Polycyclic aromatic hydrocarbon toxicity and induction of metabolism in cultivated esophageal and epidermal keratinocytes

Serially cultivated keratinocytes of human and rat epidermis and esophagus were compared with respect to their sensitivity to toxic effects of 3-methylcholanthrene and ability to metabolize benzo(a)pyrene. 3-Methylcholanthrene was highly toxic to the human keratinocytes and to early-passage rat epidermal keratinocytes, as evidenced by markedly reduced growth upon continuous exposure or reduced colony-forming ability after 1-day exposure to concentrations of 0.4 to 40 microM in the culture medium. Rat esophageal and late-passage rat epidermal cells appeared insensitive to 3-methylcholanthrene by these criteria. All the cell types except late-passage rat epidermal cells metabolized benzo(a)pyrene at comparable rates, and expressed aryl hydrocarbon hydroxylase maximally inducible by similar concentrations of 3-methylcholanthrene. Biotransformation in the human cells was greatly inhibited by alpha-naphthoflavone, a specific inhibitor of aryl hydrocarbon hydroxylase. The lack of toxicity of 3-methylcholanthrene toward late-passage rat epidermal cells can be attributed to the low constitutive rate of biotransformation these cells exhibit. The insensitivity of rat esophageal cells despite substantial metabolic activity reflects the importance of intrinsic differences among keratinocytes derived from different epithelia and species in determining toxic response. Human cervical and monkey esophageal keratinocyte cultures also actively metabolized benzo(a)pyrene, illustrating further the utility of the culture system for exploring differences among species and epithelial cell types.     

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.     

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.     

Induction of transformation by six classes of chemical carcinogens in adult rat liver epithelial cells

A transformation assay system using two clonal strains of adult rat liver epithelial cells is described. The carcinogens N-methyl-N'-nitro-N-nitrosoguanidine, methylmethanesulfonate, N-2-fluorenylacetamide, aflatoxin B1, benzo(a)pyrene, dimethylnitrosamine, nitrosopyrrolidine, and dimethyl-4-aminoazobenzene induced anchorage independency in one or both clonal strains, whereas noncarcinogenic analogues were inactive. In the absence of carcinogen exposure, the cell strains exhibited no spontaneous transformation to anchorage independency. These results demonstrate the reliability of anchorage independency as an in vitro end point and the specific responsiveness of adult rat liver epithelial cells to a wide range of carcinogens. Thus, the assay of transformation in adult rat liver epithelial cells is potentially useful for detection of chemical carcinogens and the study of their mechanisms of action.     

Interspecies comparison of human and rat mammary epithelial cell-mediated mutagenesis by polycyclic aromatic hydrocarbons

In order to compare the interactions of procarcinogens with mammary cells from humans and rats, a uniform set of mediated mutagenesis assays has been established. In these assays, species-specific mammary epithelial cells activate procarcinogens, and specific locus mutations are quantitated in cocultured V-79 cells. Mutations were induced in the rat mammary cell coculture system exposed to 7,12-dimethylbenz(a)anthracene but not benzo(a)pyrene. In contrast, in the human mammary cell coculture system benzo(a)pyrene was much more effective than 7,12-dimethylbenz(a)anthracene in the induction of mutations. These results suggest caution in extrapolating carcinogenesis data between rodents and humans. They also suggest that the relationship between the ubiquitous environmental xenobiotic benzo(a)pyrene and the etiology of human breast cancer requires further exploration.     

Interspecies comparison of polycyclic aromatic hydrocarbon metabolism in human and rat mammary epithelial cells

Our laboratory has developed optimized and uniform methods for the isolation and culture of normal mammary epithelial cells from both rats and humans. We have reported that, in a cell-mediated mutagenesis assay, treatment of rat mammary epithelial cells with 7,12-dimethylbenz(a)anthracene, but not benzo(a)pyrene, resulted in significant rates of mutagenesis in cocultured V-79 cells. An opposite mutation pattern was found with human cells under identical conditions. To determine the mechanism of this species-specific difference in polycyclic aromatic hydrocarbon-induced mutagenesis patterns, we then studied the abilities of the human and rat mammary epithelial cells to metabolize benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene. Quantitative levels of carcinogen metabolism were found to be highly dependent on the cell culture densities, although this factor appeared to have little qualitative effect. The most significant qualitative difference in polycyclic aromatic hydrocarbon metabolism between the two species was the ability of the rat, but not the human, mammary epithelial cells to conjugate significant amounts of either polycyclic aromatic hydrocarbon to glucuronic acid. Other aspects of carcinogen metabolism, including production of the precursors to known active metabolites of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene, were similar though not identical. These results, which address only primary metabolism of the polycyclic aromatic hydrocarbons, do not indicate a simple metabolic explanation for the species-specific pattern found in the mammary cell-mediated mutagenesis assay. They do suggest that the effects of cell culture density must be carefully considered in order to properly analyze either interindividual or species differences in carcinogen metabolism.     

Biochemical activation of aryl hydrocarbon hydroxylase activity, cellular distribution of polynuclear hydrocarbon metabolites, and DNA damage by polynuclear hydrocarbon products in human cells in vitro.

Carcinogenic polynuclear hydrocarbons [7,12-dimethylbenzanthracene, 3-methylcholanthrene, and benzo(a)pyrene] were added to human skin fibroblast cell cultures. Only benzo(a)pyrene at 10 microgram/ml or above induced mixed-function hydroxylase activity, altered cell proliferation kinetics, and caused DNA damage as measured by altered grain count and bromodeoxyuridine incorporation. 3-Methylcholanthrene at concentrations as high as 15 microgram/ml was ineffective. 7,12-Dimethylbenzanthracene at 6 microgram/ml or above induced mixed-function oxygenase and stimulated DNA synthesis and cell proliferation, but at those concentrations little or no cytotoxicity or DNA damage was detected. The noncarcinogenic analogs 6,8,12-trimethylbenzanthracene, 5-fluorodimethylbenzanthracene, anthracene, and phenanthrene had no detectable effect on the human cells. It was concluded that benzo(a)pyrene can initiate all the biochemical events in human cells probably necessary to initiate transformation of human cells in vitro.     

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.     

Metabolism of benzo(a)pyrene by variant mouse hepatoma cells

Four mouse hepatoma cell lines, a parent (Hepa-1c1c7) and three variants (MUL12, BPrc1, and TAOc1BPrc1) which had been derived from Hepa-1c1c7 by the fluorescence-activated cell sorter, were incubated with benzo(a)pyrene, and the metabolites were analyzed by high-pressure liquid chromatography. Among these four cell lines, Hepa-1c1c7 and MUL12 metabolized benzo(a)pyrene the most quickly and to the greatest extent, and BPrc1 had the weakest metabolic activity for this substrate. TAOc1BPrc1 had intermediate benzo(a)pyrene-metabolizing activity, depending on cell density and incubation time. At low cell density, the active variant TAOc1Bprc1 resembled the weakly active Bprc1 in accumulating a low amount of ethyl acetate-soluble metabolites in the medium while, at high cell density, TAOc1Bprc1 resembled the parent clone Hepa-1c1c7 and the highly active variant MUL12. At short incubation times, TAOc1Bprc1 also had low conjugating activity while, at longer incubation times, the conjugating activity approached that of Hepa-1c1c7 and MUL12. At low cell density, Bprc1 was able to produce phenols, but this variant did not seem to have this ability at high cell density. When the substrate concentration was 4 microM and the incubation time was 24 h, beta-glucuronidase treatment of water-soluble metabolites released about 5.3 times more pmol of quinones compared with phenols. But when the substrate concentration was 25 nM, beta-glucuronidase released about 2.0 times as many phenols compared with quinones. The parent and the two more actively metabolizing variants showed differences in the peak times of accumulation of 9,10-diol and 7,8-diol of benzo(a)pyrene, which may have implications for binding to DNA and nuclear proteins. It was concluded that BPrc1 has basal but not easily inducible aryl hydrocarbon hydroxylase activity, whereas Hepa-1c1c7, MUL12, and TAOc1Bprc1 have basal and inducible aryl hydrocarbon hydroxylase activity. These results show that variants of a single parent cell line can exhibit significant differences in the rate and extent of metabolism of benzo(a)pyrene.     

Age- and sex-dependent induction of liver microsomal benzo[a]pyrene hydroxylase activity in rats treated with pregnenolone-16{alpha}-carbonitrile (PCN)

The present studies were undertaken to resolve conflicting reportson the inducibility of liver microsomal benzo[a]pyrene hydroxylaseactivity in rats treated with pregnenolone 16-carbonitrile (PCN).Several studies have shown that treatment of Long Evans ratswith PCN causes a 5- to 10-fold induction of benzo[a]pyrenehydroxylase activity, whereas little or no induction occursin Sprague-Dawley or Wistar rats. Studies with one-month-old-maleLong Evans, Sprague-Dawley, Wistar and Holtzman rats failedto reveal an anticipated strain difference in the inducibilityof benzo[a]pyrene hydroxylase activity by PCN. Studies withimmature and mature male and female Long Evans rats revealedthat the inducibility of benzo[a]pyrene hydroxylase activitydecreases with age in male but not female rats, i.e., PCN inducedbenzo[a]pyrene hydroxylase activity 5- to 8-fold in immaturemale, immature female and mature female rats but only 2-foldin mature male rats. The age-dependent decrease in inducibilityby PCN in male rats coincided with an age-dependent increase(2.4-fold) in the basal activity of benzo[a]pyrene hydroxylase.These sex-dependent developmental changes can be explained byan age-dependent increase in the constitutive levels of themajor PCN-inducible form of cytochrome P-450 (cytochrome P-450-PCN)in male but not female rats. Electrophoresis of liver microsomesand studies on the binding of metyrapone to dithionite-reducedcytochrome P-450 provided additional evidence for age- and sex-dependentdifferences in the levels of microsomal cytochrome P-450-PCN.In addition to reconciling the conflicting literature reports,the age- and sex-dependent differences in cytochrome P-450-PCNlevels account, at least in part, for age and sex differencesin certain liver microsomal enzyme activities, including benzo[a]pyrenehydroxylase activity.     

Activation of the colon carcinogen 1,2-dimethylhydrazine in a rat colon cell-mediated mutagenesis assay

Suspensions of rat colon epithelial cells metabolized the potent colon carcinogen, 1,2-[14C]dimethylhydrazine (DMH), into 14C-labeled, alkali-soluble volatile products, presumably CO2. The colon cell suspensions, however, were less effective than rat hepatocyte suspensions. In addition, we used a cell-mediated mutagenesis assay to test rat colon epithelial cells grown from tissue explants for their ability to metabolize DMH into products mutagenic for human P3 teratoma cells. Mutagenesis in the P3 cells was indicated by an acquired resistance to 6-thioguanine. Cocultivation of the colon cells with the P3 cells in the cell-mediated assay resulted in mutagenesis, whereas in the absence of the colon cells, no mutagenesis by DMH was observed. Similar results were obtained in a hepatocyte-mediated mutagenesis assay. Colon cells were also able to activate another carcinogen, benzo(a)pyrene, into products mutagenic for the P3 cells. Individual epithelial clonal populations isolated from the colon cultures grown from tissue explants, however, expressed different capacities to activate DMH and benzo(a)pyrene into mutagens, and a high degree of DMH activation by cells from a colon clone was not necessarily associated with a similar degree of benzo(a)pyrene activation. Our results indicate that the colon itself contains epithelial cell types capable of effectively converting DMH into mutagenic (and presumably carcinogenic) products without necessarily involving intermediary metabolism by hepatocytes as previously thought.     

Enhancement of benzo(a)pyrene metabolism mediated by retinol acetate in cultured human bronchial epithelial-cells

As suggested by animal studies and human epidemiological data, retinoids possess significant cancer chemopreventive activity. Although the majority of studies in this area have focused on the ability of retinoids to prevent the promotion or progression of carcinogenesis, a significant amount of data suggest retinoids can alter initiation events. In the current report, we have evaluated the potential of retinol acetate to modulate benzo(a)pyrene metabolism in low-passage human bronchial epithelial cells in monolayer cultures, Of 16 different cell cultures, benzo(a)pyrene metabolism was increased in 14, decreased in one, and unchanged in one, when retinol acetate was added to the media, In a preliminary study with one of the cell cultures in which retinol acetate significantly enhanced benzo(a)pyrene metabolism, binding of carcinogen metabolites to;DNA was unaffected, Since retinoids are known cancer chemopreventive agents and carcinogen binding to DNA is the key event in the initiation of carcinogenesis, these results suggest that retinoids may decrease carcinogenic risk by increasing the detoxification of procarcinogens such as benzo(a)pyrene ina manner that does not yield a concomitant increase in damage to critical cellular targets such as DNA.     

Lower induction of p53 and decreased apoptosis in NQO1-null mice lead to increased sensitivity to chemical-induced skin carcinogenesis

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a cytosolic protein that catalyzes metabolic detoxification of quinones and protects cells against redox cycling and oxidative stress. NQO1-null mice deficient in NQO1 protein showed increased sensitivity to 7,12-dimethylbenz(a)anthracene- and benzo(a)pyrene-induced skin carcinogenesis. In the present studies, we show that benzo(a)pyrene metabolite benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide and not benzo(a)pyrene quinones contributed to increased benzo(a) pyrene-induced skin tumors in NQO1-null mice. An analysis of untreated skin revealed an altered intracellular redox state due to accumulation of NADH and reduced levels of NAD/NADH in NQO1-null mice as compared with wild-type mice. Treatment with benzo(a)pyrene failed to significantly increase p53 and apoptosis in the skin of NQO1-null mice when compared with wild-type mice. These results led to the conclusion that altered intracellular redox state along with lack of induction of p53 and decreased apoptosis plays a significant role in increased sensitivity of NQO1-null mice to benzo(a)pyrene-induced skin cancer.     

Independent regulation of two types of aryl hydrocarbon (benzo(a)pyrene) hydroxylase in mammalian cells.

Aryl hydrocarbon (benzo(a)pyrene) hydroxylase induced by dibutyryl cyclic AMP (dcAMP), plus aminophylline (AHH I) can be ditsinguished from the hydroxylase induced by benz (a) anthracene (AHH II) by its lower Km for benzo (a) pyrene. Treatment with the combination of benzo (a) anthracene and dcAMP plus aminophylline induced both AHH I and AHH II activities. After optimal induction of AHH II activity by benz (a) anthracene, the addition of dcAMP plus aminophylline gave an induction of AHH I. Although AHH I activity declined to an almost basal level 24 h after treatment with dcAMP plus aminophylline, the addition of benz (a) anthracene prevented this decline. Inducibility by dcAMP plus aminophylline or by benz (a) anthracene varied in different cell lines. Some cell lines were induced by both substances, with a higher induction by benz (a) anthracene, while other lines were inducible only by benz (a) anthracene, and a third cell type was not inducible by either. Selection for resistance to benzo (a) pyrene of a cell line inducible by both compounds resulted in a fourth cell type which was more inducible by dcAMP plus aminophylline than by benz (a) anthracene. The results suggest that there is an independent regulation of hydroxylase AHH I and AHH II and that the induction of these two enzyme activities is determined by different genetic controls.     

Toxicity of metabolic benzo(a)pyrenediones to cultured cells and the dependence upon molecular oxygen.

The three quinone metabolites of carcinogenic benzo(a)pyrene, the isomeric benzo(a)pyrenediones (6, 12; 1,6; 3,6), are toxic to cultured hamster cells at low concentrations. The reduction in cell number, observed after treatment with these metabolites, is the result of both direct cell killing and the inhibition of growth, since DNA synthesis is inhibited very early after treatment with benzo(a)pyrene 1,6-dione when little cell death has occurred. The rate of RNA synthesis was also inhibited by treatment of cells with benzo(a)pyrene 3,6-dione. These actions of the benzo(a)pyrenediones toward hamster cells can be eliminated or substantially reduced by the removal of oxygen from the growth medium and atmosphere in which the cells are incubated. In contrast, anaerobic conditions do not reduce the cytotoxicity observed with the alkylating agent ethyl methanesulfonate. These results support the hypothesis that benzo(a)pyrenediones, and other biologically active quinones, owe their activity to oxidation-reduction cycles involving quinone, hydroquinone, and molecular oxygen; the reactive reduced oxygen radicals and semiquinone radical formed during these cycles may be responsible for the observed cellular injury and inhibition of cellular processes.     

Oxidative stress promotes the development of transformation: involvement of a potent mutagenic lipid peroxidation product, acrolein

The effect of intracellular oxidative stress on the development of cell transformation was studied. Mouse embryo C3H/10T1/2 fibroblasts pre-treated with benzo[a] pyrene, developed transformed foci on exposure to free radical generators, such as 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and 3-morpholinosydnonimine hydrochloride (SIN-1). These compounds generate peroxyl radicals and peroxynitrite, respectively. Neither AAPH nor SIN-1 alone induced transformation. The level of intracellular antioxidants, such as alpha-tocopherol and glutathione (GSH), decreased with time of exposure to the free radical generators, whereas the addition of exogenous alpha-tocopherol, GSH and ebselen showed a reduction in the frequency of transformation. An early event during exposure to AAPH and SIN-1 was the generation of acrolein, a highly mutagenic lipid peroxidation product, which was suppressed by the addition of alpha-tocopherol. Furthermore, it was confirmed that acrolein induced the transformation of cells which were pre-treated with benzo[a]pyrene but not of the untreated cells. These results suggest that acrolein may act as an important mediator of cell transformation under oxidative stress.     

Benzo(a)pyrene phenol production by perfused rat liver and its inhibition by ethanol

A rapid and inexpensive method has been developed to estimate rates of benzo(a)pyrene phenol production by perfused rat liver. This method is based on the measurement of benzo(a)pyrene phenols utilizing a simple fluorometric procedure. Within 2 to 3 min after infusion of benzo(a)pyrene bound to serum albumin, phenols are excreted into the perfusate, primarily as glucuronide and sulfate conjugates. Maximal rates of phenol release were 8 to 10 nmol/g/hr in livers from control rats and 40 to 42 nmol/g/hr in livers from 3-methylcholanthrene-treated rats. Fasting of 3-methylcholanthrene-treated rats for 24 hr prior to perfusion experiments did not affect either the rate of phenol production or the extent of their conjugation. Ethanol (20 mM) inhibited rates of phenol formation by 50% in livers from fasted, 3-methylcholanthrene-treated rats but had no effect on benzo(a)pyrene hydroxylase activity in isolated hepatic microsomes. These data indicate that ethanol inhibits phenol formation from benzo(a)pyrene in intact liver, probably by diminishing the supply of the cofactor reduced nicotinamide adenine dinucleotide phosphate.     

Benzo(alpha)pyrene metabolism and DNA-binding in cultured explants of human bronchus and in monolayer cultures of human bronchial epithelial cells treated with ellagic acid

Ellagic acid, a plant phenolic compound present in certain foods eaten by humans, has been reported to possess antimutagenic and anticarcinogenic properties. To evaluate the potential anticarcinogenic effect of ellagic acid in humans, we investigated the effect of nontoxic concentrations of ellagic acid on the metabolism of benzo(alpha)pyrene and binding of benzo(alpha)pyrene metabolites to DNA in cultured explants of human bronchus and in human bronchial epithelial cell cultures. Ellagic acid at concentrations of 10, 25, or 50 microM did not significantly alter the metabolism of benzo(alpha)pyrene in the bronchial explant cultures and in only one of four bronchial cell cultures. However, binding of metabolites of benzo(alpha)pyrene to DNA was inhibited in all explant and cell cultures of human bronchus by 26 to 77%. These results support the work of other investigators and suggest that ellagic acid may be an inhibitor of polycyclic aromatic hydrocarbon-induced carcinogenesis in humans.     

Human breast cell-mediated mutagenesis of mammalian cells by polycyclic aromatic hydrocarbons

A system has been developed in which human breast cells activate chemical procarcinogens to mutagenic compounds. The degree of activation is quantitated by the estimation of induction of 6-thioguanine-resistant specific locus mutants in a cocultured Chinese hamster V-79 cell population which does not activate carcinogens. Both mammary stromal and parenchymal cells could activate the procarcinogen 7,12-dimethylbenz(a)anthracene. In addition, it is shown that the two mammary cell populations converted both 7,12-dimethylbenz(a)anthracene and benzo(a)pyrene to water-soluble metabolites. The stromal cells produced substantial amounts of glucuronic acid conjugates, but the parenchymal cells did not. Both cell types metabolize benzo(a)pyrene to the organic-soluble metabolites 9,10- and 7,8-dihydrodiol and both 9- and 3-hydroxybenzo(a)pyrene. These results suggest that the human breast may be a target for polycyclic aromatic hydrocarbon carcinogenesis.