Chem. Res. Toricol. 1990, 3, 150-156
150
Differences in DNA-Guanine Alkylation between Male Sprague-Dawley Rats and Syrian Hamsters following Exposure to a Single Dose of Pancreatic Nitrosamine Carcinogens Demetrius M. Kokkinakis Department of Pathology, Northwestern University Medical School, and The Cancer Center, 303 East Chicago Avenue, Chicago, Illinois 60611 Received September 27, 1989
N-Nitrosobis(2-oxopropy1)amine (BOP) and N-nitroso(2-hydroxypropyl) (2-oxopropy1)amine (HPOP) are potent pancreatic carcinogens for the Syrian hamster, while in rats they primarily target the esophagus and respiratory tract. Both species metabolize these carcinogens to yield methylating and 2-hydroxypropylating agents. Ratios of N7-methylguanine (N7-MeG) to W-(2-hydroxypropyl)guanine(W-HPG) in hepatic DNA of BOP- and HPOP-treated hamsters are 84 and 7, respectively. Similar ratios are observed in DNA of rat liver and hamster kidney, while in the lung and pancreas of both species and also in rat kidney, such ratios are significantly lower. Differences between hamsters and rats regarding the extent of DNA alkylation are observed with either BOP or HPOP. At 50 mg/kg BOP levels of W-MeG are 3428,1907,457, and 260 Fmol/mol of guanine in hamster liver, kidney, lung, and pancreas, respectively. In rats treated with the same dose of BOP, levels of this adduct in corresponding tissues are 4616,451, 372, and 105 Fmol/mol of guanine. HPOP like BOP alkylates kidney and pancreas DNA more extensively in hamsters than in rats while levels of N7-MeG in liver and lung DNA are similar in both species. Levels of the 06-methylguanine (06-MeG) are 3.6 and 5.5 times greater in the DNA of hamster pancreas than in that of the rat after an injection of 50 mg/kg BOP and HPOP, respectively. Such differences suggest t h a t initial alkylation of hamster pancreas is a critical factor in the induction of pancreatic cancer in this species by the above nitrosamines. Persistent inhibition of DNA synthesis in hamster liver, lung, and kidney, and also in rat lung, after a single injection of BOP is in agreement with the organotropic properties of this nitrosamine in the above two species.
Introduction A major implement in studying the mechanism of initiation of pancreatic cancer is the different response of hamsters and rats to treatment with &oxidized derivatives of N-nitrosodiisopropylamine.Exposure of Syrian golden hamsters to N-nitrosobis(2-oxopropy1)amine(BOP),’ N nitroso(2-hydroxypropyl)(2-oxopropyl)amine(HPOP), N-nitroso-2,6-dimethylmorpholine(NNDM), or N nitrosobis(2-hydroxypropy1)amine (BHP) induces a high incidence of pancreatic ductal adenocarcinomas (1-4). The carcinogenic potency of the above nitrosamines parallels their specificity for the hamster pancreas. Thus BOP, the most effective pancreatic carcinogen in the Syrian hamster, when injected sc weekly for life, induces primarily pancreatic ductal adenocarcinomas and, to a much lesser extent liver, gallbladder and kidney tumors ( I ) . Less potent pancreatropic nitrosamines such as NNDM or BHP ( 3 , 4 ) and its acetylated derivative (5),although they primarily target the pancreas, also induce a significant incidence of nasal cavity and lung tumors. HPOP, which is more toxic and carcinogenic than BHP or NNDM, but still 3-4 times less potent than BOP, has a tumor spectrum similar to that of BHP (1-4). Abbreviations: BOP, N-nitrosobis(2-oxopropy1)amine;HPOP, Nnitroso(2-hydroxypropyl)(2-oxopropyl)amine; BHP, N-nitrosobis(2hydrox ropy1)amine;NNDM,N-nitroso-2,6-dimethylmorpholine; N-
MeG, zmethylguanine; Os-MeG, Os-methylguanine;N-HPG, “42hydroxypropy1)guanine; Os-HPG, @-(2-hydroxypropyl)guanine; HPLC, high-pressure liquid chromatography.
In terms of toxicity and carcinogenicity, rats are more resistant than hamsters to chronic treatment with the above nitrosamines and they develop pancreatic neoplasms only when treated a t an early stage of their development (6). Weekly sc injections of BOP or BHP fail to induce liver tumors in male rats, while in females such treatment results only in a modest incidence of hepatocellular carcinomas or cholangiocarcinomas (7-9). Thus lung and especially nasal cavity, which are secondary target organs in Syrian hamsters (1, 2, 4), become major sites for the induction of tumors in male rats treated with the above nitrosamines. The high incidence of lung tumors in male Wistar rats following treatment with BHP (10) suggests a high specificity of this and perhaps of other p-hydroxynitrosamines for the rat lung. The different organotropic effects of BOP, HPOP, and BHP in rats and hamsters may be attributed to metabolic differences in the activation and/or detoxication of these nitrosamines and the consequent variations in the extent of DNA alkylation between respective organs of the above species. Such differences in the metabolism of BOP and HPOP between hamsters and rats have been established from HPLC analysis of the urine and animals treated with these carcinogens (11). However, since intermediates of nitrosamine activation are unstable and decompose to products that are ultimately oxidized to C02in vivo ( I 2 ) , comparison of the metabolite composition of urines could only reflect differences in the detoxication of these nitrosamines by the two species. On the basis of the analysis of excreted metabolites, rats detoxify HPOP via gluc-
OS93-228~/90/2703-015Q$02.50/0 0 1990 American Chemical Society
Alkylation of R a t a n d Hamster D N A by BOP a n d HPOP
uronidation, while hamsters could also facilitate excretion of this compound by sulfating the A isomer of HPOP (11). In general detoxication of HPOP is more extensive in rats than hamsters, but not as extensive as to explain the differences in its toxicity and carcinogenicity between the two species. On the other hand, differences between rats and hamsters in activating pancreatropic nitrosamine carcinogens remain unknown due to our poor understanding of the enzymology of such reactions and failure to reproduce activation of BOP and HPOP in vitro by using subcellular fractions (12,13). In this regard, a comparison of the two species in terms of their capacity to activate the above carcinogens could be better accomplished from measurements of the levels of DNA alkylation in target and nontarget tissues. For many rodent tissues the incidence and location of tumors closely correlate with the initial extent of DNA alkylation and the persistence or accumulation of promutagenic adducts in target cell's. In certain organs initial alkylation in whole-tissue DNA could be similar to that of individual cell types; however, persistence of DNA alkylation is more likely to vary among its various cell types (14,151. Since cells of the exocrine pancreas are alkylated to the same extent by BOP (16), it is assumed that quantitation of DNA adducts in whole pancreatic DNA could give an estimate of the initial alkylation of acinar and also ductal cells. However, the elucidation of the mechanism of induction of pancreatic cancer should also require an assessment of the repair of promutagenic adducts in individual cell types of this organ.
Materials and Methods Chemicals. [ 1-14C]BOP and [ 1-14C]HPOPwere purchased from Amersham Inc. (Arlington Heights, IL) and were purified on a Supercosil LC-18-DB (25 cm X 10 mm) (Supelco, Inc., Bellefonte, PA) reverse-phase HPLC column according to a previously published method (17). [methyL3H]Thymidine was purchased from ICN (Irvine, CA), and its specific activity was determined by HPLC. "-Methylguanine (M-MeG), guanine, adenine, and thymine were purchased from Sigma. "-(2hydroxypropy1)guanine (N"-HPG) was synthesized as described by Lawley and Jarman (18) and purified by HPLC (19), while 06-methylguanine (06-MeG) was synthesized as described by Balsiger and Montgomery (20). OB-(2-Hydroxypropy1)guanine (06-HPG) was synthesized according to the following method: Metallic sodium (700 mg) was added slowly and under stirring in 30 mL of 1,2-propanediol in a nitrogen atmosphere. The solution was then heated at 80 OC, and 500 mg of 6-chloro-2aminopurine (Sigma) was added under continuous stirring. The reaction was allowed to take place for 72 h at 80 "C, and products were precipitated with the addition of 700 mL of CH2C12. Os-HPG was purified by HPLC to homogeneity and its structure verified by UV and NMR spectroscopy. UV spectra of 06-HPG were as follows: at pH 2, ,A, 286.7,230 nm, Ami, 252.2 nm; at pH 7, A, 280,239.6 nm, Ad" 253.4 nm; a t pH 12, A, 280.9,239.6 nm, A256.7 nm. The 'H NMR spectrum of the above compound in CD30D taken on a Varian XL-400 spectrometer was as follows: 6 1.291 (d, 3 H), 4.245 (m, 1 H), 4.487 (d of d, 1 H), 4.527 (d of d, 1 H), and 8.360 (s, 1 H). Decoupling of the 'H NMR spectra by irradiating at 6 1.291, 4.245, 4.487, and 4.527, respectively, confirmed the presence of the 2-hydroxypropyl group. Since BOP and HPOP are potential carcinogens for humans, it is recommended that contact of their solutions with the skin should be avoided and that handling of the pure compounds should be performed under a well-ventilated chemical hood. Animals. Male Syrian hamsters and Sprague-Dawley rats weighing 65-70 g were purchased from Charles River (Wilmington, DE) and were given access t o Agway Prolab Feed RMH 3000 (Agway Inc., Waverly, NY) and deionized water. Animals with body weight between 100 and 110 g were injected (sc) with [l14C]BOP or HPOP and placed in metabolic cages. When metabolism of the nitrosamine was nearly completed a t 3 and 7 h
Chem. Res. Toxicol., Vol. 3, No. 2, 1990 151
Table I. Metabolites in the Urine"of Rats and Hamsters Injected sc with 50 m d k g BOP metabolites, % of total dose HPOPHPOPglucspecies HPOP + BHP sulfate uronide 10.2 f 1.1 7.5 f 0.4 0.1 f 0.1 ratC 13.1 f 0.9 7.9 f 0.8 0.1 f 0.1 ratd 2.2 f 0.3 3.0 f 0.4 hamsterc 9.5 f 0.5 2.6 f 0.3 2.3 0.2 9.8 f 0.9 hamsterd
*
total* 26.5 f 2.8 28.1 f 2.8 22.1 f 3.1 23.1 f 2.2
"Urine was withdrawn from bladder and combined with that excreted. Percent of total dose excreted in urine. Killed 3 h following injection. Killed 7 h following injection. after the administration of BOP and HPOP, respectively, the animals were killed and their pancreases, livers, kidneys, and lungs were removed, in that order, and stored at -70 'C for further use. Tissue Fractionation and Isolation of DNA. The cytosolic, microsomal, mitochondrial, and nuclear fractions were isolated from livers, pancreases, lungs, and kidneys of animals treated with BOP or HPOP, and DNA was extracted from nuclei according to published procedures (19,21). Protein concentrations in the above fractions were estimated according to the method of Lowry (22),and DNA was determined by the method of Labarca and Paigen (23).
Measurement of Protein- and DNA-Bound Metabolites. Alkylation of proteins and DNA in various tissues resulting from the activation of [1-14C]BOPor HPOP was determined as it has been previously described (19) and expressed as nanomoles of bound metabolite per milligram of protein or DNA. Measurement of DNA Adducts. DNA samples were hydrolyzed under neutral and acid conditions according to published procedures (19). Hydrolysates were supplemented with 50 nmol of thymine, as internal standard, concentrated by lyophilization to approximately 0.5 mL, their pH adjusted to 3.0 with HC1 or NaHC03, and centrifuged at 15000g. Adducts were analyzed by HPLC using an Absorbosphere HS C-18 (25 cm X 10 mm) column (Alltech Inc., Deerfield, IL) eluted with 0.05 M sodium phosphate buffer, pH 3.5 (0-6 min), 2% methanol in the above buffer (6-16 min), followed by a linear gradient of 1% methanol/min (16-30 min), and 30% methanol in the same buffer (30-40 m i d . Under these conditions guanine eluted a t 8.9 min, adenine at 11.1min, thymine at 15.6 min, N - M e G at 19.3 min, M - H P G a t 27.1 min, 06-MeG a t 29.5 min, and 06-HPG at 34.8 min. Measurement of the Rate of DNA Synthesis. In order to measure rates of de novo DNA synthesis, 12 Sprague-Dawley rats weighing 110-120 g and Syrian hamsters weighing 80-85 g were injected sc with 50 mg/kg BOP, and another 12 Syrian hamsters of similar weights were injected with 20 mg/kg BOP. Controls (9 rats and 12 hamsters) were injected with saline. Control and treated animals were starved for 6 h prior t o injecting (ip) with 0.5 nmol/g [methyL3H]thymidine(sp act. 4.0 Ci/mmol) and were killed 2 h following this injection at the intervals indicated. DNA was extracted from livers, kidneys, lungs, and pancreases according to previously published methods (21). One-milliliter aliquots containing 300-700 pg of purified DNA were added t o 10 mL of 3a70B scintillation counting fluid (rpi, Mount Prospect, IL) in glass vials. Samples were kept in the dark for 24 h and were counted in a scintillation counter.
Results Hamsters and rats metabolized BOP to yield HPOP and BHP, their glucuronic acid conjugates, and a number of other products which are mainly derived from the activation of the above nitrosamines (11,17). Utilization of BOP, as determined from levels of excreted metabolites, was nearly complete 3 h following its sc injection in both hamsters and rats (Table I). In this regard, disposition of BOP was faster than that of HPOP, the complete metabolism of which required at least 6 h (11). A comparison of the composition of the urine excreted by the two species following the administration of 50 mg/kg BOP showed that rats and hamsters excreted 21 and 14% of the total dose,
152 Chem. Res. Toxicol., Vol. 3, No. 2, 1990
Kokkinakis
Table 11. Bound Metabolites in Cellular Fractions and DNA of Liver, Kidney, Lung, and Pancreas of Sprague-Dawley Rats and Syrian Hamsters Injected sc with 50 mg/kg BOP or HPOP nmol of bound metabolite/mg of protein or DNA BOP HPOP rat hamster rat hamster liver 1.5 f 0.4' 1.9 f 0.2' 1.0 f 0.1 1.0 i 0.1 cytosol microsomes 6.0 0.9 5.6 f 0.3 2.8 f 0.4 2.2 f 0.3 mitochondria 3.9 f 0.8 3.9 f 0.8 1.2 i 0.2 1.4 f 0.3 6.8 i 0.3? 5.4 i 0.2 2.0 f 0.2d 1.6 i 0.1 DNA kidney cytosol 0 . 6 f 0 . 2 1 . 5 f 0 . 2 0.4f0.1 0 . 5 i 0 . 1 microsomes 1.0 i 0.2e 2.0 f 0.3 0.6 f 0.1 0.6 i 0.1 mitochondria 0.8 f 0.1 1.8 f 0.3 0.5 i 0.2 0.6 f 0.1 DNA 1.5 f 0.2' 3.6 f 0.3 0.4 f 0.1 0.5 i 0.1 lung cytosol 0.4 f 0.1 0.5 i 0.1 0.3 f 0.1 0.3 i 0.1 microsomes 0.7 f 0.18 1.2 f 0.2 0.8 f 0.3 0.6 f 0.1 mitochondria 0.6 i 0.1 1.9 f 0.1 0.6 f 0.2 0.5 f 0.1 DNA 1.0 f 0.1 0.9 f 0.2 0.5 i 0.1 0.3 f 0.1 pancreas cytosol 0.8 f 0.3" 2.2 f 0.4 0.8 f 0.1 0.6 f 0.2 microsomes 1.3 f 0.2 1.5 f 0.3 0.6 f 0.1 0.7 i 0.2 mitochondria 1.6 f 0.3 1.6 i 0.6 0.8 f 0.2 0.6 i 0.2 DNA 0.6f0.1 0.5f0.1 0 . 2 i 0 . 1 0 . 3 f 0 . 1
*
'Values
represent the average of three determinations f SD.
'Data for the binding of metabolites to cytosolic, microsomal, and
mitochondrial proteins in hamsters were reproduced from ref 19. 'P < 0.005 compared with BOP-hamster liver DNA. d P < 0.05 compared with HPOP-hamster liver DNA. e P < 0.01 compared with BOP-hamster kidney microsomes. ' P < 0.005 compared with BOP-hamster kidney DNA. P < 0.01 compared with BOP-hamster lung microsomes. P < 0.005 compared with BOP-hamster pancreas cytosol.
"
f
respectively, as free or conjugated HPOP and BHP. Glucuronidation of HPOP, which occurs mainly in the liver (II), appeared to be the major pathway for detoxication of this nitrosamine in rats, while in hamsters this mode of detoxication was not as extensive. Similar results have been obtained for the in vivo metabolism of HPOP by these two species (21). Administration of [1-14C]BOP or HPOP in rats and hamsters resulted in labeling of macromolecules in various organs of these species (Table 11). Labeling was more extensive in all hamster organs and also in liver, kidney, and pancreas of the rat following the administration of BOP than that of HPOP. However, differences between BOP and HPOP observed in the labeling of the rat lung were not significant. Liver microsomes were targeted more extensively than other cytoplasmic fractions of this or any other tissue by both BOP and HPOP. Although labeling
of the microsomal fraction by BOP was not significantly different in the livers of the two species, such labeling was 2 and 1.7 times greater in hamster than rat kidney and lung, respectively. Similarly, pancreas and kidney cytosolic fractions were labeled more extensively in hamsters than in rats after BOP, but not HPOP, administration. Differences in DNA labeling obtained after the injection of [1-14C]BOPin rats and hamsters (Table 11) paralleled those determined for the covalent binding of metabolites to cytoplasmic fractions in tissues of the above species. Thus alkylation of hepatic DNA was 20% greater in rats than in hamsters, while that of kidney DNA was over 2 times more extensive in hamsters than in rats. Differences in the labeling of pancreas and lung DNA between these two species were not significant. Chromatography of hydrolyzed DNA from various organs of BOP- or HPOP-treated rats and hamsters indicated that there were no differences between these two animals regarding the nature of adducts formed, The major adduct found in both species was N7-MeG, while other adducts such as W-HPG and 06-MeG, which have been previously identified in hamster DNA (191,were also present in that of rat. Levels of W-MeG and 06-MeG found in various tissues of hamsters and rats treated with BOP are shown in Table 111. Methylation of guanine at the N7 position in hepatic DNA was 1.3 times greater in rats than in hamsters. Similar results were obtained for 06-MeG a t doses of HOP greater than 20 mg/kg. Levels of N7-MeG were 4 and 3 times greater in hamster than rat kidney and pancreas, respectively, while the methylation of the lung was similar in the two species. DNA alkylation of rat liver, kidney, and lung by 20 mg/kg BOP was 34, 5, and 3 times, respectively, more extensive than that of pancreas, while in hamsters given a similar dose of this carcinogen, respective ratios were 8, 5, and 1. Ratios of W -to 06-MeG varied from 12.5 in the liver of rats treated with the low dose of BOP to 3.8 in the pancreas of hamsters injected with a high dose of this carcinogen. However, in the lung and the kidney of both animals, and also in hamster liver, methylation of guanine was approximately 9 times more extensive at the N7 than at the O6 position. Levels of M - and 06-MeG in tissues of animals treated with 20 or 50 mg/kg HPOP are shown in Table IV. At 50 mg/kg HPOP, levels of N7-MeG were greater in rat than hamster hepatic DNA, although those of 06-MeG were comparable in the two species. Levels of guanine methylation were 2 and 3 times higher in hamster than in rat kidney and pancreas, respectively, while methylation of lung DNA was similar in both species. DNA alkylation of rat liver, kidney, and lung by 20 mg/kg HPOP was 36, 7, and 4 times more extensive, respectively, than that of pancreas, while in hamsters treated with a similar dose of
Table 111. Concentration of Guanine Methyl Adducts in Liver, Kidney, Lung, and Pancreas DNA 3 h following sc Injection of 20 or 50 m d k g BOP in Male Soranue-Dawlev Rats and Svrian Hamsters 20
sDecies rat hamster rat hamster rat hamster rat hamster
orean liver liver kidney kidney lung lung pancreas pancreas
W-MeG 1581 i 267' 1398 f 189 229 i 19 929 f 99 132 f 20 193 i 13 47 i 1 2 c
185 f 29
06-MeG 126 f 1 2 164 i 21 27 f 4 81 i 9 15 i 3 23 f 5 NDd 41 i 11
50
WI@-MeG 12.5 i 1.2 8.5 f 0.6 8.5 i 0.4 11.5 f 0.6 8.8 i 0.7 8.4 i 0.3 4.5 f 0.3
W-MeG 4616 f 405' 3428 i 418 451 f 59 1907 i 34 372 i 111 457 f 28 105 i 31e 260 f 38
@-MeG 384 f 53 323 47 53 f 11 200 i 41 37 f 6 51 f 6 19 f 4 68 i 15
*
W/06-MeG 12.0 f 0.5 10.6 0.7 8.5 f 0.6 9.5 f 0.1 10.1 f 1.5 8.9 f 0.3 5.5 i 0.8 3.8 f 0.3
*
"Values (nmol/mmol of guanine) represent the average of three determinations i SD. ' P < 0.05 compared with hamster liver. P < 0.005 compared with hamster pancreas. dNot detectable. The detection limit for positive identification of methyl adducts was 5 pmol. < 0.01 compared with hamster pancreas.
Alkylation of Rat and Hamster D N A by BOP and HPOP
Chem. Res. Toxicol., Vol. 3, No. 2, 1990 153
Table IV. Concentration of Guanine Methyl Adducts in Liver, Kidney, Lung, and Pancreas DNA 7 h following sc Injection of 20 or 50 mg/kg HPOP in Sprague-Dawley Rats and Syrian Hamsters dose of HPOP.mz/kp 20 50 N"-MeG 06-MeG N'/06-MeG species organ N'-MeG 08-MeG N'I08-MeG rat liver 365 f 2 2 O 28 f 5 13.0 f 0.4 689 f 3gb 61 f 10 11.3 f 0.3 liver 312 f 41 26 f 8 12.5 f 0.8 hamster 580 f 45 17 f 8 7.5 f 0.3 70 f 11' 5 f l 14.0 f 1.1 rat kidney 12.5 f 0.6 103 f l Z d 9f3 kidney 120 f 15 10 f 3 12.0 f 0.8 hamster 222 f 31 28 f 8 7.9 f 0.5 rat lung 40 f I 10 f 3 4.0 f 0.3 111 f 13 37 f 5 3.0 f 0.2 lung 43 f 6 6f2 7.2 f 0.5 hamster 104 f 10 23 f 3 4.5 f 0.3 rat pancreas 10 f 5e NDf 15 f 48 4f2 3.8 f 0.5 32 f 4 7f3 4.5 f 0.3 2.5 f 0.3 56 f 6 22 f 4 hamster pancreas I, Values (nmol/mmol of guanine) represent the average of three determinations f SD. P < 0.05 compared with hamster liver. 'P < 0.005 compared with hamster kidney. P < 0.005 compared with hamster kidney. e P < 0.01 compared with hamster pancreas. 'Not detectable. The detection limit for positive identification of methyl adducts was 5 pmol. gP < 0.005 compared with hamster pancreas.
Table V. Ratios of Methylation to 2-Hydroxypropylation of Guanine in the DNA of Various Tissues of Rats and Hamsters Given a Single Injection of 50 mg/kg B O P or HPOP N7-MeG/ 06-MeG/ N7-HPG 06-HPG species organ BOP HPOP BOP HPOP liver rat 67 f 20a 9 f 2 22f6 3f2 hamster liver 84 f 30 7 f 3 28i7 3 f 1 rat 26 f lgb 7 f l 10f5' 2 f l kidney hamster kidney 72 f ISd 7 f 2 23fY 2 f 2 lung 4f2 10f3 rat 10 f 7 4 f 2 hamster lung 11f9 4 f l 1Of6 5f2 NDI 4 f 2 ND rat pancreas 5f3 hamster pancreas 3 f l ND 3 f 2 ND
I
A
a Values represent the average of three determinations f SD. P 0.05 compared with rat liver. P < 0.05 compared with rat liver. P < 0.05 compared with rat kidney. e Not significantly different compared with rat kidney. fNot determined due to low levels of 2-hydroxypropyl adducts. Detection limit for positive identification of hydroxypropyl adducts was 5 pmol.
this carcinogen respective ratios were 10, 4,and 1.3. Hydroxypropylation of DNA by either BOP or HPOP was evident but was not a i extensive as methylation (Table V). Ratios of N-MeG to N - H P G were higher following BOP than HPOP administration and varied from organ to organ. The highest ratios were induced by BOP in tissues where DNA alkylation was more extensive, such as the rat and hamster liver and also the kidney of the latter species. Significantly lower ratios were observed in tissues with low levels of DNA alkylation, such as the pancreas and lung of both animals and also the rat kidney. Similar organ and species differences were observed in ratios of 06-MeG to 06-HPG, although large experimental erros involved in estimating the 06-HPG reduced the clarity of such comparisons. Unlike BOP, HPOP induced similar ratios of N-MeG to N - H P G in the kidneys of both species. The effect of a single sc injection of BOP at doses of 20 or 50 mg/kg in hamsters and 50 mg/kg in rats on the rate of DNA synthesis in liver, kidney, lung, and pancreas is shown in Figure 1. Eight hours after the administration of 50 mg/kg BOP in hamsters, hepatic DNA synthesis was reduced to only 30% of that of controls, while a smaller dose of BOP resulted in less predictable patterns of inhibition. Recovery of DNA synthesis was slow and required at least 25 h for animals treated with the low dose of this carcinogen and more than 40 h for those treated with the high dose. An initial moderate decline in the rate of DNA synthesis was also observed in rats. In this species, however, BOP eventually stimulated hepatic DNA synthesis, which doubled at 30 h and continued to rise for at
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n w (harr) F i g u r e 1. Effect of BOP on [3H]thymidine incorporation in pancreas (A), lung (B), liver (C), and kidney (D) in hamsters and rats. Hamsters were injected sc with 50 ( 0 )or 20 (A)mg/kg BOP, while rats were injected with 50 mg/kg (0) of this carcinogen. Treated and control animals were starved for 6 h, then injected (ip) with 0.5 nmol/g [methyL3H]thymidine,and killed 2 h after this injection. Points, means for three or four animals SD.
*
least 40 h after the injection of 50 mg/kg of this carcinogen. DNA synthesis was not affected in the kidneys of rats and hamsters treated with 50 and 20 mg/kg BOP, respectively. However, 50 mg/kg BOP strongly inhibited DNA synthesis in hamster kidney, which reached a minimum between 8 and 25 h after treatment. A decline in pulmonary DNA synthesis was also observed in both rats and hamsters t r e a t e d with BOP. Inhibition of DNA synthesis by BOP in the pancreas was dose dependent in hamsters and persisted for a t least 40 h after administration of this carcinogen. Statistically significant differences in pancreatic DNA synthesis in rats and hamsters were observed a t 40 h after BOP treatment.
Discussion On the basis of the extent of labeling of various tissue fractions following administration of BOP or HPOP, it is
154 Chem. Res. Toxicol., Vol. 3, No. 2, 1990
concluded that in both species the major site for the metabolism of these nitrosamines is the liver. Cytoplasmic fractions from other tissues such as the pancreas in both hamster and rat and also those of the kidney of the former species are labeled extensively by BOP, but such labeling is not always proportional to DNA alkylation. In the pancreas, alkylation of DNA, as determined from either the total 14Clabeling of DNA or from quantitation of DNA adducts, is less extensive than that of any other organ. This may result from selective labeling of cytoplasmic components of the pancreas with alkylating agents formed in other tissues and transported there via the circulation. This view is supported by poor metabolic activation of BOP or HPOP by pancreatic cells in vitro, which is at least 50 times slower than that catalyzed by isolated hepatocytes (25). The most significant difference between hamsters and rats in tissue and DNA labeling, and also in levels of adducts generated by administration of HPOP and BOP, is found in the kidney. This suggests that hamster kidney activates these carcinogens more effectively than that of the rat. Alternatively, alkylating agents formed in hamster liver may have a greater half-life than those generated in that of the rat; they concentrate in the kidney and induce its extensive alkylating damage. This second possibility would require that hepatic activation of BOP or HPOP proceeds via two pathways which yield different alkylating agents in the two species. In the rat such agents should have a relatively short half-life and should alkylate macromolecules near the site of their formation. On the other hand, metabolism of BOP and HPOP in the hamster should yield compounds with the potential to decompose to electrophiles or even electrophiles with long half-lives which could diffuse to extrahepatic tissues. Differences between hamsters and rats in metabolizing HPOP have been documented with the finding that hamster liver has a greater capacity than that of the rat to sulfate HPOP (11). Since HPOP-sulfate has the potential to decompose into alkylating agents and it can also diffuse from the site of its formation (13),it could account for at least a fraction of DNA alkylation in extrahepatic tissues in hamsters treated with HPOP. However, the induction of similar levels of DNA alkylation in rat and hamster kidney by HPOP suggests the involvement of additional pathways in the activation of this nitrosamine. Furthermore, the fact that BOP is a more effective pancreatic carcinogen for the Syrian hamster than HPOP ( I , 2) excludes the possibility that the HPOP-sulfate plays an important role in BOPinduced carcinogenesis in this species. Ratios of methyl versus hydroxypropyl adducts are similar between respective tissues of rats and hamsters treated with HPOP. However, such uniformity between species is not observed following administration of BOP. Since hydroxypropylation of DNA follows the reduction of BOP to HPOP (19), the ratio of methyl to hydroxypropyl adducts in various tissues of animals treated with BOP depends on the extent of exposure of these tissues either to HPOP or to its more proximate metabolites. Thus activation of BOP via the intermediate formation of HPOP seems to be a contributing factor in determining DNA adduct composition in rat kidney. However, this pathway appears to be only marginally involved in DNA alkylation of hamster kidney. Hydroxypropylation of DNA and the participation of HPOP as an intermediate in the activation of BOP are more extensive in pancreas and lung than in the liver of either species. The above suggests that, although reduction of BOP is more effective in the liver than in any other organ in both hamsters and rats (13), the contribution of reduced metabolites in DNA alkylation
Kokkinakis
in this tissue is not significant. This could be attributed to effective detoxication of HPOP via conjugation with glucuronic acid and sulfate ( I I ) , and also to effective secretion of this nitrosamine in the cirulation (13). The latter process appears to be contributing to the formation of hydroxypropyl adducts in extrahepatic tissues and especially in the kidney of the rat. The carcinogenicity of BOP and HPOP is probably due to DNA alkylation and in particular to the formation of promutagenic adducts such as 06-MeG and 06-HPG. Since hydroxypropylation is the result of activation of HPOP, which is a weaker carcinogen than BOP, it is suggested that this type of alkylation is not as significant as methylation in contributing to the carcinogenicity of the latter. However, the contribution of hydroxypropyl adducts in tumor initiation can be argued on the basis of following: Due to the presence of the bulky 2-hydroxypropyl moiety in 06-HPG, this adduct may not be repaired as fast as 06-MeG by 06-alkylguanine-DNA alkyltransferase (25, 26). Substantial differences in repair of these two adducts may result in accumulation of higher levels of 06-HPG than 06-MeG during chronic treatment of animals with HPOP. In addition, HPOP and especially BHP, which yield higher ratios of hydroxypropyl to methyl adducts than BOP (19, 27), are less specific for hamster pancreas and target the upper respiratory tract in both hamsters and rats (2, 4, 7, 10). In this regard, although hydroxypropylation may not significantly contribute to pancreas and liver carcinogenesis, it may be an important factor in the development of tumors of the respiratory tract. Unfortunately, repair of hydroxypropyl adducts and in some cases that of methyl adducts cannot be studied according to available methodology since levels of hydroxypropyl adducts are near their detection limits in the DNA of extrahepatic tissues of animals treated with a single injection of I4C-labeled BOP and HPOP. The elucidation of the reasons for the different organotropic effect of BOP and HPOP in rats and hamsters would require the understanding not only of the extent of alkylation of various tissues, but also of the capacity of these tissues and their component cells to repair adducts and to synthesize DNA during carcinogenic regimens. The significance of repair of promutagenic adducts has been recognized in explaining differences in the carcinogenic potency of nitrosamines among various species (28). Saturation of the 06-MeG hepatic repair system in rats and hamsters occurs at levels of 100 and 10 nmol/mmol of guanine, respectively (29). Such differences in the repair of 06-MeG are in agreement with the greater susceptibility of hamster than rat liver to BOP carcinogenesis (1,30,7-9). Furthermore, the resistance of the rat liver to weekly BOP injections even at doses that are twice as potent as those required to saturate the 06-MeG repair system suggests that such chronic treatment could induce an adaptive enhancement of the 06-alkylguanine-DNA alyltransferase activity in this species (31,32). In this regard, the greater resistance of rat versus hamster liver to BOP is not due to differences in the initial levels of DNA alkylation in these two species, but rather to the ability of the former species to extensively repair 06-MeG in its hepatic DNA. Patterns of DNA synthesis in rat and hamster liver are in agreement with the notion that nitrosamine carcinogens inhibit DNA template activity in target tissues (33), and they further suggest that in the rat liver such activity is not suppressed due to fast and effective DNA repair. The resistance of the hamster kidney to BOP-induced carcinogenesis (1)cannot be the result of rapid repair of 06-MeG adducts in that organ, since such repair is slow
Alkylation of Rat and H a m s t e r D N A b y BOP a n d HPOP
(34)and parallels that observed in rat kidney following the administration of a carcinogenic single dose of DMN (35). In this regard the failure of BOP and HPOP to induce a large incidence of renal tumors in hamsters is not directly related to the ability of these carcinogens to damage kidney DNA. The resistance of hamster kidney to develop tumors following the administration of methylating carcinogens has been noted by Lijinsky (36,37) and has been ascribed to unknown biological factors. Patterns of DNA synthesis in the kidney of hamsters treated with BOP suggest that although a toxic effect is evident for up to 24 h following the administration of a 50 mg/kg concentration of this carcinogen, such inhibition is lifted much sooner than it is in the pancreas, lung, and liver of this species which are targeted by BOP. The concentration of 06-MeG in pancreases of BOPtreated hamsters is comparable to that measured in lungs and only about a third of that detected in the kidneys of this species. Levels of this adduct in pancreases of BOPand HPOP-treated rats are 4 and 5 times, respectively, lower than those detected in pancreases of hamsters treated with the same dose of these carcinogens. In this regard, initial levels of 06-MeG are in accordance with differences between rat and hamster pancreas in responding to carcinogenic regimens of BOP and HPOP. Patterns of DNA synthesis observed in hamsters and rats after the injection of 50 mg/kg BOP, a dose that can induce neoplastic transformation only in the pancreas of the former species, suggest that DNA damage may be significant and also persistent in hamster, but it may be quickly repaired in the rat. A more rapid removal of 06-MeG in rat than hamster pancreas (27) suggests that the different carcinogenicity of BOP in the above species is a function of the efficiency of the process responsible for the repair of DNA alkylation (27). However, since the rate of 06MeG repair could also be a function of the degree of saturation of the @-alkylguanine-DNA alkyltransferase (35), the above conclusion cannot be unequivocally supported by experiments designed to induce dissimilar initial levels of DNA alkylation in the pancreases of these two species.
Acknowledgment. This work was supported by NCI Grants 42983 and 34051. I thank Drs. W. Lijinsky and R. C. Moschel for helpful discussions and Mr. S. Lines for technical assistance in completing this project. I also appreciated the advice and support of Dr. D. G. Scarpelli.
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