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

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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»ATand AT —¿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 +4°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 histológica!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).
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