Isolation of oval cells and transitional cells from the livers of rats fed the carcinogen DL-ethionine.

For the characterization of the metabolic and biologic properties of oval cells (i.e., cells emerging in the livers of rats treated with chemical carcinogens due to proliferation of bile ductular and/or duct cells) and transitional cells (i.e., cells having properties intermediate between those of oval cells and hepatocytes), these cells were isolated from the livers of Sprague-Dawley rats fed DL-ethionine for 4-5 weeks. The livers were dissociated into single cells by perfusion in situ with collagenase, and total cell suspensions were allowed to stand at unit gravity for 10 minutes to separate parenchymal (hepatocytes) from nonparenchymal cells. Nonparenchymal cells were centrifuged in linear gradients of Metrizamide (8-24% wt/vol), and 2-ml fractions were collected from the gradients. The cells in the fractions were defined by light microscopy, electron microscopy, and histochemical and immunofluorescence methods. A cell isolate was thus obtained consisting of Kupffer's cells (approximately 20%), bile ductular and/or duct cells and oval cells (approximately 30%), and transitional cells (approximately 50%). A twofold enrichment of bile ductular and/or duct cells and their derivatives was achieved over that found in the nonparenchymal cell fraction before isopyknic gradient centrifugation.

Suppression of choline-deficient diet-induced hepatocyte membrane lipid peroxidation in rats by the peroxisome proliferators 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-beta-hydroxyethyl)- acetamide and di(2-ethylhexyl)phthalate.

Many hypolipidemic peroxisome proliferators have been shown to induce liver tumors in rats after long-term feeding. In short-term assays, however, some of them prevent the development of gamma-glutamyl transpeptidase-positive foci, the putative preneoplastic lesions, in the liver of carcinogen-initiated rats and inhibit the promoting effect of a choline-deficient (CD) diet on these lesions. The CD diet-induced lipid peroxidation in the liver has been implicated as one of the underlying mechanisms of the promoting effect. In the present study, the effects of 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-beta-hydroxyethyl)acetamid e (BR931) and di(2-ethylhexyl)phthalate (DEHP) on CD diet-induced liver membrane lipid peroxidation were investigated by determining the extents of conjugated diene formation. No evidence of lipid peroxidation was detected in the microsomal lipids of the liver after administration of BR931 or DEHP at concentrations of 0.16% and 1%, respectively, for 1, 2, and 4 wk. When added to a CD diet, both BR931 and DEHP effectively protected against the diet-induced lipid peroxidation. There was an increase in cellular glutathione levels after 4 wk and an increase in catalase activity after 2 wk in the liver of rats fed BR931 or DEHP. The levels of activity of the glutathione peroxidases and glutathione-s-transferase were significantly reduced. The results suggest that, in the acute stage, hypolipidemic peroxisome proliferator-induced effects of excess production of H2O2 and potential lipid peroxidation are balanced by stimulation of some cellular detoxifying systems. The inhibition of lipid peroxidation by hypolipidemic peroxisome proliferators may account for their inhibitory effects on the CD diet-induced promotion of preneoplastic foci.

Lipid peroxidation of liver microsome membranes induced by choline-deficient diets and its relationship to the diet-induced promotion of the induction of gamma-glutamyltranspeptidase-positive foci.

The effects of varying the type of dietary fat in the choline-deficient (CD) diet on the development of gamma-glutamyltranspeptidase (GGT)-positive foci in the liver of carcinogen-treated rats were investigated, and the results were correlated with the extent of membrane lipid peroxidation induced by the diets. Male Sprague Dawley rats were initiated with a single dose of diethylnitrosamine. Thereafter, groups of rats were fed choline-supplemented or CD diets in which the amount of saturated fat was varied by using hydrogenated vegetable oil (Primex) and corn oil (CO), either alone or in combination. The number and size of GGT-positive foci induced by the CD diet with CO as the sole source of fat were larger than those induced by the diet containing mixtures of Primex and CO. The CD diet with Primex alone was the least effective in inducing GGT-positive foci. Peroxidation of liver microsomal membrane lipids in rats fed regular CD or CD:CO diets was examined by determining the formation of conjugated dienes. The generation of diene conjugate in rats fed a CD:CO diet was evident after 2 days of the diet feeding, and the levels increased at 1 and 2 weeks. No significant diene conjugate was demonstrated in rats fed a regular CD diet for 2 days. However, after 1 and 2 weeks, there was generation of diene conjugate, the levels of which were lower in rats fed the CD diet than those on a CD:CO diet. Addition of an antioxidant, 0.25% butylated hydroxytoluene, to both CD and CD:CO diets abolished the generation of diene conjugate in rat liver microsomal membranes and markedly inhibited the promotion of GGT-positive foci in the liver of diethylnitrosamine-initiated rats. The results suggest that membrane lipid peroxidation in the liver may be related to the promotion of the induction of GGT-positive foci by a CD diet. The enhanced promotion by the inclusion of a higher level of polyunsaturated fat in the diet may be, in part, due to its greater susceptibility to peroxidation.

Immunocytochemical and morphological evidence for the origin of N-nitrosomethylurea-induced and naturally occurring primary lung tumors in F344/NCr rats.

Naturally occurring and N-nitrosomethylurea-induced lung tumors were studied in male F344/NCr rats by sequential histological, electron microscopic, and immunohistochemical methods. Rats were given one injection at 6 weeks of age of N-nitrosomethylurea at a dosage level of 41.2 mg/kg body weight i.v. Groups of rats were sacrificed at 20, 33, and 52 weeks, while some were sacrificed while moribund. Nine lung tumors from aged F344/NCr male rats were also studied. For determining localization of pulmonary antigens, sections of lungs were stained by the avidin-biotin-peroxidase complex immunocytochemical technique using antibodies to rat surfactant apoprotein or rat Clara cell antigen. At 20 weeks, in rats receiving N-nitrosomethylurea, focal alveolar type II cell hyperplasia, adenoma in focal alveolar type II cell hyperplasia, and adenoma were found in 15 (100%), 1 (7%), and 2 (13%) of 15 rats, respectively. At 33 weeks, there were 19 rats (95%) with focal alveolar type II cell hyperplasias, 10 rats (50%) with adenoma in focal alveolar type II cell hyperplasia, and 2 rats (10%) with adenomas in 20 rats. In 53 rats allowed to live up to 52 weeks, there were 10 (19%) adenomas and 3 (6%) carcinomas, as well as 49 (92%) rats with focal hyperplasia and 31 (58%) with adenomas in focal type II cell hyperplasia. Rat surfactant apoprotein was found in the cytoplasm of normal alveolar type II cells and the majority of cells in focal alveolar type II cell hyperplasias, adenomas in hyperplastic lesions, adenomas, and carcinomas. The ultrastructure of these lesions supported immunocytochemical findings with evidence of lamellar bodies. All nine naturally occurring lung tumors studied contained rat surfactant apoprotein. Rat Clara cell antigen was found, however, only focally within one adenoma induced by N-nitrosomethylurea and one adenoma in a hyperplastic lesion, and also focally in three neoplasms which occurred naturally. This study provided morphological, immunohistochemical, and ultrastructural evidence that the vast majority of N-nitrosomethylurea-induced and naturally occurring pulmonary neoplasms of F344 rats are alveolar type II cell adenomas and carcinomas and that a portion of these tumors arise within focal alveolar type II cell hyperplasias.

Induction of cellular DNA synthesis in the pancreas and kidneys of rats by peroxisome proliferators, 9-cis retinoic acid, and 3,3',5-triiodo-L-thyronine.

We recently suggested that peroxisome proliferators (PPs), 3,3',5-triiodo-L-thyronine (T3), and 9-cis retinoic acid (9-cis RA) induce hepatocyte proliferation in rats through the activation of their nuclear receptors, PP-activated receptors, T3 receptors, and retinoid X receptors. To test whether nuclear hormone receptor-mediated cell proliferation can be observed in organs other than liver, we examined the effects of these agents on the pancreas and kidneys of male Wistar rats using BrdUrd immunohistochemistry. A single s.c. injection of T3 (2 mg/kg) and single intragastric administration of 9-cis RA (40 mg/kg) or 4-chloro-6-(2, 3-xylidino)-2-pyrimidinylthio-(N-beta-hydroxyethyl) acetamide (200 mg/kg) induced a wave of DNA synthesis in the pancreatic acinar cells and in the proximal tubular epithelial cells of the kidneys, peaking after 24 h. No stimulation of DNA synthesis was observed in ductal or islet cells of the pancreas and in glomeruli of the kidneys. All-trans-retinoic acid, a ligand for retinoic acid receptor, at a dose (200 mg/kg) that induced hepatocyte proliferation, had no effects on cell proliferation of the pancreas and the kidneys. The results suggest that T3, 9-cis RA, and PP activate genes that regulate cell proliferation in target cells through receptor-mediated pathways and initiate cellular DNA synthesis.

Some biochemical characteristics of rat liver and Morris hepatoma nuclei and nuclear membranes.

Nuclei prepared from host liver and from Morris hepatomas 7777 and 7800 have been compared with respect to some of their biochemical characteristics. Several criteria were used to ensure that liver and hepatoma nuclei were of equal purity. These criteria include equal specific activity ratios (homogenate nuclei) for several marker enzymes. Phospholipids, proteins, and sialic acid content were compared in liver and hepatoma sucrose nuclei and in membrane and chromatin fractions obtained from liver or hepatoma nuclei. As determined by sodium dodecyl sulfate polyacrylamide electrophoresis, the only qualitative difference in protein that could detected was in 2 of the 4 nuclear fractions. There was an extra band in each of the 2 hepatoma fractions. Sialic acid was increased in hepatoma nuclei. In addition, a fraction containing most of the inner nuclear membrane from liver nuclei had no sialic acid, whereas the equivalent hepatoma fraction did have sialic acid. Total phospholipids were increased in hepatoma nuclei. This increased phospholipid concentration in hepatoma nuclei as compared to liver nuclei was apparent with sucrose nuclei, citric acid nuclei, membrane-denuded nuclei, chromatin, and nuclear fractions. Determination of the percentages of individual phospholipids making up the total phospholipids extracted revealed that the only significant change in the phospholipid composition of hepatoma nuclei was an increase in sphingomyelin. A large amount of this sphingomyelin was found to be associated with chromatin. The possible significance of chromatin-associated phospholipids is discussed.

Mouse papillary lung tumors transplacentally induced by N-nitrosoethylurea: evidence for alveolar type II cell origin by comparative light microscopic, ultrastructural, and immunohistochemical studies.

A histogenetic study was designed to evaluate controversial findings on the cell of origin of tubular/papillary lung tumors in mice, i.e., bronchiolar Clara cell versus alveolar type II cell. N-Nitrosoethylurea (0.5 mmol or 0.74 mmol/kg) was given to pregnant C3H (C3H/HeNCr MTV-) and Swiss Webster [Tac:(SW)fBR] mice as a single i.p. injection on Day 14, 15, 16, or 18 of gestation. The offspring were studied at various ages ranging from 7 days to 52 wk. Serial sections of the whole lung (100 to 200 sections per mouse) showed that solid/alveolar and papillary tumors arose from the pulmonary acinus, invading the bronchioles only as the tumors grew. Furthermore, a mixture of solid and papillary patterns within a single module did not represent a merging of two tumors but a progression from the solid to the papillary form. By use of two rabbit antisera against mouse lung surfactant apoproteins found in normal alveolar type II cells, it was shown by the avidin-biotin peroxidase complex procedure, by the peroxidase-antiperoxidase technique, and by indirect immunofluorescence that both solid and papillary tumors contained these proteins that are specific markers for alveolar type II cells. With a rabbit anti-rat Clara cell antiserum, none of the tumors studied was immunoreactive while normal Clara cells were reactive. The nitroblue tetrazolium formazan stain for dehydrogenase enzymes, found particularly in Clara cells, did not reveal these enzymes in any lung tumors from either strain. Ultrastructurally, no typical features of the mature Clara cell were detected in papillary or other pulmonary neoplasms. However, all tumors showed characteristic alveolar type II cell structures such as various stages of lamellar body formation, although these features were less well differentiated in the papillary tumors. Argentaffin dense bodies, representing lysosomes and immature forms of lamellar bodies, were commonly observed in papillary tumors. Some features of the papillary tumors such as cell shape, high glycogen content, and primary cilia were equivalent to those seen in pulmonary epithelial precursor cells during fetal development. With age, the papillary tumors became invasive, accumulated neutral lipids, and developed bizarre cleaved nuclei and lamellated nuclear pseudoinclusions. In conclusion, the papillary lung tumors of the mouse, at least those induced transplacentally by N-nitrosoethylurea, constitute less well-differentiated or poorly differentiated alveolar type II cell adenomas or carcinomas with fetal morphological and biochemical properties.

Analysis of pulmonary surfactant apoproteins by electrophoresis.

Surfactant apoproteins were prepared from detergent-solubilized rat surfactant by immunoaffinity chromatography. SDS-polyacrylamide gel electrophoresis of the apoproteins, without prior chemical reduction, revealed several bands of molecular weights 50 000-78 000, 140 000 and 160 000. Following treatment with dithiothreitol, the apoproteins were resolved into three bands of molecular weights 38 000, 32 000 and 26 000. Further analysis of the apoproteins by two-dimensional polyacrylamide gel electrophoresis showed that each of the proteins of molecular weights 38 000, 32 000 and 26 000 were crosslinked by disulfide bridges and formed homopolymers. Based on periodic acid-Schiff staining, the 38 000 daltghts 38 000, 32 000 and 26 000. Further analysis of the apoproteins by two-dimensional polyacrylamide gel electrophoresis showed that each of the proteins of molecular weights 38 000, 32 000 and 26 000 were crosslinked by disulfide bridges and formed homopolymers. Based on periodic acid-Schiff staining, the 38 000 daltghts 38 000, 32 000 and 26 000. Further analysis of the apoproteins by two-dimensional polyacrylamide gel electrophoresis showed that each of the proteins of molecular weights 38 000, 32 000 and 26 000 were crosslinked by disulfide bridges and formed homopolymers. Based on periodic acid-Schiff staining, the 38 000 dalton protein appeared to be the richest in carbohydrates, followed by the 32 000 and 26 000 dalton proteins. Partial proteolysis of the 38 000 and 32 000 dalton proteins showed similarity in the sizes of peptides generated. Surfactant-associated proteins from human and monkey lungs were also analyzed by polyacrylamide gel electrophoresis. A non-serum glycoprotein of molecular weight 38 000 was found. In different systems of polyacrylamide gel electrophoresis, this protein showed an electrophoretic mobility similar to that of the 38 000 dalton protein of rat surfactant. However, it formed polymers of molecular weight higher than those of polymers found in rat surfactant.

Analysis of pulmonary surfactant apoproteins by isoelectric focusing.

Apoproteins of Mr 38 000, 32 000 and 26 000 are found in surfactant isolated from rat lungs. The surfactant isolated from monkey lungs, on the other hand, contains the 38 kDa apoprotein and not the 32 and 26 kDa apoproteins. These preparations of pulmonary surfactant contain, in addition, several serum proteins. We have used a combination of salt- and sucrose-density gradient centrifugations to isolate and further purify surfactant from the washings of rat lungs. Thus, a preparation of pulmonary surfactant was obtained which contained exclusively the 38, 32, 26 and 10-12 kDa apoproteins, and which was rich in phosphatidylcholine and phosphatidylglycerol. Using an immunoassay and an immunoblotting technique, it was established that the 38, 32 and 26 kDa apoproteins are not serum proteins. The surfactant apoproteins of rat and monkey were further subjected to the high-resolution of isoelectric focusing. Thus, rat surfactant apoproteins resolved into 11 bands in the pH range 4.64-5.53. A second-dimensional electrophoresis in a sodium dodecyl sulfate system led to the migration of the 11 bands, separated by first-dimensional isoelectric focusing, into three distinct groups with apparent molecular weights of 38 000, 32 000 and 26 000, respectively. Upon isoelectric focusing, the apoproteins from monkey lung surfactant also separated into several bands in the pH range 5.18-5.82. After electrophoresis in the second dimension as above, these bands migrated as a single group with an apparent molecular weight of 38 000. Neuraminidase treatment of rat surfactant apoproteins, and subsequent IEF, led to the disappearance of several low-pI variants with a concomitant increase in the amounts of higher-pI variants. Thus, the sialic acid content of surfactant apoproteins accounts for, in large part, the observed charge heterogeneity.

Antigenic, molecular and functional heterogeneity of Clara cell secretory proteins in the rat.

In an earlier publication we had reported the preparation of a rabbit antiserum specific for rat Clara cell secretory proteins. This rabbit anti-rat Clara cell serum was found to react with two proteins in rat lung lavage by crossed-immunoelectrophoresis. Immunoblotting of rat lung lavage proteins, after sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis, disclosed three bands of reactivity with anti-Clara cell serum. The relative molecular masses of these three proteins were about 200 (protein A) 55 (protein B) and about 12 kDa (protein C). Anti-Clara cell antibodies eluted from Sepharose-4B-linked protein C (as well as the antiserum raised by immunizing rabbits with protein C) reacted with proteins A and C. Anti-Clara cell antiserum unbound to proteins A and C (as well as antiserum raised by immunizing rabbits with protein B) reacted with protein B only. In non-SDS polyacrylamide gel electrophoresis, protein B migrated as a single band, slightly cathodic to albumin; protein C resolved into three bands, all anodic to albumin. Immunoblots of isoelectric focusing gels showed three bands (pI 5.2-5.7) that reacted with antibody to protein C, and four bands corresponding to protein B were seen in the pI range 4.6-5.0. As determined by immunoperoxidase staining of paraformaldehyde fixed methacrylate embedded 1 micron thick sections of rat lung, protein(s) A (and protein C) and protein B were present in the same cells and in the same granules. Protein B was resistant to trypsin digestion, whereas proteins A and C were readily degraded by trypsin. Rat Clara cell secretory proteins consist of at least two antigenic types that appear to be functionally distinct, and each antigenic type displays charge microheterogeneity.

Amino-acid and cDNA nucleotide sequences of human Clara cell 10 kDa protein.

A human lung cDNA expression library was screened by using a rabbit antiserum specific for a human Clara cell 10 kDa protein. The cDNA from two positive clones was sequenced by the dideoxy chain termination method. The nucleotide and primary amino-acid sequence deduced therefrom are presented. The N-terminal amino-acid sequence of the Clara cell 10 kDa protein, purified from bronchoalveolar lavage, was also determined. The deduced and experimentally determined sequences were identical where data for both were available. From the amino-acid composition, deduced and experimentally determined amino-acid sequences, it was determined that the 10 kDa protein in bronchoalveolar lavage consists of two identical 70-amino-acid long polypeptide chains joined by two cystine residues. The size of mRNA for the protein was found to be about 0.6 kb and the monomeric nascent protein, obtained by in vitro translation of lung mRNA was about 7.3 kDa in size. The 10 kDa protein recovered from bronchoalveolar lavage has 61% sequence identity with rabbit uteroglobin, the two proteins have common predicted secondary structures with marked surface differences when comparing predicted and actual structure determined by X-ray diffraction. The differences imply similarity of structure but, not identity of function.

Clara cell 10 kDa protein (CC10): comparison of structure and function to uteroglobin.

The cellular localization, functional activities and structures of rat and human Clara cell 10 kDa proteins (CC10) are compared to rabbit uteroglobin. CC10 is present exclusively in the non-ciliated cells of the surface epithelium of the pulmonary airways, whereas uteroglobin is reported to be present in the lung and reproductive organs. There is about 55% identity between the amino acid sequences of rat CC10 and either rabbit uteroglobin or human CC10. The latter two have 61% identity. Using the known structure of uteroglobin as the model, correlations between the structure and function for this group of proteins are made. Substitution of the residues for the rat and human CC10 into the structure of uteroglobin suggests that these proteins may be members of a structurally homologous family. Some of the functional differences may be due to distortion of the hydrophobic pocket in the dimeric protein and a surface hypervariability located on one contiguous helix and beta turn. Rat CC10 and rabbit uteroglobin both, nearly equally, inhibit papain and bind progesterone. Human CC10 does not inhibit papain and has markedly lower progesterone binding (4.6% of rabbit uteroglobin). Antiinflammatory activity of synthetic peptides corresponding to a homologous sequence region of uteroglobin and the two Clara cell proteins was tested. The region chosen has sequence similarity to lipocortin I. The peptides not only failed to inhibit carrageenan-induced foot pad swelling but exacerbated it. All three proteins inhibit pancreatic phospholipase A2. The phospholipase A2 inhibitory effect of CC10 may be important in regulating the inflammatory responses in the lung.

Cell proliferation, cell death and hepatocarcinogenesis.

The carcinogenic process in the liver is a multistep process, characterised by an altered ratio between cell proliferation and cell death. In the last few years, we have undertaken studies aimed at determining the possible differences exhibited by two different types of cell proliferation, namely compensatory regeneration and direct hyperplasia at a molecular and cellular level. These two types of proliferative stimuli appear to play different roles in liver carcinogenesis. The scope of this article is to summarise the present knowledge about the differences in the expression of genes involved in the entry of liver cells into cell cycle, between liver regeneration following cell loss and/or cell death and direct hyperplasia induced by primary mitogens.

Papillary adenoma of type 2 pneumocytes.

A case of papillary adenoma of type 2 pneumocytes is reported. A 57-year-old man had an unusual coin lesion in the periphery of the right lung without any symptoms. When detected in a mass survey examination, it was approximately 1.5 cm in diameter, well circumscribed, and located in S4, involving the sixth-order bronchus of B4. Light-microscopic examination revealed cuboidal tumor cells arranged in a papillary pattern. Ultrastructurally, the cells had characteristic osmiophilic lamellar bodies. By immunoperoxidase staining, the tumor cells were shown to have intracytoplasmic surfactant apoproteins. The postoperative course was uneventful, and there is no evidence of disease 8 years later.

So-called sclerosing hemangiomas of lung. An immunohistochemical study supporting a respiratory epithelial origin.

Sclerosing hemangiomas are benign pulmonary neoplasms. They were initially believed by Liebow and Hubbell to be of endothelial origin; however, subsequent ultrastructural studies have suggested an alveolar pneumocyte and mesothelial derivation. Using a panel of various antibodies on eight cases, the authors found that sclerosing hemangiomas expressed cytokeratin (seven cases), epithelial membrane antigen (seven cases), carcinoembryonic antigen (five cases), vimentin (seven cases), surfactant apoprotein (eight cases), and Clara cell antigen (five cases). These results support the hypothesis that sclerosing hemangiomas represent an epithelial tumor showing simultaneous bronchiolar epithelial and alveolar pneumocyte differentiation.

An immunologic study of the secretory products of rat Clara cells.

Lungs of adult rats were lavaged with normal saline containing 0.25 mM phenylmethylsulfonyl fluoride. The surfactant pellet was removed by centrifugation and serum proteins in the lavage were removed by affinity chromatography using rabbit anti-rat whole serum antiserum. The residual proteins, thought to represent products of secretory cells, were used as the immunogen to inject rabbits. The resulting antiserum was absorbed with affinity columns of rat serum and rat liver extract. The gamma globulin fraction of the unbound antiserum was found to react with two proteins in the lavage by immunodiffusion and crossed immunoelectrophoresis. The antiserum specifically stained, by the immunoperoxidase method, a subpopulation of cells consistent in morphology with Clara cells lining the bronchioles and bronchi. The antigens were detectable, by immunohistochemistry, in rat fetus at 19 days of gestation, a progressive increase in the antigen content was noted with increasing gestational age and an adult pattern was noted at 2 weeks of age. In adult animals the intracellular content of the antigens appears to be about twofold greater than their content in the lavage fluid.

Ultrastructural localization of rat Clara cell 10 KD secretory protein by the immunogold technique using polyclonal and monoclonal antibodies.

Using a monoclonal antibody and affinity-purified polyclonal antiserum against a 10 KD protein isolated from rat pulmonary lavage, we have localized the protein within Clara cells by a post-embedment protein A-gold technique. The gold particles were localized over the secretory granules of rat Clara cells. Ultrastructural immunolocalization was abolished when the primary antibodies were previously absorbed with purified 10 KD protein. Other pulmonary cells, including type II pneumocytes and ciliated cells, were negative with this technique. These results demonstrate the presence of the 10 KD protein in the secretory granules of the Clara cell and support the concept that this protein constitutes a specific and unique secretory product of Clara cells.

Identification, cellular localization, isolation, and characterization of human Clara cell-specific 10 KD protein.

Human lung lavage proteins were fractionated by centrifugation and molecular sieving. An antiserum to the post-albumin fraction of the soluble proteins reacted with a 10 KD protein and this protein was isolated by conventional chromatography. The protein, which has a pI of 4.8, consists of two 5 KD polypeptides and is rich in glutamic acid, leucine, serine, and aspartic acid amino acids. The protein does not bind to concanavalin A, pancreatic elastase, leukocyte elastase, or trypsin, and lacks anti-protease activity. It constitutes about 0.15% of the soluble proteins in lung lavage. Antibodies to the 10 KD protein specifically and exclusively stain Clara cells in human, dog, and rat. Staining of granules of Clara cells was prominent in the distal bronchioles; however, the non-ciliated cells of respiratory bronchioles did not stain for the 10 KD protein. This 10 KD protein appears in fetal lungs at 21 weeks of gestation, and was present in about 10% of the primary pulmonary adenocarcinomas. As a specific marker for Clara cells, this protein could be useful in the study of development, regulation of secretion, and pathobiology of these cells.

Secretory proteins of the lung in rodents: immunocytochemistry.

The reactivity of rabbit antisera to rat lung secretory proteins with other rodent species was evaluated by immunocytochemistry. Rabbit anti-rat surfactant apoprotein antiserum reacts with the cytoplasm of rat, mouse, and hamster type II pneumocytes and is specific for these cells. Rabbit antiserum to rat Clara cell secretory proteins stains rat, mouse, and hamster Clara cells. Rabbit antisera specific to the two antigenic types of rat Clara cell antigens were also both reactive with rat, mouse, and hamster Clara cells. An antiserum to the non-serum proteins of hamster lung lavage was also prepared and shown to be specifically reactive with hamster Clara cells. The availability of specific reagents for secretory proteins of rodent lungs is expected to facilitate studies of the respective cell types in various pathologic states.

Induction of foci of altered hepatocytes by a single injection of azaserine to rats.

The induction of foci of altered, gamma-glutamyltranspeptidase (GGT)-positive hepatocytes by azaserine was investigated. After injection of a single dose of azaserine, many foci developed in male Wistar rats fed a choline-devoid (CD) diet containing acetylaminofluorene (AAF), but only a few in rats fed a choline-supplemented (CS) diet containing AAF. Similar results were obtained in rats fed a plain CD diet or a plain CS diet and injected with a single dose of azaserine after a partial hepatectomy. These findings indicate that azaserine is an effective initiator of liver carcinogenesis in rats, and that a CD diet acts as a strong promoter of the evolution initiated liver cells to foci of altered, GGT-positive hepatocytes.