5-Bromodeoxyuridine-Induced Carcinogenesis and Its Modification by Persistent Estrus Syndrome, Unilateral Nephrectomy, and X-Irradiation in Rats N

(CANCER RESEARCH 49. 318-323, January 15. 1989] 5-Bromodeoxyuridine-induced Carcinogenesis and Its Modification by Persistent Estrus Syndrome, Unilateral Nephrectomy, and X-Irradiation in Rats N. P. Napalkov, V. N. Anisimov, A. J. Likhachev, and L. Tomatis1

N. N. Pelrov Research Institute of Oncology, Leningrad, USSR [N. P. N., K N. A., A. J. L.]; and International Agency for Research on Cancer, Lyon, France [L. T.J

ABSTRACT normal pair for thymine and BU. Assuming that there is no We showed earlier that 5-bromodeoxyuridine (BrdUrd), which can BU repair in rat DNA (17), and as BrdUrd after incorporation into BrdUrd pairs with guanine when present as the enol substitute for thymidine during DNA synthesis, inducing transition mu tations, is incorporated into DNA of various tissues when administered tautomer, base pair substitution mutations are expected to occur to newborn rats and is not subjected to repair processes. The main (GC â€”¿Â»ATand AT â€”¿Â»GCtransitions) during subsequent DNA purpose of the present experiment is to verify if a direct perturbation of replication. DNA would be sufficient to initiate carcinogenesis. Rats aged 1,3, 7, and Since BrdUrd induces mutations due to its incorporation into 21 days were given BrdUrd s.c. at a dose of 3.2 mg/animal. At 2 months DNA, and in view of the correlation between mutagenic and some of the females were subjected to treatment known to induce per carcinogenic potential (18), exposure to BrdUrd might be ex sistent estrus; at 1 month a group of males underwent removal of one pected to result in the appearance of tumors in long-term tests. kidney, and groups of males and females were exposed to a single total- However, its carcinogenicity has not yet been adequately studied body \-irradiation at a dose of 1.5 Gy (150 rails) per rat. In females, (2, 4), either because of the short duration of the observation treatment with BrdUrd induced tumors of the ovaries, polyps in the period or the small number of animals used. uterus, and tumors of the soft tissues, nervous system, forestomach, glandular stomach, and salivary gland; no such tumor occurred in control Syrian hamster cells underwent neoplastic transformation in females. Induction of persistent estrus increased the incidences of ovarian vitro in the presence of BrdUrd only when they were also tumors and of malignant tumors of the uterus. Treatment with BrdUrd subjected to near-UV irradiation; neither treatment alone had also increased the carcinogenic effect of \-rays on the mammary gland. a transforming effect (9). This indicates that a direct perturba In males, BrdUrd induced tumors of the testis (seminomas) and adenomas tion of DNA might be sufficient to initiate carcinogenesis, of the thyroid gland; solitary tumors of the kidney, nervous system, soft making BrdUrd a unique tool for studies of the role of DNA tissues, and bladder were also found. Unilateral nephrectomy reduced damage in malignant transformation. the incidences of tumors in the testis and pituitary gland, whereas The mechanism of this effect may be that UV irradiation of subsequent treatment with \-rays did not alter the incidences of tumors BrdUrd-substituted DNA induces a photodissociation of the induced by BrdUrd. These studies demonstrated for the first time that a bromine atom, producing a uracil radical, which is an anoma nucleoside analogue, BrdUrd, has carcinogenic potential. Possible molec ular mechanisms for its carcinogenicity and for the effects of the pro lous base for DNA and induces its decomposition (19). This moting factors are discussed. suggests that the resulting DNA deletion gives rise to a type or rate of mutation that is a more tumorigenic event than that occurring in DNA due to BrdUrd tautomerization. INTRODUCTION We proposed that the carcinogenic potential of BrdUrd in long-term studies might be considerably enhanced by further The thymidine analogue, BrdUrd,2 widely used in biological exposure of animals to promoting stimuli. The stimuli we chose studies, produces a variety of effects. Thus, it induces viruses were unilateral nephrectomy (UN) for males, and bilateral in cell culture (1) and teratogenic effects in mice and hamsters ovariectomy followed by s.c. transplantation of one of the (2, 3), and delays growth and shortens the life-span in rats (4). ovaries into the tail (TOT) for females. Assuming that the It has been reported both to enhance (5) and to inhibit (6) cell persistence of BrdUrd in the cellular genome has an initiating differentiation. BrdUrd is widely used for measuring the rate effect, we thought that compensatory hyperplasia in the re of DNA synthesis (7) and sister chromai id exchange (8), as it maining kidney would have a promoting effect in malignant is incorporated exclusively into cellular DNA in the place of transformation of nephrocytes. The choice of TOT for females thymidine (9, 10). Since it enhances the sensitivity of cells to was based on the tumor-promoting effect of persistent estrus X-rays, it is also used in the therapy of malignant tumors (11). (20, 21), as demonstrated by the finding that the transplacental BrdUrd is also mutagenic, as has been shown in cellular effect of /V-methyl-jV-nitrosourea or 7,12-dimethylbenz(a)an- systems (12,13), it produces second generation mutants in the thracene was considerably enhanced by subsequent TOT at 2 eukaryotic algae Volvox cortesi (14), and it appears to have a to 3 months (22, 23). In view of the sensitizing effect of X- miscoding effect in cell-free systems (15). The mechanism of irradiation on cells containing BrdUrd (11), we also studied the mutagenic effect of BrdUrd has been proposed to be the carcinogenesis in rats exposed perinatally to BrdUrd and sub following. When it is incorporated into replicating DNA in sequently to X-irradiation. place of thymidine, it may occur in not only the usual keto form but also in an enoltautomeric form (16). The latter forms hydrogen bonds with guanine instead of adenine, which is the MATERIALS AND METHODS Chemicals. BrdUrd, from Sigma Chemical Co. (St. Louis, MO), 100% pure, was stored at +4Â°C. Received 6/20/88; revised 9/19/88; accepted 9/29/88. The costs of publication of this article were defrayed in part by the payment Animals. Outbred female rats, from the Animal Department of the of page charges. This article must therefore be hereby marked advertisement in N. N. Petrov Research Institute of Oncology (24), were used. After accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mating and detection of pregnancy, they were kept one per polypropyl ' To whom requests for reprints should be addressed, at IARC, 150 cours ene cage until delivery. The offspring were kept with the dam for 4 Albert Thomas, 69372 Lyon Cedex 08, France. 2The abbreviations used are: BrdUrd, 5-bromodeoxyuridine; BU, 5-bromour- weeks in housing conditions that have been described elsewhere (25). acil; UN. unilateral nephrectomy; NDMA. /V-nitrosodimethylamine; TOT, trans Experiments. All rats at the age of 1, 3, 7, and 21 days were injected plantation of ovaries into the tail. s.c. with BrdUrd, dissolved in distilled water ex tempore, at a volume 318

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1989 American Association for Cancer Research. BrdUrd-INDUCED CARCINOGENESIS AND ITS MODIFICATION of 0.1 ml containing 3.2 mg BrdUrd, or the same volume of solvent. females (P < 0.0002 and P < 0.0001, respectively); TOT TOT was performed under ether anaesthesia according to the method considerably reinforced this carcinogenic effect (Tables 2 and of Bielschovsky and Hall (20) and the modifications of Anisimov (21) 3). Furthermore, whereas animals exposed to BrdUrd had only at the age of 2 months. UN (right side) was performed under ether polyps in the uterus, addition of TOT led to the appearance of anaesthesia through the back at the age of 1 month. For total-body X-irradiation at the age of 1 month, animals were malignant uterine tumors in six out of 57 animals; four of these placed in Plexiglass boxes surrounded by paraffin on all sides except were endometrial adenocarcinomas. Three out of 43 rats ex for the top. and were irradiated using an RUM-17 apparatus (USSR), posed solely to TOT developed leiomyosarcomas of the uterus. at 200 kW and 15 mA; filters, 0.5 mm Cu and 1 mm Ah focal distance, Thyroid tumors also developed more frequently in rats given 50 cm. The exposure dose was 0.38 x 10~3 A/kg, and the radiation BrdUrd and TOT than in rats given either treatment alone. dose was 1.5 Gy (150 rads). The incidence of tumors at other sites did not differ substan Experimental groups and treatment schedules are given in Table 1. tially from those in controls. All animals were kept under observation until spontaneous death or Carcinogenesis in Female Rats Exposed to BrdUrd and Total- were killed when moribund. Body X-Irradiation. Exposure to X-irradiation alone signifi Pathomorphological Investigation. All dead animals were autopsied, cantly increased the yield of both total tumors and malignant and tumors were classified as fatal or incidental. All tumors and some tumors, in comparison with controls. Combined exposure to organs (kidney, gonads, thyroid, liver) were fixed in 10% neutral BrdUrd and X-irradiation increased total tumor yield over that formalin and, after routine histolÃ³gica!treatment, embedded in paraf fin. Sections 5-7 ^m thick were stained with hematoxylin & eosin and, in all other groups (Tables 2 and 4). More ovarian, thyroid, in some cases, with oil red in order to stain cell lipids. The neoplasms malignant mammary and uterine tumors were seen in rats were classified according to the International Agency for Research on exposed to BrdUrd and X-irradiation, in comparison with Cancer classification (26, 27). animals exposed to BrdUrd alone (Table 3). The incidences of Statistics. Student's /, x2*and Peto's tests were used for statistical malignant mammary tumors were 1.8% in rats exposed to analysis of the results (28,29). Life-table methods were used to calculate BrdUrd, 4.5% in those given X-irradiation alone, and 14.3% in cumulative tumor incidence (30). AS-100 (Canon, Japan), SM-4 those exposed to BrdUrd plus X-irradiation. (USSR), and VAX Digital (France) computers were used for calcula Carcinogenesis in Male Rats Exposed to BrdUrd and UN. The tions. incidences of total and malignant tumors were much greater in males exposed to BrdUrd than in the control group or in RESULTS animals exposed only to UN or to BrdUrd and UN (Tables 4 Effect of BrdUrd and Promoting Factors on Survival. Exposure and 5). Seminomas and thyroid tumors developed considerably to BrdUrd alone had little effect on average life-span, (medial more frequently in males exposed to BrdUrd than in controls life-span in control males was 689 days and in control females or in rats exposed to UN alone (Tables 4 and 6). The incidences 687 days), but female rats exposed to BrdUrd and TOT (Group of other tumors did not differ substantially from those in 3) survived longer (median life-span, 744 days) than females controls. exposed to TOT alone (median life-span, 544 days), and ad Carcinogenesis in Male Rats Exposed to BrdUrd and Total- ministration of BrdUrd considerably shortened the life-span of Body X-Irradiation. Exposure to BrdUrd and then to X-irradi females subsequently exposed to X-irradiation (547 days) and ation did not increase the carcinogenic effect of either BrdUrd or X-rays, as measured by any of the parameters (Tables 5-7). of males subsequently undergoing UN (583 days). Kidney Polycystosis Induced by BrdUrd in Rats. Histologi- Carcinogenesis in Female Rats Exposed to BrdUrd and TOT. Exposure to BrdUrd alone was followed by a significant in cally, the convoluted tubules in kidneys of rats exposed to BrdUrd were enlarged and dilated to form micro- and crease in the yield of both total tumors and malignant tumors, in comparison with control animals. TOT itself did not alter macrocysts and were lined with eosinophilic epithelial cells tumor yields, but combined exposure to BrdUrd and TOT which either tightly filled the lumen of the tubules or protruded increased the multiplicity of tumors (Tables 2 and 3). In rats into it (Table 7). UN increased the incidence of this pathology exposed to BrdUrd alone, the incidences of ovarian tumors and in rats exposed to BrdUrd by twice as much as in rats treated of uterine polyps were significantly greater than in control with BrdUrd alone.

DISCUSSION Table 1 ExpÃ©rimentaldesign ratsGroup Number of The carcinogenic effect of BrdUrd seen in our experiments has not been reported previously (2,4, 32; our unpublished data no.I23456789101112AtSexweaningFemale on BDVI rats). We have now found that neonatal treatment 1375657432727Male with BrdUrd results in the appearance of ovarian and uterine +TOT*TOTBrdUrd tumors in females and testicular tumors in males. Some animals also developed solitary malignant tumors not seen in controls +X-rays'*X-raysControlBrdUrdBrdUrdand which are very unusual among animals of the stock used in this study (24,33). These were tumors of the kidney, soft tissues, 1375552472221Effective013755574321221365450451517TreatmentControlBrdUrd*BrdUrdforestomach, glandular stomach, peripheral nerves, salivary +UN'UNBrdUrd gland, and bladder. In comparison with DNA changes that have a high miscoding +X-raysX-rays potential, such as O6-alkylguanine and O4-alkylthymine, the Â°Survivors at the time the first tumor was observed. presence of BU in DNA results in a very low probability of * 3.2 mg s.c. at 1,3. 7, and 21 days after birth. miscoding, as discussed above (see "Introduction"). However, ' Autotransplantation of one ovary into the tail of ovariectomized rats at the in contrast to the alkylated bases which undergo intensive repair age of 2 months. Ã¤Single total-body X-irradiation with 1.5 Gy at the age of 1 month. (3, 35), BU is not a substrate for DNA repair enzymes and thus ' Unilateral nephrectomy at the age of 1 month. persists over a long period (17). Assuming a fairly even level of 319

Table 2 Tumor incidences in female rats exposed to BrdUrd and TOT or X-irradiation Number of tumor-bearing animals Number of tumors tumorsTreatmentControl All tumorsTotal105

number incidence(%)"92.4 incidence ofrats137 (%)'59.3 rat0.77 rat0.16

BrdUrd 55 35 63.6 100.0 20 36.4 90.6 63 1.15 23 0.42 BrdUrd + TOT 57 40 70.2 98.4 26 45.6 93.4 76 1.33 32 0.56 TOT 43 17 39.5 76.1 57 11.6 23.4 19 0.44 510 0.12 NE* BrdUrd + X-rays 21 18 85.7 33.3 ME 37 1.76 0.48 X-raysEffective 22No.65 18%47.4 81.8CumulativeNENo.20 9Malignant%14.640.9CumulativeNEAll 40Per 1.82MalignantTotal229Per 0.41 Â°Calculated by life-table methods as the probability of tumor occurrence after correction for intercurrent mortality. * NE, not estimated.

Table 3 Tumor localization and type in female rats exposed to BrdUrd with and without TOT or X-irradiation + + typeEffective Tumor site and BrdUrd137 TOTTOT57 X-ray213733112(9)32225112X-ray2240414(3)"114(8)11116111111 ratsNumbernumber of 55105 4376 oftumorsPituitaryThyroid 6329 1910 124 746215(11) 33

glandMammary 21 146(39)

glandOvaryHaematopoetic 15(11)3 51134 11 111215 theca-cellTheca-cell tumorLeydigomaAndroblastomaCarcinomaCilioepithelial 1221211

cystLeukemiaLymphomaReticulosarcomaThymomaMesenchymal

systemKidneyAdrenal 14 512 2111241311211111111BrdUrd

12118111

tumorCortical

glandUterusSoft adenomaPheochromocytomaEndometrial

polypAdenocarcinomaStromogenic

sarcomaLeyomyosarcomaSquamous-cell carcinomaCervical leyomyomaCervical papillomaLymphomaMlg

tissuesSkinSmall hisiocytomaPapillomaCylindromaHemangiopericytomaSquamous-cellfibrous

cancerAdenocarcinomaAdenomaLeuomyosarcomaHemangiopericytomaAdenocarcinomaMlg111121BrdUrd

intestineColonForestomachStomachSalivary

glandLungOptic hemangiopericytomaAdenomaAngiosarcomaMlg

nerveEyeLiverAdenomaAdenocarcinomaAdenomaCarcinomaFibroadenomaAdenocarcinomaSarcomaGranulesneurinomaMelanomaSarcomaControl

' Numbers in parentheses, number of rats. 320

Table 4 Statistical analysis (P values) of tumor incidence in female and male rats exposed to BrdUrd with or without additional treatment FemalesPair

uterine mammary comparisonControlfor tumors0.0003 tumors0.0001 tumors0.0002 tumors0.001 tumors0.36 tumors0.34 vs. BrdUrd Control vs. TOT 0.34 0.54 0.180.005Â° 0.085 0.28 Control vs. X-rays 0.001 0.009 0.015 0.31 0.005 BrdUrd vs. BrdUrd + TOT 0.058 0.032 0.24 0.27 0.62 BrdUrd vs. BrdUrd -1-X-rays 0.0003 0.02 0.03 0.009 0.009 0.024 TOT vi. BrdUrd + TOT 0.018 0.019 0.02 0.88 0.31 0.03 X-rays vs. BrdUrd +X-raysPair 0.01Malignant 0.097Total 0.016Malignant 0.11MalesTesticular 0.10Thyroid0.19Thyroid

comparisonControlfor tumors0.0001 tumors0.0001 tumors0.0001 tumors0.01 vs. BrdUrd Control vs. UN 0.69 0.68 0.46 0.28 Control vs. X-rays 0.003 0.0005 0.06 0.49 BrdUrd vs. BrdUrd + UN 0.42 0.73 0.61 0.56 BrdUrd vs. BrdUrd + X-rays 0.27 0.46 0.87 0.36 UN vs. BrdUrd + UN 0.009 0.0006 0.003 0.06 X-rays vs. BrdUrd + X-raysTotal 0.37Ovarian 0.60Malignant 0.19Malignant 0.07 Â°For incidental tumors only.

Table 5 Tumor incidences in male rats exposed to BrdUrd and UN or X-irradiation ratsTreatmentControlBrdUrdBrdUrd Number of tumor-bearing tumorsAll of

tumorsNo.37301311910%27.255.626.024.460.058.8Cumulative"%69.990.382.475.0NE*NENo.17239457Malignanttumors%12.542.618.08.933.341.2Cumulative"%53.184.866.729.3NENENumbertumorsTotal404121141412Perrat0.290.760.420.310.930.71Malignant tumorsTotal182711468Perrat0.130.500.220.090.400.47 ofrats1365450451517All

+UNUNBrdUrd +X-raysX-raysEffectivenumber

" Calculated by life-table methods as the probability of tumor occurrence after correction for intercurrent mortality. * NE, not estimated.

BrdUrd incorporation into the DNA of various neonatal tissues reinforce a possible carcinogenic effect of BrdUrd in this organ, (10, 17), those cell populations with the greatest, most pro in view of the affinity of this agent for renal tissue.3 However, longed proliferative activity will be more likely to undergo this was not the case. UN appears to bring about predominantly malignant transformation. In our study, tumors appeared hypertrophy of the parenchyma, and the cell number is in mainly in gonadal tissues in both female and male rats. creased by only 10-20% in rats (40) and in mice (41). Such TOT is known to induce persistent estrus (20, 21), which is moderate cellular proliferation in the remaining kidney was characterized by acyclic production of estrogens, an increased apparently insufficient for replication of BU-containing DNA level of gonadotropins and alterations in lipid and carbohydrate to induce mutation and subsequent malignant transformation. metabolism (21, 36). TOT treatment of females given BrdUrd Earlier attempts to demonstrate a possible carcinogenic effect neonatally reinforced the carcinogenic effect of BrdUrd. Thus, of BrdUrd by additional exposure to other agents were unsuc in these animals the incidence of ovarian tumors was signifi cessful. Skin application of BrdUrd to mice, followed by paint cantly higher than that in rats exposed to either BrdUrd or ing with croton oil, did not induce skin or other tumors (32); TOT alone. 12-0-tetradecanoylphorbol 13-acetate did not increase the mu- The appearance of uterine adenocarcinomas and malignan tagenic effect of BrdUrd in vitro-in vivo using 10TWcells (42). cies at some other sites in female rats exposed to both BrdUrd A partial hepatectomy performed 20-24 h before administra and TOT is also of interest. After TOT, follicular cysts and tion of BrdUrd also failed to lead to development of liver tumors hyperplasia of thecal tissue develop in the ovary, followed by in rats (4). Moreover, simultaneous neonatal administration of an acyclic secretion of estrogens and of more polar "nonclassic" BrdUrd and NDMA was not followed by an increase in tumor phenolsteroids (37). Some of these possess carcinogenic activity incidence compared with the effects of NDMA alone. Further, (38). These findings, together with the resulting artificial obsta NDMA and partial hepatectomy did not have an additive effect cle to ovulation, make this model similar to the Stein-Leventhal (4). Thus, only TOT has so far been shown to increase the syndrome. The latter, which is characterized by a sclerocystic carcinogenic effect of BrdUrd. enlargement of the ovaries (resulting in sterility), hirsutism and It has been shown in several studies that incorporation of some disturbances in fat and glucose metabolism, is followed BrdUrd into nuclear DNA sensitizes cells to induction of chro by a high risk of endometrial adenocarcinoma (39). mosomal aberrations by X-rays or UV light and may contribute In exposing male rats to UN, we hoped that a compensatory hyperplasia of the parenchyma in the remaining kidney would ' Our unpublished data. 321

Table 6 Tumor localization and type in male rats exposed to BrdUrd with or without UN or X-irradiation

typeEffectiveTumor she and UN502117(6)"6(4)12211BrdUrdUNX-rays45 + ratsNumbernumber of 1514

oftumorsPituitaryThyroidTestisHaematopoietic 146

33 MalignantBenign 12 S715(13)11111

4(3)1 MalignantMalignantMalignantMalignantMalignantBenign2191

systemKidneyBladderSkinSoft 11

MalignantBenign 1211111BrdUrd54415

tissuesPeripheral MalignantMalignantMalignantMalignantMalignantMalignantBenign31BrdUrd+ 1111

nervesLungSmall

111

intestineColonBoneAdrenal

glandMammary MalignantMalignantControl1364016 1X-rays17122111111

glandBenign Â°Numbers in parentheses, number of rats.

Table 7 Incidence of renal micro- and polycysts in rats exposed to BrdUrd with regard to histological slides; to Dr. E. Cardis, Dr. A. M. Mikheev, and and without other stimuli M. G. Yakovleva for skillful assistance in computer treatment of the of results; and to E. A. Sokolova and N. A. Kuzmina for excellent technical SexFemaleMaleTreatmentControlBrdUrdBrdUrdrenal cysts(%)0/13714/55 assistance. We would like to thank E. Heseltine for editing the manu script and S. Anthony for typing it. (25.5)8/57(14.0)0/435/21 +TOTTOTBrdUrd REFERENCES +X-raysX-raysControlBrdUrdBrdUrd(23.8)0/220/13611/54(20.4)20/50

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N. P. Napalkov, V. N. Anisimov, A. J. Likhachev, et al.

Cancer Res 1989;49:318-323.

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