Metallothionein in the extracellular fluids as an index of cadmium toxicity.

In rats injected with 5 micron mole CdCl2/kg, 5 days/week, metallothionein was detected in plasma by gel filtration chromatography as early as four weeks. The mean renal concentration of cadmium was 80 microgram/g. The excretion of cadmium in urine at this time was rather low and amounted to 0.01% of the total dose. The amount of metallothionein in plasma, as determined by 109Cd-binding to the 10,000 molecular weight fraction, increased markedly during week 14. Its excretion in urine, however, did not start until about 10 weeks, when the cadmium concentration in kidney approached a mean value of 212 microgram/g. Signs of renal toxicity were evident from glucosuria and proteinuria which became severe during the next four weeks. The excretion of cadmium in urine increased markedly and the majority of it was in the form of metallothionein. It is suggested that the appearance of metallothionein in plasma and urine can be used as specific indices of cadmium poisoning and that the assay of the protein in these fluids may be useful in screening for excessive cadmium exposure.


Introduction
The chronic exposure to cadmium is known to produce renal tubular dysfunction in animals as well as man when the concentration of cadmium in renal cortex exceeds 200 ug/g wet weight (1). Measurement of cadmium concentration in urine or blood usually reflects the extent of recent exposure. Blood concentrations especially do not correlate with the body burden. There is, therefore, no threshold value for blood or urine cadmium concentration that can be used to predict the onset of renal dysfunction (1). Once the tubular damage has occurred in suspected populations of factory workers, the confirmation is usually made by measuring the urinary f32-microglobulin level (2,3).
Most of the absorbed cadmium in the body is sequestered by metallothionein which under normal circumstances is an intracellular protein. However, after long-term exposure to low levels of cadmium, the presence of metallothionein in plasma and urine has been observed by others (4)(5)(6) and also in our laboratory (7). The physiological functions of the protein are not yet fully understood. Of the various functions that have been ascribed to the protein, the two that have been advocated by Piscator and Nordberg are protection from the toxicity of cadmium and transport of the metal from liver to the kidneys (1). Contrary to this theory is the observation that intravenous administration of the protein into animals results in nephrotoxicity, characteristic of cadmium poisoning (8,9). It could be postulated, therefore, that there is a connection between the presence of metallothionein in the plasma and subsequent renal toxicity in animals exposed to cadmium. The study described here provides for the first time some insight into the existence of such a relationship.

Materials and Methods
The study was carried out in Wistar rats injected subcutaneously with 5 ,umole '"CdCl2/kg, 5 days per week, for up to 14 weeks. The amount of cadmium in whole-body, tissues, and excreta was determined by counting the radioactivity and converting the values to mg cadmium using the specific activity of the injection solution. It was assumed that dietary intake of the metal resulted in negligible increase in body burden. Glucose in urine was tested with Multistix (Ames Company) and protein was assayed by Tsuchiya's reagent as described by Piscator (10). Analysis of metallothionein was car-February 1979   (12).
The relationship between the injected dose of cadmium and the body burden of the rats is shown in Figure 1. With increasing dosage, smaller quantities of cadmium were retained. Between weeks 12 and 13, the rats accumulated and maintained the maximum quantity of cadmium (8 mg). Thereafter, the total retention declined although the exposure continued for another week. It thus appears that the maximum quantity of cadmium that can be accumulated by 300 to 350 g rats is 8 mg. The rats were probably suffering from severe renal toxicity during the last two weeks.
The rats were sacrificed at periodic intervals and their liver and kidneys were assayed for '09Cd (Table 1). It was found that the liver contained about 60o of the injected dose during the first 12 weeks of exposure. However, during the next two weeks there was a significant (20%) reduction in 4 6 8 10 12 cadmium content of the liver. A similar phenomenon was observed in the kidneys although the total DOSE (mg Cd) cadmium content of the kidneys was almost ten times lower than the liver. The above observations tetween the injected dose and the body indicate that the reduction in body burden during ,ach point is a mean of 4 to 19 animals. weeks 12-14 was caused mainly by the lower retention of cadmium by the liver and kidneys. Interestrof cadmium in liver and kidney.
ingly, at all times about 80%o of the liver cadmium and 70%o of the kidney cadmium were bound to Dose, %5a metallothionein. This suggests that the decrease in ver Kidney tissue,retention of cadmium is not caused by de-* 3. 4 6.3 ± 0.4 creased binding by metallothionein. * 3. 3 6.6 ± 0.4 When the cadmium content of the kidneys was * 3. 3 6.8 ± 0.3 expressed as Ag/g wet tissue weight, a rather in-* 3.5 6.9 ± 0.4 teresting phenomenon was observed. As shown in * 1.4 6.7 ± 0.3 increased linearly up to ten weeks of exposure and 'alues.
reached a mean value of 212 ,ug/g. The cadmium from 4 week values (p < 0.05).
concentration remained above 200 ,ug/g during the next four weeks. In chronic cadmium toxicity kidney, plasma, and urine by the ney is regarded as the target organ and 200 ,g/g of eviously (11).
renal cortex is considered as the critical concentration (1). In the present study, the kidneys were not Niscussion dissected into the cortex and the medulla. Assuming that the cortex contains about three times as much ure to cadmium seemed to afcadmium as the medulla (13) it is possible that these the rats. Although the mean rats contained more than 300 ,mg cadmium/g cortex il and cadmium-injected groups at 10 weeks and thereafter. The symptoms of cadicantly except after 14 weeks, mium toxicity became evident after 10 weeks, as the cadmium-injected animals seen by the presence of glucosuria and proteinuria. wer than that of the control During week 14, the urine contained as much as 100 week. Due to the small number mg protein/24 hr. ;his study, this point cannot be It was of interest to determine at what time period wever, it does appear that the during the course of continuous exposure does the mium may have started after metallothionein begin to appear in the plasma. The sure. Loss of body weight has samples of plasma collected from rats after only :ribed in rabbits by Nomiyama four weeks of exposure, when analyzed by gel filtration chromatography, showed a small but distinct Environmental Health Perspectives jium peak in the area where standard rat origin of the protein in plasma is not known. It is lothionein is eluted (Fig. 3). The correspond-possible, though, that it may have been released rhole kidney cadmium concentration was 80 from the liver and to some extent from other tissues With the duration of exposure, the amount of as well. Lium in plasma increased. After 10 weeks, al-The elimination of cadmium took place mainly via rh some cadmium was bound to metallothio-the fecal route (Fig 4). At the end of the second most of it was associated with larger molecu-week 6.4% of the total dose was excreted in the Sight proteins, probably albumin and globulins. feces. There was a linear increase in fecal excretion 14 weeks, however, about one-third of the of cadmium with the increase in exposure up to ia cadmium was bound to metallothionein. The week 10, and 9.7% of the dose was excreted in feces. The fecal excretion was relatively high during 250 the next two weeks and was much more pronounced during the final weeks.  (14,15). Excretion of cadmium in urine was relatively 50 small, but it increased linearly during the eight weeks of exposure (Fig. 5). The amount excreted in urine was 0.01% of the total dose after four weeks urine sample was separated by gel filtration chromatography. As seen in Figure 6, the urine from an animal injected with cadmium for ten weeks separated into three cadmium-binding peaks. Of these, the middle peak corresponded with the elution volume of standard rat metallothionein. No such peak of cadmium was detected in urine samples analyzed prior to 10 weeks. The excretion of cadmium associated with metallothionein increased as the plasma metallothionein level increased and the renal damage progressed. After 14 weeks about 90%o of the cadmium in urine was excreted bound to metallothionein. Previous work reported from our laboratory has shown that intravenously injected 109Cd or 35S-labeled metallothionein is excreted in rat urine as intact protein, and its excretion is dependent on the injected dose (16,17).

Conclusions
Under conditions of continuous exposure, it appears that more cadmium is excreted as the duration of exposure is increased. Before the onset of renal dysfunction, both fecal and urinary excretion are proportional to the exposure. After the toxicity symptoms appear, the excretion is considerably higher through both routes. There is less retention of the dose by liver, kidney and other tissues.
Renal toxicity occurs when the concentration of cadmium in whole kidney exceeds 200 ,g/g. Metallothionein appears in the urine along with other proteins and its concentration increases with the severity of the damage. In plasma, however, the protein is present long before the onset of nephrotoxicity. If metallothionein is monitored in plasma of exposed individuals, it would be an excellent screening tool for excessive exposure. Its presence may serve as a warning against potential renal toxicity of cadmium. By measuring metallothionein in urine from industrial workers and severely exposed populations it may be possible to determine the extent of damage. Such a test would be more meaningful and more specific than the measurement of 32microglobulin in urine. More work is needed to test the applicability of the observations in animals to the human situations. There is also a need to develop a sensitive and selective method to measure metallothionein in biological fluids.