Immunohistochemical Analysis of P-gp, LC3-II, and Cathepsin-D Associated with Histological Changes in Fish Liver: from the Impacted Environment to Clean Water

Abstract Herein we evaluated the histopathological alterations and expression patterns of multixenobiotic resistence (MXR) and autophagic proteins in liver samples of fish chronically exposed to anthropogenic contaminants in a highly polluted river, and then again after they had been transferred to good quality water. Two groups were established: euthanized on the day of capture (0 h), and maintained for 30 days in a tank (30 d). The fish of 0 h presented liver with vacuolated and hypertrophic hepatocytes. Also, it was observed strong immunostaining of cathepsin-D, LC3-II and P-gp. Necrosis and apoptosis were also observed throughout the liver. Conversely, the second group (30 d) showed recovery of the liver normal histology and weak immunoreaction of the studied proteins. So, our results indicated that there was a hepatic recovery in the fish kept in good quality water, as showed by the decreased expression of cathepsin-D, LC3-II, and the MXR (P-gp). Therefore, the alterations here observed could be proposed as potential biomarkers to be tested for following the impacts of remediation or mitigation measures to environmental impacts.

Impacto de diferentes fontes proteicas sobre o desenvolvimento celular hepático de ratos recém-desmamados submetidos à dieta restritiva seguida de recuperação alimentar / Impact of different protein sources on the development of hepatic cells in newly-weaned rats submitted to a restriction diet followed by food repletion

The aim of this study was to evaluate the effect of different protein sources with different amino-acid profiles on liver cell development in "Wistar" rats submitted to a food restriction and recovery model. The food restriction model was based on a 50% ingestion restriction for the rats fed with the control diet (21 days) and "ad libitum" recovery (a period of 21 days). The protein sources used in this study were: Yeast autolysate (YA), whey protein concentrate from bovine milk (WPC), a mixture containing the YA and WPC in the proportion of 64:36 (protein base), commercial casein (CC) which was used as the experimental treatment (EC) and control treatment (CP). The following parameters were evaluated in this study: the amino-acid profile of the protein sources, the development of liver cells (liver weight and growth rates - RNA, DNA, protein), weight and number of hepatocytes in the whole organ. The results showed that the treatment with (YA) was the most affected by the food restriction, showing an incomplete (leucine-deficient) amino-acid profile, a lower development of liver cells, lower growth of the liver due to a lower growth by cellular hyperplasia (number of cells), lower capacity of cell division and DNA synthesis. However, it showed a higher ability for RNA synthesis, thus indicating that growth in the restricted phase was mainly due to increase in the size of hepatocytes (cell hypertrophy). During the repletion period, all food treatments showed normal liver development, i.e. cell growth and organ hyperplasia and hypertrophy.

El objetivo de este estudio fue evaluar el efecto que diversas fuentes de proteínas con diferentes perfiles de aminoácidos ejercían en el desarrollo de las células hepáticas en ratas "Wistar" sometidas a restricción y reposición de la ingestión de alimentos. El modelo de restricción alimentar consistía en disminuir 50% del consumo de los animales control (período de 21 días) y la recuperación con ingestión "ad libitum" (período de 21 días). Las fuentes de proteínas utilizadas en este estudio fueron: autolisado de levadura (ATL); concentrado proteico de suero de leche bovino (CPL); mezcla de CPL y ATL, en la proporción de 64:36 (base proteica), caseína comercial (CC), que fue utilizada como tratamiento experimental (CE) y como estándar (CP). Fueron evaluados el perfil de aminoácidos de las proteínas, el desarrollo de la célula hepática (peso del hígado y las tasas de crecimiento: ARN, ADN y proteína total), el peso y número de hepatocitos en el órgano. Los resultados mostraron que el grupo tratado con (ATL) fue el más afectado por el proceso de restricción de alimentos, la proteína presenta un perfil incompleto de aminoácidos(deficiente en leucina). Había menor desarrollo de las células del hígado, menor crecimiento del hígado debido a un menor crecimiento por hiperplasia celular (número de células), menor capacidad de división celular y de síntesis de ADN, sin embargo, mostraron una mayor capacidad para sintetizar ARN indicando que el crecimiento en la fase de restricción se debió principalmente al aumento en el tamaño de los hepatocitos (hipertrofia celular). Durante la fase de recuperación alimentar de todos los tratamientos hubo un desarrollo hepático normal, o sea crecimiento de las células y del órgano por hiperplasia e hipertrofia.

O objetivo deste estudo foi avaliar o efeito de diferentes fontes proteicas com diferentes perfis de aminoácidos sobre o desenvolvimento celular hepático de ratos "Wistar" submetidos à restrição e recuperação alimentar. O modelo de restrição alimentar foi baseado na restrição de 50% da ingestão dos animais controle (período de 21 dias), e recuperação ad libitum (período de 21 dias). As fontes proteicas utilizadas neste estudo foram: autolisado de levedura (ATL), concentrado proteico de soro de leite bovino (CSL), mistura contendo CSL e ATL na porcentagem de 64:36 (base proteica), caseína comercial (CC), a qual foi utilizada como tratamento experimental (CE) e como tratamento padrão (CP). Avaliouse, neste estudo, o perfil de aminoácidos das fontes proteicas, o desenvolvimento celular hepático (peso do fígado e dos índices de crescimento - RNA, DNA, proteína total), peso dos hepatócitos e número de hepatócitos em todo órgão. Os resultados mostram que o tratamento com (ATL) foi o mais afetado pelo processo de restrição alimentar, apresentando um perfil de aminoácido incompleto (deficiência em leucina); apresentou menor desenvolvimento celular hepático; menor crescimento do fígado em função do menor crescimento por hiperplasia celular (número de células), menor capacidade de divisão celular e síntese de DNA. Entretanto, apresentou maior capacidade de síntese de RNA, indicando que o crescimento na fase de restrição ocorreu principalmente por aumento no tamanho dos hepatócitos (hipertrofia celular). Durante o período de restauração alimentar todos os tratamentos apresentaram desenvolvimento hepático normal, ou seja, crescimento de células e do órgão por hiperplasia e hipertrofia.

Changes in Glycogen and Glycosaminoglycan Levels in Hepatocytes of Iduronate-2-Sulfatase Knockout Mice before and after Recombinant Iduronate-2-Sulfatase Supplementation

PURPOSE: Mucopolysaccharidosis II (MPS II) is a lysosomal storage disorder caused by a deficiency of iduronate-2 sulfatase (IdS), which is involved in the degradation of glycosaminoglycan (GAG). In this study, the frequency of fasting hypoglycemia in patients with MPS II was investigated and changes in accumulation of glycogen and GAG in the hepatocytes of IdS-knockout (KO) mice were evaluated before and after recombinant IdS enzyme replacement therapy (ERT). MATERIALS AND METHODS: Plasma glucose levels were evaluated after an 8-hour fast in 50 patients with MPS II. The IdS-KO mice were divided into three groups (group 2; saline, group 3; 0.15 mg/kg of IdS, and group 4; 0.5 mg/kg of IdS); wild-type mice were included as controls (group 1). ERT was initiated intravenously at four weeks of age, and continued every week until 20 weeks of age. RESULTS: The mean glucose level after an 8-hour fast was 94.1 +/- 23.7 mg/dL in the patients with MPS II. Two (4%) out of 50 patients had fasting hypoglycemia. For the mice, GAG in the lysosomes nearly disappeared and glycogen particles in the cytoplasm were restored to the normal range in group 4. CONCLUSION: Glucose metabolism in patients with MPS II appeared to function well despite hepatocytic GAG accumulation and hypothetical glycogen depletion. A higher dose of IdS infusion in MPS II mice led to disappearance of lysosomal GAG and restoration of glycogen to the cytoplasm of hepatocytes.

Oxidative stress and hepatotoxicity in rats induced by poisonous pufferfish (Lagocephalus lagocephalus) meat

This study was undertaken to evaluate the effect of pufferfish (Lagocephalus lagocephalus) meat poisoning on hepatic functions of Wistar rats. For this purpose, groups of rats (Lcr, Lcu+b and Lcu-b) received diet supplemented with 10 percent of raw or cooked meat, respectively, with or without cooking water of L. lagocephalus while groups Mcr and Mcu+b received diet supplemented with 10 percent of raw or cooked meat of Liza aurata, which were used as a negative control. In Lcu+b group, ALT, AST and ALP rates (hepatic enzyme markers) decreased after two months of treatment, indicating liver damage. We also observed an increase of 54 and 65 percent of thiobarbituric acid reactive substances (TBARS) in their livers respectively 48 hours and two months after treatment compared to controls. The catalase (CAT) activity in group Lcu+b decreased (p < 0.05) after two periods of treatment, whereas metallothionein (MT) level significantly increased and decreased, respectively after 48 hours and two months. In fact, in the histological analysis of the livers from Lcu+b treated group, we observed an increase in vacuolisation, necrosis, hepatocytes ballooning and sinusoids dilation. These results indicate that L. lagocephalus meat cooked with water produces hepatotoxicity and oxidative damage.(AU)

Heterogeneous Composition of Voltage-Dependent K+ Currents in Hepatic Stellate Cells

PURPOSE: Hepatic stellate cells (HSC) are a type of pericyte with varying characteristics according to their location. However, the electrophysiological properties of HSC are not completely understood. Therefore, this study investigated the difference in the voltage-dependent K(+) currents in HSC. MATERIALS AND METHODS: The voltage-dependent K(+) currents in rat HSC were evaluated using the whole cell configuration of the patch-clamp technique. RESULTS: Four different types of voltage-dependent K(+) currents in HSC were identified based on the outward and inward K(+) currents. Type D had the dominant delayed rectifier K(+) current, and type A had the dominant transient outward K(+) current. Type I had an inwardly rectifying K(+) current, whereas the non-type I did not. TEA (5mM) and 4-AP (2mM) suppressed the outward K(+) currents differentially in type D and A. Changing the holding potential from -80 to -40mV reduced the amplitude of the transient outward K(+) currents in type A. The inwardly rectifying K(+) currents either declined markedly or were sustained in type I during the hyperpolarizing step pulses from -120 to -150mV. CONCLUSION: There are four different configurations of voltage-dependent K(+) currents expressed in cultured HSC. These results are expected to provide information that will help determine the properties of the K(+) currents in HSC as well as the different type HSC populations.

Protective trials against aflatoxin B[1] induced toxicity in albino rat hepatocytes. Histological and histochemical study

Is to investigate the effect of aflatoxin B[1] on hepatocytes and the possible protective effect of administration of selenium and high protein diet. The stud was carried out on 32 albino rats divided into 4 equal groups; a control group, a group receiving aflatoxin B[1] a group receiving selenium with aflatoxin and a fourth group receiving high protein diet with aflatoxin. Specimens were taken from the liver of all groups and subjected to histological, histochemical and ultrastructural studies. Animals receiving aflatoxin B[1] showed periportal hepatic degeneration in the form of ballooning of cells and vacuolation of the cytoplasm. Some cells revealed hepaloceliular necrosis. Portal tract showed bile duct proliferation. Fibrosis was occasionally seen in the portal tract. Ultrastructural results revealed irregularities in the nuclei with dilatation of the perinuclear cisternea. Some nuclei showed separation of the fibrillar and granular component of their nucleoli. The cytoplasm showed proliferation and dilatation of smooth and rough endoplasmic reticulum, degenerated mitochondria, decreased number of ribosomes and glycogen granules with the increase of lysosomes and appearance of fat droplets. Evident decrease in the DNA and RNA content were seen by methyl green pyronin stain. Administration of selenium showed evident improvement in the cellular structure and liver architecture. The electron microscopic study showed nearly normal nuclei and nucleoli. The cytoplasm showed more or less normal mitochondria, moderate number of ribosomes and glycogen granules Administration of high protein diet revealed moderate improvement. Administration of selenium antagonizes the deleterious effect induced by aflatoxin B[1] while high protein diet has less protective effect on the toxicity, of aflatoxin on the liver