Protection by indole-3-carbinol against covalent binding of benzo[a]pyrene metabolites to mouse liver DNA and protein

doi:10.1016/0278-6915(83)90265-X

Food and Chemical Toxicology

Volume 21, Issue 1, February 1983, Pages 31-35

https://doi.org/10.1016/0278-6915(83)90265-X Get rights and content

Abstract

Indole-3-carbinol (I-3-C) is a compound present in many cruciferous vegetables that has been shown to reduce aryl hydrocarbon-induced neoplasia in experimental animals. We examined the relationship between the ability of I-3-C to alter the activity of hepatic aryl hydrocarbon hydroxylase (AHH), and its ability to inhibit the covalent binding of benzo[a]pyrene (BaP) metabolites to DNA and protein. Using an in vitro system and a hepatic postmitochondrial fraction from mice that had been treated by gavage with I-3-C, we found that up to 90% of the covalent binding of BaP metabolites to macromolecules was eliminated, while AHH activity was unchanged. In experiments in vivo, treatment of mice by gavage with I-3-C before [¹⁴C]BaP resulted in up to an 80% decrease in covalent binding of ¹⁴C to DNA or protein with no concomitant decrease in hepatic AHH activity. These results suggest that I-3-C administered in vivo confers protection against the binding of BaP oxidation products to hepatic cellular macromolecules.

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Effects of analogs of indole-3-carbinol cyclic trimerization product in human breast cancer cells
2005, Chemico-Biological Interactions
Citation Excerpt :
Indole-3-carbinol (I3C) is an autolysis product of glucobrassicin found in Brassica vegetables and is under study as a potential breast cancer chemopreventive agent [1–5].
5,6,11,12,17,18-Hexahydrocyclonona[1,2-b:4,5-b*:7,8-b**]triindole (CTr) is a major digestive product of indole-3-carbinol (I3C) from Brassica vegetables and exhibits strong estrogenic activities. CTr increases proliferation of estrogen-dependent breast tumor cells, binds with strong affinity for the estrogen receptor-alpha (ERα), and activates expression of estrogen (E₂)-dependent genes. To begin to examine the structural features that determine the biological activity of CTr, we prepared and studied the effects of two analogs, 9,18-dihydro-12H-[1,2,5]trithionino[3,4-b:6,7-b*:9,8-b**]triindole (S₃CTr) and 5,6,11,12,17,18-hexahydro-5,11,17-trimethylcyclonona[1,2-b:4,5-b*:7,8-b**]triindole (Me₃CTr). N-Methylation of CTr completely ablated the estrogenic activities of CTr. In the dose range in which CTr was clearly estrogenic, Me₃CTr exhibited no detectable effect on cell growth, ERα binding to E₂, or ERα-responsive gene expression. S₃CTr showed mixed ERα agonist activities. It bound to the ERα and activated receptor binding with DNA, weakly activated expression of transfected E₂-responsive reporter gene constructs, and strongly inhibited the E₂-induced activation of these reporter constructs. S₃CTr activated aryl hydrocarbon receptor (AhR)-mediated pathways, consistent with the moderately strong binding affinity of S₃CTr for the AhR. Comparisons of the conformational characteristics among CTr and its two analogs indicated that the estrogenic effects of CTr are highly sensitive to apparently minor structural modifications, and further supported the hypothesis for a central role of hydrogen bonding around the nitrogen atom in CTr binding to the ligand binding site of ERα.
Distribution and macromolecular binding of benzo[a]pyrene and two polychlorinated biphenyl congeners in female mice
2001, Chemico-Biological Interactions
Citation Excerpt :
Despite higher radioactive counts in DNA samples obtained from the livers of animals treated with radiolabeled compound compared to the corn oil-treated group, no significant DNA binding could be observed in any treated group. This result is especially unexpected for B[a]P, which was used as a positive control since its ability to bind to DNA in vivo was already documented in similar experiments [56–58]. Differences in experimental animals (female C57BL6 mice in our study vs male Sprague-Dawley derived SIV-50 rats [56,57] or male Swiss ICR mice [58]), routes of exposure (ip in our study vs iv [56,57] or po [58]), time between exposure and euthanasia (24 h in our study vs 16 h [56,57] or 0–24 h [58]) and doses used (37.6 nmol/g in our study vs 7.6 nmol/g [58] or 2.5 nmol/g [56,57]) may bring partial explanation for this result.
PCBs are complete rodent carcinogens and their potent tumor promoting activity has been reported, but their tumor-initiating activity remains controversial. Macromolecular binding of PCB metabolites has been demonstrated in vitro, but this issue remains unclear in vivo. The purpose of this study was to determine the binding affinity of 4-chlorobiphenyl and 3,3′,4,4′-tetrachlorobiphenyl to proteins and DNA in vivo. C57/BL6 female mice were treated intraperitoneally with hepatic enzyme inducers (phenobarbital and β-naphthoflavone) and then with ¹⁴C-labelled polychlorinated biphenyls or benzo[a]pyrene. The short-term distribution of labeled compounds into liver, lungs and kidneys and into different sub-cellular fractions of these tissues was assessed and the DNA and proteins from the 700×g pellet were further purified to assess covalent binding. All compounds were distributed in low amounts into the liver, kidneys and lungs, with the greatest accumulation in the liver, and the lowest in lungs. In all tissues, test compounds were mostly found in cytosols and organellar pellets (10,000×g), and lower amounts were present in nuclear pellets (700×g) and microsomes. In lungs and kidneys, only benzo[a]pyrene showed significant covalent binding to proteins. In the liver, protein binding indices were significant for all compounds (P<0.05), but no significant binding of the test compounds to DNA could be demonstrated with this approach. Our results suggest that at the 24 h time point, all compounds were activated to electrophilic intermediates prone to macromolecular binding. Hepatic proteins apparently act as a sink for PCB-derived electrophiles, thus preventing detectable levels of covalent binding to hepatic DNA or to proteins in less metabolically active tissues.
Ligand-independent activation of estrogen receptor function by 3,3'-diindolylmethane in human breast cancer cells
2000, Biochemical Pharmacology
3,3′-Diindolylmethane (DIM), a major in vivo product of acid-catalyzed oligomerization of indole-3-carbinol (I3C), is a promising anticancer agent present in vegetables of the Brassica genus. We investigated the effects of DIM on estrogen-regulated events in human breast cancer cells and found that DIM was a promoter-specific activator of estrogen receptor (ER) function in the absence of 17β-estradiol (E₂). DIM weakly inhibited the E₂-induced proliferation of ER-containing MCF-7 cells and induced proliferation of these cells in the absence of steroid, by approximately 60% of the E₂ response. DIM had little effect on proliferation of ER-deficient MDA-MB-231 cells, suggesting that it is not generally toxic at these concentrations. Although DIM did not bind to the ER in this concentration range, as shown by a competitive ER binding assay, it activated the ER to a DNA-binding species. DIM increased the level of transcripts for the endogenous pS2 gene and activated the estrogen-responsive pERE-vit-CAT and pS2-tk-CAT reporter plasmids in transiently transfected MCF-7 cells. In contrast, DIM failed to activate transcription of the simple E₂- and diethylstilbesterol-responsive reporter construct pATC2. The estrogen antagonist ICI 182780 (7α-[9-[(4,4,5,5,5-pentafluoropentyl)sulfonyl]nonyl]-estra-1,3,5(10)-triene-3,17β-diol) was effective against DIM-induced transcriptional activity of the pERE-vit-CAT reporter, which further supports the hypothesis that DIM is acting through the ER. We demonstrated that ligand-independent activation of the ER in MCF-7 cells could be produced following treatment with the D1 dopamine receptor agonist SKF-82958 [(±)6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepinehydrobromide]. We also demonstrated that the agonist effects of SKF-82958 and DIM, but not of E₂, could be blocked by co-treatment with the protein kinase A (PKA) inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide). These results have uncovered a promoter-specific, ligand-independent activation of ER signaling for DIM that may require activation by PKA, and suggest that this major I3C product may be a selective activator of ER function.
Inhibition of CYP1A1 enzyme activity in mouse hepatoma cell culture by soybean isoflavones
1999, Chemico-Biological Interactions
The mechanisms by which soybean- and soybean isoflavone-enriched diets inhibit carcinogenesis are not known. We found that the isoflavones genistin and daidzin, and their respective aglucone forms daidzein and genistein, block 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin)-induced CYP1A1 enzyme activity. This inhibition is correlated with the capacity of the isoflavones to prevent CYP1A1-mediated covalent binding of benzo[a]pyrene (BaP) metabolites to DNA. We further evaluated daidzein and genistein, believed to be the active forms of the isoflavones, for the mechanism of the inhibitory process. Although daidzein and genistein appear structurally similar to known aromatic hydrocarbon receptor (AHR) agonists and antagonists, gel mobility shift assays indicated that the isoflavones do not inhibit dioxin-induced activation of the AHR or the accumulation of CYP1A1 mRNA, suggesting that the isoflavones do not act at the transcriptional level. We therefore evaluated the isoflavones for direct effects on the CYP1A1 enzyme. Daidzein and genistein are non-competitive with the CYP1A1 substrate BaP for microsomal BaP hydroxylation, with apparent K_i values of 325 μM and 140 μM, respectively. The extent of CYP1A1 inhibition increases with time of preincubation at 37°C, but not at 4°C, in the presence of isoflavone plus NADPH; after 60 min preincubation the inhibition remains non-competitive, with apparent K_i values of 55 μM and 50 μM, respectively. Inhibition is neither prevented nor reversed by the thiol antioxidant dithiothreitol, nor by the iron chelator deferoxamine. Repeated washing of the microsomes does not reverse the inhibition. The dependency on NADPH, temperature and time for inhibition of CYP1A1 suggests that metabolism of either isoflavone or molecular oxygen to reactive species is required. Isoflavone-mediated inhibition of CYP1A1 activity may contribute to the mechanism by which these soybean isoflavones protect against carcinogenesis.
A review of mechanisms underlying anticarcinogenicity by brassica vegetables
1997, Chemico-Biological Interactions
The mechanisms by which brassica vegetables might decrease the risk of cancer are reviewed in this paper. Brassicas, including all types of cabbages, broccoli, cauliflower and Brussels sprouts, may be protective against cancer due to their relatively high glucosinolate content. Glucosinolates are usually broken down through hydrolysis catalyzed by myrosinase, an enzyme that is released from damaged plant cells. Some of the hydrolysis products, viz. indoles and isothiocyanates, are able to influence phase 1 and phase 2 biotransformation enzyme activities, thereby possibly influencing several processes related to chemical carcinogenesis, e.g. the metabolism, DNA-binding and mutagenic activity of promutagens. A reducing effect on tumor formation has been shown in rats and mice. The anticarcinogenic action of isothiocyanates and indoles depends upon many factors, such as the test system, the target tissue, the type of carcinogen challenge and the anticarcinogenic compound, their dosage, as well as the timing of the treatment. Most evidence concerning anticarcinogenic effects of glucosinolate hydrolysis products and brassica vegetables has come from studies in animals. Animal studies are invaluable in identifying and testing potential anticarcinogens. In addition, studies carried out in humans using high but still realistic human consumption levels of indoles and brassica vegetables have shown putative positive effects on health.
Effects of 1-isothiocyanato-3-(methylsulfinyl)-propane on xenobiotic metabolizing enzymes in rats
1993, Food and Chemical Toxicology
The glucosinolate hydrolysis product 1-isothiocyanato-3-(methylsulfinyl)-propane (IMSP), also known as iberin, is consumed in the average human (US) diet at approximately 1 μmol/kg/day. The chemoprotective effects observed with the consumption of cruciferous vegetables may be due to the presence of specific glucosinolate hydrolysis products either within the crucifers, or formed after ingestion of the crucifers. The mechanism of chemoprotection may be through selective induction of components of Phase II xenobiotic metabolizing enzymes. The influence of repeated administration of low concentrations of IMSP by gavage on components of Phase I and Phase II xenobiotic metabolizing systems was examined in the liver and small intestine of male Fischer 344 rats. Doses of 1, 10 and 100 μol IMSP/kg, administered by gavage for 7 days, did not alter weight gain, or hepatic and renal weights, relative to body weight, and did not cause any histological lesions. Intestinal glutathione S-transferase (GST) activity and NAD(P)H:quinone reductase (QR) activities were significantly elevated to 3.1 and 8.1 times control values, respectively, at the 100 μmol/kg dose only. The administration of IMSP at 1, 10 or 100 μmol/kg had no significant effect on hepatic Phase I enzyme activities (cytochrome P-450 concentrations, ethoxycoumarin O-deethylase [ECD] and aminopyrine N-demethylase [AND] activities) or Phase II enzyme activities (GST, QR and UDP-glucuronosyltransferase [UDP-GT] activities towards 1-naphthol or 4-hydroxybiphenyl), at any of the doses tested and no effect on intestinal enzyme activities at doses below 100 μmol IMSP/kg. It is concluded that IMSP does not have a significant influence on induction of the Phase I or Phase II xenobiotic metabolizing enzymes in rats when tested at doses approximating those found in the human diet.

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Protection by indole-3-carbinol against covalent binding of benzo[a]pyrene metabolites to mouse liver DNA and protein

Abstract

Fd Cosmet. Toxicol.

Meth. Enzymol.

Meth. Enzymol.

J. biol. Chem.

Analyt. Biochem.

Toxic. appl. Pharmac.

Chemico-Biol. Interactions