Endogenous carbon monoxide (CO) as a modulating factor of molecular biological clock in the hypothalamic structures involved in light transmission in pig and wild boar hybrid during long and short day season.

Mature males of a wild boar-pig crossbreed, during the long and short day season, were used for the study which demonstrates that the chemical light carrier CO regulates the expression of biological clock genes in the hypothalamus via humoral pathways. Autologous blood with experimentally elevated concentrations of endogenous CO (using lamps with white light-emitting diodes) was infused into the ophthalmic venous sinus via the right dorsal nasal vein. Molecular biology methods: qPCR and Western Blot were used to determine the expression of genes and biological clock proteins. The results showed that elevated endogenous CO levels, through blood irradiation, induces changes in genes expression involved in the functioning of the main biological clock located in suprachiasmatic nuclei. Changes in the expression of the transcription factors Bmal1, Clock and Npas2 have a similar pattern in both structures, where a very large decrease in gene expression was shown after exposure to elevated endogenous CO levels. The changes in the gene expression of PER 1-2, CRY 1-2, and REV-ERB α-ß and ROR ß are not the same for both POA and DH hypothalamic structures, indicating that both structures respond differently to the humoral signal received. The results indicate that CO is a chemical light molecule whose production in an organism depends on the amount of light. An adequate amount of light is an essential factor for the proper functioning of the main biological clock.

Carbon monoxide modulates melatonin synthesis in porcine pineal Cells in vitro: a preliminary study.

Two main isoforms of heme oxygenase (HO-1 and HO-2), the main enzyme of heme metabolism, were identified in the pineal gland. This suggests possible interactions between the melatonin synthesis pathway and the HO system. The aim of this study was to investigate the participation of carbon monoxide (CO), an HO by-product, on the melatonin synthesis pathway. Tests were carried using primary cell cultures of porcine pineal glands. The tricarbonyldichlororuthenium (II) dimer (CORM-2) compound was used as a CO donor at concentrations of 1 and 3 µM, as low concentrations of CORM-2 affect the regulation of the melatonin synthesis pathway in pineal cells in vitro. In addition, the presence of Sn-protoporphyrin-IX, an HO inhibitor, changed the melatonin response of pineal cells. These results suggest the existence of an intermediate mechanism in the pineal gland, which is associated with HO activity, that is involved in the modulation of melatonin synthesis.

Electromagnetic fields with frequencies of 5, 60 and 120 Hz affect the cell cycle and viability of human fibroblast BJ in vitro.

The impact of electromagnetic field (EMF) on humans has been described in numerous studies, but many questions are still unanswered. The aim of the experiment described in this study was to evaluate the effect of EMF on the viability of human fibroblast BJ in vitro and the percentage of cells in different phases of the cell cycle (G1/G0, S, G2/M) after 2 hours of exposure to sinusoidal continuous and pulsed EMFs with frequency of 5 Hz, 60 Hz and 120 Hz at a magnetic induction of 2,5 mT. The viability of BJ cells exposed to an EMF was estimated immediately after completion of exposure and after 24 hours. Metabolic activity of cells was assessed by MTT assay and compared to a control culture not exposed to EMFs. Cell cycle analysis was performed by BrdU incorporation. The analysis of the viability demonstrated significant differences in field efficiency, depending on its nature. Exposure of cells to pulse EMFs resulted in a decrease in their viability for each of the analyzed frequencies. Reduced viability was maintained for a further 24 hours after the end of exposure of cells to pulsed EMF. In the case of continuous field, reduced BJ cell viability was observed only at the highest applied frequency - 120Hz, and this effect maintained for the next 24 hours. Although there was no significant effect on cell viability (metabolic activity) of cells immediately after exposure to continuous EMF with a frequency of 5Hz, a significant increase was observed after 24 hours of incubation.

Changes in gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor gene expression after an increase in carbon monoxide concentration in the cavernous sinus of male wild boar and pig crossbread.

Previous studies indicate that there are at least a few regulatory systems involved in photoperiodic synchronisation of reproductive activity, which starts with the retina and ends at the gonadotropin-releasing hormone (GnRH) pulse generator. Recently we have shown indicated that the amount of carbon monoxide (CO) released from the eye into the ophthalmic venous blood depends on the intensity of sunlight. The aim of this study was to test whether changes in the concentration of carbon monoxide in the ophthalmic venous blood may modulate reproductive activity, as measured by changes in GnRH and GnRH receptor gene expression. The animal model used was mature male swine crossbred from wild boars and domestic sows (n = 48). We conducted in vivo experiments to determine the effect of increased CO concentrations in the cavernous sinus of the mammalian perihypophyseal vascular complex on gene expression of GnRH and GnRH receptors as well as serum luteinizing hormone (LH) levels. The experiments were performed during long photoperiod days near the summer solstice (second half of June) and short photoperiod days near the winter solstice (second half of December). These crossbred swine demonstrated a seasonally-dependent marked variation in GnRH and GnRH receptor gene expression and systemic LH levels in response to changes in CO concentration in ophthalmic venous blood. These results seem to confirm the hypothesis of humoral phototransduction as a mechanism for some of bright light's effects in animal chronobiology and the effect of CO on GnRH and GnRH receptor gene expression.

The impact of electromagnetic field at a frequency of 50 Hz and a magnetic induction of 2.5 mT on viability of pineal cells in vitro.

The impact of electromagnetic fields (EMF) on the pineal gland has been described in numerous studies, but many questions still remain unanswered. The aim of the experiment described in this study was to evaluate the effect of EMF on the viability of the pineal gland cells of pig in vitro. Primary culture of the pineal gland cells has been exposed to the influence of an EMF at a frequency of 50 Hz with 1, 2 or 3 hours and for 3 hours every 2 or 3 days. After the experiment, viability of cells was assessed by MTT assay and compared to a control culture not exposed to electromagnetic fields. We noticed that in respect to the control, exposure of the cells to the EMF induced a significant increase in viability of cells at 2 and 3 hours of exposure. After three days of 3-hour exposure to EMF, we observed a significant decrease in cell viability in relation to the control. The results of these studies suggest that EMF can have a significant biological effect on the cells of the pineal gland in a time-dependent exposure to its action.

Changes in melatonin synthesis parameters after carbon monoxide concentration increase in the cavernous sinus.

Previous studies indicate that the gaseous messenger carbon monoxide (CO) is released from the eye into the ophthalmic venous blood depending on the intensity of sunlight. This study was designed to determine whether the increased concentration of CO in ophthalmic venous blood affects the synthesis of melatonin and therefore, whether CO released from the eye under normal lighting conditions can be a carrier of light intensity information. Thirty six mature male wild boar and pig crossbreeds (n = 36) were studied. We measured the difference in the scotophase melatonin pathway response in terms of mean concentration of increased melatonin levels after 48 hours infusion of autologous blood plasma with an experimentally induced approximately 3-fold increase in the concentration of CO into the ophthalmic venous sinus. We demonstrated in this crossbreed a marked variation in the duration and amplitude of nocturnal melatonin peak in response to increased concentration of CO in ophthalmic venous blood. During the winter this treatment limited the nocturnal melatonin rise. During the summer this same experimental treatment enhanced the nocturnal melatonin rise. Changes in melatonin levels were always associated with parallel changes in AANAT protein levels. This work demonstrates that non-physiological changes in CO concentration in ophthalmic venous blood can have an acute impact on the systemic melatonin level. These results support humoral phototransduction as a mechanism for some of bright light's effects in animal chronobiology and treatment of winter seasonal affective disorder.

THE INFLUENCE OF CARBON MONOXIDE ON THE SECRETION OF MELATONIN BY PINEALOCYTES MEASURED IN VITRO.

Photoperiod is considered the most important factor entraining the circannual physiological rhythms through changing circadian patterns of melatonin (MEL) secretion from the pineal gland. The pineal gland of mammals does not respond directly to light but is controlled by light via neuronal phototransduction originating in the retina. In accordance with humoral phototransduction hypothesis, the aim of this study was to determine whether an increased concentration of CO, as a carrier of a light signal in pineal cell culture, affects the synthesis of melatonin. This study demonstrates that a commonly used carbon monoxide donor (CORM-2) markedly stimulated melatonin release from pineal cells incubated in vitro in a time-dependent manner, but the mechanism whereby CO modulates MEL release needs to be further explored.

Carbon monoxide-mediated humoral pathway for the transmission of light signal to the hypothalamus.

The gaseous messenger carbon monoxide (CO) is released from the eye into ophthalmic venous blood depending on the intensity of sunlight. Numerous neurohormones and other regulatory factors permeate from venous blood into arterial blood in the perihypophyseal vascular complex (PVC) and are transferred to the brain by the humoral pathway. This study was designed to determine whether elevated CO in ophthalmic venous blood (OphVB) affects the expression of clock genes and their transcriptional factors in the hypothalamus. Mature males of a wild boar and pig crossbreed (n=24) were used for the study. Autologous plasma with increased concentrations of CO was infused into the ophthalmic sinus (OphS) of the experimental group (n=12). The expression of clock genes (Per1, Per2, Cry1, Cry2, Rev-erb α and Rev-erb ß) and the genes of their regulators (Bmal1, Npas2, Clock, Ror ß) was estimated in two hypothalamic structures involved in the reception and transmission of light signal (the preoptic area (PA) and dorsal hypothalamus (DH)). We demonstrated that the expression of clock genes and the genes of their regulatory factors in the experimental group was altered compared with control, both in the PA and DH. The response to an increased concentration of CO differed between individual genes and the hypothalamic regions. The expression of Per1 which, according to many authors, is regulated by light, was increased in animals treated with CO both in the PA and DH, and regardless of the time of day and season. In conclusion, the current results seem to confirm the hypothesis on the function of CO in humoral transfer from the eye to structures related to the reception and transmission of light signal and the effect of CO on clock gene expression.