the carcinogenicity of intravenous nickel carbonyl …...resume of acute biochemical effects of i.v....
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(CANCER RESEARCH 32, 2253-2258, October 1972]
The Carcinogenicity of Intravenous Nickel Carbonyl in Rats1
Thomas J. Lau, Raymond L. Hackett, and F. William Sunderman, Jr.2
Department of Laboratory Medicine, University of Connecticut School of Medicine, Farmington, Connecticut 06032 ¡T.J. L., F. W. S./ andDepartment of Pathology, University of Florida College of Medicine, Gainesville, Florida 32601 ¡R.L. H.]
SUMMARY
The carcinogenicity of i.v. nickel carbonyl, Ni(CO)4, wastested in Sprague-Dawley rats. In 72 rats (Group A) thatsurvived a single 50% lethal dose injection of Ni(CO)4 (2.2 mgnickel per 100 g), the incidence of malignant tumors (8.3%)was not significantly greater than the incidence in 47 rats inthe control group (4.3%). In 121 rats (Group B) that survivedsix 5% lethal dose injections of Ni(CO)4 (0.9 mg nickel per100 g) at intervals of 2 or 4 weeks, the incidence of malignanttumors (15.7%) was significantly greater than in the controls(p<0.05). The 19 malignant tumors in Group B included 6undifferentiated sarcomas (lung, pleura, liver, pancreas, uterus,and abdominal wall), 3 fibrosarcomas (neck, pinna, and orbit),3 carcinomas (liver, kidney, and breast), 1 s.c. hemangio-endothelioma, 1 leukemia, and 5 lymphomas (lung). Thisstudy demonstrates that multiple parenteral injections ofNi(CO)4 can induce diverse malignant tumors in varied organsand tissues of the rat.
INTRODUCTION
Nickel carbonyl is a toxic, volatile liquid which is anintermediate product in the Mond process for refining nickelmatte and is used as a catalyst in the petroleum, plastic, andrubber industries. Nickel carbonyl has been implicated as apossible respiratory carcinogen in industrial atmospheres (4, 6)and in tobacco smoke (18). In rats that were exposed toinhalation of gaseous nickel carbonyl, Sunderman et al. (16,17) found increased incidence of pulmonary carcinomas, andSanina (15) observed increased incidence of malignant tumorsof the uterus, ovaries, and mammary glands. The present studywas performed in order to ascertain whether or not i.v.administration of liquid nickel carbonyl to rats results inincreased incidence of malignant tumors. This investigationwas stimulated by our findings that the acute pathological (9)and biochemical (12, 19, 26, 28) reactions that develop in ratsafter i.v. injections of nickel carbonyl are similar to those thatoccur following exposures to nickel carbonyl by inhalation.
'Sponsored by United States Atomic Energy Commission GrantAT(11-1)-3140; by USPHS Grant CA-11250 from the National CancerInstitute; and by United States Environmental Protection AgencyContract EHS-C-71-109.
2To whom requests for reprints should be addressed.
Received May 22, 1972; accepted July 5, 1972.
MATERIALS AND METHODS
The experimental animals were randomly bred albino rats ofthe Sprague-Dawley strain (Dublin Farms, Dublin, Ga.),maintained on Purina laboratory rat chow. The rats were 8 to9 weeks old at the time of the initial injection. Ni(CO)4(Matheson Chemical Co., East Rutherford, N.J.) wasadministered by i.v. injection into a tail vein by means of amicrosyringe, as previously described (10, 11). The purity ofthe Ni(CO)4 was verified by gas chromatography on 3 differentChromatographie columns, as described by Sunderman et al.(27). Group A consisted of 72 rats (26 males, 46 females) thatsurvived a single injection of Ni(CO)4 in LDS03 dosage [2.2
mg nickel per 100 g body weight, equivalent to 5 /il ofNi(CO)4 per 100 g body weight]. Group B considted of 121rats (61 males, 60 females) that survived 6 injections ofNi(CO)4 in LDS dosage [0.9 mg nickel per 100 g body weight,equivalent to 2 fi\ of Ni(CO)4 per 100 g body weight]administered at intervals of 2 or 4 weeks. The control groupconsisted of 47 rats (15 males, 32 females) that received shaminjections of 5 ¿ilof 0.9% (w/v) NaCl solution. The rats eitherdied spontaneously or were killed when they became somoribund that they could not move about in their cages andhence could not obtain food or water. The rats were autopsiedand the tissues were examined by light microscopy.
RESULTS
As indicated in Table 1, the longevity of rats in the controlgroup and in the 2 treated groups did not differ significantly.There were no significant differences between the incidencesof benign tumors in the control group and in the 2 treatedgroups. In female rats, benign fibroadenomas of the breastwere found in 13/46 (28%) of Group A, in 11/60 (18%) ofGroup B, and in 10/32 (31%) of the control group. In malerats, the only benign tumors were 2 small s.c. plexiformneuromas that were found in 2 rats of the control group. Inthe control group, the overall incidence of malignant tumorswas 4.3%, which is consistent with published statistics forspontaneous malignant tumors in Sprague-Dawley rats (5, 29).There were no significant differences between the incidencesof malignant tumors of male and female rats within thecontrol group or within either of the treated groups. In rats ofGroup A that survived a single LDSO injection of nickelcarbonyl, the overall incidence of malignant tumors was 8.3%,which was not significantly greater than that in the control
3The abbreviations used are: LD50, 50% lethal dose; LD5, 5% lethaldose.
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T. J. Lau, R. L. Hackett, and F. W. Sunderman, Jr.
Table IIncidence of malignant tumors in rats after i.v. nickel carbonyl
Sprague-Dawley rats in Group A survived 1 i.v. injection of Ni(CO)4 at LD50 dosage (2.2 mg nickel per 100 g). Rats inGroup B survived 6 i.v. injections of Ni(CO)4 at LD5 dosage (0.9 mg nickel per 100 g) at intervals of 2 or 4 weeks. Rats in thecontrol group received sham injections of 5 n\ of 0.9% (w/v) NaCl solution.
ExperimentalgroupABControlNo.
ofrats ingroup26
(M)46(F)72
(M,F)61
(M)60(F)121
(M,F)15
(M)32(F)47
(M, F)Median
ageatdeath22
(9-25)°23(6-37)23
(6-37)21
(5-30)24(8-36)23(5-36)24
(10-30)24(6-33)24
(6-33)Total
incidenceof malignanttumorsNo.33610919022%11.56.58.316.415.015.7'06.34.3PulmonarylymphomasNo.224325022%7.74.35.64.93.34.106.34.3All
othermalignanttumorsNo.I6Ie27aIe14000%3.82.22.811.511.711.6*000
0 Numbers in parentheses, range.b Fibrosarcoma (orbit).c Cholangiocarcinoma (liver).**Fibrosarcomas (orbit, pinna, neck); undifferentiated sarcoma (lung);hemangioendothelioma (s.c. tissue); undifferentiated
leukemia; carcinoma (kidney).e Undifferentiated sarcomas (pleura, liver, pancreas, uterus, abdominal wall); carcinomas (liver, breast).f p < 0.05 versus control group (x2 test).* p < 0.02 versus control group (x2 test).
group. In rats of Group B that received 6 injections of nickelcarbonyl in LDS dosage, the overall incidence of malignanttumors was 15.7%, which was significantly greater than that inthe control group (p < 0.05). There were no significantdifferences between the percentages of pulmonary lymphomasin the control group and in the 2 treated groups. However,there was a significant increase in the percentage ofnonlymphomatous malignant tumors among rats of Group B(p < 0.02). The histológica! varieties and sites of origin of thenonlymphomatous malignant tumors that were found in thisstudy are listed in the footnotes to Table 1. These tumors wereall unequivocally malignant, and distant métastases werepresent in the majority of the tumor-bearing rats. Thehistological appearances of selected tumors are illustrated inFigs. 1 to 6.
DISCUSSION
In the present investigation, a significant increase in theincidence of malignant tumors was observed in rats thatreceived 6 i.v. injections of nickel carbonyl. The malignanttumors included a variety of histological types, and developedin diverse organs and tissues. Thus, this study providesadditional evidence of the carcinogenicity of nickel carbonylin the rat. Previous investigations which have demonstrated thecarcinogenicity of various nickel compounds in experimentalanimals have been reviewed by Sunderman (24). Nickelcarcinogenesis has been documented in several strains of rats,as well as in mice, guinea pigs, hamsters, rabbits, and cats (24).In rats, the carcinogenic potency of nickel compounds appearsto be inversely related to the solubilities of the compounds in
Table 2Resume of acute biochemical effects of i.v. nickel carbonyl
in Sprague-Dawley ratsNi(CO)4, 2.2 mg/100 g, i.V., 6 to 28 hr before sacrifice.
%ofExperimental system" controlvaluesHepatic
tryptophanpyrrolaseaftercortisoneinductionHepaticbenzpyrenehydroxylaseafter
phenothiazineinductionHepaticcytochrome P-450afterphénobarbital
inductionLeucine-14C uptake inhepaticmicrosomal
proteins invivo1*C-Labeled orotic acid uptakeinhepatic
RNA invivoRNApolymerase activityinhepatic
nucleiRNAsynthesis in vitro byhepaticchromatin-RNA
polymerasecomplexTemplateactivity of hepaticDNAfor
M. lysodeikticus RNA polymerase72
±45
±48
±82
±25
±40
±49
±98
±7b8562766p
vs.controls<0.01<0.01<0.01<0.01<0.01<0.01<0.01N.S.
a References are cited in the text.b Mean ±S.E.
aqueous media. Thus, the carcinogenic nickel compounds,which include nickel dust, nickel sulfide, nickel carbonate,nickel oxide, nickel carbonyl, and nickelocene, are allsparingly soluble in water at 37°(8, 14, 24). Soluble nickel
salts, such as nickel chloride, nickel sulfate, and nickelammonium sulfate, have not been found to be carcinogenic (8,14). The biological characteristics of the malignant tumorsthat are induced in rats by administration of nickel
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Nickel Carbonyl Carcinogenesis
compounds have been summarized by Sunderman (22) andMaenza et al. ( 13).
The acute biochemical effects that have been reported tooccur in hepatocytes of Sprague-Dawley rats following i.v.injection of nickel carbonyl in LDSO dosage are listed in Table2. Nickel carbonyl inhibits enzyme induction in severalexperimental systems, including cortisone induction oftryptophan pyrrolase (20), phenothiazine induction ofbenzpyrene hydroxylase (19), and phénobarbital induction ofcytochrome P-450 (21). There is mild inhibition of proteinsynthesis, as measured by in vivo incorporation of leucine-14C
into hepatic microsomal proteins (23), and there is markedinhibition of RNA synthesis, as measured by in vivoincorporation of ' 4C-labeled orotic acid into hepatic RNA (2).
The inhibition of RNA synthesis is attributable to nickelcarbonyl inhibition of DNA-dependent RNA polymeraseactivity, as demonstrated in vitro with intact hepatic nuclei(25) and also with chromatin-RNA polymerase complexisolated from hepatic nuclei (3). Nickel carbonyl does notaffect the template activity of hepatic DNA or chromatin fortranscription in vitro by exogenous RNA polymerase fromMicrococcus lysodeikticus (1). The finding that i.v. nickelcarbonyl inhibits DNA-dependent RNA synthesis in rathepatocytes furnishes a metabolic effect whereby nickelcarbonyl can cause acute alterations of gene expression. Apossible relationship to the mechanism of nickel carbonylcarcinogenesis is suggested by similarities between the acutebiochemical alterations produced by nickel carbonyl and thoseproduced by certain nonmetallic carcinogens, such as aflatoxinB, (7). Caution should be exercised in interpreting thisanalogy, however, since aflatoxin B] is a very potent livercarcinogen, whereas nickel carbonyl is a relatively weak livercarcinogen.
ACKNOWLEDGMENTS
We are grateful to Dr. Svend W. Nielsen, Dr. Ronald M. Maenza, andDr. Francis J. C. Roe for consultations regarding the histológica!classification of certain of the malignant tumors, and to Mrs. SandraCullifer for skillful technical assistance.
REFERENCES
1. Beach, D. J. Some Aspects of the Molecular Biology of NickelCarbonyl. Ph.D. Dissertation. University of Florida, Gainesville,1969.
2. Beach, D. J., and Sunderman, F. W., Jr. Nickel Carbonyl Inhibitionof l4C-Orotic Acid Incorporation into Rat Liver RNA. Proc. Soc.Exptl. Biol. Med., 131: 321-322, 1969.
3. Beach, D. J., and Sunderman, F. W., Jr. Nickel Carbonyl Inhibitionof RNA Synthesis by a Chromatin-RNA Polymerase Complex fromHepatic Nuclei. Cancer Res., 30: 48-50, 1970.
4. Boyland, E. Occupational Carcinogenesis. Practitioner, 199:277-284, 1967.
5. Davis, R. K., Stevenson, G. T., and Busch, K. A. Tumor Incidencein Normal Sprague-Dawley Female Rats. Cancer Res., 16:194-197, 1956.
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Induction of Sarcomas in Rats by Combination of Nickel Sulfideand 3,4-Benzpyrene. Cancer Res., 31: 2067-2071, 1971.
14. Payne, W. W. Carcinogenicity of Nickel Compounds inExperimental Animals. Proc. Am. Assoc. Cancer Res., 5: 50, 1964.
15. Sanina, Y. P. Toxicology of Nickel Carbonyl. //;: A. A. Letavet andI. V. Sanotsky (eds.), Toxicology of Leningrad: New IndustrialChemicals, pp. 144-149. Meditsina Publishing House, 1968.
16. Sunderman, F. W., and Donnelly, A. J. Metastasizing PulmonaryTumors in Rats Induced by the Inhalation of Nickel Carbonyl. Am.J. Pathol., 46: 1027-1041, 1965.
17. Sunderman, F. W., Donnelly, A. J., West, B., and Kincaid, J. F.Carcinogenesis in Rats Exposed to Nickel Carbonyl. Arch. Ind.Health 20: 36-41, 1959.
18. Sunderman, F. W., and Sunderman, F. W., Jr. Implication of Nickelas a Pulmonary Carcinogen in Tobacco Smoke. Am. J. Clin.Pathol., 35: 203-209, 1961.
19. Sunderman, F. W., Jr. Inhibition of Induction of BenzpyreneHydroxylase by Nickel Carbonyl. Cancer Res., 27: 950-955,1967.
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T. J. Lau, R. L. Hackett, and F. W. Sunderman, Jr.
Fig. 1. Malignant lymphoma of lung of a female rat of Group B. X, tumor surrounding a small pulmonary artery. X 100. B, pleomorphic roundcells with prominent nuclei. X 250.
Fig. 2. Fibrosarcoma of orbit of a male rat of Group A. A, poorly differentiated primary tumor. X 250. B, pulmonary metastasis of the tumor,with infiltration of aveolar septae by neoplastic cells. X 100.
Fig. 3. Undifferentiated sarcoma of abdominal wall of a female rat of Group B. A, sarcoma cells surrounding degenerating skeletal muscle fibers.X 250. B, pleomorphic cells with hyperchromatic nuclei. X 400.
Fig. 4. Cholangiocarcinoma of liver of a female rat of Group A. A, tumor compressing fatty liver tissue (left corner). X 250. B, neoplastictubular structures lined by polyhedral cells. X 400.
Fig. 5. Hemangioendothelioma of s.c. tissues of abdomen of a male rat in Group B. A, neoplastic endothelial cells lining vascular channels. X100. B, prominent vascular spaces. X 100.
Fig. 6. Undifferentiated leukemia in a male rat of Group B. A, focus of small round cells with large hyperchromatic nuclei in the myocardium.X 450. B, focus of Undifferentiated round cells in the liver. X 450.
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Nickel Carbonyl Carcinogenesis
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1972;32:2253-2258. Cancer Res Thomas J. Lau, Raymond L. Hackett and F. William Sunderman, Jr. The Carcinogenicity of Intravenous Nickel Carbonyl in Rats
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