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Experimental Therapeutics, Preclinical Pharmacology |
1 Cancer Biomarkers and Prevention Group, Departments of Biochemistry and Cancer Studies and Molecular Medicine, University of Leicester, Leicester, United Kingdom, and 2 National Institute on Aging, Gerontology Research Center, Baltimore, Maryland
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One of the primary mechanisms by which I3C prevents tumorigenesis is by selective beneficial alteration of Phase I and Phase II carcinogen-metabolising enzymes (9
, 10)
. I3C has also been shown to have antiestrogenic activity that is proposed to account for its protective and antiproliferative effects on estrogen-responsive tissues. I3C induces estradiol 2-hydroxylase, thus increasing the metabolic ratio of 2-hydroxyestrone:16
-hydroxyestrone in rodents (3)
and humans (11)
. In addition, I3C can induce a G1 cell cycle arrest (12)
, as well as apoptosis and inhibition of Akt phosphorylation in human breast (13)
and prostate (14
, 15) tumor cells lines.
I3C is susceptible to oligomerization under acidic conditions approximating those in gastric juice (16)
and thus, after ingestion, is converted into several condensation products. It has been suggested that the observed biological activity may be attributable mainly to the acid condensation products, in particular, 3,3'-diindolylmethane (DIM; Fig. 1
). [2-(Indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane (LTr1; Fig. 1
), indolo[3,2b]carbazole (ICZ; Fig. 1
) and 5,6,11,12,17,18-hexahydrocyclonona[1,2-b:4,5-b':7,8-b'']tri-indole (cyclic trimer) have also all been identified in rodents after oral administration of I3C (17
, 18)
. Cyclic trimer, ICZ, and LTr1 have been shown to exhibit biological activity (19, 20, 21)
, and in particular, DIM has shown promising cancer chemopreventive properties in vitro (22)
and in vivo (23)
.
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Because of the paucity of detailed information on the disposition of I3C in the intact organism, a recently developed high-performance liquid chromatography (HPLC) method was used for the analysis of I3C and its acid condensation products in vivo (17) , with the aim of studying in detail the systemic absorption and tissue distribution in mice receiving I3C via the oral route.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatments.
Female CD-1 mice (25–31 g), obtained from Harlan United Kingdom (Bicester, United Kingdom) were used under license from the Home Office and after approval by the University of Leicester Ethical Committee for Animal Experimentation. During the acclimatization period (1 week), they were housed in Moredun Isolators (Moredun Animal Health, Edinburgh, United Kingdom) under negative pressure, with a 12-h light-dark cycle, a temperature range of 20–23°C and 40–60% humidity. The mice were provided with pellet RM1 diet (Special Diets Services, Witham, United Kingdom) and water ad libitum before and during the experiment. On the dosing day, mice were removed from isolators and gavaged with I3C (250 mg/kg) as a suspension in corn oil (10 ml corn oil/kg body weight). Blood samples were collected by cardiac exsanguination under halothane anesthesia at 0.25, 0.5, 0.75, 1, 2, 4, 6, and 24 h after dosing. Four mice were used for each time point. Control animals received corn oil (10 ml/kg) and were culled 0.5 h after dosing. Each blood sample was collected in a heparinized tube. The plasma fraction was immediately separated by centrifugation (10,000 x g, 5 min) and stored at –80°C until analysis. After exsanguination, liver, kidney, lung, heart, and brain were immediately removed, wrapped in aluminum foil, snap frozen in liquid nitrogen, and stored frozen at –80°C until analysis.
Plasma Extraction and Analysis.
Plasma concentrations of I3C, DIM, and LTr1 were determined using a HPLC method described previously (17)
. In brief, the internal standard (4-methoxy-indole; 2.5 µl of a 0.4 mg/ml solution) was added to plasma samples (250 µl). The samples were vortex-mixed and equilibrated at room temperature for 30 min. Samples were extracted twice with t-butyl methyl ether (750 µl), involving 2-min vortexing on each occasion. Subsequent to each extraction, the organic and aqueous layers were separated by centrifugation (2800 x g, 10 min) and the organic (top) layers transferred to a fresh 4-ml tube. The top layers of each extraction were combined, and the t-butyl methyl ether evaporated to dryness in the presence of 15 µl of DMSO. The extracted sample in DMSO was then diluted using acetonitrile/50 mM HEPES buffer (pH 7.4; 40:60 v/v; 135 µl) and transferred to a glass vial insert for analysis by HPLC. Concentrations of I3C, DIM, and LTr1 in experimental samples were determined by comparison of the peak area ratio of the analyte and internal standard against calibration curves prepared by spiking blank plasma from control mice with a known amount of stock solution. Calibration samples were extracted and analyzed in the same way as described previously for experimental plasma samples.
To estimate concentrations of I3CA in plasma, calibration curves were prepared and concentrations determined using the internal standard method, as described previously. This produced linear calibration curves (r2 > 0.99) in the range investigated (0.1–15 µg/ml). 1-(3-Hydroxymethyl)-indolyl-3-indolylmethane (HI-IM) plasma concentrations were estimated using calibration curves prepared for DIM.
Tissue Extraction and Analysis.
Each tissue was thawed, rinsed with ice-cold saline, blotted dry, weighed, and homogenized (1:1, w/v) in 50 mM HEPES buffer (pH 7.4) using a Ystral X 10/20 homogenizer (D-79282 Ballrechten-Dottingen, Germany). Aliquots of homogenates (250 µl) were transferred into clean 4-ml tubes, with the exception of heart, where 200 µl were used because of the small amount of tissue available. 4-Methoxy-indole (2.5 µl of 0.4 mg/ml) was added to the homogenate as internal standard. Samples were equilibrated on ice for 30 min. Acetonitrile (750 µl) was added to each sample and vortex-mixed for 2 min. Samples were then stored on ice for 5 min before being vortex-mixed again for a further 2 min. The tissue and solvent were separated by centrifugation (2800 x g, 10 min) and the solvent transferred to a fresh tube. The acetonitrile/water mixture was evaporated to dryness at room temperature in the presence of 15 µl of DMSO under a stream of nitrogen gas. The residue was resuspended in 135 µl of acetonitrile/50 mM HEPES buffer (pH 7.4; 40/60 v/v) and transferred to a clean 0.5-ml Eppendorf tube. Turbidity was removed by centrifugation (10,000 x g, 5 min) and the supernatant transferred to a glass vial insert before analysis by HPLC as described below.
Concentrations in experimental samples were determined by an internal standard method using calibration curves that were prepared using tissues from control mice. Calibration curves for I3C, DIM, and LTr1 were prepared for lung, heart, brain, kidney, and liver between the range 0.1–30 µg/g. Concentrations of HI-IM in tissues were estimated using calibration curves for the structurally related DIM. ICZ was quantified in liver samples using a calibration curve prepared from 0.3–75 ng/g, using an external standard. This method was found to be more accurate for quantification of ICZ in plasma (17)
. The calibration curves for each compound in every tissue were linear (r2 > 0.99) in the range investigated. The extraction efficiency of I3C, DIM, LTr1, and ICZ was determined by comparison of the slope of a calibration curve prepared in mobile phase with a calibration curve for the same concentrations extracted from each specific tissue. Using this method, the extraction efficiencies of I3C, DIM, LTr1, and ICZ were 
69% and the internal standard 70% for all of the sampled tissues. The extraction methods were validated for analysis of liver, where the precision (expressed as coefficient of variation) was in the range of 5.2–10.2%. Accuracy values ranged from 91.2% to 108.4% for two quality control samples containing I3C, DIM, LTr1, and ICZ.
Liquid Chromatography.
HPLC analysis of plasma and tissue samples was performed on a Varian Prostar HPLC system (Varian Ltd., Surrey, United Kingdom). The system consisted of a 230 pump, 410 autosampler, 310 UV detector, and 363 fluorescence detector. The extracted standard/sample (50 µl) was injected onto the HPLC system. Chromatography was achieved using a Waters Symmetry RP 18 (4.6 x 50 mm, 5 µm) column (Hertford, United Kingdom) in tandem with a Thermoquest BDS C18 (250 x 4.6 mm, 5 µm) column (Runcorn, United Kingdom) and a Phenomenex C18 octadecyl silane (4 x 3 mm) guard column (Cheshire, United Kingdom). The mobile phase consisted of water (A) and acetonitrile (B). The gradient was as follows: 15% B to 60% B from 0 to 20 min; linear gradient to 65% B from 20 to 40 min; linear gradient to 85% B from 40 to 65 min, then re-equilibration to 15% B for 5 min. Total run time was 70 min, and the flow rate was 1 ml/min. Separation was carried out at room temperature. I3C, DIM, LTr1, HI-IM, and I3CA were quantified by UV detection at 280 nm, whereas ICZ was quantified using fluorescence detection at 335:415 nm (ex./em.).
Identification of Metabonates of I3C.
The acid condensation products and oxidative metabolites of I3C were identified by mass spectrometry of the peak of interest from the plasma and tissues. All samples were evaporated to dryness and resuspended in acetonitrile before analysis. If material from a single fraction did not provide the sensitivity required for characterization of the peak of interest, then a number of HPLC fractions were combined. Electron ionization spectra were obtained via direct insertion probe technique using a VG 70-SEQ mass spectrometer (Micromass, Manchester, United Kingdom). The conditions used were 70 eV electron energy, 200 µA trap current, and a source temperature of 250°C.
Statistical Analysis.
Comparison of tissue and plasma concentrations and tissue-to-plasma ratios were performed using ANOVA and Tukey’s multiple comparison post hoc test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. However, concentrations fell below the limit of detection within the 1st h after dosing, indicating rapid clearance.
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. Peak levels here were significantly higher than in plasma and all of the other sampled tissues (P < 0.001), with the exception of kidney (P > 0.05). Similarly, peak kidney concentrations were significantly higher than levels in plasma, lung, heart, and brain (P < 0.05), which were not significantly different from one another.
Plasma Levels and Tissue Distribution of DIM and LTr1.
DIM and LTr1 were detected in plasma and tissues after I3C administration (Fig. 2)
. DIM was detected in plasma at 15 min and was still quantifiable after 6 h, whereas LTr1 was not detected in plasma until 1 h after dosing, and levels were still increasing at the last measurable time point of 6 h (Fig. 3)
. DIM appeared to reach its peak at 2 h after I3C dosing. The peak plasma concentrations of DIM and LTr1 were approximately one sixth and one tenth that of I3C, although detectable levels persisted for much longer than their precursor (Table 1
; Fig. 3
).
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. Tissue profiles were similar to those observed in plasma, suggesting that DIM and LTr1 were in equilibrium with the plasma. To test this hypothesis, mean tissue-to-plasma ratios for DIM (0.25–6 h) and LTr1 (liver, 1–6 h; other tissues, 2–6 h) for each mouse at every time point were calculated (Table 2)
.
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; Fig. 4
). Average tissue-to-plasma ratios over the full sampling time range are shown in Fig. 4
. DIM ratios were ranked in the order: liver >>> kidney >> brain 
lung heart.
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; Fig. 4
). Average tissue-to-plasma ratios of LTr1 can be ranked in the following order: liver >> kidney lung heart >> brain (Fig. 4)
.
Plasma Levels and Tissue Distribution of HI-IM.
After HPLC analysis of plasma and tissue samples from mice that had received I3C, an additional peak with a retention time of 23.2 min was detected (Fig. 2)
. The identity of this peak was confirmed by mass spectrometry to be HI-IM (Fig. 2)
. This product was identified previously, after oral administration of I3C to rats, by Stressor et al. (18)
. An HPLC standard for HI-IM was not available, but assuming HI-IM had a similar extraction efficiency and UV sensitivity to its structurally related compound DIM, estimation of the plasma and tissue concentrations of HI-IM could tentatively be made (Fig. 5)
. The estimated concentrations were similar to those of DIM, as were plasma and tissue profiles, reaching maximal concentrations 2 h after administration of I3C. As observed for DIM, HI-IM persisted in the plasma and tissues much longer than I3C, and was still detectable in the liver 24 h after I3C administration.
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. Mass spectral analysis of extracts of combined liver samples afforded the molecular ion of ICZ (m/z 256), thus providing further evidence that ICZ was present in these samples. The mean concentrations in the liver at 6 and 24 h were 9 and 27 ng/g, respectively.
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. The identity of this peak was confirmed by mass spectrometry. I3CA was a significant metabolite, and approximate plasma concentrations were 9.6, 12.6, and 1.1 µg/ml at 15, 30, and 45 min, respectively, after I3C administration. Maximal plasma concentrations of I3CA were 3-fold higher than those of I3C, but as for the parent compound, it was no longer detectable at 1 h after I3C dosing. Although a peak with the retention time of I3CA was also present in tissues of mice that received I3C, it coeluted with the large solvent front, precluding further quantification.
A second minor oxidative metabolite, I3A was detected in plasma (Fig. 2)
. Levels were much lower than those of I3CA. By combining HPLC fractions of the peak of interest, confirmation of its identity was possible using mass spectrometry. Accurate quantification of I3A in plasma was difficult, with levels being at the limit of detection. However, maximal peak levels at 30 min after I3C administration gave peak area ratios approximately equal to 50 ng/ml of a spiked I3A standard. Again, levels were undetectable 1 h after dosing. I3A was not detectable in tissues.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oral administration of I3C to mice resulted in the detection of parent compound in plasma and tissues. In general, maximal I3C concentrations observed were considerably higher than those for its acid condensation products ([I3C] > [DIM] > [LTr1] > [HI-IM] >>> [ICZ]). This is in contrast with investigations reported previously in a number of species, where condensation products of I3C were detected, but not I3C itself (18 , 26 , 28 , 29) . Our first sampling time point was 15 min, earlier than those used in previous studies. Rapid absorption and elimination of I3C was evident, possibly explaining why the presence of I3C in plasma may have been missed by others.
Additionally, oligomerization of I3C is dependent on pH (16 , 29) . Because humans have a fasting gastric acid pH of 1.5–2.0 (30) , leading to more rapid acid-catalyzed oligomerization of I3C than the pH of 3.5–4.0 found in this study with fed mice, this may be one explanation why I3C has been undetectable in human studies (26) . However, the fasting pH in mice is also around 1.5 (31) , whereas in humans the gastric pH rises after a meal, suggesting that oligomerization in the two species may be similar. Finally, I3C readily underwent oligomerization during extraction from plasma, if the method was not chosen carefully (17) . This instability may also contribute to the lack of detection of I3C in plasma.
In vitro and in vivo studies demonstrate that I3C is a pharmacologically active compound (12, 13, 14 , 32) . However, some in vivo studies show I3C to be effective only when administered orally and not by the i.p. route (9 , 33) . DIM and LTr1 have been proven to have potential antitumor activity in their own right (22 , 34 , 35) , and detection of these compounds in vivo suggests that they could indeed be partially responsible for some of the observed activity of orally administered I3C. The biological activity of HI-IM has not been reported to date.
The detection of significant quantities of I3C itself suggests that the parent compound could be partly responsible for any pharmacological effect observed in vivo. At the earliest sampling time point of 15 min, plasma and tissue concentrations had already reached an apparent maximum. Thus, the possibility exists that concentrations of I3C were even higher immediately before or after sampling at 15 min.
Not only was it the case that acid condensation products of I3C (i.e. DIM, LTr1, and HI-IM) were detected in plasma and tissues at lower peak concentrations than I3C itself, but these levels were not reached until 2 (DIM; HI-IM) and 6 (LTr1) h after I3C dosing. However, DIM, LTr1, and HI-IM persisted in plasma and tissues much longer than I3C (Table 1
; Figs. 3
and 5
). Because formation of acid condensation products is rapid at acidic gut pH (29)
, the time taken for achievement of peak levels of DIM, LTr1, and HI-IM is most likely a characteristic of the time taken for the systemic absorption of these compounds once formed within the stomach.
All of the tissues sampled in this study (i.e., liver, kidney, brain, heart, and lung) are well-perfused organs, and it follows that all compounds should reach these tissues rapidly after absorption into the systemic circulation. Indeed, high peak concentrations of I3C were observed in tissues during the first few minutes after absorption, and tissue concentration profiles of all compounds followed the same pattern as those for plasma, indicating a rapid equilibrium of all compounds between tissue and plasma. I3C, DIM, LTr1, and HI-IM were detected in all of the tissues investigated. However, concentrations of all compounds were lowest in the brain and LTr1 was detected in this tissue in only 3 of the 32 mice studied (4 and 6 h after I3C administration) making it impossible to calculate accurate tissue-to-plasma ratios. It is possible that these compounds are subject to efflux transport at the blood-brain barrier, being substrates for P-glycoprotein for example (36)
, thus limiting brain tissue exposure. Regarding LTr1, it is possible that the relatively high limit of detection of the analytical assay precludes its measurement in brain. Levels of I3C and its oligomers were generally higher in tissues than in plasma, possibly attributable to the lipid solubility of the compounds, but distribution was rapid, and there was no apparent sequestration/accumulation (Fig. 4)
. If binding of I3C and its condensation products to tissue constituents occurs, it appears to be highly reversible. Concentrations of all compounds were highest in the liver. This organ, which represents a fairly large fraction of body mass, appears to act as a sizeable reservoir for I3C and related compounds. Detection of HI-IM, DIM, and LTr1 in the liver, after oral administration of I3C, is consistent with observations of other investigators who identified these condensation products in the liver of rats (18
, 28
, 29) that received I3C. LTr1 has also been identified in the liver of rat pups from I3C-fed dams (28)
.
From the data presented in Table 2
, it is evident that DIM and LTr1 tissue-to-plasma concentration ratios are comparable, although actual concentrations of DIM were higher than those for LTr1 (Fig. 3)
. First, this may reflect the relative amounts of each compound formed from I3C in the gut, where kinetic probability indicates that formation of a dimer will occur more easily than formation of a trimer. Second, the finding indicates differences between DIM and LTr1 in the rate and extent of absorption.
It is known that ICZ is a potent inducer of cytochrome P450 enzymes in vivo. It has been shown in vitro to bind with high affinity to the Ah receptor (37) and transcriptionally activate the CYP1A1gene (38) . Taking into account the variation in I3C dosage between studies, the levels of ICZ in the liver reported in this study are higher than those reported previously (18 , 39) . However, neither of these groups fully validated their analytical method, and without taking factors such as extraction efficiency into account, it is difficult to assess absolute concentrations.
I3CA was a major oxidative metabolite of I3C in plasma samples, in agreement with unpublished data in Wall et al. (40) . I3A was detected at very low levels in the plasma up to and including 45 min. This finding is also consistent with previous studies, where I3A was found to be an oxidative product of I3C (unpublished results in Ref. 41 ). It is possible that formation of these oxidative products from I3C could contribute to its rapid clearance from plasma. I3CA and I3A are themselves rapidly formed and cleared within the 1st h of I3C administration.
I3CA and I3A appear to be detoxification products of I3C. These oxidative compounds have been identified in vitro in human breast tumor cells treated with I3C but also in the absence of cells, indicating that enzyme activity is not required for their formation (42) . It is unknown whether this oxidative process can also be enzyme catalyzed in vivo. I3CA and I3A do not exhibit the same free radical scavenging activity as I3C (43) . A study by Gamet-Payrastre et al. (44) showed that I3C and DIM significantly inhibited the growth of human colon adenocarcinoma (HT29) cells but that I3CA, at the same concentration of 100 µM, did not significantly affect cell viability. Recent work by Staub et al. (42) found I3A to be less active than I3C in cell growth and cell cycle assays, with no effect on CDK6 expression as has been reported for I3C (12) .
In conclusion, this is the most comprehensive study of the fate of I3C in vivo to date. I3C, DIM, LTr1, and HI-IM have all been shown to be present in significant levels in plasma and a number of well-perfused tissues, after administration of I3C. ICZ did not appear to be present in significant concentrations. Although there have been a few studies on the disposition of I3C in a range of species (18 , 25 , 26 , 29 , 45) , most of them used radiolabeled I3C (18 , 25 , 45) and none fully addressed the fate of I3C per se. This study has highlighted the importance of examining earlier time points in investigating the pharmacology of this potential chemopreventive agent and will aid the interpretation of studies investigating the in vivo activity of orally administered I3C.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Margaret M. Manson. Biocentre, University Road, Leicester, LE1 7RH, United Kingdom. Phone: 44-116-223-1822; Fax: 44-116-223-1840; E-mail: mmm2{at}le.ac.uk
Received 1/27/04; revised 4/30/04; accepted 5/ 6/04.
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) and LTr1(
) in plasma and tissues of mice that received I3C (250 mg/kg). Although all mice were sampled, at some time points the levels were below the limit of quantification (100 ng/g tissue, 50 ng/ml plasma). Data show the mean ± SD for three or four mice. Where no error bars are shown, measurable amounts were found in one or two of the mice within this group. DIM, 3,3'-diindolylmethane; LTr1, [2-(indol-3-ylmethyl)-indol-3-yl] indol-3-ylmethane; I3C, indole-3-carbinol.
