Serum miR-181а and miR-25 Levels in Patients with Breast Cancer or Benign Breast Disease.

Circulating miR-181а and miR-25, which reflect regulation of the expression of carcinogenesis-related genes, were assayed in patients with invasive carcinoma of no specific type (ICNT) or benign breast diseases (BBDs) and in subjects without pathologies of the mammary gland (controls). miR-181а expression level proved to be higher compared to control in patients with fibroadenoma and adenosis with low, but not high, risk of malignant transformation, as well as in patients with luminal HER2-negative type B (Lum B HER2-), HER2-positive type (HER2+), and triple-negative breast cancer (TNBC) than in the controls and luminal-type (Lum A) breast cancer. MiR-25 expression level prevailed in patients with Lum B HER2- compared to control, Lum A, and TNBC patients compared to Lum A. Thus, miR-181а and miR-25 expression levels may be risk indicators of malignant transformation in some patients with BBD, whereas in patients with ICNT, these levels reflect pathological processes of different directions within the tumor.

Effects of prolonged subchronic benzo(α)pyrene exposure on rat liver morphology and CYP1A expression during treatment with menadione, quercetin, or tocopherol.

Arylamines and polycyclic aromatic hydrocarbons (PAHs) are hazardous anthropogenic pollutants in the environment. The toxicity of PAHs, which include benzo(α)pyrene (BP), is mediated by the activation of Р450 cytochromes of the 1А subfamily (CYP1A1 and CYP1A2). Previously, we have demonstrated that tocopherol, quercetin, and menadione inhibit the expression and activity of CYP1A in the liver of male Wistar rats after administration of a high BP dose to the rats for 3 days. Here, we confirmed the effects of tocopherol, quercetin, and menadione on the expression and activity of CYP1A and on rat liver morphology during prolonged administration (90 days) of a low BP dose. We revealed that subchronic oral administration of a low BP dose has no influence on CYP1A expression as compared to controls but can cause pathomorphological changes in rat liver tissue. These changes are abrogated by tocopherol, attenuated by quercetin, and enhanced by menadione.

Quercetin Attenuates Benzo(α)pyrene-induced CYP1A Expression.

We studied effects of nutrient quercetin on cytochromes' Р450 1А (CYP1A) activities (measured spectrofluorimetrically using 7-ethoxy-resorufin for CYP1A1 and 7-methoxy-resorufin for CYP1A2 as substrates), on mRNA levels (measured by RT-PCR), and on DNA-binding activities (evaluated by an electrophoretic mobility shift assay) of proteins regulating CYP1A expression in untreated and benzo(α)pyrene (BaP)-treated rats. Wistar rats received quercetin, BaP, or both once daily for 1-3 days. Quercetin did not influence CYP1A1 in untreated rats but inhibited BaP-mediated CYP1A induction on the transcriptional level decreasing positive input (AhR functional activity) and increasing negative input (AhRR/ARNT expression and Oct-1 and C/EBP functional activities).

The influence of changes in expression of redox-sensitive genes on the development of retinopathy in rats.

Age-related macular degeneration (AMD) is a complex multifactorial disease of the elderly, with unclear pathogenesis; AMD is the leading cause of blindness. One of the destructive processes in AMD is oxidative stress, which leads to an imbalance in the processes responsible for production and detoxification of reactive oxygen species. The aryl hydrocarbon receptor (AhR) signaling pathway can participate in the development of oxidative stress, but the main regulator of antioxidant defense is nuclear factor, erythroid derived 2 (Nrf2). AhR-dependent oxidative stress can be attenuated by activation of Nrf2, and defects in the Nrf2 signaling pathway can increase sensitivity of the cell to oxidative stress. Our aim was to determine the role of the pro-oxidant (AhR-dependent) and antioxidant (Nrf2-dependent) systems in the pathogenesis of AMD using rats of OXYS strain and of OXYSb substrain with signs of AMD-like retinopathy of varying severity. We compared the retinal levels of mRNA expression of Nrf2- and AhR-dependent redox-sensitive systems between 1-, 3-, and 12- month-old senescence-accelerated OXYS rats (have been shown to be a valid experimental model of AMD) and the rat substrain OXYSb, which shows low morbidity of AMD. We uncovered interstrain differences in the expression of Nrf2 and Nrf2-dependent genes (glutathione S-reductase [Gsr] and heme oxygenase 1 [Hmox1]), in the expression of AhR-dependent genes (cytochrome P450 1A2 [Cyp1a2] and cytochrome P450 1B1 [Cyp1b1]), and in the NADPH-quinone oxidoreductase (Nqo1) expression, which is controlled by both AhR and Nrf2. Binding of AhR and Nrf2 proteins to the regulatory regions of AhR and Nrf2 genes, respectively, was detected by chromatin immunoprecipitation in the retina of 1-, 3-, and 12-month-old OXYS, OXYSb, and Wistar (control) rats. We compared the strength of DNA-protein interactions of AhR and Nrf2 with regulatory sequences and found that the level of autoupregulation of the AhR gene was higher in the retina of 1-month-old OXYSb rats in comparison with OXYS rats. An imbalance between pro-oxidant (AhR-dependent) and antioxidant (Nrf2-dependent) systems may play a crucial role in the onset and/or progression of AMD.

Stimulation of Diethylnitrosamine Metabolism Reduces Its General Toxic and Hepatocarcinogenic Effects.

The general toxic and hepatocarcinogenic effects of diethylnitrosamine after stimulation of its metabolism with 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene (TCPOBOP) were studied. The hydroxylating activity of liver microsomes of C57Bl/6Mv mice towards p-nitrophenol increased more than 4-fold 3 days after injection of TCPOBOP. Injection of diethylnitrosamine 3 days after TCPOBOP caused a lesser body weight loss and decrease of food consumption in C57Bl/6Mv mice than in response to diethylnitrosamine without preinduction. Injection of diethylnitrosamine to suckling ICR mice after TCPOBOP induction of cytochrome P450 2e1 activity led to development of 2-fold lesser number of tumors and pretumorous nodes in the liver in comparison with animals injected with diethylnitrosamine without induction. These data indicated that metabolism stimulation reduced the general toxic and hepatocarcinogenic effects of diethylnitrosamine.

[Effect of Hepatic P450 2e1 Activity Modulation on the Toxicity and Carcinogenicity of Diethylnitrosamine in Mice].

In this paper, the biological effects of diethylnitrosamine have been studied under controlled conditions of its metabolism in mice of different ages. The data presented indicate that diethylnitrosamine in a non-metabolized form exerts general toxic and hepatocarcinogenic effects while alkylating agents of this compound produce toxic liver injury. To our knowledge, the data presented impel to revise the general notion of an exceptional role of mutagenic activation in the carcinogenic effect of chemicals.

Expression of genes for AhR and Nrf2 signal pathways in the retina of OXYS rats during the development of retinopathy and melatonin-induced changes in this process.

Modulation of oxidative stress is one of the experimental approaches to the therapy of age-related macular degeneration. Melatonin holds much promise in this respect. It was hypothesized that the efficiency of melatonin in age-related macular degeneration is associated with its ability to modulate gene expression for the AhR and Nrf2 signal pathways. Experiments were performed on premature aging OXYS rats, which serve as a reliable model of age-related macular degeneration in humans. We studied the effect of melatonin on gene mRNA for the AhR and Nrf2 signal pathways. Melatonin was shown to decrease the level of mRNA for AhR-dependent genes of CYP1A2 and CYP1B1 cytochromes in the retina, but had no effect on the content of mRNA for Nrf2-dependent genes in OXYS rats.

[Inhibitory effect of ortho-aminoazotoluene on diethylnirtosamine hepatocarcinogenesis in suckling mice. Phenomenon and possible mechanism].

The modifying effect of the one compound on carcinogenicity of the other in the combined application is attributed usually to some changes in the carcinogen metabolism, i.e. its activation or inactivation. In this paper, when used separately, diethylnitrosamine (DENA) induced 4-6 times more neoplastic lesions in the liver of suckling mice than ortho-aminoazotoluene (OAT) did. However, after combined treatment with both carcinogens the total number of hepatic lesions was significantly lower than that in mice treated with DENA only. Similar effect was observed when OAT was administered 3 days before or 3 days after DENA injection. The observed protective effect is not mediated at metabolic level, because OAT has no effect on metabolism of DENA in mouse liver. Our findings can be unequivocally explained by the competition of the carcinogens for target protein molecules, presumably transcription factors, participating in hepatocyte differentiation, which differently interact with and are diversely impaired by different compounds.

Cold-induced activities of cytochromes P450 1A1 and 1A2 in rat liver: putative role of endogenous compounds in induction mechanism.

Adaptation to cold includes adaptive changes at the organism and molecular levels. One of the interesting facts is induction of cytochromes P450 subfamily 1A (CYP1A) in the liver of rats, inducible enzymes participating in biotransformation of procarcinogenic xenobiotics, under the effect of moderate cold exposure. Cold activation of CYP1A can be mediated by adaptive changes and the resultant redistribution or intensification of the synthesis of mediator compounds. This hypothesis is verified in the present study. The role of bilirubin, tocopherol, and corticosterone as mediators of cold induction of CYP1A in the rat liver was evaluated. The results indicate that these compounds can be involved in cold induction of CYP1A, but none of them is the only mediator in this process.

Serum miR-181a and miR-25 in patients with malignant and benign breast diseases.

Breast tumor diseases include a wide range of pathologies that require different approaches to their treatment. MicroRNA (miR) levels, reflecting regulation of the gene expression involved in tumorigenesis, can be diagnostic and prognostic markers of breast diseases. The levels of circulating miR-181a and miR-25 were measured in patients with benign breast diseases (BBD), patients with invasive carcinoma of a nonspecific type (ICNT) and also in conditionally healthy women. Expression of both miRs was higher in patients of both groups as compared to controls; at the same time, the content of serum miR-181a and miR-25 was higher in BBD patients than in ICNT patients. The detected changes may be of interest in the context of precancerous changes in BBD. It seems possible to use them in the future as markers of the pathological process as a part of a large diagnostic panel.

The Mitochondria-Targeted Antioxidant SkQ1 Downregulates Aryl Hydrocarbon Receptor-Dependent Genes in the Retina of OXYS Rats with AMD-Like Retinopathy.

The mitochondria-targeted antioxidant SkQ1 is a novel drug thought to retard development of age-related diseases. It has been shown that SkQ1 reduces clinical signs of retinopathy in senescence-accelerated OXYS rats, which are a known animal model of human age-related macular degeneration (AMD). The aim of this work was to test whether SkQ1 affects transcriptional activity of AhR (aryl hydrocarbon receptor) and Nrf2 (nuclear factor erythroid 2-related factor 2), which are considered as AMD-associated genes in the retina of OXYS and Wistar rats. Our results showed that only AhR and AhR-dependent genes were sensitive to SkQ1. Dietary supplementation with SkQ1 decreased the AhR mRNA level in both OXYS and Wistar rats. At baseline, the retinal Cyp1a1 mRNA level was lower in OXYS rats. SkQ1 supplementation decreased the Cyp1a1 mRNA level in Wistar rats, but this level remained unchanged in OXYS rats. Baseline Cyp1a2 and Cyp1b1 mRNA expression was stronger in OXYS than in Wistar rats. In the OXYS strain, Cyp1a2 and Cyp1b1 mRNA levels decreased as a result of SkQ1 supplementation. These data suggest that the Cyp1a2 and Cyp1b1 enzymes are involved in the pathogenesis of AMD-like retinopathy of OXYS rats and are possible therapeutic targets of SkQ1.

[All signs of metabolic syndrome in the hypertensive ISIAH rats are associated with increased activity of transcription factors PPAR, LXR, PXR, and CAR in the liver].

It is known that the metabolic syndrome (MS), which includes hypertension, dislipidemia, glucose intolerance, and obesity leads to cardiovascular diseases. The MS risk is growing catastrophically. Molecular mechanisms allowing to understand the reason of integrated dysfunctions, taking place at MS cases, have remained almost unstudied. The chronical stress plays a crucial role in MS development; therefore in the present work a hypertensive rat strain with Inherited Stress-Induced Arterial Hypertension (ISIAH) was used as a model. It was shown that ISIAH rat strain as compared with the control WAG rat strain is characterized by increased content of triglyceride, VLDL and LDL cholesterols, a decreased content of HDL cholesterol, a high level of apolipoprotein B-100, and decreased level of apolipoprotein A-I. The ISIAH rats body weight was higher as compared with WAG rats; ISIAH rats blood glucose content was higher too. Thus, strain hypertension for ISIAH rat is accompanied by dislipidemia, increased glucose content, and increased body weight, representing a whole set of MS signs. Since at MS cases the systemic abnormalities in lipid and carbohydrate metabolism take place, the functional activity of transcription factors (TFs) participating in integral regulation of lipid and carbohydrate metabolism genes in liver was measured. PPAR, LXR, PXR, CAR DNA-binding activity was increased in ISIAH rats, suggesting involvement of these TFs in MS development. Integrated investigation of PPAR, LXR, PXR, CAR regulatory mechanisms, signal transduction and transcriptional targets will provide insights into the pathogenesis of MS and offer valuable information for designing of drugs for MS treatment.

[Expression of nuclear hormone receptors PPAR, LXR and RXR in the liver and lipid and glucose levels in blood in susceptible and resistant to hepatocarcinogenesis mice strains].

Earlier it was shown that male mice of the DD/He strain were highly susceptible to ortho-aminoasotoluene (OAT) induced hepatocarcinogenesis, and resistant to spontaneous liver tumor development as compared to the CC57BR/Mv strain. In the present work we have made a comparative investigation of peroxisome proliferator-activated receptor (PPAR), liver X-receptor (LXR) and retinoic X-receptor (RXR) mRNA levels in liver as well as concentrations of corticosterone, glucose, lipids and insulin in blood of male DD/He and CC57BR/Mv mice. Using the multiplex RT-PCR method it was found that PPAR-alpha, PPAR-gamma, RXR-alpha and RXR-beta mRNA content was essentially decreased in the liver of DD mice as compared to mice of the CC57BR strain. No significant interstrain differences of LXR-alpha and LXR-beta mRNA content were found. In DD micetere was more then the 3-fold decrease of blood content of corticosterone, which is involved in PPAR and RXR regulation. DD mice demonstrated a significant decrease in blood serum glucose and insulin concentrations as well as higher reactivity to insulin as compared with CC57BR mice. Elevated blood total cholesterol and cholesterol HDL level were found in DD mice whereas triglyceride content was basically the same in both mouse strains. It is known that glucocorticoids, PPAR and RXR play crucial role in transcription regulation of inflammation response. Therefore our data allow to suggest that decreased corticosterone level in blood, PPAR and RXR mRNA content in liver of the DD strain may lead to induction of inflammation by OAT exposure, resulting in a high incidence of tumorigenesis in this strain.

Effect of cold stress on expression of genes for the AhR-dependent pathway of CYP1 regulation in rat liver.

The level of mRNA for cytochromes P450 (CYP1A1, CYP1A2, and CYP1B1) and CYP1 regulatory proteins (heat shock protein, aryl hydrocarbon receptor, aryl hydrocarbon receptor repressor, and aryl hydrocarbon receptor nuclear translocator) was measured in the liver of rats after cold stress (4 degrees C). The CYP1A1 mRNA level increased and remained high for 10 days after 5-day cold exposure. The level of mRNA for CYP1A2, heat shock protein, and aryl hydrocarbon receptor nuclear translocator decreased by the 10th day. The level of mRNA for CYP1B1, aryl hydrocarbon receptor, and aryl hydrocarbon receptor nuclear translocator remained unchanged over this period.

Activity of 20S proteosomes and content of oxidized proteins in rat liver after long-term cold exposure.

We studied the effect of long-term (5 and 10 days) cold exposure (4 degrees C) on oxidative damage to proteins and proteosomal activity in the liver of Wistar rats. It was shown that core temperature on the 10th day of cold exposure decreased by 1.1 degrees C. The content of oxidized proteins increased at this term. Chymotrypsin-like and peptidylglutamyl peptide hydrolyzing activities increased, while trypsin-like activity decreased during cold exposure.

Effect of long-term cold exposure on activities of cytochrome P450-containing monooxygenases and glutathione S-transferase in rat liver microsomes.

Long-term cold exposure (5 degrees C) was followed by induction of rat liver monooxygenases. We revealed an increase in activity of NADPH-cytochrome C reductase, total content of cytochrome P450 (CYP), and activities of its molecular forms CYP1A1, 1A2, 2B1/B2, 2E1, and 3A1/A2 in microsomes. These indexes reached maximum by the 10th day, but decreased with lengthening of cold exposure. Glutathione S-transferase activity decreased under these conditions. Changes in enzyme activity could be related to the increase in blood corticosterone concentration.