ASSESSMENT OF THE VALUE OF METHYLATION FEATURES IN DIFFERENT TISSUES FOR PREOPERATIVE IDENTIFICATION OF HIGH-RISK BREAST TUMORS.

Epigenetic changes in breast cancer patients have long been believed to be a promising marker for clinical management - early detection, prognostication, etc. Various approaches are available to test global DNA or target gene methylation status. We used some of them in different tissues of patients with breast cancer to analyze possible links between epigenetic parameters and their value for predicting clinicopathological characteristics (most importantly stage and lymph node status) of tumors. Patients with ductal and lobular invasive carcinoma were included in the study along with age-matched controls. Blood, tumor tissue and normal breast ductal epithelial cells were investigated using MS-HRM technique (for BRCA1 gene promoter methylation assessment), COBRA-PCR (for Alu element methylation quantification) and ELISA-based method (for global DNA methylation estimation). These parameters were analyzed in comparison to clinical, histologic and phenotypic characteristics of the tumors. BRCA1 promoter methylation was detected exclusively in tumor tissues and in only two cases, both of which had aggressive phenotype. Alu and global DNA methylation showed markedly low levels in all tissues of cancer patients compared to healthy controls, with extreme hypomethylation in tumor tissue. Changes in these two parameters were not always concordant. Methylation levels detected in blood showed rather weak relationship with clinicopathological characteristics of tumors. We could conclude that tested parameters are not useful for preoperative determination of tumor stage or lymph node status, neither any other clinicopathological characteristics of tumors. Epigenetics signatures of different tissues of the same patient are not homogenous. Given the uniform picture of DNA methylation in blood in some of investigated groups, additional data and very careful approach are required when considering using it as a diagnostic or predictive tool.

LINE-1 METHYLATION IN BLOOD AND TISSUES OF PATIENTS WITH BREAST CANCER.

Methylation is an epigenetic alteration proved to be involved in many disease processes including cancer. This change affects mainly gene promoters and repetitive sequences in genome. Long Interspersed Nuclear Element-1 (LINE-1) is a family of retrotransposons - repetitive elements that modify gene activity and can themselves be targeted by epigenetic mechanisms. LINE-1 methylation level is a surrogate marker for global methylation. In many conditions this parameter is found to be altered not only in affected cell groups, but also throughout other tissues. The aim of our study was to compare LINE-1 methylation pattern in DNA extracted from blood of the patients with benign and malignant breast tissue. In addition, we investigated correlation of LINE-1 methylation in blood and tissues of same patients and relationship of all variables with histopathologic and phenotypic characteristics of tumors. Patients with biopsy-proved ductal invasive carcinoma of breast and no preoperative chemo/radiotherapy were chosen for the study group. Another pool of patients with various benign breast lesions represented controls. Blood samples from both group members were collected preoperatively. Tumor tissue sections were processed for pathology report and part of remaining tissue was used for methylation study. LINE-1 methylation level was quantified using ELISA-based assay. It was analyzed in combination with histologic and phenotypic tumor parameters and compared between different tissues and different study groups. LINE-1 was found to be significantly hypomethylated in breast cancer tissue compared to blood. Blood samples of patients with malignant tumors showed slightly lower methylation level, than samples obtained from control group members. Lymphovascular invasion was the only aggressiveness-determining factor that was found to be at least weakly correlated with LINE-1 hypomethylation in blood. We can conclude, that global hypomethylation measured by LINE-1 methylation level is significant in tumor tissue. But there is no significant difference between LINE-1 methylation levels in blood of patients with benign and malignant breast tumors; therefor LINE-1 hypomethylation in blood cannot be used as a marker for early tumor detection. Neither is it valid for determination of tumor behavior.

ALTERATION IN THE CYTOKINE SECRETION PATTERN IN T CELLS OF PATIENTS WITH CYSTIC FIBROSIS CAUSED BY DNA METHYLTRANSFERASE INHIBITOR 5-AZACITIDINE.

Cystic fibrosis (CF) is the autosomal-recessive disorder caused by mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR). The Airway inflammation plays a central role in the progression of CF disease. Cystic fibrosis characterized by the overproduction of the pro-inflammatory cytokines and reduced expression of anti-inflammatory cytokines. Although the mechanisms of abnormal cytokine expression is still poorly understood, altered epigenetic regulations in T cells might contribute. In the present study we examined the expression of IFN-γ and IL-10 by CF T cells prior to and following 5-azaC treatment. In addition we investigated DNMTs levels in nuclear extracts of CD4+ T cells derived from CF and non-CF individuals. Seven CF patients (age: 5-12 years) were included in the study and compared to six age-matched healthy subjects (age: 6- 13 years). CD4+ T cells were isolated from PBMC using CD4 MicroBead kit (Miltenyi Biotec GmbH) and were cultured in RPMI 1640 medium at 37°C with 5% CO2, in presence or absence of 5-azacytidine. Concentrations of IL-10 and γ-INF in CD4+ T Cells were measured by ELISA (eBoiscience, san Diego, CA, USA). In our study we showed that 5 Azacytidine alters nuclear levels of DNMT 3a as well as modulates cytokine levels in CD4+ T cells derived from CF patients. After 5-azaC treatment secretion of IFN-γ was significantly decreased in CF T cells, while amount of IL-10 was elevated by ~2.5 times compared to untreated controls (P<0.05). In summary, data presented in this report demonstrates that epigenetic mechanisms such as DNA methylation may be considered as a one of the potential therapeutic target in a treatment of Cystic Fibrosis.

MTHFR GENE C677T POLYMORPHISM AND LEVELS OF DNA METHYLTRASFERASES IN SUBCLINICAL HYPOTHYROIDISM.

The aim of our study was to investigate the link between MTHFR gene C677T polymorphism and DNMTs levels in patients with Subclinical Hypothyroidism (SCH). In this study 19 adult patients with subclinical hypothyroidism and 19 healthy controls (mean age 31±5.5 and 33±5.1 years respectively) were recruited. All patients were diagnosed based on serum levels of TSH, FT4, anti-TG and anti-TPO antibodies. Written informed consents were obtained from all study subjects. Genomic DNA was extracted using Quick-DNA Universal Kit (Zymo Research, USA). The MTHFR C677T polymorphism was genotyped by PCR-RFLP method. Levels of DNMT1 and 3a were measured in nuclear extracts of PBMC using DNMTs assay kits (Abcam). Our data indicates that the frequency of genotypes and alleles were different among the patient and the control group. There is a significant increase in CC genotype distribution in the control group when compared to the SCH patient group, while the CT as well as TT genotype distribution were not increased significantly in SCH group versus control group. However the C allele is significantly prevalent in the control group compared to the SCH group, while T allele is prevalent in patients compared to the control group with a statically significant difference. In addition, individuals with TT and CT genotypes and hypothyroidism showed elevated amount of DNMT3a in nuclear extracts of PBMC compared with controls, while no significant difference in DNMT1 levels was observed. This study indicates the MTHFR C677T variant may contribute in alteration of epigenetic regulation such as DNA methylation mediated by DNA methyltransferases in patients with subclinical hypothyroidism and also, carriers of the T allele might have an increasing risk of developing SCH.

NON-STEROIDAL ANTI-INFLAMMATORY DRUGS'S ANTINOCICEPTION MEDIATED BY THE OPIOID MECHANISM IN THE NUCLEUS RAPHE MAGNUS.

It has been established that the midbrain periaqueductal gray matter (PAG) and rostral ventro-medial medulla (RVM) are involved in the descending pain control system. The latter involves the midline nucleus raphe magnus (NRM) and adjacent reticular formation. These brain structures are is one of important parts of CNS circuit that controls nociceptive transmission at the level of spinal cord. Here we report that microinjection of commonly used non-steroidal anti-inflammatory drugs (NSAIDs), diclofenac, ketorolac, metamizol, and xefocam into the NRM produces strong antinociception which is mediated by the opioid mechanism. The experiments were carried out on experimental and control (saline) white albino male rats. Animals were implanted with a guide cannula in the NRM and tested for antinociception following microinjection of NSAIDs into the NRM in the tail flick (TF) and hot plate (HP) tests. The analysis of variance (ANOVA) with post-hoc Tukey-Kramer multiple comparison tests were used for statistical evaluation. The obtained data show that microinjection of these NSAIDs into the NRM produced antinociception as revealed by a latency increase in the tail-flick (TF) and hot plate (HP) latencies compared to the saline control microinjected into the same nucleus. Furthermore, we definitely showed that pre-treatment with opioid antagonist naloxone in the NRM diminishes NSAID-induced antinociception expressing in significant decrease in TF and HP latencies (P<0.001). The present findings support the concept that antinociceptive effects of NSAIDs are mediated via an endogenous opioid system possibly involving the descending pain modulatory circuit.

A STUDY OF THE RELATIONSHIP BETWEEN LEVELS OF METHYLTRANSFERASES IN PERIPHERAL BLOOD MONONUCLEAR CELLS AND CHARACTERISTICS OF TUMOR IN PATIENTS WITH DUCTAL INVASIVE CARCINOMA OF BREAST.

The role of epigenetics in tumor development and progression is actively being studied. The aim of our current pilot study is to analyze correlation of changes in the levels of methyltransferases in nuclear extracts of blood cells with some morphological and phenotypic characteristics of breast cancer. The levels of DNMT1, DNMT3a and H3K4 methyltransferase were measured. The results showed that the level of DNMT1 was highest in the control group but correlation with the tumor grade was just moderate. DNMT3a was found in highest level in Grade III cancer group, followed by Grade II and Grade I groups. Correlation of DNMT1 level with tumor grade was moderate. An opposite pattern was seen for H3K4 methyltransferase. DNMT3a level was higher in larger tumors, while the level of H3K4 methyltransferase was lowest in large tumors with significant negative correlation with the tumor size. This primary study shows that there are some changes in methyltransferase levels in PBMC from breast cancer patients. These changes are most probably attributed to modification of initiation as well as sustainment of methylated status of products.

Antinociceptive tolerance to non-steroidal anti-inflammatory drugs microinjected into dorsal hippocampus of rats is due to pharmacological tolerance.

Pain is characterized as a complex experience, dependent not only on the regulation of nociceptive sensory systems, but also on the activation of mechanisms that control emotional processes in limbic brain areas such as the amygdala and the hippocampus. We have recently found that repeated microinjection of non-steroidal anti-inflammatory drugs (NSAIDs) into the dorsal hippocampus of rats for four consecutive days induces antinociceptive tolerance as revealed by a progressive decrease of the latency in the tail-flick and hot plate tests compared to controls treated with saline into the dorsal hippocampus. Here we found that on the first day microinjection of NSAIDs, ketorolac, clodifen and xefocam into the DH produced antinociception as revealed by a latency increase in the TF and HP compared to the baseline control of intact rats and a control group with saline microinjected into the same site as well. Subsequent NSAIDs microinjections, without testing on the second and third days, caused progressively less antinociception, i.e. developed tolerance. After two days resting, by day 7 antinociception was almost completely restored for all the three drugs. Thus we demonstrated that this antinociceptive tolerance is due to pharmacological tolerance to these drugs and not to conditioning by repeating testing or hyperalgesia or other nonspecific mechanisms.

Epigenetic regulation of acute inflammatory pain.

Acute pain is associated with tissue damage, which results in the release of inflammatory mediators. Recent studies point to the involvement of epigenetic mechanisms (DNA methylation) in the development of pain. We have found that during acute inflammatory pain induced by the application of 10% mustard oil on the tongues of rats, levels of DNMT3a and 3b were elevated markedly (36 and 42 % respectively), whereas the level of DNMT1 was not changed significantly. Previous injection of Xefocam with 0,4 mg/kg dose decreased levels of DNMT3a and 3b (25 and 24% respectively). The level of DNMT1 was not changed significantly compared to the control group. The findings support the idea that inhibitors of DNA-methyltransferases could be useful for pain management. Our data suggest that NSAIDs (alone or in combination with DNMT inhibitors) may be proposed as possible epigenetic regulatory agents, which may play a role in epigenetic mechanisms indirectly through altering the activity of inflammatory mediators involved in pain development.

Expression pattern of DNA-methyltransferases and its health implication (short review).

Epigenetics is heritable and reversible alterations of gene expression without direct alteration of DNA sequences. One example of epigenetic factors is DNA methylation, which prevents certain genes from being expressed. Another example is histone modifications. In addition, miRNAs can silence genes at transcriptional and posttranscriptional level. DNA methylation is regulated by DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b). Aberrant DNMTs expression is the dominant mechanism for the genome instability which associates with a wide range of diseases such as a cancer, autoimmune diseases, mental disorders. In this article we reviewed the major mechanisms of changes of DNA methylation regulated by DNMTs and the role of this changes in pathogenesis of various diseases. In addition we briefly reviewed epigenetic agents, such as inhibitors of DNA methyltransferases or HDAC (histone deacetylase) targeting oncology, hematology, immunology, and neurologic disease indications, and which are in various phases of study or have been clinically tested and approved by FDA (Food and Drug Administration).

Opioid sensitivity of nucleus raphe magnus ater analgesia by nonsteroidal anti-inflammatory drugs.

Our recent investigations have shown that microinjection of three non-steroidal anti-inflammatory drugs (NSAIDs) analgin, ketorolac and xefocam into the central nucleus of amygdala produce tolerance to these drugs and cross-tolerance to morphine. We have observed the same phenomenon in midbrain periaqueductal grey matter and nucleus raphe magnus. The medullar nucleus raphe magnus (NRM) is one of important parts of CNS circuit that controls nociceptive transmission at the level of spinal cord. It is functionally involved in descending pain modulation, and mainly consists of serotoninergic neurons. The aim of this study was to examine opioid sensitivity of NSAIDs action in NRM of male rats. For this purpose 30 minutes later of NSAIDs administrations we microinjected µ-opioid antagonist naloxone and tested rats for tail flick and hot plate latencies. Our investigation showed that microinjection of naloxone in NRM significantly decreased antinociceptive effects of NSAIDs at the first day in the TF and HP latencies. At the second day, naloxone generally had trend effects in both TF and HP tests. These results strongly support the suggestion on endogenous opioid involvement in NSAIDs antinociception and tolerance. On the other hand, our evidence confirms once more that NRM is involved in the descending pain control circuit inhibiting spinal nocifensive reflexes.

Tolerance induced by non-opioid analgesic microinjections into rat's periaqueductal gray and nucleus raphe.

Several lines of investigations have shown that the microinjection of non-opioid analgesics, non-steroidal anti-inflammatory drugs (NSAIDs) in the midbrain periaqueductal gray matter (PAG) induces antinociception with some effects of tolerance. Our recent findings also have shown the same effects of tolerance in intraperitoneal (i.p.) injections of analgin (metamizol), ketorolac, and xefocam. Moreover, just recently, we have shown that microinjection of three NSAIDs analgin, ketorolac and xefocam into the central nucleus of amygdala produces tolerance to these drugs and cross-tolerance to morphine. The present study was designed to examine whether together with analgin, microinjection of another type of NSAIDs clodifen, ketorolac and xefocam into the PAG and the nucleus raphe magnus (NRM) leads to the development of tolerance in male rats. The experiments were carried out on experimental and control (with saline) white male rats by the models of tail-flick (TF, to the stimulation of focusing light beam) and hot plate (HP, paw withdrawal) tests. For microinjections of NSAIDs stainless steel guide cannula was implanted into the PAG and NRM by the stereotaxic atlas. Latency increase of these reflexes indicated the degree of antinociception. Analysis of variance with post-hoc Dunnet Multiple Comparison Test were used for statistical evaluations. Our study showed that microinjection of NSAIDs into the PAG produced antinociception as revealed by a latency increase in TF and HP compared to the baseline control with saline microinjected into the same nucleus. However, when these drugs microinjection subsequent testing also took place in the following days the antinociceptive effects progressively diminished so that on the 4th and especially the 5th experimental days the TF and HP latencies were similar to the averaged control baseline for rats that received repeated (5 days) injections of only saline. On the 5th day, one hour after of NSAIDs testing, experimental groups of rats received i.p. injections of mu-opioid antagonist naloxone and we did not reveal significant alterations in TP and HP latencies in non-opioid tolerant rats as well as in control animals. Microinjections of NSAIDs into the NRM also produced antinociception as revealed by a latency increase in TF and HP compared to the baseline control of as in intact rats so with saline microinjected ones into the same nucleus. Subsequent NSAIDs microinjections caused progressively less antinociception, so by day 4 there was no effect, similar to saline microinjections (baseline control) for both the TF and the HP tests, except analgin. The later did not show complete tolerance even on the 5th experimental day. Special control experiments showed that post-treatment with naloxone in RVM diminished NSAID-induced antinociception on the first and second experimental days and impeded the development of tolerance to the antinociceptive effect of NSAIDs. Obtained results underscore the strong convergence of antinociceptive mechanisms of opioids and non-opioids, particularly NSAIDs in the PAG-RVM downstream circuit in the acute effect of and the development of tolerance to both types of analgesics. On the other hand, our data confirm the results of other authors that NSAIDs are in close relation with endogenous opioids and the tolerance to these non-opioid drugs probably depends on opioid tolerance.

[Morphological structure of suprarenal glands in experimental vibration-induced pathology].

Technical progress has caused development of vibration-induced pathology, which is determined by harmful factors or environmental effects. The harmful factors include physical factors--noise, mechanical vibrations, low temperature, high humidity of the air and incorrect lighting. The aim of our study was the investigation of morphological changes in suprarenal glands under condition of vibration-induced pathology. The experiment was conducted on 20 grown-up white male rats weighting 180-200 g. The animals were daily under an hour vibration during 2 months. The vibration frequency was modulated by means of a general vibration. After an experiment, animals were decapitated in condition of general anesthesia. The experiment revealed important changes in the morphological structure of suprarenal glands. The vibration pathology causes following changes: vessels' and sinusoid capillaries' uneven widening, develop the infiltrate cells, bleeding areas, necrosis and other changes. Based on above-stated it is supposed that technical progress and introduction of new technologies is one of the risk factors, which can cause neurohumoral disorders.

Study of rat's emotional state in "conflict" situations.

The trials performed in the rats revealed that the creation of conflict situations, in the presence of two different motivations simultaneously, results in increase in anxiety state in animals. In terms of evolving and stabilizing the two active avoidance responses in animals, the three cohorts of different learning abilities (good, average and poor) were identified in parental, as well as in filial F1 and F2 generations. While influencing with stress-factors the percentage of rats having a good learning ability decreased, while the number of rats having a poor learning ability increased, respectively. The rising of emotional background was observed through the generations, which confirms that the creation of conflict situations, which affects only one third of the parental individuals as a stress factor, becomes a stress-agent for more and more rats in the further generations. Development of pathologic conditions in animals develops from the anxiety disorder, which further intensifies through the next generations.

[Influense of stimulation of Substancia Nigra and Nucleus Caudatus on postsynaptic responses of cortical nociceptive neurons].

The aim of the research was to investigate the influence of electric stimulation of Substancia Nigra and Nucleus Caudatus on postsynaptic processes, evoked in the nociceptive neuron of somatosensory cortex of cats. Intracellular recordings of electrical activity of the somatosensory cortex neurons in response to stimulation of the tooth pulp and of ventro-postero-medial (VPM) nucleus of thalamus, revealed the EPSP-peak-IPSP complex. Conditioning stimulation of Substancia Nigra or Nucleus Caudatus, which preceded the test-stimulation of the tooth pulp or VPM, with intervals of 100 to 700 ms, induced 40-50% decrease of IPSP only, in both nociceptive and convergent neurons. The maximal effect in both cases was achieved at an interval of 400-700 ms between conditioning- and test-stimuli. Probably these processes are realized through either pre- or postsynaptic mechanisms.

[The reaction of hormones of hypophysis-thyroid gland's endocrinal axis on vibration pathology and on condition of it's correction with liquid oxygen and anabolic steroids].

The experiment was held on white male rats. After the tests had been finished, by means of immunofermental method the concentration of thyroid (T3 and T4) and thyrotrophic (TSH) hormones were defined in the blood of animals in condition of vibropathology and it's correction with liquid oxygen and retabolil. The research showed, that the level of TSH, T3 and T4 decreased in condition of vibropathology and it's correction with anabolic steroid compare with control groups. But against of background liquid oxygen's injection the level of TSH increased, but the concentration of T3 and T4 were reduced compare with control group, though they were increased compare with the rest groups of experimental animals. As a result of the search, it was established, that the impact of vibration on the organism of male rats causes important changes in the hypophysis-thyroid gland's endocrinal axis, particularly it takes place the hypofunction of thyroid gland. And it's worth to mention that the correction of vibropathology with liquid oxygen cause positive influence and changes in the organism. There is a positively expressed normalization of thyroid and thyrotrophic hormones' concentration, but the correction of vibropathology with anabolic steroid has not been a successful one.

[The reaction of monoamines and sex hormones on vibration pathology and correction of this pathology with anabolic steroids].

The experiment was held on two groups of 40 grown-up white male rats. After the tests had bean finished by means of high-performance liquid chromatography method the quantity of monoamines (norepinephrine, serotonin and dopamine) was defined in hypothalamus, and the concentration of sex hormones (Follicle stimulating--FSH, Luteinizing hormone--LH, testosterone--T, estradiol--E(2)) was also defined by immunoferment method in the blood. The research showed that in both groups serotonin's level increased compared with the control group. In the first group its concentration increased to 147% and in the second one to 120%. As the rate of norepinephrine in the first group was increased by 41% against the background of anabolic steroid- -retabolil injections its concentration reduced to 90%. Its especially worth mentioning that as in the first case the concentration of dopamine was too reduced to 14% in the second case it made 70%. As a result of the research it was established that the concentration as T and E(2) as their regulator gonadotrophin hormones were decreased in the first group, but in condition of vibration pathology's correction with retabolil the rate of FSH and LH were within the norm, it follows that the rate of sexual steroids T and E(2) were within the norm.

[The effect of stimulation of the central gray substance on the neuronal activity of the trigeminal nerve nucleus]. / Vliianie razdrazheniia tsentral'nogo serogo veshchestva na aktivnost' neironov iadra troinichnogo nerva.

Stimulation of different central gray matter areas (CGM) has been estimated comparatively for its study on the neuronal activity of the trigeminal nucleus during nociceptive stimulation prior to and after the section of the medial brain stem structures between the CGM caudal part and the dorsal raphe nucleus. The possible role of the opiate and nonopiate systems in the mechanism of inhibition of the nociceptive afferentation has been also considered. Experimental data show that in the regulation of the nociceptive afferentation the CGM is a functionally nonuniform structure: in addition to the pain-suppressing system, there is also the pain-relieving system there: inhibitory influence of different CGM areas is accomplished by both the opiate and nonopiate system; the opiate inhibition is mediated by activation of neurons of the raphe nucleus, while the nonopiate one by recruitment of the lateral pathways or activation of the rostral structures of the brain.

[Effect of electroacupuncture on the activity of neurons in the central gray matter of the midbrain]. / Vliianie élektroakupunktury na aktivnost' neironov tsentral'nogo serogo veshchestva srednego mozga.

The effect of electroacupuncture in locally-segment and general analgetic points on background impulse activity of central gray substance neurons and their activity caused by nociceptive stimulation of the dental pulp, infraorbital nerve and forearm skin surface was studied in acute experiments on cats. It has been established that general analgetic points are better represented in the central gray substance, as compared to locally-segment points. Different degree involvement of central gray substance in the realization of acupuncture analgetic effect in different points is postulated. The role of dorsal and ventral compartments of the central gray substance in acupunctural analgesia is discussed.