ACUTE HEPATOTOXICITY OF Crotalus durissus terrificus ( SOUTH AMERICAN RATTLESNAKE ) VENOM IN RATS

Venom of the South American rattlesnake, Crotalus durissus terrificus (Cdt), presents myotoxic and neurotoxic outcomes, but reports on its effects on the liver are scarce. This study examined the hepatotoxicity resulting from Cdt venom administration (100, 200 and 300 μg/kg) in male Wistar rats. Animals were studies at 3, 6, 9 and 12 hours after venom injection. The hepatotoxicity was assessed through serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), gamma glutamyl transferase (GGT), bilirrubin and also by histopathological evaluation. All the different concentrations of Cdt venom resulted in increased levels of hepatic enzymes, when compared with the control group, except for the 100 μg/kg dose, which presented normal levels at 9 and 12 hours after venom administration. Bilirrubin levels remained unchanged by Cdt venom. Histological analysis revealed endothelial damage, inflammatory cell infiltration, as well as sinusoidal and portal congestion. Based on these observations, we may conclude that Cdt venom causes doseand time-dependent hepatic damage in rats, characterized by elevated hepatic enzyme levels and histological alterations.

In humans, envenomation by C. d. terrificus causes hepatic lesions involving necrosis, effects attributed to release of pro-inflammatory cytokines (24)(25)(26)(27).Also, hepatic steatosis and increased serum aspartate aminotransferase (AST) levels were reported by Azevedo Marques et al. (14) in patients bitten by C. d. terrificus and extensive hepatic necrosis by Barraviera et al. (24).In addition, Barraviera et al. (25) showed increased serum levels of AST (86%) and ALT (66%), by hepatocyte electron microscopy that showed mitochondrial damage, suggesting that liver injury may occur partly in the cytoplasm and partly in the mitochondria.Bancher, Rosa and Furlanetto (28) proved that, in mice, the venom could be detected in hepatic tissue.
The liver is a multifunctional organ, responsible for vital functions and for maintenance of energy balance in the organism.The release of intracellular enzymes into circulation is frequently used as an indicator of hepatocyte damage (29), although the release of some enzymes like AST and ALT -also present in tissues other than liver -needs to be evaluated by other injury markers to confirm any hepatic damage.Therefore, in the present study, we examined the liver damage induced by C. d. terrificus venom in rats through enzymatic markers of liver function and also by histological analysis.

Venoms and Reagents
The kits for the quantification of serum enzymes and bilirubin were from Labtest Diagnóstico (Brazil).The reagents for histology were purchased from Synth (Brazil).

Experimental Protocols
To study the hepatotoxicity of C. d. terrificus venom, rats (283 per group) received an intramuscular injection of sterile saline solution only or venom (100, 200 or 300 µg/kg) with saline solution in a final volume of 100 µL.The rats were subsequently killed with an overdose of ether at 3, 6, 9 or 12 hours after saline or venom injection (7 rats/time interval in each group) and blood was collected from the inferior vena cava.Samples of hepatic tissue were also obtained for histological analysis.

Histomorphometrical Analysis
Liver fragments from rats killed at different times after venom administration were fixed in 10% formaldehyde overnight and then dehydrated in an increasing alcohol series, clarified with xylol and embedded in paraffin.Sections of 5-µm-thick were cut by a microtome (American Optical Co., USA) and stained with hematoxylin and eosin, for subsequent examination and documentation by an Olympus CX-41® microscope (Olympus Optical, USA) fitted to an Olympus PM10SP Automatic Photomicrographic® system (Olympus America Medical, USA).Twenty fields were analyzed for each slide.Using a 100-point grid with a known area (10,000 μm 2 at 1,000x magnification) attached to the microscope lens the number of points contacting the Kupffer cells and leukocytes were counted within a total area (10,000 μm 2 ).The density of congested blood vessels, endothelial area and degenerated hepatocytes were counted and the values adjusted to the total tissue area (10,000 μm 2 ).The density of Kupfer and mononuclear cells, as well as the density of congested blood vessels, endothelial area and degenerated hepatocytes were expressed as number of cells/mm 2 of tissue.For a qualitative histological evaluation, the following criteria were used to determine the histologic score (scale 0-10): 0 = negative; 0.5 to 2.0 = mild; 2.5 to 5.5 = moderate; 6.0 to 8.0 = severe; 8.5 to 10.0 = very severe (34).

Statistical Analysis
The results were expressed as mean ± SEM and statistical comparisons were performed by Student's t-test or one-way of variance (Anova) followed by the Tukey-Kramer test.The p value was considered significant (p < 0.05); very significant (p < 0.01) and extremely significant (p < 0.001).

Serum Liver Enzyme Activities and Bilirubin Levels
Figure 1A shows the changes in AST levels at various time intervals following the injection of different doses of C. d. terrificus venom.The administration of 100 μg/kg of the venom resulted in increased AST activity at 3 hours when compared to the control group (p < 0.05), but no differences after 6, 9 and 12 hours (p > 0.05).
Regarding the 200-and 300-μg/kg venom doses, we observed an increased AST activity at all times (3, 6, 9 and 12 hours) relative to the control group (p < 0.05).AP activity increased progressively after the administration of all venom doses, although a significant change was seen only with the highest dose (300 μg/kg) at 12 hours post-venom (Figure 1C).GGT levels showed a peak increase 6 hours after administration of the lowest dose, with a return to the pre-venom levels at 12 hours post-venom.A moderate and persistent rise in GGT activity was registered with the intermediate dose (200 μg/kg), while the highest one produced a progressive increase during the 12 hours after venom administration (Figure 1D). Figure 1E shows that total bilirubin levels were not changed by any venom dose or time interval.

Histological Analysis
Sections of hepatic tissue from saline-treated (control) rats showed normal hepatocytes with strongly stained nuclei and protein-rich cytoplasm.Transversal sections showed intact sinusoids, portal spaces and a lobular central vein (Figure 2A).In contrast, rats injected with 100 µg/kg showed damage caused by an inflammatory infiltrate 3 and 6 hours after venom injection (Figure 2B).At 9 and 12 hours post-venom, the appearance of the tissue had returned to normal (Figure 2C).However, the morphometric analysis did not show statistical differences between the control group and 100 µg/kg group.At the venom dose of 200 µg/kg, copious cellular infiltrate and the extent of damage augmented with time (Figure 2D).The morphometric analysis showed that at 200 µg/kg at 12 hours, the densities of Kupfer cells, endothelium and degenerated hepatocytes were increased when compared with the control group.The density of congested blood vessels at 9 hours was higher than in the control group.At the highest venom dose (300 µg/kg), the hepatocytes showed marked disorganization, with swelling, necrosis and a progressive increase in damage up to 12 hours after venom administration (Figure 2E).Moreover, the histomorphometric findings demonstrate in Table 1 that at 9 hours, the density of Kupfer cells, congested blood vessels, endothelium densities and degenerated hepatocyte were greater than in those of control group.At 12 hours, all parameters were elevated when compared with the control group.

DISCUSSION
The present study showed that the Crotalus durissus terrificus venom augments activity of hepatic enzymes and also produces histological alterations, suggesting hepatotoxicity.
The liver maintains the organism's energy supply for many functions, such as producing substances that break down fats, converting glucose to glycogen, producing urea, synthesizing certain amino acids, filtering harmful substances and maintaining a proper blood glucose level.There are some intracellular enzymes that are released into the circulation and are frequently used as an indicator of damage to hepatocytes.The principal marker enzymes include alanine (ALT) and aspartic (AST) aminotransferases, which catalyze the transfer of α-amino groups from alanine and aspartate to the α-keto group of ketoglutaric acid to produce pyruvic acid and oxaloacetic acid, respectively (29).ALT is a more specific marker of hepatocellular injury, and occurs in the liver, kidneys, heart, skeletal muscle and pancreas, whereas AST has a wider distribution, occurring in organs such as heart, liver, skeletal muscles, kidneys and pancreas.In the liver, AST occurs predominantly (80%) in mitochondria and 20% in the cytoplasm, whereas ALT is confined to the cytoplasm (29,35).Other enzymes such as alkaline phosphatase (AP) and γ-glutamyl transferase (GGT) may also be used as markers of hepatic dysfunction, although they are found in the liver, bone, the kidneys and its enhancement is related to production elevation or excretion deficit, such as obstructive jaundice and cholestasis.GGT is present in the liver, kidney and pancreas.Bilirubin is a pigment derived from the catabolism of the heme radical in hemoproteins such as hemoglobin, while increased serum levels of this pigment can indicate cholestatic processes and hepatocellular damage.
A study in dogs revealed that C. d. terrificus venom causes marked alterations in hematological, hemostatic and biochemical parameters that may also be seen, through histological examination, as intravascular coagulation in cardiac and hepatic tissues (20).These authors also showed a progressive increase of serum levels of myoglobin, creatine kinase and aspartate aminotransferase demonstrated that animals developed rhabdomyolysis.On the other hand, AP did not increase expressly.Probably, this is related to cholestatic diseases, which is plausible because, like AP, bilirubin concentration did not increase over time and, as outlined in the preceding sections, is also related to cholestatic process (35)(36)(37).
As proven by the current study, the administration of C. d. terrificus venom to rats resulted in increased circulating levels of enzymes (ALT, AST and GGT) that are frequently employed as indicators of hepatic damage and cholestasis.The increased AST levels observed in the present work are similar to elevated serum activity of the same enzyme after human envenomations (24,11).Changes in serum levels of other enzymes, including creatine kinase (CK) and lactate dehydrogenase (LDH), have also been observed in human envenomations (11).However, there are differences in kinetic profiles of these alterations.For example, CK and LDH levels present their peaks respectively before and after other enzymes.The hepatic damage caused by the smallest venom dose (100 µg/kg), assessed by enzyme release and histological analysis (Table 1), appeared to be minimal and reversible.
Higher venom doses (200 and 300 µg/kg) resulted in greater damage that was not reversible within the time scale of the present experiments.In part, this hepatotoxicity is probably mediated by PLA 2 component of crotoxin, which presents the ability to interfere in hepatic mitochondrial respiration (38).The presence of inflammatory infiltrates associated with severe damage indicates that C. d. terrificus venom and its components are capable of affecting macrophage function and stimulating cytokine production (39,40).These findings agree with the demonstrations of acute phase inflammatory reactions in humans bitten by C. d. terrificus (26).
Coagulation disturbances (19,20) leading to fibrin deposition and ischemia, mainly through the action of the thrombin-like enzyme (gyroxin) of this venom (8,9,19) could also contribute to the hepatic damage registered in the present study.In addition, considering the inter-and intrasubspecific variation in the composition and biological activities of this venom (51,(56)(57)(58)(59)(60)(61), there may be differences in hepatic The present study provides evidence that Crotalus durissus terrificus venom induces hepatotoxicity in rats, by increasing blood liver enzymes, probably derived from liver, since histological analysis revealed augmented endothelium density and hepatic damage.

C
. d. terrificus venom was obtained by manual milking of snakes (both sexes) collected in the region of São José dos Campos and maintained in the snake house of the Nature Study Center, Vale do Paraíba University.The venom was lyophilized and stored at 4°C.Stock solutions of venom were prepared in sterile 0.9% NaCl immediately before use.Animals Male Wistar rats (180-220 g) obtained from Bem-te-vi Farm (Indaiatuba, SP, Brazil) were maintained in a 12-hour-light/dark cycle at 23 ±2°C with free access to food and water.Animal procedures described herein were approved by the institutional Animal Research Ethics Committee of UNIVAP (protocol number L035/2004/CEP) and performed in accord with the guidelines of the Brazilian College for Animal Experimentation (COBEA).

Figure
Figure 1B reveals the changes in ALT levels at diverse intervals after administration of different concentrations of C. d. terrificus venom.Concerning the 100-μg/kg dose, no changes in ALT activity was found, when compared with the control group at 3, 6, 9 and 12 hours (p > 0.05).The 200-μg/kg concentration of C. d. terrificus venom augmented ALT activity only at 9 and 12 hours relative to the control group (p < 0.05).At the dose of 300 μg/kg of C. d. terrificus venom, the ALT activity was greater than in the control group at 3, 6, 9 and 12 hours (p < 0.05).

Figure 1 .
Figure 1.Serum enzyme activities and bilirubin levels.Enzymes were collected from rat sera 3, 6, 9 and 12 hours after administration of 100, 200 and 300 μg/kg of Cdt venom.(A) Alanine aminotransferase (ALT); (B) aspartate aminotransferase (AST); (C) alkaline phosphatase (AP); (D) gamma glutamyl transferase (GGT) and (E) total bilirubin concentration.Data expressed as mean values ± S.E.M for 7 rats per group; *p < 0.05 when compared to the control group and analyzed by one-way Anova, followed by Tukey-Kramer multiple comparisons test.
effects produced by venoms obtained from C. d. terrificus specimens from other regions, and among other subspecies like C. d. cascavella, C. d. collilineatus, C. d. durissus and C. d. ruruima.Finally, diverse susceptibilities of animal strains and species (18) may also contribute to variations in hepatic effects of this venom.

Table 1 .
Morphometric analysis of liver histological aspects