Abstract
The objective of the study was to investigate the (anti)estrogenic, (anti)androgenic and aryl hydrocarbon receptor (AhR) agonistic activities of a mixture of acid condensation products of indole-3-carbinol, termed RXM, and to identify the compounds most responsible for the observed effects, using in vitro receptor-reporter gene transcriptional activation bioassays. For this, HPLC-fractions of RXM were prepared and tested. LC-MS/MS analysis was carried out for the identification of some of the acid condensation products. The RXM displayed weak estrogenic and anti-androgenic, and strong AhR agonistic properties. The fraction containing 3,3-diindolylmethane (DIM) displayed a weak estrogenic and relatively strong anti-androgenic activity. DIM was confirmed to be an androgen receptor (hAR) antagonist and a partial estrogen receptor (hERα) agonist. Also the fraction containing the trimer [2-(indol-3-ylmethyl)indol-3-yl]indol-3-ylmethane (LTr1) showed anti-androgenic activities. It was shown for the first time that DIM is not only estrogenic and anti-androgenic, but also possesses anti-estrogenic properties. Though indolo[3,2-b]carbazole (ICZ) is a potent AhR activator and was detected in the RXM, it did not contribute to AhR-agonist activity. Instead, fractions containing the trimers LTr1 and 5,6,11,12,17,18-hexahydrocyclonona[1,2-b:4,5-b′:7,8-b″]tri-indole (CTr), as well as some unidentified compounds showed the highest AhR activation. The fraction, containing the linear trimer LTr1, showed a weak anti-androgenic activity which has not been reported before. The study demonstrates the importance of a bioassay directed approach for identifying compounds that contribute most to the effects of mixtures.
Highlights
The focus was on the smaller reaction products and no attempt was made to separate the larger products eluting at the end of the chromatogram
Pure standards of I3C, ICZ and DIM were analysed on the HPLC system and the retention times (RT) of the pure compounds were initially used to allocate the peaks in the RXM
These analyses showed that the parent compound I3C (4.4 min), is almost completely converted and that both DIM and ICZ were present in the RXM, at RT 14.5 and 16.0 min, respectively
Summary
Epidemiological studies show that dietary intake of cruciferous vegetables is associated with reduced risks of developing cancers, e.g. breast, cervical, prostate, lung, and colorectal cancer (Aggarwal and Ichikawa, 2005; Cohen et al, 2000; Higdon et al, 2007; Kim and Milner, 2005; Minich and Bland, 2007; Mori et al, 2017; Morrison et al, 2020; Safe et al, 2008; Verhoeven et al, 1996; Weng et al, 2008; Wu et al, 2013; Yu et al, 2006; Zhang et al, 2018). I3C is a breakdown product of the naturally occurring dietary sulfurcontaining compound glucobrassicin (3-indolylmethyl glucosinolate), which is found in cruciferous vegetables such as Brussels sprouts, broccoli, cabbage and cauliflower. I3C is converted to a reactive intermediate, 3methylideneindole (3MI) This intermediate can react with another molecule of I3C at positions 2 or 3, leading to the formation of a 2,30-adduct or a 3,30-adduct (Grose and Bjeldanes, 1992). The latter results in the formation of DIM, after the loss of formaldehyde. ICZ and the cyclic trimer CTr are stable end-products because they don’t have positions left in the indole rings that can react with 3MI and homologues
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