found to be extremely low. Cytochrome P-450 was absent from the microsomal fraction but both NADPH-cytochrome c reductase and NADH-cytochrome c reductase activities were present. A b-type cytochrome, which was probably cytochrome b 5 , was detected in the microsomal fraction. Glutathione S -aryltransferase and UDP-glucuronyl transferase activities were absent from the soluble and microsomal fractions respectively. These results indicate that alveolar macrophages may not play a significant role in the detoxication of foreign compounds by the lung.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced changes in the hydroxylation of biphenyl by rat liver microsomes

Abstract Biphenyl 2- and 4-hydroxylase activities and cytochrome P-450 concentrations in microsomes were increased by oral doses of less than 1 μg TCDD/kg. Female rats were more sensitive than male rats to the inductive effects of TCDD. since highly significant increases in biphenyl-hydroxylating activities were observed at the dose level of 0.2 μg TCDD/kg in female but not in male rats. The inductive effect was very persistent: biphenyl 2- and 4-hydroxylases remained stimulated even after 73 days following a single oral dose of 25 μg TCDD/kg. The levels to which the hydroxylases were stimulated in female rats were the same as in male rats. Rats of all ages from 10 to 335 days responded to hepatic microsomal effects of TCDD to approximately the same degree. The enzyme inductive effect was diminished by the simultaneous administration of actinomycin D. The K m of biphenyl 2-hydroxylase (1.42 mM) was not altered significantly by TCDD treatment, but the K m of biphenyl 4-hydroxylase (0.62 mM) was increased to approximately the same value (1.6 mM) as that of the 2-hydroxylase. The V max of biphenyl 4-hydroxylase was increased 4.5-fold but that of biphenyl 2-hydroxylase was increased 16.5-fold. Rates of 2β- and 16α-hydroxylation of testosterone were suppressed by TCDD but rates of 7α- and 6β-hydroxylation were unaffected. It would appear that the hepatic microsomal mixed-function oxidases responsible for the hydroxylation of biphenyl and testosterone are different.

Repopulation of Denuded Tracheas by Clara Cells Isolated from the Lungs of Rabbits

An experimental approach was examined to study the growth and differentiation potential of different epithelial cell types isolated from the airways of adult rabbits. Clara cells isolated from the lungs of rabbits and a mixed cell population obtained from rabbit tracheas were injected (separately) into denuded rat tracheas which were then grafted into nude mice. Epithelial cells were obtained from rabbit tracheas by digestion of tracheal epithelium with proteases. The resulting cell suspension contained mucous cells, Claralike cells, ciliated cells, basal cells and a small number of inflammatory and unidentified cells. This mixed cell suspension was inoculated into rat tracheas denuded of their own epithelium which were then grafted subcutaneously onto the backs of nude mice. The tracheas were recovered two weeks later and were prepared for light and electron microscopy. These grafts were found to be lined by a tall columnar pseudostratified mucociliary epithelium, indicating that the rat tracheal grafts transplanted into nude mice supported the growth and differentiation of tracheal epithelial cells. Similarly Clara cells, isolated and purified to 85.9 +/- 2.8% (n = 9) purity from rabbit lungs by a combination of centrifugation and elutriation procedures, were inoculated into rat tracheas which were transplanted into nude mice. Two weeks later the grafts were recovered and prepared for histological and ultrastructural examination. These tracheas were lined with a low cuboidal epithelium reminiscent of bronchiolar epithelium. The epithelium was composed of ciliated cells and Claralike cells rich in smooth endoplasmic reticulum but containing only a few secretory granules. These results indicate that the tracheal graft model is suited to examine the differentiative potential of specific cell types isolated from the airways. Furthermore, they show that Clara cell isolates are able to establish an epithelium resembling bronchiolar epithelium in denuded tracheas while tracheal cells containing at least four different epithelial cell types are able to establish a mucociliary epithelium resembling the pseudostratified tracheal lining.

Altered regulation of surfactant phospholipid and protein A during acute pulmonary inflammation.

Biochemical changes in the pulmonary surfactant system caused by exposure to toxicants are often accompanied by an influx of inflammatory cells into the lungs. We have investigated the possibility that the inflammatory and surfactant biochemical effects might be connected. Co-treatment with dexamethasone, a synthetic anti-inflammatory glucocorticoid, mitigated the increases in free cells and total intracellular surfactant phospholipid normally seen in animals given silica alone, suggesting a relationship between the free cell population of the alveoli and the surfactant system during alveolitis. Furthermore, we have investigated whether induction of the surfactant system is a universal response to alveolar inflammation. Inflammation was induced in the lungs by intratracheal injections of titanium dioxide, silica, bleomycin or lipopolysaccharide (LPS) suspended in isotonic saline. Inflammatory cell and surfactant responses were measured at 3 days and 14 days following injection. There was a distinct alveolar inflammatory cell profile following administration of each agent, at each time point, indicating a dynamic inflammatory cell population during the course of the study. Furthermore, surfactant phospholipid and protein A (SP-A) pools exhibited unique responses to the inflammatory agents. Only silica-treated lungs maintained elevated levels of surfactant phospholipids and SP-A throughout the course of the experiment. We conclude that both the surfactant components and the inflammatory cell population of the alveoli undergo dynamic changes following treatment with these inflammatory agents and that activation of the surfactant system is not a universal response to alveolar inflammation, since surfactant components were not always elevated during times of increased alveolar cellularity. The unique inflammatory cell infiltrate elicited by silica is of particular interest in that surfactant components were elevated throughout the course of the experiment in this group. Indeed, we have shown that the size of the intracellular pool of surfactant is directly proportional to the number of polymorphonuclear leukocytes but not alveolar macrophages or lymphocytes in the alveoli following silica treatment. Finally, our data suggest that the phospholipid and SP-A components of surfactant respond differentially to the pulmonary toxicants in this study.

Histomorphology and Ultrastructure of Spontaneous Pulmonary Neoplasms in Strain A Mice

The microscopic and ultrastructural characteristics of spontaneous pulmonary neoplasms in strain A (strA) mice are described. Fifty-one spontaneous lung tumors were identified in 34 out of 57, 11-23-month-old male strA/Hen mice. Grossly, all tumors appeared as yellow-white, discrete nodules ranging in size from 1.0-10 mm. Tumor types were randomly distributed throughout the lung; however, the right lung lobes were most frequently involved. Histologically, tumors were classified as adenoma (34/51) or carcinoma (17/51) as defined by standard histopathologic criteria. Adenomas were usually less than 4 mm in diameter and had solid (16/34), papillary (10/34), or mixed (8/34) histologic growth patterns. Carcinomas were usually greater than 4 mm in diameter and had papillary (13/17) or mixed (4/17) histologic growth patterns. Ultrastructurally, benign tumors consisted of solid or papillary areas of neoplastic type II-like cells. Cells comprising malignant tumors had varying ultrastructural characteristics ranging from well-differentiated alveolar cell types to undifferentiated cells having intracytoplasmic osmiophilic dense bodies, vacuoles, or few specialized organelles commonly observed in mature nonneoplastic pulmonary epithelial cells.

Biosynthesis and Release of Proteins by Isolated Pulmonary Clara Cells

The major proteins synthesized and released by Clara cells were identified and compared with those synthesized and released by mixed lung cells. Highly purified Clara cells (85.9 +/- 2.4%) and mixed lung cells (Clara cells 4%, Type II cells 33%, granulocytes 18%, macrophages 2.7%, ciliated cells 1.2%) were isolated from rabbit lungs, incubated with Hams F12 medium in collagen/fibronectin-coated plastic culture dishes in the presence of 35S-methionine for periods of 4 and 18 hrs. Radiolabelled proteins were isolated from the cells and from the culture medium, electrophoresed on polyacrylamide gels in the presence of SDS under reducing conditions, and then autoradiographed. After 4 and 18 hr of incubation of the Clara cells the major radiolabelled cell-associated proteins were those with molecular weights of 6, 48, and 180 Kd. The major radiolabelled proteins released by Clara cells into the medium after 4 hrs of incubation had molecular weights of 6, 48, and 180 Kd, accounting for 42, 16, and 10%, respectively, of the total extracellular protein-associated radioactivity. After 18 hr of incubation the 6 and 48 Kd proteins represented 30 and 18% of the total released radioactivity, and the relative amount of the 180 Kd protein had decreased to 3%. With the mixed lung cells, the major proteins released into the medium had molecular weights of 6 and 48 Kd. Under nonreducing conditions the 6 Kd protein released by Clara cells had an apparent molecular weight of 12 Kd. Labelling isolated Clara cells with a mixture of 14C-amino acids also identified this low molecular weight protein as the major secretory product of the Clara cell. The 6 Kd protein did not label when the cells were incubated with 14C-glucosamine indicating that it was not a glycoprotein. These data demonstrate the release of several proteins from isolated Clara cells but the major protein had a molecular weight of 6 Kd.

Production of low molecular weight cadmium-binding proteins in rabbit lung following exposure to cadmium chloride☆

Low molecular weight cadmium-binding proteins were studed in lung tissue from rabbits exposed to aerosols of CdCl2. Lungs obtained from animals exposed by inhalation to aerosols of 800 or 1600 micrograms/m3 CdCl2 for 2-hr periods/day, every other day for a 5-day period, were found to contain at least three low molecular weight cadmium-binding proteins, two of which were similar electrophoretically and spectrally to rabbit liver metallothionein. The third protein(s), which accounted for the majority of the cadmium in the soluble fraction of the tissue, did not bind to an anionic exchange gel and did not appear to be a polymerized form of metallothionein. Translocation studies of lung cadmium suggest a long half-life for cadmium in lung tissue following inhalation exposure, due perhaps to the high affinity of cadmium for specific lung cadmium-binding proteins. A small but significant redistribution of lung cadmium did occur to both kidney and liver tissue with time.