The tumor promoter TPA enhances benzo[a]pyrene and benzo[a]pyrene diolepoxide mutagenesis in Big Blue mouse skin.

The Big Blue mouse was used to investigate the role of cell proliferation in mutation fixation in the mouse back skin model of carcinogenesis. Phorbol 12-myristate 13 acetate (TPA) was applied to the dorsum of Big Blue mice to manipulate cell proliferation, and benzo[a]pyrene (BaP) or BaP-diolepoxide (BPDE) was applied to produce premutagenic DNA damage. Mutations in the lacI transgene of skin DNA were measured. BaP and BPDE elevated mutant frequency, DNA adducts, and cell damage over untreated and acetone-treated mice. BPDE-DNA adducts peaked within 30 min of exposure and DNA adducts, formed after application of both BaP and BPDE, declined rapidly with time. As the dose of BaP increased (4 to 64 microg), DNA adducts, mutant frequency, and cell damage increased in a dose-dependent manner. TPA applied after BaP and BPDE further increased mutant frequency, DNA adducts, and cell damage, while variably affecting mitotic index and other measures of cell proliferation. TPA became less effective at increasing mitotic index as the dose of BaP increased, although all measures of cell proliferation, taken together, increased. The most effective production of DNA adducts and mutations occurred when the carcinogen was applied simultaneously with or within 1 hr of TPA. Mutations induced by BPDE were predominantly base substitutions: of these base substitutions, 35% were G:C --> A:T transitions, and 36% were G:C --> T:A and 29% G:C --> C:G transversions. Approximately 88% of all mutations and 100% of base substitutions were at G:C sites; 60% of all mutations and 70% of the base substitution mutations occurred at CpG sites. A:T --> G:C transitions were not found. All of the single-base deletions were at G:C base pairs.

Stomachs of mice lacking the gastric H,K-ATPase alpha -subunit have achlorhydria, abnormal parietal cells, and ciliated metaplasia.

The H,K-ATPase of the gastric parietal cell is the most critical component of the ion transport system mediating acid secretion in the stomach. To study the requirement of this enzyme in the development, maintenance, and function of the gastric mucosa, we used gene targeting to prepare mice lacking the alpha-subunit. Homozygous mutant (Atp4a(-/-)) mice appeared healthy and exhibited normal systemic electrolyte and acid-base status but were achlorhydric and hypergastrinemic. Immunocytochemical, histological, and ultrastructural analyses of Atp4a(-/-) stomachs revealed the presence of chief cells, demonstrating that the lack of acid secretion does not interfere with their differentiation. Parietal cells were also present in normal numbers, and despite the absence of alpha-subunit mRNA and protein, the beta-subunit was expressed. However, Atp4a(-/-) parietal cells had dilated canaliculi and lacked typical canalicular microvilli and tubulovesicles, and subsets of these cells contained abnormal mitochondria and/or massive glycogen stores. Stomachs of adult Atp4a(-/-) mice exhibited metaplasia, which included the presence of ciliated cells. We conclude that ablation of the H,K-ATPase alpha-subunit causes achlorhydria and hypergastrinemia, severe perturbations in the secretory membranes of the parietal cell, and metaplasia of the gastric mucosa; however, the absence of the pump appears not to perturb parietal cell viability or chief cell differentiation.

Mice lacking the basolateral Na-K-2Cl cotransporter have impaired epithelial chloride secretion and are profoundly deaf.

In chloride-secretory epithelia, the basolateral Na-K-2Cl cotransporter (NKCC1) is thought to play a major role in transepithelial Cl(-) and fluid transport. Similarly, in marginal cells of the inner ear, NKCC1 has been proposed as a component of the entry pathway for K(+) that is secreted into the endolymph, thus playing a critical role in hearing. To test these hypotheses, we generated and analyzed an NKCC1-deficient mouse. Homozygous mutant (Nkcc1(-/-)) mice exhibited growth retardation, a 28% incidence of death around the time of weaning, and mild difficulties in maintaining their balance. Mean arterial blood pressure was significantly reduced in both heterozygous and homozygous mutants, indicating an important function for NKCC1 in the maintenance of blood pressure. cAMP-induced short circuit currents, which are dependent on the CFTR Cl(-) channel, were reduced in jejunum, cecum, and trachea of Nkcc1(-/-) mice, indicating that NKCC1 contributes to cAMP-induced Cl(-) secretion. In contrast, secretion of gastric acid in adult Nkcc1(-/-) stomachs and enterotoxin-stimulated fluid secretion in the intestine of suckling Nkcc1(-/-) mice were normal. Finally, homozygous mutants were deaf, and histological analysis of the inner ear revealed a collapse of the membranous labyrinth, consistent with a critical role for NKCC1 in transepithelial K(+) movements involved in generation of the K(+)-rich endolymph and the endocochlear potential.

The morphological and spectral phenotype of apoptosis in HeLa cells varies following exposure to UV-C and the addition of inhibitors of ICE and CPP32.

Numerous extra- and intracellular factors, including UV radiation, can initiate a programme of cell death by apoptosis. While apoptosis is commonly defined morphologically, the relationships between morphology and molecular events are not well established. To investigate these relationships in HeLa cells, eight morphometric criteria for cell proliferation and damage and 10 criteria for apoptotic phenotype were examined using light microscopy, and corroborated by ultrastructure and spectral imaging. They were identified (1) during a time course after irradiation with 0, 10 or 30 J/m2 UV-C; (2) after separation of apoptotic from normal cells on a Percoll gradient; and (3) after irradiation with UV-C plus perturbation of the apoptotic pathway by treatment with inhibitors of two caspases, ICE and CPP32. The number of cells in apoptosis increased in a dose-dependent manner after UV-C treatment. Centrifugation of irradiated cells on a Percoll gradient increased the collection of apoptotic cells tenfold. The stereotypical apoptotic phenotype, in which cells have deep cytoplasmic blebbing and highly condensed DNA, comprised only a few percent of all apoptosis, and was rarely seen in groups receiving caspase inhibitors. The most common apoptotic phenotype was a rounded cell with large spherical nucleolus and associated DNA. After treatment with UV-C plus inhibitors the apoptotic index was decreased by about 30% compared to UV-C radiation alone. These apoptotic cells had dark spherical cytoplasm with small blebs, greatly increased numbers of cytoplasmic ribosomes, abundant nucleolar material with a large separate granular component, and chromatin condensed at the nuclear membrane. Using the technique of spectral imaging, it was found that the spectrum obtained from the granular component of the nucleolus, which was elevated in apoptotic cells treated with UV-C plus inhibitors, was similar to the dense accumulation of ribosomes in the apoptotic cytoplasm. The data indicate that spectral imaging may be a useful tool for identifying and characterizing variations in the apoptotic process, and that the caspase inhibitors used here do not completely abolish UV-C induced apoptosis, but rather alter its incidence and progression.

Colcemid alters S phase and other parameters in skin during chronic exposure to benzo(a)pyrene.

The administration of Colcemid for collecting mitotic figures in a carcinogenesis study, using benzo(a)pyrene (BaP), diminished the experimental differences between exposed and control mice. A dose-related increase in noncollected mitotic index (n-mitotic index) was seen in keratinocytes in the dorsal epidermis of mice which received four weekly treatments of BaP at 16, 32 and 64 micrograms in 50 microliters of acetone. In contrast, the number of mitotic figures collected for 4 hr by Colcemid block (c-mitotic index) was depressed at 16 micrograms, unchanged at 32 micrograms, and elevated at 64 micrograms of BaP. Weekly treatments with 4,8 or 16 micrograms BaP for 3-8 months induced an elevation in both n-mitotic and c-mitotic indices. The differences in results produced by the two methods of determining mitotic index depended upon dose and duration of treatment with BaP. The administration of Colcemid to acetone-treated mice increased the labeling index (number of labeled cells) and reduced the rate of DNA synthesis (low grain count per keratinocyte nucleus). After chronic application of BaP, Colcemid abrogated the increase in labeling index, but produced no additional effect on the number of grains per labeled keratinocyte. The modifying effect of Colcemid was greatest when administered during the peak of the tissue response to BaP. A number of significant changes in morphology of the skin associated with chronic exposure to BaP were attenuated by the use of Colcemid.

Cell proliferation and nuclear abnormalities are increased and apoptosis is decreased in the epidermis of the p53 null mouse after topical application of benzo[a]pyrene.

Cell proliferation and cell death in mouse epidermis are altered by topical application of benzo[a]pyrene (BaP), a procarcinogen, which yields metabolites that can form DNA adducts. The mitotic rate, nuclear abnormalities, labelling index, grain density, necrosis and apoptosis were compared in the epidermis of TSG-p53 null (p53-/-) and C57BL wild-type (wt) mice after weekly treatments with BaP to determine whether the absence of the p53 gene altered cytokinetic responses to DNA damaging agents in vivo. Acetone alone or 64 micrograms BaP in 50 microliters acetone was applied to the clipped dorsum of mice once, or in four consecutive weekly treatments. Indices of cell proliferation and cell death were the same in both wt and p53-/- mice treated only with acetone. One application of BaP depressed mitosis and slowed the rate of DNA synthesis in both genotypes. After four applications of BaP the number of keratinocytes in S phase increased substantially, while there was no further slowing in the rate of S phase in the wt and p53-/- mice. Cell proliferation rates and numbers of cells with nuclear abnormalities were higher and there were fewer apoptotic cells and apoptotic bodies in the p53-/- mice than in the wt mice. Numbers of 'sunburn' cells were similar in both types.

Epidermal cytokinetics, DNA adducts, and dermal inflammation in the mouse skin in response to repeated benzo[a]pyrene exposures.

Few studies have investigated the chronic cytokinetic effects of carcinogen exposure in the mouse skin. We report two experiments involving the repeated application of benzo[a]pyrene (BaP) to the dorsal skin of female Ha/ICR mice. In the first experiment, the cytokinetic, inflammatory, and DNA adduct responses were studied daily over a 9-day period encompassing the fourth and fifth weekly applications of BaP at doses of 16, 32, and 64 micrograms. The second experiment involved the same cytokinetic measurements at 1, 3, 5, and 8 months, and the weekly BaP doses were 4, 8, and 16 micrograms. The first study showed that after each application of 32 or 64 micrograms BaP, there was a wave of slow DNA synthesis in the epidermis which peaked at 24 hr, in coincidence with a wave of BaP-DNA adducts, followed by the appearance of dead and damaged keratinocytes. For the first few days after BaP application there was a depression in the mitotic rate which recovered several days before the next BaP application. There was a predominantly monocytic dermal inflammation throughout the observation period. In the second experiment, at the lower BaP doses, there was proliferative depression at 1 month, without dermal inflammation. With continued exposure, the proliferative depression changed to a dose-dependent increase in the rate of proliferation and dermal inflammation. The level of BaP-DNA adducts was followed in the 4 micrograms/week dose group, which showed a threefold increase after 4 months with the appearance of inflammation and heightened cell proliferation. These results suggest that the delayed inflammatory reaction, possibly based on a cell-mediated immune reaction to BaP, might have been responsible for the late cytokinetic responses and the associated increase in the level of BaP-DNA adducts.

Relationships between the nuclear membrane, nuclear pore complexes, and organelles in the type II pneumocyte.

Functional relationships among organelles of the type II cell are suggested based upon the proximity of organelles to specialized areas of the plasma- and nuclear membranes. In a two-dimensional morphometric analysis of the profiles of organelles in type II cells of the ferret and rat (and beagle dog), lamellar bodies were more likely to be located near the nuclear membrane than at the alveolar space (where exocytosis occurs). The size of lamellar body profiles was not correlated with distance from the nuclear membrane; however, large profiles were nearer the apical membrane, and smaller ones nearer the basement membrane. Profiles of highly branched mitochondria were 10 times more frequently associated with nuclear pore complexes than with the inter-pore nuclear membrane. Forty percent of all mitochondrial profiles were within 0.25 microns of the nucleus, 5% were within 0.02 microns and half of these appeared to touch the filaments of the nuclear pore complexes. The size of mitochondrial profiles was not correlated with distribution. In the ferret and rat, 8.6% and 2.5% respectively, of the nuclear pore complexes were associated with mitochondria. Sebaceous cells, from control mice, demonstrated a spatial distribution of granules which was size dependent but unrelated to the nuclear membrane.

Time course of airway hyperresponsiveness and remodeling induced by hyperoxia in rats.

The purpose of this study was to answer two questions concerning hyperoxia-induced airway hyperresponsiveness: 1) What is the time course of the development of airway hyperresponsiveness? 2) What is the relationship between the increase in responsiveness and smooth muscle area? Segments of intrapulmonary bronchi were isolated from male Sprague-Dawley rats that had been exposed to 80-85% O2 for a period of 1, 3, 5, or 7 days and from aged-matched control animals that breathed room air. Hyperoxia increased the sensitivity (log concentration or frequency that elicited a half-maximal response) and reactivity (maximum tension developed) of the airways to electrical field stimulation (EFS) after 3, 5, and 7 days; sensitivity to acetylcholine was not affected, but reactivity was increased after 7 days. Hyperoxia increased smooth muscle area beginning 5 days after commencing the exposure. After normalizing tension responses to smooth muscle area, reactivity of the airways to the stimuli was not different between the two groups, but sensitivity to EFS was still increased. The increase in reactivity observed after 5 and 7 days of exposure can be explained by an increase in smooth muscle area that occurred at these time points. The fact that the sensitivity of the airways to EFS remained increased after normalization, together with the fact that the increase in airway responsiveness after 3 days of exposure occurred at a time when smooth muscle area was not different from control, suggests that mechanisms other than increased smooth muscle area contribute to the development of hyperoxia-induced airway hyperresponsiveness.

Behavioral, histological, and neurochemical effects of nickel (II) on the rat olfactory system.

Experimentally, inorganic, sulfated nickel compounds (Ni2+) have been shown to produce histological lesions in the nasal mucosa of rats, more specifically, atrophy of the olfactory epithelium. The present project was designed to assess the effects of inhalation of nickel sulfate hexahydrate on behavioral, histological, and neurochemical aspects of the olfactory system. Male Long-Evans rats were exposed to either background air (control) or 635 micrograms Ni/m3 for 16 consecutive days, 6 hr/day. Exposure resulted in selective lesions to the olfactory epithelium. The number of bipolar sensory receptor cells was slightly reduced and there was a significant decrease in the thickness of the olfactory epithelium. This was due primarily to a significant loss of the sustentacular cell population, with a thinning of the apical cytoplasm, concomitant with a reduction in the number of microvilli at the surface of these cells. Significant decreases in carnosine level, consistent with the nickel sulfate exposure, were observed. However, there were no changes in olfactory function as measured by either absolute threshold or two-oder discrimination tasks.

Microvillar cells of the olfactory epithelium: morphology and regeneration following exposure to toxic compounds.

In recent years microvillar cells (MVC) have been identified in the olfactory epithelium of numerous species, including rodents, canines, and primates. However, there is no consensus on the morphologic or histochemical features of this cell, nor is the function of these cells currently known. Previous studies have examined MVC during development and in the mature olfactory epithelium, but not after toxic insult. A microvillar cell, defined by specific morphologic criteria, was studied in adult male Long-Evans rats exposed via inhalation to either 200 ppm methyl bromide for 4 h/day, 4 days/week for 2 weeks, or to 635 micrograms/m3 nickel for 6 h/day for 16 consecutive days, and sacrificed serially over several months. The pattern of recovery for MVC differed according to the severity and specificity of the insult to the olfactory epithelium. With methyl bromide, all cell types were completely depleted from olfactory epithelium immediately after injury, including MVC. MVC were slow to repopulate the epithelium, and appeared only when olfactory epithelium was complete in other respects. With nickel exposure, where the major effect was a gradual decrease in sustentacular cells with a thinning of the apical cytoplasm thickness, MVC showed a decline during exposure, but reappeared during recovery. In both cases, there was no difference in olfactory function, even when MVC were absent from the olfactory epithelium. A mature olfactory epithelium appears to be necessary to support the presence of this MVC, suggesting that it is not crucial to the regeneration processes or recovery of olfactory function, but perhaps plays some role, as yet undefined, in the unperturbed olfactory epithelium.

Carcinogenicity of 7H-dibenzo[c,g]carbazole, dibenz[a,j]acridine and benzo[a]pyrene in mouse skin and liver following topical application.

N-Heterocyclic aromatics are by-products of incomplete combustion of organic material. The overall objective of this study was to determine the relative carcinogenic potencies of 7H-dibenzo[c,g]carbazole (DBC) and dibenz[a,j]acridine (DBA) in a bioassay of complete carcinogenicity on mouse skin in a sensitive strain (Hsd:ICR(Br)) which has been used in metabolism and DNA binding studies of N-heterocyclic aromatics. No-treatment, acetone and benzo[a]pyrene (BaP)-treated animals were used as negative and positive control groups. DBC (50 nmol), DBA (50 nmol), BaP (50 nmol) or DBC plus BaP (25 nmol + 25 nmol) were applied twice weekly in 50 microliters acetone to the backs of 50 female mice/group for 99 weeks or until the appearance of a tumor. DBC, DBA, BaP and DBC plus BaP produced skin tumors in 43, 32, 49 and 42 of 50 mice each based on weekly visual observations with latent periods of 55.1, 62.2, 33.4 and 33.8 weeks, respectively. The histopathology data indicated primary skin lesions in 42, 27, 48 and 47 mice for DBC, DBA, BaP and DBC plus BaP, respectively. In addition, primary liver lesions in 37 mice were present in the DBC group. The morphological and morphometric data indicated a significant increase (P < or = 0.05) in mononuclear cells in the dermis for the BaP and DBC plus BaP groups relative to the control group. Significant increases (P < or = 0.05) were observed in the nuclear area, nucleoli per nucleus and cellular area of hepatocytes in the DBC treatment group relative to the control group. These data indicate that DBC is a potent liver carcinogen as well as a skin carcinogen following topical application.

Comparative tumor-initiating ability of 7H-dibenzo(c,g)carbazole and dibenz(a,j)acridine in mouse skin.

N-heterocyclic aromatics are environmentally important carcinogenic pollutants produced by incomplete combustion of organic material. 7H-Dibenzo-(c,g)carbazole (DBC), is a potent skin and systemic carcinogen, whereas dibenz(a,j)acridine (DBA), is a carcinogen with local effects. Therefore, the overall objective of these studies was to determine the initiating ability of DBC and DBA in mouse skin using an initiation-promotion protocol. Acetone-, TPA- or BaP-treated animals were used as negative and positive controls, respectively. DBC, DBA or BaP (200 nmol) dissolved in acetone was applied once to the backs of thirty shaved Hsd:(ICR)Br female mice, followed 2 weeks later with 2 micrograms of TPA in 50 microliters of acetone applied twice a week for up to 24 weeks. Skin tumors developed in 26, 17 and 27 animals, respectively. DBC plus TPA produced a significant influx of dermal macrophages similar to that seen for BaP. Initiation with BaP, DBC or DBA moderated the effect of TPA on most other dermal parameters, particularly neutrophils. These data indicate that, DBC, with apparently different activation pathways than BaP shows similar tumor initiating ability and morphological changes as BaP.

Benzo[a]pyrene-induced skin damage and tumor promotion in the mouse.

Epidermal cell kinetics and DNA adduct levels, and skin morphological changes were measured following weekly topical applications for 29 weeks of high (16, 32 and 64 micrograms) benzo[a]pyrene (B[a]P) doses to female ICR/Harlan mice, in order to investigate the relationship of these parameters to the timing, incidence and morphology of the elicited tumors. During the tumor latency period, [3H]thymidine labeling index, mitotic index, epidermal cell stacking, incidence of pyknotic and dark basal keratinocytes and labeled mitoses were periodically measured, as were nuclear area and DNA content. DNA adducts in skin epidermis were measured by an ELISA method over a period of 9 weeks of single weekly applications of 64, 32, 16 or 8 micrograms B[a]P. There was an initial linear increase in DNA adducts with dose in the epidermis but the increase was much less steep above 32 micrograms/week. This did not correlate with the sharp rise in tumor response above the 32 micrograms/week dose rate. Cell kinetic changes in response to the 64 micrograms/week dose reached a plateau in the first few weeks of the tumor latent period. There was little epidermal hyperplasia but an associated dose-dependent increase in [3H]thymidine labeling index, mitotic index and incidence of pyknotic and dark cells. This evidence indicated that B[a]P produced extensive cytotoxicity and cell death with regenerative proliferation under these conditions. Giant keratinocytes occurred in all dose groups. Analysis of a labeled mitosis curve indicated that B[a]P produced a G2/M block. There was a marked inflammatory response in the dermis at all B[a]P doses. Mice were observed weekly for tumor formation. Virtually all of the tumors were papillomas on initial appearance and required an average of 8 weeks to convert to carcinomas. The substantial cell killing and regenerative proliferation, and the correspondence between the dose-response patterns for epidermal damage and tumors, together with the initial appearance of tumors in the benign form, a characteristic of the action of promoting agents, provided evidence that the tissue damage associated with the high dose levels of B[a]P used in this study reflected tumor-promoting activity in this mouse epidermal tumorigenesis model. The implication of the results for mathematical models of tumor formation are discussed.

Effect of tumor-promoting agents on density and morphometric parameters of mouse epidermal Langerhans and Thy-1+ cells.

Topical application of tumor-promoting agents to the dorsal skin of female SENCAR mice on a twice-weekly basis resulted in a reduction in density per unit area of bone marrow-derived Thy-1+ dendritic cells. Activity was observed for well-established tumor-promoting doses of promoting agents of several different chemical types, including 12-O-tetradecanoylphorbol-13-acetate (TPA, diterpene diester), anthralin (dihydroxyanthrone), and n-dodecane (n-alkane). A reduction in density of the same cells was also observed on the basis of the asialoGM1 lipid as a surface marker after TPA treatment. No parallel effect was observed for epidermal Langerhans (Ia+) cells, the second major epidermal immunofunctional cell type, except in the case of anthralin, a finding which is consistent with the reported toxicity of this agent. The stage 2 promoting agent mezerein was unique in inducing a consistent increase in Langerhans cell densities, but did not affect the density of Thy-1+ cells when applied for a prolonged period unless applied following four doses of TPA. In contrast to the SENCAR strain, the promotion-resistant Balb/c and C56BL/6 strains showed no response with respect to TPA-induced reduction of Thy-1+ cell density. In addition to effects on density, the above tumor-promoting agents induced morphological changes in both Thy-1+ and Langerhans cells. When these changes were placed on a quantitative basis by the calculation of shape and area fraction parameters, marked and significant effects were observed for the above agents, but not for the partial promoting agent mezerein nor the non-promoting phorbol diester 4-O-methyl-TPA. The effects of TPA were largely blocked by the potent anti-promoting agent fluocinolone acetonide, moreover. These findings further support an important role for quantitative and qualitative alterations in dendritic epidermal cells in tumor promotion.

Comparative histomorphometric changes in SENCAR mouse epidermis in response to multiple treatments with complete and stage-specific tumor promoting agents.

Responses of various cells of the epidermis and dermis to topically applied agents have been implicated in the mechanism of multistage mouse tumorigenesis. These responses have been discussed almost entirely in the context of a single promoter treatment, although tumor expression is dependent on multiple applications. Responses of keratinocytes, epidermal dendritic non-keratinocytes and dermal leukocytes were therefore recorded following multiple topical applications of the potent complete tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA). In order to assess the importance of the response of individual cell types to the mechanisms and stages of promotion, responses to TPA were compared with those to agents with low complete promoting activity, but significant activity in individual stages of multistage promotion models. These included 4-O-methyl-TPA, a stage 1 promoting agent, mezerein and n-dodecane, stage 2 promoting agents of apparently different mechanism of action, and ethyl phenylpropiolate (EPP), a highly inflammatory stage 3 promoting agent. In agreement with previous findings, TPA induced a persistent epidermal hyperplasia and an increase in dark keratinocytes, although a similar finding was made for EPP and n-dodecane. The response to n-dodecane was significantly delayed, however, and that to EPP was accompanied by focal epidermal destruction and inflammation. The response to n-dodecane contrasted with that found for mezerein, supporting the suggestion that their mechanisms of action are distinct. Multiple treatments of 4-O-methylTPA caused no increase in dark cells, and mezerein induced no increase in numbers of pyknotic cells, whereas increases were expected in both cases on the basis of single dose experiments. Of the agents examined, only TPA induced a decrease in pale dendritic epidermal cells in the absence of marked toxicity, supporting the previous proposal that prolonged effects on this cell type are important in the promotion process. Some degree of persistent dermal leukocyte infiltration was observed with all agents excepting 4-O-methylTPA, although the extent of the response and its cellular characteristics appeared strongly dependent on the agent applied. In the case of TPA small mononuclear cells, neutrophils and macrophages all provided significant contributions to the total infiltrate. A similar phenomenon was observed with n-dodecane and EPP, with an additional increase in eosinophils which was not observed with TPA. Mezerein differed from both TPA and n-dodecane in inducing a significant increase only in eosinophils. As reported previously for single applications, prolonged TPA application caused a change in morphology and a considerable decrease in numbers of Thy-1+ epidermal dendritic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Critical comparison of histological and morphometric changes in SENCAR mouse epidermis in response to n-dodecane, 12-O-tetradecanoylphorbol-13-acetate and mezerein.

n-Dodecane, a previously little-studied tumor-promoting agent and mezerein, a diterpenoid natural product, have both been reported to have activity primarily in Stage II of two stage tumor promotion in SENCAR mouse skin. Histological changes in this tissue were therefore investigated in response to these agents in order to determine whether changes could be identified which were common to Stage II promotion by both compounds, and specific in this respect compared to those induced by the complete promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA). All three agents were applied at doses which have previously been found active in multistage tumorigenesis studies in this strain. A single dose of 50 mg dodecane induced no increase in the number of interfollicular cell layers or epidermal thickness, nor any observable inflammation, 6-144 h after application. In contrast, marked increases were predictably observed with TPA and mezerein, maximal responses occurring after 48-72 h. n-Dodecane induced no increase in the number of keratinocytes with dense cytoplasm and increased affinity for basophilic dyes (dark cells), only TPA demonstrating this activity. The alkane likewise did not increase the number of pyknotic basal keratinocytes indicating that toxicity would not account for the low Stage I activity of this agent in the way proposed for mezerein which was the most active in this respect, inducing a significant increase 48 h after treatment. Like TPA and mezerein, n-dodecane induced a significant increase in large intra-mitochondrial densities. Forty-eight to seventy-two hours after application, dodecane induced a significant decrease in the numbers of dendritic epidermal cells, a response which was also observed for TPA and mezerein, although occurring somewhat more rapidly. All three agents appeared to induce these cells to retract their characteristic processes. After four applications of n-dodecane the number of epidermal cell layers and mitotic index were equal to or greater than those observed with TPA. These findings show that in SENCAR epidermis the previously uncharacterized tumor-promoting agent n-dodecane induced essentially no histologic changes in mouse skin in common with mezerein, a second agent with activity primarily in Stage II of two stage tumor promotion, which were not also shown by the complete promoter TPA. The only characteristic specific to Stage II promoting agents therefore remains an inability to induce increased numbers of dark cells. In most other respects dodecane induced responses similar to those observed for TPA, although a distinctly different temporal dependence was noted in several cases.(ABSTRACT TRUNCATED AT 400 WORDS)

Qualitative and quantitative effects on epidermal Langerhans (Ia+) and Thy-1+ dendritic cells following topical application of phorbol diesters and mezerein.

Repeated twice-weekly applications of promoting doses of 12-O-tetradecanoylphorbol-13-acetate (TPA) to the dorsal skin of female adult SENCAR mice led to a reduction in numbers per unit area of epidermal Thy-1+ dendritic cells. Although no parallel effect was observed on Ia+ Langerhans cells, a concurrent reduction of dendritic morphology of both cell types was observed. Topical administration of TPA (2 micrograms) twice weekly for 4 or 8 weeks led to reductions in Thy-1+ cell numbers of 52 and 61%, respectively, whereas a single treatment was without effect. Similar effects were observed in animals initiated with 10 nmol 7,12-dimethylbenz[a]anthracene suggesting that the response was specific to the promotion, rather than the initiation, phase of two-stage tumorigenesis. All initiated animals bore tumors after 16 promoter treatments. In comparison with the potent promoter TPA, the weak overall, but stage-specific promoters 4-O-methylTPA and mezerein showed no effects on number or morphology of either dendritic cell type. These findings are therefore consistent with an important role for quantitative and qualitative alterations in epidermal non-keratinocytes of immune function in tumor promotion.

Intracisternal protein in the type II pneumocyte of the ferret, guinea pig, and mongrel dog.

An intracisternal protein in the type II pneumocyte of the ferret, guinea pig, and mongrel dog was examined by light and electron microscopy and morphometry. The basic pattern of layering in this membrane-bound, ribosome-studded structure (cisternal body) was visualized in cross section as dense layers separated by approximately 0.1 micron with seven fine layers between. In all species the central fine band of the seven was occasionally more prominent than the other six. In the guinea pig the seven fine layers alternated in density from light to dark. The cisternal body of the dog was similar to that of the ferret, but was very much smaller and encountered infrequently. No function has been ascribed to this structure; however, its relation to lamellar bodies, the perinuclear membrane, and surfactant apoprotein is discussed.

Morphology of tracheal and bronchial epithelium and type II cells of the peripheral lung of the guinea pig after inhalation of toluene diisocyanate vapors.

Toluene diisocyanate (TDI), a polymerizing agent used in production of plastics, can cause airways disease in some exposed individuals. Using guinea pigs as a model, the response of the airways and the type II cells of the peripheral lung was monitored morphologically and morphometrically after exposure to TDI vapors at 30 ppb, 260 ppb, and 3100 ppb. The two low doses of TDI caused little change in airways epithelium. There was no gross inflammatory cell infiltrate, however, surface infoldings and intracellular ciliated cysts increased in numbers. Animals exposed to 3100 ppb TDI 4 h/day for 5 days, sustained considerable damage to the epithelium, and stratified nonkeratinizing cells lined the airways until one week after exposure. Polymorphonuclear leukocytes were present in the early period after exposure. Increased numbers of eosinophils were present between one and two weeks following exposure. Mitoses in the epithelium were common during recovery. In the peripheral lung, though a modest subjective increase in the number of type II cells was seen after 3100 ppb TDI, the volume density of type II cells, and organellar components (lamellar bodies, mitochondria, cisternal bodies) did not change significantly after any exposure level of TDI.