Effects of dietary soy and estrous cycle on adrenal cytochrome P450 1B1 expression and DMBA metabolism in adrenal glands and livers in female Sprague–Dawley rats

https://doi.org/10.1016/j.cbi.2003.09.004Get rights and content

Abstract

Cytochrome P450 1B1 (CYP1B1) has been shown to be important in the bioactivation of 7,12-dimethylbenz[a]anthracene (DMBA) to an adrenal toxin in rats. We investigated the effects of diet and stage of estrous cycle on CYP1B1 expression in rat adrenal glands and on DMBA metabolism by rat adrenal and hepatic microsomes. Female Sprague–Dawley (SD) rats were placed on either standard soy-containing NIH-31 rat chow or soy- and alfalfa-free 5K96 diet from postnatal day (PND) 21 until sacrifice at PND50±5. Stage of estrous at sacrifice was assessed by vaginal cytology and confirmed by histological examination of the vagina. Dietary soy at the level present in NIH-31 diet did not affect serum estrogen and progesterone levels.

Immunohistochemical analysis confirmed that CYP1B1 was exclusively expressed in the zona fasciculata and zona reticularis in adrenal cortex, which are the regions vulnerable to DMBA-induced adrenal necrosis. Adrenal CYP1B1 protein expression, 3H-DMBA depletion, and formation of DMBA-3,4-, and -8,9-dihydrodiols by adrenal microsomes were greater in animals fed 5K96 diet, and the stage of the estrous cycle affected these parameters only in the soy-free 5K96 diet. In hepatic microsomes, the formation of DMBA-3,4-dihydrodiol, 7-hydroxy- and 12-hydroxy-DMBA were lower in animals fed NIH-31 diet than in those fed 5K96 diet. Thus, dietary soy and the estrous cycle appear to regulate adrenal CYP1B1 expression and DMBA metabolism by both adrenal and hepatic microsomes. The use of different basal diets containing variable levels of soy components may affect certain toxicity assessments.

Introduction

Cytochrome P450 1B1 (CYP1B1) is a relatively recently discovered member of the CYP1 family present in humans [1], [2], [3]. Human CYP1B1 is constitutively expressed in various normal tissues, especially extrahepatic tissues, such as kidney, brain, breast, and prostate, and is also over-expressed in various cancers [2], [4], [5], [6]. Rat CYP1B1 was first identified as P450RAP and later confirmed as the rat form of CYP1B1 [7], [8], [9], [10]. In rats, CYP1B1 protein is constitutively expressed in steroidogenic glands such as adrenal cortex, ovary, and testis, and to a much lesser extent, mammary glands, whereas expression of CYP1A1 is extremely low in adrenal gland [8], [9], [10]. Like CYP1A1, CYP1B1 is induced by dioxin and is one of the major enzymes involved in bioactivation of various polycyclic aromatic hydrocarbons (PAHs), including 7,12-dimethylbenz[a]anthracene (DMBA), a classic carcinogen and acute adrenal toxicant in rats [2], [9], [10], [11], [12]. However, aryl hydrocarbon receptor (AhR) ligands other than dioxin are poor inducers of CYP1B1 expression or CYP1B1 DMBA metabolic activity in rat adrenal gland [10], [13]. Instead, CYP1B1 expression in rat adrenal gland can be suppressed by hypophysectomy and stimulated by adrenocorticotrophic hormone (ACTH) administration [7]. Rat CYP1B1 can be activated by both cAMP- and AhR-mediated pathways in rat adrenocortical cells [14]. Expression of CYP1B1 and DMBA metabolism are also regulated by estradiol in rat mammary and ovary [13], [15], and both CYP1B1 expression and DMBA metabolism in ovary are modulated by hormonal fluctuations during the estrous cycle [16], [17]. However, it is not known whether CYP1B1 expression or DMBA metabolic activity can be regulated by the estrous cycle in other steroidogenic glands, such as the adrenal.

Dietary soy components, especially soy isoflavones such as the phytoestrogen genistein, have received much attention for both potential cancer prevention and endocrine disruptive activities. Epidemiological studies have generally indicated a lower incidence of breast and prostate cancer in Asian populations that normally consume more soy products than Western populations [18]. Studies in animals further indicate that soy or soy components can reduce tumor formation in mammary and prostate under certain conditions [19], [20], [21], [22]. Soy isoflavones such as genistein and other phytoestrogens bind to estrogen receptors (ER) and act as estrogen agonists and/or antagonists [23]. Studies in rats have indicated lower serum estrogen levels and a lower incidence of DMBA-induced mammary tumors in rats fed soy-containing diet compared with rats fed soy-free diet [24], [25]. Decreased constitutive and DMBA-induced CYP1B1 expression in rat mammary by dietary soy were also observed in these experiments. The expression of several hepatic cytochrome P450 isoforms has been found to be regulated by dietary soy protein or genistein [25], [26], [27]. Thus, dietary soy may modulate hormone and chemical metabolism, which in turn may affect the toxicity induced by the chemicals.

CYP1B1 has been shown to be important in DMBA bioactivation and toxicity in rat adrenal glands [7], [28]. The extremely low expression of CYP1A1 and other major CYP isoforms, including CYP1A2, CYP2A1, CYP2B1, CYP2B2, and CYP3A, in rat adrenal gland, and the almost complete inhibition of DMBA metabolism by CYP1B1 antibody indicated that CYP1B1 was the main enzyme responsible for DMBA bioactivation in rat adrenal gland [7], [8], [29], [30]. The DMBA metabolic activity in extra-hepatic tissues, especially its regioselective metabolic profile that distinguishes it from CYP1A1, has been used to evaluate CYP1B1 activity in these tissues [7], [15], [31]. Furthermore, both CYP1B1 expression in rat adrenal gland and DMBA-induced adrenal toxicity are regulated by ACTH [7], [12], [32]. Expression of CYP1B1 was suppressed in developing rat adrenal glands and could not be detected in mouse adrenal glands [7], [33]. This low CYP1B1expression could be associated with the resistance of immature rats and mice to DMBA-induced adrenal toxicity [32], [34], [35].

The aims of this study were to investigate the effects of soy-containing and soy-free diets as well as hormonal modulation in estrous cycle on adrenal CYP1B1 expression and DMBA metabolic activity in female rats. In addition, hepatic DMBA bioactivation also appears to be important in DMBA metabolism and adrenal toxicity [30], [36], [37], and hepatic CYP enzymes expression and/or activities could be affected by both soy isoflavones and the estrous cycle. Thus, the effects of soy-containing diet and estrous cycle on DMBA metabolism by rat liver microsome were also assessed in this study.

Section snippets

Materials

All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise it is specified. 3H-DMBA was purchased from Amersham Radiochemicals (Arlington Height, IL). 7-Hydroxy and 12-hydroxy derivatives of DMBA (7-OH- and 12-OH-DMBA), and -3,4-dihydrodiol were acquired from the NCI Chemical Depository (Midwest Research Institute, Kansas City, MO). Benzo[k]fluoranthanene was from Aldrich (Milwaukee, WI). Ethyl acetate, acetone, methanol, K2HPO4, KH2PO4 were from Mallinckrodt Baker (Phillipsburg,

Effects of soy diet on fluctuations of serum estradiol and progesterone levels in estrous cycles

In this study, animals were sacrificed either in the morning of the first day of diestrus or early afternoon (∼1 p.m.) of proestrus. As expected, the serum estradiol levels were significantly lower (P<0.05) in diestrus than in proestrus in animals fed with either NIH31 or 5K96. No difference was found in serum progesterone level between diestrus and proestrus in animals fed either diet. Neither serum estradiol nor progesterone was affected by diet (Fig. 1).

Detection of CYP1B1 protein expression in adrenal glands by immunohistochemical and Western immunoblot analysis

Immunohistochemical analysis indicated

Discussion

Standard rodent chows normally contain varying levels of soy meal [41]. This study was undertaken to determine whether relatively low level exposure to dietary soy in NIH-31 diet, which contains approximately 5% soy components, could affect serum estradiol and progesterone levels, adrenal CYP1B1 expression and/or activity, and DMBA hepatic metabolism in female rats. The results demonstrate that both dietary soy and estrous cycle may regulate adrenal CYP1B1 expression and activity as well as the

Acknowledgements

Xin Fu was supported by an appointment to the ORAU Research Program at the National Center for Toxicological Research, administered by the Oak Ridge Associated Universities through an interagency agreement between the US Department of Energy and the US Food and Drug Administration. We thank Dr. Peter Fu of NCTR for discussions on DMBA metabolism. We also thank Dr. Natalya Sadovova from Pathology Associates International Inc. for her technical assistance in the evaluation of estrous cycles.

References (53)

  • M Christou et al.

    Expression and function of three cytochrome P450 isozymes in rat extra-hepatic tissues

    Arch. Biochem. Biophys.

    (1987)
  • U Savas et al.

    Biological oxidation and P450 reactions: recombinant mouse CYP1B1 expressed in Escherichia coli exhibits selective binding by polycyclic hydrocarbons and metabolism which parallels C3H10T1/2 cell microsomes, but differs from human recombinant CYP1B1

    Arch. Biochem. Biophys.

    (1997)
  • M.W Chou et al.

    Combined reversed-phase and normal-phase high-performance liquid chromatography in the purification and identification of 7,12-dimethylbenz[a]anthracene metabolites

    J. Chromatogr.

    (1979)
  • M Watanabe et al.

    Effects of the estrous cycle and gender differences on hepatic drug-metabolising enzyme activities

    Pharmacol. Res.

    (1997)
  • T.M Badger et al.

    The health consequences of early soy consumption

    J. Nutr.

    (2002)
  • C.L Chaffin et al.

    Estrous cycle-dependent changes in the expression of aromatic hydrocarbon receptor (AHR) and AHR-nuclear translocator (ARNT) mRNAs in the rat ovary and liver

    Chem. Biol. Interact.

    (2000)
  • A Cassidy et al.

    Biological effects of a diet of soy protein rich in isoflavones on the menstrual cycle of pre-menopausal women

    Am. J. Clin. Nutr.

    (1994)
  • T.R Sutter et al.

    Targets for dioxin: genes for plasminogen activator inhibitor-2 and interleukin-1beta

    Science

    (1991)
  • G.I Murray et al.

    Regulation, function, and tissue-specific expression of cytochrome P450 CYP1B1

    Annu. Rev. Pharmacol. Toxicol.

    (2001)
  • T Shimada et al.

    Activation of chemically diverse procarcinogens by human cytochrome P450 1B1

    Cancer Res.

    (1996)
  • L Muskhelishvili et al.

    In situ hybridization and immunohistochemical analysis of cytochrome P450 1B1 expression in human normal tissues

    J. Histochem. Cytochem.

    (2001)
  • G.I Murray et al.

    Tumor-specific expression of cytochrome P450 CYP1B1

    Cancer Res.

    (1997)
  • S Otto et al.

    A novel adrenocorticotropin-inducible cytochrome P450 from rat adrenal microsomes catalyzes polycylic aromatic hydrocarbon metabolism

    Endocrinology

    (1991)
  • S Otto et al.

    Polycyclic aromatic hydrocarbon metabolism in rat adrenal, ovary, and testis microsomes is catalyzed by the same novel cytochrome P450 (P450RAP)

    Endocrinology

    (1992)
  • N.J Walker et al.

    Rat CYP1B1: an adrenal cytochrome P450 that exhibits sex-dependent expression in livers and kidneys of TCDD-treated animals

    Carcinogenesis

    (1995)
  • N.J Walker et al.

    Induction and localization of cytochrome P450 1B1 (CYP1B1) protein in the livers of TCDD-treated rats: detection using polyclonal antibodies raised to histidine-tagged fusion proteins produced and purified from bacteria

    Carcinogenesis

    (1998)
  • Cited by (13)

    • Effect of N,N-didesmethyltamoxifen upon DNA adduct formation by tamoxifen and α-hydroxytamoxifen

      2007, Cancer Letters
      Citation Excerpt :

      A microsomal sample from an adult male rat liver was included as positive control and standard. After CYP3A2 detection, the membrane was stripped and probed for β-actin, which was used as an internal standard to verify the protein loading of each sample [41]. Hepatic nuclei were isolated from portions of the excised livers and DNA was prepared from the liver nuclei as outlined in Gamboa da Costa et al. [36].

    • Effects of endocrine-disrupting chemicals on adrenal function

      2006, Best Practice and Research: Clinical Endocrinology and Metabolism
    • Dietary modulation of 7,12-dimethylbenz[a]anthracene (DMBA)-induced adrenal toxicity in female Sprague-Dawley rats

      2005, Food and Chemical Toxicology
      Citation Excerpt :

      This statistically significant difference (P = 0.019, Fisher exact test) suggested that the soy content of the diet might modulate DMBA-induced adrenal toxicity in rats. In an earlier study, we investigated the dietary modulation of adrenal cytochrome P450 (CYP) 1B1, the enzyme primarily responsible for the metabolism of DMBA in adrenal gland (Fu et al., 2003). In this report, using the same two diets as in the previous study, we examine the dietary modulation of DMBA-induced adrenal apoptosis and necrosis as well as adrenal function as assessed by serum corticosterone levels.

    View all citing articles on Scopus
    View full text