This study indicated the presence in the molecular elements of the biological clock of a domain capable of binding heme, which when combined with carbon monoxide gas produces a biological effect. Additionally, the possible participation of carbon monoxide in regulating the cyclic functions of the biological clock mechanism demonstrated that carbon monoxide is released into venous blood flowing away from the eye in amounts dependent on diurnal and seasonal changes in environmental light .
Analysis of the results showed that elevated endogenous CO levels through blood irradiation induce numerous changes in the functioning of the main biological clock in the POA, as well as in parts of the DH. 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 PER1-2, CRY1-2, REV-ERB α-β and ROR β are not the same for both POA and DH structures, indicating that both structures respond differently to the received humoral signal.
Additionally, the DH functioning (expression) pattern of clock elements should be considered a characteristic of the "peripheral clock", which tunes to the main pacemaker in POA. The aforementioned factors are known to be elements of a positive regulatory loop that stimulate the expression of PER and CRY genes, the main clock components . Under physiological conditions, their expression varies throughout the day (oscillation), particularly concerning the bmal1 gene [3, 18]. The reductions in the expression levels of the transcription factors in question in both experimental groups obtained in the present study indicate the influence of CO on this process. It is difficult to indicate which element of the regulatory loop was blocked, and CO probably affected the PER2 protein. This protein has been shown to positively influence bmal1 expression, and PAS domains with heme prosthetic groups in its structure react with carbon monoxide [19–21].
Although our study showed no significant changes in the expression of the PER2 gene under the influence of CO and only some samples showed significant changes in this expression, it cannot be excluded that the resulting PER2 protein could bind to the CO molecule via the heme group and exert a physiological effect in the form of a lack of positive effects on the expression of the bmal1 gene contrary to the cited literature data [3, 22, 23]. Furthermore, the presence of CRY1 and 2 overexpression had some importance. The CRY protein, through the photolyase homology domain, binds to the CRY-binding domain on the PER2 protein to form a repressive complex that inhibits CLOK:BMAL1 by displacing from promoters under physiological conditions , but when PER proteins are blocked, the correct repressive complex may not form. Displacement of the TF dimer from promoters does not occur, and expression is induced; hence, the emerging overexpression of clock elements regulated by BMAL1/CLOCK or BMAL1/NPAS2 TF heterodimers.
Another explanation could be the increased NR1D2 expression compared to controls observed in both experimental groups. NR1D and especially NR1D1 are known to negatively regulate the expression of BMAL1 and CLOCK [25, 26]. However, silencing the expression of these nuclear receptors does not result in the disappearance of bmal1 or per gene oscillations . In addition, such a situation would be unclear because NR1D1 also contains PAS domains and reacts with CO [28, 29].
Experimental group animals showed high CRY1 and CRY2 expression levels. The protein product of the CRY gene is known to be the only factor able to inhibit the function of the BMAL1/CLOK dimer attached to the promoter. This occurs through the attachment of CRY-binding domains to CLOCK and to BMAL1 . In the animals of this group, the CO levels were elevated through the action of natural light on the blood, and CO is assumed to have been produced endogenously. The effect of hyperstimulation of PER1 expression was probably already inhibited by CRY proteins. Additionally, the expression of both nuclear receptors whose proteins inhibit the expression of the mentioned transcription factors increased in group II. Increased expression levels of both CRY1 and CRY2 in the animals of this group confirm a more rapid response of the prelimbic part of the hypothalamus to the experimental conditions in comparison with the control group. Due to the specificity of CRY1 gene expression, it always occurs later than other clock genes with a twelve-hour delay after transactivation with the BMAL1/CLOCK dimer. Thus, the elevated expression of CRY1 in POA and DH and CRY2 only in POA indicates an advanced response of clock elements to carbon monoxide. High CRY1 expression levels are important for maintaining the oscillation of clock elements and allowing them to be enhanced , which may have been the case in our experiment. After analysing the protein expression of transcription factors, we noticed that the gene expression of BMAL1, CLOCK and NPAS2 in both examined structures decreased in comparison to the control group. However, the analysis of the protein levels in the same samples showed that in POA, the levels of the constitutive transcription factors BMAL1 and CLOCK decreased in winter but increased in summer in comparison to the control group. This situation may be related to CO availability because levels in winter are naturally three times lower than those in summer when the length of the day and the intensity of additional light  irradiation increase the amount of CO (but only to the physiological level obtained in summer). However, such an amount accompanied by a general deficit inhibits not only gene expression but also translation. In summer, additional irradiation also increases the CO level approximately three times above the physiological level, which probably blocks the possibility of dimerization and the dissociation of dimers from the promoter, additionally increasing the amount of detected CLOCK and BMAL1 proteins. In contrast, we observed an increase in the amount of NPAS2 protein in both winter and summer, but gene expression was downregulated relative to the controls. This pattern of expression is difficult to explain, but all the presented transcription factors are likely blocked from dimerization by CO through haem groups contained in their structure. For example, CO concentration in the cellular environment has been shown to affect the formation of the NPAS2:BMAL1 protein dimer, which acts as a transcription factor in the master biological clock mechanism located in the prelimbic area of the hypothalamus (cited in ). An increase in CO concentration over 1 µM caused a breakdown of this complex . In the case of the results from the dorsal part of the hypothalamus, there was also a clear decrease in the expression of the clock transcription factor genes, whereas in the case of the protein, the changes in expression were irregular and the opposite of those obtained in the prelimbic part. Higher protein expression was observed in winter in all the factors studied (BMAL1, CLOCK, NPAS2). In contrast, the expression was statistically significantly lower in summer. This situation may be influenced by the CO-altered output from the master clock from the POA and the influence of CO itself, which reached these structures via the humoral pathway and directly affected transcription and translation.
The mechanism of the regulatory effect of carbon monoxide on biological clock components is unclear. NR1D receptor activity is known to be more strongly inhibited by NO than by CO, whereas CO exhibits only 15% NO activity . Thus, it seems impossible for CO to significantly affect the function of NR1D proteins. Our results support this concept, as NR1D expression levels were higher compared to controls, assuming that the body, in an attempt to compensate for the effect of CO blocking these receptors, increased their expression. In group II, where CO was assumed to be more available, there was an increase in the expression of both nuclear receptors, and the effect of their blocking transcription factors was also evident. This is favoured by the appearance of mRNA for both NR1Ds immediately after their expression was activated by the BMAL1/CLOCK dimer [29, 33].
It is unclear why the expression of Per and Cry and NR1D genes increased when the expression level of transcription factors of the mentioned genes drastically decreased. Similarly, Zhang et al. and Baggs et al. [34, 35] noticed an increase in the expression of PER1, CRY1 and CRY2 after BMAL1 expression was silenced using siRNA. They suggested that the expression may be activated by BMAL2 or the complexity of the posttranslational processing step of the BMAL1, CLOCK or NPAS2 gene products, as their expression was not silenced completely but drastically reduced. The same is also true for the PER2 gene, as its expression was unaffected despite the disruption of BMAL1 gene expression by REV-ERB α and β (also called NR1D1 and 2) . Other authors have demonstrated that BMAL1 can be effectively replaced by BMAL2. However, the constitutive expression of BMAL2, which is constant over the course of a day compared to BMAL1 , is also subject to transfection by the BMAL1/CLOCK dimer, and downregulation of BMAL1 expression results in the downregulation of BMAL2 expression . This situation suggests that as gene expression decreases, especially for such essential genes, assuming no mutation is present that precludes the correct sequence of events (transcription/translation), translation efficiency increases or protein ubiquitination levels decrease to maintain system function. The short lifespan of mRNA also suggests that a reduction in expression levels may be compensated for by an increase in translational efficiency.