Carbon Monoxide (CO) as a Retinal Regulator of Heme Oxygenases -1, and -2 (HO's) Expression.

Carbon monoxide (CO) has been proposed as a chemical light signal and neural system modulator via heme oxygenases -1 and -2 (HO-1 and HO-2). Many papers have proven the CO-HO circuit to be important for such physiological pathways as the molecular biological clock and the GnRH axis, but also in such pathological occurrences as ischemic injuries, or inflammation as a regenerative and neuroprotective factor. In this in vivo experiment, we used three groups of pigs: control-housed in natural conditions without any procedures; without CO-adapted and kept in constant darkness, infused with blank plasma; and with CO-adapted and kept in constant darkness infused with CO-enriched plasma. After the experiments, each animal was slaughtered and its eyes were collected for further analysis. Quantitative PCR and Western blot analysis were performed to show statistical differences in the expressions between the experimental groups. Our data revealed that exogenous CO is regulator of mRNA transcription for HO-1 and HO-2 and PCNA. Moreover, the mRNA abundance of analyzed factors in the experimental group after CO elevation revealed a restored gene-expression level similar to the control group, which we had observed in the group's restored protein level after CO elevation. In conclusion, exogenous CO regulates HO's and PCNA gene expression on transcriptional and translational levels in a similar way as a light cue.

Altered circadian dynamics of Per2 after cystathionine-ß-synthase and/or cystathionine-γ-lyase pharmacological inhibition in serum-shocked NIH-3T3 cells.

Circadian clock genes are found in almost every cell that has a nucleus; they regulate the rhythmic nature of all processes that are cyclical. Among the genes controlled by the circadian clock, there are numerous factors that regulate key processes in the functioning of the cell. Disturbances in the functioning of the circadian clock are associated with numerous disorders. A recent study has shown the key role of H2S in regulating circadian rhythm. In this study, we investigated the in vitro effect of pharmacological inhibition of cystathionine-ß-synthase (CBS) and/or cystathionine-γ-lyase (CSE) on the circadian dynamics of Per2 expression in serum-shocked NIH-3T3 cells. Alternatively, Cbs and Cse were knocked down by transfection with siRNA. The 48-h treatment of serum-shocked NIH-3T3 cells with 1 mM dl-propargylglycine (PAG), a specific CSE inhibitor, significantly decreased the amplitude and baseline expression of Per2. During exposure to an effective CBS and CSE inhibitor (aminooxyacetic acid [AOAA]), the amplitude of oscillation and baseline expression of Per2 significantly increased. Incubation of NIH-3T3 cells with both inhibitors also significantly increased the amplitude and baseline expression of Per2 messenger RNA (mRNA). siCbs or siCse knockdowan significantly reduced the baseline and amplitude of oscillation of Per2. In conclusion, we showed that CBS/CSE/H2S pathway participates in the regulation of the circadian clock system. PAG and AOAA, change the general expression and dynamics of Per2 genes, but the increase of amplitude and overall Per2 mRNA level due to exposure to AOAA is probably caused by factors other than CBS and CSE activity.

Melatonin concentration in peripheral blood and melatonin receptors (MT1 and MT2) in the testis and epididymis of male roe deer during active spermatogenesis.

Melatonin regulates male reproductive function in seasonal and non-seasonal breeder mammals. The presence of melatonin membrane receptors (MT1 and MT2) in the testis and epididymis has been demonstrated in several species. Wild roe deer are a short-day breeding species characterised by a short rutting season lasting from mid-July to mid-August. The aim of this study was to determine the concentration of melatonin in the peripheral blood and the presence of MT1 and MT2 receptors in the testis and epididymis in male roe deer during the pre-rut (May), rut (July/August) and post-rut (September) periods. The melatonin concentration was higher in May (522.50 ± 54.20 pg/mL) compared to July/August (258.50 ± 36.82 pg/mL; P < 0.05). During September, the melatonin concentration was higher (393.50 ± 36.77 pg/mL) than in July/August (P < 0.05) but lower than in May (P < 0.05). Immunohistochemical analysis showed the presence of MT1 and MT2 receptors in Leydig cells, Sertoli cells and germ cells in the testis, in addition to the epithelial cells of the epididymis caput, corpus and cauda. MT1 and MT2 receptor expression in the testis and epididymis, assessed by Western blot, was higher in May and July/August (when spermatogenic and steroidogenic activity restarts and reaches its peak, respectively) compared to September (when spermatogenic and steroidogenic activity decreases). This could indicate a stimulatory effect of melatonin on testicular (i.e., steroidogenesis and spermatogenesis) and epididymal (i.e., spermatozoa maturation) function in male roe deer through the MT1 and MT2 receptors. Our results form the basis for further studies into the detailed mechanism of action of melatonin through MT1 and MT2 receptors for optimal reproductive activity in male roe deer and other mammals.

Altered dynamics in the circadian oscillation of clock genes in serum-shocked NIH-3T3 cells by the treatment of GYY4137 or AOAA.

BACKGROUND AND PURPOSE: Several members of the core clock mechanism are equipped with a Per-Arnt-Sim (PAS) domain through which they can bind haem [Fe(II)]. Haem is a ligand for the orphan receptors REV-ERBα/ß (NR1D1/2), which regulate circadian rhythm and metabolism. The ability to bind haem sensitises these clock components to the action of small molecule gases, including NO, CO and H2S. Studies conducted with European hamsters revealed that during winter sleep, key clock genes stop oscillating. At the same time, H2S, when administered at subtoxic concentrations, can induce a hypometabolic state in the cell. We suppose that core clock components, including the nuclear receptors REV-ERBs, neuronal PAS domain protein 2 (nPAS2) and PER2, can be H2S targets. The general objective of this study was to investigate the effect of the H2S system on the expression profile of the core clock genes in cells in vitro. EXPERIMENTAL APPROACH: We analysed the expression of Per1, Per2, Per3, Bmal1, Cry1, Cry2, Nr1d1, Nfil-3 and Dbp messenger RNA (mRNA) in serum-shocked NIH-3T3 cells treated with a slow-releasing H2S donor (GYY4137) or the cystathionine beta-synthase (CBS) inhibitor (AOAA) cultured under constant darkness and collected during 3 days in 3 h interval. KEY RESULTS AND CONCLUSIONS AND IMPLICATIONS: We found that pharmacological CBS inhibition increased the general expression and dynamics of several clock genes. On the other hand, increased H2S decreased Per2 expression. These data suggest that CBS can affect circadian clock and effect on clock-controlled transcription output.

The seasonal changes of the heme oxygenase in the retina pig.

The eye is a very important organ in the human body which is affected by various external factors. One of these factors is the sunlight which can cause the visual impairment and as well as the increase in the oxidative stress. The heme oxygenase I (HO-1) plays a very important role in the fight against the oxidative stress. The HO enzyme catalyses the degradation of the heme to the ferrous iron, the biliverdin and the carbon monoxide (CO). The HO-2 is the isoform HO-1 and is mainly constitutively expressed. We have studied the changes in the HO-1 and the HO-2 in the retina on the level of the RNA and the protein in the summer and in the winter season (the biggest difference is in the length of the day light). The retina of the eye was obtained from the breeding pigs in concern (Sus scrofa f. domestica) posthumously. The expression of the HO-1 genes in the retina cells is higher in the winter and the amount of protein decreases. However, the HO enzyme concentration definitely increases in the summer, when the production of the free radicals (the oxidative stress) related to the exposition to the sunlight is greater. The obtained results suggest that various factors have the influence on the protein synthesis. One of the factors, can be the miRNA which blocks the synthesis of the HO. Another factors, influencing the HO are the biological clock, the sunlight and the UV radiation associated with it.

Extremely low frequency variable electromagnetic fields affect cancer and noncancerous cells in vitro differently: Preliminary study.

The exposure to extremely low frequency electromagnetic field (ELF-EMF) may result in various changes at the cellular level. To identify the effect of ELF-EMF exposure on viability of cells, cancer cells (U87-MG; 143B) and noncancerous cells (BJ; HEK) in exponential growth phase were exposed or sham-exposed to different values of frequency (2, 20, 30, 50 and 60 Hz), different shapes (sinusoidal, square and triangular) and time of exposure (0.5, 1, 2, 3 h) to electromagnetic field. After exposure, viability of cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). We found a different effect of exposition of cancer and noncancerous cells to ELF-EMF on viability of cells. This preliminary study revealed that electro magentic field(EMF) might serve as a potential tool for manipulating viability of cells.

Extremely low-frequency electromagnetic field (EMF) generates alterations in the synthesis and secretion of oestradiol-17ß (E2) in uterine tissues: An in vitro study.

An electromagnetic field (EMF) of extremely low frequency may affect physiological processes in mammals. The aim of the present study was to determine the effect of an EMF on the synthesis and secretion of oestradiol-17ß (E2) in the porcine uterus. Endometrial and myometrial slices were harvested on days 12-13 of the oestrous cycle and exposed in vitro to an EMF (50 and 120 Hz, 8 mT) for 2 and 4 h in the presence or absence of progesterone (P4). Subsequently, the incubation media were used to determine the concentration of E2 with RIA. Tissues fragments were used to study the expression of CYP19A3 mRNA using Real-Time PCR and the abundance of P450 aromatase using Western Blotting. The 50-Hz EMF increased E2 release from the endometrium and the myometrium at both time points of in vitro incubation. A 120-Hz EMF decreased the endometrial secretion of E2 after 2 h of incubation and did not affect E2 secretion after 4 h. In the myometrium, the 120-Hz EMF increased E2 secretion after 4 h of incubation. In P4-treated uterine fragments, no significant EMF exposition-related changes were observed. Only myometrial fragments incubated in the presence of P4 at 120-Hz EMF (4 h) released higher amounts of E2 due to EMF treatment. The 50-Hz EMF exposure did not change the CYP19A3 mRNA expression in endometrial fragments incubated in the presence or absence of P4. In myometrial fragments, the highest CYP19A3 mRNA expression was observed in fragments not exposed to the 50-Hz EMF and P4-treated tissues compared to that in fragments exposed to 50 Hz EMF and incubated with or without P4 and control (no EMF and no P4) fragments. The EMF at 120 Hz decreased basal endometrial CYP19A3 mRNA expression and did not change the expression in the P4-treated endometrium. In the myometrium, the EMF at 120 Hz increased CYP19A3 mRNA expression in slices incubated without P4 and had no effect in the presence of P4. The EMF exposure (50 and 120 Hz) did not affect P450 aromatase abundance in either the endometrium or the myometrium. In conclusion, the EMF induces changes in the synthesis and release of E2 in uterine tissues harvested during days 12-13 of the oestrous cycle. These changes are related to the EMF frequency used, the time of the exposition and the presence of P4. We suspect that this observed phenomenon might lead to changes in the intrauterine milieu of oestrogen, which is crucial for the proper activity of uterine tissues during the mid-luteal phase of the oestrous cycle.

Klotho protects human monocytes from LPS-induced immune impairment associated with immunosenescent-like phenotype.

In this study, we provide a new evidence on immunosenescent-like phenotype induction in low density monocytes due to the long-term treatment with lipopolysaccharide (LPS). We show that LPS caused oxidative and nitrosative stress through zinc downregulation and calcium accumulation. In turn, increased amounts of ROS/RNS and pro-inflammatory cytokines TNFα, IL-1ß, IL-6 led to the irreversible DNA damage, persistent DDR activation, proliferation inhibition, reduction in cell growth and immune impairment. Furthermore, we provide evidence that klotho reduced levels of ROS/RNS and pro-inflammatory cytokines as well as upregulated secretion of anti-inflammatory IL-10 in LPS-treated monocytes, thus the observed DNA damage was less severe, promptly and properly fixed and cells quickly resumed normal proliferation and maintained their immune functionality. Therefore, klotho protein could be considered as a protective factor against immunosenescent-like phenotype in monocytes an issue relevant to many immune disorders.

Long-term culture with lipopolysaccharide induces dose-dependent cytostatic and cytotoxic effects in THP-1 monocytes.

Monocytes act as a first line of defence against invading pathogens and their dysfunctions seem to be a key factor in many immune disorders. However, the data on mechanisms underlying these dysfunctions remain elusive. In this study, we evaluated the effects of long-term (168h) lipopolysaccharide exposure on monocytes at low density cultures (1×105cells/ml). Treatment with low dose LPS (≤5µg/ml) resulted in oxidative stress induction followed by p21 pathway activation, permanent cell cycle arrest and SASP development. Furthermore, high dose LPS (≥10µg/ml) induced cell death involving mitochondrial pathways, death receptors as well as p21-dependent DNA damage response activation mediated by ROS generation and TNF-α release. Additionally, exposure to high dose of LPS resulted in THP-1 monocytes differentiation to macrophages. In conclusion, long-term culture with LPS exerts in low density monocytes cytostatic/cytotoxic effects in a dose-dependent manner by inducing senescence associated with chronic inflammation at low doses and initiation of cell death at higher doses. These findings shed new light on understanding of monocytes dysfunction, an issue relevant to chronic inflammation and many immune disorders.

Retinal venous blood carbon monoxide response to bright light in male pigs: A preliminary study.

The physical mechanism by which light is absorbed in the eye and has antidepressant and energizing effects in Seasonal Affective Disorder and other forms of psychiatric major depression is of scientific interest. This study was designed to explore one specific aspect of a proposed humoral phototransduction mechanism, namely that carbon monoxide (CO) levels increase in retinal venous blood in response to bright light. Eleven mature male pigs approximately six months of age were kept for 7days in darkness and fasted for 12h prior to surgery. Following mild sedation, anesthesia was induced. Silastic catheters were inserted into the dorsal nasal vein through the angular vein of the eye to reach the ophthalmic sinus, from which venous blood outflowing from the eye area was collected. The animals were exposed to 5000lx of fluorescent-generated white light. CO levels in the blood were analyzed by gas chromatography before and after 80min of light exposure. At baseline, mean CO levels in the retinal venous blood were 0.43±0.05(SE)nmol/ml. After bright light, mean CO levels increased to 0.54±0.06nmol/ml (two-tailed t-test p<0.05). This study provides preliminary mammalian evidence that acute bright light exposure raises carbon monoxide levels in ophthalmic venous blood.