HISTOPATHOLOGICAL EFFECTS OF CHRONIC EXPOSURE TO LEAD ON KIDNEYS OF BROWN TROUT ( SALMO TRUTTA M . FARIO ) * HISTOPATOLO [ KI EFEKTI HRONI ^ NOG IZLAGANJA OLOVU NA BUBREGE POTO

The aim of the present study was to investigate the histopathological changes on the kidneys of the brown trout (Salmo trutta m. Fario) inflicted by per os lead poisoning in the 46-week experimental period. The fish were kept in well-oxygenated freshwater basins. Approximately calculated toxic doses of lead-acetate for fish were 550 mg/kg. Following the experimental period, fish organs, in particular kidneys, were examined by light microscopy. Only fish with lesions observed in kidneys were further examined by electron microscopy. An accumulation of proteinaceous material in the glomeruli of the kidneys, and thickening of the basal membrane were observed histopathologically. This proteinaceous material was also observed in the lumina of the kidney tubules. Electron microscopic examination completely supported this finding. Furthermore, to the authors' previous knowledge, undocumented damage of the brush borders of the proximal kidney tubules was also observed.

Lead is considered one of the most toxic heavy metals.Lead is present everywhere and is used in industry, in almost all products, and through history is one of the most commonly used materials.It is used in wine production for reduction of aroma and color.Today, this use is discontinued because of the health risk.However, lead is still commonly used in the producution of many things, and Introduction / Uvod ORIGINALNI RAD -ORIGINAL PAPER lead-related contamination is a widespread issue.About 50% of lead today is used as a major constituent of lead-acid batteries, as shielding material in the electrical and phone industry, and as a pigment in the industry of paints for different colors (Haschek et al., 2002b).
Lead levels in fresh waters are about 5 mg/L, and these levels have greatly increased in the last 20 years.Major sources of lead are water distribution systems, waste waters from different industrial facilities, automobile air emission etc. Lead poisoning in fish is very rare.However, if it does occur it causes ischemia and so called, erythrocytes basophilic granulation, that may be used in diagnostics as the hematopoietic system is the primary site of lead toxicity.Furthermore, lead may slow down or prevent the normal development of skeletal muscle striation, and provoke incoordination of muscular movements and colics (Kühnert, 1991).
Lead poisoning primarily causes changes in three organ systems: the hematopoietic, the central nervous system and the urinary system.Ischemia caused by inadequate hemoglobin synthesis, and increased erythrocyte hemolysis is present evenly in acute and in chronic lead poisoning.Inadequate hemoglobin synthesis results from damaged function of delta aminolevulin acid (ALK) early in heme synthesis.The cause of increased hemolysis is less clear.It is assumed to be the rupture of erythrocytes membranes related to accumulation of lead salts on the erythrocytes.Appearance of spots in the erythrocytes is the characateristic of lead poisoning (Robbinson, 1989).
Toxic effects of lead are connected with histopathologic and biochemical changes due to its accumulation in the body.Lesions characterized with disorders in neuron function are found in the brain.Lead causes changes in the glia cells, and in the capillary walls as well.Also, changes can be found in kidneys and liver where intracytoplasmic and intranuclear changes occur.Studies have shown that no changes in the brain have been found in trouts exposed to lead concentration of 200 mg/L, in a 32-week period.However, enlargement of the nucleus, intracytoplasmic inclusions are possible changes in the liver of primarily younger fish.These intracytoplasmic inclusions accumulate unevenly in the apical portions of the kidney epithelial cells.Lead complexes with proteins are also visible in the epithelial cells of the kidneys (Klobu~ar, 1993).
The early stage of the chronic lead poisoning is characterized by interstitial fibrosis of kidney along with tubular atrophy without changes in the glomeruli.Later, glomerular hyalinization, tubular atrophy and thickening of the basal membrane occur.Changes in the interstitium are first seen as edema and later progress to chronic fibrosis and tubular atrophy.The kidneys appear smaller.Principal changes in the tubular cells take place in mitochondria.Blood perfusion of the kidney is also diminished (Cheville, 1994).
High concentration of lead results in the formation of intranuclear inclusions in the proximal tubular epithelial cells of the kidney, inhibits dehydratase of delta aminolevulinic acid and causes renal edema (Ware, 1991).

Experimental fish / Eksperimentalne ribe
Thirty specimens of one-year-old brown trout (Salmo trutta m.Fario) of the average weight of 15 g, were put in a freshwater (hard water) with a water temperature of 9 o C. The fish were kept in basins during a 46-week period and fed with food pellets (at the beginning of the experiment with 2 mm Biomare granules later to be changed to 3 mm granules).The fish were fed in compliance with the requirements of a food ration of 2 % body weight/ day.The fish that had survived after 46 weeks were sacrificed and used in further investigation.

Lead analysis / Analiza olova
Following the extraction of the kidneys we used the standard atomic absorption spectrophotometry (AAS) method (Perkin Elmer 3003 spectrometer), to detect the content of lead (Pb).

Toluidine blue staining / Bojenje toluidin plavim
For the staining of semi-thin sections embedded in wax we used the 0.25 % toluidine blue solution.The sections stained with toluidine blue solution were kept on a warm plate at a temperature of 70 to 80 o C. The duration of the staining procedure, dependent on the material, ranged between 1/2 to 2 minutes.Afterwards the sections were washed in distilled water, and dried on the microscopic slides which were placed on a dry plate at a temperature of 70 to 80 o C.

Transmission Electron Microscopic Study / Istra`ivanja transmisionom elektronskom mikroskopijom
The fish that had survived the experiment were quickly anesthetized by triciane methane sulfonate for 2-3 min.The kidneys were rapidly removed and routinely processed for transmission electron microscopic study.
The kidneys were cut into 1 mm pieces and fixed in 4% glutaraldehide solution for 24 hours.One hour after initial fixation, rinsing in Cacodylated puffer with a pH value of 7.2 was performed.After that the fixation was carried out again in 1 % osmiumtetraoxide solution for 2 hours.Postfixed tissues were rinsed in filtered water and dehydrated through a graded series of ethanol.Then they were infiltrated with propylene oxide and embedded in Epon 812.Ultrathin sections (0.5 -1 mm) were cut by using a glass knife on an "Ultracut E Reichart -Jung" with a thickness of 70 nm, collected on naked copper-meshed grids, and stained with uranyl acetate and lead citrate.The sections were examined and viewed using a Zeiss 10 C electron microscope operating at 50 kV.

Scanning electron microscopic study / Istra`ivanja skening elektronskom mikroskopijom
The kidney fragments were fixed in 4% glutaraldehyde solution for 24 hours.One hour after fixation, multiple rinsing in Cacodylated puffer (pH 7.2) was performed.After being fixed in 1% osmiumtetraoxide solution for 2 hours, the sections were dehydrated in a graded series of ethanol.Then the tissue pieces were dried by the Critical Point method (model E 3000).Prior to final drying of sections by the Critical Point method, it is necessary to keep them in acetic acid -amylacetate solution for two days.The coated specimens were examined by the scanning electron microscope Zeiss under 15 kV.

Chronic Exposure / Hroni~no izlaganje
Fish in the control basin swam normally without any signs of abnormality.Fish in the experimental basins did not show any abnormality either except in the 23rd week when their swimming movements decreased, along with a reduced feeding activity.Fish that died before the end of the experiment were also investigated.
At the end of the experiment the average lead concentration in proximal kidney tubules was analyzed by the atomic absorption spectrophotometer, and it amounted to 51 mg/g wet weight.

Figure 1. TEM micrograph of the kidney tubule. Epithelial cells contain single vacuoles (V) in the apical sections and abundancy of osmophilic bodies (white arrow). There is a great number of microvilli (MV) with tennis racquet shaped tips (arrow) at the luminal side (Tl) of the epithelial cells. 3150 X Slika 1. TEM mikrofotografija bubre`nog kanali}a. Epitelne }elije sadr`e pojedina~ne vakuole (V) u apikalnom delu i dobro zastupljena osmiofilna tela{ca (bela strelica).Vidi se veliki broj mikrovila (MV) sa vrhovima u obliku teniskog reketa (strelica) na lumenskoj strani (TI) epitelnih }elija. 3150 X
wed minor degenerative changes in the form of the tips' intumescence and widening that looks like a tennis racquet (Figure 1. and 2.).Clear thickening of the glomerular membrane caused by the lead ingestion is one of the characteristic changes on the kidneys that we confirmed by electron microscopic examination.Morphologicaly abnormal and immature erythrocytes were observed after examination of the blood smears under the light microscope (Figure 4).Clear thickening of the glomerular membrane caused by lead ingestion is one of the characteristic changes on the kidneys that we confirmed by electron microscopic examination (figure 1).Glomerular membrane thickening can consequently progress to the hyalinization of the glomerular basal membranes, also characteristic for the chronic lead poisoning.Haschek et al., (2002b) reported that lead, above all, accumulates in the kidneys, ie. in the cytoplasm of apical regions of the epithelial cells of kidney tubules, and these findings were also confirmed by the results of our investigation, by means of a great number of vacuoles and osmophilic granules (Figures 1 and 2).We assume that these granules might be lead in origin and that they are not transparent to electron rays.
Furthermore, in our investigation of experimental lead poisoning, we found damages of the microvilli of proximal tubules epithelium in the form of a confined widening that looks like the end of a tennis racquet.This change has not yet been reported in literature (Figure 1).No definitive conclusion can be made in regard of the mechanisms of development of these findings.Also, we could not notice the degenerative changes in the epithelial cells of the kidney by electron microscopic examination.Reports of Robbinson (1989), Franchini (1991) and Cheville (1994), that lead causes changes in blood count such as appearance of immature, abnormal erythrocytes, by influence on the enzymes that catalyze the process of erythropoiesis, could also be confirmed by the results of our investigations (Figure 4).Experimental lead poisoning of fish caused degenerative changes on the microvilli of the epithelial cells of kidney tubules in the form of a tip confined edema that looks like a tennis racquet.To our knowledge and available literature this finding has not been reported so far.