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Abstract

In carbon monoxide (CO) poisoning, CO affects the oxygen-carrying capacity of the hemoglobin molecule. Nucleolar-organizing regions (NORs) are genetic loci on chromosomes that are composed of ribosomal DNA and proteins. NORs can be stained with silver. A total of 18 rats were exposed to CO in three different concentrations (1000, 3000, and 5000 ppm) with 6 rats as controls. The animals were euthanized 7 days after CO intoxication. Lung tissues were taken, embedded in paraffin blocks, and sectioned at 5 μm thickness. Argyrophilic nucleolar-organizing region (AgNOR) staining was carried out. One hundred nuclei per individual were evaluated, and total AgNOR number per total nuclear number and total AgNOR area per nuclear area (TAA/NA) for each nucleus were analyzed. The CO exposure groups had significantly higher TAA/NA values and AgNOR numbers than the control group (p < 0.05). Although the differences between 1000 ppm and the other two CO-exposed groups were meaningful (p < 0.05) in the TAA/NA values, there were no differences among the CO exposure groups for the AgNOR number (p > 0.05). The increase in TAA/NA value depends on the increase in the CO exposure. Significant correlations between both the AgNOR values and histopathological scoring methods were found. Therefore, AgNOR staining method may be used as an indirect indicator for evaluating the degree of cell damage rate.

Keywords

Rat lung carbon monoxide intoxication AgNOR

Introduction

Carbon monoxide (CO) is a colorless, odorless, tasteless, and nonirritating gas. CO is released from fuels that are not lit highly enough and can cause acute and chronic poisoning deaths.^1,2 CO gas is heavier than room air and can accumulate quickly, even in well-ventilated indoor environments. The most common sources of CO include gas exhaust from motor vehicles, fumes from fires, gas-powered engines, forest fires, and paints containing methylene chloride.^3,4 CO poisoning is known to be the cause of more than half of all fatal poisonings in many countries worldwide.⁵

In CO poisoning, inhaled CO gas quickly passes through alveolar capillary membranes and enters the intravascular space, which is the primary location for connecting to the hemoglobin (Hb). Most CO becomes stable after accumulating in the erythrocytes, and the affinity of Hb for CO is 200–250 times higher than for oxygen (O₂). Shifting the dissociation curve of oxyhemoglobin to the left explains the decrease in arterial O₂ transport and the hypoxic symptoms of CO poisoning. The toxic effects of CO cannot be explained with this mechanism alone. Other mechanisms are thought to be involved.^6,7 CO intoxication creates structural changes in the Hb (by entering into the erythrocyte, CO inhibits heme proteins such as myoglobins), which makes it difficult to transfer O₂ into the tissues. CO also impairs cellular respiration by inactivating reduced cytocrome-a3.^5,8 For these reasons, CO affects the O₂-carrying capacity of the Hb molecule by binding to Hb.

CO poisoning causes a variety of symptoms in different organs of the human body due to hypoxia.⁹ The symptoms caused by CO poisoning can occur in the early period, or they can be seen weeks after exposure. In addition to the main symptoms that affect the neuropsychiatric and cardiovascular systems, other systems and organs are also affected. One of the affected systems is the respiratory system, where pulmonary edema, pneumonia, hemorrhage, and unilateral diaphragmatic paralysis may occur.¹⁰

The nucleoli are prominent structures within cell nuclei. In the nucleolus, ribosomal RNAs are synthesized, processed, and assembled with ribosomal proteins. The size and organization of the nucleolus are directly related to ribosome production and reflect the functional compartmentalization of the nucleolar machinery. When this activity is blocked or disrupted, the nucleolar proteins can interact independently from the processing activity. Nucleolar-organizing regions (NORs) are genetic loci on chromosomes that are composed of ribosomal DNA (rDNA) and proteins, some of which are argyrophilic. NORs are loops of DNA, tandemly repeated with intergenic spacers and transcribed into ribosomal RNA, which is processed into preribosomes in the nucleoli, eventually becoming part of mature ribosomes in the cytoplasm.¹¹

In humans, the NORs (sites of the ribosomal genes) are located on the secondary constrictions of the five pairs of acrocentric chromosomes 13, 14, 15, 21, and 22 and can be stained with silver when they are active. Silver binds with those rDNA sites that are transcriptionally active or have already been transcribed and still retain residual rRNA nonhistone-associated proteins. Therefore, argyrophilic NOR (AgNOR)-associated proteins are one of the most reliable methods used to visualize nucleoli in interphase nuclei.¹²

Numerous studies have been performed concerning the importance of the interphase quantity of AgNOR in tumor pathology,^13,14 hair root cells of humans,^15,16 developmental stages of Down syndrome in infants,¹⁷ and so on. However, to the best of our knowledge, there are no studies in the literature about the relationship between AgNOR proteins and CO exposure in lung cells. Therefore, we carried out the current study in order to detect any possible effects of CO exposure on the NOR protein synthesis of lung cells.

In this study, we aimed to show hypoxia-induced cell proliferation, due to CO intoxication, using the AgNOR method, and investigate any possible relationship between the AgNOR staining method and histopathological evaluation as prognostic factors.

Materials and methods

In our study, 24 male albino Wistar rats, weighing 150–180 g, were used. The experimental protocols were in compliance with the Atatürk University Ethics Committee on Research in Animals as well as the internationally accepted principles for laboratory animal use and care. Three steel tubes, with each of 10 L in volume, had full of a CO–air mixture at three concentrations (1000, 3000, and 5000 ppm) were provided by Habaş Industrial and Medical Gases Production Industries Inc., Kocaeli, Turkey. CO exposure was applied in an enclosed transparent jar with a dimension of 20 cm × 40 cm × 60 cm. There were inlet and outlet openings with 2 cm diameter at opposite sides of the jar.

The rats were randomly divided into four groups: control group, 1000 ppm, 3000 ppm, and 5000 ppm groups, consisting of six animals each. The experimental groups were exposed to CO one time, each group at a different density (1000, 3000, and 5000 ppm) with a flow rate of 4 L/min for 30 min in the transparent jar. After intoxication, the rats were housed in standard plastic cages on sawdust bedding under a 12-h light/12-h dark cycle in an air-conditioned room at 20°C and provided food and water ad libitum. One week after CO exposure, the animals were anesthetized intraperitoneally with urethane at a dosage of 1.25 g/kg and euthanized by intracardiac perfusion. Fixation in a 10% formaldehyde solution was achieved. These experimental procedures were performed in the Atatürk University Pharmacology Laboratory.

The lung tissue samples (approximately 1 × 1 × 1 cm³ in size) were obtained from the front-bottom edge of the animals’ lower lobes of their left lungs. After routine histological follow-ups, the pieces of lung tissue in the paraffin blocks were cut to 5 μm thick. Before AgNOR staining, the tissue sections were deparaffinized in xylene and then rehydrated in graded alcohol solutions. The slides were air-dried at room temperature for 15 min before being fixed in absolute methanol for 5 min. AgNOR staining was carried out according to the Benn and Perle protocol and Lindner protocol, with a slight modification for all groups.^18,19

Slides containing stained tissue sections were viewed using a light microscope (Eclipse 80i; Nikon, Tokyo, Japan) and photographed using a digital camera (Digital Sight DS-fi1; Nikon). The captured images were transferred to image processing software (ImageJ ver. 1.47t, National Institutes of Health, Bethesda, Maryland, USA), and the measurements were obtained using the “freehand selections” tool. One hundred nuclei per animal were assessed, and the mean AgNOR number and total AgNOR area per nuclear area (TAA/NA) for each nucleus were estimated.

For the histopathological examination, another series of sections were obtained and stained with hematoxylin and eosin (H&E). The sections were evaluated for the degrees of alveolar hemorrhage, vascular congestion, inflammation, and edema. Each lung slide was examined, and the severity of observed changes was scored using a scale of none (0), mild (1), moderate (2), and severe (3) damage. Histopathological analyses were made by an experienced pathologist. Statistical evaluations (Kruskal–Wallis/Dunn and one-way analysis of variance) were performed using NCSS software (Ver. 9; NCSS, LCC, Kaysville, Utah, USA).

Results

The mean AgNOR number and TAA/NA values of the rats from the control and CO-exposed groups and statistical values are given in Table 1. CO-exposed groups significant at p = 0.027 had higher mean AgNOR numbers (1.643 for 1000 ppm, 1.603 for 3000 ppm, and 1.628 for 5000 ppm groups) and TAA/NA (0.052 for 1000 ppm, 0.081 for 3000 ppm, and 0.080 for 5000 ppm groups) than the control group (1.237 and 0.0255, respectively, p = 0.0007; Figure 1).

Table 1.

TAA/NA and mean AgNOR number values of groups.

	Groups	n	Mean	Standard error	Median^a	p
TAA/NA	Control	6	0.0255	0.0011	0.025363^b	0.0007
	1000 ppm	6	0.0524	0.0025	0.049641^c
	3000 ppm	6	0.0804	0.0023	0.07941^d
	5000 ppm	6	0.0796	0.0047	0.080149^d
Mean AgNOR number	Control	6	1.237	0.0880	1.228571^b	0.027
	1000 ppm	6	1.643	0.0726	1.621784^c
	3000 ppm	6	1.603	0.0725	1.579203^c
	5000 ppm	6	1.628	0.0730	1.642653^c

TAA: total argyrophilic nucleolar organizing region area; NA: nuclear area; AgNOR: argyrophilic nucleolar organizing region.

^aIf the median values of the groups have the same letter, it said that they are not significantly different.

Figure 1.

Box plots showing TAA/NA and mean AgNOR number values of groups. TAA: total argyrophilic nucleolar-organizing region area; NA: nuclear area.

While the differences between the 3000 ppm group and 5000 ppm group were not meaningful, the differences were statistically significant among the other groups for the TAA/NA value. Additionally, for the mean AgNOR numbers, there were no statistically significant differences among the CO-exposed groups (p > 0.05; Table 1). Figure 2 shows the demonstrative examples of silver-stained NORs in the lung cells of CO-exposed groups.

Figure 2.

Silver-stained NORs (arrows) in lung cells: (A) control group, (B) 1000 ppm group, (C) 3000 ppm group, and (D) 5000 ppm group (×1000 magnification). NOR: nucleolar-organizing region.

According to the histopathological scoring method, the differences between the control and CO-exposed groups were statistically significant for congestion, inflammation, hemorrhage, and edema (p < 0.001; Table 2). Figure 3 shows the demonstrative examples of H&E-stained lung tissue images, which were used for histopathological scoring.

Table 2.

The scores of histopathological evaluations.

		Groups
Parameters	Score	Control^a	1000 ppm	3000 ppm	5000 ppm^b	p
Congestion	None	6	0	0	0	<0.001
	Mild	0	3	1	1
	Moderate	0	3	4	3
	Severe	0	0	1	2
Inflammation	None	6	0	0	0	<0.001
	Mild	0	2	2	0
	Moderate	0	3	3	4
	Severe	0	1	1	2
Hemorrhage	None	6	1	0	0	<0.0001
	Mild	0	4	3	0
	Moderate	0	1	3	4
	Severe	0	0	0	2
Edema	None	6	6	3	0	<0.0001
	Mild	0	0	3	0
	Moderate	0	0	0	1
	Severe	0	0	0	5

^aComparison of control group versus other groups; the differences were statistically significant in congestion and inflammation (p < 0.001) and hemorrhage and edema (p < 0.0001), respectively.

^bComparison of 5000 ppm group versus 1000 ppm group and 3000 ppm group; the differences were not statistically significant in congestion, inflammation, and hemorrhage (p > 0.05); only difference was meaningful in edema (p < 0.001).

Figure 3.

H&E-stained lung tissue. (A) control group: a: alveoli, b: bronchiole, v: vascular structure; (B) 1000 ppm group: macrophage in alveoli (arrow); *lymphoid aggregate, mild inflammation; (C) 3000 ppm group: moderate alveolar hemorrhage (arrow); *vascular congestion; and (D) 5000 ppm group, severe alveolar hemorrhage (arrow); *edema. H&E: ×400. H&E: hematoxylin and eosin.

When the correlations between the TAA/NA and histopathological scoring methods were evaluated, a significant relationship was found (p < 0.001; R ² = 68.9%). Additionally, the correlation was statistically meaningful for the mean AgNOR number (p = 0.015; R ² = 22.8%; Figure 4).

Figure 4.

Correlations between histopathologic scoring and AgNOR values. AgNOR: argyrophilic nucleolar-organizing region.

Discussion

The physiological amounts of endogenous CO act as neurotransmitters. Low levels of CO appropriately influence direct inflammation, cell proliferation and apoptosis, and increase mitochondrial biogenesis.⁵ With increased exposure to CO, poisoning signs begin to emerge in the organs.²⁰ CO exposure affects the O₂-carrying capacity of Hb in two ways. First, CO competitively inhibits the binding of O₂ to Hb, and it prevents the transport and release of O₂ to the tissues. CO leading to relative anemia is caused by tissue hypoxia or asphyxia.^21,22 Second, by making structural changes in the Hb, CO prevents releasing O₂ to the tissues, thus impairing cellular respiration.⁸ Lung injury after CO poisoning is rare; however, when it exists, it has been thought that it is not caused by the histotoxicity of CO, but probably because of the hypoxia resulting from impaired O₂ transfer.¹⁰

In CO poisoning, prolonged hypoxia can affect capillary permeability and may cause pulmonary edema.^23,24 Pulmonary edema, pneumonia, and adult respiratory distress syndrome may occur after CO poisoning.^25,10 Pulmonary lesions are cardiogenic damage, which may develop due to CO poisoning. Additionally, pulmonary edema can be seen as cardiogenic damage.^26
–28

It was difficult to point out the damage to the lung tissue after CO poisoning. To the best of our knowledge, the literature contains no reports that showed the relationship between AgNOR protein synthesis and lung damage caused by CO intoxication. It is known that the low levels of CO direct cell proliferation, and the AgNOR staining method can be used as a proliferation marker.^5,13,14,29

The nucleolus is connected with the regulation of major physiological cellular processes, including mitosis, ribosome assembly, stress response, and the generation of ribonucleoprotein complexes.³⁰ More than 700 nucleolar proteins, most of which seem to be involved in the processing and maturation of ribosomal RNAs, and the generation of mature ribosomes, have been described in humans.^30
–32 NORs are functional subunits of the nucleolus in which actively transcribed rDNA is surrounded by a large number of regulatory proteins in interphase.³³

The number, shape, and distribution of AgNOR reactive sites counted in the cells provide important knowledge about the behavior of those cells. In tumor cells, the relationship between cell proliferation and AgNOR amounts has been investigated widely by comparing AgNOR values with kinetic data obtained by applying a panel of proliferation markers.^13,14,34

We have performed several studies using the AgNOR method,^{13

–17,29} which supply an index of cellular activity in connection with proliferation, differentiation, and secretory activities. Our study showed that rRNA gene activity, as determined by total AgNOR number per total nuclear number or TAA/NA, increased depending on the increase of CO exposure in the lung cells. In lung cells, AgNOR protein synthesis tends to increase during CO exposure, and all cells tend to protect their situation toward dangerous agents. Perhaps these proteins occur against to CO exposure or trigger the synthesis of some other proteins that have protective roles in the lung cells. Both the AgNOR values may be used as indirect indicators for evaluating the degree of cell damage (occurring hypoxia) rate. Additional studies should be conducted to obtain more certain knowledge about this topic. In this way, new therapeutic approaches may be developed in the treatment of CO intoxication in the future. This technique may also be used to detect the degree of CO exposure in forensic medicine. CO intoxication can be diagnosed early using the carboxyhemoglobin (COHb) level. However, most of the time (the half-life of room air is 4–6 h) COHb levels may not help in the diagnosis and direction of the treatment. Our method may be used when COHb levels seem normal in the intoxication doubt autopsies and therefore can be adapted for use in forensic science. Additionally, the current technique may provide indirect information about the degree of hypoxia and asphyxia in hypoxic and asphyxic babies. If these proteins play a protective role toward hypoxia, they may be helpful in developing new treatment approaches for hypoxic or asphyxic situations in the future.

A significant relationship was found between the results of the histopathological methods and both the TAA/NA and mean AgNOR number. The results of the histopathological examination can be obtained with the evaluation of alveolar hemorrhage, vascular congestion, inflammation, and edema. Therefore, it may be said that only AgNOR protein synthesis may be used to obtain knowledge about the degree of damage to the cells, instead of the histopathological scoring method requiring an experienced pathologist.

The limitations of this study include the relatively small number of groups. In our technique, both the NOR area and NA were used for the calculation of the TAA/NA proportion. Thus, more reliable knowledge about the proliferation, differentiation, and secretory activity of each cell could be obtained. Thus, indirect knowledge may be obtained about the behavior of the lung cells for protection from CO exposure. Additionally, advantages of this technique are simple, reliable, cheap, and a valuable marker to evaluate the ribosomal gene activity in different metabolic states of cells.

Conclusion

The current technique may be used to obtain knowledge about the identification of cellular changes induced by CO in the lung. Further studies with a higher number of groups, including other CO levels and different candidate genes, should be conducted in order to obtain more certain knowledge about this topic.

Footnotes

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

References

Gorman

Drewry

Huang

The clinical toxicology of carbon monoxide. Toxicology 2003; 187: 25–38.

Kandis

Katırcı

Çakır

Acil servise karbonmonoksit entoksikasyonu ile başvuran olgularin geriye dönük analizi. Akademik Acil Tıp Dergisi 2007; 5: 21–25.

Katirci

Kandis

Aslan

Neuropsychiatric disorders and risk factors in carbon monoxide intoxication. Toxicol Ind Health 2011; 27: 397–406.

Ocak

Doğan

The relationship between carbon monoxide intoxication and sister chromatid exchange in lymphocyte cells. Toxicol Ind Health. Epub ahead of print 31 October 2012. DOI:10.1177/0748233712466131.

Kandis

Katırcı

Karapolat

. Karbonmonoksit zehirlenmesi. Düzce Tıp Fakültesi Dergisi 2009; 3: 54–60.

Raphael

Elkharrat

Jars-Guincestre

Trial of normobaric oxygen for acute carbon monoxide intoxication. Lancet 1989; 8660: 414–419.

Sokal

Kralkowska

. The relationship between exposures duration, carboxyhemoglobin, blood glucose, pyruvate and lactate and the severity of intoxication in 39 cases of acute carbon monoxide poisoning in man. Arch Toxicol 1985; 57: 196–199.

Brown

Piantadosi

. In vivo binding of carbon monoxide to cytochrome-c oxidase in rat brain. J Appl Physiol 1990; 68: 604–610.

Bahcebasi

Kandis

Baltaci

Factors affecting exhaled carbon monoxide levels in coffeehouses in the Western Black Sea region of Turkey. Toxicol Ind Health 2011; 27: 195–204.

10.

Choi

. Carbon monoxide poisoning: systemic manifestations and complications. J Kor Med Sci 2001; 16: 253–261.

11.

Hernandez-Verdun

. The nucleolus: a model for the organization of nuclear functions. Histochem Cell Biol 2006; 126: 135–148.

12.

Trere

. AgNOR staining and quantification. Micron 2000; 31: 127–131.

13.

Eroz

Cucer

Karaca

The evaluation of argyrophilic nucleolar organizing region proteins in fine-needle aspiration samples of thyroid. Endocr Pathol 2011; 22: 74–78.

14.

Eroz

Unluhizarci

Cucer

Value of argyrophilic nucleolar organizing region protein determinations in nondiagnostic fine needle aspiration samples (due to insufficient cell groups) of thyroid nodules. Anal Quant Cytol Histol 2013; 35: 226–231.

15.

Eroz

Tasdemir

Dogan

. Is there any relationship between decreased AgNOR protein synthesis and human hair loosing? Biotech Histochem 2012; 87: 494–498.

16.

Eroz

Yilmaz

Cucer

. Argyrophilic nucleolar organizing region associated protein synthesis in hair root cells of humans at different developmental stages and sex. Biotech Histochem 2013; 88: 267–271.

17.

Eroz

Okur

Ozkan

Does higher NORs expression affect the developmental stages of down syndrome infants?

Genet Counsel 2012; 23: 249–253.

18.

Benn

Perle

. Chromosome staining and banding techniques. In: Rooney

Czepulskowski

(eds) Human Cytogenetics: Constitutional Analysis: A Practical Approach. London, UK: Oxford University Press, 1986 , pp. 91–118.

19.

Lindner

. Improvements in the silver-staining technique for nucleolar organizer regions (AgNOR). J Histochem Cytochem 1993; 41: 439–445.

20.

Guzman

. Carbon monoxide poisoning. Crit Care Clin 2012; 28: 537–548.

21.

Piantadosi

. Carbon monoxide poisoning. Undersea Hyperb Med 2004; 31: 167–177.

22.

Weaver

LK.

Clinical practice. Carbon monoxide poisoning. N Engl J Med 2009; 360: 1217–1225.

23.

Fein

Grossman

Jones

Carbon monoxide effect on alveolar epithelial permeability. Chest 1980; 78: 726–731.

24.

Perina

. Noncardiogenic pulmonary edema. Emerg Med Clin N Am 2003; 21: 385–393.

25.

Doherty

. History, pathophysiology, clinical presentation and role of hyperbaric oxygen in acute CO poisoning. Emerg Med 2000; 12: 55–61.

26.

Aslan

Uzkeser

Seven

The evaluation of myocardial damage in 83 young adults with carbon monoxide poisoning in the East Anatolia region in Turkey. Hum Exp Toxicol 2006; 25: 439–446.

27.

Huang

Hsia

. Acute carbon monoxide poisoning with severe cardiopulmonary compromise: a case report. Cases J 2009; 2: 52.

28.

Uzkeser

Emet

Ersunan

Acute lung injury due to carbon monoxide exposure. Konuralp Tıp Dergisi 2012; 4: 55–58.

29.

Eroz

Cucer

Unluhizarci

Detection and comparison of cutoff values for total AgNOR area/nuclear area and AgNOR number/nucleus in benign thyroid nodules and normal thyroid tissue. Cell Biol Int 2013; 37: 257–261.

30.

Boisvert

van Koningsbruggen

Navascués

The multifunctional nucleolus. Nat Rev Mol Cell Biol 2007; 8: 574–585.

31.

Scherl

Couté

Déon

Functional proteomic analysis of human nucleolus. Mol Biol Cell 2002; 13: 4100–4109.

32.

Andersen

Lam

Leung

Nucleolar proteome dynamics. Nature 2005; 433: 77–83.

33.

Ploton

Menager

Lechki

Silver staining of nucleolus organizer regions (NORs). Application to the study of nucleolar structure and value in pathology. Ann Pathol 1988; 8: 248–252.

34.

Pich

Chiusa

Navone

. Prognostic relevance of cell proliferation in head and neck tumours. Ann Oncol 2004; 15: 1319–1329.

Is one-time carbon monoxide intoxication harmless? Evaluation by argyrophilic nucleolar-organizing regions staining method

Abstract

Keywords

Introduction

Materials and methods

Results

Discussion

Conclusion

Footnotes

Funding

References