Clinical and Experimental Study of the Toxic Neuropsychiatric Effects of Formaldehyde Exposure: Has Garlic a Protective Role?

Background: The central nervous system is one of the most important systems affected by formaldehyde (FA). Aim of Work: This study was designed to assess its toxic neuropsychiatric effects both clinically and experimentally and the protective effects of garlic. Methods: Clinically: 20 workers in the gross anatomy laboratory and 20 libertarians underwent a standardized clinical assessment including medical, neurological and psychiatric examination. Experimental: 40 male rats were divided randomly into four groups. Group I is control group. Group II received 10 mg/kg of FA intraperitoneally once daily for 14 days. Group III was treated with fresh garlic juice (1 ml/100g body weight) once daily by oral gavage for 14 days. Groups VI received fresh garlic juice plus formaldehyde daily for 14 days. At the end of the experiment, the rats’ brains were obtained for histological examination and biochemical analysis. Results: Clinical and psychiatric profile of FA exposed persons’ revealed cognitive impaired, anxious and depressed persons. There were hostile persons with more hostility toward outside. Experimentally, hippocampal and frontal superoxide dismutase and reduced glutathione showed highly significant decrease while malondialdehyde and nitric oxide level showed highly significant increase in formaldehyde treated group when compared with control group. Also histopathological changes in the hippocampal and frontal cortices by light microscope revealed many distorted cells with deeply stained shrunked nuclei and cytoplasm was surrounded by vacuolated pale areas in FA exposed group. Minimization of biochemical and histopathological changes were observed in combined formaldehyde and garlic treated group. Conclusion: The profiles of personality arouse dangerous affairs about the toxic impact of FA on persons, family, and society. Formaldehyde-induced neuronal damage, oxidative stress and lipid peroxidation in brain were minimized by addition of garlic.

• Formaldehyde
• Neurotoxicity
• Neuropsychiatry
• Garlic
• Oxidative Stress
• Cognitive Impairment

1. Introduction

Formaldehyde (FA) is a ubiquitous chemical agent. It is a part of our general outdoor environment. Also, it is a part of our indoor working and residential environment. It is believed that whole civilized population is exposed to formaldehyde [1] [2] . Formaldehyde, which is an environmental pollutant, is generated in substantial amounts in the human body during normal metabolism [3] . The common commercial form of formaldehyde (formalin) contains 37% - 50% formaldehyde in water by weight and is stabilized against polymerization by the addition of 1% - 15% methanol. Formaldehyde is also commercially available as solid containing 91% - 95% formaldehyde and 5% - 9% water (paraformaldehyde) [4] . Some of the commonly used products containing either formaldehyde or formaldehyde-releasing substances are used as construction materials, fertilizers, fumigants, polish, cosmetics, paints, cleaning agents, and toiletries [5] [6] [7] .

In addition, FA can be produced as burning product of wood, coal, tobacco, natural gas, and kerosene [6] [8] . Also, foods as coffee, codfish, meat, poultry, and maple syrup naturally contain formaldehyde [6] [9] .

In the medical field, FA is often utilized in laboratories. Fixation and long-run storage of organs and cadavers are achieved by FA. Also, it's utilized in tissue fixation stage in microscopic anatomy and pathology laboratories. Additionally, it's usually utilized in dental coating materials, within the treatment of cases with persistent urinary tract infection and as a protecting agent for a few medicines. Moreover, dialysis solutions typically contain formalin [10] [11] [12] . The permissible exposure limits for formaldehyde in all workplaces which were estimated by the National Institute for Occupational Safety and Health (NIOSH) are in the range of 1.5 - 3 mg/m³. However, many studies have proved that the FA levels in occupational sites are commonly exceeding that [13] [14] .

Formaldehyde has a high propensity for reacting with RNA, DNA, and protein, which leads to many health hazard [15] [16] . It has toxic effects on the skin, eyes, respiratory tract, urogenital system, and the nervous system [4] [17] [18] . Exposure to FA has been reported to cause neurotoxicity in both humans and animals and the extent of damage depends on the dose and the duration of the exposure [19] [20] [21] [22] . Garlic (Allium sativum), that could be a member of the family Liliaceae, has been used as a medicating ingredient with physiological potential for an extended time. Garlic contains sulfur, phosphorus, potassium and zinc ions, moderate amounts of selenium, vitamin C, antiophthalmic factor and smaller amounts of calcium, magnesium, sodium, iron, B-complex vitamins and allicin. The inhibitor effects of garlic because of allicin, that entice free radicals [23] [24] . In addition, the sulfur has anti-mutagenic and anti-carcinogenic effects [25] .

This study was designed to assess the toxic neuropsychiatric effects of FA clinically and experimentally and the protective effects of garlic (as an antioxidant) against the neurotoxicity-induced by FA in rats.

2. Methods

This study consisted of two parts:

1) Clinical comparative cross section study

2) Experimental study

2.1. Clinical comparative Cross Section study

The study was conducted in Zagazig University, Faculty of Medicine in the duration from 1st of October 2015 to 31th of December 2015. Twenty workers (employees and instructors) in the gross anatomy laboratory (lab), for more than one year, and 20 librarians were included in this study.

The inclusion criteria included: Adults’ ≥21 and ≤50 years old spent at least one year working regularly in gross anatomy lab or library with normal routine laboratory investigation such as blood sugar, lipid profile (cholesterol, triglyceride, HDL and LDL), kidney function test and liver function test.

- Absence of any gross abnormality in medical examination with normal pulse, blood pressure, heart rate and temperature.

- Chest, heart, abdomen and neurological examinations are clinically free.

- No manifestations of epidemic or endemic infections.

The exclusion criteria included substance abuse, hepatic or renal disease, malignant tumor, surgery within 6 month, neurodegenerative disorder, any medical disorder such as hypertension, diabetes and any previous history or present history of psychiatric disorders.

This study was carried out within the ethics of scientific research and an ethical approval for this study was obtained from the Institutional Review Board of Zagazig University, faculty of medicine, the approval number ZU-IRB #4045-2-9-2015. All subjects were fully informed about the nature and objectives of this study and a written informed consent was taken from them. All data are confidential and used only for the research purpose and they were not exposed to any harm or risk.

All participants underwent:

・ A standardized clinical assessment: including medical, neurological and psychiatric examination.

・ Mini-Mental state examinations [26] .

・ Hostility Quantity and Hostility Direction Questionnaire (H-Q-H-D) [27] . Arabic version [28] .

・ Middlesex hospital questionnaire (MHQ) [29] . Arabic version [30] .

2.2. Experimental

The study was conducted using adult albino rats. Females’ hormones have an effect on the results [31] so adult male rats were used for this study. The rats were maintained according to the guidelines of the Institutional Animal Ethics Committee. They were kept in polypropylene cages at the temperature 24˚C, 45% relative humidity, and 12 hours light and dark cycles with free access to drinking water and food. Possible confounding factors in this study were the weight, age, environment, water and food of the rat. However, these conditions were identical for all of the rats and we controlled any confounding factors. The rats age range (6 - 8) weeks and their weights were (200 - 250) grams.

A total of 40 male rats were divided randomly into four groups. Group I (control) received an intraperitoneal (ip) injection of normal saline every day. The rats in Group II received 10 mg/kg of formaldehyde ip once daily for 14 days concentration 36.6% to 38% in water [32] . Group III: rats were treated with fresh garlic juice (1 ml/100 g/Body weights) once a day by oral gavage for 14 days [33] . Groups VI was received oral gavage of fresh garlic juice (1 ml/100 g/Body weights) plus ip formaldehyde (10 mg/kg) daily for 14 days.

At the end of the two weeks experimental period, all of the rats were scarified and their brains (frontal and hippocampus) were obtained for histological examination and biochemical analysis.

2.2.1. Rationale of Using garlic Juice

Many people cannot freely eat fresh raw garlic (FRG) because of its intense taste and smell even though they know that garlic is good for their health. In addition, the consumption of FRG is often associated with several health hazards, such as stomach and digestion problems [34] . Therefore, different formulations of garlic preparation including aged garlic extract (AGE), dehydrated garlic powder, garlic oil and garlic oil macerate, etc. were developed [35] .

So, we decided to use garlic juice in this study for easy preparation, administration and to avoid any gastric problem to rats.

2.2.2. Preparation of Garlic juice

To prepare garlic juice, garlic bulbs were separated, peeled and washed with distilled water. After drying in a shed, the clean garlic bulbs were crushed with an electric grinder and the extract was decanted carefully through muslin cloth [36] .

・ Brain tissue Sampling

At the end of the study, the rats were euthanized by decapitation and their brains (frontal and hippocampus) were taken. Each brain was divided into two parts; one was flash frozen in liquid N2 and stored at -80˚C for later use in measures of malondialdehyde (MDA) and reduced glutathione (GSH) contents, superoxide dismutase (SOD) activities and nitric oxide (NO) level. The other part was processed for histologic examination.

・ Biochemical Study

Reduced glutathione (GSH) contents in the brain were assessed according to the method of Ahmed et al. [37] based on a 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) reaction. Briefly, 0.1 g tissue was homogenized in 1 ml phosphate buffer (pH = 8) at 4˚C. An aliquot (0.5 ml of homogenate) was mixed with 0.5 ml 10% TCA in 5 ml EDTA solution and the mixture was then centrifuged at 2000 ×g for 5 min. The supernatant generated was used for the determination of GSH at 412 nm in the spectrophotometer. As total protein content was needed for calculation of GSH content in the tissues, this parameter was determined using a Biocon Diagnostic kit (GmbH, Vohl-Marienhagen, Germany). All data were expressed as nmol GSH/mg protein.

Brain Tissue SOD activity was measured according to the method of Nishikimi et al. [38] . An aliquot of splenic or thymic homogenate supernatant (50 µl) was combined to 100 µl sodium pyrophosphate buffer (pH 8.3), 0.1 ml of 0.3 M nitrobluetetrazolium, and 0.1 ml of 780 µM NADH; after mixing, 10 µl of 186 µM phenazinemethosulfate (PMS) solution was added to initiate the reaction. Because SOD enzyme inhibits PMS-mediated reduction of nitrobluetetrazolium, the rate of increase in absorbance (measured at 560 nm) was used to reflect SOD content in the sample. All data were ultimately reported as U/g tissue, with 1 U = the amount of enzyme required to produce a 50% inhibition in NBT reduction.

Brain contents of MDA (indices of lipid peroxidation) were determined spectrophotometrically using a commercial kit (DokkiBiodiagnostic, Giza, Egypt). Briefly, an aliquot of tissue extract supernatant was mixed with 1 ml 5% trichloroacetic acid and then centrifuged at 2500 ×g for 10 minutes. Supernatant (0.2 ml) was transferred to a test tube and then 0.2 ml of 8.1% sodium dodecyl sulfate (SDS), 1.5 ml of 30% acetic acid (pH = 3.5) and 1.5 ml of 0.8% thiobarbituric acid (TBA) were added. The tube was mixed, covered with glass beads, heated in water bath (at 95˚C) for 30 minutes and then cooled. After centrifugation (4000 ×g, 10 min), the supernatant was isolated and absorbance of the pink color was measured at 532 nm in Model UVD-2950 scanning spectrophotometer (Labomed Inc., Los Angeles, CA). A standard curve was generated using 1,1,3,3-tetraethoxypropane; from this curve, MDA levels in each sample were extrapolated. All data were expressed as nmol MDA/g tissue processed, Ohkawa et al. [39] .

Nitric oxide (NO) was determined using colorimetric assay where nitrate is converted to nitrite via nitrate reductase. Griess reagent then act to convert nitrite to a deep purple azo compound that can be determined using spectrophotometer [40] .

・ Histopathological Examinations

Brain tissues were embedded in paraffin wax and cut into sections (3 - 5 μm thickness) and stained with haematoxylin and eosin (H and E). Examination of the stained tissue sections was done by a pathologist, who was blinded to the protocol of the study.

・ Statistical Analysis

Continuous variables are reported as means with SD if normally distributed, an independent t test used for comparing two groups and An ANOVA with Tukey Kramer’s test used for multiple comparisons. Qualitative data were represented as frequencies and percentages, Chi-square test (χ²) and fisher exact test were used for comparing groups. The test results were considered significant when p-value < 0.05 and all p values were two-tailed. Data were analyzed using Statistical Package of Social Science (SPSS), software version 16.0 [41] .

3. Results

3.1. Clinical Study

Regarding sociodemographic characteristics, both FA-exposed persons (gross anatomy Lab. workers) and control group (librarians) were matched for age, gender, educational level and marital status (table 1).

Clinical assessment of both groups revealed that the most common physical symptom reported by FA-exposed group was: easy fatigability, headache, excessive sleep, anorexia, eye irritation, excessive lacrimation, chest tightness and cough.

Mini-mental state examination in the present study revealed that, FA-exposed persons were more cognitively impaired than control group (24.95 ± 3.4 and 27.2 ± 2.6 p = 0.03) respectively (figure 1).

Middlesex hospital questionnaire (MHQ) of studied groups had shown a statistically significant difference between the two groups. FA exposed group suffered from more anxiety, somatization and depression (table 2).

Hostility Direction and Hostility Quantity Questionnaire (HDHQ) revealed that FA exposed group was more hostile than control group. The direction of hostility in FA exposed group was more toward outside than control group (table 3).

3.2. Experimental study

3.2.1. Biochemical Study

There was a non statistical significant difference between the control group and garlic treated groups as regard superoxide dismutase (SOD) activities, reduced glutathione (GSH), malondialdehyde (MDA) and nitric oxide level in brain tissue (Table 4).

So the negative control group was chosen to compare with the FA treated group and formaldehyde and garlic treated group.

Mean values of SOD and GSH show high statistical significant decrease While MDA and NO level show high statistical significant increase in formaldehyde treated group when compared with control group (Table 5).

Table 1

Sociodemographic data among different studied groups

p value	test	Librarians (control)	Gross Anatomy lab workers (FA-exposed )
0.61	0.509*	23 - 54 36 ± 5.972	25 - 50 35 ± 6.451	Age (years) Mean ± SD
0.25	1.16*	1 - 30 16.5 ± 2.601	1 - 26 15.5 ± 2.853	Duration of work (years) Mean ± SD
0.507	0.44**	N. (%) 12 (60.0) 8 (40.0)	N. (%) 14 (70.0) 6 (30.0)	Sex Male Female
0.31	1.03**	12 (60.0) 8 (40.0)	15 (75.0) 5 (25.0)	Marital state Married Non married
0.34	3.37**	4 (20.0) 9 (45.0) 5 (25.0) 2 (10.0)	7 (35.0) 10 (50.0) 3 (15.0) 0 (0.0)	Education Basic Secondary High Postgraduate

SD: Standard Deviation; N. (%): Number (percent); *student t tests for quantitative variables; **chi square test for qualitative variables.

Table 2

Middlesex hospital questionnaire (MHQ) among studied groups

P vlue	t test	Librarians (control) Mean ± SD	Gross Anatomy lab workers (FA-exposed) Mean ± SD
0.001**	3.44	2.15 ± 1.01	3.95 ± 2.11	Free-floating anxiety
0.68	0.405	2.31 ± 1.17	2.45 ± 1.01	Phobia
0.76	0.304	5.15 ± 2.65	4.9 ± 2.55	obsessions
0.004*	3.06	3.95 ± 2.01	6.55 ± 3.22	Somatization
0.002*	3.43	3.40 ± 1.64	5.95 ± 2.89	Depression
0.32	1.007	4.28 ± 1.44	4.75 ± 1.51	Hysteria

SD: Standard Deviation; t test: Student t test; *: Significant (P < 0.05) **: Highly?significant (P < 0.001).

Table 3

Hostility Direction and Hostility Quantity Questionnaire (HDHQ) among studied groups

P value	t test	Librarians (control) Mean ± SD	Gross Anatomy lab workers (FA-exposed) Mean ± SD
0.002*	3.29	3.10 ± 1.12	4.35 ± 1.28	Self-criticism
<0.001**	5.078	1.3 ± 0.63	2.7 ± 1.06	Paranoid hostility
0.006*	2.88	3.15 ± 1.41	4.65 ± 1.85	Acting out
0.009**	2.75	4.5 ± 1.91	6.25 ± 2.11	Criticism of others
0.002*	3.39	2.75 ± 1.1	4.15 ± 1.48	Guilt feeling
0.046*	2.06	15.65 ± 6.66	19.85 ± 6.24	Total hostility degree
0.013*	2.62	0.95 ± 5.01	-2.75 ± 3.86	Direction of hostility

SD: Standard Deviationt; t test: Student t test; *: Signifiant (P < 0.05); **: Highly significant (P < 0.001).

Table 4

Statistical comparison between the negative control and garlic treated group as regard SOD (U/L), GPx (ng/ml), MDA (mmol/l) and nitric oxide levels in brain tissues along the period of the study by t test

Parameter in brain tissue	Negative control group (I) Mean ± SD	Garlic treat group (III) Mean ± SD	t test	P value
SOD (U/L)	64.46 ± 5.72	64.34 ± 5.35	0.048	0.96
GPx (ng/ml)	28 ± 4.62	27.81 ± 4.57	0.093	0.93
MDA (mmol/l)	98.35 ± 8.99	97.85 ± 5.07	0.15	0.88
Nirtric oxide (µmol/g)	45.84 ± 2.08	45.99 ± 2.04	0.16	0.87

SD: Standard Deviation. Number of sacrificed rats for each group was 10 rats. t test: Student t test; SOD: Superoxide dismutase; GPx: Glutathione peroxidase; MDA: Malondialdehyde.

Table 5

Statistical comparison among the negative control, formaldehyde group, formaldehyde and garlic treated group as regard SOD (U/L), GPx (ng/ml), MDA (mmol/l) and nitric oxide levels in brain tissues along the period of the study by ANOVA and post hoc test

Parameter in brain tissue	Negative control group (I) Mean ± SD	FA treated group (III) Mean ± SD	FA and Garlic treated group (IV) Mean ± SD	ANOVA	P value
SOD (U/L)	64.46 ± 5.72	28.85 ± 2.56a	50.88 ± 6.02a,b	128.311	<0.0001**
GPx (ng/ml)	28 ± 4.62	10.62 ± 1.6a	20.54 ± 5.77a,b	39.873	<0.0001**
MDA (mmol/l)	98.35 ± 8.99	402.34 ± 4.9 a	115.48 ± 5.18a,b	6645.477	<0.0001**
Nirtric oxide (µmol/g)	45.84 ± 2.08	109.56 ± 3.05	65.03 ± 4.17a,b	1033.506	<0.0001**

Number of sacrificed rats for each group was 10 rats. SD: Standard Deviation; ANOVA: Analysis of variance; **: Highly?significant (P<0.001); a = significant versus control group; b = significant versus formaldehyde treated group; SOD: Superoxide dismutase; GPx: Glutathione peroxidase; MDA: Malondialdehyde.

In formaldehyde and garlic treated group SOD and GSH show high statistical significant increase while MDA and NO show high statistical significant decrease when compared with formaldehyde treated group (Table 5).

3.2.2. Histopathological Study

Specimen from brain of both control and garlic treated group showing few granular cells and pyramidal cells with vesicular nuclei, basophilic cytoplasm and processes. The surrounding neuropil contains nerve fibers and blood vessels (Figure 2, Figure 3).

While specimens from brain of formaldehyde treated rats’ revealed histopathological changes in the brain by light microscope which revealed many distorted cells with deeply stained shrunken nuclei and cytoplasm surrounded by vacuolated pale areas. Few pyramidal cells appeared normally (Figure 4). Most of these changes were reversed in formaldehyde and garlic treated group (Figure 5).

4. Discussion

Formaldehyde is widely used chemical compound in industrial field. It’s a member of the aldehyde family and one in all the simplest organic molecules. FA is taken into account as a typical indoors and outdoors waste matter [42] . The perception of FA by odor and eye irritation becomes less sensitive with time because of adaptation. This result of overexposure to formaldehyde is to alert

employee (him or her) to the potential threat [43] . The CNS is one of the commonest systems affected by the FA. Occupationally subject worker to FA includes anatomists, histologists, pathologists, students, and nurses working at dialysis units [22] [44] .

Neurotoxicity is any effects on the structure or function of both the central and/or peripheral nervous system. The nervous system is of particular important as mature neurons are incapable of regeneration [45] .

Our study is a trial to evaluate the neuropsychiatric sequel to chronic FA exposure and the role of garlic (as antioxidant) in protection from FA-induced neurotoxicity your paper.

4.1. Clinical study

Both FA-exposed persons (gross anatomy Lab. workers) and control group (librarians) were matched for several factors like age, gender, educational level and marital status. Clinical assessment of both groups revealed that, the most common physical symptom reported by FA-exposed group was: easy fatigability, headache, excessive sleep, anorexia, eye irritation and excessive lacrimation, chest tightness and cough. These results came closely in agreement with those obtained by [46] [47] [48] [49] , who stated that, several manifested symptoms might be related to formaldehyde exposure like lethargy, decrease in motor activity and loss of appetite.

Mini-mental state examination in the present study revealed that, FA-exposed persons were more cognitively impaired than control group. Four FA-exposed persons suffered from mild cognitive impairment (18 - 23) [50] . Epidemiological investigations indicate that exogenous formaldehyde exposure causes human cognitive decline [44] [51] [52] . Also, inhaled FA has been shown to cause behavioral and memory disorders in rats and has been classified as ‘probable neurotoxic’ [53] [54] .

Tong et al. [55] stated that, exposure of normal mice to FA leads to marked memory decline.

Also, exogenous formaldehyde exposure causes human cognitive impairment and animal memory loss; furthermore many studies proved that formaldehyde at pathological levels induces Aβ deposition and misfolded tau protein to form globular amyloid-like aggregates [55] .

Middlesex hospital questionnaire (MHQ) of studied teams had shown a statistically important distinction between the 2 teams. FA exposed team suffered from additional anxiety, somatization and depression. Epidemiological studies have shown that work-related exposure to FA leads to headaches, fatigue, anxiety, sleep disorders, and particularly cognitive disorders [44] [56] . Different concentrations of aerosolized formaldehyde lead to completely different effects on anxiety, depression-like behavior and cognition ability which can be related to alterations in hippocampal glucocorticoid receptors and brain tyrosine hydroxylase levels [57] . FA can react with the nerve proteins that referred to as neuroamines and neurotransmitters (as catecholamine) which might impair neurological system performance and may cause endocrine disruption [58] . FA inhalation might increase activity of the hypothalamic-pituitary-adrenal (HPA) axis therefore mitigate FA neurotoxicity. The recurrent exposure to low level formaldehyde alters HPA axis functioning and the release of stress hormones [59] [60] . There are several reports of malaise, headache, dyspepsia, balance and sleep disorders, and mental and memory changes because of FA exposure [44] [47] .

The study of hostility within the studied teams by, Hostility Direction and Hostility quantity questionnaire, discovered that, formaldehyde-exposed cluster were additionally hostile than control cluster. The direction of hostility in formaldehyde-exposed cluster was higher toward outside than control cluster. Self-criticism, paranoid hostility, acting out hostility, criticism of others were statistically higher among formaldehyde-exposed cluster as compared to controls. The results of animal experiments reveal that inhaled formaldehyde induces abnormal behaviors, such as: aggression, depression, a decline in movement activity, and spatial memory deficits [61] [62] [63] . Formaldehyde has many effects on memory, learning, and behavior [3] .

4.2. Experimental study

We examined the toxic effects of exposure to FA on the brain (hippocampus and frontal cortex) of adult male albino rats and the role of garlic to minimize these effects.

Many studies have discussed the toxic effects of FA on the central nervous system [62] [64] [65] . Long-term exposure to FA can lead to irreversible neurotoxicity [51] . FA has been found to cause different changes in the rat brain [66] . It was also reported that FA is capable of damaging the prefrontal cortex, including the hippocampus of rats [48] [49] . Mohamad et al. [67] stated that FA cause severe neurodegenerative changes in rats. The results of above-mentioned studies were parallel with our results, in which the histopathological study of brain (hippocampus and frontal cortex) of rats showed many distorted cells with deeply stained shrunken nuclei and cytoplasm surrounded by vacuolated pale areas. In detail, toxicological investigations have shown clear associations between formaldehyde exposure and brain tissue damage, increases cell proliferation [68] [69] , DNA damage [70] [71] [72] , inflammation [73] [74] , changes in miRNA expression [75] and changes in gene expression signatures [74] .

FA, which causes an increase in cytotoxic effects by compromising the intracellular balance, has a tendency to bind with proteins, nucleic acids and unsaturated fatty acids. These combinations lead to inflammatory reactions, allergic reactions, cytotoxicity, necrosis, mutagenesis and carcinogenesis by proteins denaturation. In addition, increase of free oxygen radicals in the FA exposed tissues and acceleration of apoptosis that cause cell death [21] [48] .

In the present study, Formaldehyde showed decrease in the endogenous antioxidant system (glutathione peroxidase; GSH and superoxide dismutase; SOD). In contrast, malondialdehyde (MDA) activity, and nitric oxide (NO) levels were significantly increased.

In line with our results, numerous studies have stated that, formaldehyde (FA) exposure causes neuronal damage with oxidative stress which is considered as one of the most critical mechanism of its toxicity [76] .

Chang and Xu [77] and Zararsiz et al. [49] suggested that, formaldehyde causes oxidative stress and lipid peroxidation. Their study showed decrease in the activity of superoxide dismutase (SOD), GSH-Px and increase in the concentration of malondialdehyde (MDA) which was consistent with our study.

The research of Zhang et al. [78] ; Datta and Namasivayam [79] ; Tang et al. [80] indicated that exposure to formaldehyde in experimental animals causes impairment of antioxidant enzyme activity, ROS-induced membrane lipid and protein oxidation leading to delayed apoptotic or necrotic cell death. Gouriou et al. [81] explained that, oxidative stress causes neuronal cell death by attacking cellular components. As ROS open mitochondrial permeability transition pore, resulting in mitochondrial swelling and necrosis [82] .

Our study showed increase in level of NO; an explanation for this increment comes from Tang et al. [80] . They hypothesized that FA might cause neurotoxicity through the deficiency of hydrogen sulfide (H2S), which is considered as an endogenous protective antioxidant. Deficiency of H2S results from excessive generation of nitric oxide (NO) due to the inhibition of the activity of cystathionine beta synthase (CBS), a predominant H2S-generating enzyme in the central nervous system. This hypothesis was studied by Li et al. [83] ; Tang et al. [76] . They stated that H2S can be used to protect against FA induced neurotoxicity. They explained that, one of the mechanisms of FA-induced neurotoxicity involves ER stress [84] . On the other hand, and opposite to our results, the study of Mohamad [67] showed significant decrease in the levels of NO in the brain tissue of FA-treated rats, due to the effect of superoxide which reduces NO bioavailability by binding to the gaseous molecule and forming the free radical, peroxynitrite.

As a result of the above-mentioned toxic effects of FA on the brain, and that, oxidative stress is one of the proposed mechanisms, so, it was crucial to develop effective therapeutic drugs and strategies that could reverse FA induced-neuro- toxicity. The use of different antioxidants as selenium [31] , melatonin [49] , vitamin E [85] , and omega-3 essential fatty acids [86] were used to counteract FA induced neurotoxicity. Currently reliance on natural products is gaining popularity to combat various physiological threats including oxidative stress, cardiovascular complexities, cancer insurgence, and immune dysfunction. Garlic (Allium sativum) holds a unique position in history and was recognized for its therapeutic potential. Several advancements in the field of immunonutrition, physiology, and pharmacology further explored its importance as a functional food against various pathologies [34] .

The effectiveness of garlic has been related to its potent antioxidant properties [87] [88] [89] . Several studies were done on the health benefits of garlic, usually stated its sulfur containing metabolites as allicin and its derivatives. Different garlic preparations are effective against health risks and used as dietary supplements similar to aged garlic extract (AGE) and garlic oil, etc. Its components will scavenge free radicals and defend membranes from injury and maintains cell integrity [34] .

The present study investigated the results of fresh garlic juice, as a potent protector, on FA-induced neural injury and oxidative stress within the rat brain tissue. Bagheri et al. [90] mentioned that, garlic in several forms has protector properties. However, Imai et al. [91] estimated that, AGE exhibits anti-oxidative activities, whereas raw or heated garlic stimulates oxidization.

In our study, there was significant decrease in the levels of lipid peroxidation (LPO) in the brain tissue of FA and garlic treated rats compared to FA-treated rats. Moreover, the level of GSH, SOD, GPx decreased significantly in the garlic and FA-treated group compared to FA-treated rats. While MDA, NO were significantly increased.

The histological results of this study showed that garlic has a protective effect against FA-induced neuronal damage. The intensity of neurodegenerative changes was less in combined FA and garlic treated group than that in the FA-treated group.

Garlic extract components such as S-allyl-L-cysteine (SAC) have neuroprotective effects against reactive oxygen species (ROS) mediated neuronal cell damages [23] [92] .

It is observed that aqueous garlic extract acts as an antioxidant by scavenging ROS, enhancing cellular antioxidant enzymes (e.g superoxide dismutase, Catalase, Glutathione peroxidase), inhibiting peroxidation of lipid and activation of oxidant induced transcription factors [23] [93] [94] .

Garlic contains a number of sulfur and phenolic compounds, which is considered as an excellent antioxidant with antimicrobial action [95] [96] .

Sulfur containing compounds, especially alliin and allicin are considered as the most characteristic constituents of garlic. The amino acid, alliin, is the most representative sulfur compound in fresh garlic, and is converted to allicin by alliinase enzyme when garlic is crushed [97] . Allicin, one of the main biologically active compounds derived from garlic, has various anti-oxidative and anti-inflammatory effects both in vitro and in vivo studies, allicin treatment decreased the expression levels of MDA preserved the endogenous antioxidant enzyme activities, and suppressed the expression of inflammatory cytokines [98] .

Garlic juice as associate inhibitor candidate, scavenges the ROS, stimulate cellular antioxidant enzymes. These effects are due to the presence of sulfur-containing amino acids and compounds having free carboxyl (C=0) and amino (NH2) contents in their structures [33] [99] .

5. Conclusion

Clinical and psychiatric profile of FA-exposed persons revealed cognitive impaired anxious and depressed persons. There were hostile persons with more hostility toward outside. These profiles of personality arouse dangerous affairs about the toxic impact of FA on persons, family, and society. Formaldehyde-induced neuronal damage, oxidative stress and lipid peroxidation in frontal and hippocampal brain tissue were minimized by addition of garlic.

6. Recommendations

Complete prevention of FA exposure is impossible so it is recommended to minimize exposure during Lab work by following the Safety Guidelines as regular air monitoring, maintaining high room air exhaust rates, and using personal protective equipment. Also, early detection of neurotoxic effects in worker exposed to FA and we should encourage research for both new effective prophylactic and therapeutic drugs that could reverse or alleviate FA-induced neurotoxicity.

Conflict of Interest

No conflict of interest related to this article is to declare

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