Effects of fibrates on the glycine conjugation of benzoic acid in rats.

In the course of glycine conjugation, benzoic acid is successively converted into benzoyl-CoA and benzoylglycine by mitochondrial enzymes (i.e. benzoyl-CoA synthetase and benzoyl-CoA/glycine N-acyltransferase, respectively), utilizing ATP, CoA, and glycine. Large doses of benzoate deplete CoA from the liver, suggesting that the supply of CoA may limit the capacity for glycine conjugation. Because fibrates are known to increase hepatic CoA synthesis, we examined whether treatment with fenofibrate or bezafibrate enhanced the capacity of rats to conjugate benzoic acid with glycine. Dietary administration of fenofibrate or bezafibrate (2.5 mmol/kg of feed, for 10 days) increased hepatic CoA levels 8-10-fold, while not affecting hepatic ATP levels; only fenofibrate elevated, albeit moderately, the concentration of glycine in liver. Hepatic mitochondria isolated from fibrate-fed rats, compared with those from controls, exhibited unchanged benzoyl-CoA synthetase activity but higher benzoyl-CoA hydrolase and lower benzoyl-CoA/glycine N-acyltransferase activities. Feeding with either fibrate increased liver mass by 50-60%. Control and fibrate-fed rats were administered benzoate at different doses, one to produce a large demand for CoA (i.e. 2 mmol/kg, iv) and two others to produce smaller demands for CoA (i.e. 1 mmol/kg or 2 mmol/kg plus glycine, iv). Fenofibrate-fed rats, and to a lesser extent bezafibrate-fed animals, exhibited increased glycine conjugation capacity, as indicated by faster disappearance of benzoate from the blood and appearance of benzoylglycine in the blood and urine, compared with controls; however, fibrates were not more effective in rats receiving the benzoate dose that produced the greatest demand for CoA. In contrast, benzoylglycine formation from benzoate (0.1-1 mM) was not enhanced in liver slices from fibrate-fed rats; moreover, it was lower than control levels in slices from bezafibrate-fed animals. Bezafibrate, but not fenofibrate, given to rats in a single dose (0.5 mmol/kg, ip) decreased the elimination and glycine conjugation of benzoate, indicating that bezafibrate is a direct inhibitor of glycine conjugation. In summary, fibrates influence glycine conjugation in a complex manner. Some fibrate-induced alterations (i.e. increased benzoyl-CoA hydrolase and decreased glycine transferase activities and direct inhibition by bezafibrate) can potentially hinder conjugation of benzoate with glycine, thus precluding conclusions regarding whether increased CoA availability enhances glycine conjugation. Fibrate-induced hepatomegaly appears to significantly contribute to the increased glycine conjugation capacity of rats treated with fenofibrate or bezafibrate.

Induction of metallothionein as an adaptive mechanism affecting the magnitude and progression of toxicological injury.

Pretreatment of rats with low doses of Cd produces adaptive tolerance to a subsequent high dose of Cd-induced lethality, thus shifting the dose-response curve to the right. Cd pretreatment of animals also protects against the hepatotoxicity produced by high doses of Cd. This protection is attributable to the 10- to 50-fold induction of hepatic metallothionein (MT) by Cd pretreatment. As a result hepatic subcellular distribution of Cd is significantly altered, with more Cd bound to MT in the cytosol and a concomitant reduction of Cd in other critical organelles. In addition MT-transgenic animals are more resistant, whereas MT-null mice are more sensitive than controls to Cd-induced lethality and hepatotoxicity. This further demonstrates that MT is important in Cd detoxication. Induction of hepatic MT by zinc also protects mice from carbon tetrachloride (CCl4)-induced liver injury, with more 14C-CCl4 bound to MT in the cytosol. MT-null mice are more sensitive to CCl4-induced hepatotoxicity, which supports the hypothesis that induction of MT also plays a protective role for nonmetallic chemicals. These results indicate that MT is a part of cellular adaptive mechanisms affecting the magnitude and progression of toxic insults from metals such as Cd as well as from organic chemicals such as CCl4.

Effect of several metallothionein inducers on oxidative stress defense mechanisms in rats.

One mechanism by which chemicals cause cellular injury is the formation of reactive oxygen species. In vitro studies have shown that metallothionein (MT), a small metal-binding, sulfhydryl-rich, readily inducible protein, can scavenge reactive oxygen species, especially hydroxyl radicals. Nevertheless, whether or not MT protects against oxidative stress in the intact animal is not known. Experimental induction of MT could help to clarify this question, however, it is unclear whether agents that induce MT also influence known antioxidant systems. Therefore, the present study was designed to determine whether the well-known MT inducers are specific for induction of MT or whether they might also influence other hepatic systems that protect against oxidative stress. Male rats were administered cadmium chloride (Cd; 30 mumol/kg, s.c.), zinc chloride (Zn; 1000 mumol/kg, s.c.), alpha-hederin (alpha-H, 30 mumol/kg, s.c.) or lipopolysaccharide (LPS; 1 mg/kg, s.c.) 24 h prior to measurement of antioxidant systems. Zn and alpha-H increased hepatic GSH concentration 20% and 55%, respectively. Cd significantly increased, whereas LPS reduced, the activities of selenium-dependent glutathione peroxidase and glutathione reductase. Glutathione S-transferases were not altered by any of the inducers. Cd also increased DT-diaphorase activity. Cd, Zn and alpha-H all decreased catalase activity 20-35%, while the activity of superoxide dismutase was unaffected by the inducers. The amount of total cytochrome P450 enzymes and cytochrome b5 were decreased by LPS, Cd and alpha-H, while Zn appeared to have no effect. The activities of P450 enzymes towards testosterone oxidation were also decreased by LPS, Cd and alpha-H. In conclusion, all four MT inducers examined affect systems known to protect cells against oxidative stress. Therefore, using these chemicals to determine the in vivo role of MT in protecting against oxidative stress poses difficulties.

Depletion of hepatic 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and sulfate in rats by xenobiotics that are sulfated.

Sulfation is considered a high-affinity but low-capacity conjugation mechanism that is limited by the availability of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the cosubstrate for sulfation. Salicylamide, phenol and 1-naphthol are all known substrates for the sulfation reaction. This study was conducted to determine whether the xenobiotics that are sulfated when administered to rats will lower hepatic PAPS and its precursor, sulfate. Urinary sulfate excretion was reduced 85% to 95% by these compounds. Hepatic PAPS was reduced 73%, 39%, and 87% by salicylamide, phenol and naphthol, respectively, 2 hr after administration of 2 mmol/kg. These compounds also decreased serum sulfate concentrations by 45% to 86% and lowered hepatic sulfate concentrations. In summary, these studies demonstrate that salicylamide, phenol and 1-naphthol lower hepatic PAPS and sulfate concentrations, as well as serum sulfate concentrations. These findings imply that increased sulfation, as a result of the sulfation of xenobiotics, results in depletion of hepatic PAPS concentrations, possibly because the utilization of PAPS by the sulfotransferases exceeds its generation via sulfate activation. Thus the capacity-limited sulfation of high dosages of xenobiotics appears to be due to the reduced availability of hepatic PAPS, which in turn is limited by the availability of sulfate.

Effect of oleanolic acid on hepatic toxicant-activating and detoxifying systems in mice.

We have previously shown that oleanolic acid (OA) protects mice against the hepatotoxicity of carbon tetrachloride, acetaminophen, bromobenzene, thioacetamide, furosemide, phalloidin, colchicine, cadmium, D-galactosamine and endotoxin. This study was designed to examine whether OA modulates hepatic toxicant-activating and detoxifying systems as a means of protection. Mice were treated with OA (100 and 200 mumol/kg s.c.) for 3 days, and liver microsomes and cytosols were prepared 24 hr after the last dose. OA produced a dose-dependent reduction in liver microsomal cytochrome P450 (P450) levels (25-37%) and cytochrome b5 (15-21%) content, but had no effect on NADPH-cytochrome c reductase activity. OA treatment also decreased several P450 enzyme activities, such as coumarin 7-hydroxylation (45%), 7-pentoxyresorufin O-dealkylation (35%), 7-ethoxyresorufin O-dealkylation (25%) and chlorzoxazone 6-hydroxylation (20%). Treatment of mice with OA decreased caffeine N3-demethylation (40%), but had no effect on caffeine 8-hydroxylation. OA treatment decreased testosterone 6 alpha- and 15 alpha-hydroxylation (40-50%) and androstenedione formation (35%), but slightly increased testosterone 1 alpha/beta-, 2 beta- and 6 beta-hydroxylation. Consistent with enzyme activities, OA decreased the amounts of mouse liver CYP1A and CYP2A enzymes, but had no appreciable effect on CYP3A enzymes, as determined by immunoblotting with antibodies against rat P450 enzymes. OA treatment slightly increased liver glutathione (GSH) content and the activity of GSH S-transferases toward 1-chloro-2,4-dinitrobenzene, but had no effect on GSH peroxidase and GSH reductase. The activities of superoxide dismutase and DT-diaphorase were unaffected by OA treatment. At the high dose of OA, catalase activity was decreased by 20%.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of liver cytochrome P450 by alpha-hederin: relevance to hepatoprotection.

This study was designed to determine the protective effects of alpha-hederin on chemical-induced liver injury in CF-1 mice and to evaluate cytochrome P450 suppression by alpha-hederin as a means of protection. alpha-Hederin pretreatment (30 mumol/kg, sc x 3 days) protected mice from acetaminophen-, bromobenzene-, carbon tetrachloride-, furosemide-, and thioacetamide-induced liver injury, without affecting the hepatotoxicity of chloroform and dimethylnitrosamine. To examine the role of P450 in hepatoprotection by alpha-hederin, liver microsomes were prepared 24 hr following the last dose of alpha-hederin treatment (10 and 30 mumol/kg, sc x 3 days). Treatment of mice with alpha-hederin produced a dose-dependent suppression of liver cytochrome P450 (30-50%) and cytochrome b5 (20-30%) levels, as well as NADPH-cytochrome c reductase activity (15-25%). alpha-Hederin treatment also decreased the activities of P450 enzymes, such as 7-ethoxyresorufin O-dealkylation (65%), 7-pentoxyresorufin O-dealkylation (50%), coumarin 7-hydroxylation (40%), 7-ethoxycoumarin O-deethylation (45%), caffeine N3-demethylation (30-50%), chlorzoxazone 6-hydroxylation (35-55%), and the oxidation of testosterone to 2 alpha-, 6 alpha-, 15 alpha-, 15 beta-, 16 alpha-, 16 beta-, and 18/12 alpha-hydroxyltestosterone, androstenedione, and 6-dehydroxytestosterone (25-60%). Consistent with these observations, the levels of CYP1A, CYP2A, and CYP3A enzymes were also suppressed, as determined by immunoblotting with antibodies against rat P450 enzymes. These results demonstrate that treatment of mice with alpha-hederin decreases the levels and activities of several P450 enzymes. The suppression of P450 appears to be one of mechanisms by which alpha-hederin protects mice from the hepatotoxicity of some chemicals.

The effect of Chinese hepatoprotective medicines on experimental liver injury in mice.

The purpose of this study was to compare the hepatoprotective effects of seven Chinese herbal compounds/mixtures on four known hepatotoxicants in mice. These compounds include fulvotomentosides oleanolic acid, total saponins of Panax japonicus (Jgs), total saponins of Panax notoginseng (Ngs), sweroside, oxymatrine, and dimethyl dicarboxylate biphenyl (DDB). All have previously been reported to exhibit hepatoprotective effects. Acute liver injury was produced in male CF-1 mice by CCl4, acetaminophen, cadmium chloride and allyl alcohol. Liver damage was assessed by quantifying serum activities of sorbitol dehydrogenase and alanine aminotransferase, as well as by histopathological examination. Fulvomentosides markedly decreased the toxicity produced by all four hepatotoxicants; oleanic acid also remarkably decreased acetaminophen, CCl4 and Cd-induced hepatotoxicity, but had no effect on allyl alcohol; total saponins of Panax japonicus and Panax notoginseng had moderate hepatoprotective effects on these models except that total saponins of Panax japonicus markedly decreased allyl alcohol toxicity; sweroside decreased Cd and CCl4 toxicity but had no effect on the other two hepatotoxicants; oxymatrine only decreased allyl alcohol toxicity; whereas DDB did not protect against any of the hepatotoxicants. The mechanism(s) by which these compounds/mixtures protect against different types of hepatotoxicants requires further investigation. In conclusion, of the seven compounds examined, fulvotomentoside and oleanolic acid appear to be the most effective in protecting against chemical-induced liver injury.

Protective effects of zinc on cultured rat primary hepatocytes to metals with low affinity for metallothionein.

The purpose of this study was to determine if Zn pretreatment could protect rat primary hepatocyte cultures from the cytotoxicity of five metals that have little or no affinity for metallothionein (MT). Hepatocytes were grown in monolayer cultures for 22 h and subsequently treated with ZnCl2 (100 microM) for 24 h; which increased the MT concentration 15-fold. Following Zn pretreatment, hepatocytes were exposed to various concentrations of Mn, V, Cr, Se, or Fe for an additional 24 h. Cytotoxicity was assessed by enzyme leakage and loss of intracellular K+. The toxicity of all five metals was significantly reduced in the Zn-pretreated cells. Zn pretreatment had no appreciable effect on the hepatocellular uptake (1-24 h) of Mn or Se. Zn pretreatment also did not increase the distribution of Mn or Se to the cytosol and neither metal was bound to MT, suggesting the protection was not due to their binding to MT. However, Zn pretreatment significantly decreased Mn-, Cr-, and V-induced cellular glutathione depletion. In summary, Zn pretreatment of rat primary hepatocyte cultures protects against Cr-, Mn-, Fe-, Se-, or V-induced hepatotoxicity. This protection does not appear to be related to MT induction but may be due to Zn-induced thiol or membrane stabilization and/or other biological changes produced by Zn.

Hepatic isometallothioneins in mice: induction in adults and postnatal ontogeny.

The purpose of this study was to quantitate hepatic metallothionein-I (MT-I) and metallothionein-II (MT-II) in adult mice pretreated with various dosages of selected inorganic and organic compounds and in nonchemically treated neonatal mice. Male CF-1 mice received Zn (0.38-6.0 mmol/kg, sc), Cd (5-80 mumol/kg, sc), dexamethasone (10-1000 mumol/kg, sc), or ethanol (60-180 mmol/kg, po). Liver cytosol was prepared 24 hr after the administration of each compound. In another experiment, liver cytosols were prepared from male and female neonates 1 to 35 days after parturition. MT-I and MT-II in liver cytosols were isolated by high-performance anion-exchange chromatography and quantitated by atomic absorption spectrometry. Hepatic MT-I and MT-II concentrations in adult controls were 5.1 +/- 1.3 and 3.7 +/- 1.0 micrograms/g liver, respectively. All compounds increased hepatic MT levels in a dose-dependent manner over a narrow range of dosages. The lowest dosages of Zn, Cd, dexamethasone, and ethanol that produced a significant increase in total MT content (MT-I plus MT-II) were 0.38, 0.005, 0.3, and 90 mmol/kg, respectively. Maximal induction of total MT following the highest dosages of Zn, Cd, ethanol, and dexamethasone was 58, 34, 24, and 13 times the control value (8.8 +/- 2.4 micrograms total MT/g liver), respectively. The relationship between dose and hepatic MT content was linear following ethanol administration and log-linear following Zn, Cd, and dexamethasone administration. The ratio of MT-I/MT-II was approximately 2.4 following all dosages of metals. Following low and high dosages of organic compounds, the ratio of MT-I/MT-II was approximately 1.0 and 1.5, respectively. Total MT concentration in livers of 1- to 14-day-old mice was approximately 40 times that observed in adult liver (5.5 +/- 1.6 micrograms total MT/g liver) and returned toward adult levels 21 days after parturition. The ratio of MT-I/MT-II was approximately 1.8 during Postpartum Days 1 through 14 and thereafter decreased to approximately 1.0. These results indicate that MT-I is more abundant than MT-II in mouse liver following chemical exposure and during neonatal development.

Dosage-dependent absorption of cadmium in the rat intestine measured in situ.

Previous studies have shown that the disposition of cadmium (Cd) following oral administration is dosage dependent and may possibly be due to dosage-dependent intestinal absorption of Cd. Though extensively studied, the precise nature of Cd absorption by the intestine remains unclear. Similarly, the role of metallothionein (MT) in the intestinal absorption of Cd remains equivocal. The present study was designed (1) to characterize the intestinal absorption of Cd in the rat, and (2) to determine the role of MT in intestinal Cd absorption. The study has been conducted with an isolated intestinal loop preparation in situ, which allows direct measurement of intestinal absorption under nearly physiological conditions. Under urethane-induced anesthesia. Cd (0.1, 10, 100, 1000, or 10,000 micrograms/kg) was injected intraluminally into the isolated intestinal loop in situ and all mesenteric venous (portal) blood exiting from the loop was collected for 90 min. Absorption of Cd into the portal circulation was low at all dosages studied. The percentage of the dosage absorbed ranged from 0.09% at the 0.1 microgram/kg dosage to 3.4% at the 10,000 micrograms/kg dosage. At low dosages (0.1 and 10 micrograms/kg), little difference was noted in the fractional absorption of Cd (0.09 and 0.14% of the dosage, respectively). However, the fractional absorption of Cd was 10-fold greater in rats administered 100 micrograms Cd/kg (1.1% of the dosage). Administration of higher dosages of Cd (1000 and 10,000 micrograms/kg) further increased the percentage of the dosage absorbed (1.8 and 3.4%, respectively). To evaluate the role of MT in the intestinal absorption of Cd. rats were subcutaneously injected with zinc (Zn) for 4 days (30 mg/kg/day) and the absorption of an intermediate dosage of Cd (100 micrograms/kg) was subsequently assessed in situ. Zn pretreatment increased the endogenous concentration of MT in the intestine 25-fold. Following intraluminal administration. 93% of Cd in intestinal cytosol of Zn-treated rats was bound to MT whereas 40% of the cytosolic Cd was bound to MT in saline-treated (control) rats. Moreover, the amount of Cd in intestinal cytosol was 2-fold greater in Zn-treated rats than in control rats. However, the intestinal absorption of Cd in rats pretreated with Zn demonstrated no difference from that in saline-treated rats. These results indicate that the intestinal absorption of Cd is dosage independent at low dosages of Cd (less than 10 micrograms/kg) and dosage dependent at high dosages (greater than 10 micrograms/kg). Furthermore, saturation of intestinal MT is not a major determinant of the observed dosage-dependent absorption of Cd.

Induction of metallothionein-I and metallothionein-II in rats by cadmium and zinc.

In this study, induction of the isoforms of metallothionein (MT) by Cd and Zn was determined with a high-performance liquid chromatography method. Rats were injected (sc) with Cd (1-100 mumol/kg) or Zn (100-10,000 mumol/kg) and concentrations of MT-I and MT-II in liver, kidney, and pancreas were measured 24 hr later. In control rats, only MT-II was detected in liver, whereas both forms were detected in kidney. In liver, Cd treatment increased MTs at all dosages tested such that concentrations of MT-I and MT-II were approximately equal. Zn (100-1000 mumol/kg) increased concentrations of MT-II about three times higher than MT-I, but higher dosages increased both isoforms to similar levels. Time-course experiments indicated that, at all times after Cd administration (30 mumol/kg), concentrations of MT-I and MT-II in liver were similar. However, with Zn treatment (1000 mumol/kg), MT-II levels were three times higher than MT-I at all times after 6 hr. In kidney, no differences in induction of the isoforms was observed following Cd or Zn treatment. In pancreas, Zn (300-10,000 mumol/kg) induced MT-I and MT-II to similar levels. Thus, in liver and kidney, Cd induced MT-I and MT-II similarly over a wide range of dosages. However, Zn treatment preferentially increased hepatic MT-II at low dosages but induced MT-I and MT-II to similar levels at high dosages. In contrast, Zn induced MT-I and MT-II to similar levels in kidney and pancreas.

Comparative effectiveness of topical treatments for hydrofluoric acid burns.

Hydrofluoric acid (HF) burns are characterized by progressive tissue necrosis and severe pain. Numerous topical treatments have been proposed, yet few have been studied experimentally. The present study was designed to examine the comparative efficacy of recommended treatments. Hair on the hind legs of rats was removed and 48 hours later 70% HF was applied. Calcium gluconate, Zephiran (benzalkonium chloride), A + D Ointment, aloe gel, and magnesium ointment were applied topically and burn development was monitored. Calcium gluconate significantly reduced burn size as early as one hour after application. Significant protection continued for seven days after the single application. The other treatments were not effective in decreasing or delaying HF burn development. The results indicated that calcium gluconate ointment was the most effective topical treatment for HF burns.

Acetaminophen decreases adenosine 3'-phosphate 5'-phosphosulfate and uridine diphosphoglucuronic acid in rat liver.

The purpose of this investigation was to determine the effects of acute administration of acetaminophen on adenosine 3'-phosphate 5'-phosphosulfate (PAPS) and UDP-glucuronic acid concentrations in fed male rats. Acetaminophen produced a dosage-dependent decline in rat hepatic PAPS concentrations which was significant after dosages of 150, 300, or 600 mg/kg, ip. The time course of the decline in PAPS values after 600 mg acetaminophen/kg showed that PAPS concentrations reached a nadir 1 hr after dosing (40% of control values). Serum sulfate concentrations were also decreased by large dosages of acetaminophen (32 and 15% of control 2 hr after 150 and 600 mg/kg, respectively) and a significant, positive correlation between serum sulfate and hepatic PAPS concentrations was noted. In addition, hepatic cysteine and glutathione concentrations were lowered by high dosages of acetaminophen. Hepatic UDP-glucuronic acid concentrations were greatly decreased for 2 hr after a dosage of 600 mg acetaminophen/kg (15, 23, and 42% of control 0.5, 1.0, and 2.0 hr after dosing, respectively) whereas 150 mg/kg produced a less pronounced and more transient decrease. These findings demonstrate that both PAPS and UDP-glucuronic acid concentrations in liver are decreased after administration of acetaminophen and imply that capacity-limited sulfation and glucuronidation of acetaminophen are due to a decrease in co-substrate availability.

Alteration of tissue disposition of cadmium by chelating agents.

The effect of several chelating agents (diethyldithiocarbamic acid, DDC; nitrilotriacetic acid, NTA; 2,3-dimercaptopropanol, BAL; d,l-penicillamine, PEN; 2,3-dimercaptosuccinic acid, DMSA; ethylenediaminetetraacetic acid, EDTA; and diethylenetriaminepentaacetic acid, DTPA) on the toxicity, distribution and excretion of cadmium (Cd) was determined in mice. When chelators were administered immediately after Cd, significant increases in survival were noted after treatment with DMSA, EDTA, and DTPA. DTPA, followed by EDTA and then DMSA, were consistently the most effective in decreasing the tissue concentrations of Cd and increasing the excretion of Cd. NTA, BAL, DDC and PEN had no beneficial effects. The effects of increasing the time interval between Cd administration and initiation of chelation therapy was determined by using a single administration of DTPA, EDTA, and DMSA. Mice treated immediately after Cd administration excreted approximately 50% of the administered dose of Cd compared to 0.2% in controls. Treatment with chelator at later times significantly increased Cd excretion but the magnitude of the effect was much less than that seen in mice treated immediately after Cd. To determine the role of MT in the acute decrease in chelator efficacy following Cd poisoning, rats were injected IV with Cd followed by DTPA at various times after Cd. Although DTPA reduced Cd content in the various organs when given immediately after Cd, the chelator was ineffective at all later times. Increases in hepatic and renal metallothionein (MT) did not occur until 2 hr after Cd, and did not coincide with the earlier drop in chelator efficacy. Blockade of MT synthesis by actinomycin D failed to eliminate this decreased DTPA effectiveness. Therefore, it appears that MT does not play an important role in the acute decrease in efficacy of chelation therapy for Cd poisoning. The effect of repeated daily administration of chelators on the distribution and excretion of Cd was studied by administering chelators daily for 5 days starting 48 hr after Cd. DTPA, EDTA, DMSA and BAL significantly increased the urinary elimination of Cd. Thus, mobilization of Cd into urine occurs with repeated chelation therapy, which may decrease tissue concentrations of Cd and reduce the toxicity of the metal.

Minimal role of metallothionein in decreased chelator efficacy for cadmium.

Chelator efficacy in Cd poisoning drops precipitously if therapy is not commenced almost immediately after exposure. Metallothionein (MT), a low-molecular-weight metal-binding protein with high affinity for Cd, may be important for this phenomenon. To more fully assess this role of MT in the acute drop in chelator efficacy following Cd poisoning, rats were injected iv with radioisotopic Cd (1mg/kg as CdCl2; 50 muCi/kg) followed by diethylenetriaminepentaacetic acid (DTPA; 90 mg/kg ip) at various times (0, 15, 30, 60, and 120 min) after Cd. Ther percentage of the Cd dose remaining in major organs 24 hr following Cd was determined. Although DTPA reduced Cd content in the various organs when given immediately after Cd, the chelator was ineffective at all later times. Increases in hepatic and renal MT did not occur until 2 hr after Cd, and did not coincide with the earlier drop in chelator efficacy. Blockade of MT synthesis by actinomycin D treatment (1.25 mg/kg, 1 hr before Cd) failed to prolong the chelators effectiveness. Furthermore, newborn rats have high levels of hepatic MT which had no effect on the time course of chelator effectiveness since DTPA still decreased Cd organ contents if given immediately following Cd but had no effect if given 2 hr after Cd. Therefore, if appears that MT does not have an important role in the acute decrease in efficacy of chelation therapy for Cd poisoning. The quick onset of chelator ineffectiveness may be due to the rapid uptake of Cd into tissues which makes it relatively unavailable of chelation.

Isolated rat hepatocytes as a model system for screening chelators for use in cadmium intoxication.

The utility of isolated rat hepatocytes as a model system for screening potential chelators in treatment of Cd intoxication was studied. The ability of the chelators diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 2,3-dimercaptosuccinic acid (DMSA), diethyldithiocarbamic acid (DDC), d,1-penicillamine (PEN), nitrilotriacetic acid (NTA), and 2,3-dimercaptopropanol (BAL) to decrease the cellular concentration of Cd was correlated with the previously reported effectiveness of these agents in the treatment of Cd intoxication in vivo. The results of cellular studies with either control or metallothionein-induced hepatocytes were compared to the in vivo effect of the chelators administered before or after the induction of metallothionein, respectively. The effects of DTPA, EDTA, DDC, and BAL in the hepatocyte model screening system correlated well with their reported in vivo effects. The results with NTA, PEN, and DMSA were not correlated as well but were explained by the relative differences between in vivo doses versus in vitro concentrations of the respective chelators. Therefore, the proposed model for screening potential chelators for use in cadmium intoxication appears to be a system which may prove to be an economical and rapid method to facilitate the search for efficacious chelators.