Beta-carotene and canthaxanthin inhibit chemically- and physically-induced neoplastic transformation in 10T1/2 cells.

We have studied the effects of beta-carotene (beta-C), a vitamin A precursor of plant origin, and canthaxanthin (CTX), a non-provitamin A carotenoid, on the neoplastic transformation of C3H/10T1/2 murine fibroblast cells. Chemical transformation in this well-characterized cell system has previously been shown to be reversibly inhibited by retinoids, compounds with vitamin A-like activity. Here we show that both beta-C and CTX inhibit 3-methylcholanthrene (MCA)-induced transformation with ED50s of 9 x 10(-7) M and 2 x 10(-7) M, respectively. Both carotenoids failed to inhibit X-ray-induced transformation when the cells were treated prior to and during irradiation. However, when the drugs were added 1 week after X-irradiation and maintained in the medium thereafter, as in the chemical transformation protocol, both carotenoids inhibited subsequent development of transformed foci in a dose-dependent manner. Again, CTX was more effective than beta-C, such that 3 x 10(-6) M completely inhibited radiogenically-induced foci. Similar to the previously described action of retinoids, the inhibition of MCA-induced transformation was reversible; upon removal of the drug, transformed foci developed within 2 weeks, indicating that the carotenoids were not specifically toxic to initiated cells. Although both carotenoids caused a small dose-dependent decrease in the growth rate of both parental and initiated 10T1/2 cells, they did not markedly affect colony size or number when the cells were treated as in the transformation assays, nor did they influence the expression of neoplasia of two transformed cell lines. Although the actions of beta-C and CTX are similar to those of retinoids in the 10T1/2 system, we suggest that the carotenoids act via a different mechanism, since CTX cannot be converted to active retinoids in mammalian cells, and there is no evidence that 10T1/2 cells can convert beta-C to vitamin A. We suggest that the carotenoids' lipid anti-oxidant properties may be responsible for their inhibitory actions on transformation.

Alteration of methylcholanthrene-mediated suppression of cutaneous delayed hypersensitivity by benzoflavone treatment of C57BL/6J mice.

The potential role of the cytochromes P-450 in methylcholanthrene (MC)-mediated suppression of cutaneous delayed hypersensitivity (CDH) in C57BL/6J (B6) mice was evaluated indirectly by treating mice with agents known to induce or inhibit hepatic cytochromes P-450 prior to contact sensitization. Subsequent alterations in aryl hydrocarbon hydroxylase (AHH) activity and CDH, as measured by suppression of 2,4-dinitrofluorobenzene (DNFB)-induced ear swelling, were measured. MC administration resulted in a dose-dependent suppression of ear swelling and a concomitant dose-dependent induction of hepatic AHH activity. Treatment of B6 mice with phenobarbital (PB), 80 mg/kg daily X 3, a broad spectrum inducer of P-450, resulted in a 2.5-fold increase in benzo[a]pyrene (B[a]P) hydroxylase activity without affecting CDH. Animals treated with the same PB protocol prior to an ED20 dose of MC showed no difference in suppression of CDH compared to animals treated with MC alone. In contrast, successive treatment with the selective P1-450 inducer, 5,6-benzoflavone (beta NF), prior to and following an ED20 dose of MC significantly increased suppression of CDH (p less than 0.001) usually seen at this MC dose. Treatment with a known inhibitor of cytochrome P1-450 activity, 7,8-benzoflavone (alpha NF), did not prevent AHH induction when administered prior to and following MC (ED100) nor did it suppress CDH when administered alone. However, this alpha NF treatment completely prevented suppression of CDH usually seen at this MC dose. These data provide evidence suggesting that metabolic activation by cytochrome P1-450 is required for the expression of the immunosuppressive activity of MC.

Selenium inhibition of benzo[a]pyrene, 3-methylcholanthrene, and 3-methylcholanthrylene mutagenicity in Salmonella typhimurium strains TA98 and TA100.

Selenium (Se) decreased the mutagenicity of benzo[a]pyrene (BP), 3-methylcholanthrene (3MC), and 3-methylcholanthrylene (3MCE) in Salmonella typhimurium strains TA98 and TA100. Metabolism of BP, 3MC and 3MCE to mutagens was accomplished with the liver S9 fraction from Aroclor 1254-treated male Sprague-Dawley rats. Exposure of the bacteria to 4 nmoles BP, 10 nmoles 3MC, or 10 nmoles 3MCE in the presence of S9, and up to 200 nmoles Se as Na2SeO3 resulted in decreased mutagenicities up to 39, 66 and 60% of their respective control activities without Se in TA98 and up to 46, 52 and 64% of their respective control activities without Se in TA100. Se (200 nmoles) alone was not mutagenic in strains TA98 or TA100 with or without S9. BP, 3MC and 3MCE were not mutagenic in either strain without S9. None of the tested concentrations of BP, 3MC, 3MCE and Se were cytotoxic. Assays of the aryl hydrocarbon hydroxylase (AHH) activity in the S9 preparation revealed decreased AHH activity with increase in Se concentration. The decreased mutagenicity and AHH activity were Se (as Na2SeO3) dependent and could not be duplicated by sulfur (S as Na2SO3). Inhibition of AHH activity by Se provides an explanation of the mechanism of Se inhibition of BP, 3MC and 3MCE mutagenicities in S. typhimurium TA98 and TA100.

Inhibition of DNA synthesis and neoplastic cell growth by vitamin A (retinol).

The inhibitory effect of vitamin A alcohol (retinol) on the DNA synthesis and neoplastic cell growth of chemically induced carcinomas (squamous cell carcinomas in Swiss male albino mice and basal cell carcinomas in inbred SD rats) by 3-methylcholanthrene [(MCA) CAS: 56-49-5] was studied. A marked inhibition of squamous cell carcinomas and basal cell carcinomas was observed following a combined administration of MCA and vitamin A (retinol) compared to the finding for animals treated with MCA alone (P less than .001). DNA radioactivity and autoradiographic studies with the use of [3H]thymidine showed a marked inhibition of DNA synthesis (twofold to threefold) in the neoplastic cell nuclei following a concomitant administration of vitamin A (retinol) and MCA (12%) as compared to the DNA synthesis following administration of MCA alone (31%) (P less than .001). Electron microscopic and cytologic observations revealed an advanced cytolysis and disorganization of neoplastic cells with reduction of polysomes, tonofilaments, and lysosome populations and mitochondrial alterations following vitamin A and MCA administration as compared to characteristic squamous neoplastic cells and basal neoplastic cells following MCA treatment alone. In addition, scanning electron microscopy revealed advanced changes of cell surfaces with reduction of microvilli and disorganization of cytoarchitecture. The present findings demonstrate that vitamin A exerts its anticarcinogenic effect by inhibiting DNA synthesis, disrupting cell surfaces, and possibly interfering with MCA metabolism in epidermal cells.

Inhibition of 3-methylcholanthrene-induced skin tumorigenicity in BALB/c mice by chronic oral feeding of trace amounts of ellagic acid in drinking water.

Chronic p.o. feeding of small amounts of ellagic acid, a naturally occurring dietary plant phenol, to BALB/c mice in drinking water afforded significant protection against skin tumorigenesis induced by 3-methylcholanthrene, a polycyclic aromatic hydrocarbon carcinogen. A significant increase in the latent period for the development of skin tumors by 3-methylcholanthrene was observed in the ellagic acid-fed group of mice (9 wk on test) as compared to the control group of animals (6 wk on test). The observed protection against tumor induction in the ellagic acid-fed group of animals may be due to the inhibition of the metabolic activation of the polycyclic aromatic hydrocarbon since epidermal aryl hydrocarbon hydroxylase activity was found to be significantly inhibited. Our results suggest that dietary supplementation with small amounts of ellagic acid may prove useful in reducing the risk of skin carcinogenesis induced by environmental chemicals.

Cytochrome P-450 induction by 3-methylcholanthrene and its antagonism by 2,2-dimethyl-5-t-butyl-1,3-benzodioxole.

Previous studies in this laboratory have shown 2,2-dimethyl-5-t-butyl-1,3-benzodioxole (DBBD) to antagonize 3-methylcholanthrene induction of cytochrome P-450 in Dub:ICR mice yet have no effect on phenobarbital induction. In the present experiments, C57BL/6 mice, an Ah responsive strain, produced a similar response under the same experimental conditions. The hypothesis that DBBD, although not a cytochrome P-450 inducer, competes with 3-methylcholanthrene for binding to the Ah receptor was tested. Using sucrose density gradients, the Ah receptor was measured in hepatic cytosol from Dub:ICR and C57BL/6 male mice. DBBD was unable to displace either 2,3,7,8-tetra-chlorodibenzo-p-dioxin or 3-methylcholanthrene from the Ah receptor, in vitro. However, in in vivo experiments, DBBD treatment of Dub:ICR mice caused Ah receptor depression at 6 and 24 hr with complete recovery in between, while 3-methylcholanthrene treatment caused a 2-fold Ah receptor reduction at 2 hr followed by complete recovery after 12 hr. When 3-methylcholanthrene and DBBD were coadministered, the depression of the Ah receptor was additive. DBBD-pretreated mice had a 2.25-fold reduction in Ah receptor level, effectively blocking the ability of 3-methylcholanthrene to increase the cytochrome P-450 content and either benzo[a]pyrene hydroxylase or ethoxyresorufin O-deethylase activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that 3-methylcholanthrene induction of cytochrome P-450 was inhibited by DBBD pretreatment. Hence, although DBBD does not displace 3-methylcholanthrene from the Ah receptor in vitro, it does antagonize 3-methylcholanthrene induction of cytochrome P-450 and also reduces the amount of available receptor in vivo. This interaction may be due either to antagonism or to downregulation of the Ah receptor.

[Effect of epidermal chalones on the proliferation and differentiation of tumors of the lower lip in mice]. / Deistvie epidermal'nykh keilonov na proliferatsiiu i differentsirovku kletok opukholei nizhnei guby u myshei.

Methylcholanthrene solution was applied to the lower lip mucosa of 110 CC57 BR male mice during 2 months. Simultaneously with or after methylcholanthrene application part of mice was injected into the lower lip with ethanolic extract of rat skin containing epidermal G1 and G2 chalones. The remaining mice were injected with liver chalones or were left intact. Administration of the epidermal chalone produced the tumor growth retention, and the increased frequency of tumour regression, promoted tumor differentiation and decreased tumor invasion. The results indicate the possibility of using chalones as inhibitors of tumor growth.

[Inhibitory effect of remantadine hydrochloride on oncornavirus production and transformation of a continuous culture of mouse leukemia cells]. / Ingibiruiushchee deistvie solianokislogo remantadina na produktsiiu onkornavirusa i transformatsiiu perevivaemoi kul'tury leikoznykh kletok myshi.

20-methylcholantrene in a concentration of 5 micrograms/ml and exposure for 72 hours as well as in a concentration of 2.5 micrograms/ml and exposure for 72 and 240 hours caused transformation of a continuous mouse leukemia cell culture producing Rauscher leukemia virus. Remantadine hydrochloride in non-toxic concentrations (12.5 and 6.25 micrograms/ml) decreased infectious virus production by cells of this culture and prevented its transformation by 20-methylcholantrene for 6 months.

Vitamin A and its inhibitory effect as tested on Egyptian toads.

Neoplasms of the liver were induced in 18 cases out of 44 in the Egyptian toad, Bufo regularis, by injection with 20-methylcholanthrene. Dose level was 10 mg/50 g body weight once a week. However, the injection of the same dose level of this hydrocarbon together with vitamin A palmitate on the level of 20,000 IU/50 g body weight once a week was found to develop hepatomas in the liver in only 1 of 45 cases. Results of this study indicate that vitamin A palmitate inhibited the carcinogenic effect of 20-methylcholanthrene.

Inhibition of methylcholanthrene-induced skin carcinogenesis in hairless mice by dimethyl sulfoxide.

Hairless mice were given 5 topical applications of 470 nmol 20-methylcholanthrene (MCA) at one week intervals. In one experimental group the MCA was dissolved in reagent grade acetone alone, in another group it was dissolved in a mixture consisting of 50% acetone and 50% dimethyl sulfoxide (DMSO). A control group received the mixed solvent alone. The animals were observed for development of papillomas and malignant tumors during 75 weeks. The group treated with MCA in acetone/DMSO had a higher mortality than the two other groups. The admixture of 50% DMSO to the solvent had a significant inhibitory effect on tumor and cancer rates and on cancer yield, whereas no effect could be observed on the tumor yield. Hence, 50% DMSO in the solvent has a moderate, but significant, inhibitor effect on MCA-induced skin carcinogenesis. Similar inhibitory effect of DMSO on the promotion phase in two stage-carcinogenesis protocols have been reported in the literature. The biochemical mechanisms behind this effect are unknown.

Reduction in the transplacental carcinogenic effect of methylcholanthrene in mice by prior treatment with beta-naphthoflavone.

Conversion of 14C-benzo[alpha]pyrene (BP) to alkali-soluble and water-phase products, as a measure of aryl hydrocarbon metabolism, was assayed on day 18 of gestation in the livers of pregnant C57BL/6 females and their (C57BL/6 x BALB/c)F1 fetuses. BP metabolism was inducible in both maternal and fetal livers by beta-naphthoflavone (beta-NF), injected ip on day 16 of gestation at doses of 25-130 mg/kg. At 25 and 75 mg/kg beta-NF, fetal liver metabolism of BP was induced 1.5- and 4.5-fold, respectively. The corresponding results for maternal liver indicated no effect at 25 mg/kg and 2.6-fold induction at 75 mg/kg. In a complementary carcinogenesis assay, pregnant mothers were injected ip with beta-NF (25 or 75 mg/kg) on day 15 of gestation and 3-methylcholanthrene (MC) (30 or 150 mg/kg) on day 17. Appropriate vehicle-injected control mice were also obtained. The progeny were examined for lung tumors at 28 weeks of age. The average number of lung tumors per mouse caused by 150 mg/kg MC was significantly reduced by prior treatment with beta-NF, to an extent depending on the dose of the inducer. With the 75 mg/kg dose of beta-NF, the incidence of lung tumors was reduced by half. Induction of carcinogen detoxification in maternal, fetal, and/or placental tissue is a possible mechanism by which beta-NF protected against transplacental MC tumorigenesis.

Structure-activity relationships among various retinoids and their ability to inhibit neoplastic transformation and to increase cell adhesion in the C3H/10T1/2 CL8 cell line.

Various natural and synthetic retinoids have been studied for their activity in two biological systems: (a) their activity as inhibitors of methylcholanthrene-induced neoplastic transformation in the C3H/10T1/2 clone 8 mouse fibroblast line (System 1); and (b) their ability to increase the degree of adhesion of C3H/10T1/2 clone 8 cells to a plastic substrate (System 2). These activities were then compared with their known activity in maintaining epithelial differentiation (System 3). With the notable exception of retinoic acid and 13-cis-retinoic acid, which were inactive in Systems 1 and 2, an excellent correlation was observed between activities in Systems 1 and 3 for retinyl acetate, N-(4-hydroxyphenyl)retinamide, retinylidene dimedone, N-ethylretinamide, and N-benzoylretinylamine. Compounds shown to be inactive in System 1 had little or no activity in System 2. However, the ability of retinoids to cause increased adhesion could not be correlated with Systems 1 or 3 in all cases. For instance, retinyl acetate was highly active in Systems 1, 2, and 3, whereas retinylidene dimedone was highly active in Systems 1 and 3 but weakly active in System 2. Conversely, N-(4-hydroxyphenyl)retinylamide was highly active in Systems 1 and 3 but caused a decrease in System 2. The lack of activity of 3 but caused a decrease in System 2. The lack of activity of retinoic acid isomers in the C3H/10T1/2 clone 8 system is paradoxical and may provide important information on requirements for their activation and/or transport.

In vivo and in vitro inhibition of 3-methylcholanthrene-induced aryl hydrocarbon hydroxylase activity in rat liver by actinomycin D and 7,8-benzoflavone.

Using the very well investigated and sensitive microsomal monooxynogenase aryl hydrocarbon hydrolase (AHH), we investigated the dose-response relations between an inducer, 3-methylcholanthrene (3-MC), and an inhibitor of transcription, actinomycin D (ACT D), in 10 days old rats. An optimum pretreatment schedule proved to be a single dose of 1 to 40 mg/kg 3-MC ip combined with two administrations of 200 to 600 microgram/kg ACT D within 16 hr. With increasing doses of ACT D the inhibition of 3-MC-mediated AHH induction increased up to 88%. With 7,8-benzoflavone (ANF) as a relatively specific in vitro inhibitor of the cytochrome P-448 species no distinct influence on the constitutive AHH activity could be observed up to the 30th days of age. In 60 and 200 days old rats the inhibition reached a significant level of about 50%. After 3-MC induction a nearly uniform ANF-mediated inhibition by about 50% of the hepactic AHH activity was observed: in older animals the percentage became slightly higher. The investigation appears to confirm the change in cytochrome pattern,--especially as for the participation of cytochrome P-448,--according to the age and induction stimulus by 3-MC.

Modulation of cellular interactions between C3H/10T1/2 cells and their transformed counterparts by phosphodiesterase inhibitors.

It has been demonstrated previously that nontransformed C3H/10T1/2CL8 mouse embryo fibroblasts (10T1/2) can induce a state of reversible growth inhibition in cocultured malignantly transformed mouse fibroblasts and that this inhibition is modulated by serum concentration. The present study suggests that cyclic nucleotides may be implicated in this intercellular communication. The phosphodiesterase inhibitors theophylline, caffeine, and 3-isobutyl-1-methylxanthine (IBX) at concentrations of 10(-3) M, maintained continuously, were all found to inhibit the expression of 3-methylcholanthrene-induced malignant transformation when added 7 days after removal of carcinogen. IBX was the most potent, causing 100% inhibition at 10(-4) M and 70% inhibition at 10(-5) M. This inhibition was partially reversible in the former case and completely reversible in the latter case by removal of drug. Complete inhibition by 10(-4) M IBX was still observed when treatment was delayed 21 days postcarcinogen. In reconstruction experiments, utilizing confluent monolayers of 10T1/2 cells overlaid with transformed cells, IBX caused a dose-dependent inhibition of colony size of the transformed cells. Adenosine cyclic 2':3'-monophosphoric acid (cAMP) and N6,O2'-dibutyryladenosine cyclic 3':5'-monophophoric acid potentiated this response. The presence of non-transformed 10T1/2 cells was required for this effect, since a concentration of IBX (10(-4) M) inhibitory for the growth of transformed cells in mixed cultures was without effect on the growth rate, plating efficiency, or saturation density of pure cultures of 10T1/2 cells or of their transformed counterparts. Conditioned medium removed from IBX-treated 10T1/2 cells was not growth inhibitory for transformed cells, indicating a requirement for cell-cell contact. IBX caused a dose-dependent increase in intracellular cAMP in confluent 10T1/2 cells and a more pronounced increase in cAMP concentration in the culture medium of these cells. The dose-response effects of IBX on growth inhibition of malignant cells in mixed cultures appear to correlate well with its ability to elevate cAMP levels. Thus, IBX increased the capacity of 10T1/2 cells to cause reversible growth arrest of transformed cells and appears to act in a manner analogous to the previously reported effects of serum.