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Using bioinformatics analysis we selected microRNAs which could bind 3'-UTR-region of cytochrome P450 (CYP) genes. Three microRNA miR-21, -221, -222, their potential targets might be mRNA for CYP1A1, and two microRNA miR-143, miR-152 for CYP2B1 accordingly were selected for experimental verification. Expression level of these microRNAs in rat liver upon benzo(a)pyrene (BP), phenobarbital (PB), and DDT induction was determined using RT-qPCR method. In rats treated by both BP, and DDT the hepatic content of miR-21, -221, -222 significantly demonstrated a 2-3-fold decrease. The decrease in miR expression was accompanied by a considerable (5.5-8.7-fold) increase in the CYP1A1-mediated EROD activity. The expression of miR-143 remained unchanged after the PB treatment, while the expression of miR-152 increased by 2 times, however, the (10.5-fold) increase in PROD activity of CYP2B was much higher. In the DDT-treated liver PROD activity increased by 20 times, the expression of miR-152 didn't change, and the expression of miR-143 increased by 2 times. The bioinformatics analysis of interactions between microRNAs and targets showed that the studied miRs can potentially bind 3'-end of AhR, ESR1, GR, CCND1, PTEN mRNA. Thus, the expression profile of miR-21, -221, -222, -143, -152 might change under the xenobiotics exposure. In silico analysis confirmed, that microRNAs target not only cytochrome P450 mRNA but also other genes, including those involved in hormonal carcinogenesis, they also can be regulated with studied miRs.

Background: Benzo[a]pyrene (BaP) is the widespread environmental toxicant with carcinogenic activity. BaP undergoes metabolic activation in the liver microsomal monooxygenase system, and its regional lymph nodes act as peripheral filters, purifying lymph formed in the liver. It remains an open question about the significance of the integral liver and lymphatic system work in supporting processes of adaptation and resistance to the BaP effects., Objective: The purpose of our investigation was to study structural and metabolic changes in the liver and its lymph region in males Wistar rats weighing 180-220g., Methods: Animals of the experimental group (n =20) daily for 3 days was performed BaP injections: intraperitoneal with 2 mg per 100g of body weight in 0.2-0.3 ml of olive oil. Rats in the control group (n =20) received in the same mode of injection of olive oil. The light and electron microscopy morphometric study of the liver and its regional lymph nodes morphometric analysis were performed. The intensity of lipid peroxidation in the liver was measured by the number of diene conjugates (DC), ketotriene conjugates (KC) and malondialdehyde (MDA)., Results: Simultaneous increase of the hepatic sinusoids relative area and the specific area of the regional lymph nodes sinus system under the BaP effect was found. At ultrastructural level dilatation of the Disse spaces, filling of cell detritus and collagen fibers bundles of these spaces were noted. Damage of nuclear apparatus, balloon transformation of granular endoplasmic reticulum profiles were found in hepatocytes, condensation of the mitochondrial matrix was observed. The relative squares of B-dependent zones increased and T-dependentparacortical zone reduced in the regional lymph nodes. The increase in the content of KC and MDA in liver was identified., Conclusion: benzo[a]pyrene causes interrelated cascade of reactions in the liver and in its lymphatic region: a disturbance of the blood-lymph barrier morphological organization, and so obstruction of the lymphatic drainagefrom the organ. These promotes development of tissue hypoxia and apoptosis, protein synthesizing and energy cell apparatus disruptions, formation of lymphoid tissue temporary infiltrates in the liver.

Effect of carcinogenic polycyclic aromatic hydrocarbons (PAH) benzo(a)pyrene (BP) and 3-methylcholanthrene (MC) on transcription factor NF-kappaB activation was studied. The determination of NF-kappaB activity was performed by two different methods: determination of mRNA expression of NF-kappaB-dependent I-kappaB gene, and determination of transcription activity of co-transfected with the plasmid containing the luciferase reporter gene under the NF-kappaB-sensitive promoter. As a subject of inquiry the hepatoma cell cultures HepG2 expressed Ah receptor and G27 not expressed Ah receptor were used. BP and MC weekly enhanced NF-kappaB activity in proliferating HepG2 cells. The enhance of NF-kappaB activity was significantly higher in resting cells. NF-kappaB activation by BP and MC in hepatoma G27 cells was significantly higher in hepatima G27 cells than in HepG2 cells both in proliferating and resting cells. The role of Ah receptor in PAH action on NF-kappaB activation is discussed.

The rate of hepatic cytochrome P450 Cypla1 and Cyp1a2 induction was investigated in C57BL male mice during induction with o-aminoazotoluene (OAT), benzo[a]pyrene (BP) and 1,4-dihydroxyanthraquinone (AQ). The Cypla1 mPNA level determined by quantitative RT-competitive PCR increased more than three orders of magnitude during induction with OAT and BP compared with untreated animals and remained unchanged during induction with AQ. The Cypla2 mRNA level was only 8.5, 18.7 and 1.9 times higher during induction with OAT, BP and AQ respectively than in untreated mice. At the same time 7-Ethoxyresorufin-O-deethylase (EROD) and 7-Methoxyresorufin-O-demethylase (MROD) activities of Cypla were also investigated in liver. The increase of Cypla1 mRNA level correlated with the increase of EROD activity. This suggests involvement of the transcriptional mechanism of the inducibility of this enzyme. In the case of Cypla2 there was insignificant increase of its mRNA level but high catalytic activity registered in liver in response to injection of the inductor of MR metabolism. This can imply the posttranscriptional mechanism of Cypla2 regulation. During induction with AQ the Cypla1 mRNA level remained unchanged, but the EROD activity increased almost 20-fold. The latter suggests insufficient specificity of this substrate for Cypla1. Thus, on the basis of the data obtained, the mRNA level can be considered as more accurate estimation of Cypla1 and Cypla2 inducibility, than the determination of the enzyme activity.

Unlike other cytochrome P450-dependent oxygenase inhibitors, ketoconazole has been shown to suppress the murine macrophage-mediated oxidative modification of human low-density lipoproteins (LDL) in a dose-dependent manner. The benzo[alpha]pyrene-induced microsomal monooxygenase activity was accomplished by a 1,5-fold increase in LDL oxidation by macrophages, ketokonazole (20 mu), methoxalene (20 mu), and alpha-naphthaflavone (50 mu). Ketoconazole was also effective in inhibiting macrophageal NADPH oxidase and LDL autooxidation induced by Fe2+ rather than Cu+, which is likely to be associated with its ability to act as a chelator of free and heme-bound iron ions.

Thermal properties of DNA-benz(a)pyrene complex and chromatin within liver cells in BALB/c mice and Macaca fascicularis monkeys after benz(a)pyrene administration were studied using a highly sensitive differential scanning microcalorimeter designed for investigations of dilute biopolymer solutions and complex biological systems. It was shown that benz(a)pyrene (BP) had different efforts on DNA in vivo and in vitro. It was established that at a molar ratio r < 0.03 BP/DNA bp, benz(a)pyrene served as a stabilizing but at higher concentrations as a destabilizing factor of DNA. It was found that BP damaged liver DNA stronger than bone marrow and spleen DNA of a given animal in vivo. Based on analysis of heat redistribution at the heat absorption stages corresponding to denaturation of inactive and active chromatin, we concluded that BP is capable of causing specific breaks in the DNA chain of inactive chromatin and unfolding the whole domain-loop of chromatin, which should lead to uncontrolled genome activation and, therefore, to carcinogenesis.

The basal level of benzo(a)pyrene monooxygenase, epoxide hydrolase and glutathione S-transferase activity as well as the content of cytochrome P-450 were found the same in both compared benzo(a)pyrene (BP) sensitive D. simulans strain 364yv and BP-resistant wild one (Turku). Phenobarbital pretreatment resulted in the same increase level of these enzyme activities in both strains. BP-pretreatment of 364yv flies decreased the amount of the cytochrome P-450 but raised up the turnover of BP per molecule of cytochrome P-450. SDS-polyacrylamide gel electrophoresis of the microsomal proteins from BP-pretreated 364yv flies (but not from Turku) showed an increased hemoprotein content in the 56000 band. The relationship between BP-sensitivity of the strain 364yv and BP-induced aberrant isoform of the cytochrome P-450 has been discussed.

The long-term administration of xenobiotics carcinogens o-aminoazotoluene (o-AAT) and benz(a)pyrene (BP) to rats was found to cause induction of the liver cytochrome P-450 system which gradually decreases in spite of continued administration of the agents. Induction of microsomal oxygenases under these conditions is followed by induction of the immune response to o-AAT and BP. The data obtained correspond to the conception of the immunochemical functional system of homeostasis implying that the cytochrome-450 system and the immunity system are functionally linked and are elements of the common functional adaptive system of the organism.

As shown by means of Ouchterlony double immunodiffusion technique after induction with 3-methyl cholanthrene the antibodies towards cytochrome P-448 from rat liver tissue developed a clear-cut precipitation line with homologous antigen and interacted also with cytochrome P-448 from mice liver tissue although the precipitation was less distinct. Antibodies towards mice liver cytochrome P-448 reacted also with the rat liver cytochrome P-448 but less distinctly as compared with the mice cytochrome. At the same time, these antibodies inhibited similarly the benz(a)-pyrene-hydroxylase reaction, catalyzed by both mice and rat cytochromes P-448. Partial immunological identity appears to occur in the cytochromes studied. Role of total antigenic determinants in enzymatic reactions catalyzed by cytochrome P-448 is discussed.

The confluent culture of hamster embryo cells was incubated with benzo(a)pyrene for 24 hours. Then the medium was replaced by maximal lacking both the serum and benzo(a)pyrene. The process of DNA repair was observed in four nuclear fractions according to two indexes: the disappearance of metabolites of benzo(a)pyrene covalently bound to DNA and the incorporation of 3H-thymidine to DNA in the period from I min to 72 hours. Hydroxyurea at the concentration of 5 mM was added 2-19 hours before 3H-thymidine. The highest concentration of benzo(a)pyrene metabolites was found in the DNA of nuclear matrix fraction throughout all the experiment. The initial concentration of 3H-thymidine right after its addition into the cell culture medium was the highest in DNA of nuclear matrix fraction and the lowest in DNA fraction soluble in the buffer with low ionic strength. Later on, the concentration of 3H-thymidine was decreased in matrix-bound fractions and increased in other fractions up to the total DNA level. The results suggest that the repair process requires joining of benzo(a)pyrene damaged DNA region to the nuclear matrix with the following reverse transition into the fraction where the fragment was initially located.

The nuclei of cells from regenerating rat liver were incubated with benzo(a)pyrene and the concentrations of the metabolites that covalently bound to DNA of different nuclear fractions were compared. It appeared that DNA associated with nuclear matrix (containing replicating DNA) is modified most intensively. The synchronized mouse embryo cells were incubated with benzo(a)pyrene during S phase and the levels of modifications in short and long single-stranded DNA fragments were compared. It has been observed that replicating DNA is represented in short fragments. These short DNA fragments were found to be modified by benzo(a)pyrene 4-9 times more intensively than total DNA. The possible mechanisms of both the increase in the number of DNA modifications in proliferating cells and the reason for the enhancement of carcinogenic effect on dividing cells are being discussed.

Benz(a)pyrene in a concentration of 2.5 micrograms/ml produced a 3.3-fold increase in the transforming activity of simian adenovirus S5 in primary cultures of rat kidney cells. Under analogous conditions TPA increased (2.1 times) the transforming activity of DNA of human adenovirus of type I (AdI). PX5 cells (cotransformed by S5 and benz(a)pyrene) induced tumours in 100% of syngeneic and 81% xenogeneic animals. PX4 cells (transformed by S5 only) induced tumours in 60% of syngeneic animals. Cells transformed by DNA AdI and DNA AdI + TPA did not induce tumours in animals.

A study was made of the effect of 5-methylresorcinol (5-MR) on mutagenic activity of benzo(a)pyrene (BP) and of the action of gamma-radiation in in-vitro and in-vivo systems. The induction of direct gene mutations in Chinese hamster cells V-79 and micronuclei in mouse bone marrow reticulocytes was efficiently suppressed by 5-MR treatment. The antimutagenic activity of 5-MR can be explained by inhibition of free-radical processes.

The effects of the hydroxylation product 3,4-benzo(a)pyrene and the free radical scavenger 1,2,3-trioxybenzene on cytochrome P-450 degradation in isolated rat hepatocytes induced by the Fe2+-ADP + NADPH system activating lipid peroxidation (LPO) were investigated. During incubation of hepatocytes, cytochrome P-450 is destroyed due to accumulation of LPO products. Addition of the free radical scavenger 1,2,3-trioxybenzene and the monoxygenase substrate 3,4-benzo(a)pyrene to the incubation medium induces inhibition of LPO and simultaneous stabilization of cytochrome P-450. Deceleration of malonic dialdehyde production by the free radical scavenger of the monoxygenase substrate suggests that both the compounds stabilize cytochrome P-450. It is assumed that in liver hepatocytes, exogenous free radical scavengers of the phenolic type and the products of their decarboxylation protect cytochrome P-450 against the LPO-induced destruction via oxidative metabolism of hydrophobic substrates.

Participation of bone marrow cells in the production of IgM antibody forming cells (AFC) in the primary immune response to sheep red blood cells (SRBC) in C57Bl/6 and BDA/2 mice was studied. The animals of this line differed in sensitivity to preoral benz(a)pyren (BP) injection. After BP injection a toxical injury of bone marrow cells was observed for two days in DBA 2 mice but was not marked in C57Bl/6 mice. In the former it was followed by a 10-fold decrease of IgMAFC, while no profound changes were noticed in the immune response of the latter. A new model is offered for the evaluation of bone marrow cell participation. A suggestion is made concerning some connection of immunodepression in the bone marrow with the change of the stem hemopoietic precursor differentiation.

Two cytochrome P-448 fractions, B1 and B2, were isolated from liver microsomes of 3,4-benzpyrene-induced inbred C57Bl/6 mice, using chromatography on octyl-Sepharose CL-4B and on Whatman 52E. During subsequent chromatography on hydroxylapatite fraction B1 was separated into 2 subfractions, G1 and G2. Cytochrome fractions B1, G1 and G2 have similar "peptide maps" differing from that of fraction B2. Cytochrome fraction B1 is immunologically identical to G2, partly to fraction B2 but is distinct from fraction G1. Fraction G2 is identified as the form of cytochrome P-448 catalyzing the hydroxylation of 3,4-benzpyrene and 7-ethoxyresorufin and existing in a low spin form. Cytochrome fraction G1 is apparently identical to the form P3-450. Fraction B2 was not yet described in current literature, since cytochrome P-448 (Mr = 53,000 Da) was identified only after the induction of mice with 3,4-benzpyrene but not with other inducers, e.g., polycyclic aromatic hydrocarbons.