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

Norvaline Reduces Blood Pressure and Induces Diuresis in Rats with Inherited Stress-Induced Arterial Hypertension

Growing evidence suggests that increased arginase activity affects vital bioprocesses in various systems and universally mediates the pathogenesis of numerous metabolic diseases. The adverse effects of arginase are associated with a severe decline in L-arginine bioavailability, which leads to nitric oxide synthase substrate insufficiency, uncoupling, and, eventually, superoxide anion generation and substantial reduction of nitric oxide (NO) synthesis. In cooperation, it contributes to chronic oxidative stress and endothelial dysfunction, which might lead to hypertension and atherosclerosis. Recent preclinical investigations point arginase as a promising therapeutic target in ameliorating metabolic and vascular dysfunctions. In the present study, adult rats with inherited stress-induced arterial hypertension (ISIAH) were used as a model of hypertension. Wistar rats served as normotensive controls. Experimental animals were intraperitoneally administered for seven days with nonproteinogenic amino acid L-norvaline (30 mg/kg/day), which is a potent arginase inhibitor, or with the vehicle. Blood pressure (BP), body weight, and diuresis were monitored. The changes in blood and urine levels of creatinine, urea, and NO metabolites were analyzed. We observed a significant decline in BP and induced diuresis in ISIAH rats following the treatment. The same procedure did not affect the BP of control animals. Remarkably, the treatment had no influence upon glomerular filtration rate in two experimental groups, just like the daily excretion of creatinine and urea. Conversely, NO metabolite levels were amplified in normotonic but not in hypertensive rats following the treatment. The data indicate that L-norvaline is a potential antihypertensive agent and deserves to be clinically investigated. Moreover, we suggest that changes in blood and urine are causally related to the effect of L-norvaline upon BP regulation

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

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

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

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 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