Biomedical subjects

C D Klaassen

Publications and source records attributed to C D Klaassen.

At least 19 recordsLinked to original sources

PubMed1992

Tissue sulfate determination by ion chromatography.

The application of controlled-flow anion chromatography to assay inorganic sulfate in biological fluids and tissues is described. The eluent used in previous methods for analyzing sulfate in biological fluids has been modified by adding 4.5% acetonitrile to separate sulfate from a co-eluting peak. To markedly increase the life of the column, the tissue samples were further diluted, extracted with chloroform, and analyzed at a lower detection range (0.3 microS). The method has been shown to be applicable for determining sulfate in tissues as well as biological fluids.

Animals↗

PubMed1992

Effects of butyrate homologues on metallothionein induction in rat primary hepatocyte cultures.

Sodium butyrate (NaB), a 4-carbon fatty acid, has been reported to activate the metallothionein (MT) gene in certain carcinoma cell lines. Because the effects of NaB are dependent on the cell type investigated, this study was conducted to determine if NaB and its homologues induce MT in rat primary hepatocyte cultures. Hepatocytes were grown on monolayer for 12 h and subsequently treated with formate, acetate, propionate (NaP), NaB, and valeric acid for 10 to 58 h. To examine their interaction with known MT inducers, cadmium (Cd), zinc (Zn), or dexamethasone (Dex) were added to some cultures. MT protein in the cells was quantitated by the Cd-hemoglobin assay; MT-1 mRNA was analyzed by Northern blot hybridizations with oligonucleotide probes, and quantitated by slot-blot analysis. Among the 1 to 5 carbon carboxylic acids, only NaP (3 carbon) and NaB (4 carbon) induced MT. NaP and NaB alone produced a moderate increase in MT two- to fourfold over control), but when combined with Cd or Dex, an additive increase was observed. However, when combined with Zn, a synergistic increase was detected. NaB and Zn synergistically increased MT protein, but produced only an additive increase in MT mRNA, suggesting the involvement of some posttranscriptional event(s) in the NaB-Zn induction of MT. In conclusion, NaP and NaB induced MT in normal cultured rat hepatocytes, producing an additive increase in MT protein with Cd and Dex, and a synergistic increase in MT protein with Zn.

Acetates↗

PubMed1992

UDP-glucuronosyltransferase inducers reduce thyroid hormone levels in rats by an extrathyroidal mechanism.

As many microsomal enzyme inducers have been shown to reduce thyroid hormone levels, this study was conducted to determine if this reduction is produced by directly blocking the synthesis of thyroid hormones, or by indirectly increasing the biotransformation and deactivation of thyroxine (T4) by microsomal enzymes. Surgically thyroidectomized male rats received thyroid hormone replacement therapy by implanted osmotic minipumps, resulting in T4 and T3 serum levels that were similar to those observed in euthyroid controls. Three days after minipump implantation (Day 0), rats were fed diets containing four UDP-glucuronosyltransferase (UDP-GT) inducers: phenobarbital (PB), 3-methylcholanthrene (3MC), pregnenolone-16 alpha-carbonitrile (PCN), or polychlorinated biphenyls (PCB) for 10 days. PB, 3MC, and PCN reduced total (Days 3-10) and free (Days 7-10) T4 serum concentrations 30-50%, whereas PCB produced a 70-75% reduction in total and free serum T4 (Days 3-10). Treatment with PB, PCN, and PCB decreased levels of total T3 (Days 7-10). UDP-GT activity toward T4 was increased by PB, 3MC, PCN, and PCB 270, 400, 570, and 660%, respectively, and was found to correlate with serum T4 levels (total and free). These results demonstrate that reduction of thyroid hormone levels by microsomal enzyme inducers is produced in part by an extrathyroidal mechanism, quite possibly an increase in T4 glucuronidation.

Animals↗

PubMed1992

Induction of metallothionein by diethyl maleate.

Metallothionein (MT) is a sulfhydryl-rich protein whose levels are increased by administration of a variety of agents including metals, cytokines, and oxidative stress agents. Recent studies have suggested that MT is involved in protecting against various forms of oxidative stress, but little is known about the induction of MT by oxidative stress agents. Diethyl maleate (DEM) causes oxidative stress by depleting glutathione levels and is quite effective at increasing hepatic concentrations of MT. The purpose of the current study was to learn more about the relationship between induction of MT and oxidative stress by characterizing this increase in hepatic MT levels produced by DEM. Administration of DEM (3 to 9 mmol/kg, sc) increased hepatic MT concentration in mice as much as 37-fold to 213 micrograms MT/g liver, which is similar to the hepatic MT level seen after administration of other effective MT inducers, such as Cd. The maximal increase of hepatic MT took place 12 to 24 hr after administration of 5 mmol DEM/kg. This rise in MT was preceded by a 60% depletion of hepatic glutathione 3 hr after DEM and increases in both MT-I and MT-II mRNA, which reached a peak 6 to 9 hr after DEM. Administration of DEM (3-5 mmol/kg, sc) also increased MT levels in Sprague-Dawley rats. Pretreatment with DEM protected against Cd-induced hepatotoxicity in a fashion which suggested that a functional MT was being synthesized. In summary, DEM is a highly effective inducer of MT which increases MT at the mRNA level.

Animals↗

PubMed1992

Effect of lipoic acid on biliary excretion of glutathione and metals.

Several metals are excreted in bile as glutathione complexes, and their biliary excretion is facilitated by increased hepatobiliary transport of glutathione. The present study analyzed the effect of lipoic acid (LA; thioctic acid; 37.5-300 mumol/kg, iv), an endogenous disulfide which can be reduced in vivo to a dithiol, on the hepatobiliary disposition of glutathione-related thiols and the biliary excretion of metals (10 mumol/kg, iv) in rats. Administration of LA enhanced the biliary excretion of reduced glutathione in a dose-dependent fashion. Despite increasing glutathione output, LA (150 mumol/kg, iv) did not increase, but rather decreased, the biliary excretion of methylmercury, cadmium, zinc, and copper, which are transported into bile in a glutathione-dependent manner, as indicated by a marked reduction in their biliary excretion after diethyl maleate-induced glutathione depletion. In contrast, biliary excretion of inorganic mercury, which is minimally affected by glutathione depletion, was dramatically enhanced (12- to 37-fold) by LA administration. Following injection of LA, the concentrations of endogenous disulfides in arterial blood plasma (e.g., cystine, glutathione disulfide, cysteine-glutathione, protein-cysteine, and protein-glutathione mixed disulfides) were considerably diminished, while the levels of endogenous thiols (e.g., glutathione and cysteine) were increased. This finding indicates that LA, probably after enzymatic conversion to dihydrolipoic acid, can reduce endogenous disulfides to thiols. It appears that LA induces the transport of glutathione into bile by the temporary formation of dihydrolipoic acid-glutathione mixed disulfide, which after being translocated into bile is cleaved to LA and reduced glutathione. Because the glutathione molecule thus transported into bile cannot complex metals at the thiol group, this might be the mechanism for the observed failure of the LA-induced increase in biliary excretion of glutathione to enhance the hepatobiliary transport of metals that are transported into bile as glutathione complexes (i.e., methylmercury, cadmium, zinc, and copper). The observations also raise the possibility that endogenous dihydrolipoic acid, by forming a stable complex with mercuric ion, may play the role of a carrier molecule in the hepatobiliary transport of inorganic mercury.

Animals↗

PubMed1992

In vitro degradation of apo-, zinc-, and cadmium-metallothionein by cathepsins B, C, and D.

Metallothionein (MT) has been extensively studied over the past several years because of its probable role in endogenous metal homeostasis and cellular protection. A large body of knowledge now exists describing the physicochemical properties of MT as well as the mechanisms involved in MT induction. It has been well established that MT protects tissues from metal toxicity by chelating metals that would otherwise be available to interact with and disrupt vital cell functions. Information on the degradation of metal-saturated MT and the fate of the metals associated with it would be extremely important in predicting metal toxicity. Lysosomes have been targeted as a possible subcellular site for the turnover of MT; however, the susceptibility of MT to degradation by specific acidic proteases (i.e., cathepsins) has not been described. Therefore, the purpose of the present study was to examine the relative abilities of cathepsins B, C, and D to degrade Zn7-MT, Cd7-MT, and apo-MT in vitro. In so doing, the effects of metal species, degree of metal saturation, and pH on the degradation processes were evaluated. Time course experiments revealed that apo-MT was rapidly degraded by all three cathepsins. Cathepsin B degraded apo-MT approximately 36-fold more rapidly than cathepsin C and 45-fold more rapidly than cathepsin D. Therefore, under the in vitro conditions used in this study, the relative potency of the cathepsins tested was cathepsin B much much greater than cathepsin C greater than cathepsin D. In comparison, metal-saturated MT was more than 1000-fold more resistant to degradation by the cathepsins tested. In order to determine how much metal was needed to protect MT against degradation, apo-MT was reconstituted with increasing molar equivalents of Zn2+. The results suggest that as metal to apo-MT ratios increase, less apo-MT substrate is available to the protease and degradation decreases.

Animals↗

PubMed1992

Induction of metallothionein by cadmium-metallothionein in rat liver: a proposed mechanism.

Distribution of Cd to various organs following iv administration of CdCl2 (3.5 mg Cd/kg) resulted in more than 43% of total tissue Cd accumulating in the liver. In contrast, after CdMT administration (0.5 mg Cd/kg), only 1% of the Cd was found in liver. Rats administered CdCl2 (1.0 mg Cd/kg) had hepatic MT values 30-fold greater than controls and a hepatic Cd concentration of 17 micrograms/g. In comparison, rats treated with CdMT (0.4 mg Cd/kg) had hepatic MT concentrations 7-fold greater than controls and a hepatic Cd concentration of 0.80 micrograms/g. However, when hepatic MT levels were normalized to tissue Cd concentrations, induction of MT by CdMT was 5-fold greater than by CdCl2. Northern and slot-blot analyses of mRNA showed that both CdCl2 and CdMT coordinately increased MT mRNA. These data suggest that both CdMT and CdCl2 increase hepatic MT by similar mechanisms. A dose-response increase in MT produced by CdCl2 indicated a biphasic response, with low doses producing relatively more hepatic MT than higher doses. In addition, the amount of MT produced per unit Cd after CdMT treatment was similar to those observed after low doses of CdCl2 in the dose-response experiment. These data provide strong evidence to support the conclusion that the apparent potency of CdMT observed here and in previous studies is most likely due to the small amount of Cd distributed to the liver, which is relatively more effective in inducing MT than are higher concentrations.

Animals↗

PubMed1992

Distribution of cadmium chloride and cadmium-metallothionein to liver parenchymal, Kupffer, and endothelial cells: their relative ability to express metallothionein.

Acute exposure to cadmium (Cd) salts results in liver toxicity, while administration of cadmium-metallothionein (CdMT) iv, causes renal damage. When CdMT is administered iv there is a rapid accumulation of Cd in the proximal tubule cells of the kidney. In comparison, only small amounts of Cd accumulate in the liver following administration of CdMT. Thus, in order to better understand the regulation of MT as well as the toxicity of Cd, the present study has examined the ability of each of the three primary liver cells, parenchymal (PC), Kupffer (KC), and endothelial (EC), to accrue Cd after administration of either inorganic or organic forms of Cd. In addition, the relative ability of each cell type to express metallothionein (MT) mRNA and protein was examined. Following CdCl2 (3.5 mg Cd/kg) treatment, Cd concentrations increased to about the same degree in PC and KC, but EC had about 2-fold more than PC. After administration of CdCl2 (1.0 mg Cd/kg) each cell responded to the presence of Cd by increasing intracellular MT mRNA and protein. However, PC showed the greatest response, with a 30-fold increase in mRNA and a 21-fold increase in protein. Interestingly, KC and EC possessed intracellular Cd concentrations equal to or greater than that of PC, but contained less MT than would have been expected on the basis of their intracellular Cd concentrations. Thus, KC had a 7-fold increase in MT mRNA and a 2-fold increase in protein, while EC increased mRNA 3-fold and protein 2-fold over control values. In contrast, following CdMT (0.5 mg Cd/kg) administration, only low levels of Cd were detected, with similar concentrations in each cell type. After administration of CdMT (0.4 mg Cd/kg), PC again showed the greatest response, with a 3-fold increase in mRNA and a 6-fold increase in MT protein. Only slight changes were observed in KC and EC. In conclusion, the present study has shown the following: (1) Endogenous levels of MT in KC and EC are higher than those in PC. (2) Cd is readily accumulated by all three cell types, when administered as CdCl2, but not when given as CdMT. (3) PC, KC, and EC are capable of responding to intracellular Cd by increasing MT.

Animals↗

PubMed1992

Examination of potential mechanism(s) of metallothionein induction by diethyl maleate.

Diethyl maleate (DEM) is a glutathione-depleting agent that can increase the levels of the sulfhydryl-rich protein metallothionein (MT) in liver. The purpose of the present study was to examine the mechanism(s) by which DEM increases mouse hepatic MT levels. DEM appears to be an indirect MT inducer as suggested by the lack of increase in MT levels when cultured mouse hepatocytes were exposed to DEM. Four possible mechanisms by which indirect MT inducers may cause an elevation in MT concentrations in liver were examined. Zn levels did not increase prior to the increase in hepatic MT, thus, a Zn redistribution to the liver is not the cause of the liver MT induction by DEM. The adrenal gland products were not required for MT induction in liver, as adrenalectomy did not abolish the increase in hepatic MT caused by DEM. The elevation in liver MT does not appear to be due solely to the decrease in liver glutathione (60%) in the initial hour after DEM, because phorone, which decreases liver glutathione (80%), produced only a fourfold increase in hepatic MT. Activation of macrophages does not seem to account for the rise in liver MT levels, as there was no increase in abundance of cytokine mRNAs for TNF-alpha, IL-1 beta, or IL-6 in the liver. These data suggest that the induction of hepatic MT by DEM does not occur in response to (1) an increase in liver Zn that precedes the increase in liver MT, (2) release of adrenal gland products, (3) decrease in liver glutathione, or (4) increased cytokine gene expression.

Adrenalectomy↗

PubMed1992

Induction of hepatic metallothionein by paraquat.

Paraquat, a frequently used contact herbicide, produces oxidative stress by undergoing redox cycling and generating reactive oxygen species. Paraquat is also effective at increasing hepatic levels of metallothionein (MT). The mechanism(s) by which agents that induce oxidative stress produce increases in MT concentrations is not yet known. Therefore, the goal of the current study was to characterize the elevation in hepatic MT produced by paraquat administration to mice and to examine potential mechanism(s) of this increase. A dose-response study for increases in MT showed that administration of 0.1 to 0.5 mmol/kg of paraquat, sc, increased hepatic MT with a maximal increase of 36-fold. Subsequent studies were carried out with paraquat at a dose (0.3 mmol/kg, sc) that caused oxidative stress, as shown by a 35-fold increase in the biliary excretion of oxidized glutathione. There were coordinate elevations of both hepatic MT-I and MT-II mRNA of approximately 5-fold with peaks at both 6 and 24 hr after paraquat. The time course for the elevation in hepatic MT protein following paraquat treatment showed that MT levels had a maximal increase of 18-fold obtained at 36 hr. Paraquat appears to be an indirect MT inducer, in that there were no elevations in MT when cultured mouse hepatocytes were exposed to paraquat. No rise in liver Zn was observed prior to the increase in hepatic MT, thus, a Zn redistribution to the liver did not cause the increase in hepatic MT following paraquat administration. Adrenalectomy did not abolish the increase in MT produced by paraquat, suggesting that adrenal gland products are not required for the increase in MT produced by paraquat. In conclusion, the chemical mediator responsible for the increase in hepatic MT after paraquat was not determined, but the elevation in MT concentration appears to be due to increased transcription.

Animals↗

PubMed1992

Accumulation and degradation of the protein moiety of cadmium-metallothionein (CdMT) in the mouse kidney.

Of major concern in Cd toxicity is its ability to produce renal damage after chronic exposure in humans and experimental animals. Renal injury affects predominantly the proximal tubules and more specifically the first segments of these tubules. Similar toxic effects to the kidneys are observed after administration of cadmium bound to metallothionein (CdMT). Therefore, CdMT was used in this study as a model to understand the mechanism(s) of Cd nephrotoxicity. It has been recently demonstrated that Cd from CdMT was preferentially taken up by the proximal convoluted tubules. Therefore, the purpose of these studies was to determine if the organic portion of the complex was also accumulated in these tubules. [35S]CdMT prepared from rat liver was administered intravenously to mice at a nonnephrotoxic dose (0.1 mg Cd/kg). The radioactivity in the kidney showed maximum level (80% of the dose) 15 min after the injection. This preferential renal uptake was also observed after administration of various doses of [35S]CdMT. In contrast to the earlier observed persistency of 109Cd in the kidney after 109CdMT administration, 35S disappeared rapidly (with a half-life of approximately 2 hr), and 24 hr after injection of [35S]CdMT, there was very little 35S left in the kidneys. These observations indicate that the protein portion of CdMT is rapidly degraded after renal uptake of CdMT and the released Cd is retained in the kidney. Within the kidney, 35S distributed mainly to the cortex. Light microscopic autoradiography showed that [35S]CdMT preferentially distributed to the proximal convoluted tubule (S1 and S2), which is the site of nephrotoxicity. Within the S1 and S2 segments, a greater distribution of 35S to the apical portion of the cells was observed after administration of both a nonnephrotoxic (0.1 mg Cd/kg) and a nephrotoxic (0.3 mg Cd/kg) dose. 109Cd administered as 109CdMT also distributed to the apical portion of the S1 and S2 cells. Therefore, both the organic (35S) and inorganic (109Cd) portions of CdMT are rapidly and efficiently taken up by the S1 and S2 cells of the proximal tubules, the site of nephrotoxicity. These observations support the concept that CdMT is readily taken up by the proximal tubular cells as a complex, and then its protein portion is rapidly degraded to release Cd that binds permanently to intracellular sites and produces nephrotoxicity.

Animals↗

PubMed1992

Degradation and metal composition of hepatic isometallothioneins in rats.

Recently, our laboratory demonstrated that metallothionein-1 (MT-1) is degraded faster than metallothionein-2 (MT-2) in liver of Zn-treated adult rats; however, it is not clear whether this phenomenon is unique to Zn treatment or the age of the animal. Furthermore, many investigators maintain that the degradation of MT is regulated by its metal composition. The objective of this study was twofold: (1) to determine if MT-1 is more susceptible than MT-2 to proteolytic breakdown regardless of age or chemical pretreatment and (2) to examine the hypothesis that the amount and type of metals bound to MT influences its resistance to degradation. Pulse-labeling experiments were conducted to determine the half-lives of MT-1 and MT-2 in liver of adult rats (75-day-old), immature rats (1-day-old), and mature rats treated with single dosages of Zn (1 mmol/kg, sc), Cd (10 mumol/kg, sc), or ethanol (109 mmol/kg, po). Atomic absorption spectrometry was utilized to measure the Zn, Cu, and Cd contents of MT-1 and MT-2 obtained in selected experimental groups. MT-1 had a shorter half-life than MT-2 in Zn-treated adults (21 vs 33 hr) and in nontreated immature rats (49 vs 73 hr). In contrast, the half-life values of MT-1 and MT-2 were identical in nontreated adults (4 hr) and ethanol-treated adults (9 hr) and nearly identical in Cd-treated adults (58 and 61 hr, respectively). Both isoforms obtained from immature rats and adults treated with Zn or ethanol contained approximately 6.0 g atoms Zn/mol MT, trace levels of Cu, and nondetectable quantities of Cd. In Cd-treated rats, both isoforms contained approximately equal amounts of Zn and Cd (3.2 g atoms metal/mol MT) and trace levels of Cu. These results indicate that MT-1 is either as susceptible or more susceptible than MT-2 to intracellular degradation depending on age or chemical pretreatment. Furthermore, factors unrelated to the metal composition of MT appear to regulate the degradation of MT-1 and MT-2.

Animals↗

PubMed1992

Effect of pregnenolone-16 alpha-carbonitrile and dexamethasone on acetaminophen-induced hepatotoxicity in mice.

Recently, we demonstrated that a microsomal enzyme inducer with a steroidal structure, pregnenolone-16 alpha-carbonitrile (PCN), markedly decreased the hepatotoxicity of acetaminophen (AA) in hamsters. Therefore, it was of interest to determine if PCN, as well as another steroid microsomal enzyme inducer, dexamethasone (DEX), would decrease the toxicity of AA in mice, another species sensitive to AA hepatotoxicity. Mice were pretreated with PCN or DEX (100 and 75 mg/kg, ip, for 4 days, respectively) and were given AA (300-500 mg/kg, ip). Twenty-four hours after AA administration, liver injury was assessed by measuring serum activities of sorbitol dehydrogenase and alanine aminotransferase and by histopathological examination. Neither PCN nor DEX protected markedly against AA hepatotoxicity in mice; PCN tended to decrease AA-induced hepatotoxicity, whereas DEX was found to enhance AA-induced hepatotoxicity and it produced some hepatotoxicity itself. DEX decreased the glutathione concentration (36%) in liver and increased the biliary excretion of AA-GSH, which reflects the activation of AA, whereas PCN produced neither effect. Thus, whereas PCN has been shown to markedly decrease the hepatotoxicity of AA in hamsters, apparently by decreasing the isoform of P450 responsible for activating AA to N-acetyl-p-benzoquinoneimine, this does not occur in mice after induction with either PCN or DEX. In contrast, DEX enhances AA hepatotoxicity apparently by decreasing liver GSH levels and increasing the activation of AA to a cytotoxic metabolite.

Acetaminophen↗

PubMed1992

Effects of microsomal enzyme inducers upon UDP-glucuronic acid concentration and UDP-glucuronosyltransferase activity in the rat intestine and liver.

This study was conducted to evaluate UDP-glucuronosyl-transferase (UDP-GT) activity, UDP-glucuronic acid (UDP-GA) concentration, and UDP-glucose (UDPG) concentration in the rat intestine and liver following oral administration of butylated hydroxyanisole (BHA), benzo[a]pyrene (BaP), 3-methylcholanthrene (3MC), phenobarbital (PB), pregnenolone-16 alpha-carbonitrile (PCN), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or trans-stilbene oxide (TSO). Microsomal UDP-GT activity was assayed in vitro with acetaminophen (AA), harmol (HA), and 1-naphthol (NA) as the aglycones. Intestinal HA and AA glucuronidation were enhanced by BHA, BaP, and TSO, whereas 3MC, PB, PCN, and TCDD augmented hepatic HA-glucuronide formation and BHA, PB, PCN, TCDD, and TSO significantly increased hepatic AA glucuronidation. All inducing agents except PB and PCN markedly increased both intestinal and hepatic NA glucuronidation. PB, PCN, and TCDD paradoxically decreased intestinal glucuronidation of AA and HA. A similar effect upon hepatic glucuronidation was not observed with any of the agents studied. Hepatic UDP-GA concentration was increased significantly by all inducers studied except PCN and TCDD, whereas hepatic UDPG concentration was increased only by BHA. In the intestine, significant increases in UDP-GA concentration were produced only by BHA and BaP, which also elevated intestinal UDPG. These results demonstrate that microsomal enzyme inducers evoke different effects upon intestinal and hepatic glucuronidation. These differences are manifested with regard to induced changes in UDP-GT activity as well as treatment-induced alterations in UDP-GA content. Thus, the present study further underscores the marked variance of intestinal and hepatic xenobiotic glucuronidation.

Administration, Oral↗

PubMed1992

Rat liver microsomal UDP-glucuronosyltransferase activity toward thyroxine: characterization, induction, and form specificity.

Glucuronidation of thyroxine (T4) by liver microsomal UDP-glucuronosyltransferase (UDP-GT) is a predominant pathway by which T4 is deactivated. This study was conducted to characterize in vitro T4 UDP-GT activity in rat liver microsomal preparations, to determine if T4 glucuronidation is mediated by a particular form of UDP-GT, and to determine if T4 glucuronidation can be increased by microsomal enzyme inducers. Characterization of microsomal T4 UDP-GT activity led to the establishment of optimal assay conditions. UDP-GT activity toward T4 was determined in hepatic microsomal preparations from Wistar and Gunn rats, a mutant strain of Wistar rats deficient in several forms of UDP-GT. Hepatic microsomal preparations from Gunn rats glucuronidated T4 at one-third the rate catalyzed by microsomal preparations from Wistar rats. To determine the effect of four inducers that each increase a separate class of UDP-GT, phenobarbital (PB), 3-methylcholanthrene (3MC), pregnenolone-16 alpha-carbonitrile (PCN), clofibrate (CLO), saline, or corn oil was administered to male Sprague-Dawley rats ip for 4 days. T4 UDP-GT activity was increased by PB, 3MC, PCN, and CLO 88, 150, 100, and 160%, respectively on a per-milligram-microsomal-protein basis and 138, 125, 100, and 145% on a per-kilogram-body-weight basis, respectively. Therefore, all four classes of UDP-GT inducers increase T4 glucuronidation, suggesting that T4 is not a selective substrate for a particular form of UDP-GT.

Animals↗

PubMed1992

Role of hepatic lysosomes in the degradation of metallothionein.

The degradation of metallothionein (MT) by rat liver was examined. Degradation of MT by liver homogenate was greater than by cytosol. In addition, MT degradation by the homogenate at pH 5.5 was more than that at pH 7.2. Because lysosomal proteases function at acidic pH, these findings suggest the importance of lysosomes in MT degradation. The degradation by the lysosomal fraction was about 400-fold greater than that by the cytosol. Because cathepsins are the principal lysosomal proteases, we used cathepsin-specific inhibitors, such as leupeptin, E-64 and pepstatin, to determine the relative importance of different cathepsins in degrading MT. The study reveals that cathepsin B and/or L is (are) probably the most important enzyme(s) in degrading hepatic MT, because leupeptin, which blocks cathepsin B and L activity, inhibited the degradation of apo-MT by about 80%. Cathepsin D appears to be of least importance in MT degradation, because inhibition of this enzyme by pepstatin reduced degradation by only 20%. Studies on the degradation of apo-MT, ZnMT, and CdMT indicated that apo-MT is about 1500-fold more sensitive to degradation than ZnMT and CdMT. These data suggest that metals protect MT from degradation. This is further supported by a reconstitution experiment, which shows that with a progressive decrease of MT: metal ratio following titration of apo-MT by metals, there is a concomitant reduction in degradation. At a lysosomal pH of around 4.7, about 60% of Zn and 20% of Cd are displaced from MT, thereby making it susceptible to degradation. We propose, therefore, that lysosomes are probably important for MT degradation in vivo and that metal release is a prerequisite for degradation. With the release of metals, MT becomes susceptible to degradation, which is probably accomplished by the lysosomal cathepsins, in particular cathepsins B and L.

Animals↗

PubMed1992

Cadmium-induced hepatic endothelial cell injury in inbred strains of mice.

Susceptibility to cadmium (Cd) hepatotoxicity differs among inbred strains of mice. For example, C3H/HeJ mice are sensitive to Cd-induced hepatotoxicity, whereas DBA/2J mice are resistant. The mechanism of genetic predisposition to Cd hepatotoxicity is unknown. A contemporary theory for acute target organ intoxication maintains that Cd initially damages vascular endothelium and parenchymal cell injury is a secondary event that results from localized ischemia. In the present study, the hypothesis that hepatic endothelial cells (EC) of C3H mice are more susceptible to Cd toxicity than those of DBA mice was tested. Hepatic parenchymal and endothelial cells were grown separately on monolayer cultures for 22 h and subsequently treated with various concentrations of Cd. Hepatocellular toxicity was assessed by lactate dehydrogenase leakage and intracellular K+ loss, whereas endothelial cell injury was assessed by trypan blue exclusion and the inhibition of protein synthesis. The susceptibility of hepatocytes to the cytotoxic effects of Cd was identical between strains. In contrast, the vulnerability of EC to Cd intoxication was strain-dependent. When exposed to 2.5-10.0 microM Cd, EC of Cd-sensitive mice were more susceptible to the cytotoxic effects of Cd than those of Cd-resistant mice. Basal metallothionein (MT) levels as well as Cd uptake into EC were similar in the two strains. Following Cd exposure, EC of Cd-sensitive mice accumulated similar amounts of MT as EC of Cd-resistant mice. These observations suggest that the microvasculature in livers of inbred mice is the target tissue responsible for strain-dependent susceptibility to Cd-induced liver injury. The mechanisms that account for this genetic variation in endothelial cell response to Cd are unknown, but do not appear to be related to the cellular disposition of Cd nor to a defect in the metabolism of MT.

Animals↗

PubMed1992

Bromobenzene-glutathione excretion into bile reflects toxic activation of bromobenzene in rats.

This investigation was designed to determine whether biliary excretion of bromobenzene(BB)-glutathione(GSH) conjugate can be used as an index of in vivo activation of BB. In order to test this hypothesis, the effect of chemicals known to alter the toxicity and biotransformation of BB (i.e., cytochrome P-450 inducers and inhibitors) on the biliary excretion of BB-GSH was studied in rats. BB-GSH was the major BB metabolite in bile. A linear relationship was observed between the dosage of BB administered and BB-GSH excreted into bile, up to a dosage of 250 mumol/kg of BB. Of the inducers tested, phenobarbital, which is known to increase the toxicity of BB, dramatically increased (700%) the rate of biliary excretion of BB-GSH over that in control animals. In contrast, 3-methylcholanthrene, which is known to decrease the hepatotoxicity of BB, decreased the biliary excretion of BB-GSH (56%). Inhibitors of P-450, such as SKF 525-A and piperonyl butoxide which are known to decrease the activation and hepatotoxicity of BB, also decreased the biliary excretion of BB-GSH. These findings are in agreement with the hypothesis that the biliary excretion of BB-GSH reflects the formation of the reactive BB metabolite in liver and the rate of biliary excretion can be used to determine factors that are important in determining the toxicity of BB.

Animals↗