Shehab, Amal A.; Mahdy, Amal A.
Formaldehyde (CH2O) is a colorless gas that has a pungent odor. It is a physiological intermediary metabolite taking part in many biological processes in the body. A 40% solution of formaldehyde in water is known as formalin. It is also used in medicine for the treatment of some conditions [1,2].
Commercially, formaldehyde has been widely used in the production of resins with urea, phenol, and melamine. Formaldehyde-based resins are used as adhesives and impregnating resins in the manufacture of particle board, plywood, furniture, and other wood products [2,3]. Also, formaldehyde has been used for tissue fixative for preserving biologic specimens and disinfection, as well as an antimicrobial agent in consumer products such as cosmetic products, fungicides, textiles, and embalming fluids [4,5].
Formaldehyde enters drinking-water through industrial effluents and leaching from polyacetal plastic fittings. Concentrations up to 30 ?g/l have been found in ozonated drinking water [6,7]. Formaldehyde in drinking water was formed mainly by the oxidation of natural organic matter during ozonation and chlorination [8,9].
The widespread use of formaldehyde in a variety of applications is known to result in appreciable exposure of workers and of a section of the general population. It occurs in air as a product of automobile exhaust, combustion processes, and incinerators. Formaldehyde has been found in municipal and industrial effluents and is present in food either naturally or as a result of its use as a food additive .
Aim of the work
This research aimed to study the histological changes produced by oral administration of formaldehyde on albino rat stomach.
Materials and methods
Twenty adult male albino rats (200?250 g) were used in this study. They were kept under normal conditions. They were divided into two groups, each one composed of 10 animals. Group I was used as a control group. Group II was used as an experimental group and received a commercial formaldehyde (28–32%) solution orally at a dose of 80 mg/kg  daily for 4 weeks in the morning. To provide the target intake level of the previously mentioned dose per day, 1ml of formaldehyde solution was added to 20 ml of distilled water. Then 1ml of the resultant solution was diluted with 5 ml of drinking water (tap water) and given to each rat using a gastric tube.
At the appropriate date of the experiment, an abdominal incision was carried out under general anesthesia by ether. Specimens from the posterior wall of the fore-stomach and glandular-stomach were taken and washed by their own fixative.
All specimens were divided into two parts: one part was immediately fixed in 10% buffered formalin and the other part was immediately fixed in 3% glutaraldehyde in phosphate buffer (0.1 mol/l) at pH 7.4.
Formalin-fixed specimens were processed for histological examination after staining with H&E, alcian blue PAS reaction, and Feulgen reaction for DNA [12,13]. Sections were subjected to examination using an image analyzer and a statistical study was done.
Glutaraldehyde-fixed specimens were gently irrigated by a syringe filled with the same fixative at their inner aspect. Each specimen was processed and examined using an JEOL JSM-5300 scanning electron microscope, Japan [14,15] at the Faculty of Science, Alexandria University, Alexandria, Egypt.
The Leica Qwin 500 image analyzer computer system (England) at Al-Kasr Al-Einy Faculty of Medicine was used. The interactive measurement menu was applied to measure the thickness of mucosa in all sections (Fig. 1) in the same low-power field. The area % of alcian blue PAS and the optical density of the Feulgen reactions of all glandular-stomach sections were measured in high-power fields. Ten fields were measured for each specimen. Using the color detect, positive areas were masked by a blue binary color  (Fig. 2).
The data obtained (the mucosal thickness as well as the area % of alcian blue PAS and Feulgen reactions) of all groups were recorded and subjected to statistical analysis using Student's t-test at El-Gharbia Syndicate Computer Center. Differences were considered significant when the probability of difference (P-value) is than or equal to 0.05. Highly significant differences were achieved if P-value was less than or equal to 0.001. If the P-value was more than 0.05 (P > 0.05); the differences were considered nonsignificant .
Food intake and liquid consumption seemed to decrease in the experimental group compared with the control group with no death of rats.
Light microscopic results
Stomach sections of the control group revealed limiting ridge separating the fore-stomach and the glandular-stomach portions. The wall of the stomach was formed of four layers, which were, from inner to outer, mucosa, submucosa, musculosa, and serosa (Figs 3 and 4). The submucosa contained blood vessels (Figs 3 and 6). The mucosa of both the fore-stomach and the glandular-stomach consisted of epithelium, lamina propria, and muscularis mucosa (Figs 4 and 5). The epithelium lining of the mucosa of the fore-stomach had stratified squamous keratinizing epithelium with a characteristic papillated configuration (Fig. 4) whereas that of the glandular-stomach had a simple columnar epithelium (mucous-secreting cells). The lamina propria contained gastric glands comprising the isthmus, neck region, and deep basal part and opened into the surface by gastric pits. The gastric pits were lined by surface mucous cells. The isthmus of the gastric gland contained surface mucous cells interspersed with parietal cells. The neck region had mucous neck cells and numerous parietal cells whereas the basal part was composed mainly of chief cells with a number of parietal cells. The parietal cells had an eosinophilic cytoplasm and rounded nuclei. Chief cells had a strongly basal basophilic cytoplasm (Figs 5–7).
After the daily oral administration of 80 mg/kg formaldehyde for 4 weeks (experimental group), sections of the fore-stomach showed noticeable increase in the keratin layer and apparent increase in the thickness of the lining stratified squamous epithelium as well as of the fibro-muscular stroma. The basal layer of the epithelium showed hyperplasia whereas the middle layer showed vacuolated cytoplasm. There was frequent regional loss of papillation associated with inflammatory cellular infiltrations in lamina propria and submucosa (Figs 8 and 9).
Sections of the glandular-stomach of the experimental group showed loss of the epithelial lining with focal erosion and inflammatory cellular infiltration at the sloughed area (Fig. 10). In some regions, there were multiple erosions associated with interstitial mononuclear cellular infiltration and a distorted normal architecture (Fig. 11). In addition, dilated blood vessels as well as edematous spaces in the submucosa were observed. Some sections showed cytoplasmic vacuolation of the gastric gland cells with cystic dilatation (Figs 12 and 13). Other sections showed apparent hyperplasia of the gastric epithelial cells of the neck regions (Fig. 14).
Alcian blue PAS reaction
There was an apparent decreased reaction in the mucous neck cells of the experimental glandular-stomach compared with that of the control group (Figs 15 and 16).
The epithelial cells of the experimental glandular-stomach revealed an apparent decrease in the reaction compared with that of the control group (Figs 17 and 18).
Scanning electron microscopic results
In the control group, fore-stomach sections showed regular keratinized luminal surface epithelium with few keratin scales and intervening sulci (Fig. 19). The scanned longitudinal section in the glandular-stomach showed tubular gastric glands and muscularis mucosa at the base of the gland. The tubular gastric glands formed nearly the greater part of the thickness of the mucosa. The luminal epithelial lining cells of the glandular-stomach sections appeared regular and multigonal in shape with well-defined borders. Gastric pits were seen between the apical surfaces of the lining cells; some of them were filled with mucus (Figs 20 and 21).
Four weeks after formaldehyde administration (experimental group), the luminal surfaces of the fore-stomach and the glandular-stomach appeared irregular. There were numerous keratin scales over the surface epithelium of the fore-stomach (Fig. 22), scattered erosions, necrotic debris, red blood cells, and bacteria over the surface epithelium of the glandular-stomach. The surface epithelial cells appeared to have ill-defined borders (Figs 23–25).
Data recorded by the image analyzer and application of t-test revealed that there was highly significant increase in the thickness of fore-stomach and glandular-stomach lining epithelium in the experimental group as compared with that of the control group (Tables 1 and 2) and (Histograms 1 and 2). With respect to the mean area % of the alcian blue PAS reaction, there was highly significant decrease in the glandular-stomach of the experimental group as compared with that in the control group (Table 3 and Histogram 3). In addition, there was significant decrease in the mean optical density of the Feulgen reaction of the glandular-stomach in the experimental group as compared with that of the control group (Table 4 and Histogram 4).
Accidental formaldehyde intake or its mal use as a food additive for preservative purposes may lead to deleterious effects on almost all body systems.
As regards the method used in the experiment of this study, some researchers  found no gross or microscopic lesions in birds fed different levels of formalin mixed in feed. They explained that evaporation of formalin, as it is a volatile substance, might reduce its concentration to induce pathological changes. Accordingly, oral intake through drinking water rather than food ingestion was preferred in the present study to adjust the desired concentration of formaldehyde in the experimental group. Moreover, during the experiment, there was an observable decrease in food and liquid intake in the experimental group compared with the control group. This agrees with the finding of previous researchers  who found that formalin at a dose rate of less than 10 ml/kg fed to broiler chicks’ decreased feed consumption and body weight.
After daily oral administration of 80 mg/kg formaldehyde for 4 weeks, many histological gastric changes were observed. These results are in agreement with those obtained by previous researchers . In contrast, others  did not observe any adverse effect in the stomach of dogs and rats after oral administration of formaldehyde at doses up to 150 mg/kg body weight/day in drinking water for a period of 13 weeks. This discrepancy can be explained by the differences in the strains of animals used .
The present study revealed that there was highly significant increase in the thickness of the lining epithelium of the fore-stomach and glandular-stomach associated with hyperplasia. The scanned luminal surface of the fore-stomach revealed numerous keratin scale. These findings are in agreement with those of previous studies [1,22] in which the effects were attributed to the irritating, corrosive, and toxic effects of formalin.
This study showed inflammatory cellular infiltration and dilated blood vessels in the mucosa of the fore-stomach and the glandular-stomach as well as multiple erosions and edematous spaces in the glandular-stomach. Necrotic debris was detected by a scanning electron microscope. Similar results were found by some investigators  in their study on gastric mucosa. All of the previous pathological observations indicate the occurrence of gastritis as reported by various authors [24,25]. Other investigators  reported that formaldehyde ingestion induces corrosive damage and hemorrhage of the gastrointestinal tract, which depends on the duration of contact. Furthermore, the local gastrointestinal effects, including ulcers and perforation, can be attributed to the necrotic effects of formaldehyde on the mucus membrane . Moreover, some researchers  found that, in a case study of attempted suicide, ingestion of formalin causes disorders in the oral cavity and the gastrointestinal tract. The stomach shows the most severe damage in such cases because contact of formalin with the gastric mucosa is longer than in the other parts of the gastrointestinal tract. Gastric ulcers and mild hemorrhage are frequently seen. They added that ingestion of formalin could lead to peritonitis without perforation because of gastric wall inflammation. The cicatricle stricture of the stomach tends to be a major problem in the late phase of formalin ingestion.
Bacteria over the surface epithelium of the glandular-stomach were observed by scanning electron microscope in the experimental group of this study. Some investigators  reported that the upper gut and stomach are usually sterile. These data may suggest the occurrence of bacterial translocation and passage of viable bacteria from the gastrointestinal tract to normally sterile tissues, inducing inflammatory reaction in all the examined light microscopic sections. This may be due to decreased gastric acidity, leading to invasion by microbes. This phenomenon of bacterial translocation is associated with septic morbidity as it leads to deterioration or breakdown of gut barrier function [27,28].
Regional loss of papillation of the fore-stomach surface epithelium was observed in this study. Also, loss of gastric pits associated with distorted normal architecture of the glandular-stomach could be observed. In addition, highly significant decrease in alcian blue PAS reaction of the mucus neck cells was detected compared with the control. Some authors  explained that the reduction of the secretion may be due to atrophic changes. Similarly another authors  reported the presence of atrophic gastritis in their study.
The present study revealed cytoplasmic vacuolation of gastric gland cells accompanied by hyperplasia of the gastric epithelial cells. Previous studies  mentioned that these degenerative and proliferative gastric changes are most probably related to the cytotoxic properties of formaldehyde. Other studies  reported regenerative activity in the glandular stomach and increase in the number of mucus cells in deep gastric pits of the fundic mucosa of rat provided 5% formalin in their drinking water. Other researchers  reported that the highest tumor response should be expected at sites with the highest cell proliferation activity.
Some glandular-stomach sections in the present study showed gastric changes in the form of glandular cystic dilatation. Glandular cystic dilatation was observed after gastric operation by previous researchers . They attributed this result to an inflammatory cause and reactive hyperplastic change of the gastric glands that is probably not a preneoplastic state. Other authors  reported that repeated oral intake of large amounts of formaldehyde could disrupt the normal metabolic capacity of animals to convert formaldehyde into forminate, carbon dioxide, and water, producing histopathological gastric changes. Various authors  have reported that epithelial metaplasia may become dysplastic and constitutes a background for the development of carcinoma. Other authors  concluded that formaldehyde, administered with drinking water, was shown to be carcinogenic on the basis of an increased incidence of total malignant tumors and oncological lesions varying in site and histotype. These tumors included malignant mammary tumors, oncological lesions of the stomach and intestine, testicular interstitial cell adenomas, and hemolymphoreticular neoplasias. Furthermore, other investigators  have reported that rodents and nonhuman primates show dose-related cytotoxic-proliferative and metaplastic lesions, which indicates that formaldehyde might be carcinogenic for exposed humans. However, there was insufficient evidence on the carcinogenic potential of formaldehyde for humans as the data available through epidemiological studies were limited due to lack of consistency, dose– response pattern, and inadequate estimation of exposure. Moreover, the same authors reported that at irritating and cytotoxic concentrations, inflammatory and regenerative processes, which were also observed in the present study, become the dominant risk factors by promoting formaldehyde-induced genotoxic events and malignant cell transformations.
As regards the Feulgen reaction for DNA used in this study, there was a significant decrease in the reaction in the experimental group. This indicates a decrease in the DNA content of the gastric cell nuclei. This result is in disagreement with the results of a previous study , which showed no evidence of carcinogenicity of formaldehyde even after 2 years of oral administration. However this result is in accordance with that of another study , in which nuclear changes in the stomach, duodenum, ileum, and colon were found and the strongest genotoxic effect was observed in the stomach. These data suggested that formaldehyde not only causes nuclear damage at the site of application but also at distant sites. Some authors  reported a significant decrease in the nuclear DNA content of the testis and prostate. Other authors  assumed that a threshold for carcinogenicity exists after exposure by inhalation and oral administration. The rates of gastric tumor incidences in male rat administered 1500 mg/l were 4% in the original study  and increased to 14% in the re-evaluation study . The carcinogenicity of formaldehyde may result from its ability to induce DNA–protein cross-links . Some researchers  suggested that formaldehyde is genotoxic as a result of its ability to arrest DNA replication. They added that, although DNA–protein cross?links could be removed and DNA could be repaired, failure to remove the blockage before cell division or incomplete repair could cause cell death or a mutation.
On the basis of the previous data, this study concluded that oral formaldehyde intake has deleterious effects on albino rat stomach in the form of irritating and regenerative effects as well as DNA damage that may be precancerous.
Therefore, it is recommended to prohibit the mal use of formaldehyde as a food additive, and appropriate control measures or alternative additives should be used.
Conflicts of interest
There is no conflict of interest to declare.
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