Inhibition of phorbol ester-induced tumor promotion in mice by vitamin A analog and anti-inflammatory steroid.

The effects of a vitamin A analog, TMMP ethyl retinoate [or ethyl-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-trans-2,4,6,8-nonatetraenoate] (abbreviated Ro 10-9359), and an anti-inflammatory steroid, fluocinolone acetonide (or 6 alpha, 9 alpha-difluoro-11 beta, 16 alpha, 17,21-tetrahydroxypregna-1,4-diene-3,20-dione cyclic 16,17-acetal) (abbreviated FA), given alone or together were studied in a two-stage carcinogensis system. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was used as the tumor promoter in a 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin system. Two stocks of female mice, CD-1 and Sencar, which differ in their degrees of sensitivity to skin carcinogenesis, were used. A dose-dependent inhibition of carcinogenic expression, as determined by a decreased number of papillomas per animal, was observed in each mouse stock with the use of both FA and Ro 10-9359 when given alone. When FA and Ro 10-9359 were given together, an enhanced effect on the lowering of tumor incidence was noted. FA effectively inhibited tumor formation in the sensitive mouse stock even when the steroid was given 1 day prior to TPA treatment under conditions of unusually high doses of initiator (DMBA) and/or promoter (TPA). These results suggest that both anti-inflammatory steroids and retinoids inhibit tumor promotion and can be effectively used as a combination regimen for increased chemopreventive response.

Lack of involvement of 6-hydroxymethylation in benzo[a]pyrene skin tumor initiation in mice.

The skin tumor-initiating activities of benzo[a]pyrene (BP), 6-hydroxymethylbenzo[a]pyrene (6-OH-CH2-BP), and 6-methylbenzo[a]pyrene (6-CH3-BP), as well as the effects of 7,8-benzoflavone (7,8-BF), quercetin, and 1-benzylimidazole on their activity, were determined in outbred female CD-1 mice by use of a two stage system of tumorigenesis. The skin tumor-initiating activity of 6-OH-CH2-BP and 6-CH3-BP was 12.5 and 20%, respectively, of the activity of BP, 7,8-BF had little effect on the skin tumor-initiating activity of 6-OH-CH2-BP and 6-CH3-BP. However, a dose-dependent inhibition of BP tumorigenesis by 7,8-BF was noted. Quercetin and 1-benzylimidazole also inhibited BP skin tumor-initiating activity. These findings indicated that direct hydroxymethylation of BP is not an important pathway in the activation of BP in mouse skin tumor initiation.

The effects of antioxidants on skin tumor initiation and aryl hydrocarbon hydroxylase.

Butylated hydroxytoluene, butylated hydroxyanisole, and vitamins C and E are effective inhibitors of 7,12-dimethylbenz(a)anthracene tumor initiation in a two-stage system of tumorigenesis. These antioxidants did not significantly induce epidermal aryl hydrocarbon [benzo(a)pyrene]hydroxylase, nor did they have any effect when added directly to the in vitro aryl hydrocarbon [benzo(a)pyrene]hydroxylase assay. However, butylated hydroxytolene and butylated hydroxyanisole, when topically to mice, inhibited the in vitro, epidermally mediated, covalent binding of radioactive benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene to DNA. When butylated hydroxytolene and butylated hydroxyanisole were added in vitro, they did not inhibit the epidermally mediated covalent binding of the hydrocarbons to DNA. The inhibition of polycyclic hydrocarbon tumorigenesis by antioxidants may be related to the ability of antioxidants to prevent the in vivo activation of hydrocarbons to carcinogenic epoxides and/or other electrophilic intermediates or may be related to their ability to increase detoxification of the reactive intermediate that requires intact cells to be operational. In any event, the results suggest that the antioxidants have an indirect effect on the epidermal metabolizing system which leads to a decrease in covalent binding to DNA.

Metabolic conversion of 12-O-tetradecanoylphorbol-13-acetate in adult and newborn mouse skin and mouse liver microsomes.

Tritiated 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied to adult mouse skin; at specified time intervals the mice were killed, and the labeled phorbol was extracted and subjected to separation and quantitation by high-pressure liquid chromatography. After 24 hr, TPA comprised greater than 96% of the recovered label from the skin, and its apparent half-life was 17.8 hr. Pretreatment of adult skin with TPA for 4 weeks before treatment with labeled TPA resulted in an increase in the clearance rate of TPA from the skin. Skin from newborn mice was capable of converting TPA into monoesters and phorbol, but the clearance rate in the adult was about 12 times more rapid than it was in the newborn. Epidermal homogenates converted TPA into 12-O-tetradecanoylphorbol, phorbol-13-acetate, and phorbol. Hepatic homogenates were able to convert TPA to monoesters and phorbol at rates 14 to 15 times faster than were epidermal homogenates. Attempts to isolate any previously undescribed metabolites of TPA by use of liver homogenates were unsuccessful, and mixed-function oxidation did not contribute to the metabolism of TPA. From inhibitor studies it was judged that esterases were implicated in the conversion of TPA to monoesters and phorbol. The results support the hypothesis that the tumor-promoting activity of TPA is directly related to its concentration in a specific tissue and that conversion of TPA to an active metabolite probably does not occur.

Carcinogenicity and mutagenicity of benz(a)anthracene diols and diol-epoxides.

Benz(a)anthracene (BA) and its five possible trans-dihydrodiols were evaluated for determination of their skin tumor-initiating activity and their mutagenic activity in Chinese hamster V79 cells. In addition, the skin tumor-initiating abilities of five diol-epoxides of BA were tested. Results showed (+/-)-trans-3,4-dihydroxy-3,4-dihydrobenz(a)anthracene (BA 3,4-dihydrodiol) to be approximately 10 times more mutagenic than was BA and about 20 times more mutagenic than were the other possible dihydrodiols in the V79 cells cocultivated with irradiated hamster embryo cells. As a skin tumor initiator, BA 3,4-dihydrodiol was approximately 5 times more active than BA, whereas the other BA dihydrodiols were all less active tumor initiators. (+/-)-trans-3alpha,4beta-Dihydroxy-1alpha,2alpha-epoxy-1,2,3,4-tetrahydrobenz(a)anthracene was found to be approximately 20% more active as a tumor initiator than was BA 3,4-dihydrodiol, whereas the other diol-epoxides of BA were less active than BA itself. The results suggest that the bay-region diol-epoxide of BA may be the ultimate carcinogen and mutagenic form of BA.

Skin tumor-initiating activities of the twelve isomeric phenols of benzo(a)pyrene.

The skin tumor-initiating activities of the 12 isomeric phenols of benzo(a)pyrene (BP) were determined in mice by use of a two-stage system of tumorigenesis. 11-Hydroxybenzo(a)pyrene was moderately active, whereas 2-hydroxybenzo(a)pyrene and BP were strong tumor initiators when applied topically to CD-1 mice and followed by twice-weekly applications of the promoter 12-O-tetradecanoylphorbol-13-acetate. 1-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, and 12-hydroxybenzo(a)pyrene had less than 5% of the tumor-initiating activity of BP when the data were expressed as papillomas per mouse. After 30 weeks of promotion, the number of papillomas per mouse was 8.4, 8.5, and 2.8, respectively, for the animals treated with BP, 2-hydroxybenzo(a)pyrene, and 11-hydroxybenzo(a)pyrene. A 5-week latency period before the appearance of the first tumor was observed after the application of either 2-hydroxybenzo(a)pyrene or BP, whereas a slightly longer latency period of 7 weeks was observed following application of 11-hydroxybenzo(a)pyrene. The time required for 50% of the animals to develop tumors was 13 weeks for animals treated with BP and 15 weeks for animals treated with 2- or 11-hydroxybenzo(a)pyrene.

Comparison of the tumor-initiating activities of benzo(a)pyrene arene oxides and diol-epoxides.

The ability of arene oxides, and diol epoxides of benzo(a)pyrene to initiate skin tumors in mice was determined by using a two-stage system of tumorigenesis. (+/-)-7beta,8alpha-Dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene was a more effective tumor initiator than was (+/-)-7beta,8alpha-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene when applied topically to CD-1 mice and then followed by twice-weekly applications of the promotor 12-O-tetradecanoylphorbol-13-acetate. (+/-)-7beta,8alpha-Dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene was approximately 20 to 30% as active as benzo(a)pyrene was as a tumor initiator. (+/-)-7beta,8alpha-Dihydroxy-7beta,8beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, benzo(a)pyrene, 9,10-oxide, and benzo(a)pyrene 11, 12-oxide, possessed about 1, 2, and 10%, respectively, of the tumor-initiating activity of benzo(a)pyrene.

The effects of weak or non-carcinogenic polycyclic hydrocarbons on 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene skin tumor-initiation.

Benzo[e]pyrene (B[e]P) inhibited 7,12-dimethylbenz[a]anthracene (DMBA) skin tumor-initiation in mice by 84%, whereas pyrene and fluoranthene inhibited DMBA initiation by 50 and 34%, respectively. However, B[e]P, pyrene and fluoranthene had either no significant effect or a slight enhancing effect on benzo[a]pyrene (B[a]P) skin tumor-initiation. In addition, B[e]P had essentially no effect on the initiating ability of (+/-)B[a]P-7 beta,8 alpha-diol-9 alpha,10 alpha-epoxide. As a tumor-initiator, B[e]P was found to have very weak activity at a 252 microgram/level (0.4 papillomas/mouse at 40 weeks) and no activity at 100 microgram. When given at a dose of 100 microgram twice weekly, B[e]P induced 2.1 papillomas/mouse at 30 weeks, and 25% of the mice had carcinomas at 40 weeks. However, B[e]P carcinogenic activity is weak when compared to B[a]P, which can induce a comparable tumor response at a dose of 5 microgram twice weekly. When B[e]P was tested as a tumor promoter at a dose of 100 microgram twice weekly after DMBA initiation, it induced 4.5 papillomas/mouse at 30 weeks and a 45% carcinoma incidence at 40 weeks, which was approximately twice as effective as B[e]P alone. The data show that B[e]P is a very weak tumor initiator, a weak complete carcinogen, a moderate tumor promoter, possibly a weak co-tumor-initiator when given with B[a]P, and a potent anit-tumor-initiator when given with DMBA. The anti-tumor initiating and co-tumor-initiating effects of B[e]P appear to be related to its ability to modify the conversion of the tumor initiator into an electrophilic intermediate(s) which are capable of covalently binding to DNA. In addition, B[e]P induced epidermal cellular proliferation which may be related to its promoting ability.

Skin-tumor-initiating ability of benzo(a)pyrene-7,8-diol-9,10-epoxide (anti) when applied topically in tetrahydrofuran.

The skin-tumor-initiating abilities of various metabolites of benzo(a)pyrene (BP) were determined in mice by using a two-stage system of tumorigenesis. We previously reported that BP-7,8-dihydrodiol (+/- trans) was approximately as potent as BP, suggesting that it may be a proximate carcinogen, but the alleged ultimate carcinogen of BP [BP-7,8-dihydrodiol-9,10-epoxide (anti)] was a weak tumor initiator (Cancer Lett.2: 115, 1976). Because of its high reactivity, the tumor-initiating ability of the BP-7,8-dihydrodiol-9,10-epoxide (anti) was determined by using acetone, benzene, and tetrahydrofuran (THF) as the solvent vehicles. The 'diol-epoxide' of BP was found to be an effective tumor initiator when applied topically in THF. The effectiveness of the various vehicles for the 'diol-epoxide' was as follows: THF greater than benzene greater than acetone; however, acetone was the best solvent for BP tumor initiation. The BP-9,10-dihydrodiol and BP-3-hydroxy were found to be weak tumor initiators. BP-3-hydroxy was also tested for tumor-promoting ability and was found to be inactive in this capacity.

The ability of enteric bacteria to catalyze the covalent binding of bile acids and cholesterol to DNA and their in ability to metabolize benzo(a)pyrene to a binding product and to known metabolites.

The capacity of enteric bacteria (E. coli, Salmonella, Pseudomonas, Shigella and Klebsiella) to catalyze the covalent binding of benzo(a)pyrene (BP), cholic acid, deoxycholic acid and cholesterol was investigated. In general, these bacteria were incapable of activating BP to a covalently bound product with calf thymus DNA. Metabolism studies of BP by fluorometric assay failed to indicate any accumulation of BP-3-hydroxy in the incubation medium. Detailed metabolic investigation with high-pressure liquid chromatography indicated that these bacteria did not produce any known metabolites which are formed by mammalian systems. However, radioactivity was detected in all fractions, suggesting that the bacteria were readily metabolizing BP into smaller molecules for energy and carbon sources. Although the enteric bacteria did not metabolize BP into known metabolites, some were capable of activating cholesterol, cholic acid and deoxycholic acid to covalently bound products with DNA. The binding data with cholesterol and bile acids also suggested that the binding process required NADPH as a cofactor because binding level was rather low without NADPH.

Cytotoxicity-related alterations of selected cellular functions after in vitro vanadate exposure.

A bovine kidney cell line was used to monitor select cellular functions for toxicity-dependent alterations in an effort to examine the cellular response to vanadium insult. The vanadium concentrations utilized ranged between 20 and 500 microM Na3VO4 (V) and elicited 15-60% cytotoxicity. Cytotoxicity-related decreases in thymidine incorporation into DNA and leucine incorporation into protein were noted. Paradoxically, V-treated cultures exhibited increased protein and DNA content, suggestive of a decrease in precursor transport. K+-dependent phosphatase (KP), acid phosphatase (AP) and succinate dehydrogenase (SDH) were monitored in surviving cells and in a cell-free system. Significant inhibitions were detected for KP and AP; SDH exhibited slight enhancement. In the cell-free system, KP was inhibited significantly at 10(-7) M V, while AP and SDH were either unchanged or sensitive only at concentrations of 10(-4) M V or greater. Measurement of reduced glutathione (GSH) in surviving cells revealed toxicity-dependent increases of up to 500% of control values. When compared to the cellular V content, the GSH:V molar ratio decreased from 1.7 to 0.5 as cell survival decreased.

Cytotoxicity related changes in biochemical cell function following in vitro cadmium treatment.

In an effort to examine cellular responses to cadmium insult a bovine kidney cell line was used to monitor select cell functions for toxicity related alterations. Cadmium concentrations used ranged between 0.2 and 2.5 microM CdCl2 and elicited 0-85% cytotoxicity (cell attachment); 24-h incubations were used for all studies. Toxicity related inhibition of leucine incorporation into cellular protein and thymidine incorporation into DNA was noted. Decreases in protein synthesis activity closely paralleled the cytotoxicity profile; DNA synthesis was a less sensitive indicator to toxicity. K+-dependent phosphatase (KP), acid phosphatase (AP) and succinate dehydrogenase (SDH) were monitored in surviving cells and in a cell-free system. Significant inhibitions were detected for all enzyme activities following a 24 h culture with cadmium. KP and AP were most sensitive. In the cell-free system KP was significantly inhibited with 0.1 microM cadmium; AP and SDH were either unchanged or sensitive only at concentrations of 100 microM cadmium or greater. Reduced glutathione (GSH) concentration in surviving cells was elevated up to 7-fold over control cultures. The elevation occurred in a progressive toxicity-related manner.

Cadmium uptake by rat red blood cells.

Rat red blood cells were employed to study the uptake of cadmium (109Cd). Suspensions of red blood cells were exposed to Cd concentrations of 0.005-500 microM, which were representative of whole blood concentrations (both bound and free) observed following in vivo Cd administration. Cd uptake was biphasic with an initial rapid phase (less than 1 min) followed by a slower second phase. The rate of Cd uptake at 25 degrees C was one-fourth of that at 37 degrees C. The metabolic inhibitors; sodium fluoride (1 mM), potassium cyanide (1 mM) and carbonyl cyanide-m-chlorophenyl hydrazone (2 microM) and the Na+-K+-ATPase inhibitor, ouabain (1 mM) did not reduce Cd (50 microM) uptake into red blood cells. This suggests that the uptake of Cd into red blood cells was not an active process. Incubation of Cd (10 microM) with an equimolar concentration of Zn did not alter uptake of Cd into red blood cells, but at 5 and 10 times higher concentrations of Zn, Cd uptake was enhanced 5-fold. Mercury at one-tenth and equimolar concentrations of Cd increased Cd uptake by red blood cells 2-fold. N-Ethylmaleimide (0.5-5 mM), which irreversibly inactivates membrane sulfhydryl groups, decreased Cd uptake. The data indicate that Cd uptake into rat red blood cells occurs by passive transport and that alterations of sulfhydryls of red blood cell membrane may modulate the process.

Comparisons of the toxicity of CdCl2 and Cd-metallothionein in isolated rat hepatocytes.

In the intact animal, inorganic Cd distributes mainly to the liver and produces hepatotoxicity, while Cd-metallothionein (CdMT) distributes primarily to the kidney and produces nephrotoxicity. CdMT has also been demonstrated to be more toxic than Cd in cultured kidney cells, but it is not known if CdMT is more toxic to all cultured cells or if there is a good correlation between in vitro and in vivo toxicity. Therefore, hepatocytes, which were isolated and grown in monolayer culture for 24 h, were incubated with CdCl2 (1-100 microM) or CdMT (3-100 microM Cd). The intracellular K+ content was quantitated 24 h later as an index of toxicity. The K+ concentration of the hepatocytes was decreased 50% by 4 microM CdCl2, whereas 25 microM CdMT was required to produce similar injury. In the intact animal, zinc induces the synthesis of MT and decreases the hepatotoxicity of Cd. ZnCl2 added to the media (100 microM) for 24 h before exposure to Cd or CdMT increased the intracellular MT concentration 700%. This elevation in MT reduced the toxicity of CdCl2 approximately 80% but did not alter the toxicity of CdMT. In summary, CdCl2 is more toxic to cultured hepatocytes than Cd-MT, and MT induction decreases the toxicity of CdCl2 in hepatocytes, as has been observed in the intact animal. This indicates that cultured hepatocytes appear to be an excellent model for examining the hepatotoxicity of Cd.

Cadmium accumulation and subcellular distribution in relation to cadmium chloride induced cytotoxicity in vitro.

A bovine kidney cell culture system was used to assess what relationship cadmium (Cd) uptake and subcellular distribution had to cadmium chloride induced cytotoxicity. Twenty-four hour incubation with 0.1-10 microM Cd elicited 0-90% cytotoxicity. Fifty percent cytotoxicity was estimated to result from 0.8 microM Cd. A concentration-related Cd accumulation paralleled the cytotoxicity profile. The time-course for Cd accumulation was linear for the first 6 h of exposure and plateaued by 18 h post-exposure. When the degree of cytotoxicity was compared with the cellular Cd burden at 24 h post-treatment a least-squares linear regression analysis (r = 0.93) indicated a direct relationship. Subcellular distribution studies indicated greater than 90% Cd recovery from the soluble supernatant (105 000 g) at all levels of cytotoxicity studied. Metallothionein sequestered less than 25% of the cellular Cd. As a result of the correlation of the degree of cytotoxicity with the cellular Cd burden and the independence of subcellular distribution from cytotoxicity, a cumulative mechanism of toxicity for Cd in MDBK cells was suggested.

Cadmium-copper interaction in intestinal mucosal cell cytosol of mice.

In vivo as well as in vitro protein-metal interaction was studied in cytosolic fractions from intestinal mucosal cells. Female Swiss-Webster mice wre pretreated with cadmium (25 ppm) or copper (100 ppm) in drinking water for 3 weeks. Treatment groups were divided into subgroups receiving Cd or Cd+Cu for an additional 6 weeks. In the in vitro study, mucosal cytosol obtained from pretreated animals was incubated with Cd-109 or Cd-109+Cu. Proteins were separated by gel filtration chromatography and metals determined by furnace AAS or gamma-spectrometry. Cadmium-induced synthesis of metallothionein-like proteins (MTP) in cytosol was indicated by increased Cd in those eluted fractions corresponding to the molecular weight of purified equine renal metallothionein. This cadmium level reached a plateau after 3 weeks of cadmium treatment. In addition, an increased amount of cadmium bound to MTP was noted when copper was added to cadmium in drinking water of mice pretreated with copper. This was not the case for Cd-pretreated animals. The in vitro experiments produced similar results, in that MTP fractions retained a greater percentage of Cd when animals were pretreated with copper compared to controls. Cadmium pretreatment resulted in even higher amounts of cadmium bound to MTP. The existence of a Cd as well as a separate Cu MTP, each with specific metal-binding properties, is suggested.