And Nickel Acetate-Induced Lung Tumors in Strain a Mice1

Home , Calcium acetate, Magnesium acetate

[CANCER RESEARCH 44,1520-1522, April 1984]

Inhibition by Magnesium and Calcium Acetates of Lead Subacetate- and Nickel Acetate-induced Lung Tumors in Strain A Mice1

Lionel A. Poirier,2 Jeffrey C. Theiss,3 Lyle J. Arnold, and Michael B. Shimkin

Nutrition and Metabolism Section, Laboratory of Comparative Cardnogenesis, National Cancer Institute/Frederick Cancer Research Facility, Frederick, Maryland 21701 [L A. P.], and Department of Community Medicine, University of California at San Diego, La Jolla, California 92093 [J. C. T., L. J. A., M. B. S.]

ABSTRACT previous findings of the carcinogenic activity of lead salts toward the kidneys of rats and the versatile carcinogenicity of nickel and The ability of the physiologically essential divalent metals its compounds in rodents (9, 10). As a bioassay for chemical calcium and magnesium to inhibit the tumorigenic activities of carcinogens, lung adenoma formation offers the advantages of lead and nickel towards the lungs of strain A mice was investi ease of performance and relative rapidity. This system was thus gated. The tumorigenic salts lead(ll) subacetate and nickel(ll) selected to study the possible inhibitory effects of calcium(ll) and acetate were injected i.p. at their maximal tolerated doses (0.04 magnesium(ll) salts on the tumorigenicity of lead(ll) and nickel(ll). mmol/kg/injection of each metal) for a total of 24 injections, whenever possible. Calcium(ll) acetate and magnesium(ll) ace MATERIALS AND METHODS tate were administered in the same preparation along with the lead and nickel salts at molar doses of approximately 1,3, 10, Nickei(ll) acetate, lead(ll) subacetate, calcium acetate, and magnesium and 30 times the maximal tolerated dose of the tumorigen. The acetate (reagent grade) were obtained from J. T. Baker Chemical Com animals were sacrificed 30 weeks after the first injection, and pany (Phillipsburg, NJ). Tricaprylin, which was used as a vehicle for the lung tumors were counted. The lead and nickel salts, admin injection of lead(ll) subacetate, was obtained from Sigma Chemical istered alone, each produced a significant increase in the ob Company (St. Louis, MO). Equal numbers of male and female A/St mice served number of lung adenomas per mouse. When administered (L. C. Strong Research Foundation, Inc., San Diego, CA), 6 to 8 weeks with any of the doses of calcium acetate or magnesium acetate of age, were used as test animals. Doses of nickel(ll) acetate (10.7 mg/kg of body weight/injection tested, neither lead subacetate nor nickel acetate showed any [Ni(CH3COO)2 4H2O; M, 248.86] and lead(ll) subacetate (10.0 mg/kg of significant tumorigenic activity. Calcium acetate alone (total dose, body weight/injection) [Pb^CHaCOOWOH^; M, 807.75] were used 11 mmol/kg of body weight) appeared to yield a significant rise throughout these experiments to induce lung tumors. Each injection thus in lung adenomas observed. The results indicate an antagonism contained the tumorigenic metal (0.04 mmol/kg of body weight) and was between magnesium and calcium and the tumorigenic metals administered i.p. These doses had been shown to constitute the maximal nickel and lead. tolerated dose in these animals and to be tumorigenic toward the lungs of mice (20). To assess the effect of calcium on nickel-induced lung tumorigenesis, INTRODUCTION mixtures of nickel(ll) acetate and calcium acetate were prepared in The carcinogenic activity of some divalent metals has been approximately 1:1,1:3.1:10. and 1:30 molar ratios in 0.9% NaCI solution, and each mixture was injected 3 times weekly into groups of 30 mice, recognized since at least the early 1930s (9). The list of carcin for a total of 24 injections. One group of mice was given injections of ogenic divalent metals exhibiting a stable divalent state includes nickel(ll) acetate alone 24 times i.p., and one group was given injections beryllium (7), titanium (3), manganese (20), cobalt (22), nickel (9, of calcium acetate [Ca(CH3COO)2 â€¢H2O;M, 176.19] alone 24 times i.p. 10, 23, 24), cadmium (8), and lead (6). These metals share with at a dose equivalent to the amount of calcium acetate present in the the physiologically essential divalent metal cations magnesium 1:10 mixture (total dose, 11.0 mmol/kg). One group of mice was given and calcium the property that their valence electrons occupy an injections of 0.2 ml of the 0.9% NaCI solution vehicle 24 times i.p. To external s orbital. As such, the divalent metal carcinogens exhibit assess the effect of calcium on lead-induced lung tumorigenesis, mix many of the chemical and physiological properties of magnesium tures of lead(ii) subacetate and calcium acetate were suspended in 1:1, and calcium. In fact, many of the above metal carcinogens have 1:3, and 1:10 molar ratios in tricaprylin, and each mixture was injected exhibited effects that are antagonistic to magnesium(ll) or cal- 3 times weekly into groups of 30 mice for a total of 20 injections. One group of mice was given injections of lead(ll) subacetate alone 20 times cium(ll) in one or more biological systems. The physiological i.p. One group was given injections of calcium acetate alone 20 times competition of lead(ll) versus calcium(ll) and of magnesium(ll) with a total of 9.2 mmol/kg i.p. One group of mice was given injections and calcium(ll) versus cadmium(ll) has been especially well stud of 0.1 ml of the tricaprylin vehicle 20 times i.p. ied (2, 5, 11, 16-18, 25-27). Finally, magnesium deficiency has To assess the effect of magnesium on lung tumorigenesis by nickel, been shown to produce lymphomas and thymomas in rats (1, mixtures of nickel(ll) acetate and magnesium acetate |Mg(CH3COO)s- 12). 4H2O; M, 214.46] were prepared in approximately 1:1, 1:3, 1:10, and Previous studies have shown that the metal carcinogens 1:30 molar ratios in 0.9% NaCI solution; each mixture was injected 3 nickel(ll) and lead(ll) produce adenomas in the lungs of strain A times weekly into groups of 30 mice, for a total of 24 injections. The mice (20). These observations extend to the lungs of mice group of mice given injections of nickel(ll) acetate alone and the 0.9% NaCI solution vehicle-treated group from the calcium experiment de 1Supported by NIH Contract N01-CP-3-3232. scribed above were utilized in this magnesium experiment. One group 2 To whom requests for reprints should be addressed. 3 Present address: Environmental Sciences, School of Public Health, The Uni of mice was given i.p. injections of magnesium acetate 24 times at a dose equivalent to the amount of magnesium acetate present in the 1:10 versity of Texas Health Science Center at Houston, P. O.Box 20186, Houston, TX 77025. mixture (total dose, 11.0 mmol/kg). To assess the effect of magnesium Received July 6,1982; accepted January 12,1984. on lead-induced lung tumorigenesis, finely powdered mixtures of lead(ll)

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1984 American Association for Cancer Research. Inhibition of Tumorigenicity of Lead and Nickel by Calcium and Magnesium subacetate and magnesium acetate were suspended in 1:1, 1:3, and Table 2 1:10 molar ratios of the 2 metals in tricaprylin. Because of the elevated Effect of magnesiumon the pulmonary adenoma response to metal tumorigens toxicity of the lead:magnesium combinations, each mixture was injected i.p. 3 times weekly into groups of 30 mice, for a total of only 9 injections. genic As controls, one group of mice was given i.p. injections of lead(ll) Metal:magne subacetate alone 9 times, and one group was given i.p. injections of a sium ace ofi.p. no.oflung total of 4.1 mmol of magnesium acetate alone 9 times. One group of tatemolarratio*0:101:01:11:31:101:300:101:01:11:31:10No.in vors/ini tumors/mouse0.32 mice was given i.p. injections of 0.2 ml of tricaprylin vehicle 9 times. No saltNoneTumorigenic metal jections24242424242424999999Survitial25/3029/3024/3029/3029/3027/3029/3021/3029/3020/301/3013/3018/30Av. evidence of coprecipitation or of any other reaction could be observed solution)None(0.9% NaCI Â±0.1260.59 solution)Nickel(ll)(0.9% NaCI Â±0.161.50Â±0.46d0.34 with any of the combinations of metal salts used in these studies. acetate0None Thirty weeks after the first injection, all mice were sacrificed, and the Â±0.10e0.28 lungs were placed in Tellyesniczky's fluid (100 ml of 70% ethanol:5 ml Â±0.10e0.1 of formalin:5 ml of glacial acetic acid). The lungs were then examined 9Â±0.05Â°0.21 Â±0.08e0.29 under a dissecting microscope (x10), and the surface adenomas were counted. A few surface nodules were examined histologically to confirm (tricaprylin)None Â±0.110.45 (tricaprylin)Lead(ll) Â±0.091.65 the typical morphological appearance of the adenoma. The average subacetate'Tumori Â±0.36"0.08 number of tumors per lung in the groups given injections of nickel(ll) Â±0.08a0.28 acetate, lead(ll) subacetate, calcium acetate, or magnesium acetate alone Â±0.11e were statistically compared with the appropriate vehicle-treated control group by Student's f test. The average number of tumors per lung in the " Magnesiumacetate mixed with tumorigenic metal in the appropriate vehiclein mouse groups given injections of the mixtures was compared statistically the molar ratio indicated; this mixture was injected i.p. for either 24 injections (nickel)or 9 injections(lead). with the average number of tumors per lung in the mouse groups treated "Mean Â±S.E. with the corresponding tumorigenic metal by Student's f test. c Nickel (0.04 mmoi/kg/injection i.p.) in 0.1 ml of 0.9% NaCIsolution. '' Significantlyabove vehicle-injectedcontrol group (p < 0.05). 8 Significantlybelow group treated with tumorigenic metal only (p < 0.05). RESULTS ' Lead(0.04 mmol/kg/mjectioni.p.)in 0.2 ml of tricaprylinfor a total of 9 injections.

Both tumorigenic metal salts, nickel(ll) acetate and lead(ll) subacetate (Tables 1 and 2), produced a statistically significant igenic metalxalcium acetate which were investigated. lung tumor response under the exposure conditions used in When treatment with nickel(ll) acetate and lead(ll) subacetate these experiments. Calcium acetate, when administered alone occurred simultaneously with that of magnesium acetate, the (total dose, 11.0 mmol/kg), also produced a lung tumor response lung tumor response was again completely inhibited (Table 2). (Table 1). This lung tumor response was statistically significant This inhibition occurred at all molar ratios of tumorigenic metal:magnesium acetate. It is interesting to note that an equi- when calcium acetate was administered in a 0.9% NaCI solution vehicle. Magnesium acetate alone did not produce a significant molar mixture of lead(ll) subacetate and magnesium acetate was lung tumor response (Table 2). When exposure to tumorigenic very toxic to strain A mice, and this toxicity decreased as the doses of nickel(ll) acetate and lead(ll) subacetate was coupled ratio of magnesium acetate:lead(ll) subacetate in the mixture with simultaneous exposure to calcium acetate, the lung tumor increased (Table 2). response to these tumorigenic metals was completely inhibited (Table 1). This inhibition was evident at all molar ratios of tumor- DISCUSSION

The present study demonstrates that the physiologically es Tabtel sential divalent cations magnesium and calcium prevent the rÃ¬se Effect of calcium on the pulmonary adenomaresponse to metal tumorigens in lung adenoma formation seen in Strain A mice treated with genicmetal: nickel acetate and lead subacetate. Such results provide evi cal dence that the carcinogenic activity of divalent metals may occur, cium ace ofi.p. no.oflung in part, through an antagonism with divalent magnesium and/or tatemolarratÂ»*0:101:01:11:31:101:300:101:01:11:31:10No.in vors/ini tumors/mouse0.32 saltNoneTumorigenicmetal jections24242424242424202020202020Survitial25/3028/3024/3029/3030/3030/3015/3026/3026/3020/3023/3018/3016/30Av.calcium. Consistent with this proposal are the observations that solution)None(0.9% NaCI Â±0.1261.04Â±0.26C1magnesium deficiency produces thymomas in rats (1, 12) and solution)Nickel(ll)(0.9% NaCI that tumors of the bone, a tissue with a very high requirement acetate"None .50 Â±0.46C0.40 Â±0.12e0.20 for magnesium and calcium, can be produced by the divalent Â±0.12e0.27 metal carcinogens beryllium (7) and nickel (9). Biochemical an Â±0.13e0.0 Â±0.0"0.35 tagonisms between divalent metal carcinogens and physiologi (tricaprylin)None Â±0.090.62 cally essential divalent metal cations have been described pre (tricaprylin)Lead(ll) Â±0.170.86 viously (16-18, 25-27). Thus, lead(ll) competes with calcium(ll) subacetateTumori Â±0.20C0.21 Â±0.10"0.22 (17) and magnesium(ll) (2) for intestinal absorption. Cadmium(ll) Â±0.10e0.13 competes similarly with calcium(ll) for absorption (21, 26), and Â±0.09" cadmium(ll) toxicity is inhibited by zinc(ll) and calcium(ll) (5, 11, ' Calciumacetate mixed with tumorigenic metal in the appropriate vehicle in the 25). There is thus considerable biochemical evidence rendering molar ratio indicated; this mixture was injected i.p. for either 24 injections (nickel) plausible an antagonism hypothesis of metal carcinogenesis. or 20 injections (lead). 6 Mean Â±S.E. Jennette (13) has recently written a review suggesting metal '' Significantlyabove vehicle-injectedcontrol group (p < 0.05). antagonism to explain heavy metal toxicity. SÃ¼ndermanef al. " Nickel (0.04 mmol/kg/injection i.p.) in 0.1 ml of 0.9% NaCI solution. " Significantlybelow group treated with tumorigenic metal only (p < 0.05). (24) have shown that manganese(ll) inhibits the sarcomagenic ' Lead (0.04 mmol/kg/injection i.p.) in 0.1 ml of tricaprylin. activity of nickel(ll) subsulfide.

APRIL 1984 1521

The ubiquitous roles of calcium(ll) and magnesium(ll) in cellular 3. FÃ¼rst,A.,and Haro, R. T. A survey of metal carcinogenesis.Prog. Exp. Tumor Res., 12:102-133,1969. metabolism make speculation on the prospective molecular site 4. Hui, G., and Sunderman, F. W., Jr. Effects of nickel compounds on incorpo responsible for the antagonism of metal carcinogenesis simple ration of [3H]thymidineinto DNA in rat liver and kidney. Carcinogenesis(Lond.), 1: 297-304, 1980. but verification difficult. Divalent metal carcinogens have been 5. Hyde, C. L, and Poirier, L. A. Calciumreverses the inhibitionof DNA synthesis shown to penetrate to many of the sites of normal magnesium by cadmium in normal and transformed rat liver cells in culture. Fed. Proc., 40: and calcium function. Thus, nickel bound to DMA has been 714, 1981. isolated from the livers and kidneys of nickel-treated rats (4). 6. IARC Working Group on the Evaluationof the CarcinogenicRisk of Chemicals to Man. 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The elevated incidence of lung adenomas noted in the present Lyon: IARC, 1973. 9. IARC Working Group on the Evaluationof the CarcinogenicRisk of Chemicals study in one group of mice treated with a high dose of calcium to Man. Nickel and inorganic nickel compounds, in: IARC Monographs on the acetate is difficult to interpret. This effect was seen only in mice Evaluation of the Carcinogenic Risk of Chemicals to Man, Vol. 2, pp. 126- receiving a total dose of calcium (11.0 mmol/kg administered in 149. Lyon: IARC, 1973. 10. IARC Working Group on the Evaluationof the CarcinogenicRisk of Chemicals 0.9% NaCI solution (Table 1). Mice treated with a lower dose of to Man. Nickel and nickel compounds. In IARC Monographson the Evaluation calcium(ll) in tricaprylin or with either dose of magnesium(ll) of the Carcinogenic Risk of Chemicals to Man, Vol. 11, pp. 75-112. Lyon: developed lung adenoma incidences ranging from 55 to 85% IARC, 1976. 11. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1984 American Association for Cancer Research. Inhibition by Magnesium and Calcium Acetates of Lead Subacetate- and Nickel Acetate-induced Lung Tumors in Strain A Mice

Lionel A. Poirier, Jeffrey C. Theiss, Lyle J. Arnold, et al.

Cancer Res 1984;44:1520-1522.

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