- Email: [email protected]
The induction of DNA strand breakage by nickel compounds in cultured Chinese hamster ovary cells
Cancer Letters, 15 (1982) 35-40 Elsevier/North-Holland Scientific Publishers Ltd.
35
THE INDUCTION OF DNA STRAND BREAKAGE BY NICKEL COMPOUNDS IN CULTURED CHINESE HAMSTER OVARY CELLS*
STEVEN
H. ROBISON
and MAX
COSTA
Division of Toxicology, Department of Pharmacology, The University School at Houston, P.O. Box 20708, Houston, TX 77025 (U.S.A.)
of Texas Medical
(Received 10 September 1981) (Accepted ‘7 October 1981)
SUMMARY
Both NiClz and crystalline aNi induced DNA strand breaks in cultured Chinese hamster ovary (CHO) cells. Alkaline sucrose gradient analysis of [3H]thymidine radiolabelled DNA isolated from cells exposed to NiClz at 1 pg/ml for only 2 h indicated a high degree of DNA strand breakage. Similarly crystalline eNiS caused substantial strand breakage at 1 pg/ml following a 24-h treatment interval. These nickel compounds caused DNA strand breaks at concentrations which did not significantly impair normal cellular division. A concentration-dependent effect upon the number and average size of DNA fragments was obtained with both NiClz and crystalline cuNi8. Since DNA strand breakage occurred at such low concentrations, these results suggest that nickel compounds which cause cellular transformation have highly selective and specific effects upon DNA structure.
INTRODUCTION
In vivo studies using experimental animals and human epidemiological data indicate that various nickel compounds are carcinogenic [ 1,4,7,12,13,20,21]. Recent studies employing the cell transformation assay [ 51, have demonstrated that soluble nickel compounds and crystalline nickel sulfide induce cell transformation. The latter compound was found to be more potent than the former in inducing cellular transformation. The mechanism by which metals cause transformation is poorly understood. However, several observations have revealed that crystalline cuNiSbut not amorphous NiS is selectively *Supported by grant No. R808048 from the U.S. Environmental Protection Agency and by contract No. R60016 from the U.S. Department of Energy. Dr. S.H. Robison is a recipient of an NIH postdoctoral fellowship award No. CA-06570 from the NCI.
0304-3835/82/0000-+000/$02.75 0 1982 Elsevier/North-Holland
Scientific Publishers Ltd.
36
phagocytized by cells and following endocytosis particles aggregated around the nuclear envelope [ 91. Other work has shown that phagocytized particulate crystalline eNiS undergoes cytoplasmic dissolution liberating water soluble nickel ions (P.B. Harnett et al., unpublished). These ions can then bind to cellular constituents such as nucleic acids and proteins. This raises the question of whether the particle, the free ion or a yet unidentified species are the factors involved in inducing cellular transformation. Presently, there is very little information as to how metals interact with cellular nucleic acids [ 11,191. Recent studies have shown that cadmium and nickel can cause DNA-protein crosslinks and DNA strand breaks [ 2,221. The work of Sirover and Loeb [ 181 has indicated that metals cause a decreased fidelity of DNA transcription. Furthermore, experiments using cells in culture have shown that lead chromate can induce DNA strand breaks in CHO cells [S] and nickel compounds cause chromosomal aberrations in C3M mouse mammary carcinoma cells [14]. However, there is evidence that nickel carbonyl, a highly toxic agent, does not cause DNA strand breaks [lo]. It is clear that there is some question as to whether metals do induce some form of DNA damage. We have examined the effects of nickel chloride and crystalline NiS on the DNA of CHO cells by using alkaline sucrose gradient analysis. Our results suggest that these 2 nickel compounds are very selective in inducing DNA strand breaks since breakage occurred under exposure conditions which did not significantly impair normal cell proliferation.
MATERIALS
AND METHODS
Chemicals Nickel chloride was purchased from Alfa products (Beverly, MA). Crystalline nickel sulfide was prepared according to the method of Costa et al. [5,6]. Ultrapure sucrose and sodium dodecyl sulfate (BDS) was purchased from Sigma Chemical Co., (St. Louis, MO). Tritiated deoxythymidine was purchased from New England Nuclear Corp. Fetal bovine serum, McCoy’s 5a media, and trypsin were all purchased from Gibco, Inc. (Grand Island, NY). Cell culture techniques CHO cells were seeded into loo-mm tissue culture dishes in 10 ml of McCoy’s 5a media containing 0.2 pCi/ml [3H]deoxythymidine (spec. act. 55.2 Ci/mmol) and grown in this same media to confluence. The cells were then treated with the various nickel compounds for the time intervals specified in the figures. The cell monolayer was rinsed once with saline A, then scraped with a rubber policeman and the cells collected by centrifugation. The resulting cell pellet was then resuspended in sufficient saline A so that an aliquot of 50-100 ~1 contained approximately 3 X 10’ cells. Cell number was determined with a Coulter Counter particle size analyzer.
37
Separation of radiolabelled DNA in alkaline sucrose gradients Alkaline sucrose gradients (5-20%, w/v) containing 0.3 M NaOH, 0.7 M NaCl, 10 mM EDTA, 0.05 M Tris were formed over a 0.2-ml pad of 60% sucrose (w/v). The gradients were then overlayed with 0.2 ml of a lysis solution consisting of 0.2% SDS, 0.02 M EDTA and 0.2 M NaOH. An aliquot of cells was gently layered into the lysis solution and incubated for 18 h at room temperature to insure complete cell lysis. The gradients were then centrifuged in a Beckman SW 50.1 swinging bucket rotor at 40,000 rev./min for 2 h. The gradients were fractionated by collecting 10 drops per fraction onto Whatman 3 mm filter paper strips. The strips were washed 3 times in 10% trichloroacetic acid, then washed in ethanol, dried and counted using a toluene-omnifluor mixture in a Packard liquid scintillation counter.
Crystalline
aNiS
(1 pg/ml)
CrystalMine
aNiS
(20 @g/ml)
i
B
10
20
30
40
50
60
70
80
90 Percent
B
10
20
30
40
&O
60
70
80
90
of Gradient
Fig. 1. Effect of crystalline aNiS on DNA strand breaks. Cultured CHO cells were maintained and labelled with [3H]thymidine as described in Materials and Methods. Following this procedure, the cells were treated with crystalline aNiS particles (2.2 pm mean particle diameter) for 24 h at the concentrations shown in the figure. The cells were then isolated, lysed and their DNA analyzed on alkaline sucrose gradients as described in the methods section. Gradients were standardized with respect to total cpm applied and fractions collected to facilitate comparison, A 16 S DNA marker migrated to fraction 45 in this gradient system.
38
P 5
5 P 8
9-
I 5
8 7-
E
NiCI,
I ,
,..\._ ‘.k\
‘i * -
I
‘
,-
(1 pg/mll
‘
4
I
*
L.-_ 10
,’ ’ .
5-
4
.
31 .
6
(lOpg/ml)
P 9
’
L
$
NiCI,
. -i
. I._.
24
.
#
..’
..I.
,
‘ . . . . .. .
l-
~ ,_~ cm_ ,~~ ~_~~ ~~~.~_ ~__ 20
30
40
50
60
70
80
90
Percent
8
10
20
30
40
50
60
70
80
90
of Gradient
Fig. 2. Effect of NiCl, upon DNA strand breaks. All procedures were conducted as described in Materials and Methods and the legend to Fig. 1. Cultured CHO cells were treated with NiCl, at the specified concentrations for 2 h. RESULTS
The alkaline sucrose gradient technique has been utilized to determine the presence of single strand breaks and alkaline sensitive sites caused by a number of chemical and physical agents including X-rays, ultraviolet light and carcinogenic chemicals. We have monitored the effects of NiCl? and crystalline aNi on the induction of DNA strand breaks in CHO cells. We have been able to detect single strand breaks in cells treated with either toxic or non-toxic levels of crystalline aNiS. A control gradient (Fig. 1) shows that DNA not exposed to nickel sediments to the bottom of the centrifuge tube. Exposure to crystalline cvNiScauses numerous single strand breaks, as evidenced by the peak of slower sedimenting DNA as seen in the other gradients shown in Fig. 1. Exposure to aNiS at 1 pg/ml for a 24-h time interval does not interfere with the division of CHO cells and additionally under these conditions, the level of cell plating efficiency was essentially similar to that of untreated cultures [ 41. However, this concentration caused substantial DNA strand breaks (Fig. 1) suggesting that crystalline cwNiSselectively damages DNA. A concentrationdependent effect on DNA strand breaks was obtained with crystalline aNiS treatment as indicated by the positional shift in gradient sedimentation of the radiolabelled DNA fragments isolated from cells treated with either 1 pg/ml, or 5 pg/ml or 20 pg/ml crystalline aNiS. Consistent with the results obtained using crystalline aNiS, treatment of cells with NiClz at 1 pg/ml or 10 @g/ml also resulted in single strand DNA breaks after only a 2-h exposure (Fig. 2). DISCUSSION
DNA strand breaks were caused by both soluble NiCl? and insoluble
39
crystalline cuNi§. Both compounds caused strand breaks under conditions of exposure which do not impair the proliferative and survival capacity of these cells. It is possible that low concentrations of toxic metals cause strand breaks that are readily repairable by the cell, and that higher levels either saturate the cells ability to repair the damage or inactivate the necessary enzymes for DNA repair. The mechanism by which strand breaks occur also remains unanswered. The nickel ion may interact with the DNA-phosphate backbone to cause strand breakages, it may distort the helix in such a way that endonucleases cause strand scission, or base release might occur generating an apurinic site. The nickel ion could possibly interact with the DNA in such a way that an alkaline sensitive site is generated. This site would be alkaline sensitive and strand excision occurs under the alkaline centrifugation conditions employed in our studies. DNA single and double strand breaks can be induced by a number of agents including ionizing radiation [15], ultraviolet [3] and chemical carcinogens [ 161. In addition it has been shown that apurinic sites can be generated by various compounds including alkylating agents [ 161 and the antineoplastic agent bleomycin [ 171. Lesions induced by these agents can be repaired at low concentrations, however, higher levels saturate the cell’s ability to repair damage or cause cell death. The lesions that nickel induces in DNA probably contribute to the induction of cell transformation. These lesions may also be responsible for the observed chromosomal aberrations following nickel exposure. REFERENCES 1 Barton, R.T. (1977) Nickel carcinogenesis of the respiratory tract. J. Otolaryngol., 6, 412-422. 2 Ciccarelli, R.B., Hampton, J.H. and Jennette, K.W. (1981) Nickel carbonate induces NDA-protein crosslinks and DNA strand breaks in rat kidney. Cancer Letters, 12, 349-354. 3 Cleaver, J.E. (1974) Repair processes for photochemical damage in mammalian cells. In: Advances in Radiation Biology, Vol. 7, pp. l-75. Editors: T.J. Lett and H. Adler. Academic Press, New York. 4 Costa, M. (1980) Metal Carcinogenesis Testing: Principles and in Vitro Methods. Humana Press, Clifton, New Jersey. 5 Costa, M., Simmons-Hansen, J., Bedrossian, C.W.M., Bonura, J. and Caprioli, R.M. (1981) Phagocytosis, cellular distribution and carcinogenic activity of particulate nickel compounds in tissue culture. Cancer Res., 41, 2868-2876. 6 Costa, M., Nye, J. and Sunderman Jr., F.W. (1978) Morphological transformation of Syrian hamster fetal cells induced by nickel compounds. Ann. Clin. Lab. Sci., 8, 502. 7 Damjanov, I., Sunderman, Jr., F.W., Mitchell, J.M. and Allpass, P.R. (1978) Induction of testicular sarcomas in Fischer rats by intratesticular injection of nickel subsulfide. Cancer Res., 38, 268-276. 8 Douglass, G.R., Bell, R.D.L., Grant, C.D., Wytsma, J.W. and Bora, K.C. (1980) Effect of lead chromate on chromosome aberrations, sister-chromatid exchanges and DNA damage. Mutat. Res., 77, 157-163. 9 Evans, R.M., Davies, P.J.A. and Costa, M. (1981) Visualization of cellular uptake and dissolution of carcinogenic olNiS particles by Video Intensification Microscopy. Am. Sot. Cell Biol., in press.
40
10 Hui, G. and Sunderman, Jr., F.W. (1980) Effects of nickel compounds and incorporation of [“Hlthymidine into DNA in rat liver and kidney. Carcinogenesis, 1, 297-304. 11 Jacobson, K.B. and Turner, J.E. (1980) The interactions of cadmium and certain other metal ions with proteins and nucleic acids. Toxicology, 16, l-37. 12 Jasmin, G. and RiopeIIe, J.L. (1976) Renal carcinomas and erythrocytosis in rats following intrarenal injection of nickel subsulfide. Lab. Invest., 35, 71-78. 13 Kreyberg, L. (1978) Lung cancer in a nickel refinery. Br. J. Ind. Med., 35,109-116. 14 Nishimura, M. and Umeda, M. (1979) Induction of chromosomal aberrations in cultured mammalian cells by nickel compounds. Mutat. Res., 68, 337-349. 15 Ormerod, M.G. (1976) Radiation induced strand breaks in the DNA of mammalian cells. In: Biology of Radiation Carcinogenesis, pp. 67-92. Editors: J.M. Yuhas, R.W. Tennant and J.D. Regan. Raven Press, New York. 16 Roberts, J.J. (1978) The repair of DNA modified by cytotoxic mutagenic, and carcinogenic chemicals. In: Advances in Radiation Biology, Vol. 7, pp. 211-436. Editors: J.T. Lett and H. Adler. Academic Press, New York. 17 Ross, S.L. and Moses, R.E. (1978) Two actions of bleomycin on superhelical DNA. Biochemistry, 17, 581-586. 18 Sirover, M.A. and Loeb, L.A. (1976) Infidelity of DNA synthesis in vitro: screening for potential metal mutagens or carcinogens. Science, 145, 1434-1436. 19 Sissoeff, I., Grisvard, J. and GuiIle, E. (1976) Studies on metal ions-DNA interactions: specific behavior of reiterative DNA sequences. Prog. Biophys. Mol. Biol., 31, 165199. 20 Sunderman, Jr., F.W. and Maenza, R.M. (1976) Comparisons of carcinogenicities of nickel compounds in rats. Res. Commun. Chem. Pathol. Pharmacol., 14, 319-330. 21 Sunderman, Jr., F.W. (1978) Carcinogenic effects of metals. Fed. Proc., 37,40--46. 22 Tsapakor, M.J., Hampton, T.H. and Jennette, K.W. (1981) The carcinogen chromate induces DNA crosslinks in rat liver and kidney. J. Biol. Chem., 256, 3623-3626.
35
THE INDUCTION OF DNA STRAND BREAKAGE BY NICKEL COMPOUNDS IN CULTURED CHINESE HAMSTER OVARY CELLS*
STEVEN
H. ROBISON
and MAX
COSTA
Division of Toxicology, Department of Pharmacology, The University School at Houston, P.O. Box 20708, Houston, TX 77025 (U.S.A.)
of Texas Medical
(Received 10 September 1981) (Accepted ‘7 October 1981)
SUMMARY
Both NiClz and crystalline aNi induced DNA strand breaks in cultured Chinese hamster ovary (CHO) cells. Alkaline sucrose gradient analysis of [3H]thymidine radiolabelled DNA isolated from cells exposed to NiClz at 1 pg/ml for only 2 h indicated a high degree of DNA strand breakage. Similarly crystalline eNiS caused substantial strand breakage at 1 pg/ml following a 24-h treatment interval. These nickel compounds caused DNA strand breaks at concentrations which did not significantly impair normal cellular division. A concentration-dependent effect upon the number and average size of DNA fragments was obtained with both NiClz and crystalline cuNi8. Since DNA strand breakage occurred at such low concentrations, these results suggest that nickel compounds which cause cellular transformation have highly selective and specific effects upon DNA structure.
INTRODUCTION
In vivo studies using experimental animals and human epidemiological data indicate that various nickel compounds are carcinogenic [ 1,4,7,12,13,20,21]. Recent studies employing the cell transformation assay [ 51, have demonstrated that soluble nickel compounds and crystalline nickel sulfide induce cell transformation. The latter compound was found to be more potent than the former in inducing cellular transformation. The mechanism by which metals cause transformation is poorly understood. However, several observations have revealed that crystalline cuNiSbut not amorphous NiS is selectively *Supported by grant No. R808048 from the U.S. Environmental Protection Agency and by contract No. R60016 from the U.S. Department of Energy. Dr. S.H. Robison is a recipient of an NIH postdoctoral fellowship award No. CA-06570 from the NCI.
0304-3835/82/0000-+000/$02.75 0 1982 Elsevier/North-Holland
Scientific Publishers Ltd.
36
phagocytized by cells and following endocytosis particles aggregated around the nuclear envelope [ 91. Other work has shown that phagocytized particulate crystalline eNiS undergoes cytoplasmic dissolution liberating water soluble nickel ions (P.B. Harnett et al., unpublished). These ions can then bind to cellular constituents such as nucleic acids and proteins. This raises the question of whether the particle, the free ion or a yet unidentified species are the factors involved in inducing cellular transformation. Presently, there is very little information as to how metals interact with cellular nucleic acids [ 11,191. Recent studies have shown that cadmium and nickel can cause DNA-protein crosslinks and DNA strand breaks [ 2,221. The work of Sirover and Loeb [ 181 has indicated that metals cause a decreased fidelity of DNA transcription. Furthermore, experiments using cells in culture have shown that lead chromate can induce DNA strand breaks in CHO cells [S] and nickel compounds cause chromosomal aberrations in C3M mouse mammary carcinoma cells [14]. However, there is evidence that nickel carbonyl, a highly toxic agent, does not cause DNA strand breaks [lo]. It is clear that there is some question as to whether metals do induce some form of DNA damage. We have examined the effects of nickel chloride and crystalline NiS on the DNA of CHO cells by using alkaline sucrose gradient analysis. Our results suggest that these 2 nickel compounds are very selective in inducing DNA strand breaks since breakage occurred under exposure conditions which did not significantly impair normal cell proliferation.
MATERIALS
AND METHODS
Chemicals Nickel chloride was purchased from Alfa products (Beverly, MA). Crystalline nickel sulfide was prepared according to the method of Costa et al. [5,6]. Ultrapure sucrose and sodium dodecyl sulfate (BDS) was purchased from Sigma Chemical Co., (St. Louis, MO). Tritiated deoxythymidine was purchased from New England Nuclear Corp. Fetal bovine serum, McCoy’s 5a media, and trypsin were all purchased from Gibco, Inc. (Grand Island, NY). Cell culture techniques CHO cells were seeded into loo-mm tissue culture dishes in 10 ml of McCoy’s 5a media containing 0.2 pCi/ml [3H]deoxythymidine (spec. act. 55.2 Ci/mmol) and grown in this same media to confluence. The cells were then treated with the various nickel compounds for the time intervals specified in the figures. The cell monolayer was rinsed once with saline A, then scraped with a rubber policeman and the cells collected by centrifugation. The resulting cell pellet was then resuspended in sufficient saline A so that an aliquot of 50-100 ~1 contained approximately 3 X 10’ cells. Cell number was determined with a Coulter Counter particle size analyzer.
37
Separation of radiolabelled DNA in alkaline sucrose gradients Alkaline sucrose gradients (5-20%, w/v) containing 0.3 M NaOH, 0.7 M NaCl, 10 mM EDTA, 0.05 M Tris were formed over a 0.2-ml pad of 60% sucrose (w/v). The gradients were then overlayed with 0.2 ml of a lysis solution consisting of 0.2% SDS, 0.02 M EDTA and 0.2 M NaOH. An aliquot of cells was gently layered into the lysis solution and incubated for 18 h at room temperature to insure complete cell lysis. The gradients were then centrifuged in a Beckman SW 50.1 swinging bucket rotor at 40,000 rev./min for 2 h. The gradients were fractionated by collecting 10 drops per fraction onto Whatman 3 mm filter paper strips. The strips were washed 3 times in 10% trichloroacetic acid, then washed in ethanol, dried and counted using a toluene-omnifluor mixture in a Packard liquid scintillation counter.
Crystalline
aNiS
(1 pg/ml)
CrystalMine
aNiS
(20 @g/ml)
i
B
10
20
30
40
50
60
70
80
90 Percent
B
10
20
30
40
&O
60
70
80
90
of Gradient
Fig. 1. Effect of crystalline aNiS on DNA strand breaks. Cultured CHO cells were maintained and labelled with [3H]thymidine as described in Materials and Methods. Following this procedure, the cells were treated with crystalline aNiS particles (2.2 pm mean particle diameter) for 24 h at the concentrations shown in the figure. The cells were then isolated, lysed and their DNA analyzed on alkaline sucrose gradients as described in the methods section. Gradients were standardized with respect to total cpm applied and fractions collected to facilitate comparison, A 16 S DNA marker migrated to fraction 45 in this gradient system.
38
P 5
5 P 8
9-
I 5
8 7-
E
NiCI,
I ,
,..\._ ‘.k\
‘i * -
I
‘
,-
(1 pg/mll
‘
4
I
*
L.-_ 10
,’ ’ .
5-
4
.
31 .
6
(lOpg/ml)
P 9
’
L
$
NiCI,
. -i
. I._.
24
.
#
..’
..I.
,
‘ . . . . .. .
l-
~ ,_~ cm_ ,~~ ~_~~ ~~~.~_ ~__ 20
30
40
50
60
70
80
90
Percent
8
10
20
30
40
50
60
70
80
90
of Gradient
Fig. 2. Effect of NiCl, upon DNA strand breaks. All procedures were conducted as described in Materials and Methods and the legend to Fig. 1. Cultured CHO cells were treated with NiCl, at the specified concentrations for 2 h. RESULTS
The alkaline sucrose gradient technique has been utilized to determine the presence of single strand breaks and alkaline sensitive sites caused by a number of chemical and physical agents including X-rays, ultraviolet light and carcinogenic chemicals. We have monitored the effects of NiCl? and crystalline aNi on the induction of DNA strand breaks in CHO cells. We have been able to detect single strand breaks in cells treated with either toxic or non-toxic levels of crystalline aNiS. A control gradient (Fig. 1) shows that DNA not exposed to nickel sediments to the bottom of the centrifuge tube. Exposure to crystalline cvNiScauses numerous single strand breaks, as evidenced by the peak of slower sedimenting DNA as seen in the other gradients shown in Fig. 1. Exposure to aNiS at 1 pg/ml for a 24-h time interval does not interfere with the division of CHO cells and additionally under these conditions, the level of cell plating efficiency was essentially similar to that of untreated cultures [ 41. However, this concentration caused substantial DNA strand breaks (Fig. 1) suggesting that crystalline cwNiSselectively damages DNA. A concentrationdependent effect on DNA strand breaks was obtained with crystalline aNiS treatment as indicated by the positional shift in gradient sedimentation of the radiolabelled DNA fragments isolated from cells treated with either 1 pg/ml, or 5 pg/ml or 20 pg/ml crystalline aNiS. Consistent with the results obtained using crystalline aNiS, treatment of cells with NiClz at 1 pg/ml or 10 @g/ml also resulted in single strand DNA breaks after only a 2-h exposure (Fig. 2). DISCUSSION
DNA strand breaks were caused by both soluble NiCl? and insoluble
39
crystalline cuNi§. Both compounds caused strand breaks under conditions of exposure which do not impair the proliferative and survival capacity of these cells. It is possible that low concentrations of toxic metals cause strand breaks that are readily repairable by the cell, and that higher levels either saturate the cells ability to repair the damage or inactivate the necessary enzymes for DNA repair. The mechanism by which strand breaks occur also remains unanswered. The nickel ion may interact with the DNA-phosphate backbone to cause strand breakages, it may distort the helix in such a way that endonucleases cause strand scission, or base release might occur generating an apurinic site. The nickel ion could possibly interact with the DNA in such a way that an alkaline sensitive site is generated. This site would be alkaline sensitive and strand excision occurs under the alkaline centrifugation conditions employed in our studies. DNA single and double strand breaks can be induced by a number of agents including ionizing radiation [15], ultraviolet [3] and chemical carcinogens [ 161. In addition it has been shown that apurinic sites can be generated by various compounds including alkylating agents [ 161 and the antineoplastic agent bleomycin [ 171. Lesions induced by these agents can be repaired at low concentrations, however, higher levels saturate the cell’s ability to repair damage or cause cell death. The lesions that nickel induces in DNA probably contribute to the induction of cell transformation. These lesions may also be responsible for the observed chromosomal aberrations following nickel exposure. REFERENCES 1 Barton, R.T. (1977) Nickel carcinogenesis of the respiratory tract. J. Otolaryngol., 6, 412-422. 2 Ciccarelli, R.B., Hampton, J.H. and Jennette, K.W. (1981) Nickel carbonate induces NDA-protein crosslinks and DNA strand breaks in rat kidney. Cancer Letters, 12, 349-354. 3 Cleaver, J.E. (1974) Repair processes for photochemical damage in mammalian cells. In: Advances in Radiation Biology, Vol. 7, pp. l-75. Editors: T.J. Lett and H. Adler. Academic Press, New York. 4 Costa, M. (1980) Metal Carcinogenesis Testing: Principles and in Vitro Methods. Humana Press, Clifton, New Jersey. 5 Costa, M., Simmons-Hansen, J., Bedrossian, C.W.M., Bonura, J. and Caprioli, R.M. (1981) Phagocytosis, cellular distribution and carcinogenic activity of particulate nickel compounds in tissue culture. Cancer Res., 41, 2868-2876. 6 Costa, M., Nye, J. and Sunderman Jr., F.W. (1978) Morphological transformation of Syrian hamster fetal cells induced by nickel compounds. Ann. Clin. Lab. Sci., 8, 502. 7 Damjanov, I., Sunderman, Jr., F.W., Mitchell, J.M. and Allpass, P.R. (1978) Induction of testicular sarcomas in Fischer rats by intratesticular injection of nickel subsulfide. Cancer Res., 38, 268-276. 8 Douglass, G.R., Bell, R.D.L., Grant, C.D., Wytsma, J.W. and Bora, K.C. (1980) Effect of lead chromate on chromosome aberrations, sister-chromatid exchanges and DNA damage. Mutat. Res., 77, 157-163. 9 Evans, R.M., Davies, P.J.A. and Costa, M. (1981) Visualization of cellular uptake and dissolution of carcinogenic olNiS particles by Video Intensification Microscopy. Am. Sot. Cell Biol., in press.
40
10 Hui, G. and Sunderman, Jr., F.W. (1980) Effects of nickel compounds and incorporation of [“Hlthymidine into DNA in rat liver and kidney. Carcinogenesis, 1, 297-304. 11 Jacobson, K.B. and Turner, J.E. (1980) The interactions of cadmium and certain other metal ions with proteins and nucleic acids. Toxicology, 16, l-37. 12 Jasmin, G. and RiopeIIe, J.L. (1976) Renal carcinomas and erythrocytosis in rats following intrarenal injection of nickel subsulfide. Lab. Invest., 35, 71-78. 13 Kreyberg, L. (1978) Lung cancer in a nickel refinery. Br. J. Ind. Med., 35,109-116. 14 Nishimura, M. and Umeda, M. (1979) Induction of chromosomal aberrations in cultured mammalian cells by nickel compounds. Mutat. Res., 68, 337-349. 15 Ormerod, M.G. (1976) Radiation induced strand breaks in the DNA of mammalian cells. In: Biology of Radiation Carcinogenesis, pp. 67-92. Editors: J.M. Yuhas, R.W. Tennant and J.D. Regan. Raven Press, New York. 16 Roberts, J.J. (1978) The repair of DNA modified by cytotoxic mutagenic, and carcinogenic chemicals. In: Advances in Radiation Biology, Vol. 7, pp. 211-436. Editors: J.T. Lett and H. Adler. Academic Press, New York. 17 Ross, S.L. and Moses, R.E. (1978) Two actions of bleomycin on superhelical DNA. Biochemistry, 17, 581-586. 18 Sirover, M.A. and Loeb, L.A. (1976) Infidelity of DNA synthesis in vitro: screening for potential metal mutagens or carcinogens. Science, 145, 1434-1436. 19 Sissoeff, I., Grisvard, J. and GuiIle, E. (1976) Studies on metal ions-DNA interactions: specific behavior of reiterative DNA sequences. Prog. Biophys. Mol. Biol., 31, 165199. 20 Sunderman, Jr., F.W. and Maenza, R.M. (1976) Comparisons of carcinogenicities of nickel compounds in rats. Res. Commun. Chem. Pathol. Pharmacol., 14, 319-330. 21 Sunderman, Jr., F.W. (1978) Carcinogenic effects of metals. Fed. Proc., 37,40--46. 22 Tsapakor, M.J., Hampton, T.H. and Jennette, K.W. (1981) The carcinogen chromate induces DNA crosslinks in rat liver and kidney. J. Biol. Chem., 256, 3623-3626.