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Histological and immunohistochemical study on the possible cardioprotective role of acetylcysteine in oral formalin myocardial toxicity in adult albino rats

Afifi, Noha M.a; Hanon, Amani F.b

The Egyptian Journal of Histology: December 2011 - Volume 34 - Issue 4 - p 859–869
doi: 10.1097/01.EHX.0000407660.20814.6d
Original articles
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Introduction Forty percent solution of formaldehyde in water is known as formalin. Formalin is added to milk in the production of cheese and dairy products as an antimicrobial agent. It is a very reactive compound, reacting with cellular proteins and nucleic acids; thus, safety evaluation of formalin as an additive to milk and dairy products (cheese and yoghurt) must be thoroughly considered.

Aim of the study This study was conducted to investigate the effects of oral formalin ingestion on the histological structure of the myocardium of adult albino rats and the possible cardioprotective effects of the formalin antidote ‘acetylcysteine’.

Materials and methods Thirty adult albino rats were used. They were classified into three main groups as follows: group I: served as a control. Group II: rats received formalin orally. Group III: rats received formalin in addition to the specific formalin antidote ‘acetylcysteine’. Myocardial sections were stained with H&E, MT stain, and immunohistochemical staining for endothelial nitric oxide synthase (eNOS) antigen.

Results It was found that oral formalin consumption induced myocardial abnormalities in the form of disruption, vacuolation, and wide separation of cardiac muscle fibers. Such abnormalities were, to a huge extent, prevented with the use of the antidote acetylcysteine. eNOS immunoreactivity, in ventricles of formalin-exposed rats, showed a significant decrease compared with the control group and then showed a highly significant increase in group III compared with group II and the control group.

Conclusion Oral formalin consumption was shown to induce deleterious morphological changes on the myocardium, most of which were prevented with the use of acetylcysteine. The study highlights the importance of eNOS in healthy cardiovascular cells and suggests that the decrease in eNOS that occurs in formalin toxicity may lead to altered vascular reactivity and possibly impaired cardiac function.

aDepartment of Histology

bDepartments of Forensic Medicine and Clinical Toxicology, Faculty of Medicine, Cairo University, Cairo, Egypt

Correspondence to Noha M. Afifi, Department of Histology, Faculty of Medicine, Cairo University, Cairo, Egypt Tel: +169626557; e-mail: noha_afifi@windowslive.com

Received July 10, 2011

Accepted August 24, 2011

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Introduction

Formaldehyde (FA) is a physiological intermediary metabolite that participates in many biological processes in the body. It is a constituent of many items of daily use, including foods. It is also used in medicine for the treatment of some conditions. FA has been commercially produced since the early 1900s. The textile industry uses FA-based resins as finishers to make fabrics [1]. Forty percent solution of FA in water is known as formalin. It is irritating, corrosive, toxic, and is absorbed from all surfaces of the body [2].

An aqueous solution of FA (formalin) is used as a disinfectant as it kills most bacteria and fungi. It is used to inactivate bacterial products for toxoid vaccines. Some topical creams, cosmetics, and personal hygiene products also contain derivatives of FA as the active ingredients that prevent the growth of potentially harmful bacteria [3]. The real concern lies in the use of formalin as an antimicrobial agent in food. Formalin is added to milk in the production of cheese and dairy products as an antimicrobial agent [4].

FA has been classified as a ‘group 1 carcinogen’ by the International Agency for Research on Cancer [5]. A comprehensive review of cancer in industry workers and professionals exposed to FA through inhalation has shown an excess risk of nasopharyngeal and sinonasal cancers [6]. Some excess risk of leukemia has been reported from studies on pathologists, anatomists, and embalmers and other professionals exposed to FA [7].

Formalin ingestion can lead to immediate deleterious effects on almost all systems of the body including the gastrointestinal tract, the central nervous system, the cardiovascular system, and the hepatorenal system, causing gastrointestinal hemorrhage, cardiovascular collapse, unconsciousness or convulsions, severe metabolic acidosis, and acute respiratory distress syndrome [2].

What contributes to the danger of oral ingestion is the fact that FA, one of the most potent cumulative toxins, is produced by the liver from methanol, which in turn constitutes 10% of the sweetener, aspartame, used by more than 200 million people worldwide and known to be ‘the most widely used sweetener worldwide’ [8].

FA formation after aspartame ingestion is very toxic. It accumulates within the cell, reacting with cellular proteins (mostly enzymes) and DNA (both mitochondrial and nuclear). The fact that it accumulates with each dose may result in harmful effects on those who consume diet drinks and food stuffs on a daily basis [9].

Nitric oxide (NO) is an important regulator of cardiac contractile function [10]. The formation of NO from L-arginine in mammalian cells is catalyzed by three NO synthases (NOS) isoenzymes expressed either constitutively [neuronal NOS, type I and endothelial NOS (eNOS), type II] or after stimulation by cytokines [inducible NOS, type III] [11].

eNOS is expressed within the heart, in cardiac myocytes, in cardiac conduction tissue, and in the endothelium [12]. In the heart, eNOS mediates the physiologic action of NO, such as regulation of cardiac contractility, vascular tone, platelet aggregation, and endothelial function [13]. The expression of eNOS in the myocardium is modulated in patients with dilated cardiomyopathy with clinical evidence of heart failure [14].

There are multiple studies on the hazardous effects of FA inhalation on the different organs of the body but there is lack of information on the effects of oral ingestion. This study was conducted to investigate the effect of oral formalin ingestion on the histological structure of the myocardium of adult albino rats and to evaluate the possible cardioprotective effect of acetylcysteine.

The study also aims to investigate the possible underlying mechanism by which formalin may induce such myocardial effects, by studying the expression of eNOS in the heart with oral formalin toxicity and its possible contribution to cardiac dysfunction and cell damage. This might help in understanding the pathophysiology of complex disease states including atherosclerosis, systemic and pulmonary hypertension, and cardiomyopathy.

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Materials and methods

Materials

Drugs

The drugs used were as follows:

Formaline: Purchased from Sigma Chemical Company (St. Louis, Missouri, USA), in the form of 40% solution (MW: 30.03 g/mol). It was diluted in distilled water to prepare a 5% solution [15].

Acetylcysteine: Purchased from Mina Pharmaceutical Company, in the form of effervescent tablets, consisting of 600 mg acetylcysteine + 75 mg ascorbic acid. Tablets were dissolved in distilled water and administered orally to rats at a therapeutic dose of 0.27 mg/kg with the diluted FA using a gastric tube [16].

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Animals

The study was conducted at the Animal House of Kasr-Al Aini School of Medicine, according to the Guidelines for the Care and Use of Laboratory Animals.

Thirty (15–18 weeks old) adult albino rats were included in the study (15 male and 15 female rats). Their weights ranged from 150 to 250 g ± 20 g (mean: 210 ± SD: 1.93). They were housed in a temperature-controlled and light-controlled room (12-h light/dark cycle), with free access to food and water. They were housed in three groups in plastic cages. Rats were fed ad libitum.

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The animals were divided into the following groups:

Group I (served as a control): This group included 10 rats (five males and five females). They received a vehicle of distilled water orally using a gastric tube. They were sacrificed with the experimental groups.

Group II: This group included 10 rats (five males and five females) that received formalin orally using a gastric tube.

The tolerable daily intake (TDI) of FA is defined as an estimate of the amount of FA in drinking water that can be ingested daily over a lifetime without significant health risks. It is estimated to be 0.05 mg/kg/day [15].

After dilution of FA to 5% [15] and calculating the milligrams of FA (with a known molecular weight: of 30.03) in 1 l, the TDI dose was found to be 13.3 ml for each rat, oral dose taken by each rat using the gastric tube in the form of solution contains 0.05 mg/kg of formalin.

Group III: This group included 10 rats (five males and five females) that received the same dose of formalin as group II in addition to the specific formalin antidote (acetylcysteine) at a therapeutic dose of 0.27 mg/kg orally using a gastric tube.

The time of drug intake was fixed daily Its 11 a.m. The experiment was continued for 4 weeks. All the animals were sacrificed under isoflurane inhalation anesthesia at the end of the experiment. The hearts were obtained from all the animals. Samples from the left ventricles were excised and prepared for light and immunohistochemical studies.

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Methods

Light microscopic studies

The specimens obtained were fixed in 10% formol saline, processed, and embedded in paraffin sections. Sections were cut into 5 µm thickness using a microtome, mounted on slides, and subjected to the following techniques:

  1. (1) H&E stain for histological assessment.
  2. (2) MT stain [17].
  3. (3) Immunohistochemical staining for eNOS antigen using the avidin–biotin peroxidase complex technique [18].
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The ABC technique was carried out as follows for immunohistochemical expression of eNOS

Paraffin sections were deparaffinized and hydrated. After blocking the endogenous activity of peroxidase using 10% hydrogen peroxide, the sections were incubated with primary antibodies. They included monoclonal anti-eNOS (Lab Vision Corporation, Westing House, Thermont, CA, USA). Then, after washing with phosphate buffer, the secondary antibody was applied (biotinylated goat antirabbit). The slides were incubated with labeled avidin–biotin peroxidase, which binds to the biotin on the secondary antibody. The site of antibody binding was visualized after adding (diaminobenzedine) chromogen, which is converted into a brown precipitate by peroxidase.

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Morphometric studies

  1. (1) The mean area percentage of the collagen content of myocardial sections was measured using an image analyzer computer system (Leica Qwin 500, Cambridge, UK). In each specimen, 10 nonoverlapping fields were measured at a magnification of × 400. The image analyzer was used to measure the area of collagen fiber content and was expressed in an area percentage in relation to the area of the standard measuring frame of 7099.95 µm².
  2. (2) The mean area percentage for eNOS immunoreactivity was measured in the cardiac myocytes and endothelial cells of immunostained sections using an image analyzer computer system. In each specimen, 10 nonoverlapping fields were measured at a magnification of × 400.
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Statistical analysis

The mean values of the data obtained from the image analyzer were calculated and statistically compared using the SPSS (Statistical Package for Social Sciences), Windows Version 5, Chicago, USA. Differences between groups were examined for statistical significance using the analysis of variance test. This test is used to find a significant difference between more than two groups. A value of P < 0.05 was considered significant [19].

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Results

Light microscopic results

No histological variations were evident between both sexes.

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H&E stain

Light microscopic examination of cardiac myocytes in the ventricles of the control group showed a normal histological architecture. Myocytes appeared cylindrical and branching. They exhibited an acidophilic sarcoplasm and central oval single nuclei. Blood capillaries were found in the intercellular spaces (Figs 1 and 2).

Figure 1

Figure 1

Figure 2

Figure 2

In group II, marked histological alterations were observed. Disruption and fragmentation of cardiac myocytes were evident. The majority of muscle fibers exhibited cytoplasmic lysis and massive hemorrhage was observed (Fig. 3). Many myocytes exhibited peripherally situated nuclei. The separated cardiac myocytes appeared degenerated (Fig. 4). Extravasation of blood was detected between the muscle fibers (Fig. 5). Many cardiac myocytes exhibited cytoplasmic vacuolation. Lymph vessels were markedly dilated and contained a homogenous acidophilic lymph (Fig. 6). Some specimens showed complete cytoplasmic lysis of cardiac myocytes (Fig. 7). Mononuclear cellular infiltration was evident in between the cardiac myocytes (Fig. 8).

Figure 3

Figure 3

Figure 4

Figure 4

Figure 5

Figure 5

Figure 6

Figure 6

Figure 7

Figure 7

Figure 8

Figure 8

Group III, which received formalin in addition to the specific formalin antidote ‘acetylcysteine’, showed a histological pattern nearly similar to the control group. Most of the cardiac muscle fibers appeared cylindrical, with central oval nuclei. Narrower intercellular spaces were detected between cardiac myocytes. A few muscle fibers exhibited loss of striations and marked congestion was still observed in the interstitial spaces (Figs 9 and 10).

Figure 9

Figure 9

Figure 10

Figure 10

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MT stain

Ventricular sections obtained from the control group showed the presence of collagen fibers deposited in between the cardiac muscle fibers and around the walls of blood capillaries (Fig. 11). In group II, which received formalin, increased deposition of collagen fibers was detected (Fig. 12) compared with the control group. Group III, which received formalin in addition to the specific antidote ‘acetylcysteine’, showed much less collagen fiber content between the cardiac myocytes (Fig. 13) compared with the formalin-exposed group.

Figure 11

Figure 11

Figure 12

Figure 12

Figure 13

Figure 13

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Immunohistochemical results

Immunohistochemically, in the control group, positive eNOS immunoreactivity was found in the sarcoplasm of some cardiac muscle fibers as well as in the endothelium of blood vessels (Figs 14 and 15).

Figure 14

Figure 14

Figure 15

Figure 15

Ventricular sections of rats that received formalin showed decreased eNOS immunostaining in cardiac muscle fibers, with minimal staining in the endothelium (Fig. 16).

Figure 16

Figure 16

In the groups that received the specific formalin antidote ‘acetylcysteine’, ventricular sections showed diffuse eNOS immunoreactivity in most of the cardiac muscle fibers and increased endothelial immunoreactivity (Fig. 17) compared with the formalin-exposed group.

Figure 17

Figure 17

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Morphometric and statistical results

  1. The mean area percentage of the deposited collagen fibers, measured using the image analyzer, showed a highly significant increase in group II (7.69 ± 0.12) compared with the control group (2.7 ± 0.17). Measurements showed a highly significant decrease in the mean area percentage of the deposited collagen fibers in group III (3.98 ± 0.17) compared with group II and a significant decrease compared with the control group (Table 1 and Histogram 1).
  2. The mean area percentage for eNOS immunoreactivity in the sarcoplasm of cardiac muscle fibers showed a significant decrease in group II (0.84 ± 0.07) compared with the control group (1.39 ± 0.12). eNOS immunostaining then showed a highly significant increase in group III (6.29 ± 0.55) compared with group II and the control group (Table 1, and Histogram 1).
  3. The mean area percentage for eNOS immunoreactivity in the endothelium of blood capillaries showed a significant decrease in group II (2.23 ± 0.54) compared with the control group (4.35 ± 0.47). eNOS immunostaining then showed a very highly significant increase in group III (7.2 ± 0.39) compared with group II and a significant increase compared with the control group (Table 1, and Histogram 1).
Table 1

Table 1

Histogram 1

Histogram 1

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Discussion

Formalin is a very reactive compound, reacting with cellular proteins and nucleic acids; therefore, the safety evaluation of formalin as an additive to milk and dairy products (cheese and yoghurt) must take into account the toxicity of the reaction products between formalin and milk components [20]. This is very important, especially when formalin exceeds the ‘baseline levels’ to reach the ‘adulteration levels’, which results from the intentional addition of formalin beyond the approved values.

Therefore, in view of the widespread use of formalin, its potential toxicity and carcinogenicity, and its huge impact on human health, the present study was conducted to investigate the possible effects of oral formalin consumption. Although there are a large number of epidemiological studies that deal primarily with inhalation and/or dermal exposure, no studies were found that deal primarily with oral exposure. To the best of our knowledge, only very few histological studies have been conducted that examine the toxic effects of oral formalin administration on cardiac muscle; thus, this study examined the histological and morphological alterations induced in cardiac myocytes after oral formalin consumption and the possible protective effects of the antidote acetylcysteine.

In the current study, oral formalin consumption induced myocardial abnormalities and damage to cardiac myocytes. Such abnormalities were, to a huge extent, prevented by the concomitant use of the antidote acetylcysteine. Formalin-induced myocardial abnormalities were in the form of disruption, fragmentation, and wide separation of cardiac muscle fibers. The majority of cardiac myocytes exhibited peripheral nuclei and cytoplasmic lysis with separated myofibers. Such myofibrillar lysis might be due to loss of myofilaments and hence cardiac dysfunction might be a result of contractile dysfunction at the single-cell level.

It was previously reported that such myocardial abnormalities might result from the generation of reactive oxygen species (ROS) and the formation of oxidation products. ROS-mediated lipid peroxidation might be the direct cause of cytoplasmic vacuolation detected in cardiac myocytes. Radicals can cause damage to cardinal cellular components such as lipids, proteins, and nucleic acids, leading to subsequent cell death by necrosis or apoptosis [21].

A significant observation in the present study was the marked congestion of blood vessels with extravasation of blood in between the muscle fibers. Massive hemorrhage was observed between the muscle bundles. Such hematological abnormalities predispose to sluggish circulation, endothelial damage, and focal capillary occlusion [22]. In addition, small areas of capillary closure result in dilated capillaries and increased intracapillary pressure will lead to leakage through the vessel wall. Increased capillary permeability results in the rupture of blood capillaries and blood leakage.

Dilatation of lymph vessels was also demonstrated in the present study with accumulation of lymph. Lymph vessels can become clogged with protein deposits or the flow can stagnate or even stop due to infection. Lymphostatic edema results from the abnormal accumulation of protein in the lymph vessel, along with osmotically held fluids in the interstitial space (lymphedema) [23]. Consequently, as toxins accumulate, myocytes are unable to function properly, resulting in various metabolic and infectious problems.

Ventricular sections of rats receiving the antidote acetylcysteine showed effective prevention of myocardial structural damage induced by FA. Most cardiac myocytes exhibited a nearly normal architecture with narrower intercellular spaces. However, congestion of capillaries remains a finding in that group. Similarly, a cardioprotective effect of cysteine was reported, attenuating the cardiovascular failure in rats receiving formalin [16].

In the present work, using MT stain, the degenerated myocytes in the formalin-exposed group were separated with increased content of collagen fibers, with a highly significant increase in the area percentage of collagen fibers deposited between them compared with the control group. Measurements showed a highly significant decrease in the group receiving the antidote compared with the group receiving formalin alone and a significant decrease compared with the control group.

It is evident that formalin appears to augment myocardial fibrosis. Cysteine might exert its cardioprotective action against collagen formation by preventing the proliferation of fibroblasts and collagen deposition in the blood vessels of the heart.

The present work also aimed to elucidate the possible underlying mechanism by which formalin could induce such effects on the myocardium, thereby mediating changes in cardiac function. This might help in understanding the pathophysiology of complex disease states including atherosclerosis, systemic and pulmonary hypertension, and cardiomyopathy.

Studies of cardiac myocytes in culture as well as investigations in animal models and human participants have identified NO to be an important determinant of cardiac function [24]. The emerging role of NO in the maintenance of cell physiology has highlighted the importance of this interesting molecule in cytostasis. However, the balance between the cytostatic and the cytotoxic effects of NO may be regulated by the particular NOS isoform activated [25].

In this context, in the present study, we attempted to localize the distribution and expression of eNOS in the myocardium. In the control specimens, moderate eNOS immunoreactivity was detected in ventricular myocytes and endothelium of blood vessels. In the ventricles of formalin-exposed rats, immunoreactivity for eNOS in the sarcoplasm of cardiac muscle fibers as well as in the endothelium showed a significant decrease in group II compared with the control group. eNOS immunostaining then showed a highly significant increase in group III compared with group II and the control group.

Over the past several years, the expression and regulation of eNOS has been characterized in endothelial cells [12]. NO production by eNOS not only modulates the tone of the underlying vascular smooth muscle but also inhibits several proatherogenic processes including smooth muscle proliferation and migration, platelet aggregation, monocyte and platelet adhesion, and synthesis of inflammatory cytokines [26].

Reports indicate that formalin impairs NO production in the coronary endothelial cells and contractile proteins, thereby limiting myocardial function and possibly leading to heart failure [10]. It was reported that patients with heart failure have cardiovascular dysfunction and diminished eNOS capacity compared with normal humans [27].

In formalin toxicity, endothelial injury may be a consequence of peroxidative stress. The failure of vascular endothelium to induce NO-mediated vasorelaxation may be due to the decreased formation of NO, increased degradation of NO, or a combination of both processes [12]. Such changes can be attributed to the direct effect of formalin on eNOS gene expression. A defect in NO function develops in formalin toxicity and leads to altered vascular reactivity. The occurrence of atherosclerotic cardiovascular disease may be a consequence, in part, of the decrease of eNOS protein in endothelial cells [26].

However, enhanced NO production by eNOS may serve as an oxidant scavenger, thereby minimizing the deleterious effects of superoxide and other ROS. The expected improvement in cardiac function with increased eNOS expression might be related to a reduction in systemic vascular resistance [28].

Upregulation of eNOS expression within the vascular endothelium may reduce cardiac afterload and this mechanism may be responsible for an increase in the cardiac output and attenuation of pulmonary edema, thus markedly improving survival [12].

NO is one of the most important vasodilators produced by coronary endothelial cells; this paracrine substance also exerts cardioprotective effects by influencing the function of subjacent cardiac myocytes [28].

Cardiac myocytes have recently been shown to express eNOS, although the mechanism(s) responsible for the activation of eNOS and its physiologic function remain to be determined. It was found that eNOS activity was regulated in part by the contractile state of the heart and intracellular calcium activity induced by changes in the pacing frequency of rat cardiac myocytes [13].

It was demonstrated that adult rat ventricular myocyte primary isolates have calcium-sensitive NOS-enzymatic activity [29]. Extensive characterization of NOS activity was demonstrated in ventricular myocytes, confirming the presence of eNOS in these cells in vivo and in vitro by immunohistochemistry with eNOS-specific antibodies [26].

Consistent with the findings in the present study, low eNOS activity in right ventricular tissues from patients with dilated cardiomyopathy and clinical evidence of heart failure have been previously reported [30]. Diminished NO production probably contributes to such a myocardial dysfunction.

Therefore, it is essential to establish an understanding of the complex cellular and subcellular mechanisms that regulate cardiac eNOS expression. This will eventually provide answers for important questions for both basic and clinical researchers in cardiovascular medicine.

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Conclusion

Oral formalin consumption was shown to induce deleterious morphological changes in myocardium. This means that formalin levels in the body must be very tightly controlled, as a very low daily exposure may lead to health problems.

The concomitant administration of the specific formalin antidote ‘acetylcysteine’ was shown to prevent most of these histological alterations.

The present study suggests that a decrease in eNOS that occurs in formalin toxicity may lead to altered vascular reactivity and possibly impaired cardiac function.

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Recommendations

Ideally, the government should introduce a policy to limit or even prevent the addition of formalin as a preservative in dairy products. At the very least, the government should develop and thoroughly validate rapid, simple, and low-cost ‘screening methods’ that can detect and quantify very low concentrations of formalin in dairy productions. Ideally, such new screening methods can be used outside of laboratory settings by various personnel.

The ‘tolerable daily intake’ of oral formalin should be clearly written on all sold packs. However, this does not imply that adulteration of food to a level consistent with TDI is acceptable. But this would at least provide a sufficient margin of safety for dietary consumption relative to TDI.

Experiments should be performed to detect the possible protective role of adding cysteine to dairy products pasteurized by formalin.

Studies should be conducted to determine whether in-utero exposure occurs and the extent of that exposure. Studies should investigate the potential reproductive and developmental effects after formalin exposure.

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Limiting the use of the sweetener (aspartame).

Further studies should be directed toward identifying the mechanism of the NO cardioprotective effect and its correlation with the development and pathophysiology of heart failure.

Developing therapies to improve vascular eNOS function as a means to improve clinical outcomes in patients with heart failure, like the use of nitrovasodilators.

Further studies are needed to confirm the therapeutic potential of supplemental oral ‘L-arginine’, a substrate of NOS, in patients with heart failure.

Table

Table

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Acknowledgements

Conflicts of interest

There is no conflict of interest to declare.

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Keywords:

acetylecysteine; formaldehyde; myocardium; endothelial nitric oxide synthase

© 2011 The Egyptian Journal of Histology