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Estrogen-induced pituitary lesions in rats were studied in time-sequence experiments using magnetic resonance imaging (MRI), hormone determinations and light microscopy. The main purpose of the study was to evaluate the usefulness of MRI in comparison with conventional biochemical and histopathological methods for detecting the pituitary lesions as early as possible and to follow their development. Measurements were made at 15 time points, ranging from 1 h to 272 days after s.c. implantation of the estrogen pellet. High-resolution T1 weighted sagittal images with 2 mm slice thickness were made with a 2 Tesla 30 cm small-bore MRI system. Radioimmunoassay (RIA) was used to determine the different pituitary hormones. Convential histopathology and immunoperoxidase staining methods were used to characterize the pituitary lesions and visualize the hormone-producing pituitary cells respectively. The first histopathological pituitary changes (enlarged acidophilic cells with increased number of vacuoles) were seen at day 2 after initiation of the estrogen treatment, while at day 4 the first immunohistochemical changes (increased number of prolactin-positive cells) were encountered. Significantly increased prolactin levels in blood plasma occurred from day 9 onwards. Also at day 9, changes of the pituitary gland were first visible on MR images, showing rounding of the anterior edge of the gland. Gradual enlargement of the pituitary caused by hyperplasia of hypertrophic prolactin-positive cells could be followed by MRI, and later on pituitary tumors were recognized, their images being heterogeneous due to great differences in signal intensity ranging from hypo- or iso- to hyperintense. Signal intensities of hemorrhagic tumor areas varies widely due to variation in the blood flow maintained in these areas. It was concluded that MRI is a powerful tool for detecting enlargement and tumors of the pituitary gland in rats. This method allows the development of such lesions to be followed in one and the same animal, thereby reducing the need of interim kills and thus the number of animals to be used. Chemicals/CAS: prolactin, 12585-34-1, 50647-00-2, 9002-62-4; Estradiol, 50-28-2; Estrogens; Pituitary Hormones

Vitamin A and beta-carotene protect against respiratory tract cancer by inhibiting the formation of DNA damage and controlling cellular proliferation and differentiation. Recently, it has been shown that the p53 tumor-suppressor gene plays a crucial role in the etiology of respiratory tract cancer. In the present study, we investigated the relationship between benzo[a]pyrene (B[a]P)-DNA adducts, cell proliferation and p53 expression and the possible effect of beta-carotene on such a relationship in tracheal epithelium of hamsters given intratracheal instillations of B[a]P-Fe2O3 particles suspended in saline. DNA-adducts were quantified by the 32P-postlabeling assay, cell proliferation was quantified by immunocytochemical detection of incorporated BrdU during S-phase, and p53 protein was detected by immunohistochemistry with an antibody that recognized both the wild-type and the mutated protein (BioGenex, Clone BP53-12-1). A clear relationship appeared to exist between the extent of B[a]P-DNA adduct formation, the induction of cell proliferation and the expression of p53 protein in hamster tracheal epithelium. These results suggest that B[a]P induces cell proliferation in hamster tracheal epithelial cells most likely by the induction of mutations in the p53 gene. Furthermore, beta-carotene was not found to influence the formation of B[a]P-DNA adducts, which is probably due to the high B[a]P dose. Moreover, beta-carotene did not statistically significantly affect cell proliferation and p53-protein expression in hamster tracheal epithelial cells.

The present paper reports about an immunocytochemical inventory of the cell types involved in the metabolic activation of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to a DNA methylating metabolite. The formation and distribution of the methylated DNA bases O6-methylguanine (O6-meGua) and 7-methylguanine (7-MeGua) were studied in respiratory tissues, oesophagus, liver, kidneys, pancreas, small intestine, colon and prostate of rat, mouse and hamster 6 h after treatment with a single dose of 30 mg NNK/kg. The tissue- and cell-specific distribution of O6-meGua- and 7-meGua-specific nuclear staining showed the same patterns and were remarkably similar in rat, mouse and hamster in spite of the diverging spectra of NNK-induced tumours in these species. In nasal tissue, a target for NNK-induced tumourigenesis in rat and hamster, but not in mouse, adduct-specific nuclear staining was observed in all three species in sustentacular cells, Bowman glands, respiratory epithelial cells and serous glands. Both methylated DNA bases were also observed in basal cells of the olfactory epithelium of rat and (occasionally) hamster, but not in those of the mouse. In the trachea, a target for NNK-induced tumourigenesis in hamster only, substantial adduct-specific nuclear staining was found in basal epithelial and glandular cells of the hamster; in the same cells of rat and mouse only a weak nuclear staining was found. In the lung, a common target for NNK-induced tumourigenesis, the formation of O6-meGua and 7-meGua was restricted predominantly to bronchial and proximal bronchiolar epithelium. Nuclear staining in the rat was occasionally found in alveolar cells and was also observed in hepatocytes. In the three species investigated, O6-meGua- and 7-MeGua-specific nuclear staining was found in target and non-target tissues. Apparently, and in analogy with results obtained in other studies, the species-specific organotropy for tumour formation of NNK is not exclusively determined by DNA methylation. Expanding methylation data with literature data on factors considered to be involved in tumour formation, namely proliferation, toxicity and DNA repair among others, still did not lead to a satisfactory explanation for the species-specific organotropy observed. Additional factors (yet to be identified), need to be taken into account in order to explain (and predict) tumourigenic effects induced by monofunctional methylating agents.

The study described in this paper was primarily conducted to identify the cell types involved in the formation, progression and regression of metaplastic changes in the respiratory tract epithelium of hamsters after intratracheal intubations with benzo[a]pyrene. Furthermore, the role of vitamin A and beta-carotene in these processes was studied. In the course of the study a remarkable effect of dietary beta-carotene on survival of hamsters became a subject of investigation. Hamsters were fed diets with various levels of vitamin A or beta-carotene and were treated intratracheally with a suspension of benzo[a]pyrene with ferric oxide in saline. The tumour response of the respiratory tract was very low (2.8%) and hyper- and metaplasia of respiratory epithelium were virtually absent. However, an interesting observation was an exceptionally low mortality of only 2% after 69 weeks in the group of hamsters fed a high beta-carotene diet (1% w/w), whereas in the other groups mortality after 69 weeks amounted to 25%. Although the exact cause of death of most of the hamsters could not be established, a 40% reduction of lipid peroxidation in the livers was found in the high beta-carotene group. Moreover, in this group the degree and incidence of nephrosis and of focal mineralization of kidneys and heart were lower than in the other groups. These favourable effects of the high beta-carotene diet may have contributed to the unusually high survival rate in hamsters fed this diet. Further studies are planned to verify and study this observation.

The effect of estrogen withdrawal on pituitary glands of rats treated with estradiol-17 beta for various lengths of time was monitored by magnetic resonance imaging (MRI) and histological examination. Estrogen pellets were removed at seven different time points ranging from 4 to 206 days after pellet implantation. High-resolution mid-sagittal MR images of the rat head were made 1 day before pellet implantation, immediately following pellet withdrawal, and 14 and 28 days after pellet withdrawal. Twenty-eight days after pellet withdrawal pituitary glands were fixed and processed for histological examination. Enlarged pituitaries were detected by MRI from 16 days after implantation and onwards. Twenty-eight days after estrogen withdrawal the typical triangular shape of the normal pituitary had returned and pituitary morphology was indistinguishable from that of normal pituitaries in all rats that had been treated with estrogen for up to 114 days. Pituitaries of rats that had received estrogen for 186 days had a normal MR image 28 days after estrogen withdrawal, but microscopic examination revealed multifocal hyperplasias of prolactin-positive cells throughout the pars distalis. MRI of rats treated for 206 days showed tumorously enlarged pituitaries. There was no evidence of tumor regression in these rats 28 days after pellet removal. It was concluded that hypertrophic pituitaries regained a normal size, shape and morphology after estrogen withdrawal, except for a remarkable type of hyperplasia following estrogen treatment for 186 days and a recovery period of 28 days. In tumorous pituitaries no regression of lesions was noticed.