Resistance to cadmium-induced hepatotoxicity in immature rats

Because the concentration of metallothionein (MT) in perinatal rat liver is 10 to 20 times higher than levels present in liver of untreated adult rats, it was of interest to determine if immature rats are less susceptible to the hepatotoxic effects of cadmium (Cd) seen in adults. Male Sprague-Dawley adult rats received a hepatotoxic dose of 4.0 mg Cd/kg, iv, and 10-day-old rats received 4.0, 5.0,or 6.0 mg Cd/kg, iv. Ten hours following Cd injection, plasma enzyme activities in adults were elevated (aspartate aminotransferase, 50-fold; sorbitol dehydrogenase, 87-fold) and histologic examination showed extensive hepatic injury; however, no damage was evident in 10-day-old rats, even at the 6 mg Cd/kg dose. Two hours after injection of 3.5 mg Cd/kg, iv (7 μCi ¹⁰⁹Cd/mg Cd), the concentration of Cd was higher in liver, heart, and brain and lower in kidneys of 10-day-old rats compared to adults. An age comparison of the hepatic subcellular distribution of Cd revealed a higher amount of Cd in cytosol and less in the particulate fraction of 10-day-old rats. Furthermore, cytosolic Cd was predominantly bound to MT. These data support the hypothesis that presynthesized levels of MT are important in producing tolerance to acute Cd toxicity and that tolerance results from an altered hepatic subcellular distribution of Cd.     

Analysis of strain difference in sensitivity to cadmium-induced hepatotoxicity in Fischer 344 and Sprague-Dawley rats.

Acute administration of cadmium (Cd) in rats results in hepatotoxicity that appears to involve the activation of Kupffer cells and the subsequent production of proinflammatory chemokines and cytokines. However, the importance of these endogenous mediators in Cd-induced hepatotoxicity is unknown. Therefore, this study was conducted to define and utilize a rat strain difference in sensitivity to Cd-induced hepatotoxicity to elucidate the role of cytokines and chemokines in Cd-induced hepatotoxicity. Doses were selected from a dose-response study of the effect of Cd on serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities. Hepatotoxic doses of 2.0 mg Cd/kg in Fischer 344 (F344) rats and 3.0 mg Cd/kg in Sprague-Dawley (SD) rats, as well as a relatively nontoxic dose of 2.0 mg Cd/kg in SD rats, were chosen for the time-course experiment. Blood and liver from F344 (saline or 2.0 mg Cd/kg iv) and SD rats (saline or 2.0 or 3.0 mg Cd/kg iv) were collected at 0, 1, 3, 6, 10, 18, 24, and 48 h after Cd administration. Cadmium treatment caused an increase in serum ALT and SDH by 3 h and peaked between 18 and 24 h in both strains. Hepatic Cd content, metallothionein (MT) induction, and nonprotein sulfhydryl (NPSH) content were quantified and determined to be consistent with dosing rather than strain differences. Total RNA samples isolated from liver samples were analyzed for chemokine (CINC-1 and MCP-1) and cytokine (TNF-alpha, IL-1beta, IL-6, and IL-10) mRNA levels by the Quantigene branched DNA signal amplification assay. Lipopolysaccharide treatment served as a positive control for chemokine and cytokine induction. After Cd administration, F344 rat livers did not contain higher levels or earlier induction of chemokine and cytokine mRNAs than SD rats. Therefore, this study demonstrates a strain difference in sensitivity to Cd-induced hepatotoxicity that appears to be unrelated to Cd, MT, NPSH, or cytokine expression.     

Changes in hepatic glutathione concentration modify cadmium-induced hepatotoxicity

Cd has a strong affinity for sulfhydryl groups and is hepatotoxic. Thus, to further understand the mechanism of Cd-induced liver injury, the effect of increased and decreased hepatic glutathione (GSH) concentration on Cd-induced liver injury was examined. Liver GSH was lowered by pretreating rats with phorone (250 mg/kg, ip) or diethyl maleate (0.85 mg/kg, ip) 2 hr prior to challenge with various doses of Cd. Ten hours after Cd (1) 40–80% of the rats pretreated with phorone or diethyl maleate and challenged with 1.0–2.0 mgCd/kg died whereas no mortality was observed in the control group; (2) plasma enzyme activities of alanine (ALT) and aspartate (AST) aminotransferase and sorbitol dehydrogenase (SDH) were markedly increased in phorone and diethyl maleate-pretreated rats challenged with Cd (0.7–2.0 mg/kg) versus control rats; and (3) moderate changes in liver histology were observed in corn oil pretreated and Cd challenged rats, while prior depletion of GSH potentiated histopathologic changes in liver produced by Cd alone. Another group of rats received cysteine (1.9 g/kg, po) 3 hr prior to injection of a lethal dose of Cd. Cysteine pretreatment increased liver GSH levels by 22% 3 hr after administration and attenuated Cd-induced liver injury as evidenced by marked decreases in plasma ALT, AST, and SDH activities. Pathological changes in liver were also reduced. These data indicate that liver reduced GSH concentration is important in modulating Cd-induced hepatotoxicity.     

Acute hepatotoxicity after high-dose methotrexate administration to rats.

Acute hepatotoxicity after administration of 10-1000 mg/kg methotrexate (MTX) to rats was studied by monitoring serum transaminases, liver morphology, and disposition kinetics of MTX and 7-hydroxy-methotrexate (7-OH-MTX). Half the control rats and rats administered 1000 mg/kg MTX, had their bile duct cannulated. One to 2 hr after administration of 1000 mg/kg MTX, 50% of MTX treated bile-drained rats (Ebc) developed cholestasis despite similar or larger initial bile flow rates than those which did not develop cholestasis (Ebn, controls). In Ebc animals, peak serum ASAT and ALAT levels were 6- and 4-fold higher than that of the control rats, and morphologically, prominent hepatocytic changes and grossly dilated bile canaliculi were found. Immediately prior to cholestasis, the Ebc animals reached biliary 7-OH-MTX levels (8.3 +/- 1.3 mM, mean +/- S.E.M.) which were equivalent to the threshold level for precipitation of 7-OH-MTX in rat bile in vitro, and 3-fold higher than the corresponding levels of 7-OH-MTX in the bile of Ebn rats. Ninety-five % of the drug in the precipitated material was 7-OH-MTX. Hence, 7-OH-MTX may play a role in acute MTX hepatotoxicity, a dose-limiting toxicity that may not be counteracted by leukovorin rescue.     

Strain differences of cadmium-induced hepatotoxicity in Wistar-Imamichi and Fischer 344 rats: involvement of cadmium accumulation

We previously reported that Wistar-Imamichi (WI) rats have a strong resistance to cadmium (Cd)-induced lethality compared to other strains such as Fischer 344 (Fischer) rats. The present study was designed to establish biochemical and histological differences in Cd toxicity in WI and Fischer rats, and to clarify the mechanistic basis of these strain differences. A single Cd (4.5 mg/kg, s.c.) treatment caused a significant increase in serum alanine aminotransferase activity, indicative of hepatotoxicity, in Fischer rats, but did not in WI rats. This difference in hepatotoxic response to Cd was supported by pathological analysis. After treatment with Cd at doses of 3.0, 3.5 and 4.5 mg/kg, the hepatic and renal accumulation of Cd was significantly lower in the WI rats than in the Fischer rats, indicating a kinetic mechanism for the observed strain differences in Cd toxicity. Thus, the remarkable resistance to Cd-induced hepatotoxicity in WI rats is associated, at least in part, with a lower tissue accumulation of the metal. Hepatic and renal zinc (Zn) contents after administration were similarly lower in WI than in Fischer rats. When Zn was administered in combination with Cd to Fischer rats, it decreased Cd contents in the liver and kidney, and exhibited a significant protective effect against the toxicity of Cd. We propose the possibility that Zn transporter plays an important role in the strain difference of Cd toxicity in WI and Fischer rats.     

Protective role of carnosine in mice with cadmium-induced acute hepatotoxicity

The hepatoprotective effect of carnosine was investigated against cadmium-induced acute liver injury in mice. Hepatotoxicity was induced by a single i.p. injection of cadmium chloride (6.5 mg/kg). Carnosine treatment (10 mg/kg/day, i.p.) was applied for three consecutive days, starting one day before cadmium administration. Carnosine significantly decreased the cadmium-induced elevations in serum aminotransferases. Carnosine suppressed lipid peroxidation and restored the deficits in the antioxidant defense mechanisms (reduced glutathione level, and catalase and superoxide dismutase activities) in liver tissue resulted from cadmium administration. Also, the reductions in hepatic nitric oxide and zinc ion levels, and the increases in hepatic cadmium ion concentration, and myeloperoxidase and caspase-3 activities following cadmium exposure were significantly attenuated by carnosine treatment. In addition, carnosine markedly ameliorated cadmium-induced liver tissue damage as evidenced by light and electron microscopic examinations. It was concluded that carnosine can be considered a potential candidate to protect the liver against the deleterious effect of acute cadmium intoxication.     

1,2-Dichloropropane hepatotoxicity in rats after inhalation exposure

The hepatic effects of 1,2-dichloropropane (DCP) were investigated in male Wistar rats exposed to 15, 50, 100, 250, 450, 1000, 1300, 1800 or 4900 mg DCP m-3. At the end of a 4-h period of exposure, average blood DCP levels were 0.025 and 5.38 micrograms ml-1 in animals treated with 15 and 1300 mg m-3, respectively. Blood DCP concentrations were correlated with the air DCP concentrations in the inhalation chamber. At DCP concentrations of 100 mg m-3 or higher, the liver non-protein thiol (NPT) content was significantly reduced. Assays performed 20 h after 4-h DCP exposure showed that exposure to 100-1000 mg DCP m-3 had no effect on hepatic NPT levels. The NPT content increased only in the liver of rats exposed to higher (1300-4900 mg m-3) DCP concentrations. Treatment with DCP did not cause hepatic lipid peroxidation and did not modify total protein content. The observed changes in liver cell thiol homeostasis are likely to reflect the action of reactive intermediates formed during DCP metabolism. These changes can occur in rats following exposure to considerably low levels of DCP vapour.     

Effects of oestrogen on mitochondrial thymidine kinase activity in the immature rat uterus.

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway. High TK activity and the existence of cytosolic isozyme have consistently been observed in rapidly proliferating tissues. Oestrogen treatment induces the moderate increase of activities of mitochondrial TK and the fetal type TK isozyme in immature rat uterus. These results indicate that oestrogen induces the increase of uterine DNA synthesis in both cytosol and mitochondria in immature rats.     

Involvement of calcium channels in the sexual dimorphism of cadmium-induced hepatotoxicity

Cadmium toxicity has been evaluated in a number of in vivo and in vitro toxicological studies. In vivo Cd toxicity exhibits sexual dimorphism with females being more susceptible to Cd uptake, accumulation, and toxicity in the liver. Research to date does not explain why females are more sensitive to Cd-induced hepatotoxicity. Recent studies demonstrate that progesterone sensitizes female F(344) rats and TRL-1215 cells to Cd toxicity, however the mode of action is still unclear. Approximately one half of the Cd entering the cytoplasm does so through receptor operated Ca(2+) channels. Progesterone treatment of human spermatozoa and Xenopus laevis oocytes causes a rapid influx of Ca(2+) suggesting a possible mechanism. Since hepatocytes have progesterone receptors on their cellular membrane and Ca(2+) influx into the cytoplasm occurs following progesterone treatment we evaluated the hypothesis that progesterone facilitates the uptake and accumulation of Cd via Ca(2+) channels, leading to enhanced toxicity. Primary isolated rat hepatocytes were treated with Cd, progesterone, and/or verapamil for 4 h and cytolethality was measured. Pretreatment with the Ca(2+) channel blocker verapamil increased the Cd concentration producing 50% lethality (LC(50)) by 2-fold, thus decreasing Cd cytolethality. In contrast, pretreatment with progesterone decreased the Cd LC(50) by 2-fold resulting in enhanced Cd cytolethality. Verapamil treatment reversed the progesterone enhanced Cd cytolethality. Verapamil and/or progesterone in the absence of Cd did not affect hepatocyte viability. Overall, the results of this study demonstrate that inhibition of progesterone-induced Ca(2+) influx with the Ca(2+) channel blocker verapamil, decreases Cd cytolethality in primary isolated rat hepatocytes. These findings indicate that progesterone activation of receptor-mediated Ca(2+) channels is involved in the sexually dimorphic hepatotoxicity seen following acute Cd exposure.     

Effects of feeding and fasting on hepatolobular distribution of glutathione and cadmium-induced hepatotoxicity.

Relationship between hepatolobular distribution profile of glutathione (GSH) and cadmium (Cd)-induced hepatotoxicity was examined in both fed and fasted rats by computerized densitometry of histochemically stained GSH in the liver sections using an image analyzer system. In fed rats, density gradient distribution of hepatolobular GSH, which was higher in the periportal region than in the perivenous one, was always observed even at a diurnally minimal concentration of GSH. This heterogeneous distribution of GSH, however, disappeared in fasted rats, even though the hepatic GSH concentration recovered to 81% of the control level in rats fasted for 48 h. In histopathological examination on livers 24 h after oral treatment of fed and fasted rats with 60 mg Cd/kg, zonal necrotic changes were observed from the perivenous to midlobular region but not in the periportal one in fed rats even at a diurnally minimal concentration of hepatic GSH. On the other hand, necrotic changes in the liver extended to the panlobular region including the periportal one in fasted rats. These necrotic changes were greater with a longer duration of fasting. These results suggest that the density gradient distribution of hepatic GSH but not the actual concentration of the compound plays an important role in protecting rats against acute hepatotoxicity of Cd.     

Liver delta-aminolevulinate dehydratase activity in amitriptyline- or chlorpromazine-treated rats.

Amitriptyline (AMT) and chlorpromazine (CPZ) (0.5 mg per animal, i.p.) were injected into rats separately for 30 days and their effects on heme metabolism in liver were examined. Significant decreases in the delta-aminolevulinate dehydratase activity were observed following the administration of both drugs (mean value of AMT-group: 6.58 U/g tissue; and CPZ-group: 7.04 U/g tissue) in comparison to that of controls (11.71 U/g tissue); however total liver heme content was not altered. When 24-h urinary excretions of delta-aminolevulinate (ALA) and porphobilinogen (PBG) were measured on the last day of the experiment, a slight (AMT-group: 38.40 micrograms/day) to distinct (CPZ-group: 59.11 micrograms/day) increase of urinary ALA was observed, while PBG excretion tended to decline only moderately under CPZ (3.52 micrograms/day), but significantly in presence of AMT (2.16 micrograms/day). Mean values obtained from control group were 32.12 micrograms/day for ALA and 4.25 micrograms/day for PBG.     

Dietary calcium restriction enhances cadmium-induced metallothionein synthesis in rats.

Experiments were conducted with adult male rats to investigate the effects of dietary calcium (Ca) restriction upon intake and tissue distribution of cadmium (Cd), and Cd-metallothionein (Mt) synthesis. Four groups of animals were fed either a low-Ca, semisynthetic diet (0.1% Ca) or the same diet supplemented with 0.8% Ca (normal diet). The caloric intake was similar in all groups. Two groups (low-Ca and normal diet) were used as controls, and two groups (low-Ca and normal diet) received 100 mg/l Cd (as CdCl2) in drinking water. Cd levels in liver, kidney, spleen and red cells were measured in all animals after 8 weeks of treatment. Concomitantly, Mt levels in plasma, liver and kidney were evaluated by radioimmunoassay. Ca deficiency entailed marked and significant increases in accumulation of Cd and synthesis of Mt in all assayed tissues. It is concluded that dietary Ca restriction, independent of caloric intake, enhances Cd intestinal absorption and tissue accumulation, which is followed by increased tissue Mt synthesis.     

Alpha-adrenergic regulation of the activity of thymidylate synthetase and thymidine kinase during liver regeneration after partial hepatectomy

The increases in activity of hepatic thymidylate synthetase and of thymidine kinase, which catalyze the formation of thymidylate via the de novo and salvage pathways, respectively, were significantly suppressed during liver regeneration in rats which had been given alpha-adrenoceptor antagonists (phenoxybenzamine and phentolamine) or adrenergic neuron blockers (guanethidine and reserpine). These suppressions were not observed with a beta-adrenoceptor antagonist (propranolol), or an anticholinergic agent (atropine methyl nitrate). The rise in the activity of the thymidylate-synthesizing enzymes was closely correlated with the increase in the DNA content of the liver. It is concluded that catecholamine regulates the increase in the activity of thymidylate synthetase and thymidine kinase, which are key enzymes in DNA synthesis in regenerating liver. It is also suggested that sympathetic nerves play an important role in liver regeneration.     

Time-course of cadmium-induced acute hepatotoxicity in the rat liver: the role of apoptosis

Exposure to toxic metals and pollutants is a major environmental problem. Cadmium is a metal causing acute hepatic injury but the mechanism of this phenomenon is poorly understood. In the present study, we investigated the mechanism and time-course of cadmium-induced liver injury in rats, with emphasis being placed on apoptosis in parenchymal and nonparenchymal liver cells. Cadmium (3.5 mg/kg body weight) was injected intraperitoneally and the rats were killed 0, 9, 12, 16, 24, 48 and 60 h later. The extent of liver injury was evaluated for necrosis, apoptosis, peliosis, mitoses and inflammatory infiltration in hematoxylin–eosin-stained liver sections, and by assaying serum enzyme activities. The number of cells that died via apoptosis was quantified by TUNEL assay. The identification of nonparenchymal liver cells and activated Kupffer cells was performed histochemically. Liver regeneration was evaluated by assaying the activity of liver thymidine kinase and by the rate of ³H-thymidine incorporation into DNA. Both cadmium-induced necrotic cell death and parenchymal cell apoptosis showed a biphasic elevation at 12 and 48 h and peaked at 48 and 12 h, respectively. Nonparenchymal cell apoptosis peaked at 48 h. Peliosis hepatis, another characteristic form of liver injury, was first observed at 16 h and, at all time points, closely correlated with the apoptotic index of nonparenchymal liver cells, where the lesion was also maximial at 48 h. Kupffer cell activation and neutrophil infiltration were minimal for all time points examined. Based on thymidine kinase activity, liver regeneration was found to discern a classic biphasic peak pattern at 12 and 48 h. It was very interesting to observe that cadmium-induced liver injury did not involve inflammation at any time point. Apoptosis seems to be a major mechanism for the removal of damaged cells, and constitutes the major type of cell death in nonparenchymal liver cells. Apoptosis of nonparenchymal cells is the basis of the pathogenesis of peliosis hepatis. The first peaks of necrosis and parenchymal cell apoptosis seem to evolve as a result of direct cadmium effects whereas the latter ones result from ischemia.     

Hepatoprotective activity of Leptadenia reticulata stems against carbon tetrachloride-induced hepatotoxicity in rats

Objective:

To evaluate the hepatoprotective activity of ethanolic and aqueous extract of stems of Leptadenia reticulata (Retz.) Wight. and Arn. in carbon tetrachloride (CCl₄)-induced hepatotoxicity in rats.

Materials and Methods:

The toxicant CCl₄ was used to induce hepatotoxicity at a dose of 1.25 ml/kg as 1 : 1 mixture with olive oil. Ethanolic and aqueous extracts of L. reticulata stems were administered in the doses of 250 and 500 mg/kg/day orally for 7 days. Silymarin (50 mg/kg) was used as standard drug. The hepatoprotective effect of these extracts was evaluated by the assessment of biochemical parameters such as serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, total bilirubin, serum protein, and histopathological studies of the liver.

Results:

Treatment of animals with ethanolic and aqueous extracts significantly reduced the liver damage and the symptoms of liver injury by restoration of architecture of liver as indicated by lower levels of serum bilirubin and protein as compared with the normal and silymarin-treated groups. Histology of the liver sections confirmed that the extracts prevented hepatic damage induced by CCl₄ showing the presence of normal hepatic cords, absence of necrosis, and fatty infiltration.

Conclusion:

The ethanolic and aqueous extracts of stems of L. reticulata showed significant hepatoprotective activity. The ethanolic extract is more potent in hepatoprotection in CCl₄-indiced liver injury model as compared with aqueous extract.     

Acetaminophen hepatotoxicity in aging rats

Severity of liver damage 24 hr after i.p. administration of acetaminophen in doses of 0.4 and 0.8 g/kg was evaluated in male Fischer 344 rats at 4, 14 and 25 months of age. Both doses of acetaminophen produced significant elevations of serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities in 4-month-old rats. Enzyme release was somewhat diminished in old age. Hepatic glutathione (GSH) and microsomal cytochrome P-450 concentrations were decreased in rats that received 0.8 g/kg of acetaminophen. The decreases occurred in young-adult and middle-aged rats, but not in old rats. The results demonstrated that old age does not enhance the hepatotoxic effects of acetaminophen in male Fischer 344 rats.     

Attenuation of eNOS expression in cadmium-induced hypertensive rats

Cadmium (Cd) has been reported to induce hypertension in both humans and animals; however, its mechanism has not been clearly elucidated. Vascular tone is one of the factors contributing to hypertension. This study was conducted to investigate the effects of Cd exposure on vascular muscarinic receptor responses to acetylcholine (ACh) in isolated aortas. Male Sprague-Dawley rats were exposed to Cd via drinking water (5, 10 and 50 ppm) for 3 months. Cd 10 and 50 ppm exposure caused significant decreases in the sensitivity of vascular muscarinic receptors to ACh. However, Cd exposure did not alter the vascular relaxation induced by sodium nitroprusside (SNP) which is a nitric oxide donor. Consistent with the reduction of ACh-induced relaxation, treatment with Cd decreased endothelial nitric oxide synthase (eNOS) protein level in blood vessels. These results suggested that Cd suppressed ACh-induced vascular relaxation by interfering with muscarinic receptor function, and its downstream signaling pathway may be one of the contributing factors for the development of hypertension.