Modulations of P450 mRNA in liver and mammary gland and P450 activities and metabolism of estrogen in liver by treatment of rats with indole-3-carbinol

Abstract

Indole-3-carbinol (I3C), found in cruciferous vegetables, has been shown to suppress tumorigenesis at estrogen-responsive sites. This effect may be mediated through modification by I3C of the cytochrome P450 (CYP) complement and activities leading to estrogen detoxication. In this study, we examined the effects of 4- and 10-day treatments of female Sprague–Dawley rats with I3C at 5, 25, and 250 mg/kg body weight, administered by oral gavage, on CYP mRNA expression in the liver and mammary gland, CYP-dependent activities, and the metabolism of 17β-estradiol (E2) and estrone (E1) by liver microsomes. The mRNA transcripts for hepatic CYP1A1, 1B1, and 2B1/2 and mammary CYP1A1 were up-regulated after treatment with I3C at 250 mg/kg. However, the level of expression of CYP1B1 in the liver was lower than that of other CYPs. In the mammary gland, CYP1B1 mRNA levels were unaltered by treatment and similar to those of I3C-induced CYP1A1. Hepatic P450 probe activities indicative of induction of CYP1A1, 1A2, and 2B1/2 were increased by I3C in a dose-dependent manner. Treatment with I3C at 250 mg/kg increased the capacity of liver microsomes to metabolize E2 to 2-OH-E2, 2-OH-E1, 6α-OH-E2, 6β-OH-E2, estriol, and 15α-OH-E2, and E1 to 2-OH-E1, 2-OH-E2, 6(α+β)-OH-E1, and 6α-OH-E2. The magnitudes of increases of CYP-dependent activities and rates of estrogen metabolite formation achieved with I3C at 250 mg/kg were smaller after ten than four treatments. The increased rates of formation of 6α-OH-E2, 6β-OH-E2, and 15α-OH-E2 from E2 were also detected after treatment with I3C at 25 mg/kg, and, except for increased 6β-OH-E2 from E2, no other changes in E2 or E1 metabolism occurred after treatment with I3C at 5 mg/kg. The data indicate that alterations in the CYP complement and, thus, metabolite composition from E2 and E1 are I3C dose- and treatment duration-dependent, and suggest that potential biological activity of I3C administered at low doses to rats may not involve changes in estrogen metabolism.

Introduction

I3C, an autolysis product of an abundant glucobrassicin in cruciferous vegetables (e.g. broccoli, cabbage, cauliflower, and Brussels sprouts), has been shown to inhibit chemically induced tumorigenesis of the liver, mammary gland, colon, and other tissues in laboratory rodents [1]. The blocking activity of I3C administered before or concurrently with a carcinogen is thought to be mediated through alterations in the levels and activities of Phase I (e.g. P450 or CYP) and Phase II (e.g. GSH S-transferase) isozymes in the rodent liver and/or extrahepatic tissues resulting in their increased capacity for detoxication of carcinogens, and thus decreased levels of DNA adducts, and reduced tumorigenic response. In addition, I3C has been shown to suppress spontaneous carcinogenesis at estrogen-susceptible sites, e.g. mammary gland and endometrium [2], [3]. This was ascribed to significant increases in CYP-catalyzed 2-hydroxylation of E2 and E1 to the less estrogenic 2-catechol estrogens by liver microsomes of the I3C-treated rodents. Ingestion of I3C at ∼5–7 mg/kg body weight for a week or 2 months by men or women, respectively, increased the extent of 2-hydroxylation of E2 in vivo[4], and also increased urinary excretion of 2-catechol estrogens but decreased that of more active estrogens (E2, E1, E3, and 16α-OH-E1) [5]. These findings led the authors to postulate that treatment with I3C may decrease the concentration of metabolites known to activate the estrogen receptor and, thus, reduce estrogenic stimulation in women leading to prevention of breast cancer.

Orally administered I3C induces CYP1A1, 1A2, 2B1/2, and 3A1 in the liver of female rats [6], [7], [8]. The efficacy of the ingested I3C as an inducer of CYP1 family genes depends upon activation of AhR by I3C products formed in stomach acid, e.g. ICZ and DIM [6], [9]. Among the CYP1 gene family, induction of CYP1B1 in the liver and its up-regulation in extrahepatic tissues including the mammary gland of female SD rats treated with TCDD have been reported [10], [11], [12]. Whereas the increases of the hepatic CYP1B1 RNA levels were detectable after chronic treatment with TCDD at doses as low as 3.5 ng/kg/day, those of CYP1B1 protein were determined only after the higher doses of TCDD (>35.7 ng/kg/day) [11]. In response to TCDD treatment, the level of expression (number of copies/μg RNA) of CYP1A1 or CYP1A2 was much greater than that of CYP1B1, although its mRNA level was most increased. In the rat liver, CYP1A2, 2B1/2, and the 3A family are recognized as catalysts of both 2- and 4-hydroxylations of E2 and E1 [13]. In the tissues of TCDD-treated female rats, induction of CYP1A1 and 1A2 appeared to be associated chiefly with increased 2-hydroxylation of E2 and E1 and to a lesser extent with their 4-hydroxylation, whereas the latter oxidation correlated with the induction of CYP1B1 [12]. These oxidations are the major source of catechol estrogens which via oxidation to semiquinones and quinones, especially those derived from 4-catechols, may be involved in estrogen carcinogenicity [14], [15], [16]. Hence, it is important to delineate the CYP complement and metabolism of estrogen in response to treatment with modifiers such as I3C, which is consumed by humans with vegetables of the Cruciferae family or as a dietary supplement.

In our previous study, treatment of female rats with I3C at 250 or 500 mg/kg body weight for 4 days (acute) or at 250 mg/kg body weight three times per week for 12 weeks (chronic) increased CYP1A1, 1A2, 2B, and 3A probe activities of hepatic microsomes and the metabolism of E2 and E1 including the increased rates of formation of 2-, 4-, 16α-, 6α-, 6β-, and 15α-OH metabolites [17]. It is presumed that persistent effects of the increased putative carcinogenic and estrogenic 4- and 16α-OH as well as 6α- and 6β-OH metabolites of E2 and E1 might have counteracted those of the less estrogenic 2-catechols, thus failing to suppress carcinogen-initiated mammary tumorigenesis by chronic post-initiation treatment of rats with I3C [18].

It also has been shown that the ratios of acid-catalyzed condensation products of I3C formed in vitro change with the concentration of I3C [19]. Since the I3C products elicit distinct biological activities [9], [20], it is relevant to determine the effects of treatment with I3C following a broad range of dose levels including those relatively low doses that have been tested in humans [5]. In the present study, treatments of female SD rats included three dose levels of I3C, i.e. 5, 25, and 250 mg/kg body weight, administered by oral gavage in 20% ethanol in olive oil for 4 and 10 days. The dose- and treatment duration-related effects of I3C on (a) CYP mRNA expression in the liver and mammary gland, (b) CYP probe activities of liver microsomes, and (c) CYP-dependent metabolism of E2 and E1 by liver microsomes were examined.