Chem. Res. Toxicol. 1992,5, 809-815
809
Nickel(I1)-Mediated Oxidative DNA Base Damage in Renal and Hepatic Chromatin of Pregnant Rats and Their Fetuses. Possible Relevance to Carcinogenesis Kazimierz S. Kasprzak,*jtBhalchandra A. Diwan,t Jerry M. Rice,? Manoj Misra,? Charles W. Riggs,s Ryszard Olinski,ll and Miral Dizdarogld Laboratory of Comparative Carcinogenesis, National Cancer Institute, FCRDC, Frederick, Maryland 21 702, Biological Carcinogenesis Development Program, Program Resources, Inc.1 DynCorp, NCI-FCRDC, Frederick, Maryland 21 702, Data Management Services, Inc., NCI-FCRDC, Frederick, Maryland 21 702, and Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 Received May 18,1992
DNA base damage was studied in renal and hepatic chromatin of nickel(I1)-injected pregnant female F344/NCr rats and their fetuses under conditions leading to initiation of sodium barbitalpromotable renal tumors, but not liver tumors, in the male offspring. Pregnant rats were given a total of 90 or 180 pmol of nickel(I1) acetate/kg body wt in a single ip dose on day 17 or in 2 or 4 ip doses between days 12 and 18 of gestation. Control rats received 180 pmol of sodium acetate/kg body wt. The animals were killed 24 or 48 h after the last injection. Chromatin was isolated from livers and kidneys from both adults and fetuses and analyzed by gas chromatography/mass spectrometry with selected ion monitoring. Eleven products derived from the purine and pyrimidine bases in DNA bases were identified and quantified. These were the following: 5-hydroxy-5-methylhydantok1, 5-hydroxyhydantoin,5-(hydroxymethyl)uracil, cytosine glycol, thymine glycol, 5,6-dihydroxycytosine, 4,6-diamino-5-formamidopyrimidine, 2,6-diamino4-hydroxy-5-formamidopyrimidine, 8-hydroxyadenine,2-hydroxyadenine,and 8-hydroxyguanine (8-OH-Gua). Nickel(I1) exposure increased the content of these products, especially those derived from purines, in both renal and hepatic chromatin of pregnant rats. The major difference between these two organs was the content of 8-OH-Gua, which increased greatly in the kidney but remained unchanged in the liver. In the corresponding fetal organs, the relative increases in 8-OH-Gua were comparable to the findings in adults. Fetal kidney DNA was relatively higher in pyrimidine-derived products (especially thymine glycol and 5-hydroxyhydantoin) and lower in purine-derived products (except for 8-OH-Gua) than fetal hepatic DNA. No consistent dose effect of nickel(I1) on the amounts of the DNA base products recovered from either organ was observed in either the dams or their fetuses. The products determined were typical hydroxyl radical-produced derivatives of DNA bases, suggesting a role for hydroxyl radical in the induction of their formation by nickel(I1). Some of these base products have been shown previously to be promutagenic. Therefore, the present results indicate possible involvement of oxidative DNA base damage in the mechanism of nickel(I1) carcinogenesis in the rat kidney. The prevalence of 8-OH-Gua in the kidney but not in the liver is consistent with the hypothesis that 8-OH-Gua is a tumor-initiating lesion in that organ. However, the complexity of the observed response to nickel(I1) does not exclude possible roles for other DNA base products elevated by nickel(I1) treatment, especiallythymine glycol and 5-hydroxyhydantoin,in nickel(I1)-inducedcarcinogenesis in the kidney.
Introduction Solublenickel(I1)in the form of its acetate salt (NiAcet),l given to male F344/NCr rats in a single ip injection, has been found by us ( 1 ) to initiate renal cortical epithelial
* Address correspondence to this author at NCI-FCRDC, Building 538, Room 205, Frederick, MD 21702-1201. Tel: 301-846-5738; F A X 301-846-5946. National Cancer Institute. 3 Program Resources, Inc./DynCorp. 8 Data Management Services, Inc. 11 National Institute of Standards and Technology. Abbreviations: NiAcet, nickel(I1) acetate tetrahydrate; sodium barbital, 5,5-diethylbarbituricacid, sodium salt; 5-OH-5-Me-Hyd, 5-hydroxy-5methylhydantoin;5-OH-Hyd,5-hydroxyhydantoiq5-OHMe-Ura, 5-(hydroxymethyl)uracil;Cyt glycol, cytosine glycol; Thy glycol, thymine glycol; 5,6-diOH-Cyt, 5,6-dihydroxycytosine; FapyAde, 4,6-diamino-5formamidopyrimidine;FapyGua, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; &OH-Ade, a-hydroxyadenine;a-OH-Ade,2-hydroxyadenine; 8-OH-Gua,8-hydroxyguanine;8-OH-dG, 8-hydroxy-2'-deoxyguanosine. f
tumors that appear after subsequent chronic oral dosing with the multitissue tumor promoter, sodium 5,5-diethylbarbiturate (sodium barbid). We have hypothesized that the initiation of tumors could be associated with increased amounts of 8-hydroxy-2'-deoxyguanosine (8OH-dG) in DNA extracted from-kidneys of rats 16-48 h after injection (I). 8-OH-dG is potentially mutagenic (25 ) . More recently, we have also found that a single ip dose of NiAcet (90 pmol/kg body wt) given to pregnant F344/ NCr rata on day 17 of gestation, or two doses (45 pmol/kg body w t each) administered on days 16 and 18of gestation, similarly initiate a high incidence of sodium barbitalpromotable renal tumors in male offspring (6). Systemically administered nickel(I1) is known to accumulate in kidneys of rata (7, 8 ) . Given to pregnant rats or mice, nickel(I1) easily crosses the placenta and accumulates in
This article not subject to U S . Copyright. Published 1992 by the American Chemical Society
810 Chem. Res. Toxicol., Vol. 5, No. 6,1992
all fetal tissues in small amounts but in the same proportions as in the maternal tissues, i.e., preferentially in the kidney (9-11). Organ-specific accumulation of nickel(I1) in transplacentally exposed fetuses thus correlates with its carcinogenic effect in adult rats. Positive results of nickel(I1) carcinogenicity testing in the transplacental model prompted us to verify in this model the hypothesis of tumor initiation by nickel(I1) through mediation of oxidative DNA base damage. In an in vitro system (12),nickel(I1) has been shown to increase the amounts of oxidation products derived not only from guanine but also from other DNA bases in mammalian chromatin. The resulting purine and pyrimidine derivatives may be promutagenic (2, 3, 13-17). Therefore, a wide spectrum of known oxidation products of DNA bases was quantified in the present in vivo study. Pregnant rats were given NiAcet in 1,2, or 4 ip injections, at doses equal to or higher than those used in our carcinogenesis bioassay (6). They were sacrificed 1 or 2 days after the last injection. The following DNA base derivatives were measured in renal chromatin of the fetuses: 5-hydroxy5-methylhydantoin (5-OH-5-Me-Hyd); 5-hydroxyhydantoin (5-OH-Hyd);5-(hydroxymethyl)uracil (5-OHMe-Ura); cytosine glycol (Cyt glycol); thymine glycol (Thy glycol); 5,6-dihydroxycytosine(5,6-diOH-Cyt);4,6-diamino-5-formamidopyrimidine (FapyAde);2,6-diamino-4-hydroxy-5formamidopyrimidine (FapyGua); 8-hydroxyadenine (8OH-Ade); 2-hydroxyadenine (2-OH-Ade);and 8-hydroxyguanine (8-OH-Gua). For comparison, the same DNA base derivatives were also measured in fetal livers and in maternal kidneys and liver, i.e., in tissues in which nickel(11)does not initiate sodium barbital-promotable tumors (1,6 ) .
Materials and Methods Materials. NiAcet, sodium acetate trihydrate, sodium and potassium phosphates, hydrochloric acid, and other inorganic chemicals were purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ). Formic acid (88%) was obtained from Malinckrodt (Paris, KY). Acetonitrile and bis(trimethylsily1)trifluoroacetamide containing 1% trimethylchlorosilane were obtained from Pierce Chemical Co. (Rockford, IL). Tris, EDTA, Chelex-100 resin (200-400 mesh), Triton X-100, sucrose, phenylmethanesulfonyl fluoride, dithiothreitol, 5-OHMe-Ura,FapyAde, 2-OH-Ade (isoguanine),isobarbituric acid (8hydroxyuracil), 6-azathymine, and 8-azaadenine were purchased from Sigma Chemical Co. (St. Louis, MO). 8-OH-Gua was obtained from Schweitzer and Hall, Inc. (Piscataway, NJ). 5,6-DiOH-Ura (isodialuric acid) was purchased from American Tokyo Kasei, Inc. (Tokyo, Japan). 5-OH-5-Me-Hyd was a gift from Dr. W. F. Blakely (Armed Forces Radiobiology Research Institute, Bethesda, MD). Thy glycol, FapyGua, and 8-OH-Ade were synthesized as described elsewhere (1419). The gas chromatographic retention time and the mass spectrum of 5-OH-Hyd were determined using a sample of cytosine which had been y-irradiated in NzO/Oz-saturated aqueous solution. Dialysis tubing with a molecular weight cutoff of 3500 was purchased from Spectrum Medical Industries, Inc. (Los Angeles, CA); it was heated in several changes of boiling water before use. Animals a n d Treatments. Fischer F344/NCr rats of both sexes, 12-16 weeks old, obtained from the Animal Production Area, NCI-FCRDC, Frederick, MD, were housed in polycarbonate cages on a sawdust bedding (Sanichips, P. G., Murphy Forest Products Co., Mountsville, NJ) in an environment maintained a t 22 f 2 OC, 12-h light/dark cycle. They had free access to food (NIH-31 Open Formula 6 % Modified, Zeigler Brothers, Gardners, PA) and drinking water. The rats were mated, and the day the vaginal plug appeared was considered day 1 of gestation. Four groups of 4-5 pregnant
Kasprzak et al. rats were then treated as follows: the first group received a single ip injection of 90pmol of NiAcet/kg body wt on day 17 of gestation; the second group received the same amount of NiAcet but divided into equal doses injected on days 16 and 18 of gestation; rats of the third group received a total of 180 pmol of NiAcet/kg body wt divided into four equal doses administered on days 12,14,16, and 18 of gestation; the fourth, control, group of rata received a single ip injection of 180 pmol of sodium acetate/kg body wt on day 18 of gestation. Thus, the treatment regimen of the first two groups and the control group was the same as in the carcinogenesis bioassay completed before (6). The total dose and dosage regimen for the third group of rats was one that had proved lethally toxic to the offspring in our transplacental carcinogenesis study (6), but was included in the present short-term experiment for comparative purposes. All the rats were killed by exsanguination (cardiac puncture) under carbon dioxide on day 19 of gestation. Their kidneys and livers were quickly removed. The whole kidneys and approximately 2-g samples of livers (median lobe) were then frozen and stored in liquid nitrogen. Kidneys and livers of fetuses from a single dam were pooled and frozen as above. Analysis. Isolation of nuclei was accomplished according to Lilja et al. (20)with some modifications introduced by Tsapakos et al. (21). A frozen aggregate of pooled fetal kidneys or livers from one dam, or a frozen maternal organ, was crushed under liquid nitrogen into several pieces in a porcelain mortar. About 1 g of the crushed material was transferred to a glass PotterElvehjem homogenizer and disrupted with a few strokes of first a loose and then a tighter pestle in 9 mL of Tris buffer (pH 7.4). The buffer contained 0.25 M sucrose, 50 mM Tris-HC1, 3 mM CaC12, 0.1 mM phenylmethanesulfonyl fluoride, and 0.1 mM dithiothreitol. The homogenate was then filtered through a 64mesh nylon sieve and centrifuged a t 1OOOg for 10 min. The resulting pellet was resuspended in the same buffer; nuclei were recovered from it by centrifugation a t 700g through 0.35 M sucrose. The nuclei were finally washed twice in the above buffer with and without 1% Triton X-100 and recovered by centrifugation at l00Og for 10 min. Isolation of chromatin was performed according to a slightly modified procedure of Mee and Adelstein (22)as previously described (23). Chromatin was obtained as a white gel in 1mM Tris-HC1buffer (pH 7.4). It was next dialyzed extensively against 1mM phosphate buffer (pH 7.4) containing 0.2 mM EDTA followed by 1 mM phosphate buffer (pH 7.4) which had been treated with Chelex resin. After dialysis, the chromatin was analyzed for DNA, RNA, and protein contents and characterized as described by Nackerdien et al. (12). The protein/DNA ratio in our chromatin preparations was 1.7-1.8; the RNA content was less than 5% of the amount of DNA. Chromatin samples (3/rat) containing 100 pg of DNA were transferred into 1-mL conical polypropylene tubes and supplemented with 0.5 nmol of 6-azathymineand 2 nmol of 8-azaadenine (internal standards). These samples were then frozen in liquid nitrogen and lyophilized. The lyophilized samples of chromatin were hydrolyzed with 0.5 mL of 60% formic acid in evacuated and sealed glass tubes for 30 min at 140 "C. The hydrolysates were lyophilized and then trimethylsilylated with 0.12 mL of bis(trimethylsily1)trifluoroacetamide/acetonitrile (2/1 v/v) mixture, at 130 OC for 30 min. Analysis of the derivatized samples was performed by gas chromatography/mass spectrometry with selected-ion monitoring as previously described (19, 23). For each analysis, approximately 0.2 pg of hydrolyzed and derivatized DNA was injected onto the gas chromatograph column. Calculation of the results with application of molar response factors needed to account for slight losses of some modified DNA bases over the analytical procedure was completed as described previously (24). Statistical Evaluation of the Results. Preliminary examination of the initial descriptive statistics indicated that the pooled within-treatment group variances for the 11DNA base products were statistically different. Each of the DNA base products was therefore evaluated separately. In addition, the data were also analyzed separately by organ. For each organ/base product
Chem. Res. Toxicol., Vol. 5, No. 6, 1992 811
Ni(Il)-Mediated DNA Base Damage
Table I. Effect of ip NiAcet Injections into Pregnant F344/NCr Rats on Content of DNA Base Derivatives in Renal Chromatin of the Fetuses (nmol/mg of DNA f SD). treatmentb
DNA base product 5-OH-5-Me-Hyd 5-OH-Hyd 5-OHMe-Ura cyt glycol Thy glycol 5,g-diOH-Cyt FapyAde 8-OH-Ade 2-OH- Ade FapyGua 8-OH-Gua
1
2
0.040 f 0.010 0.093 f 0.031 0.015 f 0.011 0.052 f O.O1ld 0.030 f 0.010 0.014 f 0.006 0.083 f 0.018d 0.050 f 0.027 0.066 f 0.023 0.063 f 0.015 0.111 f 0.053
0.073 f 0.036 0.217 f 0.014c-d 0.016 f 0.004 0.064 f 0.005d 0.060 f O.O1lc~d 0.022 f 0.013 0.109 f 0.016 0.060 f 0.013 0.084 f 0.012d 0.129 f 0.023cvd 0.234 f 0.043c
3 0.075 f 0.043 0.191 f 0.03T 0.016 f 0.002 0.078 f 0.003c*d 0.049 f 0.01W 0.025 f 0.007 0.109 f 0.020 0.061 f 0.008d 0.072 f 0.023d 0.118 f 0.043' 0.266 f 0.057'~~
4 0.042 f 0.017d 0.187 f 0.05oC 0.014 0.007 0.056 0.010 0.050 f 0.003'~~ 0.036 f 0.045 0.107 f 0.026 0.034 f 0.015d 0.059 f 0.025d 0.077 f 0.046d 0.106 f 0.053d
*
a Kidneys of fetuses from a single dam were pooled for analysis. The results are means of determinations for fetuses of 4-5 dams/group. 1nmol of a DNA base derivative/mg of DNA = 320 derivative residues/lOGDNA base residues. 1,fetuses of control rata, given a single ip injection of 180 pmol of sodium acetate/kg body wt on day 17 of gestation and killed on day 19 of gestation. 2, fetuses of rata given a single ip injection of 90 pmol of NiAcet/kg body wt on day 17 of gestation and killed on day 19 of gestation. 3, fetuses of rata given single ip injections of 45 pmol of NiAcet/kg body wt each on days 16 and 18of gestation and killed on day 19 of gestation. 4, fetuses of rata given single ip injections of 45 pmol of NiAcet/kg body wt each on days 12,14,16, and 18 of gestation and killed on day 19 of gestation. p < 0.05 or less vs treatment 1 by Duncan's test. p < 0.05 or less vs corresponding maternal group, by Duncan's test.
Table 11. Effect of ip NiAcet Injection into Pregnant F344/NCr Rats on Content of DNA Base Derivatives in Hepatic Chromatin of the Fetuses (nmol/ma of DNA f SDP treatmentb DNA base product 1 2 3 4 0.040 f 0.013d 0.067 f 0.021' 0.061 f 0.017'~~ 0.061 f 0.012' 5-OH-5-Me-Hyd 0.251 f 0.043"~~ 5-OH-Hyd 0.138 f 0.036d 0.169 f 0.021d 0.172 f 0.05od 5-OHMe-Ura 0.014 f 0.003 0.022 f 0.007c 0.017 f 0.003 0.014 f 0.003d 0.015 f 0.004d 0.040 f 0.020' 0.020 f 0.005 cyt glycol 0.035 f 0.01oC 0.028 f 0.006d 0.030 f 0.019 Thy glycol 0.019 f 0.008d 0.024 f 0.008d 0.011 f 0.004d 0.019 f 0.005c 0.008 f O.OOld 0.014 f 0.003d 5,6-diOH-Cyt 0.031 f 0.008 0.094 f 0.029c 0.061 f 0.017' 0.093 f 0.044c FapyAde 0.032 f 0.006d 0.059 f O.OIOc 8-OH-Ade 0.034 f 0.006 0.057 f 0.018'~~ 2-OH- Ade 0.039 f 0.015 0.084 f 0.027'~~ 0.076 f 0.025c9d 0.079 f 0.024 0.040 f 0.014 0.094 f 0.046c 0.069 f 0.01W 0.089 f 0.036' FapyGua 8-OH-Gua 0.079 0.017 0.130 f 0.082 0.060 f 0.013 0.130 f 0.066d 0 Livers of fetuses from a single dam were pooled for analysis. The results are means of determinations for fetuses of 4-5 dams/group. 1 nmol of a DNA base derivative/mg of DNA = 320 derivative residues/106DNA base residues. bd Same as in Table I. ~
~~
~
*
combination, a one-way analysis of variance (ANOVA) was performed to test for the overall significance of the differences among the 8 combined treatment groups representing a combination of 4 NiAcet treatments for each of the maturity states, Le., fetus and adult. When statistically significant differences among the 8combinedtreatmentgroups were indicated,Duncan's multiple range test (25)was used to determinehow the treatments differed from each other. The data were analyzed untransformed, using PROC GLM of the Statistical Analysis System (SAS), Release 6.06. To simplify interpretation and presentation of the results, statistical tests were not made for differences between amounts of individual DNA base products in kidney and liver within a singlematurity state/NiAcettreatmentcombination. Differences in response to NiAcet noticed in that combination were obvious enough to be discussed without statistical support.
Results
The amounts of DNA base derivatives found in renal and hepatic chromatin of rats are shown in Tables I-IV. The same tables also present the results of statistical comparisons between the NiAcet treatments and their respective control and between fetal and maternal kidney (Table I) and fetal and maternal liver (Table 11) of each of the treatments. In addition, Table I11 provides the overall ANOVA significance levels @) of the differences among the 8 combined treatment groups for fetal and maternal kidney (shown in Tables I and 111). Table IV provides corresponding levels for fetal and maternal liver
(Tables I1and IV). A brief summary of NiAcet effects on the ranked increase of the four most prevalent DNA base derivatives in renal and hepatic chromatin of dams and fetuses is given in Table V. More details are presented below. Fetuses. (A) Kidney. Measurable amounts of DNA base derivatives were found in renal chromatin of the control fetuses (Table I, column 1). Administration of NiAcet markedly increased the amounts of some of these products. No consistent NiAcet dosing effect was observed, however, on the quantities of these derivatives that were recovered. Amounts of the following purine and pyrimidine derivatives were found to be significantly increased by factors of 2.4-1.5 vs the corresponding controls: 8-OH-Gua> 5-OH-Hyd > FapyGua > Thy glycol
> cyt glycol.
(B) Liver. The hepatic chromatin isolated from control fetuses also contained measurable amounts of DNA base derivatives (Table 11, column 1). NiAcet treatment increased the amounts of most of them. The same NiAcet dose administered in a single injection was apparently more effective than in two daily injections, 2 days apart. A total NiAcet dose twice as great, applied in four daily injections, was not more damaging to hepatic DNA than the lower dose (two injections). The following DNA base products were found increased by NiAcet by a factor of 3-1.6 vs the corresponding fetal controls: FapyAde > Cyt glycol > FapyGua > 2-OH-Ade > 8-OH-Ade L 5-OH-Hyd
812 Chem. Res. Toxicol., Vol. 5, No. 6, 1992
Kasprzak et al.
Table 111. Effect of ip NiAcet Injection on Content of DNA Base Derivatives in Renal Chromatin of Pregnant F344/NCr Rats Inmol/mn of DNA f SDP treatment6 DNA base product 5-OH-5-Me-Hyd 5-OH-Hyd 5-OHMe-Ura cyt glycol Thy glycol 5,6-diOH-Cyt FapyAde 8-OH-Ade 2-OH-Ade FapyGua 8-OH-Gua
overall ANOVA D~ 0.054 0.0001 0.75 0.0001 o.oO01 0.18 0.0011 0.0001 0.0001 0.0001 0.0001 The results are means of determinations for a single kidney of 4-5 rata/group. 1nmol of a DNA base derivative/mg of DNA = 320 derivative residues/l06 DNA base residues. 1,control rata, given a single ip injection of 180 pmol of sodium acetate/kg body wt on day 17 of gestation and killed on day 19 of gestation. 2, rata given a single ip injection of 90 pmol of NiAcet/kg body wt on day 17 of gestation and killed on day 19 of gestation. 3, rata given single ip injections of 45 pmol of NiAcet/kg body wt each on days 16 and 18 of gestation and killed on day 19 of gestation. 4, rata given single ip injections of 45 pmol of NiAcet/kg body wt each on days 12, 14,16, and 18 of gestation and killed on day 19 of gestation. c p C 0.05 or less vs treatment 1by Duncan's test. Overall ANOVA significance of the differences among 8combined treatment groups (fetus/mother) presented in both Table I and this table. 1
0.052 f 0.017 0.064 f 0.008 0.015 f 0.005 0.017 f 0.006 0.020 f 0.003 0.015 f 0.004 0.051 f 0.004 0.063 f 0.022 0.077 f 0.014 0.037 f 0.004 0.090 f 0.016
2 0.053 f 0.012 0.125 f 0.01lC 0.013 f 0.004 0.033 f 0.007" 0.020 f 0.005 0.010 f 0.006 0.079 f 0.017 0.057 f 0.014 0.124 f 0.022c 0.068 f 0.010 0.159 f 0.040
3 0.069 f 0.020 0.208 f 0.046' 0.013 f 0.007 0.047 f 0.007" 0.035 f 0.012 0.017 f 0.005 0.105 f 0.032" 0.115 f 0.023c 0.169 f 0.032' 0.134 f 0.034" 0.411 f 0.087c
4 0.069 f 0.018 0.201 f 0.041' 0.014 f 0.005 0.048 f 0.012" 0.026 0.012 0.015 f 0.009 0.119 f 0.016' 0.086 f 0.026 0.279 f 0.027" 0.144 f 0.036' 0.323 f 0.1W
Table IV. Effect of i p NiAcet Injection on Content of DNA Base Derivatives in Hepatic Chromatin of Pregnant F344/NCr Rats (nmol/mg of DNA f SD). treatmentb DNA base product
1
2
5-OH-5-Me-Hyd 5-OH-Hyd 5-OHMe-Ura cyt glycol Thy glycol 5,6-diOH-Cyt FapyAde 8-OH-Ade 2-OH-Ade FapyGua 8-OH-Gua
0.080 f 0.014 0.192 f 0.029 0.019 f 0.004 0.039 f 0.018 0.056 f 0.007 0.023 f 0.006 0.027 f 0.007 0.018 f 0.004 0.020 f 0.004 0.035 f 0.010 0.083 f 0.030
0.073 f 0.014 0.379 f 0.035" 0.026 f 0.003" 0.033 f 0.009 0.038 f 0.008 0.020 f 0.003 0.089 f 0.024' 0.051 f 0.024c 0.047 f 0.013" 0.127 f 0.026c 0.130 f 0.077
3 0.100f 0.014" 0.307 f 0.035' 0.021 0.003 0.035 f 0.015 0.068 f 0.017 0.023 f 0.009 0.072 f 0.013" 0.047 f 0.008" 0.036 f 0.007" 0.095 f 0.016c 0.105 f 0.043
*
4 0.079 f 0.010 0.348 f 0.025" 0.024 f 0.005 0.040 f 0.003 0.049 f 0.015 0.025 0.006 0.063 f 0.017" 0.035 f 0.009 0.059 f 0.007" 0.064 f 0.022 0.073 0.012
*
overall ANOVA p d 0.0001 0.0001 0.0003 0.0001 0.0001 0.0001 0.0001 o.oO01 0.0001 0.0001 0.012
The results are means of determinations for 4-5 rats/group. 1 nmol of a DNA base derivative/mg of DNA = 320 derivative residues/l@ DNA base residues. Same as in Table 111. Overall ANOVA significance of the differences among 8 combined treatment groups (fetus/ mother) presented in both Table I1 and this table. 0
Table V. Comparison of the Effects of NiAcet Treatment. on the Relative Increase in Content of the Four Most Prevalent Purine and Pyrimidine Derivatives in Renal and Hepatic Chromatin of Pregnant Rats and Their Fetuses* kidney liver' 8-OH-Gua > 5-OH-Hyd > FapyGua > Thy glycol FapyAde > 2-OH-Ade > FapyGua > 5-OH-5-Me-Hyd (240) (205) (190) (160) (200) (195) (170) (150) 8-OH-Gua > FapyGua > 5-OH-Hyd > Cyt glycol pregnant rata FapyGua = FapyAde > 8-OH-Ade > 2-OH-Ade (460) (360) (325) (280) (270) (270) (260) (180) 4 Two ip injections; compare columns 3 in Tables I-IV. Relative increases, in percentages of corresponding control values, are given in parentheses. The amounts of 8-OH-Gua and Thy glycol were not significantly increased. fetuses
> 5-OH-5-Me-Hyd 1 5,g-diOH-Cyt > 5-OHMe-Ura. It is noteworthy that, unlike in the kidney, the amounts of hepatic 8-OH-Gua and Thy glycol were not significantly affected in the liver by NiAcet administered at any dosing regimen (Table 11). Pregnant Rats. (A) Kidney. As shown in Table 111, measurable background amounts of derivatives of DNA bases were found in renal chromatin of pregnant control rats as was the case with fetal kidneys. Treatment of rats with NiAcet markedly increased amounts of most of the oxidation products above control levels. In these adult rats, the effect of NiAcet on the production of purine and pyrimidine derivativesapparently depended on the NiAcet dosing regimen. After a single NiAcet injection, amounts of only three products were found to be significantly increased by a factor of 2-1.6 vs corresponding control values (Table 111, column 2): 5-OH-Hyd > Cyt glycol > 2-OH-Ade. The same total NiAcet dose as above, but administered as two equally divided portions 2 days apart,
produced a much greater effect consisting of 4.6- to 1.8fold increase of the amounts of the following DNA base products (Table 111,column 3): 8-OH-Gua > FapyGua > 5-OH-Hyd > Cyt glycol > 2-OH-Ade > FapyAde > &OHAde. Interestingly, doubling both the total NiAcet dose and number of injections failed to further augment the effect, except in the case of 2-OH-Ade (Table 111,column 4). Also worth noticing is the lack of NiAcet effect on Thy glycol level in kidneys of the dams. (B) Liver. Like that in the other tissues, chromatin isolated from livers of pregnant control rats contained quantifiable amounts of purine and pyrimidine derivatives (Table IV, column 1). These amounts were generally higher for pyrimidine derivatives (especially 5-OH-Hyd) and lower for purine derivatives (especially the adeninederived products) than in renal chromatin. Administration of NiAcet increased the amounts of oxidation products, mainly the purine derivatives (Table IV). In contrast to the effects in kidneys, the same dose of NiAcet admin-
Ni(ZZ)-Mediated DNA Base Damage
istered in a single injection was apparently as effective in liver as when given in two injections (Table IV, columns 2 and 3). Doubling of the dose did not increase the damage. Treatment with NiAcet significantly increased amounts of the following products by factors of 3.6-1.4 vs the controls (Table IV, column 2): FapyGua > FapyAde > 8-OH-Ade > 2-OH-Ade > 5-OH-Hyd > 5-OHMe-Ura. The amounts of the remaining derivatives, including 8-OHGua and Thy glycol, were unchanged. Mother-Fetus Comparison. The relative increase of the amounts of oxidation products in response to nickel(11)in maternal organs was generally greater than in fetal organs. In control rats, fetal renal chromatin differed from maternal chromatin with respect to the amounts of Cyt glycol and FapyAde, which were significantly higher in the fetal DNA (Tables I and 111,column 1). In the NiAcettreated groups, differences included more products and depended on the treatment regimen. For example, renal chromatin from fetuses of rats given a single NiAcet dose contained more pyrimidine derivatives, including 5-OHHyd, Cyt glycol, and Thy glycol, than maternal chromatin. Among the purine derivatives, fetal chromatin contained more FapyGua and less 2-OH-Ade than maternal renal chromatin. No significant differences were found among the other products (Tables I and 111,column 2). In fetuses of rats given multiple NiAcet doses, renal chromatin still contained larger amounts of Thy glycol, but fewer purinederived products, 8-OH-Ade, 2-OH-Ade, FapyGua, and 8-OH-Gua, than the corresponding maternal chromatin (Tables I and 111, columns 3 and 4). In contrast to renal chromatin, hepatic chromatin of control fetuses contained significantly smaller amounts of pyrimidine derivatives (&OH-B-Me-Hyd, 5-OH-Hyd, Cyt glycol, Thy glycol, and 5,6-diOH-Cyt) and larger amounta of a purine derivative 8-OH-Adethan the hepatic chromatin of their mothers (Tables I1 and IV, column 1). A similar trend persisted in the NiAcet-treated groups, with no significant dependence on NiAcet dosing schedule.
Discussion The purine and pyrimidine derivatives identified and quantified in the present study are typical products of reactions of the hydroxyl radical with DNA (reviewed in refs 26-28). Occurrence of these products in renal chromatin of nickel(I1)-treated rats suggests possible involvement of hydroxyl radical in the mechanism(s) of nickel(I1)-mediated DNA damage. Formation of all the products, except for 8-OH-Gua,has been shown in organs of animals treated with nickel(11) for the first time to our knowledge. The results of this work confirm our previous observations indicating that chemical changes in nuclear DNA in kidneys of animals exposed to nickel(I1) are consistent with oxygen-derived radical activity (1, 29). Those observations depended on analysis of a single oxidized nucleoside, 8-OH-dG; they revealed an increased amount of 8-OH-dG in renal DNA of male Fischer rats (1) or BALB/c mice (29) 16-48 h after a single ip injection of nickel(I1). The present study shows a significant nickel(11)-related increase in contents of not only 8-OH-Gua but also other purine and pyrimidine derivatives in renal and hepatic chromatin both in mature rats and in fetuses within 2 days after treatment. Hence, for the first time, the present work reveals the potential of nickel(I1)to cause oxidative DNA damage to organs of rat fetuses via transplacental exposure.
Chem. Res. Toxicol., Vol. 5, No. 6, 1992 813
Nickel(I1) is knownto enhance oxidativedamage to DNA bases when incubated with chromatin in vitro (12). To correctly assess the results of the present study, any contribution to our results of a similar in vitro effect (i.e., any post mortem DNA base oxidation that may occur during the analytical procedure) has to be ruled out. As established before (7,8,30,31),nickel(I1) concentration in the blood and kidney reaches its maximum level shortly after the injection (15-30 min) and then diminishes rapidly. Nearly 50% of the dose is excreted in 6 h, and more than 90%is excretedin 48 h (31). Renalnickel(II)concentration drops from about 6.5 9% of the dose per gram of wet tissue at 15min to 1.6 % at 8 h after iv injection (7). The contents of nickel(I1) in other organs, including liver, are originally lower than in kidney and decrease more rapidly than in kidney (32). The in vitro effects of nickel(I1) on DNA base oxidation depend on nickel(I1) concentration and exposure time. For example, a 100 pM nickel(I1) concentration and 24-h exposure at 37 "C were needed to produce measurable amounts of some derivatives in chromatin incubated with nickel(I1) in vitro (12). Considering the total dose of nickel(I1) injected (90 pmol/kg body wt) and its rapid excretion, as well as the dilution of tissue homogenates and the low temperature maintained during the extraction of chromatin, the increase in DNA base damage observed in the present study appears solely to reflect in vivo damage by nickel(II), not a postmortem artifact. The damage that occurs in renal and hepatic chromatin in fetuses as a consequence of transplacental exposure to NiAcet is not surprising, since nickel(I1) is known to cross the placenta and to become distributed in fetal tissues (10, 11). Nickel(I1) distribution in fetal tissues is nonuniform and resembles that seen in the dam, with kidney being the main target (10, 11). However, formation of DNA base derivatives in fetal kidneys and livers depended on the NiAcet dosing in a way different from that observed in maternal organs. First, the relative increases (vs controls) of DNA base products in kidneys and livers of fetuses were generally lower, most likely due to lower nickel(I1) concentrations in fetal tissues. Second, while the divided and/or higher total nickel(I1) doses produced higher levels of oxidation products in maternal kidneys, no such effect was observed in fetal kidneys. Moreover, following administration of the higher nickel(I1)dose, the amounts of base derivatives in chromatin of fetal kidneys were often diminished. This was in concordance with identical kidney tumor incidence in the offspring of dams given a single or divided NiAcet dose and with increased toxicity of the higher dose to fetuses that we observed in our carcinogenesis bioassay (6). Dose-dependent effects of nickel(II), like these mentioned above,were not observed in livers. The cause(@ of these differences remains (remain) unknown. A systemic treatment with nickel(II), either direct or transplacental, initiates renal tumors only in male rats (1, 6). These tumors are promotable to visible size by sodium barbital, a multitissue promoting agent that includes renal cortex among ita selective target tissues (33). Most importantly, sodium barbital can also promote liver tumors (33,341. However, the latter were not observed in nickel(11)-and sodium barbital-treated rats of either sex (1,6). These organ- and gender-dependent differences in carcinogenic response provide an interesting possibility to correlate nickel(I1) carcinogenicity with the pattern of
814 Chem. Res. Toxicol., Vol. 5, No. 6, 1992
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DNA base modification observed in target (kidney) and nontarget (liver)organs of male and female rats. Generally, administration of nickel(I1) to pregnant dams resulted predominantly in production of DNA purine derivatives in both kidney and liver. Pyrimidines were affected to a much lesser extent. The major difference between these two organs in their response to nickel(I1) consisted of highly increased amounts of 8-OH-Gua in the renal chromatin versus no significant change in content of 8-OH-Gua in hepatic chromatin. The fetal organs studied responded to nickel(I1)with practically equal increases in the amounts of both pyrimidine- and purine-derived products. The most striking difference between fetal kidney and liver was again related to 8-OH-Gua,which following nickel(I1) treatment was increased significantly in renal but not in hepatic chromatin. Thus, on the basis of these differences, we might conclude that carcinogenic response to nickel(11)is related to increased production of 8-OH-Gua. This conclusion would be consistent with what we observed in the fetuses (and offspring) and in mature male rats (1,6). However, the same conclusion would be inconsistent with findings in the female rats: their kidneys also contained elevated amounts of 8-OH-Gua, but nickel(I1) did not initiate renal tumors in female rats (6). Perhaps, the carcinogenic activity of nickel(I1) depended on the production of Thy glycol? Thy glycol is a suspected promutagen (3,17). This DNA base product was elevated by NiAcet treatment in the fetal kidney, but not in liver. In the mature rats, Thy glycol levels were not affected by NiAcet in either organ. These differences would fully concur with different carcinogenic responses in the respective orgadanimal combinations, discussed above. However, marked sex differences in susceptibility of rats to chronic nephrotoxicity and/or renal carcinogenesishave been observed before with some renal carcinogens, including ethoxyquin and unleaded gasoline (35-39). Therefore, possible significance of 8-OH-Gua and Thy glycol formation in renal chromatin to nickel(I1)carcinogenicity must be viewed with caution. The results of the present investigation provide further strong evidence in support of the hypothesis that nickel(11)carcinogenesismay be mediated through active oxygen species,in particular through hydroxyl radical. Production of hydroxyl radical in reactions of certain nickel(I1) complexes with HzOz and/or 02in vitro is well documented (40,41). This radical attacks nuclear DNA producing a variety of oxidation products of both purines and pyrimidines (28)that may affect DNA replication and/or gene expression (reviewed in ref 13). More recently, nickel(I1) exposure has been associated with a single base pair mutation of K-ras oncogene (42) andp53tumor suppressor T gene (43). The mutations consisted of either G transversion (42) or T C transition (43). On the basis of the present findings and some previous observations (11, it is tempting to speculate that the occurrence of these mutations might result from respective known mispairing effects of 8-OH-Gua or Thy glycol (2-51, two products resulting from nickel(I1)exposure, as shown in the present study. However, the overall pattern of DNA base modifications described here is too complex to justify any definitive conclusions as to possible causative association between formation of a given modified base (or spectrum of modified bases) in DNA of the rat kidney and tumor initiation. Besides causing DNA base modification, oxygen-derived radicals are known to cause DNA-protein
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cross-linking in chromatin (reviewed in refs 28 and 44). Formation of DNA-protein cross-links has also been observed in calfthymus nucleohistone treated with nickel(11)-tetraglycine complex (45) and in chromatin of cells exposed to nickel(I1) in vitro and in vivo (reviewed in ref 46). Such cross-links may affect the fidelity of DNA replication and gene expression. Also, the nickel(I1)related oxygen radicals' attack on proteins may damage enzymes involved in DNA repair and replication. This, in turn, may be crucial for persistence and propagation of DNA base alterations since the DNA damage by oxygen radicals is repairable (reviewed in ref 27). Further investigationsare needed to fully recognizethe mutagenic potential of modified DNA bases found in this study and their relevance to nickel(I1) carcinogenesis. Acknowledgment. The authors are grateful to Ms. S. L. North for skillful technical assistance and to Dr. D. A. Wink for valuable critical comments on the manuscript. This project has been funded in part with Federal funds from the Department of Health and Human Servicesunder Contract N01-CO-74102with Program Resources, Inc. The content of the publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by National Institute of Standards and Technology or the U.S.Government.
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