Promotion of aflatoxin B1 carcinogenesis by the natural tumor modulator indole-3-carbinol: influence of dose, duration, and intermittent exposure on indole-3-carbinol promotional potency.

Indole-3-carbinol (13C), a secondary metabolite from cruciferous vegetables, inhibits aflatoxin B1 (AFB1) hepatocarcinogenesis in trout (Bailey et al., J. Natl. Cancer Inst., 78: 931-934, 1987) and rats (Selivonchick et al., unpublished results) when given prior to and with carcinogen but promotes carcinogenesis in both species when given continuously following AFB1 initiation. Since human 13C intake may not be continuous, and the promotional stimulation may be reversible, we have assessed 13C promotion using delayed and discontinuous exposure protocols. Following initiation with AFB1, 13C was fed to trout for varying periods of time, with varying lengths of delay after initiation and continuous or intermittent patterns of 13C treatment. Promotional enhancement of tumor incidence by 13C was found to be significant when 13C treatment was delayed for several weeks or months after the initial AFB1 challenge. Promotion also was found to increase with length of exposure to 13C treatment and to be decreased but still evident when 13C was given in alternating months or weeks, or twice per week only. These results do not support the idea that promotional stimulation in hepatocarcinogenesis is a reversible phenomenon. To quantify 13C promotional potency in terms of its dietary concentration, a series of AFB1 tumor dose-response curves was established, each with a different level of 13C fed continuously following AFB1 initiation. The resultant tumor dose-response curves, plotted as logit percentage of incidence versus log AFB1 dose, were displaced parallel toward lower AFB1 50% tumor take (TD50) values with increasing 13C concentration. The level of 13C that halves the AFB1 dose for 50% tumor incidence was calculated to be approximately 1000 ppm 13C, fed continuously, with no substantial threshold for promotion. By comparison, 13, when fed before and with AFB1, shows a 50% inhibitory value (13C concentration that doubles the dose of AFB1 for 50% tumor incidence) in trout of 1400 ppm 13C [Dashwood et al., Carcinogenesis (Lond.), 10: 175-181, 1989]. Thus the potential for 13C as a dietary additive to promote prior hepatic initiation events when fed continuously is approximately as great as its potential to inhibit concurrent AFB1 initiation.

Anticarcinogenic activity of indole-3-carbinol acid products: ultrasensitive bioassay by trout embryo microinjection.

The relative contribution of indole-3-carbinol (I3C) and its acid condensation products to the anticarcinogenic activity of this crucifer phytochemical has been studied using trout embryo microinjection. I3C was treated with 0.07 N HCl to give a reaction mixture (RXM) comprising < 0.5% parent compound and over 20 products, the most prevalent being the dimer 3,3'-diindolylmethane (I33') and a related cyclic trimer (CT). RXM, I33' or CT was injected into embryos with [3H]aflatoxin B1 (AFB1) and total embryonic DNA was isolated 1, 3, or 10 days postinjection. Compared with controls given AFB1 alone, I3C failed to inhibit carcinogen-DNA binding at any time point. In contrast I33', CT, and RXM inhibited AFB1-DNA binding by an average of 37, 51, and 65%, respectively. Coinjection of AFB1 and 350 microM I3C, RXM, or I33' into trout embryos reduced AFB1-induced hepatocarcinogenesis after 1 year from 43.4% in positive controls to 36.0, 12.2 (P < 0.05), and 24.6% (P < 0.05), respectively. No tumor data were obtained in the AFB1 plus CT group due to poor survival of the embryos posthatching. These results indicate that acid condensation products, not the parent compound, represent the anticarcinogenic species in trout and that their formation in the stomach is a likely prerequisite for I3C anticarcinogenesis.

Mechanisms of anti-carcinogenesis by indole-3-carbinol. Studies of enzyme induction, electrophile-scavenging, and inhibition of aflatoxin B1 activation.

The induction of oxidation and conjugation enzymes, the scavenging of carcinogen electrophiles, and the inhibition of aflatoxin B1 (AFB1) activation were examined as possible mechanisms of anti-carcinogenesis by indole-3-carbinol (I3C). Liver microsomal 7-ethoxycoumarin O-deethylase and 7-ethoxyresorufin O-deethylase activities were not induced significantly in rainbow trout fed diets containing 500-2000 ppm I3C for 8 days compared to trout fed the control diet. Furthermore, no detectable changes in the specific contents of cytochrome P-450 isozymes LM2 and LM4b, as measured by Western-blotting and immunoquantitation, were found in liver microsomes following dietary I3C administration. Dietary I3C had no significant effect on liver microsomal uridine diphosphate-glucuronyl-transferase activity, measured using the substrates 1-naphthol and testosterone, or on cytosolic glutathione S-transferase activity, measured using the substrate styrene oxide. The ability of I3C or its acid reaction products (RXM; generated by the reaction of I3C with HCl) to act as scavengers for the direct alkylating agent AFB1-8,9-Cl2 was examined. Addition of I3C or RXM to in vitro incubations did not inhibit the covalent binding of AFB1-8,9-Cl2 to calf thymus DNA. Kinetic analyses of microsome-mediated binding of AFB1 to DNA in vitro indicated that RXM inhibited the metabolic activation of AFB1. RXM increased the apparent Km for the AFB1-DNA binding reaction without changing the associated Vmax; the apparent Km values at 0, 3.5, 35, and 350 microM RXM were 35, 38, 66, and 86 microM for trout liver microsomes. RXM also inhibited the activation of AFB1 by rat liver microsomes, but I3C was not an effective inhibitor against AFB1-DNA binding mediated by either rat or trout liver microsomes. The results of the present study indicate that inhibition of microsome-activated AFB1 binding to DNA by I3C products may be of significant importance in I3C inhibition of hepatocarcinogenesis in trout and other species. The inhibition of carcinogen activation by I3C is contrasted with the mechanism of anti-carcinogenesis by beta-naphthoflavone, which involves induction of xenobiotic metabolizing enzymes.

Formation and accumulation of O6-ethylguanine in DNA of enriched populations of Clara cells, alveolar type II cells, and macrophages of hamsters exposed to diethylnitrosamine.

The concentration of O6-ethylguanine (O6EG) in DNA of enriched populations of hamster Clara cells, alveolar type II cells, and macrophages was determined following acute diethylnitrosamine (DEN) exposure, 100 and 200 mg/kg body wt, and during subchronic DEN exposure, 20 mg/kg body wt, 2 times/week. Pulmonary cells were separated by centrifugal elutriation. DNA was collected on polycarbonate filters and hydrolyzed by mild acid. Purines bases were separated by high-pressure liquid chromatography, and O6EG was quantitated by optical methods. O6EG levels in DNA of Clara cell-enriched populations were found to be higher than O6EG levels in DNA of alveolar type II cell-enriched populations and macrophages following acute DEN treatment. In Clara cell-enriched populations, O6EG levels found after 200 mg/kg body wt dose were approximately 50% the levels found after the 100 mg/kg body wt dose, and O6EG levels found at 24 h post-treatment were approximately 50% the levels found at 12 h post-treatment for both the 100 and 200 mg/kg body wt doses. O6EG accumulated in DNA of Clara cell-enriched populations during subchronic DEN exposure. The differential distribution of O6EG in target and non-target cell populations suggests the involvement of this promutagenic base in the initiation process of DEN pulmonary carcinogenesis.

Formation and persistence of ethylguanines in liver DNA of rainbow trout (Salmo gairdneri) treated with diethylnitrosamine by water exposure.

Diethylnitrosamine exposure via the water resulted in the formation of 7-ethylguanine and O6-ethylguanine in rainbow trout liver DNA. The modified bases were quantitated by high-pressure liquid chromatography and fluorescence spectrophotometry. In vivo 7-ethylguanine and O6-ethylguanine levels were directly proportional to DEN concentrations between 62.5 and 250 ppm. 7-Ethylguanine and O6-ethylguanine levels were approximately directly proportional to duration of exposure to DEN between 0 and 6 hr under the conditions used, with less than proportionate increases thereafter. Removal of ethylguanines from liver DNA following a 24-hr exposure to 250 ppm DEN (a known carcinogenic regimen) was biphasic; 24% of the O6-ethylguanine and 32% of the 7-ethylguanine found immediately after exposure were removed in 12 hr but no significant decline was found over the period from 12 to 96 hr after exposure. Alkyl acceptor protein activity in trout liver was examined to assess the role of enzymatic repair in the observed loss of O6-ethylguanine in vivo. Although an O6-alkylguanine repair system similar to the alkyltransferase system reported in rodents was found in trout liver, only 4% of the O6-ethylguanine lost from DNA after exposure to 250 ppm DEN can be accounted for by activity of the alkyl acceptor protein. The high incidence of liver tumours observed in DEN-treated rainbow trout may be related to the rapid formation and substantial persistence of the promutagenic O6-ethylguanine in liver DNA.

In vivo disposition of the natural anti-carcinogen indole-3-carbinol after po administration to rainbow trout.

Indole-3-carbinol (I3C), a compound found naturally as a glucosinolate in cruciferous vegetables such as broccoli and cabbage, has been shown to modulate the carcinogenic process in a number of animal species. The lack of detailed information on the disposition of I3C in vivo provided the main impetus for the study reported here, in which the distribution and metabolic fate of I3C was assessed in selected tissues and excreta after po administration to rainbow trout (Salmo gairdneri). Animals were fasted for 3 days and given [5-3H]I3C either in the diet or by single oral gavage (40 mg/kg body weight; 15 muCi/kg body weight). Following administration, 75% of the initial 3H-dose was detected within the stomach between 0.5 and 12 hr, after which it was released to distal regions of the gut for subsequent uptake, distribution and elimination. At the end of the study (72 hr) 25% of the administered dose was recovered from the water which reflected excretion through the gills and urinary tract. Significant excretion also occurred in the bile, with approximately 5% of the initial 3H-dose recovered from the bile sacs at 72 hr. Further analyses of the radioactive components in the bile indicated that one or more derivatives of I3C, but not the parent compound itself, are excreted as glucuronide conjugates using this route. Radioactivity accumulated in the liver throughout most of the study, reaching levels of 1-1.5% between 48 and 72 hr of the administered dose. High-performance liquid chromatography analyses indicated the presence of four main radiolabelled species in these livers, one of which co-eluted with the parent compound, I3C. The major radiolabelled species recovered from the liver was tentatively identified as the dimer, 3,3'-diindolylmethane (I33'), which comprised 40% of the total hepatic radiolabel. This dimer, I33', was also found to accumulate in the diet containing I3C, which reflected a time-dependent dimerization of the parent compound in vitro. These findings are discussed in view of recent postulates of a role for I3C condensation products such as I33' in the mechanism of I3C anti-carcinogenesis.

DNA ethylation in hamster tissues during subchronic diethylnitrosamine administration and in the hamster trachea after acute diethylnitrosamine administration.

Formation of O6-ethylguanine (O6EG) in trachea DNA was measured 12 h after acute i.p. administration of 50, 100 and 200 mg diethylnitrosamine (DEN)/kg body wt to Syrian golden hamsters. Purine bases were fractionated by h.p.l.c., and ethylated guanines were quantified optically by fluorescence spectrophotometry. Significant levels of this promutagenic base were found after the various single doses of DEN. O6EG in trachea DNA was also measured at various times up to 48 h after an acute i.p. dose of 200 mg DEN/kg body wt. The maximum level of O6EG was detected 12 h after DEN treatment. A rapid decrease in O6EG concentration was seen in trachea DNA between 12 and 24 h, but only a negligible decrease was detected from 24 to 48 h post-treatment. The formation and accumulation of O6EG in trachea, liver, lung and kidney DNA were determined in hamsters treated subchronically with DEN, 20 mg/kg body wt, s.c. twice weekly up to 8 weeks. O6EG accumulated to significant levels in trachea and liver DNA. 7-Ethylguanine did not accumulate in the DNA of any of the organs studied during subchronic DEN exposure. The formation and persistence of O6EG in trachea DNA of DEN-treated hamsters correlated with the sensitivity of this tissue to the carcinogenic action of DEN.

Tumor dose-response studies with aflatoxin B1 and the ambivalent modulator indole-3-carbinol: inhibitory versus promotional potency.

Indole-3-carbinol (I3C), a natural compound from cruciferous vegetables, inhibits aflatoxin B1 (AFB1) carcinogenesis in trout when administered prior to and during carcinogen exposure, but also promotes it in the same species when given after AFB1 initiation. To provide quantitative potency information for these opposing activities, detailed tumor dose-response studies were performed with AFB1 (10-400 ppb) and I3C (0-4,000 ppm). In a plot of (logit) percent tumor response vs log AFB1 exposure, the results generated a series of parallel AFB1 dose-response curves. Increasing I3C doses displaced these curves, respectively, toward higher and lower AFB1 doses in the inhibition and promotion studies. Similar potencies were observed over the dose range 0-1,500 ppm I3C; the 50% promotion and inhibition (P50 and I50) values were 1,000 vs 1,400 ppm I3C, respectively. Differences in the protocols used in the two studies suggest that the inhibitory activity of I3C is more likely to supersede promotion under human exposure conditions.

Carcinogenicity, metabolism and Ki-ras proto-oncogene activation by 7,12-dimethylbenz[a]anthracene in rainbow trout embryos.

Field studies suggest that recent epizootics of hepatic neoplasms in some feral fish populations are associated with polycyclic aromatic hydrocarbon (PAH) exposure, but attempts to induce liver tumors in these species under laboratory conditions have been unsuccessful. Several studies have shown hepatic neoplasma to be inducible in laboratory fish species following PAH exposure at the free-swimming life stage. However, neither the susceptibility of the fish embryonic life stage to tumor induction by PAHs nor the potential of these carcinogens to induce oncogenic point mutations analogous to those reported in feral fish hepatic tumors have been clearly established. To address this, rainbow trout embryos were exposed by passive water uptake to 7,12-dimethylbenz[a]anthracene (DMBA), a potent model PAH in many mammalian tumor protocols. DMBA was rapidly absorbed by trout eggs and metabolized. The major non-polar metabolites identified were 12-hydroxymethyl-7-methylbenz[a]anthracene and 3,4-dihydroxy-3,4-dihydro-DMBA, whereas approximately 25% of the water soluble metabolites were identified as glucuronides by beta-glucuronidase treatment. Embryonic DNA adduction increased with time of DMBA exposure (2.2 +/- 0.3 pmol DMBA-equivalents/mg DNA at 24 h). Liver tumor incidence nine months after DMBA treatment was found to increase with DMBA concentration and exposure period (3.8% at 1 p.p.m./2 h; 23% at 5 p.p.m./2 h; 85% at 5 p.p.m./24 h). Stomach adenomas and nephroblastomas also were observed at low incidence in the DMBA-treated trout. Among 11 hepatic tumors examined, nine carried Ki-ras alleles with activating point mutations in codon 12 (4/11 GGA-->AGA; 4/11 GGA-->GTA) or codon 61 (1/11 CAG-->CTG). This spectrum differs substantially from those reported for DMBA-initiated mouse skin papillomas or hepatic tumors. These results may have important environmental implications because they suggest that even a brief exposure to PAHs during a sensitive stage of development may adversely affect some fish populations. They also indicate considerable variation in DMBA ras gene mutations among species and target organs.

Mechanisms of anti-carcinogenesis by indole-3-carbinol: detailed in vivo DNA binding dose-response studies after dietary administration with aflatoxin B1.

Several recent reports have described inhibitor-mediated reductions in the covalent binding of various carcinogens to DNA in vivo. The majority of these studies show inhibitory effects after testing at one inhibitor and one carcinogen dose level only. Consequently, the detailed relationships between inhibitor dose, carcinogen dose, and in vivo inhibitory potency have not been clearly delineated in any species. To systematically determine these relationships in vivo, rainbow trout (Salmo gairdneri) were exposed to a range of carcinogen (aflatoxin B1, AFB1) and inhibitor (indole-3-carbinol, I3C) doses by concomitant dietary exposure. Inhibitory potencies were then assessed using in vivo covalent binding of AFB1 to hepatic DNA as an end-point. Linear increases in DNA binding occurred with increasing dose of AFB1 and with time of inhibitor/carcinogen co-treatment, at each I3C dose level. Successive increases in inhibitor dose resulted in corresponding dose-related decreases in AFB1--DNA binding such that a series of curves of decreasing slope was produced. AFB1--DNA binding was suppressed by almost 95% at the highest I3C dose tested. These studies describe for the first time such a degree of inhibition by I3C on covalent binding of AFB1 to DNA in vivo, where inhibitor and carcinogen are covariables administered repeatedly in the diet. Moreover, the linear inhibitory response observed at low I3C doses indicates the possible absence of any significant threshold for I3C protection against AFB1--DNA binding. Thus, even at low levels I3C may offer some protection against chemically-induced neoplasia.

Modulation of diethylnitrosamine-induced hepatocarcinogenesis and O6-ethylguanine formation in rainbow trout by indole-3-carbinol, beta-naphthoflavone, and Aroclor 1254.

Rainbow trout were fed a diet containing indole-3-carbinol (2000 ppm), beta-naphthoflavone (500 ppm), or Aroclor 1254 (100 ppm) for 6 weeks before a single 24-hr exposure to an aqueous solution of 250 ppm diethylnitrosamine (DEN). The fish were killed 42 weeks later to determine the carcinogenic response. DEN exposure produced an 80.2% incidence of liver tumors and an average of 3.47 tumors per tumor-bearing fish, whereas no tumors were detected in the sham-treated control fish. Tumor induction was inhibited by indole-3-carbinol (27.5% incidence, 1.89 tumors per tumor-bearing fish) but enhanced by beta-naphthoflavone (91.8% incidence, 3.60 tumors per tumor-bearing fish). Aroclor 1254 had no effect on DEN-induced hepatocarcinogenesis (80.0% incidence, 3.03 tumors per tumor-bearing fish). The effects of these modulators on O6-ethylguanine and 7-ethylguanine formation (measured by HPLC and fluorescence spectrophotometry) were examined. Liver DNA ethylguanine levels were reduced in indole-3-carbinol-pretreated fish and increased in beta-naphthoflavone-pretreated fish compared to untreated controls after DEN exposure. Aroclor 1254 pretreatment had no significant effect on DNA ethylguanine formation. Similar O6-ethylguanine to 7-ethylguanine ratios were found among the control and treated groups. The results of this study indicate that modulation of DEN hepatocarcinogenesis by indole-3-carbinol and beta-naphthoflavone may be mediated by their effects on O6-ethylguanine formation and, therefore, on the initiation phase of carcinogenesis.

Quantitative inter-relationships between aflatoxin B1 carcinogen dose, indole-3-carbinol anti-carcinogen dose, target organ DNA adduction and final tumor response.

A number of recent studies have described inhibitor-mediated reductions in the covalent DNA binding and tumorigenicity of various carcinogens, in species such as rats, mice and rainbow trout (Salmo gairdneri). Since inhibitory effects have, in most cases, been reported after testing at one carcinogen and one inhibitor level only, the detailed relationships between carcinogen dose, inhibitor dose, in vivo DNA binding and final tumor response are not well understood in any species. To determine these relationships we have employed the trout model in a combined DNA binding/tumor dose-response protocol using approximately 10,000 animals. Trout were pretreated with one of five different dose-levels of indole-3-carbinol (I3C), a naturally occurring anti-carcinogen found in cruciferous vegetables such as broccoli and cabbage. After 4 weeks, animals received the same dietary level of I3C for a further 2 weeks together with [3H]aflatoxin B1 (AFB1) in the dose-range 10-320 p.p.b. From tanks containing 150 animals (three tanks per I3C-AFB1 dose-point), 15 fish were selected at random in order to assess hepatic AFB1-DNA binding levels. Remaining animals were returned to control diet for determination of tumor response at 12 months. Linear increases in DNA binding occurred with dose of AFB1 at each I3C dose-level. Successive increases in I3C dose gave dose-related decreases in AFB1-DNA binding, resulting in a series of curves of decreasing slope. Shifts in DNA-binding slopes were compared quantitatively with horizontal displacements towards higher carcinogen dose in corresponding tumor dose-response curves. At I3C doses of less than or equal to 2000 p.p.m., the inhibitor-altered tumor response was predicted precisely by changes in dose received (DNA adducts formed) in the target organ. These data constitute the first direct evidence of pure anti-initiating activity by a natural anti-carcinogen found in human diet, where all animals were treated at the same time and under identical conditions of exposure in both DNA binding and tumor studies. The data are discussed further in view of (i) their implications for DNA binding-carcinogenicity correlations and the concept of 'molecular dosimetry', and (ii) limitations in the current database on anti-carcinogenesis as regards in vivo potency information, particularly for 'ambivalent modulators' which exhibit both inhibitory and promotional activity.

Modulation of mycotoxin and nitrosamine carcinogenesis by indole-3-carbinol: quantitative analysis of inhibition versus promotion.

The value of chemopreventive agents for reducing human response to mycotoxins and N-nitrosamines remains uncertain, especially since many such agents also can act as tumour promoters. Indole-3-carbinol (I3C) from cruciferous vegetables can inhibit DNA adduction and hepatocarcinogenesis induced by aflatoxin B1 (AFB1) or N-nitroso-diethylamine in trout if given before and with the carcinogen but promotes carcinogenesis when given after initiation. Similar results have been obtained with I3C and AFB1 in rats. In detailed studies using 10,000 trout, inhibition of AFB1 carcinogenesis was found to be saturable at high doses of I3C, approximately proportional to dose of I3C through the range of human intake and, within this range, quantitatively predicted by I3C-mediated reduction of AFB1-DNA adduction in liver. In a second study, post-initiation promotion of AFB1 carcinogenesis was approximately proportional to I3C dose, increased with duration of exposure, decreased with delayed onset of exposure, and reduced but still significant when I3C was given on alternate months or weeks or twice per week only. Hence, promotion by this common component of cruciferous vegetables required prolonged exposure but not necessarily on a daily basis.

beta-Catenin mutation in rat colon tumors initiated by 1,2-dimethylhydrazine and 2-amino-3-methylimidazo[4,5-f]quinoline, and the effect of post-initiation treatment with chlorophyllin and indole-3-carbinol.

Carcinogens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 1,2-dimethylhydrazine (DMH) induce colon tumors in the rat that contain mutations in beta-catenin, but the pattern of mutation differs from that found in human colon cancers. In both species, mutations affect the glycogen synthase kinase-3beta consensus region of beta-catenin, but whereas they directly substitute critical Ser/Thr phosphorylation sites in human colon cancers, the majority of mutations cluster around Ser33 in the rat tumors. Two dietary phytochemicals, chlorophyllin and indole-3-carbinol, given post-initiation, shifted the pattern of beta-catenin mutations in rat colon tumors induced by IQ and DMH. Specifically, 17/39 (44%) of the beta-catenin mutations in groups given carcinogen plus modulator were in codons 37, 41 and 45, and substituted critical Ser/Thr residues directly, as seen in human colon cancers. None of the tumors from groups given carcinogen alone had mutations in these codons. Interestingly, many of the mutations that substituted critical Ser/Thr residues in beta-catenin were from a single group given DMH and 0.001% chlorophyllin, in which a statistically significant increase in colon tumor multiplicity was observed compared with the group given DMH only. These tumors had marked over-expression of cyclin D1, c-myc and c-jun mRNA and c-Myc and c-Jun proteins were strongly elevated compared with tumors containing wild-type beta-catenin. The results indicate that the pattern of beta-catenin mutations in rat colon tumors can be influenced by exposure to dietary phytochemicals administered post-initiation, and that the mechanism might involve the altered expression of beta-catenin/Tcf/Lef target genes.