Histopathological and ultrastructural damages of liver in dioxin-exposed people with chronic liver diseases in Viet Nam

Journal of military pharmaco-medicine n o 1-2020 173 HISTOPATHOLOGICAL AND ULTRASTRUCTURAL DAMAGES OF LIVER IN DIOXIN-EXPOSED PEOPLE WITH CHRONIC LIVER DISEASES IN VIETNAM Pham Quang Phu1; Tran Viet Tu1; Nguyen Ba Vuong1 SUMMARY Objectives: To investigate histopathological and ultrastructural damages of liver in dioxin- exposed people with chronic liver diseases in Vietnam. Subjects and methods: 33 dioxin- exposed people with chronic liver diseases who lived around Danang airbase - a dioxin- contaminated area in Vietnam were recruited in 2014. The compared group included 15 chronic hepatitis B patients living in an uncontaminated area. Liver biopsy was collected for the histopathological and ultrastructural diagnosis. Liver fibrosis stages were evaluated based on the Metavir classification (F0, F1, F2, F3, and F4). Ultrastructural damages of liver were examined by scanning electron microscope and transmission electron microscope. Results: In the dioxin- exposed group, grade F1 was the most common observations on histopathological tests (42.4%). Grade F0 and F2 accounted for 27.3% and 30.3%, respectively. Hepatic fibrosis lesions with grade F3 and F4 were not found in this group. Consistent ultrastructural damages on liver cells and their organelles were observed in all of liver biopsy samples in dioxin-exposed group. Conclusions: There were liver fibrosis lesions on histopathological images and images of ultrastructural damage of liver in dioxin-exposed people with chronic liver diseases. * Keywords: Exposed dioxin; Chronic liver diseases; Histopathological and ultrastructural damage. INTRODUCTION Based on US studies in Vietnam, the Ministry of Health has classified primary liver cancer as a list of diseases, deformities, and defects related to exposure to toxic chemical/dioxin [1]. Thus, the process casusing dioxin primary liver cancer has affected chronic liver damage. The international and domestic studies on experimental animals showed that the liver is a major organ affected by toxins 2.3.7.8-tetrachlorodibenzo-p-dioxin (2.3.7.8-TCDD) [3, 4]. The liver is an organ system that functions to metabolize, immobilize, inactivate and eliminate the endogenous and exogenous toxins of the body. Therefore, it is a very vulnerable organ in the long-term poisoning process of exogenous toxins, including agent orange/dioxin. However, assessing the extent of liver damage in patients with agent orange/dioxin on histopathology and ultrastructure is very limited. Therefore, we conducted research on this topic with the aim: To evaluate the histopathological and ultrastructural damages of liver in patients with chronic hepatitis infected with agent orange/dioxin. 1. 103 Military Hospital Corresponding author: Pham Quang Phu (bsphu79@yahoo.com) Date received: 18/12/2019 Date accepted: 11/01/2020 Journal of military pharmaco-medicine n o 1-2020 174 SUBJECTS AND METHODS 1. Subjects. 33 patients with chronic hepatitis exposed to dioxin treated at 17 Hospital, Danang from August 2014 to December 2014. * Selection criteria: - Criteria for research group selection: + Patients aged 18 - 70 years were diagnosed with chronic hepatitis through histopathology. Having lived in dioxin "hot spot" - Danang airport for over 5 years, blood tested with high dioxin levels (TEQ > 9.4 pg/g lipid) [5]. + Based on health monitoring records, an unexplained increase in liver enzymes lasts more than 6 months (with indications for liver biopsy). + Diagnosis of chronic hepatitis: Inflammatory cell infiltration: mononuclear leukocytes with mainly lymphocytes at the portal, there may be liver fibrosis on histopathology [6]. + Volunteering to participate in the research. * Exclusion criteria: + Patients disagreed to participate in the research. + Chronic hepatitis due to other causes: Hepatitis B, C viruses, alcohol, drugs, autoimmune... + There were contraindications to liver biopsy. + The patient disagreed to participate in the research. - Selection criteria for comparison group: 15 subjects with a liver biopsy indicated at 103 Military Hospital to perform histopathology and ultrastructure of liver. * Diagnosis of chronic hepatitis B: - HBsAg (+) ≥ 6 months or HBsAg (+) and anti-HBc IgG (+). - AST, ALT increased each spell or continuously over 6 months. - Liver biopsy showed chronic hepatitis picture with various levels of necrosis or/and fibrosis [2]. 2. Methods. Advanced, cross-sectional descriptive research. Liver histopathology is consulted and unified by two experienced doctors of 103 Military Hospital and Institute 69 of Hochiminh Mausoleum Protection Command. Biopsy samples met pathological and ultrastructural reading requirements when they met the standard of ≥ 6 portal areas. The degree of fibrosis on a Metavir score: F0: no fibrosis, F1: portal fibrosis without septa, F2: portal fibrosis with few septa, F3: numerous septa without cirrhosis, F4: cirrhosis [7]. The ultrastructure was read on the transmission electron microscope and scanning electron microscope at Institute 69 of Hochiminh Mausoleum Protection Command. Ultrastructural damage to the liver at the cellular and organelle levels was noted. The collected facts and figures were checked and entered into the computer. Processing data under SPSS 20.0 program (Statistical Package for Social Sciences). Journal of military pharmaco-medicine n o 1-2020 175 RESULTS AND DISCUSSION Table 1: Distribution of patients by age groups and gender. Gender Age Male n (%) Female n (%) Total n (%) ≤ 40 7 (21.2) 3 (9.1) 10 (30.3) 41 - 60 8 (24.2) 8 (24.2) 16 (48.5) > 60 3 (9.1) 4 (12.2) 7 (21.2) Total 18 (54.5) 15 (45.5) 33 (100) The average age in the research group was 46.3 ± 12.1. The youngest was 25, the oldest was 69. The ratio of male/female: 1:2. Table 2: Hepatopathological damages of the liver of the research groups. Hepatopathological damages Yes No Total Granular degeneration 32 (97%) 1 (3.0%) 33 (100%) Vacuolar degeneration 33 (100%) 0 (0%) 33 (100%) Fatty degeneration 32 (97.0%) 1 (3.0%) 33 (100%) Lipoma 0 (0%) 33 (100%) 33 (100%) Lymphocytes and polymorphonuclear leukocytes around liver cells 33 (100%) 0 (0%) 33 (100%) Mallory 0 (0%) 33 (100%) 33 (100%) Pigmentation 0 (0%) 33 (100%) 33 (100%) Giant mitochondria 0 (0%) 33 (100%) 33 (100%) Metabolic acidosis of hepatocytes 4 (12.1%) 29 (87.9%) 33 (100%) Venous congestion 0 (0%) 33 (100%) 33 (100%) Chronic liver damage was seen on the most biopsies including hepatocellular degeneration: 32/33 cases (97.0%) fatty degeneration, 32/33 cases (97.0%) granular degeneration, 33/33 cases (100%) vacuolar degeneration. All subjects had inflammatory cell infiltrates, there were lymphocytes and polymorphonuclear leukocytes around liver cells. There were 4 cases of metabolic acidosis of hepatocytes, accounting for 12.1%. Other damages of chronic liver disease, such as mallory, pigmentation, lipoma, giant mitochondria, and venous congestion were also evaluated. However, such damages were not encountered in all research subjects. Journal of military pharmaco-medicine n o 1-2020 176 * The degree of fibrosis in the liver according to Metavir in the research groups: F0: 9 patients (27.3%); F1: 14 patients (42.4%); F2: 10 patients (30.3%); F3: 0 patient (0%); F4: 0 patient (0%). Among the research subjects, cirrhosis-related fibrosis damages with the highest rate were F1 (14/33 cases 42.4%), cirrhotic damages group F0 was 9/33 cases (27.3%) and fibrosis damages group F2 was 10/33 cases (30.3%). Cirrhosis damages of F3 and F4 were not detected. * Histological activity index (HAI) in the comparison group (n = 15): Very mild chronic hepatitis (1 - 3 points): 0 patient (0%); mild chronic hepatitis (4 - 8 points): 0 patient (0%); moderate chronic hepatitis (9 - 12 points): 9 patients (60%); severe chronic hepatitis (13 - 18 points): 6 patients (40%). * Stage of fibrosis in the comparison group (n = 15): None: 7 patients (46.7%); diseminated selerosis: 0 patient (0%); fibrosis with many splicing (from the beginning to central lobes): 6 patients (40%); clear cirrhosis: 2 patients (13.3%). Because there were no similar studies on histopathological damage in patients with dioxin-exposed chronic hepatitis, therefore, when reading the results of histopathological damages, it is necessary to have two experienced anatomists for consultation. However, the damages found in the study group were mostly nonspecific and may be seen in chronic liver disease due to other causes. Other chronic damages of chronic liver disease such as mallory, pigmentation, lipoid granuloma, giant mitochondria, venous thrombosis were also evaluated. However, those damages were not encountered in all study subjects. For people with chronic hepatitis due to other causes, histopathological damages has values that suggest the cause. For liver damage caused by hepatitis B virus in the comparison group with necrotic inflammatory damages, we applied the Knodell scale to assess liver damage through the histologycal activity index (HAI). Table 3: Image of ultrastructure of liver. Research group Comparison group Yes No Total Yes No Total Inflammatory cell infiltrates 33 (100%) 0 (0%) 33 (100%) 15 (100%) 0 (0%) 15 (100%) Intercellular space, dilated bile ducts 0 (0%) 33 (100%) 33 (100%) 0 (0%) 15 (100%) 15 (100%) 100% of cases of research and comparison groups had inflammatory cell infiltrates, no cases of intercellular space or the dilated bile ducts were detected. Journal of military pharmaco-medicine n o 1-2020 177 Figure 1: Image of biliary tract with the arachnoid villus (TEM, x5000) (patient Pham Ngoc L, 32 years old - research group). Table 4: Cirrhosis of the liver. Research group Comparison group Quantity Ratio (%) Quantity Ratio (%) No liver fibrosis 2 6.1 1 6.7 Perinatal fibrosis (with or without fibrosis around cells) 17 51.5 7 46.7 Portal fibrosis, very few fibers 14 42.4 5 33.3 Fibrosis with many splicing or bridging sclerosis 0 0.0 1 6.7 True cirrhosis 0 0.0 1 6.7 Total 33 100.0 15 100.0 - Liver fibrosis in the research group: Perinatal fibrosis accounted for the largest proportion (51.5%). - In comparison group: Perineal fibrosis had the largest number. Figure 2: Hyperstructural image of the liver. Penetration of fibers, bundle collagen fibers into the intercellular space of hepatic parenchyma cells (TEM. x 5000) (patient Vo Van T, 33 years old - research group). Journal of military pharmaco-medicine n o 1-2020 178 Table 5: Superstructure damages to liver cells. Research group Comparison group 2 Yes No Total Yes No Total Damaged cell membrane 0 (0%) 33 (100%) 33 (100%) 6 (40%) 9 (60%) 15 (100%) Damaged nuclear membrane 0 (0%) 33 (100%) 33 (100%) 9 (60%) 6 (40%) 15 (100%) Damaged nuclear 0 (0%) 33 (100%) 33 (100%) 8 (53.3%) 7 (46.7%) 15 (100%) Wide stretchrough endoplasmic reticulum 33 (100%) 0 (0%) 33 (100%) 0 (0%) 15 (100%) 15 (100%) Smooth endopla smic reticulum Wide stretch 33 (100%) 0 (0%) 33 (100%) 14 (93.3%) 1 (6.7%) 15 (100%) Multicellular torsion 33 (100%) 0 (0%) 33 (100%) 0 (0%) 15 (100%) 15 (100%) In the research group, ultrastructural damages of liver found in the dilated granular endoplasmic reticulum were 33/33 (100%), no damages were found in cell membranes, nuclear membranes, and nuclear. Unlike the research group, damaged cell membranes were found in 6/15 cases (40%), 9/15 cases (60%) were damaged in the nuclear membranes, 8/15 cases (53.3%) were damaged in nuclear, no dilated granular endoplasmic reticulum and rough endoplasmic reticulum were found. Table 6: Mitochondria damages. Research group Comparison group 2 Evenly distributed 1 (3%) 15 (100%) Gathered close to the cell membrane 32 (97%) 0 (0%) Location of mitochondria Total 33 (100%) 15 (100%) Normal (double, clear diaphragm) 26 (78.8%) 12 (80%) Fracture 7 (21.2%) 3 (20%) The structure of mitochondria membrane Total 33 (100%) 15 (100%) Normal structure 0 (0%) 15 (100%) Sparse 33 (100%) 0 (0%) Not found 0 (0%) 0 (0%) Crust mitochondria Total 33 (100%) 15 (100%) 1/3 of mitochondria in cells 0 (0%) 14 (93.3%) 2/3 of mitochondria in cells 1 (3.0%) 1 (6.7%) All mitochondria in cells 32 (97.0%) 0 (0%) The density of organelles increased electronically Total 33 (100%) 15 (100%) Yes 33 (100%) 1 (6.7%) No 0 (0%) 14 (93.3%) Dark seeds have a high electron level in the organelle Total 33 (100%) 15 (100%) Journal of military pharmaco-medicine n o 1-2020 179 Yes 31 (93.9%) 0 (0%) No 2 (6.1%) 15 (100%) Crystal sugar Total 33 (100%) 15 (100%) Normal 0 (0%) 15 (100%) Deformity (clavate form, with buds) 33 (100%) 0 (0%) Shape of mitochondria Total 33 (100%) 15 (100%) The same 0 (0%) 14 (93.3%) Not the same 33 (100%) 1 (6.7%) Giant 0 (0%) 0 (0%) Size Total 33 (100%) 15 (100%) Yes 10 (30.3%) 0 (0%) No 23 (69.7%) 15 (100%) Vitreous mitochondria Total 33 (100%) 15 (100%) All the study subjects with mitochondria damages, the damages were relatively homogeneous in the samples including sparse mitochondria, highly electronic particles in organelles, mitochondria were not uniform in size, deformed mitochondria shape; mitochondria position was concentrated in the cell membrane. In the comparison group: Mitochondria damages were rare (1/15 cases) where the mitochondria membrane structure was broken, organochromatic density increased electronically in 2/3 of the mitochondria in the cell with 1/15 cases (6.7%), mitochondria and crystal lines were not found and shape and position of the mitochondria were normal. The Ministry of Health has classified primary liver cancer in a list of diseases, defects and deformities related to exposure to toxic chemicals/dioxins [1]. In many other studies, based on the sampling method, the degree of hepatic fibrosis damage varies. In our study, the recorded degree of hepatic fibrosis damage was not as severe as other studies. However, we also found that 30.3% of F2 fibrosis cases were classified as significant fibrosis groups and should have monitoring and treatment plans to prevent the disease from getting worse. Chronic exposure to dioxin causes a long-term stressful effect on the hepatocyte detoxification response, which makes hyperstructural morphological changes of hepatocytes that are specifically recognized as mitochondria. Many previous studies on experimental animals fed with dioxins had found some changes in the hyperstructural properties of hepatocytes [8]. In the comparison group, there was a different damage on the hyperstructural image recorded as damages in the cell membrane, nuclear membrane and nucleus, with very few damages in mitochondria. Journal of military pharmaco-medicine n o 1-2020 180 Thus, we can see a clear and homogeneous damage on the study group’s image of hepatic hyperstructure. The results were similar to studies on liver damage in animals, so we think of dioxin- related liver damage in the study group. From the hyperstructural results, if there are basic studies on the combination of physiology, pathophysiology, molecular biology, etc, it will contribute to clarify the mechanism of liver damage of dioxin. CONCLUSION Through the research on the histopathological and ultrastructural damages of liver on 33 dioxin-exposed people with chronic liver diseases, we had the following conclusions: - Cirrhosis-related fibrosis damage group F1 had the highest rate: 14/33 cases (42,4%), 9/33 cases (27.3%) of liver fibrosis damages group F0 and 10/33 cases (30.3%) of fibrosis damages group F2. Cirrhosis damages of F3 and F4 were not detected. - On the research groups: Image of ultrastructure of the liver, damages in the cell membranes, nuclear and nuclear membranes were not found; however, mitochondria damages were relatively homogeneous in all cases. 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