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INDUCTION OF DNA-PROTEIN CROSSLINK IN RAT LUNG AND BLOOD BY THE CARCINOGEN NICKEL Lei Yi-xione*, Zhang Qiao** and Zhuang Zhi-xiong** I ** * Department of Hygiene, Guangzhou Medical College, Guangzhou, China Research Unit of Genotoxicology, Sun Yat-sen University of Medical Sciences, Guangzhou, China I I I I I I I I Abstract Nickel(II) compounds are common environmental contaminants and human carcinogens. One of the lesions associated with nickel(II) exposure is formation of DNA-protein crosslinks (DPC), but the biological significance of DPC by nickel(II) in vivo is presently poorly understood. In order to investigate the relationship between lung cancer and DPC induced by nickel compounds, and in an attempt to develop biomarkers for nickel exposure, we have used a rapid, simple and sensitive 1251_ postlabelling assay to detect the formation of DPC in white blood cells (WBC) and lungs from male Sprague-Dawley rats exposed intraperitoneally to nickel chloride (NiC12). The results show that 20 hr after the rats were treated with NiC12 at concentrations ranging from 10 to 30 mg/kg body wt. i.p., DPCs were found in white blood cells (WBC) and lungs in a dose-dependent manner. The formation of DPC in WBC and lungs was also observed following multiple exposure of rats to NiC12 (10 mg/kg, i.p. 3 weeks); the results were similar to those after a single dose. We consider that the DPCs found in rat lungs after NiCI2 treatment are possibly related to the carcinogenicity of nickel compounds. In addition, the DPC in the lungs and WBC may be used as biomarkers to quantitatively represent exposure to NiCl2 and genotoxic lesions induced from such exposure. In our DPC-induction studies, WBC were shown to be more sensitive than the lungs in responding to nickel; there also was a significant correlation in DPC between the two tissues, indicating that measuring DPCs in WBC may be a good surrogate for investigating human exposure of target tissues to environmental carcinogens or mutagens. Introduction t! DNA-protein crosslinks (DPC) are thought to be important genotoxic lesions induced by environmental contaminants and carcinogens such as UV light (1), y-radiation (2), aklylating agents f (3), formaldehyde (4), benzo(a)pyrene (5), and some metal compounds such as nickel (6),  chromate (7) and cis- or trans-platinum(II) diamine-dichlorides (8). These lesions, unlike DNA strand breaks and other readily-repaired DNA lesions are relatively persistent in the cells (9),(10). ~ Because they are poor repaired, DNA-protein complexes may be present during DNA replication and possibly cause a loss of important genetic material such as the inactivation of tumor suppressor genes (10),(11),(12). N - O .' 00 Nickel compounds are common environmental contaminants and human carcinogens. A v number of epidemiological and experimental studies have shown that nickel compounds cause lung cancer W = in both humans and animals (13). Recently, many studies with these agents have shown that they N I

induce DNA-protein crosslinks, mostly in intact cells in vitro; in contrast, there are fewer reports in vivo (14),(15),(16),(17). The biological significance of DNA-protein crosslinks in vivo is poorly understood at present. In order to investigate the relationship between lung cancer and DNA- protein crosslinks induced by nickel compounds, and in an attempt to develop biomarkers of nickel exposure, we have used a new rapid, simple and sensitive 1251-postlabelling assay developed recently by Zhuang et al. modified from earlier report by Lin X eta l. (17) to detect the formation of DNA-protein crosslinks in vivo in white blood cells (WBC) and lungs from male Sprague-Dawley rates exposed intraperitoneally to nickel chloride (NiC12). Materials and Methods Chemicals Nickel chloride was purchased from Guangzhou Chemical Factory; sodium dodecyl sulfate (SDS) was purchased from SERVA; protein K was purchased from E. Merck; Tris was purchased from FARCO Chemical Supplies; urea was purchased from Promega Corporation; Na1251 was purchased from the Atomic Energy Institute, Chinese Academy of Science; Q-mercaptoethanol was purchased from FARCO Chemical Supplies; male Sprague-Dawley rats were obtained from the Center of Experimental Animal, Sun-Yat-Sen University of Medical Science. Animals Male Sprague-Dawley rats were randomly assigned to exposed and control groups using weight as a factor. Each group comprised 8 rats. In the acute exposure experiment, rats weighing 175-200g were given i.p. injections 0.5 ml of 0.9% NaCl solution containing NiCl2 at doses of 10, 20, 30 mg/kg body wt. Control rats were given i.p. injections of 0.5 ml of 0.9% NaCI solution. In the subacute exposure experiment, rats weighing 150-175g were given by i.p. injections of 0.9% NaCl solution containing NiC12 at 10 mg/kg (twice a week i.p.) for 3 weeks. Control rats were given by i.p. injections of 0.5 ml of 0.9% NaCI solution. DNA isolation After exposure, rats were sacrificed, the lungs were excised and 3-5 ml blood per rat was collected in a tube using heparin as an anticoagulant and the WBC were isolated by centrifugation. The lungs collected were homogenized. DNA was extracted from lung and WBC tissues as previously described (17) with some modification. Briefly, tissue pellets were lysed in 10 mM Tris, pH 8.0 containing 100 mM NaCI and 1% SDS. The homogenate was incubated with RNase (10 mg/ml) and proteinase K (300 µg/ml) following which DNA and DNA-protein complexes isolated by repeated extraction with phenol/chloroform and precipitation with ethanol. -2- I I I I I I I I I I I I I I I

I I I I I I I I I I I I 1251-Radioactive postlabelling 125I-postlabelling of DNA-protein complexas was as described (17). DNA (100 µg) was sus ended in 100 µl of 2% SDS, 30% urea and 0.5 M Tris-HC1 pH 7.6 and mixed with 10 µCi of NaP25I and 5 µl of chloramine T solution (6 mg/ml) and incubated at room temperature for 2 min. The iodine was reduced by the addition of 10 µl of 20% 0-mercaptoethanol, and the DNA-protein complexes (with tyrosine labeled by iodination) was isolated by repeated (3x) p25cipitation with ethanol. The pellet was finally dissolved in 10 mM Tris pH 8.0. The unincorporated I I in the supernatant was discarded. The DNA samples were assayed for radioactivity in a ry-counter and their UV absorbance was measured at 260/280 nm. Efficiency of 1251-labelling was expressed as cpm/µg DNA. Results DNA-protein crosslinks induced by NiCI2 in tissues The ability of nickel chloride to induce DNA-protein crosslinks in rat WBC and lungs was analyzed using 1Z5I-postlabelling assay. The results showed that 20 hr after rats had been injected intraperitoneally with NiC12 ranging from 10 to 30 mg/kg body wt., DNA-protein crosslinks were found in WBC and lungs in a dose-dependent manner (Table 1 and Fig. 1). Similarly, the formation of DNA- protein crosslinks in the two tissues were also observed in rats exposed repeatedly to NiC12 at 10 mg/kg for 3 weeks (Table 2). Table 1. Formation of DNA-protein Crosslinks in Wlrite Blood Cell (WBC) and Lungs of Rats Following a Single Exposure to Nickel Chloride NiC12 (mg/kg) No Rats ODa X ± SD (cpm/µg DNA) DPC % of Control WBC 0 8 1.82 1281 t 256 100 10 8 1.76 2876 t 594 236* 20 8 1.75 4474 t 1455 367* 30 8 1.75 3192 + 1410 262* Lung 0 8 1.90 4063 t 658 100 10 8 1.90 6459 f 1144 159* 20 8 1.91 7804 t 1089 192* 30 8 1.90 5861 f 803 144* aThe ratio of optical density of samples measured at 260/280 *P<0.01 Compared with control (Student's t-test) 0 ao ~ V 00 W ~ W 3 p i

Figure 1. Formation of DPC in Rats WBC and Lungs Following Single Exposure to Nickel Chloride DPC % of Oontrol 400 1 I I I I 0 m 0 6 10 Wn 25 30 36 NICIs (m0/k0) Table 2. Forwation of DNA-protein Crosslinks in Rats WBC and Lungs Following Multiple Exposure to NiC12 (10 mg/kg) NiCl2 (mg/kg) No Rats ODa XtSD (cpml}tg DNA) DPC%of Control WBC Control 8 1.81 1340 ± 181 100 NiCl 8 1.76 3310 ± 906 247** Lung Control 8 1.92 4347 ± 757 100 NiC12 8 1.89 6352 ± 1538 146* * P < 0.0.5 ** P<0.01 Compared with control (Student's t-test) A Comparison of DNA-protein Crosslinks Formed in WBC With Those in Lungs To determine DNA-protein crosslinks in WBC relative to target organs of nickel toxicity of DNA- protein crosslinks between WBC and lung are analyzed and correlated. The results showed that WBCs were more sensitive to nickel than lung tissue. In addition, there was a significant correlation between the amount of DNA-protein crosslinks in WBC and in lungs (r=0.97, P<0.05) (Fig. 2). -4- I I I I I I I I I I

I I I , I I I I I I I I I I I I I I Figure 2. The Correlation of DPC Between WBC and Lungs Following a Single Exposure to NiC12 OPC (Fold of ecnhci) In lung Zff ~ y0.6768•0.3380x 0•0.97, P-0.051 1.6 t ~ OPC (FC/d cf eontrol) In W6C Discussion s The formation of DNA-protein crosslinks may be an important mechanism of chemical-mediated genetoxicity. Structural proteins that normally do not bind to DNA can become covalently crosslinked with DNA under the influence of certain chemicals, such as nickel and chromate compounds. The formation of inappropriate covalent DNA-protein crosslinks can disrupt gene expression and chromatin structure and can also lead to deletion of DNA sequences during DNA replication, since these lesions cannot be readily repaired (10),(11),(12). Previous studies have shown that DNA-protein crosslinks may be related to genetoxicity and carcinogenicity of chemical carcinogens. For example, Lam et al. (18) observed the formation of DNA-protein crosslinks by acetaldehyde in target tissues of the rat nasal cavity at concentrations similar to those that induced nasal cancer. Sugiyama et al. (10) suggested that the results from CaCrO4 induced DNA-protein crosslinks in Chinese hamster ovary (CHO) cells implicated the crosslinks as an important lesion that may be responsible for the cytotoxic and carcinogenic properties of chromate. Many studies with nickel compounds have shown that it can directly and indirectly induce DNA- protein crosslinks in vitro. Nickel(II) was thought to form stable protein-nickel(II)-DNA complexes, and a strong interaction between ttickel(II) and amino terminal residues and the imidazole group of histidine residues has been demonstrated (14). On the other hand, increasing evidence suggests that mckel(II) may generate reactive oxygen species (ROS), which may indirectly mediate DNA damage, protein oxidation and DNA-protein crosslinks formation (15),(16). The reports on the DNA-protein crosslinks in vivo by nickel compounds are fewer. Ciccarelli et al. (18) had detected DNA-protein crosslinks in kidney nuclei from nickel carbonate-treated rats using alkaline elution technique. They considered that the results tnight be related to the nephrotoxicity and carcinogenicity of nickel compound. However, the DNA- N protein crosslinks by NiClz in vivo are poorly understood. In our studies, DNA-protein crosslinks were 0 00 found in WBC and lung of rats treated with NiC12 in a dose-dependent manner. Moreover, multiple ~ exposure of rats to NiC12 also produced DNA-protein crosslinks in the two tissues. Due to the fact that 00 W W -5- I

the formation of DNA-protein crosslinks may be related to carcinogenicity of chemical carcinogens and since nickel compounds are well established as human and animal carcinogens, we consider that the DNA-protein crosslinks found in rat lungs after NiC12 treatment is possibly related to the carcinogenicity of nickel compounds. In addition, the DNA-protein crosslinks in lung and WBC may be biomarkers to represent quantitative exposure to NiC12 and the development of genotoxic lesions resulting from such exposures. A number of investigators have suggested the use of lymphocytes as targets for developing biomarkers of chemical exposure (19),(20). These cells offer a number of advantages. First, they are easily obtainable from humans and can be isolated in relatively high purity. Second, many lymphocytes are long lived in the body and therefore have the potential to be sentinels to past exposure. Third, lymphocytes are nucleated cells thus allowing the DPC to be formed. Recently, some studies have demonstrated the preferential accumulation of chemicals in lymphocytes as compared to other blood cells (21). In the present studies, WBC was found to be more sensitive to nickel inducing DPC formation than lung tissues. In addition, there was a significant correlation between DNA-protein crosslinks in WBC and in lung, indicating that DNA-protein crosslinks in WBC may be a good surrogate for investigating the exposure of human population to environmental carcinogens or mutagens. N O tb ~ V t70 W W Qi -6- -1 I i I I I I I I I I I

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15. Klein, CB et al.; The rote of oxidative prosesses [sic] in metal carcinogenesis. Chem. Res. I I I Toxicol. 1991; 4:592-604 16. Kasprazak, KS; The rote of oxidative damage in metal carcinogenicity. Chem. Res. Toxicol. I 1991; 4:604-615 17. Lin, X etal.; Analysis of residual amino acid-DNA crosslinks induced in intact cells by nickel I and chromium compounds. Carcino eg nesis 1992; 13(10):1763-1768 18. Ciccarelli, RB et al.; Nickel carbonate induces DNA-protein crosslinks and DNA strand breaks in rat kidney. Cancer Lettersl8. 1981; 12:347-354 , 19. Perera, F; The Potential usefulness of biological markers in risk assessment. Environ. Health Perspect 1987; 76:141-145 I 20. Lucier, GW and Thompson, CL; Issues in lymphocytes be used as surrogate markers? Environ. Health Perspect. 1987; 76:187-191 I 21. Coogan, TP et al.; Distribution of chromium within cells of the blood. Toxicol. Aopl. Pharmacol. 1991; 108:157-166 I I I I I I , 0 , tp ~ ~ w w w , 0) -8- ' I