Hamster cheek pouch as a bioassay system in short term screening for tumor initiating and promoting agents. Optimization of parameters and observations of effects of cytotoxicity on tumor promotion by retinyl acetate.

The effects of various conditions on tumor initiation by 7,12-dimethylbenz(a) anthracene (DMBA) and promotion by retinyl acetate (RA), both in dimethylsulfoxide, were studied by topical application in hamster cheek pouch. The variables studied were: initiation and promotion time and schedule (alternate vs consecutive day paintings), concentration of DMBA used for initiation, and cytotoxicity. Spontaneous tumor progression followed by regression was observed in control animals which had been initiated for 4 weeks with 0.2% DMBA, but not given subsequent promotion treatment. Spontaneous progression was not observed in control animals which had been given only 2 weeks of initiation treatment, although RA both promoted new tumors and caused progression of benign hyperplastic lesions (BHLs) to advanced tumors. The approximate optimal conditions for obtaining acceptable tumor yields from promotion while minimizing associated errors and time periods were: 2 weeks of initiation with 0.2% DMBA and 4 weeks of promotion with 0.05 M RA, both with alternate day painting schedules. Reducing the initiation period but painting on consecutive days caused intolerable cytotoxicity and lowered tumor yields although the total dose of DMBA was kept constant. Increasing initiation time increased tumor yield in unpromoted controls as well as promoted groups, thus increasing errors in net yield from promotion. Similar results were obtained from doubling concentration of DMBA and halving initiation time. Reducing promotion time to below 4 weeks lowered tumor yield due to insufficient time for tumor development. Increasing promotion time to 6 weeks lowered tumor yield in both control and promoted groups, possibly due to spontaneous regression. Cytotoxicity enhanced progression to advanced tumors all of which were inflamed polypoid fibrovascular lesions. The cytotoxicity of DMBA was far greater than that of RA. Evidence is presented that tumor promotion by RA is not due to cytotoxicity. The hamster pouch system may afford an excellent short term mammalian screening test for tumor initiating and promotion agents.

Rapid promotion and progression of fibrovascular polyps by inflammation and/or hyperplasia in hamster check pouch: implications for carcinogenesis assay.

Tumor initiation by topical application of 7,12-dimethylbenz[a]anthracene (DMBA) in dimethyl sulfoxide (DMSO) followed by topical application of retinyl acetate (RA), ethylphenylpropiolate, or acetic acid in DMSO at inflammatory and hyperplasiogenic dose regimens caused the rapid promotion of fibrovascular polyps with dysplastic epithelium in hamster cheek pouch. Such lesions did not occur in control animals initiated with DMBA followed by application of DMSO only, where inflammation was also minimal. At the dose regimen employed, RA caused obvious cytotoxicity and tissue destruction. With EPP and AA, there was no histological evidence of tissue destruction. At dose regimens resulting in minimal inflammation and no apparent cytotoxicity, RA promoted almost no polyps, but a higher yield of other tumor types. Thus, inflammation and/or hyperplasia apparently exerted a strong polyp-promoting and progressive influence. This and other differences between the tumorigenic responses of hamster-pouch mucosa and mouse skin suggest that the former supplement the latter in carcinogenic risk assessment.

Promotion of benign hyperplastic lesions by calcium, magnesium and cAMP, and inhibition of tumor progression by magnesium in hamster cheek pouch.

Ca, Mg (0.1 or 0.2M) and ionophore A23187 (10(-5) - 10(-4)M) promoted benign hyperplastic lesions (BHLs) to a highly variable degree when applied topically in hamster cheek pouch after initiation with 7, 12-dimethylbenz(a)anthracene (DMBA). The changes in the promoting effects of Ca and Mg between experiments were not parallel. Mg but not Ca inhibited retinyl acetate-induced progression of BHLs to advanced tumors. Cyclic adenosine 3',5'-monophosphate (cAMP) (10(-5) - 10(-3)M) promoted BHLs. The results are discussed in terms of possible mechanisms involved.

Promotion of benign hyperplastic lesions in hamster cheek pouch by membrane labilizing agents and calcium ion: evidence that intracellular calcium-induced release from growth control is an obligatory preliminary stage of tumor promotion.

Ca ion, ionophore 23187 and the membrane labilizing agents triton X-100, trypsin and phospholipase C promoted benign hyperplastic lesions (BHLs) but only rarely advanced tumors in hamster cheek pouch mucosa, which had been initiated with 7,12 dimethylbenz (a) anthracene. Retinyl acetate (RA) and croton oil markedly promoted both BHLs and advanced tumors. These results suggest that pathways modulated by intracellular Ca cause initiation-stabilized release from growth control, thus the promotion of BHLs, whereas additional mechanisms are required for the progression of BHLs to more advanced tumors. Some Ca-modulated systems which could be involved in promotion of BHLs are discussed in the light of published literature.

Effects of the differentiating agents (inducers) dimethylacetamide, di- and tetramethylurea on epidermal tumor promotion by retinyl (vitamin A) acetate and croton oil in hamster cheek pouch.

The cell-differentiating agents (inducers) dimethylacetamide, dimethylurea and tetramethylurea significantly lowered the yields and/or incidences of total tumors, benign plaques, benign hyperkeratotic lesions and advanced tumors promoted by retinyl acetate or croton oil after initiation by 7,12-dimethylbenz(a)anthracene. The percentage of plaques was increased by the inducers suggesting that they inhibited progression of plaques to more advanced tumors. These results suggest that topical application of inducers might have therapeutic potential in epidermal carcinoma.

Effects of polyphosphates on the solubility and mineralization of HA: relevance to a rationale for anticaries activity.

Preexposure of hydroxyapatite (HA) to polyphosphate reduced the near-equilibrium acid solubility of HA, the mineralization of HA, and the exchange of PO4 between medium and HA. Appreciably longer exposure times were required for maximal effects of trimetaphosphate (TMP) than of pyrophosphate (PP), tripolyphosphate (TPP), and hexametaphosphate (HMP). Calcifying solution solubilized small amounts of the HA-bound polyphosphates. This occurred to the smallest extent in the case of TMP, a fact which could have relevance for the superior anticaries effect of TMP.

Effects of adenosine and guanosine cyclic phosphates and their corresponding nucleotides and nucleosides on vitamin A-induced epidermal tumor promotion and growth in hamster cheek pouch.

Adenosine and guanosine cyclic monophosphates (cAMP and cGMP) exerted opposite effects at similar concentration and similar effects at markedly different concentrations on the yields of tumors promoted by vitamin A in hamster cheek pouch. Increasing the concentration of cAMP between 10(-5) M and 10(-3) M was associated with increases in tumor yield and diameter which partially or completely overcame the net tumor promotion inhibitory effects which were sometimes seen at 10(-5) M cAMP. In contrast, increasing the concentration of cGMP between 10(-5) M and 10(-3) M caused the net effects to change from increases to decreases in tumor yield without much change in diameter. The relative magnitudes of the tumor yield increasing and decreasing effects of the cyclic nucleotides varied substantially between experiments at constant concentration of cyclic nucleotide. Adenosine-5'-monophosphate (AMP) showed simultaneous tumor promotion inhibitory and growth stimulatory effects, the former effect being similar to that of cAMP but tumor yield decreased rather than increased as concentration of AMP was increased whereas the opposite occured with cAMP. Guanosine-5'-monophosphate (GMP) showed the tumor yield increasing effect of low concentraitons of cGMP but not the yield decreasing effect of high concentrations of cGMP. Adenosine (10(-3) M) showed a significant tumor yield increasing effect similar to that of 10(-3) M cAMP. Guanosine showed no significant effects at either 10(-3) M or 10(-5) M concentrations.

Relations between early dental calculus production and calcium and phosphate parameters of salivary fractions.

A pilot study was carried out to obtain an idea of the strength of the relations between early mandibular calculus production in humans and amounts and concentrations of bound calcium and phosphate in three salivary fractions. The salivary fractions were: (1) the spontaneous precipitate from incubated whole saliva, (2) the spontaneous precipitate from incubated centrifugal (20,000 X g) saliva supernatant and (3) the fresh centrifugal saliva sediment. Strong and highly significant correlations were found for the amount and concentration of calcium in the fresh sediment and whole saliva precipitate fractions. Although the correlations for the calcium parameters of the saliva supernatant precipitate were not significant, there were significant differences in these parameters between low and high calculus formers as determined by Student's t test. Relations between calculus production and amount and concentration of phosphate were weaker and not significant for all fractions. The data suggest that the amount or concentration of calcium in fresh saliva sediment is an important determinant of early mandibular calculus production.