Studies on the toxicity and metabolism of cadmium-thionein

Cadmium, when bound to thionein from either rat or rabbit liver, was 7 to 8 times more toxic for the rat than was ionic Cd^{2 +}. Zinc-thionein was not only non-toxic at a dose level of 2.4 mg protein-bound-Zn^{2 +}/kg, but also protected the animals against a subsequent, normally lethal dose of cadmium-thionein. In contrast with the free cation, Cd^{2 +} administered intravenously as the metallothionein accumulated to its highest concentration in the kidney and, at a lethal dose, caused severe tubular damage. After administration of non-lethal doses of ¹⁰⁹Cd^{2 +} -labelled cadmium-thionein to rats thionein-bound-¹⁰⁹Cd^{2 +} accumulated in the kidneys, but in animals that were dosed with either the ³⁵S- or ³⁵H-labelled metalloprotein, little or none of the radioactive isotope was recovered in the renal metallothionein at 48 hr. The ³⁵S and ³H isotopes, however, were incorporated into high molecular weight proteins of the kidney soluble fraction and also were excreted in the urine, both as the metallothionein and as smaller, diffusible molecules. In vitro, ³H-labelled cadmium-thionein was degraded to acid-soluble products by homogenates of rabbit kidney cortex. It is suggested that parenterally administered cadmium-thionein is taken up by the renal tubules and catabolized, probably by the lysosomes of the tubular cells, with the liberation of Cd^{2 +} ions. These cations, if present in sufficiently high concentration, cause acute renal damage. Exposure of rats, with high hepatic concentrations of cadmium-thionein, to the hepatotoxins, carbon tetrachloride and retrorsine, did not cause the transfer of Cd^{2 +} from the liver to the kidney.     

The synthesis of metallothionein and cellular adaptation to metal toxicity in primary rat kidney epithelial cell cultures

Single cells were isolated from rat (100–150 g) kidney cortex slices by mild trypsinization. They were plated on plastic culture dishes and were maintained in a selective Eagle's D-Valine Minimum Essential Media (MEM) to form an epithelial cell monolayer culture. The fibroblast growth was retarded by D-Valine. The cells in monolayer culture accumulated cadmium when they were incubated with a ¹⁰⁹CdCl₂ (10^?5 M) containing medium. The synthesis of metallothionein by these cells was demonstrated by incorporation of [³⁵S]cysteine and ¹⁰⁹CdCl₂ into a heat stable protein (mol. wt 10 000) within 8 h of exposure and also by immunoprecipitation using a specific antibody to rat liver metallothionein. The cytotoxic effects of Zn²⁺, Cu²⁺, Cd²⁺ and Hg²⁺ were studied in culture after addition of various concentrations of metal salts (10^?5 –10^?2 M). Disrupted cellular colonies with severe cell damage were observed after addition of 10^?3 M Cd²⁺ or Hg²⁺ as CdCl₂ or HgCl₂ while similar toxicity was observed only after addition of 10^?2 M Zn²⁺ or Cu²⁺ as ZnSO₄ or CuSO₄ to confluent cell cultures. The cellular damage due to Cd²⁺ was protected when the cells were pretreated with Cd²⁺ (10^?5 M) for 24 h and these cells could tolerate much higher concentrations of cadmium (10^?2 M). These results indicate a direct protective role of intracellular metallothionein in the cellular toxicity of cadmium.     

Relationships between subcellular distributions of cadmium and perturbations in reproduction in the polychaete Neanthes arenaceodentata

The accumulation and subcellular distribution of Cd in the polychaete worm, Neanthes arenaceodentata, were examined following an eleven-week period of exposure to a range of free cadmium ion activities, [Cd²⁺]. The accumulation of Cd in N. arenaceodentata was directly proportional to [Cd²⁺] in seawater at lower concentrations (10⁻¹² M to 10⁻¹⁰ M) but deviated from proportionality at higher concentrations (10⁻⁹ M and 10⁻⁸ M). This deviation in proportionality at higher [Cd²⁺] was attributable to a relative increase in the concentration of metal associated with the metallothionein and the very low molecular weight metal-ligand pools of the cytosol.Reproductive potential was also monitored in these organisms to examine the ecological significance of Cd accumulation and shifts in its subcellular distribution. Perturbations in reproduction were observed at 10⁻⁸ M [Cd²⁺] which coincided with the deviation in proportionality of Cd accumulation and increased accumulation of Cd in the cytosol.     

Cadmium resistance and content of cadmium-binding protein in two enzyme-deficient mutants of mouse fibroblasts (L-cells)

The toxic Cd²⁺ ion accumulates in mammalian organisms, the main storage organs are apparently the liver and the kidney. In these organs Cd²⁺ is bound to low molecular weight proteins (thioneins) as metallothionein. We describe here the development of resistance to otherwise lethal concentrations of Cd²⁺ by two non-epithelial cell lines, both derived from mouse fibroblasts (L-cells). One of the cell lines (clone ID) is deficient in thymidine kinase and resistant to 5-bromodeoxyuridine, the other (A9) deficient in hypoxanthine-guanine phosphoribosyl transferase and resistant to 8-azaguanine. After stepwise increase in Cd²⁺ concentration, clone 1D cells had apparently normal growth rate in the presence of 100 micromolar Cd²⁺ after 6 months of Cd treatment. The A9 cells were apparently more sensitive to Cd²⁺, after about one year's Cd treatment they had apparently normal growth in the presence of 100 micromolar Cd²⁺. This concentration of Cd²⁺ would kill cells of both cell lines not previously exposed to Cd. In the resistant A9 cells about 40 per cent of the cadmium were bound to a cadmiumbinding protein (Cd-BP) of molecular weight of about 12,000, most probably metallothionein, in the resistant clone 1D cells the corresponding figure was 60 per cent. The non-resistant cell lines had apparently no metallothionein. We have thus found that also non-epithelial cells can synthesize low molecular weight Cd-BP and that there apparently is a good correlation between cadmium resistance and content of Cd-BP.     

The time-course of cadmium-thionein synthesis in the rat

After the intravenous injection of Cd²⁺ (1.6 mg/kg body wt) the cation accumulates to slightly greater concentrations in the liver and kidneys of the 10-week-old female Wistar rat, than in these organs of the 10-week-old male. Ionic Cd²⁺ is more toxic for the female than for the male, the ld₅₀ values being 1.4 (1.2?1.7) and 2.2 (1.9?2.6) mg Cd²⁺/kg body wt, respectively. This difference in toxicity is not correlated with differences in rates of hepatic cadmium-thionein synthesis. In both sexes, uptake of Cd²⁺ by the liver is complete within 1 hr. In the male rat there is a lag phase of 3–4 hr between the administration of Cd²⁺ and the onset of the induced synthesis of thionein in the liver. Once this synthesis occurs, the content of the metallothionein increases rapidly, Cd²⁺ being transferred to the apoprotein from proteins of high molecular weight that provide the initial binding sites for the cation in the soluble fraction of the liver. At a dose level of 1.6 mg Cd²⁺/kg, synthesis of the metallothionein is mainly complete within 8 hr and appears to be unaffected by age; both the length of the lag phase and subsequent rate of formation of the hepatic metalloprotein in the 80-week-old male rat being the same as those in the 10-week-old animal. In contrast with the male, approximately 5 per cent of the total Cd²⁺ in the soluble fraction of the liver of the female rat is bound as the metallothionein within 1 hr after the administration of the cation. This incorporation of Cd²⁺ into the metalloprotein is attributed to replacement by Cd²⁺ of Zn²⁺ in zinc-thionein, which is present in low concentration in the liver of the normal female rat. This initial replacement is followed by a slow increase in the content of thionein-bound Cd²⁺ during the following 2–3 hr. Thereafter, presumably due to stimulation by the Cd²⁺-induced messenger, the rate of synthesis increases rapidly to a maximum, which is at least equal to, and occurs at the same time, as that in the male. At 24 hr after the administration of Cd²⁺ to the male rat, the content of Zn²⁺ in the hepatic metallothionein is similar to that of Cd²⁺. Replacement of this Zn²⁺ by Cd²⁺ may account for the immediate incorporation of the latter cation into the hepatic metallothionein that occurs when the animals are given a second dose of Cd²⁺. After this initial replacement the synthesis of thionein, which has been primed by the first dose of Cd²⁺, occurs without lag on exposure to the second, and both Zn²⁺ and Cd²⁺ are incorporated into the metallothionein. Intravenous injection of 1.6 mg Cd²⁺/kg body wt also leads to a rapid accumulation of the cation in the kidney, but does not induce the synthesis of the metallothionein in this organ of either the male or female rat during the following 48 hr.     

Cd2+ responses of cultured human blood cells

Cd²⁺ cytotoxicity, uptake, and partitioning, and Cd²⁺-induced metallothioneine synthesis were studied in cultured peripheral human blood cells. Mononuclear cells were found to resist relatively high levels of Cd²⁺. Few cells were killed below 50 μm Cd²⁺. Above this value, survival decreased exponentially with dose. The mean LD50 for mononuclear cells cultured in Cd²⁺ for 40 hr was 100 μm. Polymorphonuclear cells (granulocytes) were found to be more resistant, with a significantly higher threshold and LD50, and a more complex dose response.Most of the Cd²⁺ incorporated by blood cells was taken up by nucleated cells. Despite their greater resistance, polymorphonuclear cells incorporated more Cd²⁺ at higher doses (50 to 150 μm) than did mononuclear cells. No Cd²⁺ was bound to metallothioneine in polymorphonuclear cells following exposure to Cd²⁺ for even extended periods of time (18 hr) at high doses of ¹⁰⁹Cd²⁺ (25 μm). Instead Cd²⁺ appeared in a Sephadex G-75 peak of approximately 60,000 Da, as well as in the void peak.No significant amount of preexisting metallothioneine (MT) or metallothioneine mRNA was found in the mononuclear cells. However, MT synthesis was induced rapidly following exposure to Cd²⁺. [¹⁰⁹Cd²⁺]MT appeared within 1 hr following exposure to 50 μm¹⁰⁹Cd²⁺, and MT synthesis rates measured from [³⁵S]cysteine incorporation were found to be maximal within 4 hr.     

Metabolism of cadmium in the neonate: Effect of hepatic zinc,copper and metallothionein concentrations on the uptake of cadmium in the rat liver

The accumulation and subcellular distribution of Cd²⁺ (1 mg/kg body wt, i.p.) in the liver of the neonatal rat is age-dependent. At 4 hr after treatment the liver Cd²⁺ contents in the 12-day-old, 20-day-old and adult rat are similar and greater than in the 2-day-old animal. The differences in hepatic Cd²⁺ concentration in the older age groups are consistent with the nonlinear weight gain of the liver in the developing animal. In the hepatic cytosol Cd²⁺ is incorporated into a high molecular weight and metallothionein fractions and transferred from the former to the latter. This process occurs less rapidly with increasing age. The uptake of Cd²⁺ by the whole liver and the hepatic metallothionein is not related to the total liver concentration of Zn²⁺ or copper and is not significantly influenced by the concentration of pre-existing metallothionein or the concentration of thionein-bound Zn²⁺ or copper. The results are discussed in relation to the possible effects of Cd²⁺ on the liver metabolism and tissue distribution of Zn²⁺ and copper in the developing animal.     

Oxidation of sodium sulphide by rat liver,lungs and kidney

Heparinized rat blood containing sodium [³⁵S]sulphide was perfused through isolated rat lungs, kidney or liver. The rate and extent of sulphide oxidation varied from one organ to another. In the isolated perfused lung system, [³⁵S]sulphide was oxidized slowly to [³⁵S]thiosulphate; only small amounts of [³⁵S]sulphate were delectable, possibly due to the absence of sulphide oxidase. In the isolated perfused kidney system, [³⁵S]sulphide was oxidized to [³⁵S]sulphate with [³⁵S]thiosulphate as a possible intermediate. In liver perfusion experiments [³⁵S]sulphide was oxidized rapidly and almost exclusively to [³⁵S]sulphate. The addition of unlabelled thiosulphate inhibited the formation of [³⁵S]sulphate and caused the release of [³⁵S]thiosulphate from the isolated liver. This suggests that thiosulphate is an intermediate in sulphide oxidation to sulphate. A mechanism for the rapid oxidation of sulphide to thiosulphate was shown to be present in rat liver mitochondria and, in the presence of glutathione, the thiosulphate was oxidized to sulphate. These results are discussed in relation to the contribution of lungs, kidney and liver to the oxidation of sulphide in vivo.     

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.     

Cadmium-induced inhibition of hepatic drug oxidation in the rat: Time dependency of tolerance development and metallothionein synthesis

Experiments were undertaken to determine if a correlation existed between the time-dependent synthesis of hepatic metallothionein and the development of tolerance to the inhibition of hepatic drug oxidation elicited by cadmium in the male rat. Maximal rates of metallothionein synthesis were achieved within 2 hr after administering cadmium in a 0.21 mg Cd/kg (ip) dose. The total hepatic concentration of metallothionein, as well as the cadmium binding capacity of this protein, also increased rapidly with maximal values being observed from 8 to 67 hr after cadmium administration. Despite this rapid increase in hepatic metallothionein levels, the tolerance to the inhibition of in vivo drug oxidation induced by a challenge cadmium (0.84 mg Cd/kg) dose did not develop until 16 hr after treatment with the 0.21 mg/kg cadmium dose. Furthermore, hepatic metallothionein levels decreased from a maximum at 67 hr to approximately control levels at 336 hr. Although the tolerance to the inhibition of drug oxidation also decreased from a maximum at 72 hr a modest degree of protection was maintained even at 336 hr. Pretreatment with the 0.21 mg Cd/kg dose increased the hepatic uptake of a ¹⁰⁹Cd challenge dose (0.84 mg Cd/kg). This increase was associated with an increased ¹⁰⁹Cd binding to metallothionein in the cadmium-pretreated animals. While these data are suggestive of a role for metallothionein in tolerance development, the lack of correlation of the time course of metallothionein synthesis with the development of the tolerance would suggest that factors in addition to metallothionein may also participate.     

Cadmium sensitivity, uptake, subcellular distribution and thiol induction in a marine diatom: exposure to cadmium

The aims of this study were to (1) evaluate the changes in the Cd tolerance of a marine diatom after exposure under different Cd concentrations for various durations and (2) to explore the potential subcellular and biochemical mechanisms underlying these changes. The 72-h toxicity, short-term Cd uptake, subcellular Cd distribution, as well as the synthesis of phytochelatins (PCs) were measured in a marine diatom Thalassiosira nordenskioeldii after exposure to a range of free Cd ion concentrations ([Cd²⁺], 0.01-84 nM) for 1-15 days. Surprisingly, the diatoms did not acquire higher resistance to Cd after exposure; instead their sensitivity to Cd increased with a higher exposed [Cd²⁺] and a longer exposure period. The underlying mechanisms could be traced to the responses of Cd cellular accumulation and the intrinsic detoxification ability of the preconditioned diatoms. Generally, exposure to a higher [Cd²⁺] and for a longer period increased the Cd uptake rate, cellular accumulation, as well as the Cd concentration in metal-sensitive fraction (MSF) in these diatoms. In contrast, although PCs were induced by the environmental Cd stress (with PC₂ being the most affected), the increased intracellular Cd to PC-SH ratio implied that the PCs’ detoxification ability had reduced after Cd exposure. All these responses resulted in an elevated Cd sensitivity as exposed [Cd²⁺] and duration increased. This study shows that the physiological/biochemical and kinetic responses of phytoplankton upon metal exposure deserve further investigation.     

Increase in hepatic metallothionein in rats treated with alkylating agents

Male Sprague-Dawley rats were dosed (2 ml/kg, ip) with sodium iodoacetate (30 mg/kg), bromobenzene (1200 mg/kg), diethylmaleate (560 mg/kg), or iodomethane (46 mg/kg), 40 and 16 hr prior to being sacrificed and the concentration of hepatic metallothionein was determined. Treatment with the above agents resulted in a two- to fivefold increase in the concentration of hepatic metallothionein when compared to control rats fed ad libitum. In addition, treatment of rats with bromobenzene and sodium iodoacetate resulted in a two- to threefold increase in the concentration of hepatic metallothionein when compared to pair-fed control rats. Male rats were also dosed with either sodium iodoacetate (30 mg/kg, ip) or saline (ip) 40 and 16 hr prior to sacrifice and in each case with [³⁵S]cystine 35 and 11 hr prior to sacrifice. Sodium iodoacetate treatment doubled the percentage of the ³⁵S in the 100,000g supernatant incorporated into metallothionein. In addition, rats were treated with either sodium iodoacetate (30 mg/kg, ip) or sodium iodoacetate (30 mg/kg, ip) and after 3 hr cycloheximide (1.5 mg/kg, ip). Sodium iodoacetate treatment alone resulted in a significant increase in the concentration of hepatic metallothionein; however, in animals also treated with cycloheximide no increase was observed. These results indicate that the increase in hepatic metallothionein observed after treatment with sodium iodoacetate is due to an increase in the synthesis of metallothionein.     

Kinetics of cadmium-induced hepatic and renal metallothionein synthesis in the mouse

Rates of hepatic and renal metallothionein synthesis were estimated by measuring the incorporation of [³H]cysteine into metallothionein prepared from mice at various times following a single intraperitioneal injection of cadmium acetate (2 mg of Cd/kg). Tissue metallothionein concentrations were measured indirectly as a function of the total cadmium-binding capacity of the isolated metallothionein. Maximal incorporation of [³H]cysteine into hepatic metallothionein occurred 6–12 hr following cadmium exposure, while renal metallothionein synthesis was maximal after 3 hr. Incorporation of [³H]cysteine into metallothionein as well as metallothionein concentrations was greater in the liver than in the kidney. It is concluded that the liver is the primary site of cadmium-induced metallothionein synthesis.     

Renal Excretion of some Aryl Sulphate Esters in the Rat

The renal clearance of inulin, phenyl [³⁵S]sulphate, naphthyl 2-[³⁵S]-sulphate and 2-hydroxy-5-nitrophenyl [³⁵S]sulphate was measured at various plasma concentrations in the rat.

A considerable proportion of each ester was bound to plasma proteins in vivo and the ratio of ³⁵S-labelled sulphate ester/inulin clearance demonstrated that all three esters are secreted by the renal cells.

The contribution of the renal secretory process to the overall urinary excretion ranged between 22–59% (phenyl [³⁵S]sulphate), 58–87% (naphthyl 2?[³⁵S]sulphate) and 60–72% (2-hydroxy-5-nitrophenyl [³⁵S]sulphate).

These findings are discussed in relation to the detoxication of phenols by sulphate conjugation.     

Inhibition of the liberation of arachidonic acid by cadmium ions in rabbit alveolar macrophages

The effects of CdCl₂ on the liberation of arachidonic acid (20∶4) from membrane phospholipids of A23187-stimulated rabbit alveolar macrophages and on the activity of phospholipase A₂ (PLA₂) in a cytosolic fraction were studied. Alveolar macrophages were prelabeled with [³H]arachidonic acid (20∶4) and then treated with A23187. This treatment resulted in a remarkable increase in the liberation of [³H]20∶4 from their phospholipids. Exposure of cells to Cd²⁺ inhibited the liberation of [³H]20∶4 in a dose-dependent manner. Liberation of [³H]20∶4 from cell lipids was calcium dependent and the inhibitory effect of Cd²⁺ competed with the stimulatory effect of Ca²⁺. When Ca²⁺ was removed from the incubation medium, Cd²⁺ did not influence the liberation of [³H]20∶4. Entry of⁴⁵Ca²⁺ into cells was enhanced by treatment of A23187. However, Cd²⁺ did not influence the cellular uptake of⁴⁵Ca²⁺. Treatment with A23187 markedly enhanced entry of¹⁰⁹Cd²⁺ into cells. The effect of Cd²⁺ on the activity of phospholipase A₂ was determined with 1-palmitoyl-2-[¹⁴C]arachidonoyl-sn-glycero-3-phosphocholine as substrate. Calcium-dependent activation of PLA₂ was observed and Cd²⁺ inhibited activation in a dose-dependent manner. These results suggest that exposure of alveolar macrophages to Cd²⁺ causes a reduction in the rate of liberation of 20∶4 from cell lipids, as a possible result of the inhibition of PLA₂ activity by Cd²⁺.     

The effect of trifluoperazine on the phosphorylation of ribosomal small subunit protein S6 in d-galactosamine-injured and normal rat liver

The effect of the Ca²⁺-calmodulin antagonist trifluoperazine on the elevation of phosphorylation of rat liver small ribosomal subunit protein S6 induced by the hepatotoxic agent d-galactosamine has been studied. Trifluoperazine applied in various doses (1–100 mg/kg body wt) before injection of d-galactosamine into the rat does not reverse the strong increase in phosphorylation promoted by d-galactosamine. Instead, trifluoperazine has been identified as a potent stimulator of S6 phosphorylation in normal rat liver in vivo without causing significant changes in the cyclic AMP content of liver and the overall rate of liver protein synthesis. Both drugs, however, were not effective in stimulating the incorporation of [³²P]phosphate into microsomes or crude ribosomes in liver slices in vitro. The results suggest that a calmodulin-activated protein kinase probably is not primarily engaged in S6 phosphorylation produced by d-galactosamine. However, further in vitro studies are needed to reach a definite conclusion.     

Protection by Zinc-Metallothionein (ZnMT) against Cadmium-Metallothionein-Induced Nephrotoxicity

In contrast to inorganic Cd, acute iv administration of Cd boundto metallothionein (CdMT) concentrates in renal tissue. Thisuptake of CdMT produces functional and morphological changesin kidneys, similar to those observed after chronic exposureto inorganic Cd. In order to examine the importance of the metalcomponent of MT in the renal uptake of MT, the renal concentrationof ³⁵S after administration of [³⁵S]ZnMT and [³⁵S]CdMT was compared.Renal uptake of ³⁵S from both CdMT and ZnMT was very rapid,with peak concentrations observed 15–30 min after administration.³⁵S in kidneys increased in a dose-dependent manner after administrationof various doses of [³⁵S]ZnMT, up to 1.3 µmole MT/kg;however, higher doses did not further increase renal ³⁵S concentrations.A similar saturation of ³⁵S reabsorption was observed for therenal uptake of [³⁵S]CdMT. CdMT produced renal injury with dosesas low as 0.26 µmol MT/kg (0.2 mg Cd/kg). In contrast,with a dose of ZnMT as high as 5.12 µmol MT/kg (2 mg Zn/kg),no histopathological changes were observed. Therefore, ZnMTappears to be nontoxic even though ZnMT delivers more MT tothe kidney than does CdMT. Because ZnMT and CdMT are apparentlyhandled by the same renal transport mechanism, the effects ofZnMT on ¹⁰⁹CdMT renal uptake and nephrotoxicity were determined.One group of mice was given a nephrotoxic dose of ¹⁰⁹CdMT (0.51µmol MT/kg containing 0.4 mg Cd/kg, iv), and the othergroup received an equimolar dose of unlabeled ZnMT 1 min before¹⁰⁹CdMT administration. Renal function was evaluated by measuringurinary glucose and protein excretion, as well as histopathology.Marked renal toxicity was observed 24 hr after ¹⁰⁹CdMT administration.In contrast, renal function appeared normal in mice receivingZnMT before ¹⁰⁹CdMT. However, a similar concentration of ¹⁰⁹Cdwas found in kidneys of both groups. The present results demonstratethat ZnMT is not only nontoxic to the kidney at a dose as highas 5 µmol MT/kg, but can also protect against the nephrotoxiceffect of CdMT without decreasing renal Cd concentration.     

Binding of glutathione-depleting agents to metallothionein

The purpose of this investigation was to compare metallothionein to the tripeptide glutathione in their ability to sequester electrophilic agents. Following incubation of liver homogenates with high concentrations of nonradioactive iodoacetate, and a tracer amount of iodo[¹⁴C]acetate and subsequent isolation of metallothionein by ultracentrifugation and gel filtration (Sephadex G-75), ¹⁴C-derived radioactivity coeluted with metallothionein. Following in vivo administration of bromo[¹⁴C]benzene or [¹⁴C]chloroform to zinc- and phenobarbital-pretreated rats and subsequent purification of hepatic metallothionein, ¹⁴C did not appreciably coelute with metallothionein. In vitro addition of diethyl maleate, iodomethane, or iodoacetate to liver homogenate obtained from zinc-pretreated rats resulted in a concentration-related loss of glutathione- and metallothionein-like activity. However, the concentration of alkylator needed to produce decreases in metallothionein-like activity was approximately 10 times higher than that needed for glutathione loss. The results suggest that sulfhydryl groups on metallothionein can act as binding sites for possible detoxification of alkylating agents. However, the agents used in this investigation have an apparent higher affinity for glutathione than metallothionein and thus binding to this protein probably does not represent a major detoxification pathway.     

The Biodegradation of the Surfactant Undecyl Sulphate

1. The metabolism of the odd-numbered carbon chain surfactant, potassium undecyl [³⁵S]sulphate. in the rat was investigated.

2. The major route for elimination of radioactivity was the urine, regardless of the route of administration.

3. The surfactant was extensively degraded in vivo to yield propionic acid 3-[³⁵S]sulphate, the major radioactive component in urine. A second urinary metabolite was identified tentatively as pentanoic acid 5-[³⁵S]sulphate.

4. Whole-body autoradiography revealed the liver as the major site of metabolism.

5. The nature of the metabolic products of undecyl sulphate suggest that it is bio-degraded by initial ω-oxidation followed by β-oxidation.     

Axonal Transport of Cadmium in the Olfactory Nerve of the Pike

¹⁰⁹Cd²⁺ was applied in the olfactory chambers of pikes (Esox lucius) and the dynamics of the axoplasmic flow of the metal was determined in the olfactory nerves by gamma spectrometry and autoradiography. The results showed that the ¹⁰⁹Cd²⁺ is transported at a constant rate along the olfactory nerves. The profile of the ¹⁰⁹Cd²⁺ in the nerves showed a wave front of transported metal followed by a saddle region. When the nasal chambers were washed 2 hr after application of the ¹⁰⁹Cd²⁺ well-defined transport peaks for the metal were seen in the olfactory axons. The maximal velocity for the transport of ¹⁰⁹Cd²⁺, which corresponds to the movement of the wave front, was 2.38±0.10 mm hr (mean±S.E.) at the experimental temperature (10 C). The average velocity for the transport of the ¹⁰⁹Cd²⁺, which corresponds to the peak apex movement of the wave, was 2.18±0.05 mm/hr (mean±S.E.) at 10 C. The transported ¹⁰⁹Cd²⁺ was strongly accumulated in the anterior parts of the olfactory bulbs, whereas in other brain areas the levels of the metal remained low. Autoradiography of a pike exposed to ¹⁰⁹Cd²⁺ via the water showed a strong labelling in the receptor-cell-containing olfactory rosettes, whereas other structures in the olfactory chambers were only weakly labelled. The accumulation and axonal transport in the olfactory neurons may be noxious and constitute an important component in the toxicology of cadmium in fish, and this may apply also to some other heavy metals.