Hanna Szaefer

Pterostilbene is equally potent as resveratrol in inhibiting 12‐O‐tetradecanoylphorbol‐13‐acetate activated NFκB, AP‐1, COX‐2, and iNOS in mouse epidermis

Resveratrol, a phytoalexin present in grapes, has been reported to inhibit multistage mouse skin carcinogenesis. Recent studies showed that topically applied resveratrol significantly inhibited cyclooxygenase-2 (COX-2) expression and activation of nuclear factor-kappaB (NF-kappaB) induced by tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis. The aim of the present study was to further explore the effect of resveratrol on TPA-induced signaling pathways in mouse epidermis and to compare with its dimethylether, pterostilbene. Resveratrol and pterostilbene significantly reduced activator protein 1 (AP-1) and NF-kappaB activation. In the case of AP-1, the binding of c-Jun subunit was particularly affected, while only slight effect on c-Fos binding to TPA-responsive element (AP-1 binding consensus sequence) (TRE) site was observed. Both stilbenes inhibited the activation of NF-kappaB by blocking the translocation of p65 to the nucleus and increasing the retention of IkappaBa in the cytosol. The latter might be related to decreased activity of IkappaB kinase and/or proteasome 20S. Reduced activation of transcription factors decreased the expression and activity of COX-2 and inducible nitric oxide synthase (iNOS). In most assays, pterostilbene was either equally or significantly more potent than resveratrol. Pterostilbene might show higher biological activity due to its possible better bioavailability, since substitution of hydroxy with methoxy group increases lipophilicity.

Alteration in Phase I and II Enzyme Activities and Polycyclic Aromatic Hydrocarbons-DNA Adduct Formation by Plant Phenolics in Mouse Epidermis

Several naturally occurring plant phenols were shown to inhibit the mutagenicity and/or tumorigenicity of chemical carcinogens, including polycyclic aromatic hydrocarbons (PAHs). In this study, the effect of the topical application of three structurally diverse phenolic acids and trihydroxystilbene, resveratrol, on epidermal aryl hydrocarbon hydroxylase (AHH), phase II enzymes, as well as the binding of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) to epidermal DNA were compared. The single, topical application of 8 and 16 μmol of protocatechuic or chlorogenic acid increased the activity of AHH by 10-30%, whereas resveratrol in a dose of 16 μmol almost completely (99%) inhibited the enzyme activity. Phenolic acids also increased the activities of phase II enzymes. Resveratrol did not affect the glutathione S-transferase activity but induced UDP glucuronosyltransferase (by ~100-150%) and to a lesser extent NAD(P)H:quinone oxidoreductase. In a dose of 16 μmol all phenolic acids afforded 40-50% inhibition of covalent benzo[a]pyrene-diol-epoxide (B[a]PDE) binding to DNA. Resveratrol had no effect on B[a]PDE adduct formation but reduced the levels of all the major DMBA adducts. Phenolic acids, particularly tannic acid, mostly affected the formation of syn- and anti-DMBADE dAdo adducts. These results indicate that both the modulation of carcinogen activating enzymes and the prevention of their ultimate metabolites binding to DNA by naturally occurring phenolics are involved in the antitumorigenic activity of these compounds. For phenolic acids, however, their interactions with reactive PAH metabolites and/or blocking of a specific binding site in a genome seem more important. Derivatives of stilbene, such as resveratrol, affect DNA adduct formation and thus the initiation of tumorigenesis through the interaction with the Ah receptor rather than the scavenging active metabolites.

Betanin, a beetroot component, induces nuclear factor erythroid-2-related factor 2-mediated expression of detoxifying/antioxidant enzymes in human liver cell lines

Our recent study has shown that beetroot juice protects against N-nitrosodimethylamine (NDEA)-induced liver injury and increases the activity of phase II enzymes, suggesting the activation of the nuclear factor erythroid-2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway. The aim of the present study was to further explore the mechanism of the activity of beetroot by evaluating the cytoprotective effects of its major component. The influence of betanin (BET) on the activation of Nrf2 and the expression of GSTA, GSTP, GSTM, GSTT, NQO1 and HO-1 was assessed in two hepatic cell lines: non-tumour THLE-2 and hepatoma-derived HepG2 cell lines. The level of the tumour suppressor p53 in both cell lines and the methylation of GSTP in HepG2 cells were also evaluated. Treatment of both cell lines with 2, 10 and 20xa0μm of BET resulted in the translocation of Nrf2 from the cytosol to the nucleus. The mRNA and nuclear protein levels of Nrf2 and the binding of Nrf2 to ARE sequences were increased only in the THLE-2 cells and were accompanied by the phosphorylation of serine/threonine kinase (AKT), c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). BET also significantly increased the mRNA and protein levels of GSTP, GSTT, GSTM and NQO1 in these cells. Conversely, besides the translocation of Nrf2 from the cytosol to the nucleus, BET did not modulate any of the other parameters measured in the HepG2 cells. BET did not change the methylation of GSTP1 in these cells either. These results indicate that BET through the activation of Nrf2 and subsequent induction of the expression of genes controlled by this factor may exert its hepatoprotective and anticarcinogenic effects. Moreover, the activation of mitogen-activated protein kinases may be responsible for the activation of Nrf2 in the THLE-2 cells.

Beetroot juice protects against N-nitrosodiethylamine-induced liver injury in rats

Red beetroot, a common ingredient of diet, is a rich source of a specific class of antioxidants, betalains. Our previous studies have shown the protective role of beetroot juice against carcinogen induced oxidative stress in rats. The aim of this study was to examine the effect of long term feeding (28 days) with beetroot juice on phase I and phase II enzymes, DNA damage and liver injury induced by hepatocarcinogenic N-nitrosodiethylamine (NDEA). Long term feeding with beetroot juice decreased the activities of enzymatic markers of cytochrome P450, CYP1A1/1A2 and CYP2E1. NDEA treatment also reduced the activities of these enzymes, but increased the activity of CYP2B. Moreover, combined treatment with beetroot juice and NDEA enhanced significantly CYP2B only. Modulation of P450 enzyme activities was accompanied by changes in the relevant proteins levels. Increased level and activity of NQO1 was the most significant change among phase II enzymes. Beetroot juice reduced the DNA damage increased as the result of NDEA treatment, as well as the biomarkers of liver injury. Collectively, these results confirm the protective effect of beetroot juice against oxidative damage shown in our previous studies and indicate that metabolic alterations induced by beetroot feeding may protect against liver damage.

Effect of naturally occurring plant phenolics on the induction of drug metabolizing enzymes by o-toluidine

Plant phenolics modify the metabolic activation of several carcinogens, including aromatic amines. In this study, we have evaluated the effects of three structurally diversified plant phenolics, protocatechuic acid (PCA), tannic acid (TA) and ellagic acid (EA) on cytochrome p450-dependent enzymes and glutathione S-transferase (GST) activities after oral administration alone or in combination with o-toluidine in rat liver and kidney. Protocatechuic and ellagic acids significantly decreased the activities of ethoxy- (EROD), methoxy- (MROD) and penthoxyresorufin (PROD) dealkylases in liver. In kidney, all phenolics inhibited only the activity of PROD. Enzyme modulation in liver correlated with CA metabolism measured in plasma. Treatment of rats with ellagic acid 1 h before o-toluidine administration diminished the activities of all hepatic alkoxyresorufine dealkylases induced by o-toluidine but increased renal EROD. In contrast to EA, protocatechuic and tannic acids increased the activities of p450-dependent enzymes in liver. All phenolics administered in combination with o-toluidine increased the activity of GST, which was reduced after the treatment with o-toluidine alone. In addition, CA metabolism in plasma resulting from oral treatment with CA was measured. The formation of CA metabolites was reduced by PCA and EA, and the metabolism of CA induced by o-toluidine was depressed by administration of all three phenolics. Our results indicate that plant phenolics, especially EA, may modulate the genotoxic effects of o-toluidine by modifying pathways leading to the formation of its reactive metabolite. Moreover, as the result of CYP1A modification these compounds may affect the metabolism of CA.

The activation of the Nrf2/ARE pathway in HepG2 hepatoma cells by phytochemicals and subsequent modulation of phase II and antioxidant enzyme expression.

Previous studies have shown that naturally occurring phytochemicals, indole-3-carbinol, phenethyl isothiocyanate, protocatechuic acid, and tannic acid increased the activity and protein level of hepatic phase II enzymes in animal models. In order to further explore the mechanism of this activity, we investigated the effect of these compounds on the activation of nuclear factor erythroid-2-related factor 2 (Nrf2)-regulated transcription in human hepatocellular carcinoma HepG2 cells. Treatment with all the tested compounds resulted in the translocation from the cytosol and nuclear accumulation of active phosphorylated Nrf2. Furthermore, phenethyl isothiocyanate and indole-3-carbinol increased the transcript and protein levels of GSTA, GSTP, GSTM, GSTT, and NQO1. On the other hand, protocatechuic and tannic acids enhanced only the expression of GSTA, GSTM, and GSTT. The expression of genes encoding antioxidant enzymes CAT, SOD, GR, and GPx was increased after the treatment with all the tested phytochemicals. These results indicate that isothiocyanates/indoles and protocatechuic and tannic acids induce phase II and antioxidant gene expression in HepG2 cells through the Nrf2-Keap1-ARE signaling pathway. Moreover, the results of this study confirmed that the degradation products of glucosinolates are more effective inducers of phase II and antioxidant enzymes than protocatechuic and tannic acids.

Evaluation of the Effect of Beetroot Juice on DMBA-induced Damage in Liver and Mammary Gland of Female Sprague–Dawley Rats

Red beetroot contains a specific class of antioxidants collectively named betalains, which have been shown to have anticarcinogenic and anti‐inflamatory potential. We investigated the effect of beetroot juice on the hepatic and mammary gland carcinogen metabolizing enzymes, DNA damage and liver injury, altered by 7,12‐dimethylbenz[a]anthracene (DMBA). In the liver, pretreatment with beetroot juice significantly decreased levels and activities of the majority of tested biochemical parameters, elevated by DMBA. Feeding with beetroot juice decreased the activities of CYP1A1 and 1A2 and increased phase II enzymes. The activities of all enzymes tested were enhanced in the animals treated with DMBA alone and in combination with beetroot juice. The most significant changes in the level of the enzymes tested were observed for NAD(P)H:quinone oxidoreductase‐1. In mammary gland, beetroot juice induced the level of glutathione S‐transferase pi, enzyme involved in active metabolites of DMBA detoxification. The final effects of beetroot juice are tissue specific and depend on the class of carcinogen. Copyright

Modulation of CYP19 expression by cabbage juices and their active components: indole-3-carbinol and 3,3′-diindolylmethene in human breast epithelial cell lines

PurposeThe aim of this study was to evaluate the effect of white cabbage and sauerkraut juices of different origin and indole-3-carbinol (I3C) and diindolylmethane (DIM) on expression of CYP19 gene encoding aromatase, the key enzyme of estrogen synthesis.MethodsHuman breast cell lines (MCF7, MDA-MB-231 and MCF10A) were examined to compare the action of cabbage juices versus their active components (I3C, DIM). Real-time PCR and Western blot were used in order to analyse CYP19 mRNA and protein, respectively.ResultsRemarkable differences in the effect on CYP19 transcript and protein level were found between the cabbage juices (in 2.5–25xa0mL/L concentrations) and indoles (in 2.5–50xa0μM doses) in the three cell lines. While cabbage juices at the lower doses diminished the aromatase expression in nontumorigenic/immortalized MCF10A breast cells (0.25–0.86-fold change, Pxa0<xa00.05), I3C and DIM were more efficient in decreasing the aromatase expression in estrogen-dependant MCF7 breast cancer cells (0.24–0.82-fold change, Pxa0<xa00.05). Inhibition of aromatase by juice obtained from cabbage grown on industrial farm was correlated with the induction of apoptosis (1.7–1.8-fold change, Pxa0<xa00.01) in MCF10A cells. In estrogen-independent MDA-MB-231 cells, up-regulation of CYP19 expression by I3C and DIM (1.5–2.0-fold change, Pxa0<xa00.05) was observed. Similarly, in MCF7 cells juices increased aromatase expression (1.1–2.2-fold change, Pxa0<xa00.05).ConclusionThese results, particularly that obtained in nontumorigenic/immortalized MCF10A cells, suggest that chemopreventive activity of cabbage against breast cancer observed in epidemiological studies may be partly explained by inhibition of the aromatase expression.

Modulation of rat hepatic and kidney phase II enzymes by cabbage juices: comparison with the effects of indole-3-carbinol and phenethyl isothiocyanate.

The effect of raw cabbage and sauerkraut juices on the expression and activity of phase II enzymes, glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1), in the rat liver and kidney was compared with that of two commercially available products of glucosinolate degradation: indole-3-carbinol (I3C) and phenethyl isothiocyanate (PEITC). Male Wistar rats were treated by oral administration with cabbage juices, I3C or PEITC for 4, 10 and 30 d. The results showed that juices, particularly sauerkraut juice as with I3C and PEITC, significantly increased GST and NQO1 activities in the rat liver. The only exception was the 30 d time point of feeding with raw cabbage juice. Cabbage juices, I3C and PEITC affected the hepatic GST μ to the greatest extent and GST α to a lesser extent. The results of the present study also showed that the treatment of rats with juices and compounds tested caused the translocation of the NF-E2-related transcription factor (Nrf2) active subunit from the cytosol to the nucleus, providing an argument for the involvement of this transcription factor in the induction of GST and NQO1. In contrast to the liver, cabbage juices affected only the renal GST θ, while treatment with I3C and PEITC significantly increased the activity of NQO1. Thus, the results of the present study indicate that induction of the key detoxifying enzymes by cabbage juices, particularly sauerkraut, may be responsible for their chemopreventive activity demonstrated by epidemiological studies and in animal models. However, the final effects might be organ or tissue dependent.

Modulation of CYP1A1, CYP1A2 and CYP1B1 Expression by Cabbage Juices and Indoles in Human Breast Cell Lines

Epidemiological studies have shown that consumption of cabbage and sauerkraut is connected with significant reduction of breast cancer incidences. Estrogens are considered a major breast cancer risk factor and their metabolism by P450 enzymes substantially contributes to carcinogenic activity. The aim of this study was to investigate the effect of cabbage and sauerkraut juices of different origin on the expression profile of the estrogen metabolism key enzymes (CYP1A1, CYP1A2, CYP1B1) in breast cell lines MCF7, MDA-MB-231, and MCF10A. The effects of cabbage juices were compared with that exerted by indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM). The treatment with cabbage juices or indoles for 72 h affected the expression of CYP1 family genes in cell-type dependent manner. Their induction was found in all cell lines, but the ratio of CYP1A1 to CYP1B1 was 1.22- to 10.6-fold in favor to CYP1A1 in MCF7 and MCF10A cells. Increased levels of CYP1A2 in comparison with CYP1B1 were also observed in MCF7 cells. In contrast, in MDA-MB-231 cells CYP1B1 was preferentially induced. Since the cell lines investigated differ in invasion capacity, these results support epidemiological observations and partly explain the mechanism of the chemopreventive activity of white cabbage products.