Inhibition of hepatic aryl hydrocarbon hydroxylase by 3-methylcholanthrene, 7,8-benzoflavone and other inducers added in vitro.

A close correlation has been observed between the ability of aromatic polycyclic hydrocarbons and 7,8-benzoflavone (7,8-BF) to induce hepatic aryl hydrocarbon hydroxylase (AHH) in vivo and to inhibit the induced enzyme system in vitro. The activity of this mono-oxygenase was measured by the conversion of 14C-labeled dimethylbenz(a)anthracene (DMBA) or benzo(a)pyrene (BP) to water-soluble products by rat liver preparations (8000 X g supernatant). DMBA as substrate had the advantage over BP in giving a wider range of ethyl acetate-soluble metabolites and allowing the observation of changes in the pattern of these products following injection or addition of the inducing agents. This property was used to detect low concentration (0.1 muM) of polycyclic hydrocarbons which are strong AHH inducers and which may also be carcinogenic. The liver preparation was active for several months when stored at --20 degrees. A possible mechanism of action for the in vitro behaviour of polycyclic hydrocarbons and 7,8-BF towards AHH is proposed.     

Effect of various chemicals on the metabolism of benzo(a)pyrene by cultured rat colon

The effect of various co- and anti-carcinogens of colon carcinogenesis on the metabolism of benzo(a)pyrene (BP) in cultured rat colon is reported. Rat colon enzymatically converted BP into metabolites which bind to cellular macromolecules i.e., DNA and protein. Activity of aryl hydrocarbon hydroxylase (AHH) activity and binding levels of BP to macromolecules were higher in the descending colon when compared to other segments. The major metabolites of BP, extractable with ethylacetate, were quinones, tetrols, 7,8-diol and a peak containing 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene and 7,8,9-trihydroxy-7,8-dihydrobenzo(a)pyrene. The binding levels of BP to DNA and protein in the explant was lowered by co-incubation with 7,8-benzoflavone (7,8-BF) (3.6 and 18.0 μM), a known inhibitor of AHH, and with disulfiram (100 μM), an anti-oxidant. The absence of vitamin A in the media also resulted in a lower level of BP binding to DNA and protein and in lower activity of AHH. Pretreatment with known inducers of AHH such as phenobarbital (PB) or benz(a)anthracene (BA), did not have any significant effect on the binding levels of BP to DNA or on the AHH activity. of the bile acids investigated only taurodeoxycholic acid significantly increased the binding level of BP to DNA.     

Formation of 7,12-dimethylbenz[alpha] anthracene-DNA adducts in 7,8-benzoflavone-treated hamster embryo cells.

Pretreatment of secondary cultures of Syrian hamster embryo cells with 7,8-benzoflavone (7,8-BF) inhibited both the metabolism of 7,12-dimethylbenz[alpha] anthracene (DMBA) and the formation of DMBA-DNA adducts. The DMBA-deoxyribonucleoside adducts from 7,8-BF-treated cultures had the same elution profiles on Sephadex LH-20 columns as those from cultures exposed to DMBA alone, but 7,8-BF-treated cultures contained smaller amounts of DMBA-DNA adducts per mg DNA. As the concentration of 7,8-BF was increased, the decrease in the amount of DMBA-DNA adducts per mg DNA was logarithmic with respect to the decrease in the amount of DMBA metabolized. The results suggest that more than one metabolic step is required for the binding of DMBA to DNA in hamster embryo cells.     

Aryl hydrocarbon hydroxylase in mouse mammary gland: In vitro study using mammary cell lines

The effects of 3-methylcholanthrene (MCA), 5,6-benzoflavone (βNF), 7,8-benzoflavone (αNF) and pregnenolone 16α-carbonitrile (PCN) upon aryl hydrocarbon hydroxylase (AHH) were determined in primary mammary gland epithelial cell cultures prepared from the C3Hf^?/Ki mouse. MCA elevated AHH activity by 3–4 fold after 24 h of treatment; αNF produced a 50% inhibition. The specific activity of AHH in these cells was elevated by 6 h after exposure to MCA; enzyme activity was still maximally elevated after 48 h. The effects of MCA were also investigated in a group of mammary cell lines, one of which was derived from a control virgin mouse, the MCG V14; 3 of which arose from mammary tumors, MCG T10, MCG T14 and MCG T19; and 2 of which were sublines developed from hyperplastic alveolar nodules, HAN-1 and HAN-2. Induction was seen in all lines at 24 h, with the MCG T14 being the most responsive and the HAN-2, the least. Although the MCG T19 tumor cells did respond in culture, when implanted in the mouse, the AHH of the subsequent tumor was not elevated upon administration of MCA in vivo.     

Evidence for translational control of the binding of 7, 12-dimethylbenz[a]anthracene to DNA of murine epidermal cell in culture.

The effects of various inhibitors of aryl hydrocarbon hydroxylase (AHH), antioxidants, inhibitors of DNA, RNA, and protein synthesis, and protease inhibitors on the binding of [7,12-3H]dimethylbenz[a]anthracene ([3H] DMBA) to DNA of murine epidermal cells in culture have been investigated. 7,8-Benzoflavone, 5,6-benzoflavone and methyrapone (inhibitors of AAH) and antioxidants, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), efficiently reduced the binding of [3H] DMBA to cellular DNA. Inhibitors of DNA and RNA synthesis did not affect this process whereas inhibitors of protein synthesis suppressed the binding of [3H] DMBA to cellular DNA. Protease inhibitors p-tosylamide-2-phenylchloromethyl ketone (TPCK) and p-tosyl-L-lysine chloromethyl ketone (TLCK) also reduced the interaction between DMBA and DNA. Thus, it appears that binding of DMBA to cellular DNA is regulated at the level of translation or/and post translation.     

In vitro effects of L-ascorbic acid (vitamin C) on aryl hydrocarbon hydroxylase activity in hepatic microsomes of mice.

When aromatic hydrocarbon (Ah)-responsive and -non-responsive strains of mice were pretreated with 3-methylcholanthrene (MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), vitamin C reduced the microsomal aryl hydrocarbon hydroxylase (AHH) activity. The AHH inhibitors 7,8-benzoflavone (7,8-BF) and 3-methylsulfonyl-3',4,4',5-tetrachlorobiphenyl (3-MSF-3',4,4',5-tetraCB) showed various inhibitory effects depending upon the types of microsomes, whereas vitamin C exhibited inhibition irrespective of the types of microsomes. 7,8-BF and 3-MSF-3',4,4',5-tetraCB as well as vitamin C suppressed the reverse mutation of the Salmonella typhimurium tester strains TA98 and TA100 induced by benzo[a]pyrene.     

In vitro effects of l-ascorbic acid (vitamin C) on aryl hydrocarbon hydroxylase activity in hepatic microsomes of mice

When aromatic hydrocarbon (Ah)-responsive and -non-responsive strains of mice were pretreated with 3-methylcholanthrene (MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), vitamin C reduced the microsomal aryl hydrocarbon hydroxylase (AHH) activity. The AHH inhibitors 7,8-benzoflavone (7,8-BF) and 3-methylsulfonyl-3′,4,4′,5-tetrachlorobiphenyl (3-MSF-3′,4,4′,5-tetraCB) showed various inhibitory effects depending upon the types of microsomes, whereas vitamin C exhibited inhibition irrespective of the types of microsomes. 7,8-BF and 3-MSF-3′,4,4′,5-tetraCB as well as vitamin C suppressed the reverse mutation of the Salmonella typhimurium tester strains TA98 and TA100 induced by benzo[a]pyrene.     

Effects of various inducers on diethylstilbestrol metabolism, drug-metabolizing enzyme activities and the aromatic hydrocarbon (Ah) receptor in male Syrian golden hamster liver

In order to elucidate the role of metabolic activation of the synthetic estrogen, diethylstilbestrol (DES), in the mechanism of liver tumor formation in male Syrian golden hamsters observed after combined treatment with DES and 7,8-benzoflavone (7,8-BF), the metabolism of DES and the concentrations and activities of various drug-metabolizing enzymes were studied in hamster liver microsomes after various pretreatments. The levels of the hepatic aromatic hydrocarbon (Ah) receptor were also determined. Pretreatment with 7,8-BF increased both P450 and cytochrome b5 levels, whereas phenobarbital (PB) and 3-methylcholanthrene (MC) induced P450 but not cytochrome b5. 7,8-BF pretreatment increased 7-ethoxyresorufin-O-deethylase (EROD) 3-fold and 7-pentoxyresorufin-O-dealkylase (PROD) 2.5-fold, whereas aromatic hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin-O-deethylase (ECOD) activities were only slightly induced by 7,8-BF. MC pretreatment increased EROD 8-fold and PROD activity 7-fold, whereas PB pretreatment enhanced AHH 4.5-fold and PROD activity 4-fold. In contrast to PB, pretreatment with 7,8-BF and MC reduced the oxidative metabolism of DES in hepatic microsomes, but the pattern of metabolites was identical with that in untreated controls. Treatment of hamsters with the inducers changed the hepatic Ah receptor level. PB and MC-pretreatment resulted in an increase of the receptor level 1.5-fold and 1.3-fold, respectively, whereas 7,8-BF-pretreatment leads to a 1.5-fold decrease. The dissociation constant Kd is 170 nM for the reaction of 7,8-BF with the hamster Ah receptor compared to 70 nM for 5,6-BF and 38 nM for 2,3,7,8-tetrachlorodibenzofuran (TCDF). The Kd-value is 3.6 nM for TCDF with the rat receptor protein. It is concluded from these data that metabolic activation of DES is not involved in the mechanism of hepatocarcinogenesis in this animal tumor model.     

Characteristics of aryl hydrocarbon hydroxylase activity in rat mammary epithlial cells grown in primary culture.

Rat mammary epithelial cells grown in primary culture contain the microsomal enzyme, aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH), which catalyses the oxidative conversion of polycyclic aromatic hydrocarbons (PAH) to more polar derivatives. Constitutive AHH activity, measured with an established fluorometric method, was 46 pmol/mg protein/h in homogenates of rat mammary epithelial cells after 5 days in culture. The addition of dimethylbenz[a]anthracene (DMBA), benz[a]anthracene (BA), or 3-methylcholanthrene (3-MC) to the cell culture medium increased AHH activity 5.3-, 4.7- and 2.4-fold, respectively. Kinetic studies revealed that maximal hydroxylase induction occurred 16 h after 1 micro M DMBA was added to the culture medium. The decay of the DMBA-induced hydroxylase was biphasic: one component had a t1/2 of 15--30 min and another a t1/2 of 4 h. Norepinephrine, 17 beta-estradiol and 5,6-benzoflavone also increased AHH activity in mammary epithelial cells in vitro, however, sodium phenobarbital had no effect. Fetal bovine serum (FBS), previously shown to be a potent in vitro inducer of AHH activity, had no effect on either constitutive or DMBA-induced mammary epithelial hydroxylase activities following treatment with 1% activated charcoal. Metyrapone and 7,8-benzoflavone, inhibitors of microsomal mixed function oxidase activity, reduced both constitutive and DMBA-induced AHH activities when added to homogenates of untreated and DMBA-treated mammary epithelial cells. The addition of 7,8-benzoflavone reduced both constitutive and DMBA-induced hydroxylase activities by approx. 80%, whereas metyrapone addition inhibited these activities by 20%. The study demonstrates several in vitro factors which alter AHH activity in primary cultures of rat mammary epithelial cells.     

Mutagenicity of benzo[a]pyrene 7,8-dihydrodiol and 7,12-dimethylbenz[a]anthracene 3,4-dihydrodiol in S. typhimurium mediated by microsomes from rat liver and mouse skin

The mutagenic activities of trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene (BP 7,8-diol) and of trans-3,4-dihydroxy-7,12-dimethylbenz[a]-anthracene (DMBA 3,4-diol) towards S. typhimurium TA100 were measured in assays that were carried out on a micro-scale in liquid medium in the presence of microsomal fractions prepared from mouse skin or rat liver. In the presence of an NADPH-generating system, microsomal enzymes converted both diols into mutagens that were probably the respective 'bay-region' diol-epoxides. The rate of the enzyme-catalysed conversion of the BP 7,8-diol into mutagens by microsomal preparations from mouse epidermis was similar to that occurring with microsomes from rat liver. Pretreatment of mice by the topical application of benz[a]anthracene (BA) or 7,12-dimethylbenz[a]-anthracene (DMBA) increased the mutagenic activity of BP 7,8-diol mediated by mouse skin microsomal preparations by 2-fold and this was paralleled by a 4-fold increase in epidermal aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity. The results are discussed in relation to the high susceptibility of mouse skin to polycyclic aromatic hydrocarbon (PAH) carcinogenesis.     

Effect of 7,8-benzolfavone on the formation of benzo(alpha)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.     

The carcinogen benzo(e)pyrene is metabolized by DM15 cells without an uncoupling effect on their gap junctions

Benzo(e)pyrene (B(e)P) promotes carcinogenesis in the skin. Unlike some other promoters however, B(e)P does notproduce an uncoupling effect on gap junction permeability in DM15 transformedfibroblasts. This study demonstrates thatDM15 cells exhibit a relatively high level of B(e)P metabolism. Moreover, although pretreatment of DM15 cells with benz(a)anthracene results in an 8-fold increase of arylhydrocarbon hydroxylase activity and a 2-fold increase in the rate ofB(e)P metabolism, it did not enable B(e)P to affectLucifer Yellow transfer between DM15 cells. We conclude that neitherB(e)P nor its metabolites are capable of uncoupling gap junction permeability in DM15 cells.Abbreviations AHH aryl hydrocarbon hyroxylase - BA benz(a)anthracene - B(a)P benzo(a)pyrene - B(e)P benzo(e)pyrene - LY Lucifer Yellow - MFO mixed-function oxidases - PAH polycyclic aromatic hydrocarbons     

Inhibitory effects of snuff extract on ornithine decarboxylase and aryl hydrocarbon hydroxylase activities in relation to cell proliferation of mouse tongue epithelial cells

Effect of snuff extract (SE) on cell proliferation as measured by 3H thymidine (TdR) uptake, induction of ornithine decarboxylase (ODC) and aryl hydrocarbon hydroxylase (AHH) was studied in primary embryonal mouse tongue cultures. Cultures treated with SE in combination with 7,12-dimethylbenz(a)anthracene (DMBA) showed inhibition of cell proliferation and decrease of ODC and AHH activities, compared to control, DMBA, and DMBA + 12-O-tetradecanoylphorbol 13-acetate treated cultures.     

The binding of benz(a)anthracene derivatives to glyceraldehyde-3-phosphate dehydrogenase and mammary tissue following exposure to laboratory light.

7,12-Dimethylbenz(a)anthracene (DMBA) and 7-methoxymethyl-12-methylbenz(a)anthracene (MeO-DMBA) are converted to a number of products during short exposures in aqueous suspension to laboratory illumination. The mixture of products binds to glyceraldehyde-3-phosphate dehydrogenase (GPDH) while inhibiting its activity but there is no apparent relationship between the binding and inhibition of enzyme activity. There is little, or no, binding or enzyme inhibition when the compounds are protected from light. 7-Bromomethyl-12-methylbenz(a)anthracene (Br-DMBA) binds to GPDH whether photoactivated or not but enzyme inhibition depends upon light exposure. The binding of light-exposed DMBA by surviving rat mammary tissue was five-times greater than with the unchanged hydrocarbon. Binding of MeO-DMBA products also occurred after light exposure but not in the dark.     

Phenobarbital induces cytochrome P-450- and cytochrome P-448-dependent monooxygenases in rat hepatoma cells

The induction by phenobarbital (PB) of aldrin epoxidase (AE) and aryl hydrocarbon hydroxylase (AHH), markers of cytochrome P-450- and cytochrome P-448-dependent monooxygenases, was studied in cell lines derived from Reuber H35 rat hepatoma which differ widely in their degree of differentiation. The following results were obtained: (1) PB induced AE 2-6-fold and AHH 2-4-fold in the differentiated clones, Fao, 2sFou, and C2Rev7 during an exposure period of 72 h. The barbiturate increased AHH but not AE in the dedifferentiated clone H5, the poorly differentiated line H4IIEC3/T, and in the well differentiated line H4IIEC3/G-. (2) Continuous presence of the barbiturate was required for maintaining the induction of the two monooxygenase activities in C2Rev7 cells. (3) Maximum induction of AE was observed at a PB concentration of 1.5-3.0 mM. (4) The effects of 7,8-benzoflavone on AHH-activities induced by phenobarbital in C2Rev7 and H5 cells suggested that they are mediated by cytochrome P-450- and cytochrome P-448-dependent monooxygenase forms, respectively. Thus, the flavonoid had only a slight inhibitory effect on PB-induced AHH in C2Rev7 cells, but strongly inhibited PB-induced AHH in H5 cells. The induction of AE and of 7,8-benzoflavone-inhibitable AHH in 2sFou cells indicated that PB is capable of inducing cytochromes P-450 and cytochrome P-448 in the same cell.     

Difference in liver homogenates from Donryu, Fischer, Sprague-Dawley and Wistar strains of rat in the drug-metabolizing enzyme assay and the Salmonella/hepatic S9 activation test

Comparison studies for detecting differences between liver microsome and S9 preparations from 4 strains (Donryu, Fischer, Sprague-Dawley, Wistar) of young male rats were carried out with pretreatment of the animals by inducers such as PCBs and PB plus 5,6-BF. Each microsome fraction was assayed for the enzymic activity of metabolism of model substrates such as aniline, benzophetamine, BP, DMN and 7-ethoxycoumarin. The hepatic S9 sample was also compared, as regards its metabolizing ability to activate 9 pre-mutagens (2AA, AAF, o-AAT, BP, DAB, DMBA, DMN, m-PDA, quinoline) to directly acting mutagens in the Salmonella/hepatic S9 activation test by using TA98, TA100 and TA1537 strains with or without cytochrome P450 inhibitors (SKF-525A, metyrapone, 7,8-benzoflavone).

In the enzymic assay with PCBs-induced microsomes, BP hydroxylation revealed a strain-specific difference: the microsomes from Fischer and Wistar rats were more effective for metabolizing BP than those from the other strains of rat. The effect of induction by PB plus 5,6-BF for Fischer rats showed relatively higher enzymic activity in the same induction group. Other microsomes prepared from rats with and without induction by PB plus 5,6-BF did not show a clear-cut strain dependency in the enzymic activities assayed.

In the mutation experiments with hepatic S9 samples, the examination of DAB and quinoline revealed a marked strain difference when S9 samples prepared from PCBs-pretreated and PB-plus-5,6-BF-induced rats were used: the S9 sample from Fischer rats was available for activating the two pre-mutagens to directly acting mutagens. No marked difference in the metabolic activation of the remaining 7-pre-mutagens was observed on other S9 preparations.

In examinations of mutagenicity activities with the use of three inhibitors, the two S9 preparations made with the two induction methods showed inhibition profiles closely similar to each other. However, there were minor differences in the profiles by these inhibitors.

From these findings it was concluded that Fischer rat-liver S9 is useful for detecting mutagens in the metabolic activation test, when induction by PB plus 5,6-BF was used in the Ames Salmonella test.     

The effect of complete carcinogens on intercellular transfer of lucifer yellow in fibroblast culture

Summary The effect on permeability of gap junctions of complete powerful carcinogens, 3-methylcholanthrene (MC), 7, 12-dimethylbenz(a)anthracene (DMBA), ethyl methanesulfonate (EMS), and weak carcinogens, benz(a)anthracene (BA), benzo(e)pyrene (B(e)P) as well as the arylhydrolase inhibitor 7,8-benzoflavone (7,8-BF) has been studied with the use of a dye-coupling technique and transformed Djungarian hamster DM15 fibroblasts. MC, EMS and 7,8-BF were found to exert a strong inhibitory effect on cell-to-cell dye transfer. BA and DMBA had the uncoupling activity only in 2 out of 4 experiments. B(e)P was not shown to affect LY transfer between DM15 cells. The uncoupling effect of MC, 7,8-BF and EMS (only when EMS used at the concentration of 600 µg/ ml but not 1000 µ/ ml) appeared reversible. The causes of failure to detect DMBA and B(e)P effects on gap junctions are discussed.Abbreviations B(a)P benzo(a)pyrene - B(e)P benzo(e)pyrene - BA benz(a)anthracene - 7,8-B,F 7,8-benzoflavone - DMBA 7,12,dimethylbenz(a)anthracene - MC 3-methylcholanthrene - EMS ethyl methanesulfonate - LY Lucifer Yellow - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PAH polycyclic aromatic hydrocarbons     

A class of strong inhibitors of microsomal monooxygenases: the ellipticines.

Ellipticine (E) and its 9-hydroxy derivative inhibit strongly various liver monooxygenase activities mediated by microsomes from control and phenobarbital (PB), benzo[alpha]pyrene (BP) or Aroclor 1254 (Aroclor)-pretreated rats. The inhibition constants, Ki, are remarkably low, and often smaller than 1 micron, particularly in the case of microsomes containing cytochrome P-448. The inhibitory potency (I50) of 9-hydroxyellipticine (9-OHE) is larger (about ten-fold) than the one of classical inhibitors (metyrapone or 7,8-benzoflavone (7,8-BF)), whatever the activities studied and the induction of microsomes. Differences exist between the mechanisms of inhibition according to the form of cytochrome P-450 present in microsomes of differently pretreated rats; whichever the activities studied, one observes: (a) a competitive inhibition towards the activity of non-induced or PB-induced microsomes and (b) a non-competitive inhibition towards the activity of Aroclor or BP-induced microsomes, at variance with 7,8-BF. These results are in good agreement with the interaction properties of the ellipticines with microsomal cytochromes P-450.     

Detection of aryl hydrocarbon hydroxylase activity in normal and neoplastic human breast epithelium

Studies were conducted to determine whether normal and/or neo-plastic (MCF-7) human breast epithelial cells contain the microsomal aryl hydrocarbon hydroxylase (AHH) which catalyses the conversion of polycyclic aromatic hydrocarbons (PAH) to carcinogenic intermediates. Low constitutive levels of AHH activity were found in homogenates of both normal human breast epithelial and MCF-7 cells. The addition of 7,12-dimethylbenz(a)anthracene (DMBA) to the culture medium of either cell type significantly increased AHH activity. Peak induction of hydroxylase activity occurred following the $\text{in}$$\text{vitro}$ addition of 10 μM DMBA. A time course of DMBA-induced AHH activity in both normal human breast epithelium and MCF-7 cells revealed maximal induction 16 hr after 10 μM DMBA was added to the culture medium. Benzo(a)pyrene (BP), 3-methylcholanthrene (MCA) and benz(a)anthracene (BA) also induced AHH activity in normal and MCF-7 cells. For example, the addition of 10 μM BP to the culture medium of either normal human breast epithelial or MCF-7 cells for 16 hr increased AHH activity 13.8 and 65.3-fold, respectively. For all PAH, the magnitude of AHH induction was substantially greater in MCF-7 than normal breast epithelial cells. Finally, α-naphthoflavone inhibited BA-induced AHH activity in MCF-7 cells. The study demonstrates the presence of a PAH-inducible AHH enzyme(s) in normal human breast epithelial cells grown in primary culture and in the human breast tumor cell line, MCF-7.     

Protection against 3-methylcholanthrene-induced skin tumorigenesis in Balb/C mice by ellagic acid

Topical application of ellagic acid, a naturally occurring dietary plant phenol, to Balb/C mice resulted in significant protection against 3-methylcholanthrene (MCA)-induced skin tumorigenesis. Ellagic acid was found to be an effective inhibitor of tumor formation whether the tumor data are considered as percent mice with tumors, cumulative number of tumors, tumors per mouse or tumors per tumor bearing animal as a function of the number of weeks on test. By 8, 10, 12, 14, and 16 weeks of testing, the number of tumors per mouse in the group receiving MCA alone was 2.0, 3.4, 4.0, 4.9 and 5.3, respectively, whereas the corresponding numbers in the group receiving MCA plus 2 mumol ellagic acid were 0, 0.3, 0.4, 0.6 and 1.2, respectively. At the termination of the experiment (16 weeks) aryl hydrocarbon hydroxylase (AHH) activity in skin and liver and the extent of 3H-BP-binding to skin, liver and lung DNA were determined and both of these parameters were found to be significantly inhibited in the animals treated with ellagic acid. These results indicate that ellagic acid can inhibit the metabolism of polyaromatic hydrocarbons and modulate skin carcinogenesis induced by these chemicals.