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 . 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 .
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 . 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) . 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 .
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 . 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 .
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 . 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 .
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 . It was reported that patients with heart failure have cardiovascular dysfunction and diminished eNOS capacity compared with normal humans .
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 . 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 .
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 .
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 .
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 .
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 .
It was demonstrated that adult rat ventricular myocyte primary isolates have calcium-sensitive NOS-enzymatic activity . 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 .
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 . 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.
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.
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.
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.
Conflicts of interest
There is no conflict of interest to declare.
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Keywords:© 2011 The Egyptian Journal of Histology
acetylecysteine; formaldehyde; myocardium; endothelial nitric oxide synthase