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Abstract

The aims of the study are to detect whether there are any possible effects of chronic carbon monoxide (CO) exposure on the argyrophilic nucleolar-organizing region (AgNOR)–associated protein synthesis and evaluate any possible relationship between the amount of AgNOR protein and the level of myocardial injury also and between AgNOR and histopathological evaluation methods. Adult male albino Wistar rats (n = 18) were randomly divided into three groups (groups A, B, and C). Group A served as control, while groups B and C were rats exposed to CO gas chronically (1000 and 3000 ppm CO concentration with a flow rate of 4 L/min for 30 min/day for 7 days, respectively). Total AgNOR area/nuclear area (TAA/NA) and the mean AgNOR numbers for each myocyte nucleus were determined. There were significant differences among all groups for TAA/NA ratio. These differences were not significant for mean AgNOR numbers. According to the histopathological evaluation scores, there were significant differences between the groups. The differences were significant among the groups for loss of sarcomere pattern. A strong positive correlation between histopathological injury scores and TAA/NA ratio was found (R_sq = 0.48; p = 0.002), however, the correlation was not significant for mean AgNOR numbers (R_sq = 0.08; p = 0.25). In conclusion, TAA/NA ratio can be used as an indicator for obtaining information about the level of myocardial damage instead of histopathological evaluation scores.

Keywords

Chronic carbon monoxide intoxication AgNOR myocardium

Introduction

Hazardous effects of carbon monoxide (CO) on human body have been known since the 19th century. Affinity of CO to hemoglobin (Hb) is 200–280 times greater than that of oxygen. CO gas binds to Hb rapidly, leading to the formation of carboxyhemoglobin (COHb). Then, the oxygen-carrying capacity of the blood decreases causing tissue hypoxia.^1

–5 It has been known that CO poisoning is the cause of more than half of the fatal poisonings in many countries. The number of deaths caused by CO poisoning is not exactly known. The deaths shown to be caused by CO poisoning are less than that they actually occur. In Turkey, especially in winter, CO poisoning as a result of the use of coal stoves, heaters, or grills in areas with inadequate ventilation is very common, and these patients often present to emergency departments.^1,6

CO poisoning causes hypoxia in the body, and heart is one of the organs most sensitive to hypoxia. CO gas binds to intracellular myoglobin in the myocardium and impairs the oxygen supply to the mitochondria. Patients with underlying cardiac conditions have a higher risk of death from arrhythmias, and fatal heart attacks may occur as a result of CO poisoning. Anginal chest pain attacks, arrhythmias, and increased level of cardiac markers frequently occur after CO exposure. Thus, it was thought that there should be morphological changes that could be related to CO, especially since the myocardial cells bind more CO gas than skeletal muscle cells. Ultramicroscopic lesions were shown, but the relative roles of general tissue hypoxia and specific CO toxicity are unknown.³

Chronic CO poisoning can be defined as CO poisoning resulting from recurrent, long-term exposure to small amounts of CO. Chronic CO exposure may be seen in many occupational groups such as traffic policemen, coal mine workers, heavy vehicle operators, mine workers, firefighters, and kitchen workers. It is usually difficult to diagnose CO poisoning in the case of chronic exposure, and it can generally be diagnosed after several emergency department admissions. Even though the COHb levels are low in chronic CO poisoning, clinical condition of the patient may be worse than that of acute CO poisoning patients with higher COHb levels. In the case of chronic intoxication, in addition to symptoms seen in acute poisoning, progressive neuropsychiatric symptoms and cognitive defects may develop.²

Nucleolar organizer regions (NORs) are loops of ribosomal DNA (rDNA), tandemly repeated with intergenic spacers and transcribed into ribosomal RNA (rRNA), which is processed into pre-ribosomes in the nucleoli, and finally become parts of mature ribosomes in the cytoplasm.^7,8 These structures are also frequently named as rRNA or rDNA gene family. NORs are functional subunits of the nucleolus in which actively transcribed rDNA is surrounded by different regulatory proteins.⁹

There are a lot of studies evaluating the importance of the interphase argyrophilic NOR (AgNOR) quantity in tumor pathology,^10
–12 hair root cells of humans,^13,14 developmental stages of infants with Down syndrome and healthy individuals,^15,16 possible effects of CO exposure on the NOR protein synthesis of lung cells,¹⁷ and acute CO exposure in heart cells.¹⁸

In the literature, there is only one study about the relationship between acute CO exposure and AgNOR proteins in the heart cells that was performed by us.¹⁸ To the best of our knowledge, there are no studies in the literature about the relationship between chronic CO exposure (rats were exposed to CO gas 7 times, each group at a different CO concentration with a flow rate of 4 L/min for 30 min a day for 7 days) and synthesis of AgNOR proteins in heart cells. Therefore, we carried out the current study to detect any possible effects of chronic CO exposure on the NOR protein synthesis of heart cells and to evaluate whether there is any relationship between the amount of AgNOR protein and the level of myocardial injury and between the AgNOR evaluation methods and histopathological evaluation methods used for the detection of myocardial damage caused by chronic CO exposure.

Methods

Adult male albino Wistar rats (n = 18), weighing 200–300 g, were used in our study. The study was approved by Atatürk University Animal Experiments Ethics Committee (18.12.2013/153), and it was in compliance with the internationally accepted principles for care and use of laboratory animals.

The rats were randomly divided into three groups consisting of six rats each:

Group A: Control group (exposed to ambient room air).

Group B: Exposed to CO gas at a low concentration (mixture of room air and 1000 ppm CO gas).

Group C: Exposed to CO gas at a high concentration (mixture of room air and 3000 ppm CO gas).

Two steel tubes, 10 L each (containing mixture of room air and 1000 ppm or 3000 ppm CO gas) were provided by Habaş Industrial and Medical Gases Production Industries Inc., Kocaeli, Turkey. Rats were exposed to CO in an enclosed transparent jar with dimensions of 20 × 40 × 60 cm³. There were two apertures (inlet and outlet) with 2 cm diameter at opposite sides of the jar. The experimental groups (Groups B and C) were exposed to CO gas chronically (each group at a different CO concentration (1000 and 3000 ppm) with a flow rate of 4 L/min for 30 min a day for 7 days in the transparent jar). After each exposure, they were removed to breathe room air. All the rats were housed in a standard cage in a quiet and temperature-controlled room (20 ± 2°C) in a 12-h light/12-h dark cycle, receiving food and water ad libitum.

AgNOR detection

After the 7th day, animals were anesthetized intraperitoneally with urethane at a dosage of 1.25 g/kg and killed with intracardiac perfusion. Fixation was achieved in a 10% formaldehyde solution. The heart tissue samples (with dimensions of approximately 1 × 1 × 1 cm³) were embedded in paraffin blocks. After routine histological examination, 5 µm thick sections were obtained from the paraffin blocks. These tissue sections were deparaffinized in xylene and then rehydrated in graded alcohol solutions. Then, the slides were air-dried at room temperature for 15 min and fixed in absolute methanol for 5 min. AgNOR staining method was performed according to the protocol followed by Benn and Perle¹⁹ and Lindner²⁰, with a slight modification for each slide containing stained tissue sections of all groups. The slides were viewed using a light microscope (Eclipse 80i, Nikon, Japan), and myocytes were photographed using a digital camera (Digital Sight DS-fi1, Nikon). Captured images were transferred to image processing software (ImageJ version 1.47t, National Institutes of Health, Bethesda, Maryland, USA), and the evaluation was carried out using the “freehand selections” tool. One hundred nuclei per animal were evaluated, and the ratio of total AgNOR area (TAA)/nuclear area (NA) and the mean AgNOR numbers for each myocyte nucleus were determined and noted.

Histopathological examination

After routine histological examination performed by an experienced pathologist, 5 µm thick slices were stained with hematoxylin and eosin (H&E) for light microscopic evaluation. The slides including myocytes were viewed using a light microscope (Eclipse 80i, Nikon), and myocytes were photographed using a digital camera (Digital Sight DS-fi1, Nikon).

Each specimen was scored for the degree of severity of histopathological changes as follows: myocardial fiber swelling and vacuolation (+1); myocytolysis/necrosis of myocardial fibers (+1); disorganization of myocardial fiber (+1); and when no damage is recorded (0), as it was done in the study performed by Saad et al.²¹ The above changes were judged as significant if seen in three or more high-power fields. In this study, we also evaluated the presence of hemorrhage and nuclear enlargement (nucleomegaly) evaluated in our previous study.¹⁸ In addition to standard histopathological evaluation, loss of sarcomere pattern which was found to be related to myocardial injury by Armstrong et al.²² was also evaluated in the current study.

Statistical analysis

Statistical Package for Social Sciences (SPSS, Inc., Chicago, Illinois, USA) for Windows 11.5 was used for statistical analysis. While evaluating the study data, in addition to descriptive statistical methods (mean, standard deviation (SD)), Kruskall–Wallis and Mann–Whitney U tests were used to compare groups. Statistical correlation between histopathological injury scores and both the TAA/NA ratio and mean AgNOR numbers in interphase nuclei of heart tissue cells were performed using Pearson correlation test, and correlation between loss of sarcomere pattern and TAA/NA ratio was performed by polynominal regression test. Results were given as mean ± SEM, median in 95% confidence interval, and p < 0.05 was considered to be statistically significant.

Results

AgNOR evaluation

There were significant differences among all groups for TAA/NA ratio (p = 0.01; Table 1). The differences between Group A (control group) and group B (mixture of room air and 1000 ppm CO gas) and group A and group C (mixture of room air and 3000 ppm CO gas) were significant (p = 0.037 and p = 0.004, respectively). Conversely, no significant differences were found between groups B and C for TAA/NA ratio (p = 0.337). For mean AgNOR numbers, there were no significant differences between the groups (p = 0.368; Table 1). Figure 1 shows the demonstrative examples of silver-stained NORs in the heart tissues samples of each groups.

Table 1.

TAA/NA ratio and mean AgNOR values of groups.

	Groups	N	Mean	SEM	Median	p
TAA/NA	A (Control)	6	0.0486	0.0023	0.0468	0.01
	B (1000 ppm)	6	0.0616	0.0053	0.0590
	C (3000 ppm)	6	0.0666	0.0018	0.0669
Mean AgNOR numbers	A (Control)	6	1.6644	0.0294	1.6552	>0.05
	B (1000 ppm)	6	1.7440	0.0984	1.7736
	C (3000 ppm)	6	1.7426	0.0459	1.7409

TAA: Total argyrophilic nucleolar-organizing region area; NA: nuclear area; AgNOR: argyrophilic nucleolar-organizing region; SEM: standard error of mean.

Figure 1.

Silver-stained NORs (arrows) in heart tissue cells: (a) control group, (b) 1000 ppm group, and (c) 3000 ppm group (×1000 magnification). NOR: nucleolar-organizing region.

Histopathological evaluation

When we took the histopathological evaluation scores into consideration, there were significant differences between the groups (p = 0.001). The differences between groups A and B, groups A and C, and groups B and C were significant (p = 0.003, p = 0.003, and p = 0.006, respectively). Figure 2 shows the demonstrative examples of H&E-stained heart tissue images, which were used for histopathological scoring.

Figure 2.

(a) Control group: cardiac myocytes with sarcomeres are seen, but no swelling, necrosis, or disorganization is seen. (H&E, ×1000); (b) 1000 ppm group: mild disorganization, cellular swelling, and focal sarcomere loss are seen in myocytes (H&E, ×1000; arrows: sarcomere loss); and (c) 3000 ppm group: myocytes with total loss of sarcomere pattern and severe disorganization are seen (H&E, ×1000; arrows: sarcomere loss).

When the groups were evaluated for loss of sarcomere pattern, the differences among all groups were significant (p < 0.0001). The differences between groups A and B, groups A and C, and groups B and C were significant (p = 0.001, p = 0.002, and p = 0.006, respectively).

Correlations

There was a strong positive correlation between histopathological scores and TAA/NA ratio (R_sq = 0.48; p = 0.002). However, the correlation between histopathological scores and mean AgNOR numbers per cell was not statistically significant (R_sq = 0.08; p = 0.25; Figure 3). A statistically significant positive correlation was seen between loss of sarcomere pattern and TAA/NA ratio (R_sq = 0.496; p = 0.006; Table 2, Figure 4).

Figure 3.

Correlation between histopathological scores and both TAA/NA ratio and mean AgNOR numbers. AgNOR: argyrophilic nucleolar-organizing region; TAA: total argyrophilic nucleolar-organizing region area; NA: nuclear area.

Table 2.

Model summary and parameter estimates for relationship between loss of sarcomere pattern and TAA/NA ratio.

Cubic	Model summary					Parameter estimates
Cubic	R ²	F	df1	df2	Significance	Constant	b1	b2	b3
	0.496	7.373	2	15	0.006	−9.607	249.058	0.000	−17,282.703

TAA: Total argyrophilic nucleolar-organizing region area; NA: nuclear area.

Figure 4.

Polynominal regression graphics for loss of sarcomere pattern and TAA/NA ratio. TAA: total argyrophilic nucleolar-organizing region area; NA: nuclear area.

Discussion

The current study shows that TAA/NA ratio increases, and histopathological changes related to myocardial damage was prominent in the groups exposed to CO gas. Besides, it was seen that TAA/NA ratio correlates well with the histopathological injury scores (in terms of both histopathological scores and loss of sarcomere pattern). These findings may be helpful in the process of diagnosing chronic CO poisoning when specially determining the exact cause of a sudden death of an otherwise healthy patient who was suspected to be exposed to CO gas.

Deterioration of cardiovascular functions is commonly encountered in CO poisoning because myocardium has an increased oxygen requirement and extracts a significant fraction of oxygen in the blood.^23,24 Moreover, oxygen-carrying capacity of myoglobin, which plays its role in the intracellular transport of oxygen, decreases after binding to CO, resulting in decreased oxygen transport to myocardial cells.^25,26 In addition to the effect of myocardial hypoxia, CO has a direct toxic effect on the heart mediated by reversible inhibition of mitochondrial respiration and augmentation of oxidative stress.²⁷ Myocardial dysfunction, left ventricular failure, acute myocardial ischemia, and arrhythmias causing permanent myocardial damage are commonly seen cardiovascular complications of CO poisoning. Myocardial injury which is commonly seen in CO poisoning increases the mortality rate. Chronic cardiotoxicity secondary to CO poisoning may not be clinically apparent and often remains undiagnosed since prominent symptoms may be lacking, and specific ischemic changes may not be evident.²³ Additionally, COHb levels may be normal in the case of chronic poisoning.² Thus, the diagnosis of CO cardiotoxicity becomes even more difficult. Hence, we hypothesized some new methods to confirm the diagnosis of chronic CO poisoning and conducted the current study.

It had been shown that TAA/NA ratio increases and a histopathologically evident damage occurs in tissues of heart and lung in the case of acute CO poisoning.^17,18 It was seen in the current study that chronic CO exposure also leads to histopathologically evident damage and increases TAA/NA ratio in cardiomyocytes. We suggest that the increase in TAA/NA ratio might be a result of “self-protective” responses of cardiomyocytes against CO poisoning.

In our previous studies,^17,18 we found out that the amount of AgNOR proteins increases depending on the level of CO exposure in the lung cells and in cardiac cells exposed to acute CO poisoning.

Histopathological scores include the evaluation of myocytolysis/necrosis of myocardial fibers, myocardial fiber vacuolation, myocardial fiber swelling and interstitial edema, and disorganization of myocardial fiber with or without fibroblastic proliferation. According to our results, there was a strong positive correlation between TAA/NA ratio and histopathological scores. Thus, it may be said that detection of TAA/NA ratio only may give information about the degree of myocardial damage and be used instead of histopathological evaluation scores in chronic CO poisoning.

Armstrong et al.²² showed that the loss of sarcomere pattern is related to myocardial injury. The differences among all groups were significant for the loss of sarcomere pattern in the current study, too. Besides, strong positive correlations were found between loss of sarcomere pattern and TAA/NA ratio. Therefore, it may be said that TAA/NA ratio can be used instead of the loss of sarcomere pattern as well. Thus, a significant time can be spared and unnecessary use of laboratory resources can be prevented.

Contrary to the findings of our previous studies,^17,18 we did not find significant differences between the groups for mean AgNOR numbers. Additionally, the correlation between the histopathological scores and mean AgNOR numbers per cell was not significant. We wonder when the cellular stress increases, as in the case of CO exposure, whether the AgNORs form clusters or overlap or stay as single dots. If they have such a behavior, would this behavior of the cells make them more resistant against dangerous agents? Also, would this behavior give information about the effects duration and degree of the stress? New studies are needed to obtain more certain knowledge about the topic.

Hypoxia caused by CO poisoning may cause cardiac dysfunction and lead to cardiac arrest. Increased AgNOR protein synthesis caused by CO exposure may have protective roles or trigger the synthesis of some other proteins that have similar functions in the heart cells. Therefore, these proteins may be used for the prevention of the cardiac dysfunction and cardiac arrest. In the future, new therapeutic approaches may be developed in the treatment of CO intoxication.

It was shown in the study of Mamaev et al. that AgNOR numbers rapidly decline in cardiomyocytes at the stage of total myocardial ischemia, followed by a rapid rise of cold chemical cardioplegia at the stage of reperfusion and myocardial rewarming.²⁸ Mamaev et al. in another study reported that mean AgNOR number per nucleus increases in patients with ischemic heart disease not complicated by severe heart failure; however, cases involving severe heart failure had progressively decreasing cardiomyocyte AgNORs. They suggested that the significant decline in AgNORs in cases with severe ischemic heart disease complicated with heart failure seems to be related to cardiomyocyte adaptation (a type of hibernation) to a decreased circulation.²⁹ The mean number of AgNORs per nucleus, the size of AgNORs, and the percentage of the area occupied by AgNORs in the nucleus in doxorubicin-treated rats were found to be increased in the study of Leblanc et al.³⁰ They suggested that those changes might be due to a defect of nucleolar association related to the pharmacologic effects of the drug. However, increased amount of AgNORs might result from a “self-protective” mechanism of cardiomyocytes. They also found that AgNOR staining technique showed nuclear changes that were overlooked in H&E staining.³⁰ Mean AgNOR numbers in cardiomyocytes¹⁸ and lung tissue¹⁷ of rats were seen to be increased in the case of acute CO poisoning. No significant changes were detected in mean number of AgNORs per cell in the current study, which may be, hypothetically, explained by a mechanism causing an increase of AgNOR numbers in the acute phase to activate protective mechanisms against CO intoxication, but when exposure to CO continues, AgNOR numbers are diminished in order to adapt the cells of myocardium to prolonged ischemia as it was proposed by Mamaev et al.²⁹ for ischemic heart disease complicated by severe heart failure. Another possible explanation for that condition may be the failure of the protective mechanisms of myocytes in the case of prolonged CO exposure.

Armstrong et al.²² saw that ischemia had led to loss of sarcolemmal dystrophin and spectrin, and they suggested that the loss of these proteins may be responsible for the transition of the reversible myocardial damage to an irreversible one. Loss of sarcomere pattern as a result of ischemia was seen in the current study, too. Loss of normal sarcomere pattern may lead to the death of myocardial cells and irreversible myocardial damage. Then, acute heart failure may develop. Finally, death may ensue.

Limitations

The most important limitation of our study is the absence of similar studies to compare with. Actually, there are a few studies about AgNORs in acute CO poisoning, but there are no studies evaluating AgNORs in the case of chronic CO poisoning.

In summary, TAA/NA ratio can be used as an indicator to obtain information about the degree of myocardial damage and be used instead of histopathological evaluation scores and loss of sarcomere pattern in chronic CO poisoning. We suggest that TAA/NA ratio may be helpful in the process of diagnosing chronic CO poisoning when specially determining the exact cause of a sudden death of an otherwise healthy patient who was suspected to be exposed to CO gas. More studies including large number of series should be performed to obtain more certain knowledge about this topic.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Are there any effects of chronic carbon monoxide exposure on argyrophilic nucleolar-organizing region–associated protein synthesis in rat myocardium?

Abstract

Keywords

Introduction

Methods

AgNOR detection

Histopathological examination

Statistical analysis

Results

AgNOR evaluation

Histopathological evaluation

Correlations

Discussion

Limitations

Footnotes

Declaration of Conflicting Interests

Funding

References