Toxicogenomic analysis of N-nitrosomorpholine induced changes in rat liver: comparison of genomic and proteomic responses and anchoring to histopathological parameters.

A common animal model of chemical hepatocarcinogenesis was used to examine the utility of transcriptomic and proteomic data to identify early biomarkers related to chemically induced carcinogenesis. N-nitrosomorpholine, a frequently used genotoxic model carcinogen, was applied via drinking water at 120 mg/L to male Wistar rats for 7 weeks followed by an exposure-free period of 43 weeks. Seven specimens of each treatment group (untreated control and 120 mg/L N-nitrosomorpholine in drinking water) were sacrificed at nine time points during and after N-nitrosomorpholine treatment. Individual samples from the liver were prepared for histological and toxicogenomic analyses. For histological detection of preneoplastic and neoplastic tissue areas, sections were stained using antibodies against the placental form of glutathione-S-transferase (GST-P). Gene and protein expression profiles of liver tissue homogenates were analyzed using RG-U34A Affymetrix rat gene chips and two-dimensional gel electrophoresis-based proteomics, respectively. In order to compare results obtained by histopathology, transcriptomics and proteomics, GST-P-stained liver sections were evaluated morphometrically, which revealed a parallel time course of the area fraction of preneoplastic lesions and gene plus protein expression patterns. On the transcriptional level, an increase of hepatic GST-P expression was detectable as early as 3 weeks after study onset. Comparing deregulated genes and proteins, eight species were identified which showed a corresponding expression profile on both expression levels. Functional analysis suggests that these genes and corresponding proteins may be useful as biomarkers of early hepatocarcinogenesis.

Workshop report. Children as a special subpopulation: focus on immunotoxicity. Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV), 15-16 November 2001, Berlin, Germany.

An international symposium on the impact of environmental hazards, chemicals and drugs on the developing immune system of children was held in Berlin (Germany) organized by the BgVV. Epidemiological evidence indicates that an immature immune system challenged early in life by bacterial antigens may prevent, to some extent, allergic reactions including asthma bronchiale triggered by environmental pollutants. However, the prevalence for infectious disease is increased in childhood, especially when exposure to contaminants takes place in the period of pregnancy and breast-feeding. The effects of chlorinated biphenyls, dioxin, endotoxins, hexachlorobenzene, and direct and indirect in utero tobacco smoke exposure are examples. All participants recommend comparative and follow-up epidemiological studies and clinical examination of infants and children at risk during upbringing. There is ample evidence from experimental studies that indicates adverse effects on the developing immune system after in utero and postnatal exposure to chemicals and drugs. The adverse reactions of aciclovir, benzodiazepines, hexachlorobenzene, organotins (di-n-octyltin dichloride, tributyltin oxide), pesticides (methoxychlor, heptachlor) and polyhalogenated aromatic hydrocarbons (2,3,7,8-tetrachlorodibenzo-p-dioxin) are presented and reviewed. To determine the predictive value of test data in risk assessment for neonates and children, development, differentiation and maturation of the immune system in humans and laboratory rodents is compared in their pre- and postnatal stages. Considering some differences in immunocompetence at birth and after lactation, and differences in the time frame for maturation of the immune system, reaction types are thought to be common, comparable and similar in human childhood and early adolescence and the postnatal lifetime of laboratory rodents. The participants of the symposium felt strongly that regulatory steps urgently need to be initiated to incorporate some relevant aspects into existing test guidelines for testing developmental immunotoxicity. In this context, it is recommended that animals culled otherwise in one- and two-generation studies be examined for developmental immunotoxicity according to the valid methods and parameters discussed. The majority of participants agreed that a safety factor of 10 is too low in risk assessment and management to protect a sensitive subpopulation of children against man-made environmental pollutants.

Results of a cyclosporin A ringstudy.

The aim of the study was to find out whether an extended subacute toxicity study (additional organ weights, histopathology and immune functional tests), routinely employed in testing of chemicals, would shown indications of (adverse) effects on the immune system below general toxicity. Therefore, a five laboratory ring study on the basis of an oral 28-day repeated dose study in rats (OECD guideline 407) was carried out with 1, 5 and 25 mg/kg of cyclosporin A (CsA) per day by gavage. Besides some toxic effects such as reduced body weight gain and increased kidney calcification in the high-dose group, the results of the additional pathologic examinations revealed that CsA caused a pattern of specific morphological alterations of the lymphoid tissues in mid- and high-dose groups. Selected immune parameters such as immunoglobulin determination, plaque-forming assay, flow cytometry, activation status of macrophages and natural killer cells (NK), and proliferative response of spleenocytes and cells from mesenteric lymph nodes (concanvalin A (ConA) and pokeweed mitogen (PWM) stimulation) were also investigated. Some results compared to the controls revealed alterations down to the low-dose group. The extended methodology consistently indicated the potential detection of effects on the immune system below general systemic toxicity. The study will be continued by investigating a second compound with primarily immunostimulating effects. Results from those studies should further contribute to the current discussion of up-dating of repeated dose toxicity guidelines with respect to immunotoxicity.

Classification of cystic keratinizing squamous lesions of the rat lung: report of a workshop.

An international workshop of toxicologic pathologists reviewed cystic keratinizing squamous lesions of the rat lung. These lesions develop in response to the chronic inhalation of diverse particulate materials. Controversy exists over the biological significance of these changes and their relevance to humans. For the first time, in one place, a group of pathologists analyzed slides from all available studies. The workshop reached a consensus as to classification of these unique pulmonary tissue responses and offers diagnostic criteria for application. Although additional research is needed, this working classification scheme should serve as a practical interim approach for pathologists and regulatory agencies.

Anchorage independent colony growth of fetal hamster lung epithelial cells after treatment with diepoxybutane.

To test the reliability of a new cell transformation assay, a cloned fetal Syrian hamster lung epithelial cell line (M3E3/C3) was used. The target cells originating from the respiratory tract were treated in vitro over a concentration range of 0-10(-5) M/l with diepoxybutane, cultured during the expression period of 28 or 35 days and then transferred into soft agar. Anchorage independent colony growth in soft agar occurs only if cells are transformed. Growth and number of colonies were taken as a score of the carcinogenic potential of the test substance. Under the conditions of this cell transformation assay it was possible to detect the carcinogenic potential of diepoxybutane unequivocally.

Effects of nitroso compounds and aromatic amines on fetal tracheal explants.

Tracheas were excised from fetal Syrian golden hamsters on the 15th day of gestation. Tracheal explants were cultured in vitro and exposed to different dose-levels of well known carcinogens. We chose two nitroso compounds, N-Methyl-N-nitro-N-nitrosoguanidine (MNNG) and Diethylnitrosamine (DEN) and two aromatic amines, Aminofluorene (AF) and Acetylaminofluorene (AAF). The tracheal explants were treated for 24 h in vitro, then the carcinogens were washed off and the tracheas were kept for 21, 28 or 35 days in culture. After fixation tracheal explants were transversely cut with serial section techniques and scored for morphological changes of the epithelium by light microscopy. Most of the control explants completed differentiation and had a normal morphology at the end of the in vitro culture period. Occasionally we found a decrease of the number of ciliated cells and some areas with squamous metaplasia in the respiratory epithelium. Carcinogen treatment with nitroso compounds led to a significant increase of the morphologic changes of the epithelium. These effects were especially obvious after DEN treatment. Morphologic changes of the epithelium such as metaplasia and hyperplasia were discussed as carcinogen-related events. In vitro exposure with aromatic amines did not induce marked metaplastic or hyperplastic changes in the respiratory epithelium of tracheal explants.

View of the regulatory use of rat liver foci data in the Federal Republic of Germany.

Altered hepatocellular foci (AHF) are suspected of being phenotypic markers of a stage in the sequential process of tumor development in the liver. A number of short-term and mid-term tests, including some that use AHF as an endpoint, have been reviewed by the Federal Health Office to determine if they are suitable to replace long-term animal experiments for predicting the carcinogenic potential of chemicals. Although regulatory authorities recognize the advantages of short-term tests, including a possible reduction in the number of animals used, it is still premature to make a general recommendation regarding the suitability of these tests for regulatory decision-making. At this time, the Commission of European Communities (CEC) follows a decision-tree approach for regulating chemicals coming to the market. Since it is unrealistic to expect a reasonable number of AHF to evaluate in 28-day or 90-day studies, AHF can only be practically evaluated in the conventional long-term bioassay. There is still insufficient knowledge regarding the biological significance of the various phenotypic forms of AHF to use them as a relevant endpoint for regulatory purposes. However, data on AHF derived from routine hematoxylin and eosin (H&E) work can be used to aid in the interpretation of the study and in recommending additional investigations.

Effects of BHA and related phenols on the forestomach of rats.

To determine the pathogenesis of BHA-induced forestomach lesions, the nature and time course of the early lesions in the forestomach of Wistar rats were studied. The rats were given BHA at a dose level of 2% in a powdered diet or by oral intubation of 1 g BHA/kg body weight/day in arachis oil. The hyperplastic changes in the mucosa were visible 1 day after the second application. The localization was dependent on the mode of application. Dietary exposure yielded changes in the area of the limiting ridge; oral intubation of BHA produced lesions in the apex of the forestomach. In a subchronic 90-day feeding study in rats, no recognizable effect was observed when 0.125% BHA was incorporated into the diet as a solution in arachis oil. In reversibility studies, severe forestomach lesions observed after feeding 2% BHA for 6, 12 or 15 months regressed almost completely following withdrawal of the BHA for a period of 7 months. BHA induced similar forestomach damage in NMRI mice and Syrian golden hamsters, whereas guinea-pigs, a species having no forestomach, did not show comparable lesions. Substances with similar chemical structure were tested in short-term feeding studies (tert-butylhydroquinone, 4-methoxyphenol, 1,4-dimethoxybenzene, hydroquinone, 3-methoxyphenol, 2-methoxyphenol, anisole, p-cresol, phenol and BHT). Only 4-methoxyphenol strongly affected the forestomach mucosa in a manner similar to that associated with BHA. The methoxy group in the para position seems to be important for the hyperplasiogenic activity.

Scanning electron microscopical investigations on the respiratory epithelium of the Syrian golden hamster. VI. In vitro effects of different polycyclic aromatic hydrocarbons.

In a comparative study, the biological effects of different polycyclic aromatic hydrocarbons (PAH) on the fetal respiratory epithelium of Syrian golden hamsters were examined (light and scanning electron microscopy) in tracheal explants exposed in vitro to benz(a)anthracene (BaA), benz(a)acridine (BaAC), benzo(b)fluoranthene (BbF), benzo(e)pyrene (BeP), benzo(k)fluoranthene (BkF) and pyrene (PYR). Concentrations of the compounds related to the solubility in dimethylsulphoxide in the tissue culture medium (0.5% DMSO). Focally slight inhibition of epithelial differentiation and/or circumscribed simple metaplasia with an incidence of up to 10% were found in the controls (0.5% DMSO), BeP and PYR groups as well as in the explants exposed to low levels of BaA and BaAC. The frequency of these lesions increased with raising (doubling) the dose (50%: BaAC, BbF, BfK; 100%: BaA). Following exposure to the latter compounds a similar dose response relationship was observed for dysplastic alterations of the respiratory epithelium. In some of these cases (10%) the epithelial change was associated with focally hyperplastic and/or proliferative exophytic growth. Morphologically, the alterations were comparable to those representing early changes associated with the development of epidermoid and mucoepidermoid neoplasms found after in vivo PAH exposure. In terms of transformation, the data support the information obtained from observations on mixed fetal hamster lung cells exposed to the same compounds.

Transplacental effect of N-diethylnitrosamine on chromosomal aberrations in fetal tracheal cells of the Syrian hamster: comparison between epithelial and mesenchymal cells.

Chromosomal aberrations were investigated in tracheal cells of fetal Syrian hamsters after transplacental administration of N-diethylnitrosamine (DEN). On the 15th day of pregnancy, Syrian hamsters were injected s.c. with a tumorigenic dose of DEN (50, 100 and 200 mg/kg body weight). Two hours later, the fetal tracheae were isolated, the epithelial tissue was separated from the mesenchymal tissue by collagenase-treatment, and then each cell population was transferred to cell culture after Dispase treatment. At 24, 48 and 72 h after the cell cultivation, chromosomal damage was examined. The results clearly showed that a high incidence of chromosomal aberrations, especially chromatid-type exchanges, was seen in the epithelial cells of DEN-treated groups. However, significant induction of chromosomal aberrations was not observed in the mesenchymal cells from DEN-treated groups.

Assessment of nitrosamine transplacental carcinogenesis in the Syrian golden hamster.

The effects of diethylnitrosamine (DEN) and vinylethylnitrosamine (VEN) in the F1 generation of Syrian golden hamsters are described after the compounds were administered to the P generation during the second half of pregnancy. Morphologic findings revealed that the upper respiratory tract (i.e., trachea) was most affected. VEN appeared highly effective because the first neoplastic alterations (papillary polyps) were seen in both the P and the F1 animals at the 30th postnatal day. The fetal tracheal epithelium also showed chromosomal aberrations after DEN exposure, and VEN enhanced the cloning efficiency of these cells. Combined in vivo - in vitro studies are discussed with special emphasis on the early detection of transformation and neoplastic growth.

[Scanning electron microscopical investigations on the respiratory epithelium of the Syrian golde4n hamster. V. Dose-related in vitro effects of benzo(a)pyrene].

The biological effects of in vitro exposure of fetal hamster tracheal explants to three dose levels of benzo(a)pyrene (BaP:25, 50, 100 microgram/ml medium) in 0.5% dimethylsulphoxide (DMSO) were studied by light and scanning electron microscopy. A dose-related difference was seen in a quality of the observed pathological alterations in the specimens examined after 28 days of culture. Whilst in the explants treated with 50 or 100 microgram/ml BaP the respiratory epithelium revealed necrotic areas and extended metaplastic alterations, explants exposed to 25 microgram/ml BaP showed normal differentiation in some areas of the respiratory epithelium. In explants treated with the highest dose (100 microgram/ml BaP), hyperplastic foci were seen in the epithelium and tended to proliferate towards the tracheal lumen. These observations show similarity with early neoplastic alterations.

Scanning electron microscopical investigations on the respiratory epithelium of the Syrian golden hamster. IV. In vitro effects of dimethylsulphoxide and benzo(a)pyrene.

The biological effects of in vitro exposure of cultured fetal hamster tracheal epithelium to benzo(a)pyrene in 0.5% dimethylsulphoxide (BaP: 5 and 10 micrograms/ml medium) were studied by light and scanning electron microscopy. The solvent, DMSO, alone induced a slight, temporary retardation in development and differentiation of cilia. At day 28 of culture, however, differences in growth and maturity disappeared between DMSO-exposed and untreated explants. No dose-related differences found in the surface morphology of alterations were seen in BaP-treated tracheae; the explants revealed metaplastic foci and reduced, incomplete ciliogenesis compared to DMSO- and non-treated controls. Towards the end of the culture period, ciliogenesis tended slowly to replace epithelial alterations.

Scanning electron microscopical investigations on the respiratory epithelium of the Syrian golden hamster: transplacental effects of vinylethylnitrosamine.

The transplacental effect of vinylethylnitrosamine (VEN) on the respiratory epithelium of Syrian golden hamsters was tested after administration of 5 and 10 mg VEN/kg b.w. dissolved in olive oil at the 12th or 15th day of gestation. The morphological data, obtained mainly by scanning electron microscopy on tracheae revealed metaplastic and hyperplastic epithelial changes, as well as focal exophytic papillary proliferations at the 30th postnatal day in the group treated at the 15th day of gestation. No comparable lesions were found in the trachea of the F1 generation when mothers were treated with VEN at day 12 of pregnancy or in animals of the control group (olive oil only). The biological effects of VEN after transplacental exposure were compared to those of other nitrosamines.

Chromosomal aberrations in tracheal epithelial cells of the fetal Syrian golden hamster after transplacental N-diethylnitrosamine administration.

Transplacental effect of N-diethylnitrosamine (DEN) on chromosomal morphology, was investigated in fetal tracheal epithelium of Syrian hamsters. At the 15th day of pregnancy, Syrian golden hamsters were injected subcutaneously with a tumorigenic dose of DEN (50, 100 and 200 mg/kg body wt). Two hours later, the fetal tracheae were isolated, epithelial cells of the respiratory mucosa were separated from mesenchymal tissue and were transferred to cell culture. At 24, 48 and 72 h after cultivation, chromosomal damages were examined. The results showed clearly that a high incidence of chromosomal aberrations, particularly chromatid-type exchanges, were seen in the epithelial cells from DEN-treated groups.

A fetal respiratory epithelial cell line for studying some problems of transplacental carcinogenesis in Syrian golden hamsters.

Using repeated cloning and treatment with cis-HPL (200 micrograms/ml), an analogue of a procollagen precursor inhibitory to the growth of collagen-synthesizing cells of mesenchymal origin, clonally premature epithelial cell lines were isolated from fetal SGH lungs cultured on the 15th day of gestation. One of the cell lines, M3E3/C3, which has been extensively studied for biological characterization, developed poorly differentiated carcinomas in injected hamsters after transformation by MNNG. Moreover, when grown on collagen gel, this cell line indicated an obvious potency for in vitro differentiation in response to vitamin A by developing activated Golgi regions, well developed rER and a number of mucus-like granules. Since such a differentiative responses is expected to be definable in the light of respiratory epithelium developing in utero, this cell line may be useful for studying mechanisms of differentiation-dependent sensitivity of fetal organs to transplacental carcinogen exposure.

Scanning electron microscopical investigations on the respiratory epithelium of the Syrian golden hamster III. Regeneration after traumatic injury.

To investigate the extent to which intratracheal intubation may alter the respiratory epithelium of the Syrian golden hamsters, single and repeated intubations were undertaken and the resulting injury and subsequent epithelial regeneration were examined by scanning electron microscopy. Generally, epithelial injury as a result of a single intubation had healed ad integrum within 20 to 40 days. On the other hand, repeated treatment often caused tracheitis and led to prolonged regeneration which sometimes persisted as papillary hyperplasia 40 days after the final intubation. The appropriateness of intratracheal instillation as a method of administering chemical carcinogens and the similarity of the epithelial regeneration processes to early neoplastic alterations of the epithelium are discussed.