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Abstract

Formaldehyde (FA) is one of the most widely used chemical compounds in industrial field. It is described as toxic, particularly to the nervous system, the urogenital system, and the respiratory tracts. In this study, we determined the effects of acute oral exposure to FA in rabbit brain tissue. A total of 16 rabbits were selected and divided into 2 groups: formaldehyde group (group F) and control group (group C). FA was administered to group F at a rate of 40 mg/kg/day via a nasogastric tube for 5 days. Saline was similarly administered to the eight controls. All the animals were euthanized after 5 days of exposure, and brain tissue samples were collected in 10% neutral formalin and embedded in paraffin. To investigate the effects of FA on the apoptotic process, we examined active caspase-3, Bax, and Bcl-2 immunohistochemical expression and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate –biotin nick-end labeling (TUNEL) reactivity in the rabbit brains. In addition, glial fibrillary acidic protein (GFAP) was biochemically assessed in brain tissue samples for neurotoxicity. We found that FA treatment caused a significant decrease in Bcl-2 expression and an increase in active caspase-3 and Bax expressions as well as an increase in the number of TUNEL-positive apoptotic cells. The GFAP level was found to be significantly higher in group F. In conclusion, acute oral exposure to FA caused DNA damage, apoptosis, and neuronal injury in the rabbit brains.

Keywords

Formaldehyde apoptosis oral toxicity glial fibrillary acidic protein rabbit brain

Introduction

Formaldehyde (FA) is a chemical compound that is commonly used in cleaning and cosmetic products, textile products and polishers, the paint and coatings industry, and the plastics industry; it is known to be toxic to human cells.¹ FA is also used for the fixation of the pathology specimens in the medical field.² Nevertheless, FA is added to foods (e.g. fruits, vegetables, seafood, and honey) and drugs as a preservative.³ In addition, FA can be introduced to the body via various routes such as the respiratory system, the digestion system, and the skin. FA is then converted to formic acid in erythrocytes and the liver by FA dehydrogenase enzymes. Thereafter, formic acid decomposes to carbon dioxide, water, and bicarbonate before being excreted via urine.^1,3

In many studies, FA has been described as a toxic component particularly to the nervous system, the urogenital system, and the respiratory tracts.^1,4 The neurotoxicity mechanism of FA has not been elucidated in detail. The metabolite of FA, formic acid, can readily cross the blood–brain barrier, leading to central nervous system (CNS) toxicity by increasing cerebral glutathione concentration and decreasing 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity, which is an important enzyme of the myelin-forming cells. It can also generate disturbance of glutathione peroxidase and succinate dehydrogenase activities.^5,6 Small amounts of unmetabolised FA can pass through the blood–brain barrier and interact directly with nervous system cells. Moreover, FA causes an increase in neurofilaments, intraaxonal swelling, and mitochondrial disruption.^6–8 It is well known that nervous system tissue damage can be followed using a specific indicator, represented as glial fibrillary acidic protein (GFAP). GFAP, which is elevated after neuron injury, is thought to be an important molecule that provides structural stability to astrocytic processes.⁹

Numerous studies have demonstrated that apoptosis is one of the mechanisms resulting in cell death during brain injury.^10–12 Two major pathways control apoptosis activation: the extrinsic (death receptor-mediated) pathway, initiated by tumor necrosis factor family members through the death-inducing signaling complex, as an activating complex for pro-caspase-8; and the intrinsic (mitochondria-mediated) pathway through the apoptosome, as an activating complex for pro-caspase-9, which ultimately activates caspase-3 for the execution of cells.^13,14 The latter pathway is regulated primarily by Bcl-2 family proteins, which are distinct regulators of early-stage apoptosis; an interaction between pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 is necessary for the apoptotic process.¹⁵ Bax has been suggested as being necessary for developmental cell death,¹⁶ and Bcl-2 has been indicated for cell survival. Decreased Bcl-2 immunoreactivity and increased Bax immunoreactivity has been observed in injured neurons.¹⁷

A systematic search of the literature revealed that limited data are available regarding the effects of FA in experimental animals following acute oral exposure. Although there are a large number of animal studies related to FA exposure and its detrimental effects on different tissues, Bcl-2 family proteins together with GFAP expression have not been evaluated. For these reasons, we aimed to investigate the effects of acute oral exposure to FA on GFAP concentration and the immunohistochemical expression of pro- and anti-apoptotic proteins, as well as the number of apoptotic cells, labeled by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate–biotin nick-end labeling (TUNEL) staining in brain tissue of rabbits.

Methods

Animals

The study was performed upon the approval of the Gaziosmanpasha University Experimental Animals Ethics Committee at the Gaziosmanpasha University Experimental and Clinical Research Centre (DETAB), Turkey. A total of 16 New Zealand white rabbits, ranging in age from 8 to 10 months and weighing 1.2–1.8 kg were used for this experiment. The animals received food and water ad libitum and were kept in an air-conditioned animal house at 25 ± 2°C and 50−60% humidity, with a 12-h light–dark cycle. Their body weight was recorded daily during the experimental period. The animals were divided into two groups (group FA (F) and group control (C)) consisting of eight rabbits in each group. Nasogastric tubes were inserted into all the rabbits before FA or saline administration. FA was dissolved in triple distilled water, and the dose was calculated in weight/volume. For 5 days, group F received 40 mg/kg/day of FA via nasogastric tubes, while group C received saline (0.9% sodium chloride) at the same dose and route. The animals were observed daily. All the animals were euthanized after exposing for 5 days, and the brains were removed. The left hemisphere was collected in a 10% formalin solution and embedded in paraffin. The cortex was harvested from the right hemisphere and homogenized in ice-cold 0.05 M phosphate buffer (pH = 7.1) using an Ultra-Turrax^® T18 Basic homogenizer (IKA Werke GmBH & Co., Staufen, Germany). The samples were centrifuged at 1000g for 20 min, collected, and stored at –80°C until the measurements were taken.

Immunohistochemical staining of caspase-3, Bax, and Bcl-2

The immunohistochemical procedure has been described elsewhere.¹⁸ Serial sections were collected on poly-l-lysine-coated slides (Sigma-Aldrich, St Louis, Missouri, USA). The tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol. The sections were then treated in a microwave oven in a 10 mM citrate buffer, pH 6.0, for 5 min, twice. After three washes in phosphate-buffered saline (PBS), endogenous peroxidase activity was quenched with 3% hydrogen peroxide in PBS for 20 min. The sections were incubated in a blocking serum (ScyTek Laboratories, Logan, Utah, USA) for 10 min and then incubated overnight at 4°C with mouse monoclonal active caspase-3 (ab13847, 1:1200 dilution, Abcam, Germany), Bax (ab77566, 1:200 dilution, Abcam, Germany), and Bcl-2 (ab692, 1:100 dilution, Abcam, Germany) in a humidified chamber. After incubating with primary antibodies at 4°C overnight, sequential incubations were performed with biotinylated polyvalent antibodies (BioGenex, Fremont, California, USA) and peroxidase-labeled streptavidin (Biogenex). Immunohistochemistry was performed using a horseradish peroxidase-labeled streptavidin biotin kit (Biogenex) according to the manufacturer’s instructions.

Bound peroxidase was developed with 3-amino-9-ethylcarbazol (AEC) chromogen (ScyTek Laboratories), and the sections were counterstained with Mayer’s hematoxylin (ScyTek Laboratories). The sections from the controls were treated with the appropriate isotype mouse immunoglobulin Gs. Photomicrographs were taken with a light microscope (model DM2500, Leica, Nussloch, Germany).

Evaluation of immunohistochemistry

The intensities of active caspase-3, Bax, and Bcl-2 immunoreactivity were semi-quantitatively evaluated using the following intensity categories: 0 (no staining), 1+ (weak but detectable staining), 2+ (moderate or distinct staining), and 3+ (intense staining). For each tissue, an H-score value was derived by calculating the sum of the percentages of cells that stained at each intensity category and multiplying that value by the weighted intensity of the staining, using the formula $H - s c o r e = \sum P i (i + 1)$ , where i represents the intensity scores and Pi is the corresponding percentage of the cells. In each slide, five randomly selected areas were evaluated under a light microscope (40× objective). The percentage of cells for each intensity within these areas was determined at different times by two investigators, who were not informed about the type or source of the tissues. The average score of both observers was used.

TUNEL assay

TUNEL staining was carried out using an in situ cell death detection kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. The paraffin sections were deparaffinized in xylene, rehydrated, and treated in a microwave oven in a 10-mM citrate buffer, pH 6.0, twice for 5 min, and left to cool for 20 min. After three washes in PBS, endogenous peroxidase activity was inhibited with 3% hydrogen peroxide. The sections were then incubated with an equilibration buffer for 10–15 s, and terminal deoxynucleotidyl transferase enzymatic labeling of nuclear DNA strand breaks was carried out in a humidified atmosphere at 37°C for 60 min. TUNEL-positive cells were visualized and counterstained using an alkaline phosphatase substrate kit and nuclear fast red (F4648, Sigma-Aldrich). Each procedure was followed by several rinses in 100 mM of PBS. A negative control was similarly performed, except that the TUNEL reaction mixture was omitted. Counter staining was performed in Mayer’s hematoxylin, and the percentage of apoptotic cells stained pink was determined. Only cells showing nuclear condensation/fragmentation and apoptotic bodies in the absence of cytoplasmic TUNEL reactivity were considered apoptotic.

Measurement of apoptosis

The slides stained with the TUNEL technique were evaluated using a light microscope (model DM2500, Leica). The average number of apoptotic cells was determined by counting the TUNEL-positive cells in the five neighboring medium-power fields; the maximum number of stained cells was observed, and the total was divided by five. Cells in areas with necrosis, poor morphology, or margins of sections were not included.

Determination of GFAP

GFAP levels were measured on the prepared homogenate from brain tissue samples using a GFAP enzyme-linked immunosorbent assay kit (USCN Life Science Inc., Houston, Texas, USA) according to the manufacturer’s instructions. Briefly, 100 μl of standards (1000, 500, 250, 125, 62.5, 31.2, and 15.6 pg/ml) and samples were added to the anti-GFAP precoated plate and incubated for 2 h at 37°C. After incubation, the solutions in the plate were discarded, and 100 μl of detection reagent A was added to each well and further incubated for 1 h at 37°C. The solutions in the plate were , and the plates were washed three times using 350 μl of wash buffer supplied by the kit. Then, 100 μl of detection reagent B was added and incubated for 30 min at 37°C. The solutions were discarded, and the plate was washed five times using 350 μl of wash buffer. Afterward, 90 μl of substrate solution was added, and the plate was incubated for 15 min at 37°C in a dark area, after which 50 μl of stop solution was added, and absorbance measurement was conducted immediately at 450 nm. The sample values were calculated using a standard curve and expressed in picogram per milliliter fresh tissue.

Statistical analysis

Normality and variance were tested using the one-sample Kolmogorov–Smirnov test, skewness, kurtosis, and histograms for each variable. Quantitative data are presented as mean and standard deviation and qualitative data as frequency and percentage. Depending on these results, nonparametric analysis was undertaken for each variable. Biochemical value differences between groups were analyzed using the Mann–Whitney U test. Analyses were completed using the Statistical Package for Social Sciences (SPSS Inc., Chicago, Illinois, USA) version 20.0 program. Statistical significance for all analyses was set at p < 0.05.

Results

Apoptosis in cortex and hippocampus

TUNEL staining was performed to determine apoptotic cells, and TUNEL-positive cells were detected in both group C and in group F (Figure 1). Apoptotic cells appeared less frequently in group C (Figure 1(e) and (f)), while the number of apoptotic cells was significantly higher in group F (Figure 1(a) to (d)). When compared with the cortex (Figure 1(b) and (d)), more TUNEL-positive cells were observed in the hippocampus (Figure 1(a) and (c)); and this result was found to be significant (p = 0.003). The percentages of the TUNEL-positive cells in the cortices of groups C and F were 3.7 ± 0.6 and 16.8 ± 1.7, respectively, and 5.7 ± 0.9 and 27.8 ± 2.5, respectively in the hippocampus (Figure 2).

Figure 1.

Representative photos of TUNEL staining in the hippocampus ((a), (c), (e)) and cortex ((b), (d), (f)) of rabbit brains. The black arrows indicate TUNEL-positive apoptotic cells. TUNEL staining indicates a large number of TUNEL-positive apoptotic cells in group F ((a) to (d)) compared with group C ((e) and (f)). Scale bars: 50 μm. TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling.

Figure 2.

Semi-quantification for TUNEL staining in the cortex and hippocampus of rabbit brains. The number of TUNEL-positive apoptotic cells significantly increased in group F compared with that in group C, both in the hippocampus and cortex of rabbit brains. Asterisks indicate the significant differences between group C and group F (p < 0.001). TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling.

Alterations in active caspase-3, Bax, and Bcl-2 immunoreactivities following FA exposure

In the control animals, active caspase-3, Bax, and Bcl-2 immunoreactivity were observed as diffuse patterns of staining in the perinuclear cytoplasm (Figure 3(a) to (h)), and the neurons appeared round and healthy. In addition, no immunoreactivity was observed either when sections were preincubated with immunizing antigen peptides or when the primary antibody was omitted from the protocol (Figure 3(c), (f), and (i)). In contrast to the dramatic reduction in cytoplasmic Bcl-2 immunostaining (Figure 3(h)), neurons in the cortex were characterized by a pronounced increase in the cytoplasmic active caspase-3 and Bax immunostaining (Figure 3(b) and (e)) in group F. According to H-score analysis, in group F, the number of Bcl-2 immunoreactivity showed a substantial decrease, since caspase-3 and Bax immunoreactivity were significantly increased (Figure 4).

Figure 3.

Representative photographs of active caspase-3 ((a) to (c)), Bax ((d) to (f)), and Bcl-2 ((g) to (i)) immunohistochemistry. The black arrows indicate immunopositive cells. The number and intensity of caspase-3 and Bax immunopositive cells significantly increased in group F ((b) and (e)) compared with group C ((a) and (d)). Bcl-2 immunostaining demonstrates strong immunoreactivity in group C ((g)), while moderate downregulation in group F (h) is seen. Negative controls of each immunostaining are also seen ((c), (f), and (i)). Scale bars: 50 μm.

Figure 4.

H-score values of caspase-3, Bax, and Bcl-2 immunostaining intensities in groups C and F. Cytoplasmic caspase-3 and Bax immunoreactivities significantly increased in group F (129 ± 10.9 and 148 ± 15.9, respectively) compared with those in group C (31.25 ± 5.2 and 35 ± 6.3, respectively), while cytoplasmic Bcl-2 staining significantly decreased in group F (50 ± 10.2) compared with group C (125 ± 12.5). The data are represented as mean ± SEM. Asterisks: p < 0.001, group C versus group F for caspase-3, Bax, and Bcl-2.

GFAP concentration

The mean GFAP values of the groups are presented in Table 1. Group F had a significantly higher mean GFAP value than group C (p = 0.006).

Table 1.

Mean GFAP values of the fresh brain tissue samples.

		Group F (n = 6)	Group C (n = 6)	p Value
GFAP	Mean ± SD	633.66 ± 183.66	314.33 ± 93.50	0.006^a,b
GFAP	CI	440.92 − 826.41	216.20 − 412.46

CI: confidence interval; GFAP: glial fibrillary acidic protein.

^aMann Whitney U test.

^b p < 0.01.

Discussion

The present study revealed that FA lead to apoptosis associated with the changes in active caspase-3, Bax, and Bcl-2 expression and DNA damage in the rabbit brains. Our results also demonstrated that GFAP values, an indicator of nervous system toxicity, significantly increased in group F.

Although the mechanism of toxicity has not been elucidated in detail, recent studies have demonstrated that oxidative damage is judged as the most critical effect of FA exposure.^19–23 It is mediated by providing a balance between prooxidant and antioxidant systems.^24,25 The neuron cytotoxic action of FA can be regulated by the activation of free-radical-producing enzymes and by the inhibition of antioxidant systems, which results from an increase in the production of the reactive oxygen species (ROS). Moreover, FA is a substrate for cytochrome P-450 monooxygenase system II E1 isozyme and can be oxidized in the endoplasmic reticulum by peroxidase, aldehyde oxidase, and xanthine oxidase.^26,27 Activity of these enzymes leads to a higher rate of ROS formation, which causes damage to biomolecules, including membrane lipids, proteins, and nucleic acids. Furthermore, the CNS has a higher content of readily peroxidizable unsaturated fatty acids in cell membranes, and they are easily attacked by ROS, resulting in the peroxidation of lipids. Membrane lipid peroxidation can impair membrane fluidity and cell structure, which can lead to cell lysis. Consequently, lipid peroxidation and protein oxidation associated with oxygen radicals might participate in impaired cellular function and necrosis.²⁸ Therefore, FA causes neuronal injury, which is associated with the accumulation of ROS formation and results in oxidative damage.²²

It has been reported that FA has toxic effects on the CNS and causes many morphological changes in rat brains.^20,29 In addition, inhaled FA has been shown to cause behavioral and memory disorders in rats and has been classified as “probably neurotoxic”.³⁰ However, in a recently published investigation on the anti-apoptotic role of FA in cultured hippocampal neurons,¹³ the authors showed that activity of apoptosis-associated caspase-3 and -7 was suppressed by FA, which is in contrast to our findings.

In a report, Restani and Galli indicated that the effects of FA are related to the volume and concentration of the ingested solution.¹ To explain the long-term toxicity, Tobe et al. conducted a 24-month drinking water study, administrating FA to 20 male and 20 female Wistar rats at concentrations of 0 (control), 0.02, 0.10, and 0.50%. All rats in the 0.50% group died by 24 months, and they showed a no-observed-effect level of 10 mg/kg/day for drinking water (0.02%).³¹ Thus, it can be suggested that the FA concentration and duration of exposure time are critical in order to see the detrimental effects of FA. In the present study, high doses of FA were given to rabbits via nasogastric tubes for 5 days and not inhaled. Because of the high dose and the different application route, we observed a substantial number of detrimental effects of FA on the brains.

Numerous studies have demonstrated that FA induces apoptosis and disturbs tissue integrity,^32,33 and several mechanisms have been suggested for FA-induced apoptosis. Changes in the expression of Bcl-2 family proteins are usually thought to lead to apoptosis through the intrinsic route.³³ In the present study, Bcl-2 expression decreased, since caspase-3 and Bax expression increased in group F. Consistent with our results, Tsukahara et al.³⁴ also reported that the inhalation of FA increased Bax/Bcl-2 expression in the hippocampus. In another study, the prefrontal cortex of rats was affected by the inhalation of FA, and melatonin was used to prevent this toxic effect.²⁰

Although neuronal death has been associated with changes in Bcl-2 and Bax gene expression, in vitro models of apoptotic cell death indicate that an alteration in the cellular localization of these proteins could result in cell death.³⁵ In response to a death signal, Bax may translocate to the mitochondria and may be associated with a release of Bcl-2 and cytochrome C from the inner mitochondrial membranes.³⁶ In our analyses on the nature of the immunohistochemical staining with antibodies to Bcl-2 and Bax, we observed the punctate intracellular labeling characteristic of mitochondria. In fact, Bcl-2 immunoreactivity was characterized by a diffuse, perinuclear cytoplasmic pattern. The results of the present study suggest that reduced Bcl-2 reactivity in group F appeared to show overt cell loss.

In addition to its role as an anti-apoptotic protein during development, Bcl-2 can prevent cell death caused by multiple pathways.^11,37,38 Bcl-2 overexpression in neuronal cultures has been reported to prevent, in vitro, necrotic and apoptotic cell death in vitro induced by calcium ionophores, glutamate, hydrogen peroxide, and hypoxia.^38–40 The reduction or loss of Bcl-2 in group F was associated with cell death or cell loss. It is tempting to speculate that the disappearance of Bcl-2 in neurons of group F might exacerbate their vulnerability to this toxic substance. We do not know whether this reduction or cell loss can be regenerated by another mechanism. Indeed, there are studies showing that the overexpression of Bcl-2 leads to neuroprotection following axotomy in vivo.⁴¹

Using TUNEL assays to detect DNA fragmentation, we also observed more apoptotic neurons in the cortex and hippocampus of group F. In accordance with our findings, other researchers have also detected TUNEL-positive cells after FA treatment in animals.^20,34

The term “astrogliosis” is also important to mention when explaining the effects of FA exposure. Astrogliosis is a typical response of the CNS resulting from trauma, disease, genetic disorders, or chemical insult. Astrogliosis is also characterized by the rapid synthesis of GFAP.⁴² GFAP first isolated by Eng et al. in 1971 is the principal intermediate filament and represents the major part of the cytoskeleton of astrocytes.^9,42,43 Various studies have shown the complex responses of astrocytes to injury. Increased protein content and immunostaining of GFAP have been found in experimental studies following gliosis, in which trichloroethane, xylene, dichloromethane, colchicine, 6-hydroxydopamine, 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine, ethanol, and 3,3′ iminodipropionitrile were tested.⁹

Inhalation, dermal, and local contact are all forms of exposure that can lead to FA toxicity. The oral route has been taken into account less frequently, as it is thought that human exposure to FA is relatively unimportant. In relation, low-birth weight, increased abnormal sperm incidence, and tumor-promoting activity in the glandular stomach are the effects of oral exposure to FA that have been identified by various experimental studies.^44–47 As a consequence of using FA in food containers, food packaging materials, and food preservatives, FA is found in fresh and preserved fish, seafood, honey, roasted foods, tomatoes, apples, cauliflower, spinach, and carrots;¹ therefore it must not be underestimated that a significant number of people are orally exposed to FA.^3,48

In conclusion, this model suggests that acute oral FA exposure can lead to neuronal damage in the cortex and hippocampus. However, further experimental and clinical studies are required to elucidate the effects of acute oral exposure to FA.

Footnotes

Authors Note

Authors SA, SC, AA, and MS contributed to the study design, conduct of the study, data collection, data analysis, and manuscript preparation, SK served as a scientific advisor, SD reviewed the original study data and data analysis and also served as a scientific advisor, and authors TK and ZK helped with critical editing.

Conflict of interest

The authors declared no conflicts of interest.

Funding

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

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Central nervous system toxicity after acute oral formaldehyde exposure in rabbits

Abstract

Keywords

Introduction

Methods

Animals

Immunohistochemical staining of caspase-3, Bax, and Bcl-2

Evaluation of immunohistochemistry

TUNEL assay

Measurement of apoptosis

Determination of GFAP

Statistical analysis

Results

Apoptosis in cortex and hippocampus

Alterations in active caspase-3, Bax, and Bcl-2 immunoreactivities following FA exposure

GFAP concentration

Discussion

Footnotes

Authors Note

Conflict of interest

Funding

References