In vitro investigation of cytochrome P450-mediated metabolism of dietary flavonoids.

Human and mouse liver microsomes and membranes isolated from Escherichia coli, which expressed cytochrome P450 (CYP) 1A2, 3A4, 2C9 or 2D6, were used to investigate CYP-mediated metabolism of five selected dietary flavonoids. In human and mouse liver microsomes kaempferol, apigenin and naringenin were hydroxylated at the 3'-position to yield their corresponding analogs quercetin, luteolin and eriodictyol, whereas hesperetin and tamarixetin were demethylated at the 4'-position to yield eriodictyol and quercetin, respectively. Microsomal flavonoid metabolism was potently inhibited by the CYP1A2 inhibitors, fluvoxamine and -naphthoflavone. Recombinant CYP1A2 was capable of metabolizing all five investigated flavonoids. CYP3A4 recombinant protein did not catalyze hesperetin demethylation, but showed similar metabolic profiles for the remaining compounds, as did human microsomes and recombinant CYP1A2, although the reaction rates in general were lower as compared to CYP1A2. CYP2C9 catalyzed the 4'-demethylation of tamarixetin, whereas CYP2D6 did not seem to play any role in the metabolism of the selected flavonoids. The major involvement in flavonoid metabolism of human CYP1A2, which mediates the formation of metabolites with different biochemical properties as compared to the parent compound and furthermore is known to be expressed very differently among individuals, raises the important question of whether individual differences in the CYP enzyme activity might affect the beneficial outcome of dietary flavonoids, rendering some individuals more or less refractory to the health-promoting potential of dietary flavonoids.

Dietary phytoestrogens and estrogen inhibit experimental atherosclerosis.

OBJECTIVE: The effect of phytoestrogen intake in combination with estrogen replacement therapy (ERT) on atherogenesis is largely unknown. The aim was thus to study the impact of phytoestrogens alone, or combined with oral estrogen, on experimental atherosclerosis in cholesterol-fed rabbits. METHODS: Two separate studies were performed in ovariectomized, cholesterol-fed female rabbits. In Study A, 45 rabbits were randomized to either a soy-free diet with or without oral 17 beta-estradiol (E2) 4 mg/day, or a soy-rich diet without any hormone for 14 weeks. In Study B, 100 rabbits were randomized into five groups (oral E2 0.5, 1, 2 or 4 mg/day, or no hormone) based on a soy-rich diet for 30 weeks. RESULTS: By the end of treatment in Study A, aortic cholesterol content was twice the amount in the group treated with the soy-free diet compared with the soy-rich group and with the soy-free plus E2 group (p < 0.001). In Study B, aortic cholesterol content showed no significant difference between the groups (ANOVA, p = 0.49), but a tendency towards a lower aortic cholesterol content in the E2-treated animals compared with placebo was observed. CONCLUSION: Dietary phytoestrogens significantly reduce aortic cholesterol content with a potency comparable to that of ERT, and seem to enhance (although mildly) the antiatherogenic effect of E2 in this model.

17beta-Estradiol but not the phytoestrogen naringenin attenuates aortic cholesterol accumulation in WHHL rabbits.

The effects of 17beta-estradiol (17beta-E(2)) or the phytoestrogen naringenin on spontaneous atherosclerosis were studied in 36 ovariectomized homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits receiving a semisynthetic control diet; this diet added 0.0040% 17beta-E(2;) or 0.20% naringenin, for 16 weeks. The uterine weight was increased (P < 0.001) and the concentration of estrogen receptor alpha was decreased (P < 0.001) in the 17beta-E(2) group compared with the controls. Total plasma cholesterol and triglycerides were not different from those in the controls. In lipoproteins, HDL cholesterol was increased (P < 0.01), and LDL triglyceride and IDL triglyceride were lowered (P < 0.05). The oxidation (as concentration of malondialdehyde) was increased in LDL (P < 0.05) but not in plasma. The cholesterol accumulation was decreased (P < 0.05) in the ascending aorta and in the total aorta but the ratio of intima to media and area of intima in ascending, thoracic, and abdominal aorta were not significantly different. In the naringenin group the only differences, compared with the control group, were increased HDL cholesterol (P < 0.001) and decreased activity of glutathione reductase (P < 0.05). In conclusion, 17beta-E(2), but not naringenin, attenuated aortic cholesterol accumulation independently of plasma and LDL cholesterol. Further, these results support previously suggested pro-oxidant ability of 17beta-E(2) toward LDL and a possible connection between the pro-oxidant nature of 17beta-E(2) and its antiatherogenic effect.

The influence of simple sugars and starch given during pre- or post-initiation on aberrant crypt foci in rat colon.

The aim of the present study was to investigate the enhancing effect of dietary sugar on the development of aberrant crypt foci (ACF) in male F344 rats initiated with azoxymethane (AOM). The potential role of sugar as either a co-initiator or a promoter was investigated by giving diets high in sucrose and dextrin (61%) during either the pre-initiation, the initiation, and/or the post-initiation stage of the ACF development. The colonic cell proliferation, activity of colonic phase II enzymes, and a biomarker of lipid peroxidation were additionally examined in order to obtain information on the specific mechanisms involved in the suggested effect of sucrose and dextrin on ACF development. The number of large sized and the total number of ACF were significantly increased by feeding sucrose and dextrin in the post-initiation period. No positive association between colonic cell proliferation and ACF was seen. The level of oxidative stress in the cytosol from the proximal colon and colonic glutathione transferase and quinone reductase was not affected by the sugar treatments. The overall results from this study show that sucrose and dextrin enhance the number of preneoplastic lesions in AOM-initiated rats, and act primarily as promoters in the development of ACF.

Biotransformation of the citrus flavone tangeretin in rats. Identification of metabolites with intact flavane nucleus.

The present study was carried out in order to investigate the in vivo biotransformation and excretion of the flavone, tangeretin, found in citrus fruits, by analysing urine and faeces samples from rats after repeated administration of 100 mg/kg body weight/day tangeretin. The formed metabolites were separated and identified by HPLC and the structures elucidated by LC/MS and 1H NMR. Ten new, major metabolites with intact flavonoid structure were identified. The metabolites identified were either demethylated or hydroxylated derivatives of the parent compound and metabolic changes were found primarily to occur in the 4' position of the B-ring. The total urinary excretion of tangeretin metabolites with intact flavan nucleus was about 11% of the administered daily dose.

Estrogenic activity of flavonoids in mice. The importance of estrogen receptor distribution, metabolism and bioavailability.

The in vivo estrogenic potential of the flavonoids apigenin, kaempferol, genistein and equol was investigated in immature female mice. Genistein and equol, administered by gavage for 4 consecutive days [post-natal day (PND) 17-20, 100 mg/kg body weight], was found to significantly increase uterine weights and the overall uterine concentration of estrogen receptor alpha (ERalpha). In kaempferol- and equol-exposed mice the cytosolic ERalpha concentration was significantly increased as compared to the solvent control, which is speculated to result in an increased sensitivity of the uterus to subsequently encountered estrogens. Oral administration of equol, genistein, biochanin A and daidzein to 6-week-old female mice revealed a great variation in their systemic bioavailability. The urinary recovery of equol was thus over 90% of a single gavage administered dose, whereas the urinary recoveries of biochanin A, genistein and daidzein were 16, 11 and 3%, respectively. Most of the metabolites were either hydroxylated or dehydrogenated forms of the parent compounds. The in vitro estrogenic potency of some of the metabolites was greater than that of the parent compounds, whereas others were of similar or lower potency. Bioavailability, metabolism, the ability to alter ERalpha distribution in the uterus and the estrogenic potential of parent compound and metabolites may thus contribute to the differences in in vivo estrogenicity of dietary flavonoids.

Dose-response effects of lycopene on selected drug-metabolizing and antioxidant enzymes in the rat.

The administration of lycopene to female rats at doses ranging from 0.001 to 0.1 g/kg b.w. per day for 2 weeks was found to alter the drug-metabolizing capacity and antioxidant status of the exposed animals. An investigation of four cytochrome P450-dependent enzymes revealed that benzyloxyresorufin O-dealkylase activity in the liver was significantly induced in a dose-dependent fashion at all lycopene doses investigated. Likewise, ethoxyresorufin O-dealkylase activity was induced, although only at the two highest lycopene concentrations tested. An investigation of selected phase 2 detoxification enzymes provided evidence that lycopene was capable of inducing hepatic quinone reductase, approximately two-fold, at doses between 0.001 and 0.05 g/kg b.w. per day, whereas no effect was observed at the remaining doses tested. Glutathione transferase, using the two substrates, 2,4-dichloronitrobenzene and 1-chloro-2, 4-dinitrobenzene, was significantly induced at the 0.1 g/kg b.w. per day dose, whereas no effect was observed at the remaining lycopene doses. Analysis of the antioxidant status of the blood compartment revealed that three out of four antioxidant enzymes were affected by lycopene treatment. The activity of superoxide dismutase was thus significantly induced at lycopene doses of 0.005 and 0.05 g/kg b.w, whereas glutathione reductase and glutathione peroxidase was only induced at the 0.005 g/kg b.w. per day dose. For all antioxidant enzymes investigated, the activities seemed to return to the control level after exerting peak induction at doses between 0.005 and 0.05 g/kg b.w. per day. The explanation for this remains unknown. The plasma concentration of lycopene at dietary levels of 0.001, 0.005, 0.05 and 0.1 g/kg b.w. per day was estimated to be 16, 32, 71 and 67 nM, which is barely within the lower range of the mean human plasma concentration of lycopene, which ranges from 70-1790 nM. Oxidative stress induced by the heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), and investigated by analyzing for malondialdehyde in plasma, was not found to be affected by prior lycopene exposure. The level of PhIP-DNA adducts in the liver or colon was likewise not affected by lycopene at any dose. Overall, the present study provides evidence that lycopene administered in the diet of young female rats exerts minor modifying effects toward antioxidant and drug-metabolizing enzymes involved in the protection against oxidative stress and cancer. The fact that these enzymatic activities are induced at all of these very low plasma levels, could be taken to suggest that modulation of antioxidant and drug-metabolizing enzymes may indeed be relevant to humans, which in general exhibit a plasma lycopene level several fold above the effective levels observed in this study.

Screening of selected pesticides for inhibition of CYP19 aromatase activity in vitro.

Many pesticides are able to block or activate the steroid hormone receptors and/or to affect the levels of sex hormones, thereby potentially affecting the development or expression of the male and female reproductive system or both. This emphasizes the relevance of screening pesticides for a wide range of hormone-mimicking effects. Twenty-two pesticides were tested for their ability to affect CYP19 aromatase activity in human placental microsomes using the classical [(3)H](2)O method. Prochloraz, imazalil, propioconazole, fenarimol, triadimenol, triadimefon (all fungicides), and dicofol (an acaricide) gave rise to a statistically significant inhibition of aromatase activity. The IC(50)s of prochloraz, imazalil, propioconazole fenarimol, triadimenol, and triadimefon were calculated from dose-response curves to be 0.04, 0.34, 6.5, 10, 21 and 32 microM, respectively. The IC(50) of dicofol was greater than 50 microM. The positive control 4-hydroxyandrostendione (1 microM) caused an inhibition of aromatase activity by 74%. The compounds, which did not affect the aromatase activity, were bromopropylate, chlorfenvinphos, chlorobenzilate, chlorpyrifos, diuron, heptachlor, iprodion, linuron, pentachlorphenol, procymidon, propyzamide, quintozen, tetrachlorvinphos and tetradifon. With the purpose of comparing the results for fenarimol obtained with the microsomal system with data from an intact cell system, an aromatase assay based on JEG-3 cells was established. 4-Hydroxyandrostendione (1 microM) inhibited the aromatase activity in JEG-3 cells by 94%. The IC(50) for fenarimol in this system was 2 microM, slightly lower than that observed in the microsomal system. For the first time, fenarimol has been demonstrated to inhibit aromatase activity in human tissues and, furthermore, propioconazole, triadimefon, and triadimenol were identified as weak aromatase inhibitors. In conclusion, seven out of 22 tested pesticides turned out to be weak to moderate aromatase inhibitors in vitro, indicating the relevance of elucidating the endocrine effects in vivo of these- compounds.

Chlorophyllin chemoprevention in trout initiated by aflatoxin B(1) bath treatment: An evaluation of reduced bioavailability vs. target organ protective mechanisms.

Chlorophyllin (CHL) is known to inhibit DNA adduction and hepatocarcinogenesis in trout when administered at doses up to 4000 ppm in the diet with aflatoxin B(1) (AFB(1)). The principal protective mechanism is believed to involve CHL:AFB(1) complex formation, which may reduce systemic carcinogen absorption. However, mechanisms operative within the target organ in situ have not been ruled out. The present study used alternative CHL and AFB(1) exposures as well as hepatic metabolism studies to distinguish these mechanisms. Duplicate lots of 150 rainbow trout each were initiated by brief water bath exposure to 0.1 ppm AFB(1), with or without 500 ppm CHL in the water. The addition of 500 ppm CHL to the water bath, under conditions where AFB(1) is calculated to be >99% sequestered as the CHL:AFB(1) complex, reduced hepatic AFB(1)-DNA adduction by 95% and reduced hepatocarcinogenesis from 20.5% to 2%, compared with exposure to AFB(1) alone. Inclusion of 500 ppm CHL in the water bath also significantly reduced total body burden and hepatic levels of AFB(1) as well as AFB(2), a structural analogue of AFB(1) unable to directly form the 8,9-epoxide proximate electrophile but equally capable of complexing with CHL. By contrast, internal target organ CHL loading by pretreatment of trout with 4000 ppm dietary CHL for 7 days prior to (and 2 days following) AFB(1) waterbath exposure had no effect on AFB(1)-DNA adduction or tumorigenicity. Dietary CHL up to 8000 ppm had no effect on hepatic CYP2K1, CYP1A, glutathione transferase, UDP-glucuronosyl transferase, or, with one exception, the relative ratios among hepatic AFB(1) metabolites in vivo. These results support the hypothesis that CHL:AFB(1) complex formation and reduced systemic AFB(1) bioavailability is a principal mechanism for CHL chemoprevention in this model and that in situ target organ inhibitory mechanisms are relatively insignificant.

Time-related bisphenol-A content and estrogenic activity in saliva samples collected in relation to placement of fissure sealants.

It was recently reported that estrogenic activity was detected in saliva samples collected during 1 h after placement of one fissure sealant (Delton) and this related to Bisphenol-A (BPA) content. The aim of the present study was to determine the time-related BPA content and estrogenic activity in saliva samples collected before and after placement of two fissure sealants each with a different monomer composition. Eight healthy male volunteers with no history of prior placement of fissure sealants or composite resin fillings had four molars sealed with either Delton LC (four people) or Visio-Seal (four people). Base-line saliva samples were collected preexperimentally, in the morning when fasting. Fissure sealants were placed and saliva samples collected immediately, 1 h and 24 hs after placement of the fissure sealant. BPA was found in saliva samples collected immediately after placement of Delton LC (range 0.3-2.8 ppm). No detectable amounts of BPA were determined 1 h and 24 h after Delton treatment (detection limit < or = 0.1 ppm). In base-line samples and in all samples collected from Visio-Seal treated individuals, no BPA was detected. In a recombinant yeast cell assay, significantly increased estrogenic activity was found in saliva samples collected immediately after placement of Delton LC sealant (P < 0.05; ANOVA) whereas no statistically significant estrogenic activity was observed in the remaining groups. In conclusion, minute amounts of BPA, however considerably lower than previously reported, were detected in saliva samples collected immediately after but not 1 and 24 h(s) after placement of Delton LC fissure sealant. BPA was not detected after placement of Visio-Seal fissure sealant.

Differential effects of dietary flavonoids on drug metabolizing and antioxidant enzymes in female rat.

1. Gavage administration of the natural flavonoids tangeretin, chrysin, apigenin, naringenin, genistein and quercetin for 2 consecutive weeks to the female rat resulted in differential effects on selected phase 1 and 2 enzymes in liver, colon and heart as well as antioxidant enzymes in red blood cells (RBC). 2. Glutathione transferase (GST) activity assayed by use of the substrate 1-chloro-2,4-dinitrobenzene was significantly induced by apigenin, genistein and tangeretin in the heart but not in colon or liver. 3. In RBC chrysin, quercetin and genistein significantly decreased the activity of glutathione reductase (GR), catalase (CAT) and glutathione peroxidase (GPx), whereas superoxide dismutase (SOD) was only significantly decreased by genistein. 4. The oxidative status of the animal, measured as plasma malondialdehyde, revealed that chrysin, quercetin, genistein, and beta-naphthoflavone (BNF) significantly protected against, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP)-induced oxidative stress. Hepatic PhIP-DNA adduct formation was not affected by any of the administered flavonoids, whereas PhIP-DNA adduct formation in colon was slightly, but significantly, inhibited by quercetin, genistein, tangeretin and BNF. 5. The observed effects of chrysin, quercetin and genistein on antioxidant enzymes, concurrently with a protection against oxidative stress, suggest a feedback mechanism on the antioxidant enzymes triggered by the flavonoid antioxidants. 6. Despite the use of high flavonoid doses, which by far exceed the human exposure levels, the effect on drug metabolizing and antioxidant enzymes was still very minor. The role of singly administered flavonoids in the protection against cancer and heart disease is thus expected to be limited.

Screening of selected pesticides for oestrogen receptor activation in vitro.

Twenty pesticides were tested for their ability to activate the oestrogen receptor in vitro using an MCF7 cell proliferation assay and a Yeast Oestrogen Screen. The fungicides fenarimol, triadimefon, and triadimenol were identified as weak oestrogen receptor agonists, which at 10 microM induces a 2.0, 2.4, and 1.9-fold increase in proliferation of human MCF7 breast cancer cells (E3 clone). The relative proliferation efficiency (RPE) was 43-69%, indicating partial agonism at the oestrogen receptor. Several pesticides did not have any effect on the proliferation response after 6 days of exposure, including: chlorpyrifos, diuron, iprodion, linuron, pentachlorphenol, prochloraz, propioconazol, propyzamine, quintozen, tetrachorvinphos and tetradifon. Some pesticides resulted in a negligible proliferation response, which was not statistically significant under the present experimental conditions. These were: bromopropylate, chlorfenvinphos, chlorobenzilate, dicofol, heptachlor, and imazalil. Fenarimol and dicofol also gave rise to a positive oestrogenic response in yeast cells transfected with the oestrogen receptor alpha, whereas the remaining compounds resulted in a negative response due either to biological inactivity or cytotoxocity to the yeast cells. The EC50 for fenarimol was estimated to be 13 microM in the yeast cells, compared with an EC50 of 3 microM in the MCF7 cells, indicating higher sensitivity of the latter assay. No in vivo data for fenarimol, triadimefon or triadimenol have previously been published that support oestrogenic activity in the intact animal. Thus, from the present results we suggest that oestrogen receptor activation may not be an important mode of action for these compounds. The need to include at least two bioassays in a screening procedure and for combining in vitro and in vivo data is emphasized.

Chemopreventive properties of chlorophylls towards aflatoxin B1: a review of the antimutagenicity and anticarcinogenicity data in rainbow trout.

The anticarcinogenic activity of chlorophyllin (CHL), a water-soluble derivative of chlorophyll, was first reported in rainbow trout. This review describes certain experiments which set the stage for long-term tumor bioassays, in trout and other species, using CHL and various food-borne carcinogens. Initial work with trout and rat liver enzymes in the Salmonella assay showed that CHL was a potent antimutagen towards heterocyclic amines, polycyclic aromatic hydrocarbons, aflatoxins and other classes of mutagen. Antimutagenic activity was further demonstrated using the corresponding direct-acting mutagens in the absence of an exogenous metabolizing system. Mutagen-inhibitor interaction (molecular complex formation) was identified in spectrophotometry studies, suggesting that CHL acts as an 'interceptor molecule'. In vivo, CHL reduced hepatic AFB1-DNA adducts and hepatocarcinogenesis when the inhibitor and carcinogen were co-administered in the diet. Finally, co-injection of inhibitor and AFB1 into trout embryos established that CHL was more effective than chlorophyll a in reducing AFB1-DNA adducts 2 weeks after injection, and liver tumors after 1 year.

Detection of weak estrogenic flavonoids using a recombinant yeast strain and a modified MCF7 cell proliferation assay.

A newly developed recombinant yeast strain, in which the human estrogen receptor has been stably integrated into the genome of the yeast, was used to gain information on the estrogenic activity of a large series of dietary flavonoids. Among 23 flavonoids investigated, 8 were found to markedly stimulate the transcriptional activity of the human estrogen receptor in the yeast assay increasing transcriptional activity 5-13-fold above background level, corresponding to EC50 values between 0.1 and 25 microM. Five compounds increased the transcriptional activity 2-5-fold over the control, with EC50 values ranging from 84 to 102 microM, whereas the remaining flavonoids were devoid of activity. The most potent flavonoid estrogens tested were naringenin, apigenin, kaempferol, phloretin, and the four isoflavonoids equol, genistein, daidzein, and biochanin A. With the exception of biochanin A, the main feature required to confer estrogenicity was the presence of a single hydroxyl group in the 4'-position of the B-ring of the flavan nucleus, corresponding to the 4-position on phloretin. The estrogenic potency of the flavonoids was found to be 4 000-4 000 000 times lower than that observed for 17beta-estradiol, when compared on the basis of EC50 values. The estrogenic activity of the dietary flavonoids was further investigated in estrogen-dependent human MCF7 breast cancer cells. In this system several of the flavonoids were likewise capable of mimicking natural estrogens and thereby induce cell proliferation. Similar structural requirements for estrogenic activity were found for the two assays. The present results provide evidence that several of the flavo-estrogens possess estrogenic properties comparable in activity to the well-established isoflavonoid estrogens. The use of Alamar Blue, a vital dye which is metabolically reduced by cellular enzymes to a fluorescent product, was found to greatly simplify the MCF7 cell-based estrogen screen, making this mammalian assay applicable as a large-scale screening tool for estrogenic compounds.

In vitro biotransformation of flavonoids by rat liver microsomes.

1. Sixteen naturally occurring flavonoids were investigated as substrates for cytochrome P450 in uninduced and Aroclor 1254-induced rat liver microsomes. Naringenin, hesperetin, chrysin, apigenin, tangeretin, kaempferol, galangin and tamarixetin were all metabolized extensively by induced rat liver microsomes but only to a minor extent by uninduced microsomes. No metabolites were detected from eriodictyol, taxifolin, luteolin, quercetin, myricetin, fisetin, morin or isorhamnetin. 2. The identity of the metabolites was elucidated using lc-ms and 1H-nmr, and was consistent with a general metabolic pathway leading to the corresponding 3',4'-dihydroxylated flavonoids either by hydroxylation or demethylation. Structural requirements for microsomal hydroxylation appeared to be a single or no hydroxy group on the B-ring of the flavan nucleus. The presence of two or more hydroxy groups on the B-ring seemed to prevent further hydroxylation. The results indicate that demethylation only occurs in the B-ring when the methoxy group is positioned at C4', and not at the C3'-position. 3. The CYP1A isozymes were found to be the main enzymes involved in flavonoid hydroxylation, whereas other cytochrome P450 isozymes seem to be involved in flavonoid demethylation.

Mechanisms of chlorophyllin anticarcinogenesis against aflatoxin B1: complex formation with the carcinogen.

Chlorophyllin (CHL), a food-grade derivative of the green plant pigment chlorophyll, has recently been shown in this laboratory to be a potent inhibitor in vivo of hepatic aflatoxin B1 (AFB1)-DNA adduction and hepatocarcinogenesis (Breinholt et al. (1995) Cancer Res. 55, 57-62). We report here that CHL forms a strong noncovalent complex with AFB1 in vitro (dissociation constant (Kd) by Scatchard analysis = 1.4 (+/- 0.4) microM based on copper chlorin content), which may contribute to its anticarcinogenic activity. Kd values for the related porphyrins chlorine e6, protoporphyrin IX, and zinc protoporphyrin IX were also of the same order of magnitude as that of the commercial CHL. Mole ratio analysis provided evidence that all porphyrins examined associate with AFB1 at an approximate one to one stoichiometric ratio. Energy minimization computer modeling of the complex indicates a favorable association energy of -20 kcal/mol, independent of oxidation state of the 8,9-double bond of AFB1. AFB1 incubated in vitro with liver microsomes in the presence of added CHL showed comparable levels of inhibition in the production of several phase 1 metabolites, including the postulated procarcinogenic metabolite AFB1 8,9-epoxide. Kinetic analysis of microsome-catalyzed AFB1-DNA adduction suggested a CHL inhibition constant near 10 microM chlorin. In vivo, addition of CHL to concentrated AFB1 solutions followed by gavage administration resulted in dose-dependent inhibition of hepatic AFB1-DNA adduction, whereas the same dosages of AFB1 and CHL incorporated into a single bolus of trout diet for gavage provided less protection at all CHL doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary chlorophyllin is a potent inhibitor of aflatoxin B1 hepatocarcinogenesis in rainbow trout.

Epidemiological and experimental evidence indicates a strong relationship between diet and cancer. The purpose of this study was to examine the potential of chlorophyllin (CHL), a food-grade derivative of the ubiquitous green plant pigment chlorophyll, to inhibit experimental carcinogenesis. We report that CHL is a potent, dose-responsive inhibitor of aflatoxin B1 DNA adduction and hepatocarcinogenesis in the rainbow trout model when fed with carcinogen. CHL neither promoted nor suppressed carcinogenesis with chronic postinitiation feeding. By molecular dosimetry analysis, reduced aflatoxin B1-DNA adduction accounted quantitatively for reduced tumor response up to 2000 ppm dietary CHL, but an additional protective mechanism was operative at 4000 ppm CHL. The finding of potent inhibition (up to 77%) at CHL levels well within the chlorophyll content of some green leafy vegetables may have important implications in intervention and dietary management of human cancer risks.

Chemopreventive properties of chlorophyllin: inhibition of aflatoxin B1 (AFB1)-DNA binding in vivo and anti-mutagenic activity against AFB1 and two heterocyclic amines in the Salmonella mutagenicity assay.

Chlorophyllin (CHL), a sodium/copper derivative of chlorophyll, has been used to treat a number of human conditions with no toxic effects being reported. Recent studies have described the anti-mutagenic activity of CHL in several short-term genotoxicity assays; however, this compound has not been reported to inhibit carcinogen--DNA binding in vivo, and it has yet to be evaluated as an anti-carcinogen in any species. The chemopreventive properties of CHL were studied in trout using inhibition of aflatoxin B1 (AFB1)--DNA binding as an end-point. Chlorophyllin and AFB1 were coadministered in the diet, and carcinogen--DNA binding levels were determined in liver after 1, 3, 5 and 7 days. Linear increases in AFB1--DNA binding occurred with time of treatment at each CHL dose level (0, 500, 1000 and 2000 p.p.m.). Each increase in CHL dose produced a concomitant decrease in AFB1--DNA binding, resulting in a series of curves of decreasing slope. At the highest CHL dose level of 2000 p.p.m., AFB1--DNA binding was inhibited by 70%. These results suggest that CHL should be a potent inhibitor of AFB1-induced hepatocarcinogenesis in this model. In the Salmonella assay, CHL exhibited potent anti-mutagenic activity against AFB1 and two heterocyclic amines when incubated in the presence of trout liver activation systems. CHL also inhibited the mutagenic activity of AFB1-8,9-epoxide in the absence of a metabolic activation system. Dietary CHL substantially inhibited liver AFB1-DNA binding in vivo, even when AFB1 was given by i.p. injection to avoid direct AFB1--CHL interaction in the diet or gut. Collectively, these studies support a CHL inhibitory mechanism involving complex formation with the carcinogen in the gut coupled with electrophile scavenging or further complexing in the target organ.