Comparison of toxicity and disposition of cadmium chloride and cadmium-metallothionein in rats

In Chapter 1 of this thesis a general introduction is presented with a survey of the literature. It gives a brief overview of the factors involved in the absorption, metabolism and toxicity of Cd after oral intake.In short, the main source of environmental exposure to cadmium for nonsmokers is food. Oral Cd-studies in rodents have shown that the Cd-dose, the Cd- speciation and the mineral composition of the diet have an enormous impact on the final absorption and organ distribution of Cd. Although most toxicity studies have been performed with inorganic Cd, this is clearly not the chemical form in which Cd occurs in the diet. In animals one of the main Cd- binding ligands is the protein metallothionein, an inducible, cysteine-rich protein of low molecular weight. In plants the main Cd-binding ligands are phytochelatins, proteins wich are functionally analogous to metallothioneins. Metallothioneins can survive, at least partially, the gastro-intestinal digestion. It has been suggested that some of the exogenous Mt passes the intestinal barrier and reaches the kidneys directly. Since CdMt is a potent nephrotoxic agent when injected intravenously, this might indicate that dietary CdMt gives more reason for health concern than inorganic Cd. However, information on the bioavailibility and toxicity of dietary CdMt is currently lacking. Therefore the objectives of this thesis were:- To compare toxicity of inorganic Cd and CdMt in rats and in cell cultures of target organs,- To compare the dose-dependent kinetics of Cd- uptake from CdMt and inorganic Cd.- To establish differences in metabolic pathways between CdMt and CdCl 2 to predict disposition and toxicity at environmentally relevant doses.The studies described in Chapter 2 and Chapter 3 examined the toxicity and disposition in rats fed diets containing either pig's liver incorporated Cd or cadmium salt for 4 weeks. For a meaningful interpretation of the results the cadmium-binding ligand in pig's liver was first identified as a mixture of two metallothionein iso forms. The identification was based on molecular weight, Cd-binding properties, heat-stability and spectral analysis. It appeared that over 90 % of the Cd present in the pig's liver was bound to metallothionein. It was shown that signs of toxi- city (e.g. anemia and hepatotoxicity) were less pronounced in rats exposed to 30 ppm Cd as CdMt than in rats exposed to a similar dose of inorganic Cd-salt. The fact that CdMt is less toxic than CdCl 2 correlates well with thefinding that the intestinal and hepatic uptake of Cd after exposure to 30 ppm Cd as CdMt was lower than after exposure to 30 ppm Cd as CdCl 2 .However, in spite of the lower total uptake of CdMt, the renal Cd accumulation from CdMt was relatively higher than from CdCl 2 . Thus, the kidney to liver ratio of Cd concentration was significantly higher in rats fed CdMt than in the rats fed CdCl 2 .Since it is well known that CdMt after intravenous administration preferentially accumulates in the kidneys, the results might suggest that CdMt can pass the intestinal barrier and is released in the systemic circulation. This was supported by the fact that the metallothionein concentration in the kidneys in the first week of the experiment was higher after exposure to CdMt than after exposure to CdCl 2 .However in the introduction (Chapter 1) we have emphasized that rats exposed to low doses of CdCl 2 show, similar to dietary CdMt, a selective renal Cd accumulation, which was indicated by their high kidneylliver Cd ratio. Indeed, the study of chapter 3 revealed that at lower dietary Cd levels (1.5 and 8 ppm), relatively more Cd is deposited in the kidneys than at the 30 ppm level. The difference between the differential disposition of CdCl 2 and CdMt between kidneys and liver was less pronounced at the lower doses, but even at these doses the kidneylliver ratio of Cd is still higher with CdMt than with CdCl 2 . In the discussion the question was raised of what the impact of Cd redistribution from liver to kidneys will be during longterm oral intake in the final level of renal intoxication. This question was adressed to in chapter 8.Chapter 4 presents a comparative study on the accumulation and toxicity of CdCl 2 and two isoforms of CdMt in cell lines and primary cultures of the intestine, liver and kidneys. Exogenous CdMt added to the culture medium was much less toxic to all cells tested than CdCl 2 and no cell type was specifically sensitive to CdMt. Even when the Cd-content of CdMt was artificially raised (5 mol Cd/mol Mt), the cells were not affected by CdMt treatment. In general the difference in cytotoxicity between CdMt and CdCl 2 corresponds well with the difference in cellular uptake of Cd. This is in agreement with the in vivo results described in chapter 2, 3 and 8, showing a difference in toxicity between CdMt and CdCl 2 due to a difference in uptake and organ distribution.The studies described in Chapter 5 and Chapter 6 deal with the influence of minerals on the Cd absorption from CdCl 2 and CdMt. Most investigations have focused on single Cd-mineral interactions, and no real attemps have been made to compare the actual mineral status of other trace elements in the same study. Eight minerals were taken into account, all of which had been suggested to interact with the Cd accumulation in the body. The mineral combinations were chosen such that the effect of individual components could be analysed with multiple analyses of variance. The protection against Cd-accumulation and toxicity by mineral combinations was mainly due to the presence of iron (Fe). The question whether the protective effect of iron also applies for organic Cd, was examined in the study of chapter 6. Rats were fed CdMt supplemented with a mineral mixture of Ca/P, Zn and Fe which according to the observations in chapter 5 was the most effective against Cd accumulation. The total uptake of Cd from CdMt was significantly decreased although the protection of the mineral mixture was lower thanfor CdCl 2 . Since the Cd accumulation is mainly influenced by Fe and not by other minerals, the studies of chapter 5 and 6 raise the question whether the intestinal transport of Cd in rats is specifically mediated by the Fe transfer system. The fact that the mineral supplement also protects against CdMt, albeit less pronounced than against CdCl 2 might be explained by the partialdegradation of CdMt in the gastrointestinal tract. Once degraded, Cd from CdMt will show the same metabolic behaviour as inorganic Cd-salt and the uptake in liver and kidneys will be decreased by mineral suppletion. However a small fraction of the ingested CdMt is not degraded and it seems that the mineral supplement does not affect the absorption of the intact exogenous CdMt, which will then reach the kidneys unhampered. This explains why the kidneylliver ratio of Cd concentration was higher in the rats fed a CdMt diet supplemented with Fe in comparison to rats fed a CdC12 diet supplemented with Fe. After addition of the mineral supplement, rats fed CdCl 2 or CdMt showed relatively more Cd accumulation in the kidneys than in the liver. Since addition of the mineral supplement decreases the Cd absorption, it will prevent to some extent the overload of the intestinal metallothionein pool (see chapter 3) and as a consequence the kidneylliver ratio will increase.Chapter 7 deals with the metabolic fate of endogenous (synthesized by tissue) and exogenous (taken up by tissue) CdMt. Purified Mt isoform 1 and 2 from liver and intestine eluted as two single peaks on RP-HPLC. However, both kidney isoforms eluted as one single peak. The fact that renal Mt isoform-2 was not separated on HPLC and the fact that intestinal Mt-isoform 2 is hardly present in control rats, offers the unique possiblity to study the metabolic fate of purified CdMt isoform 1 in intestine and kidneys. A rapid exchange of 109Cd from exogenous CdMt (=hepatic 109CdMt isoform 1) towards endogenous CdMt was shown in the kidney and the intestinal mucosa after in vivo and in vitro addition of exogenous CdMt. The Cd redistribution was dependent on the ratio between the endogenous and exogenous CdMt concentration. Only if the oral or intravenous exposure to exogenous CdMt is high enough to lead to high exogenous/endogenous CdMt ratio, the 109Cd remains principally bound to exogenous CdMt in the intestinal mucosa and kidney. If we consider that at environmentally relevant Cd doses (less than 2 ppm in the diet) the exogenous CdMt concentration is much lower than the endogenous CdMt content, this implies that at low dietary CdMt doses Cd will mainly be bound to endogenous, intestinal CdMt and other (high molecular weight) Cd binding proteins.Finally, a long-term feeding study was performed and the results are given in Chapter 8. It was shown that the kidney/liver ratio of Cd concentration was inversely proportional with dose. Thus as the oral dosage increased, relatively more Cd was found in the liver than in the kidneys. At the dietary concentration of 30 and 90 ppm the Cd ratio was higher in rats fed CdMt than in rats fed CdCl 2 , However, at low dietary concentrations (0.3 and 3 ppm) the difference in renal disposition between CdCl 2 and CdMt did not occur. The first signs of renal injury indicated by slight signs of enzymuria were seen in rats exposed to CdCl 2 . Gradually the effects became more severe and histopathological changes were observed (i.e. glomerulonephrosis and an increase in basophilic tubules). The finding that 90 ppm CdCl 2 induced slight renal dysfunction whereas 90 ppm CdMt does not can be explained by the fact that the renal Cd concentration after 10 months was much lower for CdMt than for CdCl 2 (60 mg/kg tissue and 170 mglkg tissue respectively). These findings imply that in contrast to intravenous administered CdMt and CdCl 2 renal effects of oral administered CdMt and CdCl 2 . are dependent on the difference in degree of Cd accumulation in the tissue. Thus, there is no indication that CdMt in spite of its higher kidney/liver Cd ratio athigh oral doses is more nephrotoxic than CdCl 2 .To discuss the consequences for human consumer it will he essential to take into account all metabolic routes. Figure 1 shows in a simplified model the major metabolic routes of Cd after oral intake of CdCl 2 or CdMt. The model is based on the results of this thesis together with available information from the literature. Fig 1A depicts the metabolic pathways after oral exposure to CdCl 2 . At low oral CdCl 2 doses the majority of the Cd will he bound to the endogenous metallothionein of the intestine, which will be released in the systemic circulation and then be deposited in the kidneys. At a high oral CdCl 2 dose the available endogenous intestinal Mt pool is overloaded and Cd will be bound to high molecular plasma proteins and then be deposited in the liver. Cd will be bound to hepatic CdMt and with time some of the hepatic CdMt will reach the kidneys. Fig. 1B. shows the metabolic routes of Cd after oral exposure to CdMt. A large part of the CdMt will not survive the gastro-intestinal digestion and "liberated" Cd will show the same metabolic behaviour as Cd-salts. This explains why Fe interacts with the uptake of CdMt. Moreover at low dietary CdMt doses, the Cd from dietary CdMt which has reached the intestinal mucosa, is quickly redistributed towards endogenous CdMt. This implies that at environmentally relevant Cd doses the transport form of CdCl 2 and CdMt across the intestinal wall is similar (cf. Fig 1A). The similar Cd disposition observed for both CdCl 2 and CdMt in chapter 8 after long term exposure to low (<3 ppm) dietary doses is in agreement with this theory. At a high oral dose of CdMt relatively more CdMt will survive the gastro-intestinal digestion and more CdMt will reach the intestine intact. In this case it is plausible that exogenous CdMt is present in excess and Cd will remain bound, at least initially, to the dietary CdMt in the intestinal mucosa. Subsequently, the dietary CdMt will, similar to endogenous CdMt, be released in the systemic circulation and taken up in the kidneys. Thus at high oral Cd doses the metabolic routes of CdCl 2 and CdMt will differ.The critical renal Cdconcentration used in therisk assessment of Cd is in part based on laboratory animal experiments with inorganic Cd-salts. However, man is generally exposed to low dietary levels of organic Cd. In terms of risk assessment for the compound CdMt, the present study shows that at low dietary Cd doses, Cd is released from dietary CdMt after passage through the intestinal tract and uptake in the intestinal mucosa. This implies that at environmentally relevant oral Cd doses there is no difference in metabolic pathways between Cd-salt and CdMt. At high doses the metabolic pathways for both Cd- forms slightly differ, but the total Cd-uptake from CdMt at these levels was lower than for CdCl 2 .Although CdMt is more nephrotoxic than inorganic Cd after parenteral administration, we found no evidence that CdMt is more nephrotoxic than CdCl 2 after oral administration.Therefore this thesis shows that toxicity data obtained from studies with rodents exposed to low levels of CdCl 2 are also applicable for the risk assessment of Cd-intake from CdMt. Another important finding was that Cd accumulation from organic and inorganic Cd is mainly influenced by Fe and not by other minerals. Special consideration should therefore be given to an adequate Fe intake when assessing the health risk of the Cd intake.