Effects of cyclophosphamide and polycyclic aromatic hydrocarbons on cell growth and mixed-function oxidase activity in a human colon tumor cell line

Human colon tumor cells (cell line LS174T) retain a cytochrome P-450-containing drug metabolism system capable of hydroxylating polycyclic hydrocarbons and the anticancer drug cyclophosphamide. The hydroxylation of benzo(a)pyrene by human colon tumor cells is highly inducible. Phenobarbital plus hydrocortisone induce benzo(a)pyrene hydroxylation activities 10-fold, while benz(a)anthracene induces the rate of hydroxylation 30-fold. Cytochrome P-450 specific content is increased 2- to 3-fold by treatment with phenobarbital plus hydrocortisone and benz(a)anthracene, respectively. Addition of cyclophosphamide alone results in no increase in hydroxylation activities but causes a decrease in cell growth rate. The combination of cyclophosphamide with either of the inducers phenobarbital plus hydrocortisone or benz(a)anthracene results in markedly enhanced inhibition of cell growth as judged both by a decrease in the number of cells per plate and in the incorporation of [3H]thymidine into DNA. Thus, these data show that cyclophosphamide is cytotoxic to human colon tumor cells and that the cytotoxicity is enhanced by simultaneous administration of benz(a)anthracene or phenobarbital plus hydrocortisone to the tissue cultures.     

Genetic control of the regulation of cell susceptibility to carcinogenic polycyclic hydrocarbons by cyclic AMP

The metabolism of benzo (a) pyrene (BP) in normal golden hamster and BHK cells in culture was increased by treating the cells with dibutyryl cyclic AMP (dcAMP), prostaglandin E¹, theohylline or aminophylline. The largest increase, 6-fold for the normal cells and 20-fold for the BHK cells, was obtained by treatment with both dcAMP and aminophylline. Treatment with aminophylline also stimulated the metabolism of 20-methylcholanthrene and 7,12-dimethylbenz (a) anthracene. The increased metabolism of these three carcinogenic polycyclic hydrocarbons was associated with an increased cytotoxicity. Treatment with aminophylline increased the cytotoxicity of five other potent and weak carcinogenic polycyclic hydrocarbons, but not of two non-carcinogenic polycyclic hydrocarbons. The amount of BP metabolism in 27 different cell types from various mammals, including humans, ranged from less than 0.1 μg to 2.3 μg metabolized BP per 10⁶ cells. Treatment of these different cell types with aminophylline gave either an increase in BP metabolism, an induction of metabolism in cells that did not metabolize without aminophylline, or no induction after treatment with aminophylline and dcAMP. The existence of responding and non-responding cell lines indicates that the regulation of the level of polycyclic hydrocarbon metabolism by dcAMP is genetically controlled. The induction of metabolism in cells that did not metabolize without aminophylline resulted in the conversion of cell resistance to cell susceptibility to the cytotoxic effect of BP. Treatment with dcAMP and aminophylline can therefore be used to increase the sensitivity of screening tests for chemical carcinogens.     

Genetic differences in metabolism of benzo(a)pyrene in cultured epidermal and dermal cells of responsive and nonresponsive mice

Genetic differences in metabolism of benzo(a)pyrene (BP) were investigated using cultured epidermal keratinocytes and dermal fibroblasts isolated from four inbred strains of mice: C3H/HeJms and C57BL/6J that are responsive; and DBA/2 and AKR/Jms that are nonresponsive in terms of inducibility of aryl hydrocarbon hydroxylase (AHH) activity. Primary cultures of epidermal and dermal cells isolated from newborn mice were treated with benz(a)anthracene for 24 hr. In both types of cells, AHH activity was induced in all four strains of mice, irrespective of their responsiveness in vivo. In the epidermal cells, basal AHH activity varied within a relatively small range of 4.6 to 8.8 pmol per mg protein per hr, and the activity was increased 4.4 to 8.7 times by benz(a)anthracene treatment. There was no difference in the extent of the induction in different strains of mice. In dermal cells, the basal level of AHH activity was in the range of 3.6 to 9.1 pmol per mg protein per hr, and benz(a)anthracene treatment induced AHH, but to various degrees depending on the responsiveness in vivo. Epidermal cells of the responsive mice metabolized more than 90% of the added BP in 48 hr, while those of nonresponsive mice metabolized only 60 to 70%. Dermal cells also metabolized BP, but to a lesser extent than did epidermal cells, and there was no strain difference in its metabolism. Time-course studies revealed that epidermal cells of responsive mice metabolized BP more rapidly than did those of nonresponsive mice. The activities of epidermal and dermal cells to metabolize BP were further demonstrated by a cell-mediated mutation assay. Consistent with the results of time-course analysis of the metabolites, shorter treatments with BP (less than 24 hr) showed a clear difference in BP metabolism associated with responsiveness.     

Binding of polynuclear aromatic hydrocarbons to the rat 4S cytosolic binding protein: structure-activity relationships

The relative competitive binding affinities of benzo[a]pyrene (B[a]P), benzo[e]pyrene, benzo[g, h, i]perylene, picene, 7,12-dimethylbenz [a]anthracene, 1,2,3,4-dibenz[a]anthracene, 1,2,5,6-dibenz[a]anthracene, perylene, 4H-cyclopenta[d,e,f]-phenanthrene, benz[a] anthracene, triphenylethylene and triptycene for the rat hepatic cytosolic 4S binding protein were determined using [3H]benzo[a]pyrene as the radioligand. With the exception of triphenlethylene, triptycene and 4H-cyclopenta[d,e,f]phenanthrene, the EC50 values for the remainder of these compounds were between 1.25 X 10(-7) and 2.5 X 10(-8) M with 1,2,5,6-dibenz[a]anthracene being the most active ligand. A comparison of the relative cytosolic Ah (9S) receptor binding affinities and aryl hydrocarbon hydroxylase (AHH) induction potencies of these hydrocarbons with their 4S protein binding affinities demonstrated the following: five compounds, namely 1,2,5,6-dibenz[a]-anthracene, 1,2,3,4-dibenz[a]anthracene, picene, benzo[a]pyrene and 3-methylcholanthrene exhibited high to moderate binding affinities for the 4S and 9S cytosolic proteins (EC50 values less than 10(-6) M) and induced AHH in rat hepatoma cells; three compounds, namely perylene, benzo[e]pyrene and benzo[g,h,i]perylene exhibited high affinities for the 4S binding protein (1.25 X 10(-7), 4.4 X 10(-8) and 2.9 X 10(-8) M, respectively) and low affinities (EC50 values greater than 10(-5) M) for the Ah receptor protein; moreover these three compounds did not induce AHH in rat hepatoma H-4-II E cells in culture. These data suggest that the 4S binding protein may not play a significant role in AHH induction although the results do not rule out a function for this protein in the transregulation of AHH and its associated cytochromes P-450.     

Metabolism of the carcinogenic hydrocarbon benzo(a)pyrene in human fibroblast and epithelial cells. II. Differences in metabolism to water-soluble products and aryl hydrocarbon hydroxylase activity

Aryl hydrocarbon (benzo(a)pyrene) hydroxylase (AHH) activity and metabolism of benzo(a)pyrene to water-soluble products were measured in cultures of body fibroblasts and kidney epithelial cells from different human embryos. AHH activity at 24 h after treatment with or without benz(a)anthracene was determined in cultures from 23 embryos, and 3 days' accumulated metabolism of benzo(a)pyrene to water soluble products was measured in cultures from 18 embryos. The body fibroblasts from the different embryos could be divided into three groups according to the amount of water-soluble products, but not according to the AHH activity. These three groups were not found by either assay in the cultures of kidney epithelial cells. In both fibroblast and epithelial cells, high metabolism to water-soluble products was not necessarily associated with high AHH activity. The results extend our previous finding (Huberman and Sachs, 1973) of three presumably genetic groups for BP metabolism to water-soluble products in human fibroblast but not in epithelial cells and indicate that this grouping was not found in these cells by measuring AHH activity.     

Characterization of the Ah receptor and aryl hydrocarbon hydroxylase induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin and benz(a)anthracene in the human A431 squamous cell carcinoma line

Certain human cell lines previously have been shown to exhibit substantial induction of aryl hydrocarbon hydroxylase (AHH, cytochrome P450IA1) when treated in culture with aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benz(a)anthracene. Yet the Ah receptor, which is known to mediate the AHH induction process in rodent cells and tissues, has not previously appeared to be present at a significant level in any human cell line. In the human A431 squamous cell carcinoma line we found that cytosolic Ah receptor was present in high concentration (approximately 200 fmol/mg cytosol protein at maximal saturation); this corresponds to approximately 10,000 Ah receptor sites per cell in the human A431 line compared with about 35,000 receptor sites per cell in the mouse Hepa-1 hepatoma cell line in which Ah receptor previously has been extensively characterized. Detection of Ah receptor in A431 cytosol required modification of assay techniques, especially reduction in the amount of charcoal used to adsorb nonspecifically bound radioligand. The specific binding peak from A431 cytosol sedimented approximately 9S on sucrose gradients, the same as the cytosolic receptor from the well-characterized mouse Hepa-1 hepatoma cell line. In addition to [3H]TCDD, specific binding to Ah receptor in A431 cytosol also was detected with [3H]3-methylcholanthrene and with [3H]benzo(a)pyrene as radioligands. A specific [3H]TCDD-Ah receptor complex was extracted from nuclei of A431 cells incubated in culture at 37 degrees C with [3H]TCDD. The nuclear form of Ah receptor sedimented approximately 5S, the same as the nuclear receptor from mouse Hepa-1 cells. AHH activity was induced in A431 cells treated in culture with TCDD or benz(a)anthracene. The maximum level of induced AHH activity that could be achieved in A431 cells was about 20% of the maximally induced level in the mouse Hepa-1 cell line. However, the dose-response curves for AHH induction by TCDD or benz(alpha)anthracene in A431 cells were shifted about one log unit to the right of the curves for Hepa-1 cells. The lower sensitivity of A431 cells to AHH inducers was in proportion to the lower affinity with which cytosolic Ah receptor in A431 cells bound [3H]TCDD. The saturation curve for binding of [3H]TCDD to cytosolic Ah receptor in A431 cells also was shifted about one log unit to the right of the curve for saturation of the cytosolic receptor from mouse Hepa-1 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of an assay for aryl hydrocarbon (benzo(a)pyrene) hydroxylase in human peripheral blood monocytes.

An assay has been developed and measurements of aryl hydrocarbon [benzo(a)pyrene] hydroxylase have been made in peripheral blood monocytes from a human population. Treatment with benz(a)anthracene in cell culture increased aryl hydrocarbon hydroxylase activity from 6.5 to 37-fold in monocytes from each of 25 apparently healthy donors. A weak correlation (r = 0.38) was observed between the induction ratios obtained with monocytes and lymphocytes from the same donors. Reproducibilities of the monocyte and lymphocyte assays were comparable. In monocytes, the measurement of basal and induced aryl hydrocarbon hydroxylases activity does not require pretreatment with mitogens as is the case with lymphocytes. Monocytes also exhibit a much wider range of induction ratios than do lymphocytes.     

Inter-individual variations of arylhydrocarbon-hydroxylase activity in cultured human epidermal and dermal cells

Arylhydrocarbon hydroxylase (AHH) activity was determined in cultured human epidermal and dermal cells with and without induction by benz[a]anthracene (BA). Large inter-individual variations were found in the basal and induced AHH activities and the induction ratio. The average basal AHH activity of 29 epidermal cultures in primary culture was 3.9 units (SD, 4.2; range, 0-16.3) (one unit was defined as 1 pmol of 3-hydroxybenzo[a]pyrene formed/mg protein/hr). AHH activity was induced time- and dose-dependently by BA. The average AHH activity induced by treatment with 13 microM BA for 24 hr was 97.4 units (SD, 69.4; range, 2.7-216.9). The induction ratio ranged from 0.6 to 262.0, but in the majority of cultures (15 of 27 cultures, 55%) it was less than 30. The mean induction ratio and SD was 51.1 +/- 63.0. The frequency distributions of basal AHH activity and the induction ratio were asymmetrical with a right tail. No correlation was found between the AHH activity or induction ratio and the age or sex of the donors or their residential district. The basal and induced AHH activities of dermal fibroblasts were both lower than those of epidermal cells; the average basal AHH level of 14 cultures was 1.5 units (SD, 1.4; range, 0-5.2), while the average induced AHH activity was 9.6 units (SD, 6.3; range 0-23.5). The induction ratio of dermal fibroblasts was lower than that of epidermal cells, the mean and SD being 7.5 +/- 7.4 (range, 1.9-27.0). There was no correlation between the AHH activities or induction ratios in epidermal and dermal cells isolated from the same donors.     

Induction and properties of aryl hydrocarbon hydroxylase in bovine pancreatic ducts.

Inducibility and characteristics of aryl hydrocarbon hydroxylase (AHH) in cultured bovine pancreatic ducts were studied by the fluorometric method. AHH was present and inducible in all the pancreatic ducts studied when they were exposed to 20 microgram benz[a]anthracene (BA)/ml medium. AHH activity in the control tissue ranged from 1.0 to 3.0 U/mg DNA, whereas the activity in the BA-treated tissue was 4.2--28.5 U/mg DNA, which resulted in the induction of 5- to 18-fold activity. At 12 hours of BA exposure, AHH activity in the treated tissue was 12-fold that in the control tissue and continued to increase to 15-, 19-, and 31-fold that in the control tissue at 24, 48, and 72 hours, respectively. The BA-induced AHH activity had a broad pH optimum between 7.1 and 7.7, and the maximum activity was found at pH 7.4. The AHH activity was linear with respect to the incubation time up to 30 minutes. The effect of the benzo[a]pyrene concentration on AHH activity was studied on the BA-induced enzyme. The apparent Michaelis constant for the substrate was 0.5 microM, and the maximum velocity was 8.6 U/mg DNA. BA-induced AHH activity was inhibited 65% by 100 microM 7,8-benzoflavone, whereas the control enzyme activity was stimulated 100% by the same concentration of 7,8-benzoflavone.     

Polycyclic hydrocarbon-produced toxicity, transformation, and chromosomal aberrations as a function of aryl hydrocarbon hydroxylase activity in cell cultures

The cytotoxic effect of benzo[a]pyrene alone in fetal rat hepatocytes in culture is prevented by phenobarbital. Benzo[a]pyrene is not toxic to HTC, A9, or HeLa cells in which aryl hydrocarbon hydroxylase activity is either absent or very low; however, benzo[a]pyrene is cytotoxic to each of these three established cell lines grown together with the liver cells in the presence of phenobarbital. Polycyclic hydrocarbon-produced cytotoxicity is associated with chromatid breaks, whereas aneuploidy is more closely correlated with malignant transformation of hamster secondary cultures. Benz[a]anthracene or a-naphthoflavone competitively inhibits the hydroxylation of other polycyclic hydrocarbons such as the carcinogens 7,12-dimethylbenz[a]anthracene or benzo[a]pyrene. The exposure of fetal hamster secondary cells to excessive amounts of benz[a]anthracene prior to, during, and following treatment with 7,12-dimethylbenz[a]anthracene prevents malignant cell transformation from occurring.     

Correlation between transformation potential and inducible enzyme levels of hamster embryo cells

When benzo(a)pyrene was used to evaluate the transformabilityof 129 hamster embryo cell preparations from pooled or individualembryos, approximately 50% of the cultures were transformable.A transformable and a non-transformable cell culture were furthertested with other carcinogens (3-methylcholanthrene [MCA], benzylchloride, ethyl-p-toluenesulfonate, 2-naphthylamine, and aflatoxinB₁). The transformable culture responded to all of the carcinogenswhile the non-transformable culture always gave negative results.Aryl hydrocarbon hydroxylase (AHH) and epoxide hydrase (EH)levels were compared in the two cell cultures using ß-naphthoflavone(BNF), benz(a)anthracene (BA), sodium phenobarbital (PB) orMCA as microsomal enzyme-inducing agents. It was found thatAHH levels and the degree of induction following treatment ofthe cells with BNF or BA were consistently higher in the transformablethan in the non-transformable cell culture. EH levels were greaterin the transformable cells following treatment with either BNF,BA, PB or MCA. Inducible AHH and EH levels might, therefore,be useful as predictors of the transformation potential of hamsterembryo cell cultures.     

The effect of modifiers of microsomal enzymes on chemical oncogenesis in cultures of C3H mouse cell lines.

Two cell lines, both derived from the C3H mouse and each having different responses (oncogenic and cytotoxic) to polycyclic aromatic hydrocarbon oncogens, were studied with respect to their drug-metabolizing enzymes. The 10T1/2CL8 cells (a C3H mouse embryo fibroblastic cell line) were much more effective in converting 3-methylcholanthrene (3-MC) to 3-MC water-soluble metabolites, 3-MC phenols, and 3-MC-bound cellular macromolecules than were CVP3SC6 cells (a new line of C3H mouse adult ventral prostate fibroblasts). Basal aryl hydrocarbon hydroxylase activity was higher in 10T1/2CL8 cells than in CVP3SC6 cells, while the reverse was found for epoxide hydrase activity (using 3-methylcholanthrene-11, 12-oxide as substrate. 3-MC or benz(a)anthracene induced epoxide hydrase activity in both cell lines to about the same extent. 3-MC did not induce aryl hydrocarbon hydroxylase activity in CVP3SC6 cells. Aryl hydrocarbon hydroxylase activity was markedly induced in both cell lines by benz(a)anthracene and was slightly induced in 10T1/2CL8 cells by 3-MC. In a chemical oncogenesis cell culture system, transformation of 10T1/2CL8 cells mediated by 3-MC could be increased two- to threefold by treating the cell cultures with: either benz(a)anthracene, styrene oxide, cyclohexene oxide, or 1,2,3,4-tetrahydrona=phthalene-1,2-oxide; or with cyclohexene or 1,2-dihydrona-phthalene, alkene precursors of cyclohexene oxide and 1,2,3,4-tetrahydronaphthalene-1,2-oxide, respectively. When 10T1/2CL8 cells were treated with a combination of benz(a)anthracene and cyclohexene, 3-MC-mediated transformation was increased 7.8-fold. CVP3SC6 cells that were not transformed by 3-MC or other hydrocarbon oncogens were transformed by a combined treatment with benz(a)anthracene, 1,2-dihydronaphthalene, and 3-MC.     

Purification and characterization of cytochrome P-450 induced by benz(a)anthracene in mouse skin microsomes

Topical application of benz(a)anthracene to mouse skin elicited a 2-fold increase in cytochrome P-450 content, with accompanying increases in monooxygenase activities such as benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and acetanilide 4-hydroxylation, in the microsomes. A major form of cytochrome P-450 was purified from skin microsomes of mice treated with polycyclic aromatic hydrocarbon. A specific content of 1.95 nmol/mg of protein, which corresponded to 48-fold purification from the microsomes, was observed. The purified protein produced a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a molecular weight of 55,000. Using Western blotting, the band immunochemically cross-reacted with antibody which had been raised against rat liver cytochrome P-450MC-1. The purified preparation efficiently catalyzed benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation when reconstituted with NADPH-cytochrome P-450 reductase. These activities were inhibited by 7,8-benzoflavone as well as anti-cytochrome P-450MC-1 antibody, but not by P-450PB-1 antibody. The results indicate that, in mouse skin microsomes, a cytochrome P-450 induced by benz(a)anthracene is enzymatically and immunochemically similar to rat liver cytochrome P-450MC-1. It is suggested that this enzyme plays an important role in the activation of carcinogenic polycyclic aromatic hydrocarbons.     

Activation of carcinogenic polycyclic hydrocarbons in polyoma-virus-transformed cells as a prerequisite for polyoma virus induction.

Polyoma-virus (PV)-transformed cell clones, which are inducible for virus synthesis by various physical and chemical agents, metabolize the chemically non-reactive carcinogen benzo(a)pyrene (BP) into water soluble products. In cultures of such clones, which metabolize BP to a level of 30-6-% of that of normal cells, up to 10.4% of the cells were induced for PV synthesis by BP, 20-methylcholanthrene (MCA) and 7,12-dimethylbenz(a)anthracene (CMBA). No PV induction was observed with the non-carcinogenic polycyclic hydrocarbons pyrene chrysene and benz(a)-anthracene. A proportion of subclones, isolated from a PV- inducible clone, which metabolized 0.1 mug or less BP per 10-6 cells were all inducible for PV synthesis by these carcinogens. Subclones isolated from an inducible clone pretreated with BP were shown to metabolize less than 0.1 mu BP per 10-6 cells and were resistant to virus induction by the carcinogenic polycyclic hydrocarbons. Benzoflavone, which inhibited the metabolism of BP in clones metabolizing high levels of this carcinogen, also prevented the induction of PV antigen and infectious virus synthesis in these clones. The data indicate a relationship between the carcinogenicity of polycyclic hydrocarbons and their ability to induce virus in the PV-transformed cells and suggest that virus induction depends on metabolic conversion of these hydrocarbons into similar reactive compounds that are responsible for malignant transformation and mutagenesis.     

Population distribution of placental benzo(a)pyrene metabolism in smokers

Human placental microsomes isolated from term placentas derived from nonsmoking women and women smoking 1 to 40 cigarettes a day were analyzed for the metabolism of benzo(a)pyrene measured as various metabolites by HPLC and/or as aryl hydrocarbon hydroxylase (AHH)6 activity. In accordance with other reports, AHH activity was several times higher in smokers than in nonsmokers. Regression analysis on 13 different placental tissues from women smoking from 1 to 40 cigarettes demonstrated a high correlation (r = 0.8 to 0.9) between AHH activity (or the formation of benzo(a)pyrene phenols resolved by HPLC) versus the formation of the procarcinogenic benzo(a)pyrene-7,8-diol. Subsequent studies on placentas derived from 67 women who smoked 10 to 40 cigarettes per day demonstrated a definite dose-response relationship between AHH activity and the number of cigarettes smoked/day. The dose-response curve was sigmoidal in shape; however, when the data were plotted on a semi-log scale the curve assumed a linear shape, reaching saturation of AHH induction beyond 20 to 25 cigarettes/day. While mean AHH activity was dependent upon the number of cigarettes smoked/day, considerable interindividual variability in AHH (ranging more than 1,000-fold in some cases) was observed among individuals with comparable smoking histories, i.e. smoking the same number of cigarettes. Population distribution suggested clustering of the population in the low-AHH-activity region while cord-blood thiocyanate analysis and twin studies suggested that genetic factors contributed to a major portion of the inter-individual variability in AHH activity observed among smokers.