A mechanism of protection against intoxication with various metals

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

A Mechanism

13,30-36

(1968)

of Protection with Various

against Metals

Intoxication

H. SELYE, A. SOMOGYI, AND I. MBcs Institut de Mkdecine et de Chirurgie expbrimentales Universitt de Montrkal, Montreal, Canada Received January 15,1968

A Mechanismof Protection Against Intoxication with Various Metals. HANS, SOMOGYI, ARPAD, and MBcs, IRENE. (1968). Toxicol. Appl. Pharmacol. 13, 30-36. Calcergensare compounds that causetopical calcification upon subcutaneousinjection in unpretreatedrats, whereascalciphylactic challengersdo so only after systemicpretreatmentwith a conditioning factor (e.g., vitamin D compounds,parathyroid hormone). Both the mortality and the spleniccalcification, commonly elicited by the intravenous injection of various calcergens(rare earth metal chlorides), are prevented,or at leastgreatly diminished,by pretreatmentwith calciphylactic challengers(ferric dextran, chromium dextran and, to a lesserextent, aluminum dextran). This prophylactic effect is due to the challenging metalmoiety of the chelates,sincedextran alonedoesnot protect. Thesefindingsare reminiscentof earlier observationsthat had shownthat the topical calcifying effect of calcergensis likewiseinhibited by the admixture of calciphylactic challengers.It ispossiblethat the antagonismdepends upon changesin calcium or phosphorusmetabolismsince the eliciting agentsare limited to metalshaving specificcalcinotic effects. SELYE,

Several earlier communications from this laboratory have called attention to the fact that calciphylactic challengers can block certain effects of calcergens (Selye et al., 1962; Gabbiani et al., 1962, 1966). By definition, calciphylactic challengers produce calcification of soft tissues at the site of their administration only after sensitization by systemic treatment with such calcium-mobilizing factors as parathyroid extract, vitamin D, or dihydrotachysterol; calcergens exert the same effect without any previous sensitization. Thus, calciphylaxis is, whereascalcergy is not, dependent upon sensitizing alterations in systemic calcium and phosphate metabolism. However, the end result of the two reactions is the same, namely, the crystallization of hydroxylapatite which forms a readily visible, white disk at the site where the provocative substance was injected (Selye, 1962). Recently we observed that in rats the topical calcification normally induced by

calcergens fails to occur if these are injected simultaneously with calciphylactic challengers; conversely, agents that do not cause calcification upon subcutaneous injection (either in unpretreated or in calciphylactically sensitized rats) offer no protection against the topical calcium deposition elicited by caicergens. This apparently paradoxical fact further emphasizes the essential dissimilarity of calciphylaxis and calcergy (Selye et al., 1968). The question arose whether it would be possibleto protect animals against otherwise 30

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fatal doses of calcergens (lead, scandium, rare earth metals) by particularly potent calciphylactic challengers (iron-, chromium-, or aluminum-dextran chelates) both types of compounds being injected intravenously. METHODS

For all our experiments we used female Sprague-Dawley rats with a mean body weight of 100 g (90-l 10 g), subdivided into groups of 10-30 animals each. They were given toxic doses of metallic compounds (calcergens or noncalcergens) either without pretreatment or after the administration of potentially protective calciphylactic challengers. As such, we used ferric dextran (Fe-Dex), chromium dextran (Cr-Dex), or aluminum dextran (Al-Dex); these were kindly prepared by Fisons Pharmaceuticals,3 who also supplied a sample of the low molecular weight (6000) dextran used in the manufacture of the metallic chelates. The following compounds, used either as toxic agents or as potential protectors were all chemically highly purified preparations as distributed by the manufacturers: aluminum dextran “Al-Dex”3 (an aluminum chelate, 20.4 mg Al/ml); cerium chloride* (CeCl, .7H,O); cesium chloride* (CsCl); chromium chloride* (CrCI, .6H,O); chromium dextran “Cr-Dex”J; cobalt chloride* (CoC12 .6H,O); dextrani ; dysprosium chloride4 (DyCl,); Escherichia coli 08 endotoxin6; erbium chloride4 (ErC13.6H20); europium chloride4 (EuCl,); ferric dextran “Fe-Dex”s [Imferon@ (==50 mg Fe/ml)]; ferrous chloride* (FeC12.4H20); gadolinium chloride4 (GdCl,); holmium chloride4 (HoCl,); lanthanum chloride* (LaC13 *7H,O); lead acetate “Pb-ac”* [Pb(C2H302)*. 3H,O]; lutetium chloride4 (LuC13); magnesium chloride* (MgC12.6H,0); mercuric chloride* (HgCl,); neodymium chloride4 (NdCls); nickel chloride* (NiC12.6H20); potassium chloride* (KCl); praseodymium chloride4 (PrCl,); samarium chloride4 (SmCl,); scandium chloride4 (ScCl,); selenium dioxide4 (Se02); silver nitrate2 (AgNO,); sodium platinic chloride4 (Na2PtC16*4H20); terbium chloride” (TbCls); thulium chloride4 (TmCl,.7H,O); ytterbium chloride4 (YbCl,.6H,O); yttrium chloride4 (YCls-6H20). Preliminary experiments were done with all agents to determine the minium dose of the toxic agents just sufficient to produce mortality and the optimum time interval between the administration of these compounds and pretreatment with the potential protectors. Consequently, in the final experiments which are summarized in our tables, the individual doses of both types of compounds were administered under light ether anesthesia into the jugular vein in 1 ml of water at moderate speed to avoid high mortality during the injection. The pretreated animals received Fe-Dex (= 30 mg Fe), Cr-Dex (= 10 mg Cr), or Al-Dex (= 10 mg Al), 5 hours before the toxic metals. The dosages of the latter are given in the tables. The animals were maintained exclusively on Purina Laboratory Chow (Purina Co. of Canada) and tap water. The survivors were killed on day 5 with chloroform and their 1 Abbott Laboratories, North Chicago, Illinois. 2 Fisher Scientific Co., Fair Lawn, New Jersey. 3 Fisons Pharmaceuticals, Holmes Chapel, England. 4 K & K Laboratories Inc., Plainview, New York. 5 Matheson Coleman & Bell, Morwood, Cincinnati, Ohio. 6 Dr. 0. Westphal, Max-Planck-Institut fiir Immunbiologie,

Freiburg, Germany.

32

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MfiCS

organs inspected with a binocular loupe. Splenic calcification, which occurs frequently following intravenous injection of calcergens, was gauged in terms of an arbitrary scale in which 0 = no changes, 1 = slight, 2 = moderate, and 3 = marked degree of calcification (Selye, 1962). The calcific nature of the splenic lesions was verified histochemically after fixation in alcohol-form01 by means of the von Kossa and chloranilic acid techniques (Carr et al., 1961). The statistical significance of the mortality was calculated by the chi-square test. In the case of splenic calcification, the significance of the apparent difference between the experimental and the corresponding (unpretreated) control values was calculated by the application of Student’s t test. Only when one of the two results to be compared was “0” did we estimate the significance of the difference on the basis of the confidence limits. RESULTS

In the first experiment we wanted to determine whether the mortality and the characteristic splenic calcification produced by various typical calcergenscan be prevented by two potent calciphylactic challengers, namely Fe-Dex and Cr-Dex. As shown in Table 1, at the dosesused, the mortality causedby any one of the calcergens TABLE

1

PROTECTION BY FE-DEX AND CR-DEX AGAINST CALCERGENS

Mortality (%) Treatment CeCl,, DyC13, ErC13, EuC13, GdC&, HoC13,

LaC13, LuC13,

NdC&, Pb-ac, Pb-ac, PrC13, ScCl,,

25 mg 16mg 16mg 25 mg 20 mg 13 mg 30 mg 14 mg 20 mg 20 mg 16mg 20 mg 6mg

SmC13, 25 mg TbC13, 15mg TmC13, 14mg YbC13, 16 mg YC13,

12 mg

Spleniccalcification (scale: O-3)

Control Control (unpretreated) Fe-Dex” Cr-Dex” (unpretreated) Fe-Dex” _____ 70 50 70 92 95 80 85 70 55 86 73 60 40 90 35

(20) (20) (20) (30) (20) (20) (20) (20) (20) (30) (15) (20) (20) (20) (20)

0” (10) Ob (10) oc (70) 1Od (10) Od (10) 10” (10) 1Od (10) 0’ (10) lob (10) Od (10) 10’ (10) 30 (10) 0’ (20) 10” (10) 10 (10)

0’ (10) Ob (10) 0” (10) 10d (10) Od (10) 10d (10) 30 (10) Ob (10) 90 (10) 60 (10) lob (10) 0 (10) 10’ (10) 10 (10)

60 (20) 70 (20) 80 (10)

0’ (10) 10” (10) 20’ (10)

0’ (10) 30 (10) 10” (10)

10” (10)

0.5 + 0.22 2.8 + 0.34 2.0 + 0.26 3.0 2.0 + 1.0 2.5 i 0.16 1.2 It 0.49 0 1.3 zt 0.41 0

0 1 .O + 0 2.5 f 2.2 f 1.5 f 1.3 f 2.5 f

0.29 0.50 0.24 0.22 0.28 0.50

(1Given 5 hours before treatment at the dose level indicated in the text. b 0.05 z P z 0.01 = 0.01 > P > 0.001. d 0.001 > P.

e Number of animals given in parentheses.

Cr-Dex”

0 ;:

0 Od

Od 0 ;: 0 0.3 i 0.24 0

;: 0 Od Ob 0 Ob 0

0

0

0’ 0

0’ 0

;.l 0.1 + O.ld

8: Od

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tested was significantly diminished or even abolished by Fe-Dex as well as by Cr-Dex in most cases. However, curiously Cr-Dex, unlike Fe-Dex, failed to protect against lead acetate, both at the 20-mg and the 16-mg levels. Not all calcergens produce splenic calcification. Among those tested here, the salts of lutetium, lead, and scandium failed to do so. It may be said, however, that pretreatment with either Fe-Dex or Cr-Dex completely or almost completely protected the spleen against the calcifying action of all those calcergens of the present series which normally do exhibit this activity. This protection was statistically significant except in a few groups where early mortality in the control group too seriously reduced the number of animals available for calculation.

FIG. 1. Top: Massive calcification of the spleen in a rat given ErC13 iv. Borrom: Absence of calcification in the spleen of a similarly treated rat which had received an intravenous injection of Fe-Dex 5 hours before the ErC13.

The typical splenic calcification after treatment with ErC13 and its prevention by Fe-Dex as seen macroscopically, are illustrated in Fig. 1. Histologically, we note that here, as after treatment with other calcergens, calcification is most pronounced in the peripheral regions of the Malpighian corpuscles where phagocytes are especially abundant. As shown by Fig. 2, this calcinosis can be totally prevented by Fe-Dexpretreatment so that even histologic investigation reveals no demonstrable calcium deposit. We performed a similar series of experiments with the same calcergens using Al-Dex (10 mg elementary aluminum per dose) as a possible prophylactic agent. The results of this series are not tabulated because we found Al-Dex to be much less efficacious than Fe-Dex or Cr-Dex in protecting against mortality, although it was almost as active in preventing splenic calcification. It will be recalled that we undertook these experiments with a bias, because we anticipated an antagonism between calcergens and calciphylactic challengers on the 7

34

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MkS

basis of earlier observations. These had shown that the local calcifying action, exhibited by calcergens at the site where they are subcutaneously injected, is prevented by concurrent local treatment with calciphylactic challengers (Selye et al., 1968). Consequently, in the first experimental series, we tested only the possibility of preventing the systemic toxicity of calcergens. A second experiment was required, therefore, to verify whether an active calciphylactic challenger such as Fe-Dex could also protect against compounds previously proven not to be calcergens (Selye, 1962). As shown in Table 2, no noteworthy protection was offered by Fe-Dex against the mortality induced by

FIG. 2. Left: Histologic appearance of the spleen following treatment with ErCl,. Heavy calcification is observed particularly in the periphery of the Malpighian corpuscles. Right: Complete absence of calcification in the spleen of a rat which received Fe-Dex prior to ErCI, injection. von K6ssa x 29.

the noncalcergens tested, with the exception of CrCls and FeCls, the only two calciphylactic challengers in this ,series. The toxicity of AgNOs was actually very much enhanced by Fe-Dex. Since none of these toxic agents produced splenic calcification, an antagonistic interaction between the compounds given could not be tested in this respect. It is evident, however, that although calciphylactic challengers can protect against toxic doses of calcergens, they do not do so against noncalcergens, with the possible exception of calciphylactic challengers. However, among the latter an insufficient number has been tested up to now to arrive at a generalization. When given intravenously, iron, chromium, and aluminum are not as well tolerated as inorganic salts as in the form of their dextran chelates; that is why we have used Fe-Dex, Cr-Dex and Al-Dex rather than the corresponding chlorides. However, the question remained whether, in these experiments, it was not the dextran moiety that offered protection. Therefore, a third experimental series was performed in which we

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TABLE LACK

OF PROTECTION

BY FE-DEX

35

INTOXICATION

2

AGAINST

VARIOUS

Mortality Control unpretreated’

Treatment AN%, %.IOtLg CoC12, 4 mg CrCl,, 18 mg CsCI, 100 mg E. coli 08 endotoxin, FeCl*, 6 mg &Clz, 800wit KCl, 12 mg MgC12,17 mg Na$Q, 6.5 mg NiCI,, 8 mg Se%, 400 cLg

NONCALCERGENS

(%) Fe-Dex”, e

0 60 60 40 60 55f 100 40 60 loo 90 70

500 pg

load 60 Ob 30 80 5’. / 100 70 50 70 90 90

’ Given 5 hours before treatment at the dose level indicated in the text. bO.Os~P>O.O1. c 0.01 > P 10.001. d 0.001 > P.

e Ten rats per group except where indicated. f Twenty rats per group. the effect of dextran upon the toxicity of six calcergens whose toxicity was particularly well prevented by metal dextrans in the first experiment. For this purpose, we employed the same dextran (mol. wt. 6000) at the same dose (120 mg/ml) as was contained in the Fe-Dex of the first experiment. Again the injection was performed intravenously under light ether anesthesia, 5 hours before administration of the calcergens. Table 3 shows that dextran itself offers no significant protection either against mortality or against splenic calcification.

examined

TABLE LACK

OF PROTECTION

Mortality

-

Treatment

CeC13, EuCIJ, GdC13, LaCI,, Pb-ac, Pb-ac.

25 mg 25 mg 20 mg 30 mg 16mg 20 mg

Control, unpretreated’ 80 100 60 100 60 80

3

BY DEXTRAN

AGAINST

(%) Dextran’, 40 90 30 90 50 90

C

CALCERGENS

Splenic calcification

(scale : O-3)

Control, unpretreated

Dextran”

0 -b 3.0 It 0 -” 0 0

a Given 5 hours before treatment at the dose level indicated in the text. * Since all animals died before termination of the experiments, splenic calcification assessed. ’ Ten rats per group.

0.8 3.0 3.0 1.0

zt 0.31 It 0 &0 & 1.0 0 0

could not be

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MlkS

DISCUSSION

It may be concluded from these experiments that Fe-Dex, Cr-Dex, and, to a lesser extent, Al-Dex offer considerable protection against the lethal effects of various calcergens in the rat. The intense splenic calcification produced by many of these calcergens is even more constantly and completely prevented by the metallic chelates than the mortality. The toxicity of other metals, which do not possess calcifying properties, failed to be similarly counteracted. Earlier work had shown that aluminum, chromium, and iron are strong calciphylactic challengers even when given as inorganic salts, whereas dextran is not. Hence, it was postulated that in the iron-, chromium-, and aluminum-chelates the prophylactic effect also resides in the metallic moiety. In agreement with this assumption, it could be shown that dextran itself fails to offer noteworthy protection either against the lethal or the splenic calcifying effect of the calcergens tested. Additional experiments revealed that Fe-Dex offers no protection against intravenous injections of toxic doses of various other unrelated agents such as epinephrine, serotonin, vasopressin, or colloidal carbon. Furthermore, pretreatment with just tolerable doses of calcergens (EuCl,, GdCl,) or of a calciphylactic challenger (Fe-Dex) likewise failed to offer protection against subsequent treatment with toxic amounts of the same compound. All these observations suggest that there exists some antagonism between the systemic effects of calciphylactic challengers and calcergens, reminiscent of the previously demonstrated prevention by the former of the topical calcification elicited by the latter. The mechanism of this interaction remains to be elucidated; however, it is reasonable to suspect that the antagonism, being limited to metals having specific calcinotic effects, depends upon changes in calcium or phosphate metabolism. ACKNOWLEDGMENTS This work was supported by the U.S.P.H.S., Child Welfare Division (Grant No. HD02612-01). The authors wish to thank Professor Dr. 0. Westphal of the Max-Planck-Institut fur Immunbiologie, Germany, for a supply of E. coli endotoxin 08, and the following companies for kindly supplying the compounds used in these experiments: Fisons Pharmaceuticals (aluminum dextran and chromium dextran), and Abbott Laboratories (dextran). REFERENCES L. B., RAMBO, 0. N., and FEICHTMEIR, T. V. (1961). A method of demonstrating calcium in tissue sections using chloranilic acid. J. Histochem. Cytochem. 9, 415-417. GABBIANI, G., SELYE, H., and TUCHWEBER, B. (1962). Prevention of indium intoxication by ferric dextran. Brit. J. Pharmacol. 19, 508-5 12. GABBIANI, G., JACQMIN, M. L., and RICHARD, R. M. (1966). Soft-tissue calcification induced by rare earth metals and its prevention by sodium pyrophosphate. Brit. J. Pharmacol. 27,1-9. SELYE, H. (1962). Calciphylaxis. Univ. of Chicago Press, Chicago, Illinois. SELYE, H., TUCHWEBER, B., and GABBIANI, G. (1962). Prevention by ferric dextran of the topical calcification induced by direct calcifiers. Med. Exptl. 7, 180-186. SELYE, H., SOMOGYI, A., and MBcs, I. (1968). Calcergy inhibited by calciphylactic challengers. Science 159, 1361-1362. CARR,