Cd-pretreated Rats Research Articles

Efficient tissue repair underlies the resiliency of postnatally developing rats to chlordecone + CCl 4 hepatotoxicity

It is often assumed that at a younger age populations are at higher risk of toxic effects from exposure to toxic chemicals. Recent studies have demonstrated that neonate and postnatally developing rats are resilient to a wide variety of structurally and mechanistically dissimilar hepatotoxicants such as galactosamine, acetaminophen, allyl alcohol, and CC1 4. Most interestingly, young rats survive exposure to the lethal combination of chlordecone (CD) + CCl 4 known to cause 100% mortality in adult male and female rats. In a study where postnatally developing (20- and 45-day), and adult (60-day) male Sprague Dawley rats were used, administration of CCl 4 (100 μl/kg i.P.) alone resulted in transient liver injury regardless of age as indicated by plasma alanine transaminase (ALT), sorbitol dehydrogenase (SDH) levels and histopathological lesions. In CD-pretreated rats, CCl 4-induced toxicity progressed with time culminating in 25 and 100% mortality by 72 h after CCl 4 in 45- and 60-day rats, respectively, in contrast to regression of CCl 4-induced toxicity and 0% mortality in 20-day rats. [ 3H]thymidine ( 3H-T) incorporation and proliferating cell nuclear antigen (PCNA) studies revealed an association between delayed and diminished DNA synthesis, unrestrained progression of liver injury, and animal death. Time-course studies revealed that the loss of resiliency in the two higher age groups might be due to inability to repair the injured liver rather than due to infliction of higher injury. Intervention of cell division in 45-day CD rats by colchicine (CLC, 1 mg/kg, i.p.) 30 h after CCl 4 challenge increased mortality from 25 to 85%, confirming the importance of stimulated tissue repair in animal survival. In contrast, efficient and substantial DNA synthesis observed in 20-day rats allows them to limit further progression of liver injury, thereby leading to full recovery of this age group with 0% mortality. Examination of growth factors and proto-oncogene expression revealed a 3- and 3.5-fold increase in transforming growth factor-α (TGF-α) and H- ras mRNA expressions, respectively, coinciding with maximal hepatocyte DNA synthesis in 20-day normal diet (ND) rats, as opposed to only 2- and 2.5-fold increases observed in 60-day ND rats, respectively. Increased expression of c- fos (10-fold) in 20-day rats occurred l h after CCl 4 compared to less than a 2-fold increase in 60-day rats. These findings suggest that prompt stimulation of tissue repair permits efficient recovery from injury during early postnatal development of rats.

The Nephrotoxicity of Intravenously Administered Cadmium-Metallothionein: Effect of Dose, Mode of Administration, and Preexisting Renal Cadmium Burden

Exogenously administered cadmium-metallothionein (Cd-MT) is highly nephrotoxic, producing renal damage similar to that seen following chronic Cd exposure. However, nephrotoxicity following Cd-MT administration occurs at a much lower renal Cd concentration than that following chronic Cd exposure. In the present study, the sensitivity of female rats to bolus and infused doses of Cd-MT was evaluated. The Cd-MT was administered via the jugular vein either as a bolus or infused over a 24-hr period via osmotic minipumps in naive rats and in rats which had been pretreated with 5 μM CdCl 2/kg, sc, five times/week, for 9 weeks. Renal toxicity was evaluated by urinary lactate dehydrogenase, protein, and MT excretion. In naive rats, a bolus dose of 0.15 mg Cd/kg as Cd-MT was nephrotoxic. In comparison, a two-fold higher infused dose was required to cause nephrotoxicity. In Cd-pretreated rats, a bolus dose of 0.45 mg Cd/kg as Cd-MT was necessary to produce nephrotoxicity, presumably because Cd pretreatment yielded renal MT levels that were about 15 times higher than the levels in naive animals. Although a bolus dose of 3 mg Cd/kg as CdCl 2 resulted in a renal Cd concentration about 1.5 times that found after administration of 0.15 mg Cd/kg as Cd-MT, it did not produce any nephro-toxicity, The results of this study suggest that a renal Cd concentration, which is nephrotoxic if the Cd-MT is administered as a bolus dose, is well tolerated if the Cd-MT is delivered at a sustained level over a 24-hr period. Furthermore, the preexisting renal Cd burden reduces the sensitivity to nephrotoxicity of Cd-MT, presumably due to elevated MT levels available for sequestration of incoming Cd. We conclude that mere accumulation of Cd in the kidney does not necessarily result in nephrotoxicity; instead, the circulating Cd-MT level, as well as the intracellular MT concentration, appear to be the more important determinants of nephrotoxicity.

Loss of Calcium Homeostasis Leads to Progressive Phase of Chlordecone-Potentiated Carbon Tetrachloride Hepatotoxicity

Earlier work has shown increased hepatocellular free Ca2+ levels in rats receiving a single subtoxic dose of CCl4 after dietary pretreatment with nontoxic (10 ppm, 15 days) levels of chlordecone (CD), indicating a significant perturbation of Ca2+ homeostasis in the interactive toxicity of CD + CCl4 combination treatment. In the present study, the mitochondrial and microsomal ability to sequester Ca2+ as well as plasma membrane translocase activity was investigated, since it is known that cells maintain normal Ca2+ homeostasis by these mechanisms. Hepatic plasma membrane Ca2+-ATPase (high and low affinity components) as well as 45Ca uptake by mitochondria and microsomes was measured using a range of calcium concentrations in Ca2+-EGTA-buffered medium at different time points after a single ip administration of CCl4 (100 μl/kg). Male Sprague-Dawley rats were maintained for 15 days either on a normal diet or on a diet containing 10 ppm CD prior to CCl4 injection. Hepatic plasma membranes, devoid of microsomal and mitochondrial contamination, were prepared using polyethyleneimme-coated beads. CD treatment alone did not significantly decrease the plasma membrane Ca2+ATPase activity. Similarly, CCl4 treatment alone did not alter Ca2+-ATPase in hepatic plasma membranes at any concentration of free Ca2+ in assay medium employed in this study. The interactive combination treatment, however, resulted in significant, irreversible, and specific inhibition of the high affinity component of the hepatic plasma membrane Ca2+-ATPase at early time points. Low affinity Ca2+-ATPase was not affected with any treatment protocol. CD pretreatment alone significantly inhibited 45Ca uptake by mitochondria and microsomes when incubated at 10 μM and higher, concentrations much higher than normal cytosolic levels, but not at lower concentrations of Ca2+. CCl4 administration to both normal and CD-pretreated rats resulted in significant inhibition of microsomal and mitochondrial 45Ca uptake as early as 1 hr at all concentrations of free calcium. While the extent of inhibition was greater and irreversible after CD + CCl4 treatment, it was reversible after normal diet + CCl4 treatment. Phosphorylation of proteins was determined in order to investigate if the inhibition of microsomal 45Ca uptake during CD + CCl4 toxicity might be correlated to decreased phosphorylation of any particular protein involved in Ca2+ transport. SDS-polyacrylamide gel electrophoresis of microsomal protein revealed at least 30 Comassie blue stainable bands. Of these, 6 bands were phosphorylated when microsomes were incubated with [32P]ATP. After CCl4 treatment, the band corresponding to ∼20 kDa protein in liver microsomes decreased in its intensity in both normal and CD rats, suggesting that CCl4-induced inhibition of microsomal 45Ca uptake was due to decreased phosphorylation of ∼20 kDa protein. The decrease was significantly greater after CD + CCl4 combination treatment. These results suggest that an irreversible perturbation of Ca2+ homeostasis is a critical event in the liver injury associated with the hepatotoxicity of CD + CCl4 combination treatment.

Protection from chlordecone-amplified carbon tetrachloride toxicity by cyanidanol: Regeneration studies

Previous work has shown that chlordecone (CD)-amplified CCl 4 hepatotoxicity and lethality can be mitigated by pretreatment with cyanidanol. These studies also revealed that stimulated hepatocellular regeneration might play an important role in the cyanidanol protection of CD-amplified CCl 4 toxicity. The present studies conducted over a time course of 0 to 120 hr after CCl 4 challenge describe sequential changes in hepatic [ 3H]thymidine incorporation into hepatocellular nuclear DNA, polyamines and related enzymes, and histomorphometry of liver sections from variously treated rats. Male Sprague-Dawley rats (125–150 g) were maintained on a control diet or on a diet contaminated with CD (10 ppm) for 15 days and/or pretreated with cyanidanol (250 mg/kg, ip) at 48, 24, and 2 hr befor a single ip injection of either a standard protocol dose (100 μl/kg) or a low dose (50 μl/kg, L) of CCl 4 on Day 16 of the dietary protocol. Cyanidanol pretreatment significantly stimulated the hepatic [ 3H]thymidine incorporation into hepatocellular nuclear DNA of control rats irrespective of CD pretreatment. Similarly, polyamine metabolism was altered favorably for cell division, although mitotic index (metaphase) was not increased. Cyanidanol-stimulated [ 3H]thymidine incorporation was highly suppressed in rats receiving the CD + CCl 4 standard dose combination treatment up to 36 hr, but after this time point a marked increase was observed. Hepatocellular regeneration, quantified histomorphometrically as volume density of cells in metaphase, was progressively increased in rats protected from CD + CCl 4 interaction by cyanidanol, starting at 36 hr and lasting until 72 hr. Favorably altered polyamine metabolism was evident from the stimulated ornithine decarboxylase, as well as from the stimulated interconversion of the higher polyamines to maintain increased concentration of putrescine. Challenge by the same dose of CCl 4 (100 μl/kg) to CD-pretreated rats not protected by cyanidanol failed to cause any increase in [ 3H]thymidine incorporation up to 36 hr and resulted in animal death starting at 36 hr. In the surviving rats, [ 3H]thymidine incorporation at 48 hr was increased, but was less than 50% of the increase observed in the cyanidanol group. In these rats, attenuation in the stimulation of cell division and insufficiently increaed putrescine levels were observed, which are consistent with the inadequate level of hepatocellular regeneration. With rats receiving CD + CCl 4(L) combination, the [ 3H]thymidine incorporation at 48 hr was less than 50% of the increase of cyanidanol-protected rats. Cyanidanol pretreatment to the CD + CCl 4 group of rats prevented the decrease in the hepatic DNA levels. CCl 4 challenge to control rats did not show any discernible alterations in the hepatic DNA levels. However, CCl 4 administration to CD-pretreated rats caused a significant and progressive decrease in hepatic DNA starting at 6 hr until animal death. Similarly, CD + CCl 4(L) rats showed a significant decrease in hepatic DNA at 24 and 48 hr, whereas, in the surviving rats a recovery in DNA levels was observed. These results suggest that the major event in the cyanidanol protection of CD-amplified CCl 4 toxicity is the stimulation of hepatocellular regeneration targeted for tissue repair.

Involvement of sulfhydryl metabolism in tolerance to cadmium in testicular cells

Cadmium (Cd)-induced acute testicular toxicity and testicular interstitial cell (IC) tumors can be prevented by low-dose Cd pretreatment. The mechanism of this self-tolerance is unknown. In this regard glutathione (GSH) may play a role in protecting cells from damage by Cd. Therefore, possible mechanisms of self tolerance to Cd in ICs were investigated with emphasis on sulfhydryl metabolism. Rats were pretreated with low-dose Cd (3.0 μmol/kg, sc). Such low-dose Cd pretreatment prevented the necrotizing effects of normally testopathic doses of Cd (20.0 μmol/kg, sc) given 24 hr later. ICs were isolated by collagenase dispersion 24 hr after pretreatment and incubated with Cd (1.0 m m) for 1 hr. In vivo Cd-pretreatment alone increased GSH levels (as determined by HPLC) of whole cells (17%) and cytosol (17%) compared to nonpretreated control. When ICs from nonpretreated rats were exposed to Cd in vitro, GSH in whole cells declined 8% compared to nonpretreated control and 21% compared to cells from in vivo Cd-pretreated rats. In ICs isolated from pretreated rats and exposed to Cd in vitro, GSH levels were normal in whole cells and slightly increased in cytosol. In whole testes low-dose Cd reduced GSH overall, both in cytosol (34%) and in nuclei (14%). Changes in cysteine levels were also seen, similar to those of GSH in whole ICs and cytosol. Neither low-dose in vivo Cd-pretreatment nor in vitro Cd exposure greatly altered levels of the low M r testicular Cd-binding proteins as assessed by electrophoresis. These results indicate that sulfhydryl metabolism, specifically increased GSH, may be a factor in self tolerance to Cd in ICs.

Chlordecone (kepone®)‐potentiated carbon tetrachloride hepatotoxicity in partially hepatectomized rats — a histomorphometric study

Our previous studies indicated the involvement of some unidentified mechanisms, apart from the bioactivation phenomenon, in chlordecone (CD)-potentiated CCl4 hepatotoxicity and lethality. Recent studies revealed that hepatocellular regeneration is suppressed in CD + CCl4 toxicity. The present work is a continuation of our earlier work employing a partial hepatectomy model for stimulating hepatocellular division, in normal (N) or CD-treated (10 ppm for 15 days) rats. Male Sprague-Dawley rats maintained on an appropriate dietary protocol and undergoing sham (SH) or partial hepatectomies (PH) were employed. Hepatocellular regeneration was assessed by measuring the percentage mitotic figures and by autoradiography of liver sections from rats given 3H-thymidine in vivo. Hepatotoxicity was assessed by examining liver sections for necrotic cells, swollen cells and cells having lipid droplets. CCl4 (100 microliters kg-1)-induced histopathological alterations in CD-pretreated rats were significantly decreased in rats 2 days post-PH (PH2) as compared to SH rats or rats 7 days post-PH (PH7), indicating that amplification of CCl4 toxicity is significantly reduced when there is a greater regenerative activity. The percentage of mitoses as well as the percentage of labelled cells were significantly elevated at 2-6 h after CCl4 administration in N rats but remained suppressed in CD rats. In CD-pretreated PH2 rats where the percentage of mitoses and the percentage of labelled cells were many-fold greater when compared to SH or PH7 rats, a portion of the stimulated hepatocellular division decreased significantly at 2-6 h after CCl4 administration, but remained significantly greater when compared to basal level of regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon tetrachloride metabolism in partially hepatectomized and sham‐operated rats pre‐exposed to chlordecone (kepone)

The potentiation of CCl4 toxicity by pre-exposure to chlordecone (CD) is well established. Chlordecone-induced metabolism of CCl4 and suppressed hepatocellular repair have been offered as possible mechanisms for this potentiation. Recent work using the partially hepatectomized (PH) rat as a model for an actively regenerating liver has provided supportive evidence for the latter hypothesis. The present study was initiated to determine if metabolism and disposition of 14CC14 is altered in the PH rat, and if this is a contributing factor to the reported protective effect afforded by the PH procedure. Male Sprague-Dawley rats (150-175 g) maintained on dietary CD (10 ppm) for 15 days were partially hepatectomized or sham-operated (SH) on day 15. Another group of CD-pretreated rats received 0.9% CoCl2 (60 mg/kg, sc, qd for 2 days) in lieu of the surgical procedure. On day 16 the rats were challenged with a single dose of CCl4 (100 microL/kg, ip) containing 20 muCi 14CCl4. A radiolabel inventory consisting of exhaled 14CCl4, 14CO2 production, total hepatic 14C, free 14CCl4 and covalently bound 14C was taken over a 6-hr time period. Lipid peroxidation and serum enzyme activities [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)] were measured in indices of toxicity. Neither CD pretreatment alone nor CoCl2 treatment alone produced significant alterations in metabolism of low dose (100 microliters/kg) CCl4. No significant difference in 14CCl4 recovery or 14CO2 production was detected for PH versus SH rats. Hepatic 14CCl4-derived 14C (per gram tissue) was greater in PH rats. Values for free 14CCl4, covalently bound 14C, and lipid peroxidation were similar for SH and PH rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Metal constitution of metallothionein influences inhibition of δ-aminolaevulinic acid dehydratase (porphobilinogen synthase) by lead

This study was undertaken to evaluate the effect of Zn and Cd pretreatment on the inhibition of delta-aminolaevulinic acid dehydratase (ALAD; porphobilinogen synthase, EC 4.2.1.24) by Pb. Male CD rats were pretreated with 200 mumol of Zn/kg s.c. (subcutaneously) or 18 mumol of Cd/kg s.c., 48 and 24 h before assay of ALAD. Pretreatment with Zn resulted in activation of hepatic and renal ALAD and attenuated the inhibition of this enzyme by Pb in vitro. Pretreatment with Cd increased hepatic ALAD activity, and the inhibitory effect of Pb on the hepatic enzyme was attenuated in this group. In contrast with the situation in liver, pretreatment with Cd did not affect the activity of renal ALAD and did not alter the inhibitory effect of Pb on the renal enzyme. The Pb IC50 (concentration causing half-maximal inhibition) values for hepatic and renal ALAD in Zn-pretreated rats and for hepatic ALAD in Cd-pretreated rats were increased above control, whereas the IC50 for renal ALAD in Cd-pretreated rats was unchanged. Cytosolic binding patterns for the three metals were assessed by gel-filtration chromatography and disclosed that 203Pb was co-eluted with Zn and Cd bound to liver and kidney Zn-thioneins and liver Cd,Zn-thionein, although minimal binding of 203Pb to kidney Cd,Zn-thionein was observed. Estimation of the molar ratio of metals bound revealed Cd/Zn ratios of 2 and 5 for Cd,Zn-thioneins from liver and kidney respectively. The inhibition of purified ALAD by Pb was also attenuated by addition of purified Zn-thioneins and Cd,Zn-thioneins from liver and kidney in the following order: liver Zn-thionein = kidney Zn-thionein greater than liver Cd,Zn-thionein much greater than kidney Cd,Zn-thionein. Thus liver and kidney Zn-thioneins and liver Cd,Zn-thionein with a low Cd/Zn ratio readily decrease the free pool of Pb available to interact with ALAD. These data also demonstrate that the capacity of metallothionein to alter the intracellular distribution of Pb and mediate the inhibition of ALAD by Pb is dependent on the tissue source and relative metal constitution of the metallothionein.

Comparative changes in hepatic DNA, RNA, protein, lipid, and glycogen induced by a subtoxic dose of CCl 4 in chlordecone, mirex, and phenobarbital pretreated rats

Male Sprague-Dawley rats (200–250 g) were maintained on a normal powdered diet or on a similar diet containing 10 ppm chlordecone (CD), 10 ppm mirex (M) or 225 ppm phenobarbital (PB) for 15 days. On day 15, the animals received a single i.p. administration of CCl 4 (100 μ kg ). Hepatic DNA, RNA, protein, lipid and glycogen were determined 1, 4, 6, 12, 24 and 36 h after CCl 4 administration. A significant decrease (18 %) in hepatic protein was observed 24 h after CCl 4 challenge in the CD-pretreated rats; significant changes were not observed in the other treatment groups. Hepatic RNA was decreased (37%) in CD-pretreated rats at 36 h; no changes were observed in the DNA content. Hepatic RNA and DNA were increased (20% and 16%, respectively) 6 h after exposure to CC1 4 alone. Lipid content was increased at all time points starting at 4 h in response to CCl 4 challenge in the CD-pretreated rats. In the M- and PB-pretreated rats increases in hepatic lipid (22 and 28%, respectively) were observed only at the 6-h time point. Only a transient increase occurred after CCl 4 alone at 4 h. While depletion of hepatic glycogen was manifested in all groups at all time points following CCl 4, that in the CD + CCl 4 group was the greatest. A recovery of glycogen beginning at 12 h was observed in the rats receiving CCl 4 alone and in the M and PB pretreated animals. No such recovery was evident in CD + CCl 4 group. These studies indicate that biochemical changes compatible with cellular death are more pronounced in the CD-pretreated rats than in those receiving CCl 4 alone, suggesting that the metabolic and supportive biochemical mechanisms for hepatocellular repair are suppressed in rats receiving CD + CCl 4.

The effect of cadmium pretreatment on the disposition and excretion of methylmercury and trace elements (zinc, copper) in rats.

Cadmium chloride (Cd) was injected s.c. into male rats at a dose rate of 3 mg Cd/kg 3 times a week for 4 weeks. The animals were maintained for administration of methylmercury (203 Hg) chloride at a dose of 3 mg CH3Hg/kg given p.o. 3 times a week for 2 weeks, followed by 3 weeks of recovery period. Animals were sacrificed 24 h after the final dose of MeHg, or 5 weeks after cessation of Cd administration. Cd-pretreatment significantly decreased total Hg concentration in the kidney and RBC and almost completely inhibited demethylation of MeHg in the kidney (from 32% to 3% of inorganic Hg). Cd-pretreatment did not affect urinary excretion of total Hg, but significantly increased daily excretion of total Hg in feces. MeHg given alone significantly increased renal but not hepatic copper levels and decreased copper in the plasma and brain. In Cd-pretreated rats, both renal and hepatic copper concentration were in the normal ranges. Zinc levels in Cd-pretreated rats significantly increased in the kidney, liver and brain but decreased in plasma (compared to control and MeHg-alone treated animals). From these results it can be concluded that Cd-pretreatment may decrease MeHg toxicity by increasing the fecal mercury excretion and by inhibiting the formation of inorganic mercury in the kidney, which is a more potent renal toxin than MeHg.

Tolerance to cadmium-induced hepatotoxicity following cadmium pretreatment

Tolerance to several toxic effects of Cd, including lethality, has been shown following pretreatment with Cd. This study was designed to determine if tolerance also develops to Cd-induced hepatotoxicity. Rats were challenged with Cd (2.0, 3.0, 4.0, or 5.0 mg/kg, iv) 24 hr after pretreatment with saline (2 ml/kg, sc) or Cd (2.0 mg/kg, sc). Ten hours following challenge, plasma enzyme activities were dramatically elevated in control rats (sorbitol dehydrogenase 30- to 300-fold and aspartate aminotransferase 3- to 40-fold). In addition, histologic examination revealed moderate to severe hepatic injury, evidenced by cell swelling, cytoplasmic eosinophilia, pyknosis, karyorrhexis, and necrosis. In Cd-pretreated rats, plasma enzyme levels were similar to control values and only slight morphologic changes were evident. This tolerance to Cd-induced hepatotoxicity is probably due to the increase in hepatic metallothionein induced by Cd pretreatment, which has been shown to alter the hepatic subcellular distribution of Cd such that less binds to subcellular organelles and more binds to metallothionein located in the cytosol.

Tolerance to cadmium-induced toxicity depends on presynthesized metallothionein in liver.

Tolerance to Cd-induced toxicity following pretreatment with Cd is well documented, yet the exact mechanism of tolerance and role of metallothionein (MT) remains equivocal. In this study it was determined if the rapid induction of MT following injection of a challenge dose of Cd or the concentration of presynthesized MT induced by Cd pretreatment is more important for development of tolerance by ascertaining (1) whether the rates of synthesis of MT differ following injection of the challenge dose of Cd in control and Cd-pretreated rats and (2) if a relationship exists between presynthesized hepatic or renal MT and tolerance to Cd-induced lethality. Rates of hepatic and renal MT synthesis did not differ following injection of a challenge dose of Cd (2.0 mg Cd/kg, iv) in control and Cd-pretreated (2.0 mg Cd/kg, sc) rats. However, the progressive increase in concentration of MT in liver after Cd pretreatment correlated with the increase in tolerance to a lethal dose of Cd (4.0 mg Cd/kg, iv), as evidenced by a decrease in mortality in pretreated rats. A similar correlation was not observed between kidney MT levels and tolerance. Therefore, it appears that presynthesized MT in liver rather than increased synthesis of MT following injection of a second dose of Cd is responsible for Cd-induced tolerance.

Altered subcellular distribution of cadmium following cadmium pretreatment: Possible mechanism of tolerance to cadmium-induced lethality

Animals pretreated with cadmium (Cd) subsequently develop tolerance to an otherwise lethal dose of Cd. Possible mechanisms for this tolerance include reduced absorption, an altered tissue distribution, and an altered subcellular distribution of Cd. Male rats received a single Cd pretreatment (2.0 mg/kg, sc) 24 hr prior to administration of a typically lethal challenge dose of Cd (3.9 mg/kg, iv). Tolerance was evident because no mortality was observed in Cd-pretreated rats. Since Cd induces synthesis of hepatic metallothionein (MT), a higher percentage of the challenge dose might be sequestered in the liver of Cd-pretreated animals with less distributed to target organs of toxicity. At 2 and 24 hr following Cd challenge, no marked changes in organ distribution of the challenge dose of Cd were observed as a result of Cd pretreatment. However, isolation of hepatic subcellular fractions 2 hr following injection of the challenge dose revealed less Cd in nuclei, mitochondria, and endoplasmic reticulum, and more in cytosol as a result of Cd pretreatment. The increased cytosolic Cd was bound primarily to MT which had been induced markedly by Cd pretreatment. These data indicate that differences in absorption or tissue distribution of Cd are unlikely explanations for development of tolerance to Cd. Rather, tolerance appears to result from an MT-related change in the hepatic subcellular distribution of Cd, evidenced by lower concentrations of Cd in nuclei, mitochondria, endoplasmic reticulum, and cytosolic high-molecular-weight proteins and higher concentrations bound to MT in cytosol.

Biliary excretion of cadmium in rat. V. Effects of structurally related mercaptans on chelation of cadmium from metallothionein.

The effects of several mono-, di-, and trithiols (400 mumol/kg) in mobilizing Cd from metallothionein were studied in rats 24 h after a single injection of 109CdCl2 (1 mg kg/Cd). BAL (2,3-dimercaptopropanol) and propanetrithiol (1,2,3-trimercaptopropane) were the most effective mercaptans in increasing the biliary excretion of Cd (3.1 and 5.5% of administered dose, respectively, compared to 0.04% in control injected with propylene glycol) in in situ experiments with a significant decrease in hepatic Cd. Propane-1-thiol was inactive and propanetrithiol was the most toxic of the compounds studied. A number of propane dithiols having sulfhydryl groups at different carbon positions with and without substituents (OH or SO-3) and dimercaptoethane were also tested for effectiveness in removing Cd. All the lipophilic compounds with two adjacent sulhydryl groups were effective in increasing the biliary excretion of Cd, but were less effective than BAL and propanetrithiol. The major form of Cd in liver cytosols of rats pretreated with CdCl2 and injected with mercaptans was metallothionein. A small amount of Cd in liver cytosol and a major portion of biliary Cd in propanetrithiol-injected rats were bound to high-molecular-weight proteins when fractionated on Sephadex G-75 columns. On the other hand, after injection of BAL, most of the Cd in the bile was associated with a fraction of molecular weight 10,000. Even though Cd was present mainly as metallothionein in livers of Cd-pretreated rats, the biliary forms of Cd after injection of BAL and propanetrithiol were different. Similar results were obtained when Cd was added in vitro to bile samples collected from control rats that were injected with these chelating agents alone. However, the Sephadex G-75 elution profile of Cd-BAL and Cd-propanetrithiol after direct addition to control rat bile showed Cd complexes of identical molecular weight (less than 6000). These results suggested that the Cd-binding ligands present in bile after injection of BAL and propanetrithiol were different from and had a higher molecular weight than the complexes in vitro with Cd and the respective chelating agents.