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Apoptotic and antiproliferative activities of small heterodimer partner (SHP) nuclear receptor ligand (E)-4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC), which was derived from 6-[3'-(1-adamantyl)-4'-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN), and several carboxyl isosteric or hydrogen bond-accepting analogues were examined. 3-Cl-AHPC continued to be the most effective apoptotic agent, whereas tetrazole, thiazolidine-2,4-dione, methyldinitrile, hydroxamic acid, boronic acid, 2-oxoaldehyde, and ethyl phosphonic acid hydrogen bond-acceptor analogues were inactive or less efficient inducers of KG-1 acute myeloid leukemia and MDA-MB-231 breast, H292 lung, and DU-145 prostate cancer cell apoptosis. Similarly, 3-Cl-AHPC was the most potent inhibitor of cell proliferation. 4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorophenyltetrazole, (2E)-5-{2-[3'-(1-adamantyl)-2-chloro-4'-hydroxy-4-biphenyl]ethenyl}-1H-tetrazole, 5-{4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorobenzylidene}thiazolidine-2,4-dione, and (3E)-4-[3'-(1-adamantyl)-2-chloro-4'-hydroxy-4-biphenyl]-2-oxobut-3-enal were very modest inhibitors of KG-1 proliferation. The other analogues were minimal inhibitors. Fragment-based QSAR analyses relating the polar termini with cancer cell growth inhibition revealed that length and van der Waals electrostatic surface potential were the most influential features on activity. 3-Cl-AHPC and the 3-chlorophenyltetrazole and 3-chlorobenzylidenethiazolidine-2,4-dione analogues were also able to inhibit SHP-2 protein-tyrosine phosphatase, which is elevated in some leukemias. 3-Cl-AHPC at 1.0 microM induced human microvascular endothelial cell apoptosis but did not inhibit cell migration or tube formation.

A critical component in controlling the production of biological products derived from human and animal cell lines is the characterization and testing of banked cell substrates. The objective is to confirm the identity, purity, and suitability of these cells for manufacturing use. Quality concerns for biological products derived from cell lines originate from the presence of cellular and adventitious contaminants. Well-characterized cell banks not only permit a consistent source of production cells throughout the life of a product but also decrease the likelihood of contamination by other cell lines and adventitious agents. An important part of qualifying a cell line is choosing the appropriate testing for the presence of adventitious contaminants. The qualification of cell banks includes tests for cell identity and endogenous and adventitious microbial contaminants (bacteria, fungi, mycoplasmas, and viruses). For cells producing recombinant deoxyribonucleic acid-derived products, analysis of the expression construct at the nucleic acid level (genetic stability) is also a primary concern. The strategy for designing a safety-testing program for banked cells should be based on sound scientific principles and current regulatory guidance.

The surface characteristics of cultured distal colonic mudosa from adult male Fischer 344 rats have been studied under the scanning electron microscope (SEM). Colonic mucosa, physically separated from the submucosa and muscle, was cultured on a matrix of human fibrin foam. The luminal surface of uncultured colonic mucosa formed repeating circular units of cells around individual crypt openings. After 2 days in organ culture, this normal arrangement of surface epithelium was lost. Circular arrangement of cells surrounding the crypts reappeared after 7 days in culture but did not show the well-demarcated crypt units. By 14 days in vitro fewer crypt units were seen; many of these showed a slightly convex contour. Organ cultures retained a variable number of glandular crypts after 21 to 28 days. Occasional epithelial cells were found attached to the interstitial surfaces of the fibrin foam. The organ culture system described appears to be applicable to experimental investigations into the direct effects of various substances on adult colonic epithelium.

The ability of cyclic AMP-elevating agents to induce normal differentiation has been investigated in retinoid-deficient hamster tracheal epithelium in organ culture. Dibutyryl cAMP (dbcAMP) and other cAMP-regulating agents alone caused disappearance of keratin and regeneration of normal mucociliary epithelium in retinoid-deficient cultures. Incubation of retinoid-deficient cultures with dbcAMP, isoproterenol, and cholera toxin (CT) (without addition of exogenous retinoid) reversed keratinization in a dose-dependent manner. The ED50 of cultures treated with dbcAMP was 4 X 10(-6) M; ED50 of isoproterenol was 7 X 10(-5) M; and CT, 0.6 micrograms/ml. Phosphodiesterase inhibitors and other cAMP analogs were inactive. Dibutyryl cAMP in combination with theophylline enhanced normal differentiation. Retinoid-deficient tracheas pretreated for 20 h with 10(-9) M all-trans-retinoic acid (RA) responded to 10(-6) M dbcAMP by potentiating normal differentiation; this concentration of dbcAMP alone was inactive. Isoproterenol showed a similar response but to a lesser degree. These cAMP-elevating agents applied in combination with theophylline did not increase activity.

An in vitro human tumor cell assay was used in an attempt to culture head and neck tumors from patients with squamous cell carcinomas. Initially, specimens from nine head and neck tumors were disaggregated by mechanical methods and assayed in soft agar. Five of nine tumors grew in the soft-agar system yielding a cloning success rate of 56%. Plating of 5 X 10(5) cells resulted in 12 to 255 colonies per plate after 21 days in culture, with a cloning efficiency between 0.002% and 0.08%. Recently, the authors replaced the agar with an agarose culture matrix. Of 10 specimens with positive pathology, 9 have shown colony growth (greater than 20 cells). Cloning efficiency in agarose improved approximately 2-fold. Morphologic assessment of tumor colonies in culture showed the same characteristics as those of the original tumor. Overall success rate of growing head and neck tumors in agar and agarose has been 14 of 19 patients (74%). The development of a soft agarose assay for head and neck tumor cells should provide an in vitro technique for predicting in vivo response to anticancer drugs and other therapeutic modalities such as radiotherapy and hyperthermia.

The effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on rat colonic mucosa were investigated in organ culture. Distal colonic mucosa separated from the muscle layers was cultured on a substrate of human fibrin foam. Exposure of colonic organ cultures to 0.1, 1.0, and 10.0 micrograms MNNG/ml of medium on a multiple discontinuous basis, e.g., every third day for 3 hr, produced significant carcinogen effects. [3H]Thymidine was incorporated throughout crypts and in surface epithelium of carcinogen-treated explants. Outgrowth of epithelioid cells into the fibrin foam matrix was observed in all treated explants 9 days after initial MNNG exposure. Control untreated cultures showed limited outgrowth. After 15 and 21 days in culture, epithelioid outgrowth was still observed in 1.0 microgram MNNG/ml-treated cultures.

Normal values for intestinal flora were determined on four adult, two juvenile, and four infant agoutis (Dasyprocta sp) maintained at our institution. Serologic, hematologic, biochemical, and histologic observations were also made on these same agoutis, and serologic, microbiologic, and endoparasitic tests were made on serum and fecal samples from agoutis maintained at other institutions. Streptococci, lactobacilli and nonenteropathogenic Escherichia coli were isolated from cecal contents of all age groups. Gaffkya tetragena and Bacillus sp were recovered from infant agoutis, Proteus mirabilis from juvenile agoutis, and Proteus mirabilis and Micrococcus sp from adult agoutis. Infant agoutis showed marginal antibody titers to reovirus type 3 and Toolan's H-1 viruses, and Theiler's GDVII antibodies were detected in juvenile agoutis, but no antibody titers to murine viruses were found in the adult agoutis maintained at our institution. Hematologic, biochemical, and histologic data for these agoutis were comparable to those of other hystricomorph rodents. Serologic tests of the agoutis maintained at other institutions disclosed antibodies for GDVII virus in all of 17 animals tested, while marginal titers were found for Sendai virus in four and for reovirus type 3 in eight of the 17 animals. Only low incidences of pathogenic bacteria (Salmonella, Pseudomonas, and Citrobacter) were isolated from the agoutis maintained elsewhere. The endoparasites found in these agoutis were Ascaris, Toxascaris, Strongyloides, Trichuris, and Trichomonas.

Studies were initiated to determine the practicality of using various tissue cultures for the propagation of murine viruses isolated from laboratory animals. The cytopathogenic effects of 10 murine viruses known to cause disease in laboratory rodents were compared in monolayer cultures of L929, BHK-21, WI-38, BSC-1, and Vero cells. The susceptibility of primary hamster embryo, hamster kidney, mouse embryo, mouse kidney, and rat embryo cell cultures was also tested. Seven of the viruses produced effects in at least 1 of the cell substrates. The remaining 3 viruses, namely H-1, K, and mouse hepatitis, produced no effects in the cell cultures tested.

The ultrastructural surface features of tracheal epithelium at various times of development in organ culture were compared with those in the trachea of Syrian golden hamsters of similar age using scanning electron microscopy. Whole tracheal organ cultures, prepared from 3-day-old hamsters, were maintained in HEPES buffered CMRL 1066 medium with 0.2% bovine serum albumin. The ventral epithelial surface of trachea from 3-day-old animals was characterized by numerous microvillous cells, occasional well-developed cilia, and cells containing short cilia representing various stages of ciliary differentiation. After seven days in culture, an increased number of ciliated cells as well as developing cilia were seen. The epithelium after 14 days in culture appeared to have equal numbers of ciliated and microvillous cells, a pattern strikingly similar to that observed in vivo. After 21 days in culture, groups of well-developed cilia were interspersed with nonciliated cells covered with short, poorly developed surface microvilli. A similar pattern was found in the trachea of comparable age (24 days), with the exception of the microvillous cells; many being dome-shaped containing cluster of microvilli with prominent outlines. The tracheal organ culture, as developed in this investigation, appears to represent an excellent model for studying age-related effects of toxic and infectious agents on ciliated epithelial cells.

Exposure of hamster tracheal organ cultures to gas phase singlet delta oxygen, O2, at atmospheric pressure produced significant alterations in the mucociliary epithelium resulting in changes in ciliary activity and cellular morphology.

A system for maintaining adult rat colonic mucosa in organ culture for up to 28 days is described. Distal colonic mucosa physically separated from the muscle layers was cultured at 37 degrees C on a substrate of human fibrin foam in HEPES- and bicarbonate-buffered Waymouth's MB 752/1 medium supplemented with 10% fetal bovine serum, L-glutamine, bovine albumin, ascorbic acid, hydrocortisone, insulin, and ferrous sulfate; the optimal atmostphere for culture was 95% O2 and 5% CO2. Viability of explants was demonstrated by tissue morphology with light microscopy, incorporation of [3H]thymidine and [3H]leucine into DNA and protein, [14C]glucosamine and [3H]fucose incorporation, and glycoprotein synthesis. Two days after initiation of culture, degeneration of surface and crypt cells was observed. Secreted mucosubstances covered the explants. Explants maintained in 95% O2 retained a variable number of glandular crypts with normal columnar epithelium for 14 to 21 days in culture. At 28 days, explants contained a single layer of cuboidal surface epithelium and a rare cryptlike gland.

Human fibrin foam as a matrix for three-dimensional organ cultures was successfully employed for the cultivation of newborn rat and hamster colon tissue. Colonic tissue was maintained under different oxygen atmospheres and with various synthetic media. Explants of rat and hamster colon, maintained in Leibovitz's L-15 medium buffered with HEPES in the presence of 5% CO2 and air, retained their normal columnar epithelial architecture for 24 days in culture. Outgrowth of epithelial cells from colonic explants similar to those of the mother fragments grew into the foam. Viability of the explants was indicated by radioactive thymidine incorporation, and by the ability of the fragments to support viral replication.

A series of conformationally restricted retinoids was synthesized and screened in two assays used to measure the ability of retinoids to control cell differentiation, namely, the reversal of keratinization in tracheal organ culture from vitamin A deficient hamsters and the inhibition of the induction of mouse epidermal ornithine decarboxylase by a tumor promoter. These compounds had bonds corresponding to selected bonds of the E-tetraene chain of retinoic acid (1) held in a planar cisoid conformation by inclusion in an aromatic ring. The meta-substituted analogue 3 of 4-[(E)-2-methyl-4-(2,6,6-trimethylcyclohexenyl)-1,3-butadienyl+ ++]benzoic acid (2) was far less active than 2 in both assays. In contrast, the vinyl homologue of 2 (4) and the 7,8-dihydro and 7,8-methano analogues (5 and 6) had activity comparable to that of 2. Analogues of 4-[(E)-2-(1,1,4,4-tetramethyl-1,2,3,4-tetrahydro-6-naphthyl)propenyl] benzoic acid (7) were also screened. Replacement of the tetrahydronaphthalene ring of 7 by a benzonorbornenyl group (9) significantly reduced activity, as did removal of the vinylic methyl group from 9 (10). Replacement of the propenyl group of 9 by a cyclopropane ring (12) also reduced activity. Replacement of the tetrahydronaphthalene ring of 7 by 4,4-dimethyl-3,4-dihydro-2H-1-benzopyran and -benzothiopyran rings (13 and 14) also decreased activity. Inclusion of the 7,9 double bond system of 1 in an aromatic ring (15 and 16) reduced activity, whereas inclusion of the 5,7 double bond system in an aromatic ring enhanced activity (7 and 19). Inclusion of the 11,13 and 9,11,13 double bond systems in aromatic rings (2 and 18) also reduced activity below that of 1. Retinoic acid, 7, 13, 14, and 19 inhibited papilloma tumor formation in mice. Toxicity testing indicated that 7 was more toxic than 1, 13, 14, and 19, 19 was more toxic than 1, and 13 and 14 were less toxic than 1.

A series of conformationally restricted retinoids has been synthesized and assayed for biological activity. These compounds have aromatic rings in place of selected double bonds of the tetraene side-chain of retinoic acid and could be considered as analogues of retinoic acid in which some of the double bonds possess s-cis topology. Thus far, analogues in which the bonds corresponding to the (5,7E)-, (7,9E)-, (9,11,13E)- and (11,13E)-double bond systems of retinoic acid are restricted to a cisoid conformation have been studied. Analogues were screened for their ability to reverse keratinization in hamster tracheal organ culture and to inhibit the induction of ornithine decarboxylase in mouse epidermis. Selected compounds were also screened in the antipapilloma assay in mice. The toxicity of some analogues on intraperitoneal injection in mice was determined.

6-[(E)-2-(2,6,6-Trimethyl-1-cyclohexen-1-yl)ethen-1-yl]- and 6-(1,2,3,4-tetrahydro-1,1,4,4-tetramethyl-6-naphthyl)-2-naphthalenecarboxylic acids (4 and 8) have been synthesized and show significant activity in reversing the keratinization process in hamster tracheal organ culture and in inhibiting the induction of ornithine decarboxylase in mouse skin by 12-O-tetradecanoylphorbol-13-acetate, two assays used to measure retinoid activity. The 2-naphthalenecarboxylic acid 8 was more active than 4.

There is reported the first four members of heteroarotinoids, the names of which are ethyl (E)-p-[2-(4,4-dimethylthiochroman-6-yl)propenyl]benzoate (1b), ethyl (E)-p-[2-(4,4-dimethylchroman-6-yl)propenyl]benzoate (1c), ethyl (E)-p-[2-(4,4-dimethyl-1-oxothiochroman-6-yl)propenyl]benzoate (1d), and (E)-p-[2-(4,4-dimethylchroman-6-yl)propenyl]benzoic acid (1e). IR, 1H NMR and 13C NMR data have been recorded for each compound and support the structural assignments. To provide a firm basis for comparison purposes of future analogues, an X-ray analysis was performed on a single crystal of ethyl (E)-p-[2-(4,4-dimethylthiochroman-6-yl)propenyl]benzoate (1b) and a precursor 4,4-dimethylthiochroman-6-yl methyl ketone 1,1-dioxide (18). These data for the heteroarotinoid 1b revealed that the two aryl ring systems were nearly perpendicular in each of the two molecules present in the unit cell (86.37 degrees and 84.17 degrees, respectively). The space group for both molecules was P1 in triclinic systems. Unit cell dimensions (at 15 degrees C) are as follows: for 1b, a = 20.568 (6) A, b = 14.760 (3) A, c = 7.679 (2) A, alpha = 113.33 (2) degrees, beta = 79.45 (2) degrees, gamma = 79.98 (2) degrees, Z = 4; for 18, a = 9.292 (5) A, b = 9.291 (5) A, c = 7.951 (3) A, alpha = 102.16 (3) degrees, beta = 77.49 (3) degrees, gamma = 79.60 (4) degrees, Z = 2. The sulfur-containing ring is in a distorted half-chair in 1b and the methyl carbon C(12) is shown to be trans to H(13) at the C(11)-C(13) bond. The biological activity of these arotinoids was determined in the tracheal organ culture assay and compared with trans-retinoic acid for ability to reverse keratinization in vitamin A deficient hamsters. The ester 1b displayed activity about one-half log unit less than that of the reference while 1c and 1e had activity nearly one log until less than trans-retinoic acid. The sulfoxide was the least active of the heteroretinoids.

Hamster tracheal organ cultures were used to study the in vitro effects of vanadium and oil-fired fly ash on mucociliary respiratory epithelium. Two vanadium compounds, VOSO4 and V2O5, and fly ash from an oil-fueled power plant were dissolved or suspended in culture medium over a range of concentrations and epithelia were exposed for 1 hr/day, for 9 consecutive days. At intervals during this period, alterations in cilia-beating frequency, cytology, and histology were documented by light microscopy. Explants treated with VOSO4 either decreased ciliary activity or produced ciliostasis depending upon the concentration and length of exposure. Early morphological alterations consisted of vacuolization of both nuclei and cytoplasm. After multiple exposures, cytology of VOSO4-treated respiratory mucosa was markedly affected. Similar changes were observed in cultures exposed to V2O5; however, the cytotoxicity appeared earlier and was more pronounced. Fly ash-treated explants produced similar biological effects when compared to both vanadium compounds. Thus, the data indicate that the extent of vanadium toxicity depends, at least in part, on the vanadium content of the compound tested, and that exposure to this metal and vanadium-rich fly ash can inhibit normal mucociliary function, a vital clearance mechanism in the respiratory tract.

N-(all-trans-Retinoyl)amino acids were synthesized via all-trans-retinoyl chloride and an ester of the amino acid. The retinoyl derivatives of leucine, phenylalanine, alanine, tyrosine, and glutamic acid were prepared. The 13-cis-retinoyl derivatives of leucine, phenylalanine, alanine, and glycine were prepared similarly from 13-cis-retinoic acid. In assays of the retinoylamino acids for reversal of squamous metaplasia in hamster trachea organ cultures, these compounds were less active than retinoic acid, but the leucine, alanine, and phenylalanine derivatives were similar in activity to several retinamides that suppress bladder carcinogenesis in vivo. Two of the retinoylamino acids, as well as two simple retinamides, were shown to be moderately cytotoxic to murine leukemia and human epidermoid carcinoma cells in culture.

The toxic effects of gas-phase singlet oxygen, 1O2, on the ciliated respiratory epithelium of hamster trachea have been demonstrated. Tracheal explants treated with 1O2 showed a dose-dependent decrease in cilia beating frequency and focal ciliostasis. A statistically significant decrease in ciliary activity occurred at 1O2 concentrations as low as 154 ppb after a 2-hr exposure. Cytological alterations in the mucociliary epithelium were observed in explants exposed to 235 ppb 1O2 or greater. When cytotoxic effects were related to the time of exposure to 1O2, maximum effects occurred after a 4-hr exposure. In vitro recovery studies indicate that ciliary activity returned to normal between 4 and 8 hr after exposure.

The mechanism of action of retinoid in reversing keratinization in hamster trachea is yet unknown. The purpose of this study was to determine if cellular retinoic acid binding protein (CRABP) is present in tracheal epithelium following incubation in serum-free, vitamin A-deficient culture medium for 10 days, and if the effectiveness of a retinoid in reversing keratinization in organ culture is correlated with its ability to compete for CRABP sites. The cytosol prepared from tracheal cultures contained CRABP at a concentration of 2.61 pmoles per mg protein. Of the four retinoids with carboxyl end group selected for the study, two of the biological active retinoids competed for the CRABP sites. However, no correlation was observed between the biological activity of the inactive retinoids and their ability to associate with the CRABP sites. These results indicate that even though the action of retinoid may be mediated by retinoid binding protein, it cannot be used as a sole predicator of retinoid response in hamster trachea.

Various health effect parameters were compared to determine which tests were the most sensitive indicators of toxic effects of exposure to metallic sulfate aerosols. Inhalation studies were conducted involving either single 3-hr exposure to various concentrations of cupric sulfate (0.43-2.64 mg/m3 SO4), aluminum sulfate (1.65-2.75 mg/m3 SO4), and aluminum ammonium sulfate (1.47-3.81 mg/m3 SO4) or multiple (five and ten) daily 3-hr exposures to cupric sulfate (0.1 mg/m3 SO4). The test parameters studied in male and female CD1 mice were changes in mortality after respiratory infection with Group C Streptococcus zooepidemicus; pulmonary bactericidal activity; pulmonary cell number, type, viability, and ATP content; and pulmonary morphology by scanning electron microscopy. Tracheal ciliary beating frequency and morphology were also studied in both CD1 mice and Syrian golden hamsters. Differences in bacteria-induced mortality rate appeared to be the most sensitive and consistent indicators of pollutant damage. The other parameters produced evidence of damage but generally only at the higher pollutant concentrations. Cupric sulfate was the most toxic of the three sulfates, but the differences between the toxicity of the aluminum sulfate and aluminum ammonium sulfate were less clear.

The effects of Mount St. Helens volcanic ash on rings of hamster tracheal epithelium in organ culture were studied. Volcanic ash samples with mass median aerodynamic diameters (MMAD) of 7.7 micrometers and 1.6 micrometers caused markedly different alterations in the tracheal mucosa. Examination by SEM of the ventral epithelial surface of tissue from untreated control explants after 2 weeks in culture showed equal numbers of ciliated and microvillous cells. Examination by SEM of tracheas exposed to the smaller size particles revealed that ash concentrations as low as 1 microgram/ml increased mucous secretion after one 2-hr exposure. After four or nine 2-hr exposures, cells contained cilia that were short and blunt. Ciliary activity after these exposures showed a significant depression in beating frequency. Tracheal ring cultures exposed to the larger volcanic ash particles exhibited moderate cytomorphological changes after one 2-hr exposure at concentrations of 1, 10 and 100 micrograms/ml. As the number of exposures increased, most of the columnar cell layer was lost, resulting in exposure of the basal cells. After nine exposures at the two highest concentrations of ash (10 and 100 micrograms/ml), only a few ciliated cells were remaining. Statistically significant reductions in ciliary activity paralleled the epithelial damage. The degree of epithelial damage and changes in the cilia beating frequency were related to the dose and the number of exposures to the volcanic ash.

The effects of fly ash from a coal-fired power plant on tracheal epithelium of crRGH (SYR) Syrian golden hamsters were studied in organ cultures and after in vivo exposures. The tracheal epithelium of animals receiving 5-9 daily (5 d/wk) 3-h exposures to 2 mg fly ash per cubic meter showed large areas of basal cell hyperplasia and stratification. Surface alterations characteristic of stratified metaplasia were observed. Exposure to 1 mg/m3 produced diffuse basal cell hyperplasia. Hamster tracheal ring cultures exposed in vitro to 50 micrograms/ml fly ash for 1 h/d or to 10 micrograms/ml for 3h/d showed epithelial changes similar to those observed in vivo. Whole suckling hamster tracheas in organ culture exposed to fly ash at concentrations of 10 and 50 micrograms/ml for 1 or 3 h/d exhibited cornifying epidermoid metaplasia after 7 exposures. The most characteristic findings in surface cells were broad metaplastic areas with keratin formation.

Replication of Sendai virus, pneumonia virus of mice, and SV5 was investigated in tracheal organ cultures from 2- to 4-day-old and 2- and 4-week-old hamsters, and viral infectivity in tracheal explants was compared with that in tissue culture monolayers. Explants from 2- to 4-day-old hamsters produced higher titers of the three paramyxoviruses, as detected by hemadsorption with guinea pig and murine erythrocytes in primary rhesus monkey kidney cells. Tracheal cultures from 2- and 4-week-old hamsters yielded 1.5 and 2.5 log10 lower infectivity titers. Infected explants exhibited cytopathological changes that correlated well with cessation of ciliary activity. Viral titers in BHK-21, Vero, and BS-C-1 monolayer cells, the systems commonly used for isolation and propagation of murine paramyxoviruses, were lower than those in 2- to 4-day-old hamster tracheal explants. These observations suggest that hamster trachea organ culture could have practical application as an aid for primary isolation of paramyxoviruses from clinical specimens from rodents with respiratory ailments.

Aromatic analogues of (E)-1-(4-carboxyphenyl)-2-methyl-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)butadiene (1b) and its ethyl ester (1a) were synthesized as potential chemopreventive agents for the treatment of epithelial cancer and such skin diseases as psoriasis and cystic acne. The phenyl ring of 1 was replaced by 2-fluorophenyl, 2-methoxyphenyl, thienyl, furanyl, and pyridyl groups. The 1-fluorobutadiene analogue of 1 was also synthesized. With exception for the furanyl analogue, these compounds demonstrated good activity in reversing keratinization in hamster tracheal organ culture and in inhibiting the induction of ornithine decarboxylase in mouse epidermis by a tumor promoter.

Binding affinities of a new and unusual series of retinoic acid analogues to cellular retinoic acid-binding protein, a possible mediator of their biological function in the control of differentiation and tumorigenesis, and to serum albumin, their plasma transport protein, were determined. Also, biological activity of these retinoids in the reversal of keratinization in hamster tracheal organ cultures was assessed and compared with their binding affinities. Analogues that possessed high biological activity showed high binding efficiency to cellular retinoic acid-binding protein. Those that were biologically less active were poor binders to the binding protein. Three retinoids, 4657-57, 3920-59, and 4445-75, which showed 90 to 100% binding efficiency of that of retinoic acid for cellular retinoic acid-binding protein expressed high biological activity detectable in the range of 10(-10) M as against 10(-11) M for retinoic acid. The correlation noticed in these two activities not only enhances the confidence in the two assay procedures but also paves the way for design and development of potential chemopreventive agents. No apparent differences were observed in the binding affinities of the retinoids to binding proteins of a normal tissue or a tumor tissue. No correlation existed between the binding affinities of these retinoids to serum albumin and their biological activity. Structure-activity relationships of the retinoids in relation to their binding affinities and biological activities have been discussed.

The toxicity of fly ash from a coal-fired power plant for respiratory tract epithelium was studied in heterotropic tracheal grafts. Hamster tracheal grafts were continuously exposed to beeswax-cholesterol pellets containing 100, 1000 and 5000 micrograms fly ash and evaluated at 1, 2, 4 and 14-15 months of exposure. Histopathologic effects and the autoradiographic pattern of [3H]thymidine incorporation were determined. In all concentrations of fly ash, an early mild submucosal inflammatory response was seen. Morphologic response of the tracheal epithelium was characterized by hyperplasia followed by squamous metaplasia and atrophic lesions. Although a rare papillomatous structure with cellular atypia was seen in grafts receiving 1000 micrograms fly ash, no carcinomas appeared during the 15-month observation period. Varying degrees of submucosal toxicity were also observed during this time period. Autoradiographic studies showed a significant increase in [3H]thymidine incorporation in grafts receiving fly ash treatment.

Effects of various oxygen concentrations and culture media on the maintenance of 4-day-old hamster trachea organ cultures and the yield of Sendai virus were studied. The basic media used were Eagle minimal essential medium, medium 199, and CMRL 1066 supplemented with glutamine and antibiotics and buffered with NaHCO3 or N-2-hydroxyethyl-piperazine-N'-2'-ethanesulfonic acid (HEPES). In addition, each medium was evaluated under a gas phase of 5% CO2 and 95% O2, 5% CO2 and 45% O2 and 50% N2, or 5% CO2 and 95% air. Culturing of explants with CMRL 1066 and medium 199 buffered with HEPES in the presence of 5% CO2 and air proved most efficient; ciliary movement and ciliated surface epithelium were maintained for periods up to 27 days. No significant difference in the rate of replication of Sendai virus was seen in the three different media with the two buffer systems in the three different gaseous phases. The addition of 0.2% bovine serum albumin to the media yielded greater quantities of virus, up to 200-fold increase in titer without producing changes in ciliary function. A distinctive pattern of morphological changes was observed in explants of trachea epithelia inoculated with Sendai virus. These results suggest the practical application in the use of whole hamster trachea explants as a diagnostic aid in the isolation of Sendai virus from laboratory rodents.

Autoradiographic identification of unscheduled DNA synthesis (UDS) in short-term organ culture of hamster tracheal epithelium has been used as a predictive test for mutagenic and/or carcinogenic compounds. Tracheal explants were treated for 2 h with singlet delta oxygen plus [3H]thymidine. Silver grains over the nuclei of epithelial cells from the superficial layer of the mucosa were observed, indicating UDS. Control cultures, exposed to the gas phase without singlet oxygen, failed to elicit UDS.

The reported occurrence of synergistic false-positive coliform reactions on M-Endo MF medium was investigated. The objectives of the study were (i) to isolate populations of bacteria which produced such false-positive reactions, (ii) to determine whether false-positive sheen reactions are a result of synergistic interaction, and (iii) to determine the metabolic intermediates involved in false-positive sheen production. Samples of river water were subjected to coliform analysis by the membrane filter technique with M-Endo MF medium. Suspect sheen-forming colonies were analyzed to determine purity and identity of cultures. Mixed cultures were separated, and individual isolates were examined for sheen production. The isolates not producing a sheen were recombined and tested further for sheen production. Those mixtures reproducing the sheen were characterized biochemically and tested to determine the metabolic intermediates involved. Chromatographic analysis of the metabolites showed that individual isolates produced an assortment of neutral organic compounds including 2-butanone, 2,3-butanedione, formaldehyde, and butyraldehyde, whereas acetaldehyde or propionaldehyde was produced only by the mixed cultures. Tests showed that both propionaldehyde and acetaldehyde could react to produce a sheen on M-Endo medium. The conclusion was reached that synergistic false-positive coliform reactions do indeed occur on M-Endo MF medium.