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The effects of sodium arsenite exposure on the hepatic maturation period of cellular and functional reorganization in developing rat livers were evaluated. Animals received intraperitoneal injections of sodium arsenite (1.5 mg/kg body weight) or distilled water on days 9 to 28 after birth. On day 29, the animals were sacrificed either by cervical dislocation or by perfusion fixation. The perfusion fixed liver tissue was processed for paraffin embedding, sectioning and hematoxylin and eosin staining. The fresh liver tissue was processed for cryo-sectioning followed by Sudan Black B staining and for biochemical estimation of reduced glutathione. Microscopic observation revealed comparable preserved hepatic lobular patterns and distributions of uninucleate and binucleate hepatocytes in the control and the experimental groups. The mean nuclear area and diameter of the hepatocytes was increased in the experimental group. Lipid droplet distribution pattern in Sudan Black B stained sections revealed higher staining intensity towards the centrilobular area in both groups. Semiquantitative estimation of staining intensity showed lower mean gray values in zone 3 than in zones 2 and 1 (suggestive of the setting in of the adult pattern) in both groups. The reduced glutathione levels in the liver tissue and the altered nuclear size of the hepatocytes in the experimental group suggested the impairment of morphological and biochemical processes induced by arsenic exposure during the postnatal period.
Arsenic is an environmental pollutant of global concern. Inorganic arsenic in polluted drinking water is the chief source of exposure to arsenic in the Ganga-Meghna-Brahmaputra basin of India and Bangladesh [
Pregnant female Wistar rats were from the Experimental Animal Facility of the All India Institute of Medical Sciences. The study was approved by the Institute Ethics Committee.
The animals were provided with a standard rodent diet and drinking water
The day of birth of the pups was considered postnatal day zero (PND 0). The mother-reared pups were divided into a control group (I) and an experimental group (II) (n=12/group). They received intraperitoneal injections of double distilled, pyrogen-free, sterile water (group I) or aqueous sodium arsenite (1.5 mg/kg body weight, group II) from PND 9 to 28. The animals were sacrificed on PND 29. During the experimental period, the animals were weighed daily and various developmental signs were noted (eye opening, development of fur, etc.). Each group was further divided into subgroups "a & b" (n=6). The experimental design is summarized in
The animals of groups Ia and IIa (
The animals of groups Ib and IIb (
The pups were randomly assigned to the control or experimental group, each containing 12 pups. These groups were further randomly divided into subgroups, each with 6 pups, for the obtaining of fresh and paraformaldehyde fixed liver tissue post sacrifice.
The middle lobe of the liver (fresh unfixed; groups Ib and IIb) was weighed, sliced and homogenized at a final concentration of 20% in water [
H&E and SBB staining procedures were employed.
Animals in groups Ia and IIa (
Animals in groups Ib and IIb (
Sections were observed using a Nikon Eclipse E600 microscope and photographed using a digital camera (DS-Fi1, Nikon, Melville, NY, USA).
The H&E and SBB stained sections were separately morphometrically analyzed. Taxonomy, the counting of uni- and bi-nucleate cells, and nuclear area, and equivalent nuclear diameter, were assessed in groups Ia and IIa. Optical density (OD), a marker of the density of lipid accumulation, was measured in groups Ib and IIb.
For morphometric analysis of H&E stained sections, digital photomicrographs were captured to measure the area and the equivalent diameter of hepatocytes' nuclei under 40× (pixel size, 0.24 µm/pixel). One randomly chosen section of each sample, incorporating both right and left lateral lobes, and ten randomly chosen fields/sections were considered for measurement, so that a minimum of fifty hepatocytes per sample were measured [
The SBB stained sections were observed by optical microscopy with image analysis software (NIS-Elements AR 3.1, Nikon). The chosen fields were converted to gray scale of 0 (black)-255 (white). In each field, three rectangular grids of equal area were placed between the central vein and the portal triads in question (half area of a classical liver acinus). The grid areas were numbered 3, 2, 1 from the central vein towards the portal triad (
The morphometric parameters' mean±SD were computed for each lobe (right and left lateral) of each liver. The values were compared between the control and the experimental groups using Mann Whitney U and Kruskalwallis tests. The mean reduced glutathione levels of the individual samples of each group were treated as clustered data, and their differences were compared between the groups by Mann Whitney U testing. The OD data were subjected to Mann Whitney U testing and Kruskalwallis testing to evaluate the difference in staining intensity on zonal basis, if any, between the control and the experimental animals. SPSS ver. 17 (SPSS Inc., Chicago, IL, USA) was used for the analysis and
All reagents and stains were from Sigma Aldrich (St. Louis, MO, USA).
Various developmental features (ear unfolding, development of fur and spontaneous quadruped walking with the ventral surface of body off the floor) appeared at the scheduled times in both groups of animals. There was no observed difference in the two goups' general activities such as food intake, alertness and movement.
The groups' growth, measured daily by body weight, showed progressive and comparable increases in body weight from PND 1 to 9, before arsenic was first introduced to group II (
No apparent gross abnormality in the liver's shape or lobar pattern was observed in either group.
The lobular pattern, with the central vein in the center and the portal triads in the periphery, was clearly evident in both groups (
The hepatocytes' mean nuclear area was 28.32±0.83 µm2 in group I and 34.42±3.28 µm2 in group II. Their mean nuclear diameter was 5.97±0.83 µm in group I and 6.56±0.31 µm in group II. The differences were significant (
The expression of SBB staining, a measure of lipid droplet distribution, was mostly confined to the cytoplasm of the hepatocytes, with some hepatocytes showing more intense staining (
The GSH level of wet liver tissue in group II (11.23±0.98 µg/g) was significantly (
The effects of postnatal exposure (PND 9-28) to sodium arsenite on the hepatic maturation parameters of rats were assessed. The conversion of double cell plates to single cell plates [
The observation of no significant difference in body weight between the two groups is in accordance with earlier work [
Arsenic-induced distortion of liver histology, such as sinusoidal dilatation, cellular edema, megalocytosis, vacuolation and distorted hepatocyte nuclei, have been reported [
The conversion of double to single cell (hepatocyte) plates is an important parameter of hepatic maturation. The initial irregular and thick arrangement of cell plates [
Hepatocyte nuclear diameters in group I of this work were similar (4-6 µm) to those found in other works [
Sodium potassium adenosine triphosphatase (Na/K ATPase) has been reported to be responsible for the energy dependent extrusion of sodium ions (Na+) and uptake of potassium ions (K+), an important part of the maintenance of ionic homeostasis [
In the present study, group I (89.26±0.82%) and group II (90.99±1.77%) showed mainly uninucleate cells with a small remainder of binucleate cells. This is in partial agreement with another study [
Lipid droplet distribution shows a definitive pattern with age: there is an even distribution of lipid droplets in the centrilobular and periportal areas of newborn mice [
The initiation of oxidative stress following exposure to hepatotoxic agents is an important factors underlying chronic liver diseases associated with fibrosis. Disturbed membrane molecular properties through damage to such as membrane lipids, building blocks of proteins (amino acids), sugars (carbohydrates) and nucleic acids, is likely the primary factor of chemical induced hepatocellular injury. The liver is the center of metabolism of various chemicals including inorganic arsenic in a number of animals including mammals [
Arsenic's imbalancing of redox systems is also associated with its role as a potent inhibitor of glutathione reductase. Animals fed arsenic have been reported to show reduced levels of hepatic GSH with decreased enzymes of GSH regeneration, suggesting that arsenic induced exacerbation of toxic effects with depleted GSH levels in the liver [
Biochemical and morphological evidence of arsenite induced hepatotoxicity was observed in developing rat livers. The evidence was reduced levels of GSH (leading to increased oxidative stress), increased size of hepatocyte nuclei, sporadic vacuolation of hepatocyes and sinusoidal dilatation in the animals administered arsenic. To understand the exact mechanisms underlying arsenic induced toxicity at cellular and molecular levels, broader studies using various dosages of sodium arsenite and varied durations of exposure at different developmental periods should be considered.
We acknowledge the financial and technical support provided by the Department of Anatomy, All India Institute of Medical Sciences (AIIMS), New Delhi, India.
Experimental design. GSH, glutathione; i.p., intra-peritoneal; PND, post natal days.
High magnification photo micrographs of (A) hematoxylin and eosin stained and (B) Sudan Black B stained sections of liver (postnatal days [PND] 29). (A) A standard rectangular grid (270 µm × 80 µm) between the central vein (black asterisk) and the portal triad (black triangle). The nuclear area and equivalent diameter of the nuclei were generated by encircling the nuclear outline after selecting tool AREA (circle). Uninucleate (
Linear growth curve based on daily recorded weight (postnatal days [PND] 1 to 29) (A); bar diagram showing body weight at PND 9 (beginning of the treatment period) and PND 29 (day of sacrifice) (B) of the control and the experimental groups. Values expressed as mean±SD.
Low (A, B) and high (C-F) magnification photomicrographs of hematoxylin and eosin stained sections of liver (postnatal days 29) showing: preserved classical lobular pattern (dotted outline) with the central vein (black asterisk) in the center and the portal triads (black triangles) at the corners of the lobule in (A) control and (B) experimental animals. Apparent increase in hepatocyte size (
High magnification photo micrographs of hematoxylin and eosin stained sections of liver (postnatal days 29) from control (A) and experimental (B) group. Intracytoplasmic vacuolization (
Bar diagrams of (A) mean nuclear area (µm2), (B) mean nuclear diameter (µm), and (C) percentage of uni and binucleate hepatocytes in different lobes of the liver of hepatocytes of the control and the experimental groups. Values expressed as mean±SD, *
High magnification photo micrographs of Sudan black B stained sections of liver (postnatal days [PND] 29) from (A) the control and (B) the experimental groups. More (
Bar diagram showing the levels of reduced glutathione (GSH, µg/g of liver tissue) in the control and the experimental groups. Value expressed as mean±SD, *