How to develop globular proteins into adhesives.

To make globular proteins suitable for application in adhesives, the specific bonds and interactions which shape their structure have to broken. Only then, a layer of relatively large, flexible and interwoven polymer chains, which are firmly attached to the solid surface by adsorption, can be created. Such a network layer is essential to save the adhesive bond under an applied force, because it can distribute the concentration of stresses generated at the interface into the bulk. Unfolding and swelling of a protein can be achieved by changing the solvent quality. For the globular whey protein beta-lactoglobulin, the optimal conditions for unfolding and swelling is found with 98% formic acid as a solvent. In formic acid, beta-lactoglobulin looses its amphoteric character (it is protonated, probably for approximately 20%). In addition, formic acid is less polar than water and thus a better solvent for the apolar parts of the protein. The swelling and unfolding behaviour of beta-lactoglobulin is studied by viscosity and CD-spectroscopy measurements. For the interpretation of the results we apply the Kuhn formalism that the conformation of a protein can be described in terms of a statistical chain which consists of segments of an average persistence length P. The statistical segment length P, which varies with the experimental conditions, is directly related to the adsorption energy required for a strong adhesion between coil and surface. It determines the depletion energy kT P(-2) m(-2) which must be overcome by specific attraction between side groups of the protein chain and the surface. For beta-lactoglobulin in 98% formic acid, we find a P value of approximately 2.2 nm, pointing at a relatively flexible chain. The minimum net adsorption energy kT P(-2) is then approximately 1 mJ m(-2), a relatively small value to be exceeded. Preliminary results of destructive adhesion tests on beech wood lap-shear joints reveal promising tensile strengths of approximately 2.9+/-1.1 N mm(-2), indeed.

Comparison of five incubation systems for rat liver slices using functional and viability parameters.

Precision-cut liver slices are presently used for various research objects, e.g. to study metabolism, transport, and toxicity of xenobiotics. Various incubation systems are presently employed, but a systematic comparison between these incubation systems with respect to preservation of slice function has not been performed yet. Therefore, we started a comparative study to evaluate five of these systems: the shaken flask (an Erlenmeyer in a shaking water bath), the stirred-well (24-well culture plate equipped with grids and magnetic stirrers), rocker platform (6-well culture plate with Netwell insert rocked on a platform), the roller system (dynamic organ culture rolled on an insert in a glass vial), and the 6-well shaker (6-well culture plate in a shaking water bath). The liver slices were incubated in these incubation systems for 0.5, 1.5, and 24.5 h and subsequently subjected to viability and metabolic function tests. The viability of the incubated liver slices was evaluated by: potassium content, MTT assay, energy charge, histomorphology, and LDH leakage. Their metabolic functions were studied by determination of the metabolism of lidocaine, testosterone, and antipyrine. Up to 1.5 h of incubation all five incubation systems gave similar results with respect to viability and metabolic function of the liver slices. However, after 24 h, the shaken flask, the rocker platform, and the 6-well shaker incubation systems appeared to be superior to the stirred well and the roller incubation systems.

Effects of saponins and glycoalkaloids on the permeability and viability of mammalian intestinal cells and on the integrity of tissue preparations in vitro.

The effects of potato and tomato glycoalkaloids and a saponin mixture from Gypsophila were investigated in cytotoxicity studies (neutral red uptake, mitochondrial MTT reduction and release of lactate dehydrogenase), using cultured cell lines of rat and human intestinal mucosal epithelium. Experiments to assess the effects of these compounds on the integrity of the intestinal epithelium were also carried out using preparations of isolated rat jejunum in vitro. By investigating the effect of these compounds on cultured cells and on intestinal tissue preparations, changes in membrane integrity, as evidenced by lactate dehydrogenase leakage in cell culture, could be confirmed in a system more relevant to the whole gut. Of the compounds tested, alpha-tomatine was consistently the most potent in all tests, and indications of a synergistic effect on membrane depolarization were observed between alpha-chaconine and alpha-solanine at total glycoalkaloid concentrations of less than 1 mM (< 0.86 mg/ml), with an optimum when the former comprised 25% of the mixture. An increase in the apparent permeability of the brush border was observed at sublethal concentrations of the compounds, and this may have important implications with respect to enhanced uptake of macromolecules, such as allergens, whose passage through the epithelium is normally somewhat restricted.

Influence of growth factors and medium composition on benzo[a]pyrene- and vitamin A-induced cell proliferation and differentiation in hamster tracheal epithelium in organ culture.

Tracheal organ cultures and isolated tracheal epithelial cells are frequently used to study effects of carcinogens and retinoids on both proliferation and differentiation of respiratory tract epithelial cells. For each of these in vitro models, optimal culture conditions have been established, varying in type of culture medium and composition of growth factor and hormone supplementation, which by themselves may influence cellular proliferation and differentiation. In this study, we investigated the influence of medium composition and growth factor supplementation on the effect of benzo[a]pyrene (B[a]P) and vitamin A on cellular proliferation and differentiation in hamster tracheal epithelium in organ culture. In tracheae cultured in Ham's F12 medium, cell proliferation was decreased by B[a]P relative to untreated controls, whereas vitamin A in combination with B[a]P increased cell proliferation compared with that in tracheae treated with B[a]P alone. The effects in tracheae cultured in CMRL-1066 medium were just the opposite: B[a]P increased cell proliferation and vitamin A decreased B[a]P-induced proliferation. To explain this difference in cell proliferation, the effects of various growth factors (epidermal growth factor and transferrin) and medium components (nucleotides, NAD(+)/NADP and CaCl(2).2H(2)O) on B[a]P and vitamin A-induced cell proliferation were investigated. The main factor responsible for the different effects on cell proliferation appeared to be the concentration of Ca(2+) in the culture medium; addition of CaCl(2).2H(2)O to Ham's F12 medium resulted in effects of B[a]P and vitamin A on cell proliferation comparable with those observed in tracheae cultured in CMRL-1066 medium. These results clearly show that the composition of the culture medium, and particularly the concentration of Ca(2+), strongly influences the effect of vitamin A and B[a]P on cell proliferation in hamster tracheal epithelium in organ culture.

Cell proliferation and DNA adducts in hamster tracheal epithelium exposed to benzo[a]pyrene in organ culture.

The effect of benzo[a]pyrene (B[a]P) on cell proliferation in cultured hamster tracheal epithelium was studied in relation to the formation of B[a]P-DNA adducts. To this end, tracheae were isolated from Syrian golden hamsters, cut into rings and cultured in Ham's F12 medium. Then, the tracheal rings were exposed to 2 or 20 mumB[a]P, either continuously for 7 days or for 2 days followed by a 5-day recovery period without B[a]P. At intervals rings were sampled for determination of cell proliferation (by means of the labelling index). In addition, B[a]P-DNA adduct levels were determined in the continuous exposure experiment by means of in situ detection by immunofluorescence microscopy. After 2 days of exposure to 2 or 20 mum B[a]P, there was a significant increase in B[a]P-DNA adduct level. A further, linear increase in B[a]P-DNA adduct level, however, was only observed after continuous exposure to 20 mum B[a]P, whereas the adduct level in the 2 mum continuous exposure group remained virtually the same. In unexposed tracheal epithelium an initial peak of cell proliferation was observed. This initial proliferation was significantly lower in the exposed samples. Only continuous exposure to 20 mum B[a]P steadily decreased the labelling index from day 2 to 7. It is concluded that the increase in B[a]P-DNA adduct level is correlated with the reduction of cell proliferation in hamster tracheal epithelium exposed to B[a]P in Ham's F12 medium.

Epidermal cell proliferation and terminal differentiation in skin organ culture after topical exposure to sodium dodecyl sulphate.

Epidermal cell proliferation and differentiation were investigated in vitro after exposure to the anionic surfactant sodium dodecyl sulfate (SDS). Human skin organ cultures were exposed topically to various concentrations of SDS for 22 h, after which the irritant was removed. Cell proliferation was measured immunohistochemically by incorporation of bromodeoxyuridine (BrdU) into the DNA of cells during S-phase, while the expression of transglutaminase and involucrin were used as markers of differentiation. Cell proliferation was moderately increased at concentrations of SDS that did not affect the histomorphology (0.1% and 0.2% SDS). A marked increase of cell proliferation was observed 22 to 44 h after removal of SDS at a concentration (0.4%) that induced slight cellular damage. Exposure of human skin organ cultures to a toxic concentration of SDS 91.0% led to decreased cell proliferation. Transglutaminase and involucrin were expressed in the more basal layers of the epidermis after exposure to 0.4% or 1.0% SDS. Moreover, intra-epidermal sweat gland ducts were positive for transglutaminase at these irritant concentrations. These in vitro data demonstrate that SDS-induced alterations of epidermal cell kinetics, as described in vivo are at least partly due to local mechanisms and do not require the influx of infiltrate cells. However, we were unable to relate to altered cell kinetics to the release of interleukin-1 alpha or interleukin-6. Furthermore, supplementation of the culture medium with 12-hydroxyeicosantetraenoic acid did not affect epidermal cell proliferation. Rabbit skin cultures appeared more sensitive to SDS than human skin. At nontoxic doses, the irritant induced an increase of epidermal cell proliferation, similar to that observed in human skin discs.

Expression of insulin-like growth factor II in spontaneously immortalized rat mesothelial and spontaneous mesothelioma cells: a potential autocrine role of insulin-like growth factor II.

Insulin-like growth factors (IGFs) are polypeptides that play an important role in cellular proliferation and differentiation. The present study examines the role of IGFs in the growth of mesothelial cells. Cell lines derived from normal rat mesothelium as well as lines derived from spontaneous rat mesotheliomas were found to express RNA transcripts for IGF-II. In contrast, cell lines derived from asbestos-induced rat mesotheliomas did not express this growth factor. All cell lines expressed receptors for IGF-I and IGF-II, as well as insulin receptors. Coexpression of IGF-II and its cognate receptor suggested that IGF-II was acting as an autocrine growth factor in the spontaneously immortalized cells and the cells derived from the spontaneous tumors. The biological activity of IGF-II secreted by the cell lines into conditioned medium could be neutralized using an IGF-II-specific antibody. Growth was inhibited in a dose-dependent manner; at the highest antibody concentration used (100 micrograms anti-IGF-II/ml), cell growth was decreased to 47% of control values. This inhibition was partially reversible by treatment of the cultures with IGF-II (91% of the control). These data suggest that IGF-II expression may be involved in the spontaneous alteration of rat mesothelial cells and may function as an autocrine or paracrine growth factor to modulate the growth of these cells in vitro and in vivo. Ubiquitous expression of IGF-II by cells that have not been exposed to asbestos and the lack of IGF-II expression by asbestos-transformed cells suggest that the mechanisms of changes in growth factor expression differ in mesothelial cells transformed by different pathways.

Relation between benzo[a]pyrene-DNA adducts, cell proliferation and p53 expression in tracheal epithelium of hamsters fed a high beta-carotene diet.

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.

Vitamin A and beta-carotene influence the level of benzo[a]pyrene-induced DNA adducts and DNA-repair activities in hamster tracheal epithelium in organ culture.

Although most studies concerning the effect of vitamin A and beta-carotene on chemical carcinogenesis are focused on tumour promotion and progression, these compounds may affect initiation as well. In this study the influence of vitamin A and beta-carotene on unscheduled DNA synthesis (UDS) was investigated in hamster tracheal epithelium in organ culture exposed to benzo[a]pyrene (B[a]P). DNA-repair activities were compared with the level of B[a]P-DNA adducts as measured both by 32P-postlabeling and by immunocytochemical detection. In hamster tracheal epithelial cells, both vitamin A and beta-carotene significantly increased B[a]P-induced UDS, with 40% and 45%, respectively. At the same time, vitamin A and beta-carotene decreased the level of B[a]P-DNA adducts in these cells with 18% and 40%, respectively as measured by 32P-postlabeling and with 12% and 35%, respectively as measured by immunocytochemistry. The effect of vitamin A on B[a]P-induced UDS and DNA-adduct levels in hamster tracheal epithelium appeared to depend on the dose of B[a]P vis-à-vis the concentration of vitamin A. The results of the present study show that both vitamin A and beta-carotene cause a decrease in B[a]P-DNA adduct levels by enhancing DNA-repair activities. Because the formation of B[a]P-DNA adducts is considered to be an early step in respiratory tract carcinogenesis, it is suggested that enhancement of DNA-repair activities by vitamin A and the subsequent removal of DNA adducts may be one of the mechanisms involved in vitamin A-mediated protection against cancer.

Release of arachidonic and linoleic acid metabolites in skin organ cultures as characteristics of in vitro skin irritancy.

In vitro techniques make a major contribution to the development of alternatives to the in vivo "Draize" skin irritation test, and the development of sensitive and generally applicable in vitro endpoints of cutaneous toxicity is an area of intensive research. To investigate in vitro characteristics of cutaneous irritation, skin explants of rabbit and human origin were topically exposed to chemical irritants, after which the culture medium was analyzed for the presence of metabolites of both arachidonic and linoleic acid. In rabbits exposed to the potent irritant benzalkonium chloride, a direct relation was established between clinical signs of irritation and in vitro release of the proinflammatory mediator 12-hydroxyeicosatetraenoic acid (12-HETE) by the exposed skin. Histological examination revealed varying degrees of epidermal damage. 12-HETE was also the predominant hydroxy fatty acid released in a dose-dependent way by rabbit skin cultures after in vitro exposure to sodium dodecyl sulfate (SDS), benzalkonium chloride (BC), and formaldehyde (FA). Human skin cultures released, in addition to 12-HETE, predominantly 15-HETE and 13-hydroxyoctadecadienoic acid (13-HODE), omega-6 oxygenase products of arachidonic acid and linoleic acid, respectively. The irritant-induced release of hydroxy fatty acids was strongly inhibited by the lipoxygenase inhibitor eicosatetraynoic acid, indicating enzyme-mediated generation of these bioactive lipids. Comparison of hydroxy fatty acid release to more established markers of cytotoxicity (leakage of the cellular enzymes, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH)) revealed that increased levels of 13-HODE, 9-HODE, 12-HETE, and ALT were specific markers of cutaneous irritancy in rabbit skin cultures.

A critical appraisal of intratracheal instillation of benzo[a]pyrene to Syrian golden hamsters as a model in respiratory tract carcinogenesis.

Several experimental models have been developed to study respiratory tract carcinogenesis. The most widely applied in vivo model uses Syrian golden hamsters which receive intratracheal instillations of a suspension of benzo[a]pyrene (B[a]P) particles attached to ferric-oxide (Fe2O3) particles in saline; it was first described by Saffiotti et al. [1]. This model has several benefits compared with other experimental models; however, the large number of variables affecting the tumour response is a clear disadvantage because the tumour response is difficult to control. In this review, we describe a systematic analysis of various variables that may influence the tumour response of the respiratory tract with the aim to further standardize the method and increase, through that, its suitability and predictability. The most important variables influencing the tumour response, as shown by statistical analysis of 29 representative studies, turned out to be the administered dose and the particle size. Both these variables influence the actual dose and the contact-time of the B[a]P particles with the target cells. The present study does not support the widespread opinion that ferric-oxide particles enhance the tumour response of the respiratory tract. In conclusion to the present analysis, some recommendations are made which probably increase the predictability of the model.

Benzo[a]pyrene-induced respiratory tract cancer in hamsters fed a diet rich in beta-carotene. A histomorphological study.

The effect of a high dietary level of beta-carotene on the formation of preneoplastic and neoplastic respiratory tract lesions was studied in hamsters intratracheally treated with benzo[a]pyrene (B[a]P) attached to ferric oxide (Fe2O3) and suspended in saline. In addition to conventional histopathological examinations, the expression of cytokeratins and the glutathione S-transferase isoenzyme Pi (GST-Pi) was determined in tracheal epithelium using immunocytochemical techniques. B[a]P treatment increased the expression of cytokeratins in tracheal mucous and ciliated epithelial cells as detected by antibody RCK102 (cytokeratins 5 and 8), which normally recognizes basal cells only. The expression of cytokeratins in mucous and ciliated cells as detected by antibody RGE53 (cytokeratin 18) was decreased by B[a]P treatment. Furthermore, the expression of the cytokeratin detected by antibody RKSE60 (cytokeratin 10), characteristic of metaplastic squamous cells, and the expression of the GST-Pi, characteristic of metaplastic changes, was increased in trachael epithelium of hamsters treated with B[a]P. There was no evidence for dietary beta-carotene affecting the expression of cytokeratins or GST-Pi. The incidence of preneoplastic changes and tumors of the respiratory tract was not reduced by dietary beta-carotene. On the contrary, the tumor response of the respiratory epithelium was almost twice as high in hamsters fed the high beta-carotene diet than in hamsters on the low beta-carotene diet. However, this difference was not statistically significant (P = 0.15); hence, the present study did not produce evidence for a clear effect of beta-carotene on B[a]P-induced respiratory tract cancer in hamsters.

Differential effects of chemical irritants in rabbit and human skin organ cultures.

The toxicity of well known irritants was investigated in rabbit and human skin organ cultures. Test chemicals were selected from various categories of irritants and included both water-soluble and water-insoluble compounds. Using a highly standardized protocol, test chemicals were applied topically at concentrations relevant to the in vivo situation. Toxicity was assessed by histomorphological examination, inhibition of conversion of the tetrazolium salt MTT, inhibition of epidermal cell proliferation and release of pro-inflammatory hydroxy fatty acids. Chemicals that are known to induce irritation in vivo invariably caused histopathological changes and inhibition of cell proliferation, whereas non-irritating chemicals did not; however, inhibition of MTT conversion and release of hydroxy fatty acids occurred with only a limited number of irritants. The response to the chemical irritants was different in rabbit and human skin cultures. Rabbit skin was slightly more sensitive to sodium dodecyl sulfate and benzalkonium chloride than human skin. Moreover, mineral oil enhanced epidermal cell proliferation in rabbit skin, but not in human skin. This study demonstrates that chemical irritants cause substance- and species-specific effects in skin organ cultures. It is therefore unlikely that irritant potential of chemicals from all irritant categories can be detected in vitro using one single parameter. A multiple endpoint approach and the inclusion of human tissue are recommended for optimal in vitro irritancy testing of chemicals.

Cytotoxicity of retinoic acid, menadione and aflatoxin B(1) in rat liver slices using Netwell inserts as a new culture system.

Precision-cut rat liver slices were used to develop a new dynamic incubation system in which histomorphology and measurement of the release of lactate dehydrogenase (LDH) and the conversion of MTT were applied to evaluate cytotoxicity. Liver slices, precision-cut using a Krumdieck tissue slicer, were cultured in a new system using 200-mum polyester mesh Netwell inserts in six-well cell-culture clusters on a rocker platform at 37 degrees C and 40% O(2). The major advantage of this new culture system is the easy way in which slices can be manipulated and the culture medium be sampled or changed. Rat liver slices were exposed for 4 hr to retinoic acid (RA), menadione or aflatoxin B(1) (AFB(1)). Directly after treatment and after an additional 20-hr recovery period, histomorphological observations of slices were made, and LDH release and MTT conversion were measured. Slices exposed to RA showed dose-related cytotoxicity in the MTT assay only. The cytotoxic response to AFB(1) was more pronounced in the assay of LDH release than in the MTT assay. Histomorphology, LDH release and the MTT assay revealed cytotoxic effects induced by menadione. We conclude that culturing liver slices using Netwell inserts is a good alternative to other culture systems for testing non-volatile compounds.

Mesothelial cell proliferation induced by intrapleural instillation of man-made fibers in rats and hamsters.

Long-term inhalation exposure to a biopersistent man-made ceramic fiber (RCF 1) results in a high incidence of pleural mesotheliomas in Syrian golden hamsters but not in identically exposed rats. To understand better the mechanisms involved in the intraspecies pathobiology of fiber-exposed mesothelium, the ability of the two different man-made fibers to induce cell proliferation in hamster and rat pleural mesothelial cells was investigated. Three dose levels of either glass fibers (MMVF 10) or ceramic fibers (RCF 1) were instilled intrapleurally into male Fischer 344 rats and male Syrian Golden hamsters. Rats and hamsters were exposed to approximately equal numbers of long thin fibers per kilogram of body weight using a single intrapleural instillation. Bromodeoxyuridine (BrdU) was administered via an implanted osmotic pump, and mesothelial cell proliferation was assessed at 7 and 28 days postinstillation (PI) using immunocytochemical visualization of labeled S-phase cells. Both rats and hamsters exhibited dose-dependent increases in proliferation of pleural mesothelial cells following exposure to both fiber types. Interspecies differences in mesothelial cell proliferation were noted for fiber type and pleural site. At 28 days PI, RCF-induced mesothelial cell proliferation was found to be more pronounced in hamsters than in rats in the caudal visceral pleural. Comparing both fibers either by equal mass or by equal fiber numbers, mesothelial cell proliferation in RCF 1-treated animals was higher than in animals exposed to MMVF 10, especially in hamsters, and may be a factor in the difference in mesothelioma induced by the two fibers. The higher sustained (28 day) mesothelial cell proliferation in the visceral pleural of hamsters exposed to RCF may contribute to the species-specific differences in mesothelioma incidence found in long-term rodent inhalation studies.

Pleural lesions in Syrian golden hamsters and Fischer-344 rats following intrapleural instillation of man-made ceramic or glass fibers.

The mesothelium is a target of the toxic and carcinogenic effects of certain natural mineral and man-made fibers. Long-term inhalation of a ceramic fiber (RCF-1) results in a high incidence of pleural mesotheliomas in Syrian golden hamsters but not in identically exposed Fischer-344 rats. The present study compared the histopathology of the early pleural response in rats and hamsters instilled with artificial fibers. Groups of Syrian golden hamsters and Fischer-344 rats were instilled with ceramic (RCF-1) or glass (MMVF-10) fibers directly into the pleural space. Each species received approximately equal numbers of long, thin fibers per g body weight. Fiber-induced lesions were compared 7 and 28 days postinstillation. Both hamsters and rats developed qualitatively similar dose-dependent inflammatory lesions that were not fiber-type specific. Both species developed fibrosis in conjunction with inflammation in the visceral pleura, but a striking interspecies difference was noted in the pattern of mesothelial cell response. Hamsters developed greater surface mesothelial cell proliferation and had focal aggregates of mesothelial cells embedded deep within regions of visceral pleural fibrosis. It is hypothesized from the present study that the marked fiber-induced proliferative mesothelial cell response of the hamster visceral pleura may explain the high number of pleural mesotheliomas found in long-term fiber studies in this species.

DNA adduct formation and repair in hamster and rat tracheas exposed to benzo[a]pyrene in organ culture.

Syrian golden hamsters are much more susceptible than Wistar rats to the induction of tracheal tumors by benzo[a]pyrene (B[a]P). To investigate whether this difference is reflected in the pattern of DNA adduct induction and removal, tracheas from either species were isolated and exposed to B[a]P (5 micrograms/ml) in organ culture. At various time-points B[a]P-DNA adducts were quantified by 32P-postlabeling; unscheduled DNA synthesis (UDS) and cell proliferation were determined by [3H]thymidine incorporation during the 18 h before sampling. In an induction-repair experiment tracheas were exposed to B[a]P for 2 days, and cultured for another 4 days without B[a]P. After 2 days of exposure total B[a]P-DNA adduct levels were 10 times higher in hamster compared to rat tracheas. In hamster tracheas one major adduct was formed (95%), namely the adduct between (+)-anti-benzo[a]pyrene diolepoxide and deoxyguanosine (BPDE-N2dG). In rat tracheas BPDE-N2dG comprised approximately 60% of the total B[a]P-DNA adduct level. The other major adduct found in rat tracheas is probably derived from interaction of syn-BPDE and deoxyadenosine. During exposure to B[a]P in hamsters the adduct level increased to 36 +/- 19 adducts/10(6) nucleotides (add/10(6)n) on day 2. Two days after removal of B[a]P the B[a]P-DNA adduct level had decreased to 60% of that on day 2; there was no further decrease in the B[a]P-DNA adduct level, despite considerable cell proliferation at the end of the 6 day culture period. UDS increased during exposure to B[a]P and decreased after removal of B[a]P. In rats removal of B[a]P did not lead to a decrease in the B[a]P-DNA adduct level, which agreed with the observed absence of UDS. In a second experiment tracheas were exposed to B[a]P continuously for 15 days. In hamster tracheas the total B[a]P-DNA adduct level increased from 11 +/- 0.7 add/10(6)n after 1 day of exposure to 105 +/- 2 add/10(6)n after 15 days; also UDS increased with increasing exposure until day 11. Cell proliferation was low at the end of the culture period. In rat tracheas no progressive increase in the B[a]P-DNA adduct level was seen, UDS was not increased and cell proliferation had increased significantly at the end of the exposure period. The extent of adduct induction in the trachea of the two species corresponded with the different susceptibilities to B[a]P-induced tumor formation.

High survival rate of hamsters given intratracheal instillations of benzo[a]pyrene and ferric oxide and kept on a high beta-carotene diet.

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.

In vitro dermal toxicology using skin organ cultures.

In order to reduce animal discomfort and to obtain more quantitative endpoints, there is a need for reliable, preferably simple and inexpensive in vitro alternatives to skin toxicity testing. An in vitro model was developed in which full-thickness skin from various species can be cultured (rabbit, pig and human). Subsequent to topically applied test compounds, parameters of dermal toxicity were investigated, including cytotoxicity (MTT assay) and the release of inflammatory mediators (HETE's). Moreover, percutaneous absorption and concurrent biotransformation of compounds was studied. MTT conversion was inhibited in a dose-dependent manner following topical application of a wide range of irritants. Repair of initial damage was observed to some extent. The eicosanoid 12-HETE, which is thought to play an important role in chemotaxis, is released. Interestingly, the anti-inflammatory mediator 15-HETE was released only after a prolonged culture time of 48 hr, possibly indicating repair of the induced damage. The metabolic fate of the pesticide propoxur was investigated. Permeation rates were comparable in human and rabbit skin, while pig skin was found to be twice as permeable. Extensive cutaneous metabolism was observed in all three species.

Effect of coffee drinking on cell proliferation in rat urinary bladder epithelium.

A possible effect of freshly brewed drip coffee on urinary bladder carcinogenesis was investigated in male Wistar rats using cell proliferation in urinary bladder epithelium as the indicator of tumour promotion. Male rats were given either undiluted coffee brew (100% coffee), coffee diluted 10 times (10% coffee) or tap water (controls), as their only source of drinking fluid for 2 or 6 wk. Uracil, known to induce cell proliferation in urinary bladder epithelium, was included in the study as a positive control. In rats receiving 100% coffee, body weights, liquid intake and urinary volume were decreased. Neither histopathological examination of urinary bladder tissue nor the bromodeoxyuridine labelling index revealed biologically significant differences between rats receiving coffee and the tap water controls. Uracil increased the labelling index and induced hyperplasia of the urinary bladder epithelium, as expected. It was concluded that these results produced no evidence that drinking coffee predisposes to tumour development in the urinary bladder.