Relationship between binding affinities to cellular retinoic acid-binding protein and biological potency of a new series of retinoids.

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.

N-(Retinoyl)amino acids. Synthesis and chemopreventive activity in vitro.

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.

Aromatic retinoic acid analogues. 2. Synthesis and pharmacological activity.

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.

Synthesis and pharmacological activity of 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.

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.

Synthesis and characterization of selected heteroarotinoids. Pharmacological activity as assessed in vitamin A deficient hamster tracheal organ cultures. Single-crystal X-ray diffraction analysis of 4,4-dimethylthiochroman-6-yl methyl ketone 1,1-dioxide and ethyl (E)-p-[2-(4,4-dimethylthiochroman-6-yl)propenyl]benzoate.

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.

Conformationally restricted retinoids.

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.

Cytotoxic effects of singlet oxygen.

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.

Pathologic changes induced by coal-fired fly ash in hamster tracheal grafts.

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.

Evidence of DNA repair in organ cultures of hamster tracheal epithelium following exposure to gas phase singlet oxygen.

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.

Replication of murine paramyxoviruses in hamster tracheal organ culture and comparison with standard tissue culture methods.

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.

Organ cultures of rat and hamster colon.

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.

Influence of oxygen and culture media on maintenance of whole hamster trachea organ cultures and replication of Sendai virus.

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.

Organ culture of adult rat colonic mucosa on fibrin foam.

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.

Effects of cyclic adenosine 3':5'-monophosphate-elevating agents and retinoic acid on differentiation in retinoid-deficient tracheal cultures.

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.

Growth stimulation of rat colonic cells in organ culture with N-methyl-N'-nitro-N-nitrosoguanidine.

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.

Cytotoxic effect of vanadium and oil-fired fly ash on hamster tracheal epithelium.

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.

Growth of human head and neck squamous cell carcinoma stem cells in agarose.

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.

Surface characteristics of cultured adult rat colon using scanning electron microscopy.

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.

Fly ash-induced changes in hamster tracheal epithelium in vivo and in vitro.

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.