Menadione Suppresses Benzo(a)pyrene-Induced Activation of Cytochromes P450 1A : Insights into a Possible Molecular Mechanism

Oxidative reactions that are catalyzed by cytochromes P450 1A (CYP1A) lead to formation of carcinogenic derivatives of arylamines and polycyclic aromatic hydrocarbons (PAHs), such as the widespread environmental pollutant benzo(a) pyrene (BP). These compounds upregulate CYP1A at the transcriptional level via an arylhydrocarbon receptor (AhR)-dependent signaling pathway. Because of the involvement of AhR-dependent genes in chemically induced carcinogenesis, suppression of this signaling pathway could prevent tumor formation and/or progression. Here we show that menadione (a water-soluble analog of vitamin K-3) inhibits BP-induced expression and enzymatic activity of both CYP1A1 and CYP1A2 in vivo (in the rat liver) and BP-induced activity of CYP1A1 in vitro. Coadministration of BP and menadione reduced DNA-binding activity of AhR and increased DNA-binding activity of transcription factors Oct-1 and CCAAT/enhancer binding protein (C/EBP), which are known to be involved in negative regulation of AhR-dependent genes, in vivo. Expression of another factor involved in downregulation of CYP1A-pAhR repressor (AhRR)-was lower in the liver of the rats treated with BP and menadione, indicating that the inhibitory effect of menadione on CYP1A is not mediated by this protein. Furthermore, menadione was well tolerated by the animals: no signs of acute toxicity were detected by visual examination or by assessment of weight gain dynamics or liver function. Taken together, our results suggest that menadione can be used in further studies on animal models of chemically induced carcinogenesis because menadione may suppress tumor formation and possibly progression.Peer reviewe

Aryl Hydrocarbon Receptor in Oxidative Stress as a Double Agent and Its Biological and Therapeutic Significance

The aryl hydrocarbon receptor (AhR) has long been implicated in the induction of a battery of genes involved in the metabolism of xenobiotics and endogenous compounds. AhR is a ligand-activated transcription factor necessary for the launch of transcriptional responses important in health and disease. In past decades, evidence has accumulated that AhR is associated with the cellular response to oxidative stress, and this property of AhR must be taken into account during investigations into a mechanism of action of xenobiotics that is able to activate AhR or that is susceptible to metabolic activation by enzymes encoded by the genes that are under the control of AhR. In this review, we examine various mechanisms by which AhR takes part in the oxidative-stress response, including antioxidant and prooxidant enzymes and cytochrome P450. We also show that AhR, as a participant in the redox balance and as a modulator of redox signals, is being increasingly studied as a target for a new class of therapeutic compounds and as an explanation for the pathogenesis of some disorders

The Role of Aryl Hydrocarbon Receptor (AhR) in Brain Tumors

Primary brain tumors, both malignant and benign, are diagnosed in adults at an incidence rate of approximately 23 people per 100 thousand. The role of AhR in carcinogenesis has been a subject of debate, given that this protein may act as either an oncogenic protein or a tumor suppressor in different cell types and contexts. Lately, there is growing evidence that aryl hydrocarbon receptor (AhR) plays an important part in the development of brain tumors. The role of AhR in brain tumors is complicated, depending on the type of tumor, on ligands that activate AhR, and other features of the pathological process. In this review, we summarize current knowledge about AhR in relation to brain tumors and provide an overview of AhR’s potential as a therapeutic target

The Role of CYP3A in Health and Disease

CYP3A is an enzyme subfamily in the cytochrome P450 (CYP) superfamily and includes isoforms CYP3A4, CYP3A5, CYP3A7, and CYP3A43. CYP3A enzymes are indiscriminate toward substrates and are unique in that these enzymes metabolize both endogenous compounds and diverse xenobiotics (including drugs); almost the only common characteristic of these compounds is lipophilicity and a relatively large molecular weight. CYP3A enzymes are widely expressed in human organs and tissues, and consequences of these enzymes’ activities play a major role both in normal regulation of physiological levels of endogenous compounds and in various pathological conditions. This review addresses these aspects of regulation of CYP3A enzymes under physiological conditions and their involvement in the initiation and progression of diseases

Menadione changes DNA-binding activity of some nuclear proteins from the rat liver (AhR/ARNT, Oct-1, and C/EBP).

<p>(A, B) EMSA analysis of binding of AhR/ARNT to XRE3, (C–F) Oct-1 to NRE, and (G–J) C/EBP to the C/EBP response element in the nuclear extracts from the liver of untreated rats (control) and the rats treated with menadione (15 mg/kg for four days), benzo(α)pyrene (BP; 25 mg/kg), or both BP (25 mg/[kg body weight] for three days) and menadione (15 mg/kg for four days). The specificity of the bands was confirmed by means of mutated oligonucleotides or an excess of an unlabeled oligonucleotide. The density of the bands was measured in pixels using Totallab software. The data are presented as mean ± SEM (n = 3); *p < 0.05 according to ANOVA with the Newman–Keuls <i>post hoc</i> test.</p

Menadione inhibits CYP1A1 activity <i>in vitro</i> via a noncompetitive mechanism.

<p>(A) Menadione inhibits CYP1A1 activity <i>in vitro</i> in the hepatic microsomes of benzo(α)pyrene-treated rats by a (B) noncompetitive mechanism with K_i = 3.24 μM. All measurements were performed in duplicate in three independent experiments. CYP1A1 activity was measured by means of the rate of O-dealkylation of ethoxyresorufin as described by Burke and colleagues [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155135#pone.0155135.ref028" target="_blank">28</a>].</p

Primer sequences for analysis of gene expression.

<p>Primer sequences for analysis of gene expression.</p

Menadione does not change mRNA expression of <i>AhR</i> and <i>ARNT</i> but reduces benzo(α)pyrene (BP)-induced <i>AhRR</i> expression.

<p>Menadione does not influence the expression of genes <i>AhR</i> (panel A) and <i>ARNT</i> (panel B), but reduces the mRNA level of AhR repressor (<i>AhRR</i>) (panel C) that is increased by BP. Rats received BP at 25 mg/(kg body weight) once a day for three days, menadione (15 mg/kg for four days), or both BP (25 mg/[kg body weight] for three days) and menadione (15 mg/kg for four days). The control group received vegetable oil. The data are presented as mean ± SEM (n = 4 to 5); *p < 0.05 according to ANOVA with the Newman–Keuls <i>post hoc</i> test.</p

Possible mechanisms of the menadione-driven inhibition of the benzo(α)pyrene (BP)-induced AhR-dependent signal transduction pathway.

<p>Menadione decreases DNA-binding activity of the AhR–ARNT complex thus reducing positive input, and at the same time, activates Oct-1, which inhibits expression of AhR-dependent genes. red triangle—Benzo(α)pyrene, green oval with brown, blue, yellow and red ovals inside—inactive AhR in complex with its protein partners, green oval with a triangle inside—AhR activated by benzo(α)pyrene, pink oval—ARNT, yellow circle—menadione, blue oval—mediator protein, turquoise–light gray oval—Oct-1, NRE: negative regulatory element, XRE: xenobiotic-responsive element, AhR: aryl hydrocarbon receptor, ARNT: aryl hydrocarbon receptor nuclear translocator.</p

Menadione inhibits benzo(α)pyrene (BP)-induced CYP1A activity <i>in vivo</i> via a transcriptional mechanism.

<p>(A, C) Administration of menadione increases activity of CYP1A1 and CYP1A2. (B, D) Coadministration of BP and menadione suppresses activity of CYP1A1 and CYP1A2 and (E) their protein and (F, G) mRNA expression that was induced by BP. Rats received BP at 25 mg/(kg body weight) once a day for three days, menadione (15 mg/kg for four days), or both BP (25 mg/[kg body weight] for three days) and menadione (15 mg/kg for four days). The control group received vegetable oil. The data are presented as mean ± SEM (n = 4 to 8); *p < 0.05 according to ANOVA with the Newman–Keuls <i>post hoc</i> test, #p < 0.05 according to Student’s <i>t</i> test. CYP1A1 activity was measured by means of the rate of ethoxyresorufin O-dealkylation, whereas CYP1A2 activity by means of the rate of methoxyresorufin-O-dealkylation as described by Burke and colleagues [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155135#pone.0155135.ref028" target="_blank">28</a>]. Panel C: to visualize CYP1A2 on all lanes, we loaded the samples (corresponding to the treatment with BP and BP+menadione) at 0.5 μg of total protein per well and the control sample at 80 μg of total protein per well.</p