Leila Diamond

Alpha-Naphthoflavone: An Inhibitor of Hydrocarbon Cytotoxicity and Microsomal Hydroxylase

Alpha-naphthoflavone inhibits the metabolism of 3,4-benzopyrene and 7,12-dimethylbenz(a)anthracene in hamster enlbryo cell cultures and protects the cells against the inhibition of cell multiplication by these carcinogens. Alphla-nalphthoflavone also inhibits the aryl hydrocarbon hydroxylase activity in homogenates of induced hamster embryo cells and in liver microsomes from rats previously treated with polycyclic aromatic hydrocarbons, but not in microsomes from control rats.

Dimethylbenzanthracene Tumorigenesis and Aryl Hydrocarbon Hydroxylase in Mouse Skin: Inhibition by 7,8-Benzoflavone

Mouse skin contains aryl hydrocarbon hydroxylase which is highly inducible. The enzyme system is inhibited when 7,8-benzoflavone is added to homogenates of skin epidermis. 7,8-Benzoflavone also inhibits mouse skin tumorigenesis caused by repeated treatment with 9,10-dimethylbenzanthracene or by a single treatment with this chemical followed by weekly treatment with croton oil. These findings suggest that this enzyme system may be responsible for the activation of 9,10-dimethylbenzanthracene to its carcinogenic form.

Tumor promoters: Effects on proliferation and differentiation of cells in culture

Abstract Tumor promoters enhance tumor formation when administered after an initiating action by a carcinogen. The phorbol diester class of tumor promoters has been shown to affect many biochemical and biological processes in mouse skin and cell culture. The effects of these compounds on the proliferation and differentiation of cells in culture are reviewed herein; the possible relation of these effects to the mechanism of tumor promotion are discussed.

The nature of benzo(a)pyrene-DNA adducts formed in hamster embryo cells depends on the length of time of exposure to benzo(a)pyrene.

Summary Most of the benzo( a )pyrene-DNA adducts found in hamster embryo cells after 4 or 6 hr of benzo( a )pyrene treatment resulted from reaction with (±)7α,8β-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo( a )pyrene (the syn -isomer) whereas most of the adducts found at 24 or 72 hr resulted from reaction with (±)7α,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo( a )pyrene (the anti -isomer).

Metabolism of benzo(a)pyrene by fish cells in culture

Benzo[a]pyrene (BaP) metabolism was studied in cell lines derived from rainbow trout (RTG-2), bluegill fry (BF-2), and fathead minnow (FHM). Confluent cultures were exposed to 3H-BaP (0.5 nmol/ml), and, after various exposure times, metabolites were extracted from the media with an organic solvent and analyzed by high-pressure liquid chromatography. BF-2 and RTG-2 cells converted 63% of the BaP to water-soluble metabolites within 24 h, while FHM cells converted only 12%. BF-2 and RTG-2 cells metabolized more than 90% of the BaP by 48 h, while only 67% of the BaP was converted to water-soluble metabolites by FHM cells after 96 h. The major organic-solvent-extractable metabolites in all three cell lines were 9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene and unidentified polar metabolites. Of the water-soluble metabolites formed by BF-2, FHM, and RTG-2 cells, 67, 42, and 19%, respectively, were converted to ethyl-acetate-extractable metabolites by treatment with beta-glucuronidase. All three cell lines formed a glucuronide of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol); in BF-2 and FHM cells, the 7,8-diol represented almost half of the metabolites released by beta-glucuronidase treatment. Thus, cell lines derived from three widely distributed species of freshwater fish have the capacity to metabolize BaP to a form that is a proximate carcinogen in rodents and to produce a water-soluble conjugate of this metabolite.

Effect of 7,8-benzoflavone on the formation of benzo[a]pyrene-DNA-bound products in hamster embryo cells

The hydrocarbon-deoxyribonucleoside products present in enzyme digests of DNA from hamster embryo cultures that had been treated with[3H]-benzo[alpha]pyrene (BP) were isolated by chromatography on Sephadex LH20 columns. The products isolated from cells treated with 7,8-benzoflavone (7,8-BF) for 18 h prior to the addition of [3H] BP were indistinguishable from the products isolated from untreated cultures, but the amounts of these products decreased with increasing concentrations of 7,8-BF. The amount of BP metabolized was also decreased in 7,8-BF-treated cultures. The decrease in the amounts of hydrocarbon-deoxyribonucleoside products per mg DNA was logarithmic with respect to the decrease in BP metabolism. The findings are consistent with the hypothesis that 7,8-BF inhibits both an initial and a later metabolic step involved in the conversion of BP to a reactive species that binds to cellular DNA.

Inhibition of adipose conversion of BALB/c 3T3 cells by interferon and 12-O-tetradecanoylphorbol-13-acetate

The adipose conversion of BALB/c 3T3 preadipose cells is inhibited by interferon; this inhibition is directly correlation with the interferon concentration. In cultures treated with low doses of interferon and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, another inhibitor of adipose conversion (Diamond et al., 1977), the two compounds act synergistically to block differentiation. Several lines of evidence suggest that the compounds differ in the mechanism by which they inhibit adipose conversion.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate stimulates lactate production in BALBc 3T3 preadipose cells

Abstract The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which reversibly inhibits the adipose conversion of BALB c 3T3 preadipose cells, increases lactate production by these cells. The stimulation of lactate production requires 4–7 days for optimal effect. Once TPA is removed from the cultures, the rate of lactate production falls to control levels. The concentration dependence for the TPA-mediated stimulation of lactate production is similar to that for its inhibitory effect on adipose conversion. Exogenous lactate in the absence of TPA also inhibits adipose conversion. These results suggest that the ability of TPA to interfere with the normal pattern of glucose metabolism may be important in the inhibitory effect of TPA on triglyceride accumulation in these cells.

Mutagenic activity of methyl- and fluoro-substituted derivatives of polycyclic aromatic hydrocarbons in a human hepatoma (HepG2) cell-mediated assay

Several series of methyl- and fluoro-substituted polycyclic aromatic hydrocarbon (PAH) derivatives were tested for mutagenic activity in cell-mediated assays with cells of the human hepatoma cell line, HepG2, as PAH activators. The mutagenic activity of dibenz[a,h]anthracene [DB(a,h)A] increased progressively with the substitution of a methyl group at one or both non-benzo bay-region sites. At a concentration of 0.25 micrograms/ml, the mutation frequencies induced by DB(a,h)A, and 7,14-diMeDB(a,H)A were 1.3, 13.1 and 59.0 6-thioguanine-resistant colonies/10(5) viable V79 cells, respectively. Methyl groups at non-benzo bay-region sites in 3-methylcholanthrene and benzo[e]pyrene had little effect on mutagenic activity at the highest concentration which could be tested (2 micrograms/ml). The presence of a fluorine atom on the bay-region A-ring of 7,12-dimethylbenz[a]anthracene (DMBA) drastically drastically reduced mutagenic activity. At a concentration of 1.0 micrograms/ml, the mutation frequencies induced by DMBA, 1-fluoro-DMBA and 4-fluoro-DMBA were 50.5, 6.8 and 1.6, respectively. On the other hand, the mutation frequency was increased 6-fold when the fluoro substituent was in the 10-position of the D-ring of DMBA. Thus, for the most part, the relative mutagenic activities of these compounds in the HepG2 cell-mediated assay paralleled their skin tumor-initiating activity in SENCAR mice reported earlier (DiGiovanni et al., 1982, 1983a, b). These studies demonstrate the value of the HepG2 cell line as an exogenous, intact human cell activation system in short-term assays designed to evaluate the genotoxic effects of PAHs.

Analysis of metabolism of carcinogenic polycyclic hydrocarbons by position-sensing proportional counting of thin-layer chromatograms.

Abstract A procedure which combines thin-layer chromatography with position-sensing proportional counting has been developed for analyzing the metabolism of carcinogenic polycyclic aromatic hydrocarbons. The profiles of the metabolites of [3H]benzo(a)pyrene and 7,12-[12-14C]dimethylbenz(a)anthracene produced in cell culture were comparable when obtained by this procedure and by standard methods. However, position-sensing proportional counting allows simultaneous counting of all components of a sample within 10–20 min, and thereby permits the analysis of many hydrocarbon samples in a short time. In addition, the procedure eliminates the necessity of cutting or scraping carcinogen-containing thin-layer chromatograms.