Effect of naturally occurring coumarins on the formation of epidermal DNA adducts and skin tumors induced by benzo[a]pyrene and 7,12- dimethylbenz[a]anthracene in SENCAR mice

Several naturally occurring coumarins previously found to be potent inhibitors of mouse hepatic ethoxyresorufin-O-deethylase (EROD) and/or pentoxyresorufin-O-dealkylase (PROD) were examined for their effects on formation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) DNA adducts in mouse epidermis, as well as, their effects on skin tumor initiation by these polycyclic aromatic hydrocarbons (PAH). Bergamottin, a potent inhibitor of hepatic EROD, given topically 5 min prior to an initiating dose of B[a]P, significantly decreased total covalent binding of B[a]P to DNA in a dose-dependent manner 24 h after treatment. A dose of 400 nmol bergamottin reduced covalent binding of B[a]P by 72%. Coriandrin, at a dose of 400 nmol also significantly reduced total covalent binding of B[a]P by 59%. In addition, formation of the major (+)anti-B[a]P-diol epoxide-N2-dGuo adduct was selectively reduced by both of these coumarins. In contrast, bergamottin and coriandrin did not significantly decrease covalent binding of DMBA to epidermal DNA at doses of either 400 nmol or 800 nmol. Imperatorin and isopimpinellin, which are more potent inhibitors of hepatic PROD activity, significantly reduced overall binding of DMBA to epidermal DNA by 67% and 52%, respectively, when applied at doses of 400 nmol. These two coumarins also inhibited B[a]P-DNA adduct formation at similar doses but to a lesser extent. Imperatorin at a dose of 400 nmol dramatically decreased formation of covalent DNA adducts derived from both the anti and syn diol epoxides of DMBA. Bergamottin was a potent inhibitor of tumor initiation by B[a]P while coriandrin was less effective in this regard. Imperatorin was an effective inhibitor of skin tumor initiation by DMBA and also inhibited complete carcinogenesis by this PAH. At dose levels higher than those effective against DMBA, imperatorin also inhibited tumor initiation by B[a]P. The results demonstrate that several naturally occurring coumarins possess the ability to block DNA adduct formation and tumor initiation by PAHs such as B[a]P and DMBA. The mechanism for reduced DNA adduct formation and tumor initiation appears to involve inhibition of the P450s involved in the metabolic activation of these hydrocarbons. Finally, the differential effects of certain coumarins on B[a]P vs DMBA DNA adduct formation and tumor initiation may be useful for dissecting the role of specific cytochromes P450 in their metabolic activation.      

Oral administration of the citrus coumarin,isopimpinellin, blocks DNA adduct formation and skin tumor initiation by 7,12-dimethylbenz[a]anthracene in SENCAR mice

The current study was designed to evaluate the effects of oral administration of the citrus coumarin, isopimpinellin, on skin tumor initiation by topically applied benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA). To evaluate the effects of orally administered isopimpinellin on skin tumor initiation by B[a]P and DMBA, its effects on DNA adduct formation were first evaluated. Female SENCAR mice were pre-treated twice with corn oil, or isopimpinellin (70 mg/kg body wt per os) at 24 h and 2 h prior to topical treatment with B[a]P or DMBA. Another citrus coumarin, imperatorin, was also included in these experiments for comparison. Orally administered isopimpinellin and imperatorin significantly inhibited B[a]P-DNA adduct formation by 37 and 26%, respectively. Imperatorin also blocked DMBA-DNA adduct formation by 43%. In a second dose-response study, orally administered isopimpinellin (35, 70 and 150 mg/kg) blocked DMBA-DNA adduct formation by 23, 56 and 69%, respectively. For the tumor study, mice were pretreated orally with corn oil or isopimpinellin at 24 and 2 h prior to initiation with DMBA, and 2 weeks later promotion began with 12-O-tetradecanoylphorbol-13-acetate (TPA). Isopimpinellin significantly reduced the mean number of papillomas per mouse by 49, 73 and 78% compared to corn oil controls at 30, 70 and 150 mg/kg body wt, respectively. Orally administered isopimpinellin also significantly reduced the percentage of mice with papillomas at the highest dose tested (150 mg/kg). The effectiveness of isopimpinellin given topically over a broad dose range against DMBA tumor initiation was also evaluated for comparison. As part of this study, several parameters of systemic toxicity were evaluated following oral dosing with isopimpinellin and imperatorin. Mice were treated orally with corn oil, isopimpinellin or imperatorin (35, 70 and 150 mg/kg body wt per os) once daily for four consecutive days, killed at 24 h after the last dose, and livers, lungs, and kidneys evaluated histologically. In addition, urinary parameters of nephrotoxicity, blood parameters of liver and kidney function, and thrombin clotting time were assayed. No significant changes in blood clotting, or renal or hepatic function were observed. There was, however, a significant increase in liver wt accompanied by cytoplasmic vacuolation of hepatocytes. There were no histopathological changes in lungs or kidneys. Overall, these data indicate that isopimpinellin (and imperatorin) have chemopreventive effects when administered orally on skin tumor initiation by DMBA.     

Naturally occurring coumarins inhibit 7,12-dimethylbenz[a]anthracene DNA adduct formation in mouse mammary gland

Naturally occurring coumarins (NOCs) are anti-carcinogenic in the mouse skin model. To characterize the chemopreventive potential of NOCs against breast cancer, we first examined their effects on 7,12-dimethylbenz[a]anthracene (DMBA)-DNA adduct formation in mouse mammary gland. We hypothesized that those NOCs that both inhibited cytochrome P450 1A1/1B1 and induced hepatic glutathione S-transferases (GSTs) would be the most effective in blocking DMBA-DNA adduct formation in mouse mammary gland. To address this hypothesis, simple coumarins (e.g. coumarin and limettin, which induced mouse hepatic GSTs but had little effect on P4501A1/1B1) and linear furanocoumarins (e.g. imperatorin and isopimpinellin, which induced hepatic GSTs and were potent inhibitors of P4501A1/1B1) were compared. Mice were pretreated with NOCs (150 mg/kg body wt, by gavage) prior to either a single dose of DMBA (50 microg) or multiple doses of DMBA (20 microg daily for 3 and 6 weeks). Mammary DMBA-DNA adduct formation was quantitated by the nuclease P1-enhanced 32P-postlabeling assay. With the single dose of DMBA, coumarin, limettin, imperatorin and isopimpinellin inhibited DMBA-DNA adduct formation by 50, 41, 79 and 88%, respectively. Coumarin, limettin and imperatorin blocked DMBA-DNA adduct formation by 36, 60, and 66% at 3 weeks, and by 0, 49 and 55% at 6 weeks of DMBA dosing, respectively. In a 6 week dose-response study of select NOCs and 7,8-benzoflavone (a potent P4501 inhibitor that had little effect on GSTs), DMBA-DNA adduct formation was inhibited by 0, 43 and 24% in the limettin groups; by 26, 26 and 69% in the isopimpinellin groups; and by 80, 96 and 97% in the 7,8- benzoflavone groups at 35, 70 and 150 mg/kg, respectively. Taken together, these results suggest that linear furanocoumarins had a greater inhibitory effect on DMBA-DNA adduct formation in mouse mammary glands compared with simple coumarins, and that the predominant effect may be P4501 inhibition.     

Inhibitory effects of naturally occurring coumarins on the metabolic activation of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in cultured mouse keratinocytes

Several naturally occurring coumarins to which humans are routinely exposed have been previously found to be potent inhibitors and inactivators of cytochrome P450 (P450) 1A1-mediated monooxygenase in both murine hepatic microsomes and in a reconstituted system using purified human P450 1A1 [Cai et al. (1993) Chem. Res. Toxicol., 6, 872- 879 and Cai et al. (1996) Chem. Res. Toxicol., 9, 729-736]. In the present study, several of these coumarins were investigated for their inhibitory effects on the metabolism and metabolic activation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in cultured mouse keratinocytes. Initial analysis of B[a]P metabolism in cultured keratinocytes showed that imperatorin, isoimperatorin, coriandrin, and bergamottin, at concentrations of 2 nM equal with B[a]P, reduced the formation of water-soluble metabolites of B[a]P by 33% to 57%. Bergamottin and coriandrin were the most potent inhibitors of the compounds examined. HPLC analysis of organic solvent-soluble metabolites of B[a]P indicated that all the coumarins tested significantly reduced the formation of individual B[a]P metabolites (including phenols, diols and tetraols). However, the greatest effect was on the formation of B[a]P tetraols. Additional experiments determined the ability of selected coumarins to block covalent binding of B[a]P and DMBA to DNA in keratinocytes. Bergamottin preferentially inhibited the binding of B[a]P to DNA by 56%, while coriandrin preferentially inhibited the binding of DMBA to DNA by 48%. Notably, analysis of individual DNA adducts formed from B[a]P and DMBA indicated that both bergamottin and coriandrin specifically inhibited the formation of anti diol-epoxide DNA adducts derived from both hydrocarbons. The preferential inhibitory effect of bergamottin and coriandrin on the formation of anti diol-epoxide adducts derived from DMBA was further confirmed by separation of anti- and syn-diol-epoxide- DNA adducts using immobilized boronate chromatography. The current study demonstrates that certain naturally occurring coumarins inhibited metabolic activation of B[a]P and DMBA in cultured mouse keratinocytes and specifically inhibited the formation of DNA adducts derived from the anti diol-epoxide diastereomers from either hydrocarbon. The current data also suggest that certain naturally occurring coumarins may possess anticarcinogenic activity toward polycyclic aromatic hydrocarbons.      

Inhibition of benzo[a]pyrene-DNA adduct formation by Aloe barbadensis Miller

The antigenotoxic and chemopreventive effect of Aloe barbadensis Miller (polysaccharide fraction) on benzo[a]pyrene (B[a]P)-DNA adducts was investigated in vitro and in vivo. Aloe showed a time-course and dose- dependent inhibition of [3H]B[a]P-DNA adduct formation in primary rat hepatocytes (1x10(6) cells/ml) treated with [3H]B[a]P (4 nmol/ml). At concentrations of 0.4-250 microg/ml aloe, the binding of [3H]B[a]P metabolites to rat hepatocyte DNA was inhibited by 9.1-47.9%. Also, in rat hepatocytes cultured for 3-48 h with aloe (250 microg/ml) and [3H]B[a]P (4 nmol/ml), [3H]B[a]P-DNA adducts were significantly reduced by 36% compared with [3H]B[a]P alone. Aloe also inhibited cellular uptake of [3H]B[a]P in a dose-dependent manner at a concentration of 0.4-250 microg/ml by 6.3-34.1%. After a single oral administration of B[a]P to male ICR mice (10 mg/mouse), benzo[a]pyrene diol epoxide I (BPDE-I)-DNA adduct formation and persistence for 16 days following daily treatment with aloe (50 mg/mouse) were quantitated by enzyme- linked immunosorbent assay using monoclonal antibody 8E11. In this animal model, BPDE-I-DNA adduct formation was significantly inhibited in various organs (liver, kidney, forestomach and lung) (P < 0.001). When mice were pretreated with aloe for 16 days before B[a]P treatment, inhibition of BPDE-I-DNA adduct formation and persistence was enhanced. Glutathione S-transferase activity was slightly increased in the liver but cytochrome P450 content was not affected by aloe. These results suggest that the inhibitory effect of aloe on BPDE-I-DNA adduct formation might have a chemopreventive effect by inhibition of B[a]P absorption.      

Effects of phenethyl isothiocyanate, a carcinogenesis inhibitor, on xenobiotic-metabolizing enzymes and nitrosamine metabolism in rats.

Phenethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables, has been shown to inhibit chemical carcinogenesis, possibly due to its ability to block the activation or to enhance the detoxification of chemical carcinogens. The present study was conducted to elucidate the biochemical mechanisms involved by characterizing the effects of PEITC on phase I and phase II xenobiotic-metabolizing enzymes. A single dose of PEITC to F344 rats (1 mmol/kg) decreased the liver N-nitrosodimethylamine demethylase (NDMAd) activity (mainly due to P450 2E1) by 80% at 2 h and the activity of NDMAd remained decreased by 40% at 48 h after treatment. The liver pentoxyresorufin O-dealkylase (PROD) activity and P450 2B1 protein level were elevated 10- and 7-fold at 24 h after treatment respectively. The liver microsomal ethoxyresorufin O-dealkylase (EROD) (mainly due to P450 1A) and erythromycin N-demethylase (mainly due to P450 3A) activities were decreased at 2-12 h after treatment and recovered afterwards. The lung microsomal PROD and EROD activities were not significantly affected; whereas, the nasal microsomal PROD and EROD activities were decreased by 40-50%. After a treatment with PEITC, the rates of oxidative metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were decreased in liver microsomes by 40-60% at 2 h and recovered gradually; the rates in lung microsomes were markedly decreased by 60-70% at 2 h and remained at the decreased level at 24 h; and the rates in nasal mucosa microsomes were decreased gradually with the lowest activities observed at 18 h (50%) followed by a gradual recovery. Furthermore, the treatment with PEITC resulted in a maximal 5-fold increase of NAD(P)H:quinone oxidoreductase and 1.5-fold increase of glutathione S-transferase activities in the liver, but the activities of these two enzymes were not significantly affected in the lung and nasal mucosa. The sulfotransferase activity in the liver was decreased by 32-48% at 24-48 h after treatment; the nasal activity was increased by 1.8- to 2.5-fold, but the lung activity was not significantly changed. The hepatic UDP glucuronosyltransferase activity was slightly decreased at 2 h but slightly increased at 48 h after treatment, but no changes were observed for the lung and nasal activities. The study demonstrates that PEITC selectively affects xenobiotic-metabolizing enzymes in the liver, lung and nasal mucosa and it is especially effective in inhibiting the P450-dependent oxidation of NNK in the lung and of NDMA in the liver.     

Inhibition of the binding of 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene to DNA in mouse skin epidermis by 1-ethynylpyrene

The effects of 1-ethynylpyrene (EP), 1-vinylpyrene (VP) and 2-ethynlnaphthalene (EN) on the covalent binding of 7,12-dimethylbenz[a]anthracene (DMBA) and of benzo[a]-pyrene (B[a]P) to the epidermal DNA in mouse skin were investigated. When applied topically, 5 min before an initiating dose of 10 nmol DMBA or of 200 nmol B[a]P, EP was an effective inhibitor of the formation of the covalent complexes of these procarcinogenic polycyclic aromatic hydrocarbons (PAHs) with the epidermal DNA. VP, applied under the same conditions, was a significantly less effective inhibitor of the binding of DMBA to DNA and showed even weaker inhibition of the binding of B[a]P. EN was ineffective as an inhibitor of the binding of either DMBA or B[a]P. These results establish that both the pyrene nucleus and the ethynyl substituent of EP contribute to the effective inhibition of the binding of DMBA and B[a]P to the epidermal DNA of mouse skin. No significant changes in the ratios of the anti- to the syndiol epoxide-DNA adducts of DMBA or of B[a]P were produced by doses of EP that produced inhibitions of the binding to DNA. At doses of VP that inhibited covalent binding of both DMBA and B[a]P, no changes in DMBA-DNA adduct distributions were observed but changes in the relative proportions of several B[a]P-DNA adducts were noted. These data are discussed in terms of the potential of aryl acetylenes to act as suicide inhibitors (mechanism-based inactivators) of cytochrome P450-dependent monooxygenase isozymes.     

Exposure-route-dependent DNA adduct formation by polycyclic aromatic hydrocarbons

Understanding the kinetics of aromatic-DNA adducts in target tissues and white blood cells (WBC) would enhance the applicability of DNA adducts in WBC as surrogate source of DNA in biomonitoring studies. In the present study, rats were acutely exposed to benzo[a]pyrene (B[a]P; 10 mg/kg body wt) via intratracheal (i.t.), dermal and oral administration. DNA adducts were analyzed in relevant target organs and WBC by nuclease P1 enriched (32)P-post-labeling at 1, 2, 4, 11 and 21 days after exposure. Additionally, the internal dose was assessed by measurement of urinary excretion of 3-hydroxy-B[a]P (3-OH-B[a]P). Total B[a]P-DNA adduct levels in WBC were highest after i.t. and oral administration, whereas DNA adducts were hardly detectable after dermal exposure. Highest adduct levels were reached at 2 days after exposure. In lung tissue, DNA adduct levels reached maximal values at 2 days and were highest after i.t., oral and dermal exposure, respectively. DNA adduct levels were significantly lower in WBC as compared with lung. Nonetheless, overall B[a]P-DNA adduct levels in WBC were significantly correlated with those in lung. In target organs, highest DNA adduct levels were observed in skin after topical application, and lowest in stomach after oral administration of B[a]P. Furthermore, DNA adduct levels in WBC were correlated with DNA adduct levels in skin after dermal exposure and stomach after oral administration of B[a]P. Two-fold higher levels of 3-OH-B[a]P were excreted after i.t. administration of B[a]P as compared with dermal or oral exposure. Urinary 3-OH-B[a]P concentrations were correlated with DNA adduct levels at the site of B[a]P application. Overall, it can be concluded that aromatic-DNA adduct levels in WBC can be applied as a surrogate source of DNA for the site of application of B[a]P and reflect binding to lung DNA, independently of the exposure route.     

Mechanism(s) of turmeric-mediated protective effects against benzo(a)pyrene-derived DNA adducts.

The effects of turmeric feeding before and after benzo(a)pyrene [B(a)P] exposure on the levels of B(a)P-derived DNA adducts were studied in tissues of Swiss mice employing (32)P-postlabelling analysis. A reduction in the levels of B(a)P-derived DNA adducts in liver, lung, and forestomach was observed in animals pre-treated with 0.2 or 1% turmeric diet and exposed to B(a)P by oral intubation when compared to animals receiving standard laboratory diet and B(a)P. The observed decrease was not due to dilution caused by nascent DNA synthesis. Comparative evaluation of levels of B(a)P-derived DNA adducts in tissues of animals shifted to 0.2 or 1% turmeric diet after 24 h of oral intubation of B(a)P with those continued on standard laboratory diet did not suggest enhanced disappearance/repair of B(a)P-derived DNA adducts due to exposure to turmeric. Further, pre-treatment of mice with 1% turmeric diet significantly reduced the B(a)P-induced increase in activity of cytochrome P450 (CYP450) isozymes CYP 1A1 and 1A2 in liver, lung, and forestomach of mice. In addition, hepatic glutathione S-transferase (GST) was found to be elevated in turmeric pre-treated mice. Thus turmeric-mediated decrease in induction of phase-I enzymes in liver, lung, and forestomach of mice and enhancement of hepatic GST appear to play an important role in reducing the B(a)P-induced DNA damage in target and non-target tissues.     

Benzo(e)pyrene-induced alterations in the binding of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene to DNA in Sencar mouse epidermis

Benzo(e)pyrene [B(e)P] cotreatment slightly increases the tumor-initiating activity of benzo(a)pyrene [B(a)P] and greatly decreases the tumor-initiating activity of 7,12-dimethylbenz(a)anthracene (DMBA) in Sencar mice (DiGiovanni et al., Carcinogenesis 3: 371-375, 1982). The effects of B(e)P on the binding of B(a)P and DMBA to Sencar mouse epidermis were investigated using a protocol similar to the mouse skin tumorigenicity studies. After 12 h of exposure to 50 nmol [3H]B(a)P and low or high doses of B(e)P, the level of [3H]B(a)P bound to mouse epidermal DNA increased by 30%. However, after 24 h exposure to 50 nmol [3H]B(a)P and after 12 or 24 h of exposure to 200 nmol [3H]B(a)P, B(e)P had no effect on the amount of [3H]B(a)P bound to DNA. The ration of anti-(the isomer with the epoxide and benzylic hydroxyl on opposite faces of the molecule) B(a)P-7,8-diol-9,10-epoxide [B(a)PDE]-deoxyribonucleoside adducts to syn- (the isomer with the epoxide and benzylic hydroxyl on the same face of the molecule) B(a)PDE-deoxyribonucleoside adducts did not change at either initiating dose of B(a)P or at any time regardless of the dose of B(e)P. After 12 h of exposure to high doses of B(e)P and a 50-nmol initiating dose of B(a)P the level of [3H]B(a)P bound to DNA increased but there was no change in the proportion of particular B(a)PDE-deoxyribonucleoside adducts present. In contrast, B(e)P inhibited the binding of initiating doses of DMBA (5 and 20 nmol) to DNA after 12 and 48 h of exposure to all dose ratios of B(e)P:DMBA tested. The three major adducts, tentatively identified as anti-DMBA-3,4-diol-1,2-epoxide (DMBADE):deoxyguanosine, syn-DMBADE:deoxyadenosine and anti-DMBADE:deoxyadenosine, decreased to the same relative extent as the dose of B(e)P increased. Thus, the effects of B(e)P on the total binding of these hydrocarbons to DNA in epidermis correlate with the cocarcinogenic and anticarcinogenic effects of B(e)P on B(a)P and DMBA, respectively, in a mouse skin initiation-promotion assay. These results indicate that the mechanism of the co- or anticarcinogenic action of hydrocarbons such as B(e)P involves alteration of the binding of carcinogenic hydrocarbons to DNA. They also suggest that measurement of carcinogenic hydrocarbon-DNA adducts formed during cotreatment with other hydrocarbons will provide a rapid method for predicting the co- or anticarcinogenic effect of the other hydrocarbons.     

Effects of sex and age on DMBA:DNA binding in epidermis of SENCAR mice following topical administration of dimethylbenz[a] anthracene

Epidermal DMBA:DNA adduct formation was determined in male and female SENCAR mice following short-term exposure to 7,12-dimethylbenz[a]anthracene (DMBA). While qualitatively the adduct profiles are similar in both male and female mice, male mice generally had higher levels of each individual adduct as well as a greater total binding of DMBA to DNA at 6 and 48 h after topical administration. Additionally, the effect of age on total DMBA:DNA binding was examined in epidermis of male mice. While there appeared to be some effect of age on total DMBA:DNA binding at 6 h after administration, these differences were not statistically significant. However, DMBA:DNA binding at 48 h was found to vary significantly with age. The levels of binding were greatest between 40 and 54 days of age. These studies demonstrate the importance of considering both the age and sex of animals when examining the metabolism and binding of DMBA and in muscle skin tumor induction experiments using DMBA as an initiator.     

Effects of benzyl isothiocyanate and phenethyl isothiocyanate on benzo[a]pyrene metabolism and DNA adduct formation in the A/J mouse

Benzyl isothiocyanate (BITC) inhibits lung tumorigenesis induced in A/J mice by benzo[a]pyrene (B[a]P). In contrast, phenethyl isothiocyanate (PEITC) does not. We tested the hypothesis that BITC inhibits B[a]P tumorigenicity in mouse lung by inhibiting DNA adduct formation, and compared the effects of BITC and PEITC. In mouse liver or lung microsomal incubations, BITC and PEITC inhibited formation of 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (B[a]P-7, 8-diol) and some other B[a]P metabolites. The metabolism of B[a]P was compared in mouse lung and liver microsomes, 6 or 24h after treatment with BITC or PEITC. In lung, 6 h after treatment, B[a]P-7, 8-diol and some other metabolites were inhibited by BITC and PEITC. However, 24 h after treatment, no inhibition of B[a]P-7,8-diol was observed in microsomes from BITC-treated mice, whereas it was substantially increased in mice treated with PEITC. Effects on B[a]P metabolism in liver microsomes were generally modest. Conversion of B[a]P-7,8-diol to mutagens by mouse liver microsomes was more strongly inhibited by BITC than PEITC. Effects on 7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA adduct formation were evaluated in DNA from mice treated with isothiocyanates and B[a]P, and killed 2-120h later. The area under the curve (AUC) for BPDE-DNA adducts in lung was 29.5% less (P = 0. 001) in the BITC-B[a]P treated mice and 19.0% less (P = 0.02) in the PEITC-B[a]P mice than in the mice treated with B[a]P alone. Similar results were obtained in liver DNA. There were no significant differences between the reduction of BPDE-DNA AUC values by BITC versus PEITC. The results of this study support the hypothesis that BITC inhibits B[a]P-induced lung tumorigenesis in A/J mice by inhibiting the metabolic activation of B[a]P to BPDE-DNA adducts. However, differences in BPDE-DNA adduct formation do not appear to explain fully the contrasting effects of BITC and PEITC on B[a]P-induced lung tumorigenesis.     

Tissue distribution of DNA adducts in CDF1 mice fed 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ)

Male and female CDF1 mice were administered a single oral dose of 3 mumol of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) or 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and killed 24 h later. DNA was isolated from the livers, lungs, kidneys, colon and forestomach and analysed by 32P-postlabelling for the presence of IQ and MeIQ adducts. Several adduct-enrichment procedures were investigated, including ATP-deficient labelling conditions, butanol extraction and nuclease P1 digestion, and only the ATP-deficient procedure was found to produce the same adduct pattern on polyethyleneimine--cellulose TLC as the standard procedure. Up to nine adduct spots were detected in liver DNA from IQ-treated mice, two of which were not detected in other tissues. The levels of binding in both male and female mice were in the order liver greater than kidney greater than colon greater than forestomach greater than lung. Analysis of DNA from MeIQ-treated mice revealed the presence of up to seven adducts, one of which was detected in liver but not in other tissues. The relative order of DNA binding was kidney greater than liver greater than or equal to colon greater than forestomach greater than lung. As dietary feeding of IQ induces liver, lung and forestomach tumours, and MeIQ induces liver and forestomach tumours in this mouse strain, these binding levels do not correlate with the susceptibility of the organs to carcinogenesis induced by these compounds; the results may indicate the importance of additional factors in determining organ specificity of carcinogenicity.     

Effects of dietary and intraperitoneally administered beta-naphthoflavone on mutagenicity and tissue distribution of Trp-P-1 in the rat

The effect of dietary beta-naphthoflavone (BNF) on tissue retention of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was studied in the rat. Female rats, 3 weeks old, were fed a BNF-containing diet for 3 days before being dosed orally or i.v. with 14C-labelled Trp-P-1. The rats were killed at 4, 24 or 48 h after dosage and subjected to tape-section autoradiography. The tissue localization of Trp-P-1-derived radioactivity was compared to that observed in untreated rats and in rats given BNF i.p. Ethoxyresorufin-O-deethylase (EROD) activity and mutagenicity of Trp-P-1 in the Ames test, using S9 prepared from forestomach, glandular stomach, small intestine, liver and lung, were used as in vitro assays to measure the degree of cytochrome P450IA1 and/or P450IA2 induction. Dietary BNF treatment caused a 30- to 40-fold increase in EROD activity in the small intestine, but only a 2-fold increase in the liver and the lung. These inter-organ differences were not observed after i.p. administration of BNF. The increase in mutagenicity of Trp-P-1 in the Ames test could be correlated to the increase in EROD activity. The autoradiographic data showed that the route of administration of BNF as well as of Trp-P-1 were important for the tissue localization of Trp-P-1. Dietary BNF treatment caused a pronounced retention of Trp-P-1-derived radioactivity in the epithelia of the small intestine, forestomach, oesophagus and the oral cavity, regardless of the administration route of Trp-P-1; a similar though less pronounced epithelial retention was observed after i.p. injection of BNF. A clear-cut boundary of accumulated radioactivity between the forestomach and the glandular stomach where the levels were almost non-detectable was observed in rats fed the BNF-containing diet. It is concluded that dietary inducers may be important determinants of metabolism and tissue distribution of toxic compounds.     

Inhibition of rat liver cytochrome P450 isozymes by isothiocyanates and their conjugates: a structure-activity relationship study

A series of arylalkyl and alkyl isothiocyanates, and their glutathione,cysteine, and N-acetylcysteine conjugates were used to studytheir inhibitory activity toward the dealkylation of ethoxyresorufin(EROD), pentoxyresorufin (PROD), and methoxyresorufin (MROD)in liver microsomes obtained from the 3-methylcholanthrene orphenobarbital-treated rats. These reactions are predominantlymediated by cytochrome P450 (P450) isozymes 1A1 and 1A2, 2B1and 1A2, respectively. All isothiocyanates inhibited PROD morereadily than EROD. Increases in the alkyl chain length of arylalkylisothiocyanates to C₆ resulted in an increased inhibitory potencyin these assays; at longer alkyl chain lengths (C₈-C₁₀) theinhibitory potency declined. The IC₅₀s for phenethyl isothiocyanate(PEITC) were 47, 46 and 1.8 µM for EROD, MROD and PROD,respectively. Substitution of an additional phenyl group onPEITC also increased the inhibitory potency; the IC₅₀s for 1,2-diphenylethyl isothiocyanate (1, 2-DPEITC) and 2, 2-diphenylethylisothiocyanate (2,2-DPEITC) were 0.9 and 0.26 µM for EROD,and 0.045 and 0.13 µM for PROD, respectively. The relativeinhibitory potency of PEITC and its conjugates was N-acetylcysteine-PEITC(PEITC-NAC) < glutathione-PEITC (PEITC-GSH) < cysteine-PEITC(PEITC-CYS) < PEITC. The observations that the parent isothiocyanateswere more potent inhibitors than the conjugates suggest thatdissociation of the conjugate is required for activity. Naturallyoccurring alkyl isothiocyanates, sulforaphane (SFO) and allylisothiocyanate (AITC), were very weak inhibitors in the assays.These results suggest the potential of isothiocyanates as structuralprobes for studying P450 isozymes. In addition, the inhibitoryactivity of isothiocyanates for PROD correlated with the previouslydemonstrated tumor inhibitory potency in (4-methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) induced A/J mouse lung tumor bioassays, which supportsearlier findings that P450 2B1 is one of the major isozymesinvolved in NNK activation and that inhibition of this isozymeis an important mechanism for the chemopreventive activity ofisothiocyanates.     

中药蛇床子水溶性提取物中化学成分的抗诱变性研究

本文采用Ａｍｅｓ、体内试验小鼠骨髓细胞染色体畸变和骨髓多染红细胞微核试验,对蛇床子水溶性提取物中９种化学成分（蛇床子素,佛手柑内酯,异虎耳草素、欧芹属素乙、花椒毒酚,花椒毒素,甲基嘧啶,尿嘧啶和１种待定化合物）进行了抗诱变性研究。结果表明,蛇床子素,佛手柑内酯,异虎耳草素,欧芹属素乙,花椒毒酚和花椒毒素在典曲霉菌素Ｂ１诱变性的抑制作用中具有较高活性。蛇床子素,佛手柑内酯、异虎耳草素和欧芹属素乙在环     

Cancer initiation by polycyclic aromatic hydrocarbons results from formation of stable DNA adducts rather than apurinic sites.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants with high carcinogenic potencies that have been linked to the etiology of human cancers through their presence in cigarette smoke and environmental mixtures. They are metabolically activated in cells by cytochrome P450 enzymes and/or peroxidases to reactive intermediates that damage DNA. One pathway of activation forms dihydrodiol epoxides that covalently bind to exocyclic amino groups of purines in DNA to form stable adducts. Another pathway involves formation of radical cations that bind to the N7 or C8 of purines to form unstable adducts that depurinate to leave apurinic (AP) sites in DNA. In the present study the proportions of stable DNA adducts and AP sites formed by the carcinogenic PAHs dibenzo[a,l]-pyrene (DB[a,l]P), 7,12-dimethylbenz[a]anthracene (DMBA), and benzo[a]pyrene (B[a]P) have been investigated in a target tissue for carcinogenesis, mouse epidermis. After topical application of the PAHs on the skin of female SENCAR mice epidermal DNA was isolated and the formation of stable DNA adducts was measured by (33)P-postlabeling and HPLC analysis. AP sites in DNA were measured with an aldehyde reactive probe in a slot-blot assay. At both 4 and 24 h after exposure, DB[a,l]P formed significantly higher amounts of stable DNA adducts than DMBA, and B[a]P exhibited the lowest level of binding. In contrast, the number of AP sites present in mice treated with these PAHs was in the order: DMBA > B[a]P > DB[a,l]P. The level of AP sites was significantly lower than the level of stable adducts for each PAH. The most potent carcinogen, DB[a,l]P, induced the highest level of stable adducts and the lowest level of AP sites in epidermal DNA. These results indicate that stable DNA adducts rather than AP sites are responsible for tumor initiation by carcinogenic PAHs.     

Interspecies differences in the major DNA adducts formed from benzo(a)pyrene but not 7,12-dimethylbenz(a)anthracene in rat and human mammary cell cultures

Mammary epithelial cells from rats and humans show both quantitative and qualitative species- and carcinogen-specific differences in their abilities to activate benzo(a)pyrene (B(a)P) and 7,12-dimethylbenz(a)anthracene (DMBA). Previous studies of the DNA binding of these compounds in mammary epithelial cells demonstrated that rat cells bound relatively more DMBA than B(a)P to DNA under identical treatment conditions, while the opposite pattern was exhibited by human mammary epithelial cells. The specific DNA adducts formed in these cells after 24-h incubations with [3H]DMBA and [3H]B(a)P were analyzed to determine if there were qualitative as well as quantitative differences in the amounts of individual adducts. Similar proportions of specific DMBA-DNA adducts were found in both rat and human cells, although the total amount of adducts formed was significantly higher in the rat cells. In contrast, an essentially qualitative species-specific difference was observed in the major B(a)P-DNA adduct present in the rat and human cells. The major B(a)P adduct formed in the human mammary epithelial cells was identified as the (+)-anti-B(a)P-7,8-dihydrodiol-9, 10-epoxide(BPDE)-deoxyguanosine adduct. However, this adduct was formed at very low levels in the rat mammary epithelial cells. The rat cells contained a large proportion of syn-BPDE adducts, and other unidentified B(a)P-DNA adducts. The high level of the (+)-anti-BPDE-deoxyguanosine adduct in the human but not the rat mammary cells is consistent with the potential role of (+)-anti-BPDE in the high mutagenic activity of B(a)P in the cell-mediated mutagenesis assays using the human mammary cells as activators, and the low mutagenic activity of B(a)P when rat cells were used as activators. The quantitative differences in the activation of DMBA by cells from these two species are also consistent with the cell-mediated mutagenic activities of DMBA using these cells as activators. These results suggest that the higher carcinogenic activity of DMBA compared to B(a)P in the rat mammary gland may not be indicative of the relative carcinogenic potencies of these compounds for human mammary cells.     

32P-postlabeling analysis of DNA adducts persisting for up to 42 weeks in the skin, epidermis and dermis of mice treated topically with 7,12-dimethylbenz[a]anthracene

The initial and persistent levels of 7,12-dimethylbenz[a]-anthracene (DMBA)-DNA adducts in mouse skin, epidermis and dermis after topical carcinogen application were studied by 32P-postlabeling assay. In the major experiment, a single dose of 1.2 mumol of the carcinogen was applied to the shaved backs of adult female BALB/cANN mice, and DNA was isolated from epidermis and dermis, respectively, 24 h and 1, 2, 3, 4, 8, 16, 24, 36 and 42 weeks later. Total binding at 24 h was approximately 34 and approximately 28 adducts in 10(7) normal nucleotides for epidermal and dermal DNA, respectively. (One adduct in 10(7) nucleotides equals 0.3 fmol adduct/microgram DNA.) While initial binding was higher in epidermal DNA, the adducts were approximately 10 times more persistent in dermal DNA: at 42 weeks, total binding levels were approximately 0.17 and approximately 1.7 adducts in 10(7) nucleotides for epidermis and dermis, respectively. To quantitate low levels of DMBA-DNA adducts, 32P-postlabeling assays were run in the presence of a limiting amount of carrier-free [gamma-32P]ATP; this was found to favor labeling of the adducts, thereby leading to a 20- to 100-fold enhancement of the method's sensitivity for individual adducts. One of the three major DMBA-DNA adducts was more persistent than were the others; the level of this adduct remained constant at approximately 60% of the total in epidermal and dermal DNA during the last 18 weeks of the 42-week observation period. Since a [3H]thymidine-labeling experiment showed a normal epidermal DNA turnover 40 weeks after DMBA treatment, it was concluded that the bulk of the persistent adducts was present in subpopulations of dormant cells. We have hypothesized that such cells, in the absence of a promoting stimulus, are incapable of division because of the adduction and/or mutation of genes critical for growth (proto-oncogenes), and may thus correspond to the 'latent tumor cells', as defined by Berenblum and Shubik in their classical analysis of the attributes of tumor initiation and promotion.