This study is to assess the structural lining of the stomach when naturally and artificially ripened fruit [pawpaw] is consumed since the stomach is an organ of storage and digestion. Mature unripe Pawpaw’s were plucked off from the parent plant and were dividing into two groups. Group 1, was kept to rip at normal room temperature while group two was induced with Calcium Carbide to rip. 10gram of Calcium carbide was placed in a bowl containing 5ml of water for dissolution in a closed metal bucket containing 1kg of the fruit [pawpaw] rapped with black nylon and was allowed for two days. After ripening, sampled fruits were washed and juiced. 600g of both the naturally ripened and calcium carbide ripened pawpaw were peeled separately and blended in an electric blender with deionized water. The juice was filtered and poured into clean bottles labeled [CaC2 induced ripened juice and naturally ripened pawpaw juice] for administration. A total of 21 adult Wister rats weighing between 126.9-214.3g were used. LD50 was carried out using Lorke, 1983 method. Three groups were considered; Group 1: Normal control group of 4 rats [2 males and 2 females] receive normal water and feeds only as placebo. Group 2: Treatment Group [1] of 4 rats [2 males and 2 females] received naturally ripened pawpaw juice. Group 3: Treatment Group [2] of 4 Wistar rats [2 males and 2 females] received Calcium Carbide ripened pawpaw for 4 weeks. About 5mL/kg for both the natural fruit and the CaC2 ripened fruits were administered against each body weight of the adult Wistar rats. The Wistar rats were weighed and one Wistar rat was sacrificed weekly in each group. Blood sample and organs [stomach] were collected from the three groups for histopathological analysis. The results showed hyperplasia, ulceration, atrophy of the stomach walls of the two treatment groups.
The stomach is a C-shaped muscular organ of the digestive system found in vertebrates with various parts such as Cardiac, fundus, body, pylorus. Histologically, it contains four layers which include, mucosa, submucosa, muscularis and serosa. It functions ranges from chemical and mechanical digestion, storage of food, absorption and secretion of hormones. Calcium carbide treatment of food is extremely hazardous because it contains traces of arsenic and phosphorous and once dissolved in water, it produces acetylene gas. Arsenic, phosphorous and acetylene gas may affect the different body organs and causes various health problems like headache, dizziness, mood disturbances, sleepiness, mental confusion, memory loss, cerebral edema, seizures and prolonged hypoxia [1]. A study was carried out to evaluate the effect of Irreversible Electroporation (IRE) on the stomach wall after IRE was applied on liver tissues adjacent to the anterior wall of the stomach. IRE ablation was performed in eight Tibet mini-pigs with three lesions per pig. On the Day-7 histopathological examination, inflammation and fibrosis were observed in the serosal layer of the stomach in each animal and mild inflammation of the myofibers was found in only two pigs. All the stomach layers returned to normalcy by 28 days post-IRE. Thus, IRE ablation of hepatic tissues situated close to the stomach wall cannot lead to stomach perforation. IRE is therefore a safe procedure for ablating hepatic tumors that are adjacent to the stomach [2].
Evaluate the effects of Irreversible Electroporation (IRE) on the stomach wall following the direct application of IRE onto the organ surface was performed. IRE ablation was performed in 8 Tibetan mini-pigs, which were randomly assigned into two groups based on their ablated areas: Group A, gastric cardia, fundus of stomach, gastric body and group B, lesser gastric curvature, greater gastric curvature, stomach pylorus. Here were no signs of perforation on the stomach wall. Serum aminotransferase and WBC levels increased in both groups on day 1 post-IRE and decreased gradually thereafter. The gastroscopy procedure revealed oval ulcers on day 7 post-IRE and smaller ulcers on day 28 post-IRE. Transmural necrosis, inflammation and fibrosis were observed at 7 days post-IRE. Healing ulcers were observed at 28 days post-IRE. In conclusion, IRE ablation caused damage to the stomach wall; however, IRE did not induce any perforation [3]. The use of toxic and suspicious ripening agents is of great concern as the activities of human beings have been said to contribute to exposure of food materials to heavy metal contamination [4]. It has been reported that though the cosmetic quality of artificially ripened fruits like the external color and the texture increases, organoleptic qualities, nutritional value and shelf life is reduced [5,6] Chronic alcohol consumption has been known to induce a decrease in gut wall integrity in actively drinking alcoholics and patients with alcohol-induced liver disease. Alcohol consumption caused a significant increase in serum I- and L-FABP levels, compared to water consumption. Levels increased directly post-consumption and decreased to normal levels within 4 h. LBP, sCD14 and IL-6 levels were not significantly higher in the alcohol group. Moderate acute alcohol consumption immediately damages the enterocyte but does not seem to cause endotoxemia [7]. One of the most common drug-induced injuries is irritation of the lining of the stomach caused by Nonsteroidal Anti-Inflammatory Drugs (NSAIDs). NSAIDs can irritate the stomach by weakening the ability of the lining to resist acid made in the stomach. Sometimes this irritation may lead to inflammation of the stomach lining (gastritis), ulcers, bleeding, or perforation of the lining [8]. Since the stomach is an organ for storage of various food substances, chemical reaction will occur, hence the need for this study.
Materials: The materials used for this experiment include Wistar rats, Calcium carbide, Water, Pawpaw, Syringes and Needles, Hand Gloves, Incubator, stop watch, Oven, centrifuge Model 800, cotton wool, Chloroform, 40% formaldehyde, Desiccator, Methylated spirit, EDTA bottles, normal sample bottles, Animal weighing balance, Water bath and amongst others.
Design of the Study
This is an experimental study of adult Wistar rats fed with naturally ripened and Calcium Carbide induced ripened fruits [pawpaw] to compare and investigate the effect of Calcium Carbide on the stomach architecture of the Wistar rats.
Fruit Collection
Mature unripe pawpaw’s were plucked off from the parent plant. The fruits were dividing into two groups; one group was kept and allowed to rip at normal room temperature and the other group was induced with Calcium Carbide to ripe.
Weight and Application of Calcium Carbide
Calcium carbide was bought at Swali Market, Yenagoa, Bayelsa State. About 10gram of Calcium carbide was placed in a bowl containing 5 mL of water for dissolution in a closed metal bucket containing 1kg of the fruit [pawpaw] rapped with black nylon and was allowed for two days[48 hours] for ripening. After ripening, sampled fruits were washed and juiced.
Sample Preparation
About 600g of both the naturally ripened and calcium carbide ripened fruits [pawpaw] were peeled separately and blended in an electric blender with 350 mL/1L of deionized water. The juice was filtered with a clean fine sieve and was poured into clean bottles labeled [CaC2 induced ripened juice and naturally ripened pawpaw juice] that was stored in a refrigerator for subsequent use.
Experimental Animals
A total 21 adult Wister rats of both sexes weighing between 126.9- 214.3g were used for this study. The animals were purc hased and kept in standard environmental condition, given standard rodent food (formulated) and water adlibitum in the animal house of the Bayelsa Medical University. The rats were divided into three groups for each sex, based on the body weight and then different concentrations of naturally ripened and calcium carbide forced ripened Pawpaw were administered orally. Animals were allowed to acclimatize for two [2] weeks and was fed with standard grower mash with clean water before the commencement of treatment following the protocols of [9].
Administration of Prepared Sample
LD50 was carried out using [10] method for administration of samples. A total of nine [9] Wistar rats were used for this section. About 12 Wistar rats were used for the main experiment:
Group 1: Normal control group of 4 rats [2 males and 2 females] receive normal water and feeds only as placebo
Group 2: Treatment Group [1] of 4 rats [2 males and 2 females] received 5ml/kg naturally ripened ripned fruits [pawpaw juice]
Group 3: Treatment Group [2] of 4 Wistar rats [2 males and 2 females] received Calcium Carbide ripened fruits [pawpaw] 5ml/kg for both the natural fruit and the CaC2 ripened fruits were administered against each body weight of the adult Wistar rats for one [1] Month
Organ Collection
The Wistar rats were weighed each week, then one Wistar rat was sacrificed in the groups, samples were collected from the three groups for histological assay.
The collected stomach samples were prepared histologically and analyzed from the various groups of the Wistar rats [Control, Natural and CaC2 treated groups] for two [2] and four [4] weeks and the results are shown in below.
The rodent’s fore-stomach is thin walled and serves as a storage sac for food that has been ingested and is awaiting digestion. But it is not the anatomical equivalent to the rumen of ungulates.
The inner surface is ridged with a keratinized stratified squamous epithelium and has thus been referred to as the cutaneous zone of the stomach. The lamina propria is poorly developed, the submucosa comprises rather loose connective tissue and the muscularisis well developed. Typically the stratified epithelium has a stratum germinativum, stratum spinosum, stratum granulosum and stratum corneum, although cornification is slight. Three layers of smooth muscle are found in the muscularis of the fore-stomach [11].
Results from the Wistar rat of the control group showed normal stomach architecture [Figure 1]. For both male and female Wistar rats fed with CaC2 forced ripened pawpaw for two [2] showed normal fore- stomach and glandular stomach mucosa [Figure 2 and 3]. While the Wistar rats fed with naturally ripened pawpaw for two [2] weeks showed mild and moderate mucosal atrophy [Figure 4 and 5]. Focal mucosal atrophy is characterized by decreased size and number of mucosal epithelial cells and may result from ulceration, inflammation, mineralization, or infarction. Generalized mucosal atrophy can be chemically induced. Diffuse atrophy can also be an age-related change with a diffuse decrease of mucosal height and increased mucosal fibrous connective tissue. In the aged rat, fundic glands may be diffusely atrophic, with fewer parietal and chief cells and contain cysts. Glands are replaced by fibrous connective tissue with focal lymphocytic infiltrates in the lamina propria and the glandular epithelium and a mixed inflammatory infiltrate along the limiting ridge [11,12].
Figure 1: Photomicrograph (H&E x400) of the stomach of a Wistar rat fed with normal grower mesh [Control] showing a normal fore-stomach with the Epithelium (EP), Muscularis Mucosa (MM) and Submucosa (SM). Diagnostic Lesion: Normal Architecture of the Fore-Stomach
Figure 2: Photomicrograph (H&E x400) of a Normal fore-Stomach from male Wistar Fed with CaC2 for [2 weeks] showing the Epithelium (EP), Muscularis Mucosa (MM) and Submucosa (SM). Diagnostic Lesion: Normal Fore-Stomach
Figure 3: Photomicrograph (H&E x400) of a Normal Fore-Stomach from Female Wistar fed with CaC2 for [2 weeks] Showing a Normal Glandular Stomach Showing the Gastric Epithelium (GE) and Gastric Pit (GP). Diagnostic Lesion: Normal Glandular Stomach Mucosa
Figure 4: Photomicrograph (H&E x400) of a normal fore-stomach from male Wistar fed with CaC2 for [4 weeks] showing moderate epithelial hyperplasia (arrows) with minimal inflammation. Diagnostic Lesion: Mild Inflammation
Figure 5: Photomicrograph (H&E x400) of Glandular Stomach from Female Wistar fed with CaC2 for [4 weeks]. There is Diffuse Moderate Epithelial Hyperplasia (arrows). Diagnostic Lesion: Moderate Epithelial Hyperplasia
Results from the Wistar rat fed with CaC2 forced ripened pawpaw for two [4] weeks showed mild and moderated epithelial hyperplasia for both the male and female [Figure 6 and 7]. Hyperplasia of the fore-stomach is defined as an increase in the number of cells in one or more epithelial strata (basal, spinous, or granulosum) [12]. Hyperkeratosis (increased thickness of the stratum corneum) is often seen when hyperplasia of the mucosa is present [11,12]. Hyperplasia may be diffuse or focal and results in an increased thickness of the nonglandular stomach mucosa. Hyperplasia may also be seen at the limiting ridge. Hyperplasia of the nonglandular stomach is often characterized as a uniform thickening of all layers of the epithelium without upward or downward projections. However, in more severe cases there are convolutions of the epithelium with downward projections of rete peg-like structures. There may be proliferation of the basal cells, which can extend into the submucosa [11,12]. In same vein, both male and female Wistar rats fed with natural pawpaw juice for four [4] weeks showed mild inflammation of the stomach wall [Figure 8 and 9]. Inflammation can occur in the fore stomach as elsewhere in the gastrointestinal tract. Lesions can be spontaneous (idiopathic), related to gavage procedure, or due to direct/indirect chemical toxicity. Inflammation in the fore stomach most often occurs in association with epithelial hyperplasia, ulcer, or erosion [12].
Figure 6: Photomicrograph (H&E x400) of a Glandular Stomach Male Wistar Fed with Natural Fruit for [2 weeks]. There is Mild Generalized Mucosal Atrophy. Diagnostic Lesion: Mild Mucosal Atrophy
Figure 7: Photomicrograph (H&E x400) of a Glandular Stomach Female Wistar Fed with Natural Fruit for [2 weeks]. There is Moderate Generalized Mucosal Atrophy. Diagnostic Lesion: Moderate Mucosal Atrophy
Figure 8: Photomicrograph (H&E x400) of a Fore-Stomach of a Male Wistar Fed with Natural Fruit for [4 weeks].There is Mild Inflammation with Numerous Inflammatory Cells Within the Submucosa (SM) and Smooth Muscle Layer (SML); Overlying the Stratified Epithelium (EP). Lesions is Associated with Mild Epithelial Hyperplasia (Arrows). Diagnostic Lesion: Mild Inflammation
Figure 9: Photomicrograph (H&E x400) of a Fore-Stomach of a Female Wistar Fed with Natural Fruit for [4 weeks]. Showing Moderate Epithelial Hyperplasia (arrows) with Minimal Inflammation. Diagnostic Lesion: Mild Inflammation
Consumption of naturally and Calcium Carbide for ripened fruits could cause alterations in the stomach lining. Some causing hyperplasia, ulceration, atrophy which could lead to lose of cells, nutrients. I recommend for further studies to validate this research.
Acknowledgment
I sincerely acknowledgement Dr. Allison Theodore and Mr. Preye Omoni-Ebidor Sidney for their contributions.
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