Challenge for 3D culture technology: Application in carcinogenesis studies with human airway epithelial cells.

Lung cancer is still one of the major intractable diseases and we urgently need more efficient preventive and curative measures. Recent molecular studies have provided strong evidence that allows us to believe that classically well-known early airway lesions such as hyperplasia, metaplasia, dysplasia and carcinoma in situ are really precancerous lesions progressing toward cancer but not necessarily transient and reversible alteration. This suggests that adequate early control of the precancerous lesions may lead to improved prevention of lung cancer. This knowledge is encouraging in view of the imminent necessity for additional experimental systems to investigate the causal mechanisms of cancers directly in human cells and tissues. There are many questions with regard to various precancerous lesions of the airways. For example, should cells, before reaching a stage of invasive carcinoma, undergo all precancerous stages such as hyperplasia or metaplasia and dysplasia, or is there any shortcut to bypass one or more of the precancerous stages? For the study of such questions, the emerging 3-dimensional (3D) cell culture technology appears to provide an effective and valuable tool. Though a great challenge, it is expected that this in vitro technology will be rapidly and reliably improved to enable the cultures to be maintained in an in vivo-mimicking state of differentiation for much longer than a period of at best a few months, as is currently the case. With the help of a "causes recombination-Lox" (Cre-lox) technology, it has been possible to trace cells giving rise to specific lung tumor types. In this short review we have attempted to assess the future role of 3D technology in the study of lung carcinogenesis.

Different breakage-prone regions on chromosome 1 detected in t(11;14)-positive mantle cell lymphoma cell lines and multiple myeloma cell lines are associated with different tumor progression-related mechanisms.

To better define secondary aberrations that occur in addition to translocation t(11;14)(q13;q32) in mantle cell lymphomas (MCL) and in multiple myelomas (MM), seven t(11;14)-positive MCL cell lines and four t(11;14)-positive MM cell lines were analysed by fluorescence R-banding and spectral karyotyping (SKY). Compared with published data obtained by G-banding, most chromosome aberrations were redefined or further specified. Furthermore, several additional chromosome aberrations were identified. Thus, these cytogenetically well defined t(11;14)-positive MCL and MM cell lines may be useful tools for the identification and characterization of genes that might be involved in the pathogenesis of MCL and MM, respectively. Since MCL and MM were found to have different alterations of chromosome 1, these were investigated in more detail by fluorescence in situ hybridization (FISH) and multicolor banding (MCB) analyses. The most frequently altered and deletion-prone loci in MCL cell lines were regions 1p31 and 1p21. In contrast, breakpoints in MM cell lines most often involved the heterochromatic regions 1p12-->p11, and the subcentromeric regions 1q12 and 1q21. These data are in accordance with previously published data of primary lymphomas. Our findings may indicate that different pathways of clonal evolution are involved in these morphologically distinct lymphomas harboring an identical primary chromosome aberration, t(11;14).

Molecular cytogenetic characterization of the mantle cell lymphoma cell line GRANTA-519.

Combining fluorescence R-banding, fluorescence in situ hybridization and spectral karyotyping allowed us to precisely define chromosomal breakpoints, gains, losses and a newly detected amplification in the human mantle cell lymphoma (MCL) cell line GRANTA-519. GRANTA-519 is characterized by the t(11;14)(q13;q32) resulting in overexpression of cyclin D1, a key player in cell cycle control. Hitherto unresolved complex rearrangements involve 1p, 1q, 3cen, 9p, 11q, 12p, 12q, 16p, 17p, and 18cen. Moreover, a 4- to 6-fold gain of sequences on 18q leads to a low-level amplification of the BCL2 gene and to an overexpression of the BCL2 protein. These results provide the basis for the identification of not only candidate oncogenes responsible for MCL in gained regions, but also for the identification of putative tumor suppressor genes in commonly deleted regions like 1p22, which would eventually enable functional studies of these genes.

Inhalation toxicity of propineb. Part II: Results of mechanistic studies in rats.

Previous repeated inhalation exposure studies revealed two independent organotropic effects of inhaled propineb dust: One was restricted to the lung, the other to muscle weakness of hindlimbs. These effects were believed to be causally related to the principle decomposition products of this type of dithiocarbamate in the biological milieu and related to zinc and carbon disulfide. Two mechanistic 1-wk inhalation studies were performed, each focusing on one of these findings. The 7 x 6-h/day repeated-exposure inhalation study analyzed whether the nature of the response occurring at the alveolar level is "adaptive" or "early adverse" and whether soluble zinc is the causative agent. Groups of 18 female rats were exposed nose-only to mean concentrations of 0, 1.1, 5.5, and 25.8 mg propineb/m(3) and 6.9 mg ZnO/m(3). On postexposure days 1, 3, and 15 the time course of responses was analyzed by bronchoalveolar lavage (BAL), including quantification of Zn and metallothionein (MT) in BAL cells. Clinical evidence of muscular weakness was investigated separately in 20 female Wistar rats exposed to 70 mg propineb/m(3) on 5 consecutive days (6 h/day), followed by a 2-wk postexposure period. Clinical signs, body weights, and feed and water consumption were recorded as frequently as technically feasible. Fifty percent of rats received an oral cysteine supplementation to verify/refute the hypothesis that the incapacitation observed in previous studies is the cause of emaciation and associated impairment of CS(2) detoxification. The findings of the first study are consistent with this hypothesis, namely, that soluble Zn triggers a series of pulmonary events that is consistent with the homeostasis of this essential metal. It is concluded, accordingly, that the adjusted maximal workplace level for ZnO is also valid for propineb to preclude Zn-mediated responses to occur in the lung. With respect to muscular effects, this mechanistic study demonstrates further that the increased detoxification capacity afforded by oral supplementation of cysteine mitigates markedly the toxic potency of propineb. Procedural variables specific to the inhalation bioassay appear to be decisive for the elicitation of muscular effects. The major variable is considered to be the large drop in body weights associated with each exposure session and the concomitantly decreased uptake of essential nutritional factors (e.g., cysteine) involved in the detoxification of this compound. Accordingly, the muscular deficits elicited by high concentrations of propineb are viewed to be secondary effects in an animal species likely to be more susceptible to this type of change than humans (Pauluhn & Rosenbruch, 2003).

Respiratory sensitization to diphenyl-methane-4,4'-diisocyanate (MDI) in guinea pigs: impact of particle size on induction and elicitation of response.

The impact of particle size of aerosolized polymeric diphenylmethane-4,4'-diisocyanate (MDI) for the induction and elicitation of respiratory sensitization was evaluated. Four groups of 16 female guinea pigs each received either the vehicle, repeated intradermal (id) injections (3 x 0.3% MDI), one high-level inhalation exposure of 15 min to 135 mg MDI/m(3) air using a small aerosol (MMAD approximately 1.7 microm) or large aerosol (MMAD approximately 3.8 microm). Three weeks later, animals were challenged subsequently with two ramped concentrations of MDI aerosol (average concentrations 16 and 49 mg/m(3) air, each for 15 min) and two different particle sizes, i.e., the MMAD was either approximately 1.6 microm or approximately 5.1 microm for the small- and large-size aerosol, respectively. Respiratory sensitization was assessed by two endpoints: the measurement of respiratory rate, and examination of influx of eosinophilic granulocytes into the mucosa and submucosa of the trachea, bronchi, and lung-associated lymph nodes (LALN). The recruitment of eosinophilic granulocytes into bronchial tissues was subdivided as follows: muscularis mucosae, submucosa, and perivascular. From measurements of respiratory rate, it would appear that guinea pigs sensitized by id injections or by inhalation exposure with the large aerosol tended to display a higher responsiveness than naive controls when challenged with the small aerosol. The recruitment of eosinophilic granulocytes in the bronchial tissue was greater in both inhalation induction groups as compared to the vehicle control. It appears that there was a somewhat greater response in animals sensitized by id injections or by inhalation exposure with the large aerosol and challenged with the small aerosol. Topographically, this difference was apparent only at the bronchial perivascular level and lung-associated lymph nodes (LALN), whereas at the submucosal and muscularis mucosae level the impact on particle size tended to be less pronounced. In summary, this study suggests that a brief, high-level inhalation exposure of MDI aerosol caused a sensitization of bronchial tissues in guinea pigs. The higher sensitization potency of the large aerosol may possibly be related to a dosimetric phenomenon because of the greater fraction of deposition of large particles within the upper respiratory tract. Overall, challenge exposures with this type of irritant aerosol appear to evoke more consistent effects when the MMAD is in the range of approximately 2 rather than approximately 5 microm.

Two-week inhalation toxicity of polymeric diphenylmethane-4, 4'-diisocyanate (PMDI) in rats: analysis of biochemical and morphological markers of early pulmonary response.

The pulmonary response of Wistar rats to respirable polymeric diphenylmethane-4,4'-diisocyanate (PMDI) aerosol was examined in a 2-wk repeated nose-only inhalation exposure study. Exposure concentrations were 1.1, 3.3, and 13.7 mg PMDI/m(3) (6 h/day, 15 exposures). The level of 13.7 mg/m(3) was actually a combination of an initial target concentration of 10 mg/m(3) in wk 1, which was raised to 16 mg/m(3) in wk 2, due to a lack of signs suggestive of pulmonary irritation. An acute sensory irritation study on rats served as basis for selection of these concentrations. Shortly after the 2-wk exposure period, rats were subjected to pulmonary function and arterial blood gas measurements. Lungs were examined by light and transmission electron microscopy, and labeling indices in terminal bronchioles were measured. Bronchoalveolar lavage (BAL) was performed to assess various indicators of pulmonary inflammation, including neutrophil and macrophage numbers, protein, lactate dehydrogenase (LDH), gamma-glutamyltranspeptidase (gamma-GT), alkaline phosphatase (APh), acid phosphatase (ACPh), and beta-N-acetylglucosaminidase (beta-NAG). Phosphatidylcholine in BAL fluid and BAL cells was determined as aggregated endpoint suggestive of changes in pulmonary surfactant. Rats exposed to 3.3 and 13.7 mg/m(3) experienced concentration-dependent signs of respiratory tract irritation. Determination of arterial blood gases, lung mechanics, and carbon monoxide diffusing capacity did not demonstrate specific effects. Analysis of BAL fluid and BAL cells revealed changes indicative of marked inflammatory response and/or cytotoxicity in rats exposed to 13.7 mg/m(3), and the changes were characterized by statistically significantly increased activities of LDH, beta-NAG, and protein. Phospholipid concentrations were increased in rats exposed to 1.1 mg/m(3) and above (elevated levels of lipid material in alveolar macrophages demonstrated by polychrome stain) and 3.3 mg/m(3) and above (increased intracellular ACPh activity and intracellular phospholipids). In these groups, gamma-GT was statistically significantly increased. These findings suggest that changes in phospholipid homeostasis appear to occur at lower levels than those eliciting inflammation and cytotoxicity. Light and transmission electron microscopy suggest that exposure to 3.3 and 13. 7 mg/m(3) resulted in focal inflammatory lesions and an accumulation of refractile, yellowish-brownish material in alveolar macrophages with concomitant activation of type II pneumocytes. In the terminal bronchioles a concentration-dependent increase of bromodeoxyuridine (BrdU)-labeled epithelial cells was observed in all PMDI exposure groups. In summary, it appears that respirable PMDI aerosol interacts with pulmonary surfactant, which, in turn, may stimulate type II pneumocytes to increase their production of surfactant and to proliferate.

Stem cells of the respiratory epithelium and their in vitro cultivation.

Parenchymal (epithelial or mesenchyma) stem cells are rapidly drawing both scientific and clinical attention in solid organs like the liver, skin, intestine and abdominal mesothelium, just as has been the case in the hematopoietic system. For the stem cells of these organs various definitions, markers for identification, methods of isolation and in vitro cultivation, and lineage mechanisms have been proposed and some of them are now proven to be valid and useful. In this article attempts will be made to explore whether there are stem cells in the lower respiratory system (from the trachea to the lung periphery) and what they look like. Because of its anatomical and functional complexity the stem cell concept for the respiratory system has been developing rather slowly. Nevertheless, the data available seem to indicate that in analogy to the above mentioned organs there is only one type of epithelial stem cells throughout all sections of the lower respiratory system during fetal through adult stages. They are multipotent for cell differentiation and able to yield lineage progenitors for ciliated, goblet, basal. Clara neuroendocrine, alveolar type 1 and alveolar type 2 cells.

Formation and persistence of the miscoding DNA alkylation product O6-ethylguanine in male germ cells of the hamster.

The cellular parameters which modulate trans germ-line carcinogenesis by DNA-reactive agents have not yet been studied in detail. Therefore, we have measured in this study the formation and repair kinetics of the miscoding alkylation product O6-ethylguanine (O6-EtGua) in nuclear DNA of spermatogonial cells of the Syrian golden hamster (SGH) after exposure to either of two potent N-nitroso carcinogens, ethylnitrosourea (ENU) or diethylnitrosamine (DEN). Both compounds, the spontaneously decomposing ENU, and DEN, which has to be converted by cellular enzymes to the reactive ethyl diazonium ion, induce the same pattern of alkylation products in nuclear DNA. Adduct analyses were performed at the single-cell level by using a quantitative immunocytological assay and anti-(O6-EtGua) monoclonal antibodies. 1.5 h after intraperitoneal application of ENU (100 microg/g body weight) O6-EtGua levels in the nuclear DNA of spermatogonia were similar to those in other cell types of the same hamster. About 30% of the initially formed DNA adducts were still persistent in spermatogonial cells even 4 days after ENU exposure. The presence of O6-EtGua in DNA after exposure to DEN (100 microg/g body weight) implies the capability of hamster spermatogonial cells to convert nitrosamines into DNA-alkylating metabolites. This capability of male germ cells in combination with their limited repair capacity for a critical DNA adduct and their high rate of proliferation may be considered as a major risk factor for genetic effects including carcinogenesis in subsequent generation(s).

Sensitivity of a hamster lung cell line to direct and indirect acting carcinogens.

Cytotoxicity of benzo(a)pyrene (B(a)P), 7,12-dimethylbenz(a)anthracene (DMBA), aflatoxin B1 (AB1), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was estimated in vitro by using a hamster lung cell line. The studies were conducted to assess the usefulness of an organ-specific cell culture system for demonstrating differences in the cytotoxic potency of diverse chemical carcinogens. Cytotoxicity was determined by using the succinate dehydrogenase assay (MTT assay) after different incubation times and concentrations with the corresponding carcinogens. The effective concentration EC50 as well as the slope of the regression line were used as parameters for the biological effects. The results from these studies indicate a clear dose-dependent reaction after incubation of the cells with aflatoxin B1 (EC50: 2.3 microM) and MNNG (EC50: 4.0 microM). For the polycyclic hydrocarbons benzo(a)pyrene and DMBA, a dose-independent reaction was found. These results indicate that consideration of the EC50 values only might not be sufficient to characterize differences in the cytotoxic activity of different substances. Chemicals can lead to equal values in the EC50, but cells can differ significantly in their biological sensitivity, meaning that the extent of reduction in cell proliferation depends on the chemical used. By considering the two above-mentioned parameters, a ranking for the analyzed substances will be possible in the following way: AB1, MNNG, DMBA and B(a)P. Taken together, our experiments show that it is possible to reveal differences in the cytotoxic potency of chemicals by using in vitro methods.

In vitro reconstituted tissue as an alternative to human respiratory tract.

The data obtained from in vitro systems utilizing human cells and tissues should form a basic part of the information necessary for risk assessment. The most important thing for such systems is, therefore, to simulate the structures and functions of cells and tissues in the native organ as closely as possible. In designing in vitro systems, there may be two approaches-one aiming at the growth of cells in a primarily two-dimensional fashion, the other allowing cells to form in vivo-mimicking three-dimensional architectures. In cultures in which the airway epithelial cells are growing in a two-dimensional fashion, some functional and structural characteristics can be developed to a considerable extent. However, there are some that cannot be developed or expressed under that condition but require a three-dimensional growth pattern. In this paper we explore the capacity of early to long-term passage airway epithelial cells (human and hamster) to resume architectures and functions existing in the native tissue in the specific environments given in vitro.

[Two cases of recurrent, massive hemoptysis with contralateral, concurrent hemoptysis].

Bilateral, concurrent massive hemoptysis with respiratory failure is rare and difficult to manage. We encountered two patients with this condition. One was a 51-year-old woman and one was a 63-year-old woman. Both had inactive pulmonary tuberculosis and episodes of hemoptysis. Home oxygen therapy was prescribed to the former patient and bronchial artery embolization was done in the latter. The sites of initial bleeding were in the left basal segment and in the lingular segment. Bleeding was well controlled by endobronchial tamponade with a Fogarty balloon catheter in the left main bronchus, contralateral intubation, and systemic artery embolization. Use of water instead of air in the balloon was more effective in over the long term. Concurrent bleeding from the contralateral upper lobe bronchus was controlled by compression with a tracheal tube cuff at the bronchial orifice. Afterward, an infiltrative shadow was seen on the chest X-ray film and hemoptysis occurred in case 2 occurred in case 2. Treatment with erythromycin and antituberculosis drugs brought about improvement of chest X-ray findings and disappearance of the hemoptysis.

Formation of O6-ethylguanine in spermatogonial DNA of adult Syrian golden hamster by intraperitoneal injection of diethylnitrosamine.

17-Week-old Syrian golden hamsters received a single intraperitoneal injection of diethylnitrosamine (DEN) at a dose of 100 mg/kg body weight. The DNA alkylation product O6-ethylguanine was formed in the spermatogonia. The data demonstrate that DEN can pass the blood-testis barrier and be metabolized in the spermatogonia to yield an alkylating derivative or that externally activated metabolites themselves can pass the barrier.

Increased risk of cancer in the descendants of Syrian hamsters exposed prenatally to diethylnitrosamine (DEN).

Transmission of site-specific tumorigenicity (papillomas in larynx and trachea) of diethylnitrosamine (DEN) to the 2 subsequent generations (F1 and F2) was studied using an outbred strain (Han:AURA) of pregnant Syrian golden hamsters (P generation), which were treated i.p. with 10 mg/kg b.w. of DEN on day 12, 13 or 14 of gestation. Laryngotracheal papillomas were induced by DEN in the P and F1 generations only, while these tumours did not occur in the F2 generation. Spontaneously occurring tumours, including uterine adenocarcinomas, lymphomas, and laryngotracheal neuro-endocrine cell tumours, were observed at higher incidences among the F2 animals derived from the P generation hamsters treated with DEN only on day 13 or 14 of gestation. In the same animals, the ratio of malignant to benign tumours was considerably higher than in controls. In addition, the F2 hamsters derived from the DEN-treated P generation showed more frequent multiple organ involvement in tumorigenesis than the F2 controls. Several uncommon malignant tumours were detected in the F2 offspring, possibly the result of damage caused to germ cells by the prenatal exposure of F1 Syrian hamsters to DEN.

Regulation of c-jun by lung carcinogens in Clara cells of hamsters.

In vitro differentiated hamster Clara cells were used to study the effects of lung carcinogens on the regulation of the c-jun oncogene. Northern blot analysis revealed a decrease in the expression of jun transcripts 24 h following the exposure of Clara cells to the direct acting forms of benzo[a]pyrene (BPDE*) or 5-methylchrysene (5MeCDE). To determine whether this decrease was mediated at the transcriptional level, we have used CAT reporter constructs driven by nested deletions of the 5' non-coding regulatory region of the c-jun oncogene. While BPDE was capable of activating certain regulatory domains of the c-jun promoter, this activation was not observed with either 5MeCDE or the less active lung carcinogens BADE or 6MeCDE. Analysis of enhancer elements identified the SP1 target site as a strong silencer after BPDE treatment. While positive regulatory element(s) mediating activation of c-jun by BPDE were localized within the promoter region up to -1639, further upstream sequences reduced this transcriptional activation. Thus, when the complete promoter region, up to -4500, was tested, no transcriptional activation was noted following BPDE treatment. These observations suggest that the regulation of c-jun in Clara cells exposed to potent lung carcinogens is mediated at the post-transcriptional level, possibly by reducing the stability and, in turn, the half life of c-jun mRNA. Overall, in contrast to the response of c-jun to numerous carcinogens and stress inducing agents noted in various other cell systems, our findings suggest the existence of a tissue-specific regulatory response for c-jun.