Comparative Study of DNA Damage and Repair Induced by Ten TV-Nitroso Compounds in Primary Cultures of Human and Rat Hepatocytes1

Ten carcinogenic V-niiroso compounds were assayed for DNA-damaging activity in primary cultures of human and rat hepatocytes. DNA fragmentation was measured by the alkaline elution technique, and un scheduled DNA synthesis by quantitative autoradiography. Positive doserelated responses in the range of subtoxic concentrations indicated were obtained in cells of both species with /V-nitrosodiethylamine (10-32 HIM), /V-nitrosodi-n-propylamine (1.8-10 HIM), /V-nitrosomorpholine (1-3.2 HIM),jV-nitrosopiperidine(1-3.2 IHM),\-nitnisiipyrrolidiiH(3.2-18 HIM), yV-nitroso-jV-methylurea (0.32-1.8 mM), yV-nitroso-TV-ethylurea(OJ21.8 HIM),and /V-nitroso-JV-butylurea (0.1-0 J2 mM). W-nitrosodi-ii-butylamine was practically inactive at the maximal soluble concentration (1 mM). The responses of human hepatocytes were qualitatively similar to those of rat hepatocytes, but statistically significant differences between the two species in the amounts of DNA damage and/or unscheduled DNA synthesis were observed with /V-nitrosodimethylamine, ¿V-nitrosomorpholine, /V-nitrosopiperidine, Vnitnisopyrrolidine, and A-nitroso-,\-hut\ lurea. On the other hand, quantitative differences in the genotoxic effects induced by S mM V-nitrnsudimuthylamine in cultures derived from 20 human donors and from 20 rats were greater than average interspecies differences displayed by this nitrosamine and by other yV-nitroso com pounds. These results indicate that the rat hepatocyte DNA repair assay is a valid model for predicting the genotoxic potential of .Y-nitroso compounds in human hepatocytes. INTRODUCTION NOC3 have been found to produce tumors in a large variety of animal species (1-4). Because of their potent carcinogenic activity, wide environmental occurrence, and possible in vivo formation from precursor amines and nitrosating agents, con siderable efforts have been made to assess human exposure and to correlate it to an increased incidence of cancer at specific sites (3, 5-7). Occupational and environmental exposure to preformed NOC as well as endogenous exposure through for mation of NOC in vivo have been reviewed by Bariseli and Montesano (8), who summarized also biochemical, pathologi cal, experimental, and epidemiolÃ3gica! data providing evidence that humans are presumably sensitive to the carcinogenic action of these compounds. To provide additional information on the potential genotoxic activity of NOC in man, in the present study we have examined 10 carcinogens of this chemical family for their capability of inducing cytotoxicity, DNA damage, and DNA repair synthesis in human cells. Since the liver usually Received 7/10/87; revised 4/6/88; accepted 4/8/88. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by the Consiglio Nazionale delle Ricerche, Finalized Projects Oncology (contracts 86.00326.44 and 86.00470.44) and Preventive and Rehabilitative Medicine (contract 86.01877.56), and by funds of the Public Instruction Ministry. ! To whom requests for reprints should be addressed, at Istituto di Farmaco logia dell'Università , Viale Benedetto XV, no. 2,1-16I32 Genova, Italy. 3The abbreviations used are: NOC, /V-nitroso compounds; NDMA, A'-nitrosodimethylamine; NDEA, A'-nitrosodiethylamine; NDPA, /V-nitrosodi-n-propylamine; NDBA, A'-nitrosodi-n-but)lamine; NMOR, yV-nitrosomorpholine; NPIP, A'-nitrosopipehdine; NPYR, jV-nitrosopyrrolidine; NMU, W-nitroso-A'-methylurea; NEU, /V-nitroso-Ar-ethylurea; NBU, A'-nitroso-/V-butylurea; HPC, hepatocyte primary culture(s); UDS, unscheduled DNA synthesis; WME, William's Medium E. shows a higher capacity than extrahepatic tissues of metaboliz ing nitrosamines, the experiments were carried out in primary cultures of human hepatocytes, which have been shown to represent a reliable model for the assessment of the biological activity of chemicals in human liver (9-13). DNA fragmentation was evaluated by the alkaline elution technique (14), and UDS by quantitative autoradiography as net grains per nucleus (15). Because of the successful use of screening assays with rat hepatocytes (16-18) in detecting the potential carcinogenicity of NOC, to further validate the rat model and to provide better information on its value in risk assessment, a comparison was performed by measuring the same end points in primary cul tures of rat hepatocytes. MATERIALS AND METHODS Chemicals. The 10 NOC tested were obtained from the following sources: NDMA (98% pure) and NDEA (98% pure) from E. Merck, Darmstadt, Federal Republic of Germany; NDPA (reagent grade) and NDBA (reagent grade) from Eastman Kodak Co., Rochester, NY; NMOR (100% pure), NPIP (98% pure), NMU (99% pure), NEU (99% pure), and NBU (100% pure) from Serva Feinbiochemica, Heidelberg, Federal Republic of Germany; NPYR (reagent grade) from ICN Biomedicals, Inc., NY. Tetraethylammonium hydroxide was purchased from E. Merck; WME from Flow Laboratories, Milan, Italy; collagenase type I from Sigma Chemical Co., St. Louis, MO; and [methyl-3H]thymidine (specific activity, 25 Ci/mmol) from the Radiochemical Center. Amersham, United Kingdom. All other chemicals were reagent grade. Hepatocyte Primary Cultures. Fresh human tissue was obtained from discarded surgical material during the course of prescribed surgery. Details of donors of liver samples are reported in Table 1. Hepatocytes were isolated from apparently healthy tissue as previously described by Strom et al. (19) by a collagenase perfusion through catheters inserted in the larger vessels on the cut surface. The cell yields obtained from the various donors ranged from 5 x 10* to 10 x 107/g of liver. The percentages of viable cells after perfusion, as measured by trypan blue exclusion, are listed in Table 1. Rat hepatocytes were isolated from Sprague-Dawley male albino rats (200 to 250 g) by collagenase perfusion as described by Williams (20). The percentage of viable cells ranged from 80 to 95%. Isolated human or rat hepatocytes were suspended in WME supple mented with 10% fetal calf serum and gentamycin (50 Mg/ml). Aliquots of this suspension were plated in plastic dishes as follows: 2x10'' cells in 60-in in uncoated dishes for DNA fragmentation assay and 1x10'' cells in 35-mm dishes coated with rat tail collagen for determination of cytotoxicity and UDS. After an attachment period of 3 h at 37'C in an atmosphere of 95% air-5% CO2, hepatocytes were incubated for 20 h with the test compound in serum-free WME. NOC were dissolved directly in the medium immediately before use. In cultures to be used for UDS determination, 10 nCi/ml of [mefA^/^Hlthymidine were added to the incubation medium. At the end of treatment, cells were immediately assayed for cytotox icity by trypan blue exclusion and for DNA fragmentation and UDS. DNA Fragmentation/Alkaline Elution Technique. DNA fragmenta tion was evaluated by the alkaline elution technique, as previously described (21). With this technique, the presence of DNA single-strand breaks and/or alkali-labile sites is revealed by accelerated DNA elution as compared with controls. Results are expressed both as the percentage of DNA eluted from the filter and as the elution rate over controls (A", 4144 on April 14, 2017. © 1988 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from GENOTOXICITY OF NOC IN HUMAN HEPATOCYTES Table 1 Details of patients from whom liver samples were obtained for the isolation of hepatocytes Donor NameSexAge (yr)Indication for surgeryBilirubin (mg/dl)Alkaline phosphatase(units/liter)AST1 (units/liter)ALT (units/liter)-r-GT (units/liter)CPK (units/liter)LDH (units/liter)Cell viability (%)NameSexAge (yr)Indication for surgeryBilirubin (mg/dl)Alkaline phosphatase(units/liter)AST (units/liter)ALT (units/liter)-y-GT (units/liter)CPK (units/liter)LDH (units/liter)Cell viability (%)NameSexAgeIndication for surgeryBilirubin (mg/dl)Alkaline phosphatase(units/liter)AST (units/liter)ALT (units/liter)-X-GT (units/liter)CPK (units/liter)LDH (units/liter)Cell viability (%)1E. O.M59Bile duct carci noma3028908L.G.M69Hepatic métastasesof colon carci noma0.31533929417215L.