INTRODUCTIONFormalin an aqueous solution of Formaldehyde is the most commonly used chemical

INTRODUCTIONFormalin, an aqueous solution of Formaldehyde is the most commonly used chemical for embalming of cadavers and routine histopathological techniques. Formaldehyde was introduced in 1893 by Blum as a fixative and embalming fluid. It is also a well known preservative and a sterilizer. There are long-standing concerns about the adverse health effects of Formaldehyde exposure, including carcinogenicity, for professionals exposed to Formalin-based fixatives, such as pathologists, anatomists, nurses, embalmers, anatomy students, veterinary students and for workers exposed to Formaldehyde in manufacturing. It is estimated that approximately 2.1 million workers are exposed to Formaldehyde (Occupational Safety and Health Administration, 1995).Formaldehyde (FA) (formula: HCHO; chemical name: Methanal) is a flammable, colourless gas with pungent odour and is readily polymerized at ambient temperature. It is one of the major indoor air pollutants (World Health Organization, 2000). Formaldehyde is rarely found in its original state because it has a short half-life in air (approximately 30-50 minutes) and decomposes in light to form a toxic substance. In liquid form it is stable over time. It is commercially available as Formalin, which contains 37% by weight or 40% by volume of Formaldehyde gas in water (International Programme On Chemical Safety, 1989).Formaldehyde is an important intermediate in biosynthetic pathway. It is produced in all metabolically active cells and levels in blood range from 2-3 mg/l (World Health Organisation, 2010). It is a highly reactive compound capable of covalently binding to macromolecules and causing toxicity. Although dermal contact is one of the major routes of exposure (Lyapina M et al., 2012) but following dermal contact Formaldehyde is poorly absorbed. On the other hand, upon exposure through inhalation and ingestion, exogenous Formaldehyde is rapidly and almost completely absorbed to the site of contact (Agency For Toxic Substances and Disease Registry, 1999).Formaldehyde reacts rapidly with primary and secondary amines, thiols, hydroxyls and amides, forming methylol derivatives at the site of contact. It also reacts with DNA, RNA and protein to form adducts or cross-links. Studies in experimental animals suggest that DNA-protein cross-links do not accumulate with repeated exposure (World Health Organization, 2010).Formaldehyde is rapidly metabolized to formate, mainly by Formaldehyde dehydrogenase, at the initial site of contact (Lyapina M et al., 2012). Formate may be further oxidized to form carbon dioxide or may be used in the one carbon biosynthetic pathways to form purines, thymidine and some amino acids (Agency For Toxic Substances and Disease Registry, 2010). Following ingestion of Formaldehyde solution, formate levels are seen to rise within 30 minutes and peak within an hour (International Programme On Chemical Safety, 2002)Formaldehyde is primarily eliminated by exhalation as carbon dioxide; urinary excretion (as various metabolites) is a lesser route (World Health Organisation, 2010).Gross anatomy laboratories are environments of chronic exposure to Formaldehyde vapours, this study therefore, investigates the histopathological effects of Formaldehyde on liver, kidneys and lungs of adult wistar rats, to create awareness among the personnel exposed to Formaldehyde of the impending danger of prolonged exposure.Aims and objective:• To study histopathological changes in albino rat in:a) LiverAfter exposure to 40% Formaldehyde. METHOD AND MATERIALThe present study was conducted on 20 inbred Albino rats (wistar strain) of both sexes. Animals were procured from the Central Animal House of Government Medical College, Jammu. Necessary clearance for the use of animals for experimental purpose was obtained from the Animal Institutional Ethical Committee constituted for the purpose. The animals were housed in PVC cages of size (44 x 28.6 x 30cm³) which were placed in a room of size (10 x 20 sq ft.) with cross ventilation, under laboratory conditions with controlled environmental temperature of 25±2°C, humidity (16% ±10%) and photo-periodicity of 12 hours day/12 hours night (Fig 1A and 1B) as per the guidelines of the Committee for Purpose of Control and Supervision of Experiment on Animals (CPCSEA). The animals were provided standard rodent chaw/feed and water ad-libitum. Before subjecting them to experimentation, animals were provided a week’s time to acclimatize with laboratory conditions.The animals were divided into two main groups (A and B) as detailed below:Group A: Control Group (10 animals), which were not exposed to Formaldehyde.Group B: Experimental Group (10 animals), which were exposed to 40% Formaldehyde for 5 hrs/day for 21 days consecutively.Table 1: Exposure, Dose and Duration:S.No. Group No. of Animals Dose and Duration1 A 10 No exposure2 B 10 40% Formaldehyde 5hrs/day for 21consective daysThe animals were exposed to 20ml of 40% Formaldehyde in a petridish (Fig. 2A) which placed at a distance of 30cm from the cage (Fig. 3). Commercially available 40% Formaldehyde was used for the experiment (Fig. 2A).After the experimental period of 21 days the rats were euthanized by keeping them in an inverted glass jar containing a large piece of cotton soaked in chloroform. After sacrificing the animals, tissue samples from lung, kidney and liver were taken and immediately fixed in 10% neutral buffered Formalin for 24 hours and then routinely processed by paraffin wax embedding method. Sections of 5-7 µ thickness were cut, stained by Haematoxylin and eosin stain.OBSERVATIONS:LIGHT MICROSCOPIC EXAMINATION OF LIVER:Group A:Light microscopic examination of liver sections of group A rats revealed the normal basic architecture of the liver, showing the hexagonal classical hepatic lobules with central veins located in the centre of the lobule and portal areas containing portal triad formed by portal vein, hepatic arteriole and bile ductule surrounded by connective tissue at 3 to 5 corners of the lobule. The areas corresponding to the portal lobule, portal acinus and the zone I, II and III based on the position of hepatocytes in relation to the vascular backbone of the portal acinus were identified (Fig. 10).Within the Classical hepatic lobule, the central veins had a thin connective tissue wall lined internally by endothelial cells and were present in the centre of the lobules. The cords of the hepatocytes which were one cell thick at most of the places were found to be radiating from the central veins the periphery of the lobule which contained the portal areas (Fig. 11). The sinusoids were lined mostly by endothelial cells and contained a few kupffer cells. Sinusoids present in the lacunae between the cords of hepatocytes were found to be normal caliber and contained few blood cells. The portal areas present at the corners of the classical hepatic lobule contained connective tissue, fibroblasts and stained light pink in color. Embedded in the connective tissue were seen structures such as the portal venule, hepatic arteriole and bile ductule. The portal venule was thin walled lined by endothelial cells, the hepatic arteriole was thick walled containing smooth muscle in its wall and lined by endothelial cells and the bile ductule was lined by cuboidal cells (Fig. 12). Hepatocytes were polygonal, stained pink in color and had centrally placed spheroidal, euchromatic nucleus which stained light blue in color and contained one nucleolus. Occasionally hepatocytes containing two nuclei were also seen. Two nucleoli were also seen in the nuclei of some hepatocytes(Fig. 13).Endothelial cells lining the lumen of sinusoids had scanty, pink cytoplasm with a centrally placed flattened nucleus (Fig. 13).Group B:On examination of liver of group B rats, the basic architecture of the liver was found to be preserved however various histopathological were observed. The central veins were markedly dilated and congested. The sinusoids were also dilated and congested (Fig. 14).Focal areas of hemorrhage were seen in the parenchyma (Fig. 14).Dilated and congested portal veins and hepatic arterioles were present in the portal areas (Fig. 15).Severe Inflammatory cell infiltration was seen in portal areas and around the central vein and in the parenchyma (Fig. 16).Focal areas of necrosis (patchy necrosis) were also present as evident by ground glass appearance of hepatocytes with loss of nuclear staining (Fig. 17).Hepatocytes were enlarged, swollen and edematous, with ill defined boundaries and irregularly clumped cytoplasm. Clear spaces known as cloudy swelling (vacuoles of varying sizes, seen as small and large empty spaces within the hepatocytes) was observed (Fig. 18).Apoptotic cells with hypereosinopilic cytoplasm, small shrunken condensed nuclei with increased basophilia (pyknotic nuclei) were seen at certain places(Fig. 19). The nuclei of the hepatocytes showed pleomorphism (variable appearance) and were hyperchromatic. Some of the hepatocytes showed karyomegaly (enlarged nuclei with prominent nucleoli), some had karyopyknosis (small condensed nuclei), some hepatocytes showed faintly stained nuclei suggestive of karyolysis (dissolution of their nuclei) whereas some hepatocytes showed karyorrehxis (nuclei were broken into pieces) (Fig. 20, 21). Table 2: Histological Changes in LiverS.No. Parameter Group A Group B1 Architecture Normal Preserved 2 Central Vein Normal Markedly Dilated and congested 3 Sinusoids Normal Dilated and congested4 Nucleus Normal Nuclear pleomorphism 5 Hepatocytes Normal Cloudy swelling and vacuolated cytoplasm ,ill defined boundaries6 Infiltration Absent Marked Inflammatory cell infiltration in peri-portal area, around central vein and in parenchyma.7 Hemorrhage Absent Focal areas of hemorrhage 8 Portal triad Normal Dilated and congested portal veins and hepatic arterioles9 Necrosis Absent Focal areas of necrosisDiscussion:Although Formaldehyde has been recently classified as Carcinogenic in humans, it is still widely used chemical for fixation of tissues in histology, for fumigation of operation theatres and sterilization of surgical instruments. Anatomists, pathologists, forensic surgeons, staff of dissection hall, embalming laboratory, histology laboratory and medical students are as such exposed to Formaldehyde gas. Formaldehyde is the chemical of choice for above mentioned purposes, if used with proper precautions, but it can produce harmful effects on many systems as respiratory system, central nervous system, gastrointestinal system, skin and genitourinary system producing minor to major problems ranging from burning eyes to carcinomas.Inhalation of Formaldehyde leads to formation of DNA-protein cross-links, DNA single strand breaks and gene mutation in cells. This is probably the mechanism of Carcinogenic, Mutagenic and sensitizing action of Formaldehyde (World health organization, 2010).The Current study was undertaken to evaluate the histopathological effects on organs of rats, with focus being on liver.The present study showed that exposure to Formaldehyde induced histopathologic changes in liver. Marked dilatation and congestion of the central veins, sinusoids, and portal vein and hepatic arterioles was observed. These findings are consistent with those reported by Mehdi AH et al. (2014) who observed dilatation of the central vein and enlargement of the sinusoids following exposure to 40% formaldehyde for 21 days consecutively. Fazeli SA et al. (2006) and Afrin M et al. (2016) also reported dilatation and congestion of sinusoids and centrilobar veins following exposure to Formaldehyde.In the present study hepatocytes showed severe cloudy swelling and vacuolar degeneration. Similar findings of cloudy swelling and vacuolar degeneration was reported by Abdulqader SZ and Mustafa IA (2014) in rats after exposure to Formaldehyde .Mehdi AH et al. (2014) demonstrated similar cloudy swelling in rats exposed to 40 % Formaldehyde for 21 days consecutively.Abdulqader SZ and Mustafa IA (2014) in their study observed cytological alterations in the hepatocytes which showed degeneration (shrunken and swollen appearance). The shrunken cells had hypereosinophilic cytoplasm, condensed nucleus and blebbed plasma membrane. The increase in presence of Apoptotic cells in the liver was also observed in the current study.The nuclei of the some of the hepatocytes showed pleomorphism and were hyperchromatic and pyknotic. These results are in accordance with that of Cikmaz S et al. (2010), who observed hepatocytes with loss of cytoplasm with hyperchromatic and pyknotic nuclei as a result of chromatic coarsening in rats exposed to 40% Formaldehyde for 13 weeks.In the present study severe inflammatory cell infiltration in and around the portal area, in the sinusoids, around the central veins and in the necrotic areas was observed. These findings are in accordance with Pekmez H et al. (2008) who observed mononuclear cell infiltration in the portal area and around central vein, in rats exposed to 40% Formaldehyde gas for 14 days and Cikmaz S et al. (2010) who noted mononuclear cell infiltration in the portal areas and around central vein. Similar findings were reported by Abdulqader SZ and Mustafa IA (2014), who observed the appearance of inflammatory leucocytes in liver.In the present study, histopathological examination revealed focal areas of necrosis in liver parenchyma. Similar observations were made by Soni A et al. (2013) who observed the mean of necrotic foci in liver after treatment with Formaldehyde and Benzo (α) Pyrene.Treesh SA et al. (2014) observed that after receiving 30 mg/kg Formaldehyde intraperitonealy for duration of 5 days the hepatocytes exhibited a vacuolated cytoplasm, dilated and congested sinusoids, congested blood vessels and kupffer cells hyperplasia. These findings are comparable to the findings of present study except for kupffer cell hyperplasia which could be explained by intraperitoneal administration of Formaldehyde. Similar kupffer cell hyperplasia was observed by Uçmakli E et al. (2013) after intraperitoneal injection of Formaldehyde.Hemorrhagic foci were observed in the liver parenchyma. This is consistent with the findings of Mamun MAA et al. (2014) who reported focal areas of hemorrhage and congestion inside the liver tissue. Conclusion : Microscopic examination of sections of liver revealed that the basic architecture of the liver was preserved. The central veins were markedly dilated and congested. The sinusoids were also dilated and congested. Dilated and congested portal veins and hepatic arterioles were present in the portal areas. Inflammatory cell infiltration was seen in portal areas and around the central vein as well as in the parenchyma. Hepatocytes showed cloudy swelling and pleomorphic nucleus. Focal areas of necrosis and hemorrhage were also present.The observations of current study reinforce that Formaldehyde, which is one of the most active chemicals in embalming fluid and in some chemical industries, is highly toxic and Formaldehyde inhalation with above mentioned concentration and duration can induce several histopathological changes. Therefore it is prudent that efforts should be increased at replacing Formaldehyde with less toxic chemical and individuals who are occupationally exposed to it should be sensitized about the hazards of Formaldehyde exposure. Furthermore there should be standardized adoption of protective measures in order to mitigate the risks associated with Formaldehyde exposure.