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Unscheduled DNA synthesis of rat hepatocytes in monolayer culture
Mutation Research, 126 (1984) 205-214 Elsevier
205
MTR 03853
Unscheduled DNA synthesis of rat hepatocytes in monolayer culture Jorn A. H o l m e a n d E r i k J. S o d e r l u n d Department of Toxicology, National Institute of Public' Health, Postuttak, Oslo 1 (Norway) (Received 10 October 1983) (Revision received 13 December 1983) (Accepted 15 December 1983)
Summa~ Monolayer cultures of rat hepatocytes activated tris(2,3-dibromopropyl)phosphate (Tris-BP) more efficiently than 2-acetylaminofluorene (AAF), to genotoxic products which caused mutations in co-cultures of S. typhimuriurn. In contrast, AAF caused a greater genotoxic response in the hepatocytes than Tris-BP, as judged by the increase in DNA-repair synthesis measured by liquid scintillation counting of 3H-TdR incorporated into DNA isolated from the nuclei of the hepatocytes. Covalent binding of 0.05 mM 3H-Tris-BP to cellular proteins occurred at a similar rate as covalent binding of 0.25 mM a4C-AAF. Tris-BP was the more cytotoxic of the two compounds as determined by leakage of cellular lactate dehydrogenase into the culture medium. The observed differences in the cytotoxic and genotoxic responses between Tris-BP and AAF were probably caused by differences in the nature of their reactive metabolites with respect to stability, lipophilicity a n d / o r their interactions with various cellular nucleophilic sites. The relative DNA-repair synthesis induced by an AAF exposure for 18 h decreased with time after plating of isolated hepatocytes. Tris-BP first caused an increase in the relative DNA-repair synthesis up to 27 h after plating, whereafter the response declined reaching control values using cultures 75 h after plating. In parallel with the decreased relative response in DNA-repair synthesis with time, the background radioactivity in isolated nuclei from untreated cells increased both when the hepatocytes were incubated in the presence or absence of hydroxyurea to inhibit replicative DNA synthesis. Increased DNA-repair synthesis was demonstrated as early as 3 h after commencing exposure to the test substances. While the induced DNA-repair synthesis caused by Tris-BP remained constant after 6 h of exposure, the response caused by AAF increased with increased exposure time beyond 6 h. To assess the role of different metabolic pathways in the genotoxic and cytotoxic responses of Tris-BP and AAF, the hepatocytes were exposed to test substances in the presence of various metabolic inhibitors for 3 h, whereafter the cell medium was removed and replaced by cell-culture medium containing 3H-TdR and hydroxyurea. The cytochrome P-450 inhibitor metyrapone decreased both the genotoxic and cytotoxic effects of Tris-BP, while a-naphthoflavone reduced the genotoxic effect of AAF. The addition of glutathione (GSH) or N-acetylcysteine decreased both the cytotoxic and genotoxic effects of Tris-BP, while cellular depletion of GSH by diethylmaleate increased these effects. Manipulations in the cellular levels of sulfhydryl-containing substances in the hepatocytes by these agents had little effects on the DNA-repair synthesis caused by AAF. The results indicate that such a hepatocyte culture system may be very useful as a tool to study mechanisms involved in the formation of cytotoxic a n d / o r genotoxic metabolites from various xenobiotics. Abbreuiations: Tris-BP, tris(2,3-dibromopropyl)phosphate; AAF, 2-acetylaminofluorene; 3H-TdR, [Me-3H]thymidine; DMSO, dimethyl sulfoxide; LDH, lactate dehydrogenase; GSH, glutathione; BSA, bovine serum albumin; DEM, dieth-
ylmaleate; ANF, a-naphthoflavone; HU, hydroxyurea; cytochrome P-450, a collective term for all forms of the cytochrome P-450 polysubstrate mono-oxygenases.
206 Most chemical mutagens and carcinogens as well as many cytotoxic compounds require metabolic activation before they are converted to their ultimate reactive forms responsible for cellular damage (Miller, 1978; Weisburger and Williams, 1982). Nonreplicating cultures of primary rat hepatocytes in suspensions or as monolayers, possess a considerable capacity to metabolize both cytotoxic and genotoxic substances (Sirica and Pitot, 1980). The balance between activation and detoxification pathways of the toxic chemicals in such hepatocyte systems more closely resemble the in vivo situation than that occurring using liver subcellular fractions (Sirica and Pitot, 1980; Glatt et al., 1981; Holme et al., 1983a). Furthermore, well established methods make it easy to obtain large quantities of homogeneous liver parenchymal cells and to culture these in suspensions (Berry and Friend, 1969; Seglen, 1975; Holme et al., 1982) or as monolayers (Bissel et al., 1973; Williams, 1976; Holme et al., 1983b). These are some of the main reasons why hepatocyte systems are very valuable for both in vitro screening for cytotoxic and genotoxic chemicals and for studying the mechanisms of action of such compounds. Isolated hepatocytes in culture have been shown to exhibit DNA repair, measured as induced unscheduled DNA synthesis after exposure to genotoxic chemicals (Williams, 1977; Stich et al., 1981). Unscheduled DNA synthesis has been detected by incorporation of radiolabelled thymidine into hepatocyte DNA, measured either by autoradiography (Williams, 1977; Stich et al., 1981) or by liquid-scintillation counting of DNA isolated on cesium chloride gradients (Sirica et al., 1980; Michalopoulos et al., 1978), on polycarbonate filters (Hsia et al., 1983) or by other analytical techniques (Yager and Miller, 1978). Recently, Althaus et al. (1982) measured unscheduled DNA synthesis by liquid-scintillation counting of 3HTdR incorporated into the DNA of isolated ratliver nuclei. We used this method to characterize unscheduled DNA synthesis and to study further cytotoxic effects caused by the flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) and the aromatic amine 2-acetylaminofluorene (AAF) in monolayers of hepatocytes. To assess the role of different metabolic pathways in the formation of toxic metabolites from
these precarcinogens, we determined their cytotoxic and genotoxic responses in hepatocytes in the presence of various metabolic inhibitors. Materials and methods
Chemicals. 3H-Tris-BP was prepared as described previously (Soderlund et al., 1981). 9-14CAAF (47.6 mCi/mmole) was purchased from New England Nuclear Chemicals GmbH (Dreieichenhain, West-Germany). Other chemicals were obtained from the following sources: Tris-BP and ANF from Aldrich Europe, Beerse (Belgium); AAF, N-acetyl-L-cysteine, from Koch-Light, Coinbrook (Great Britain); mycostatin from Squibb, Twickenham (United Kingdom); foetal calf serum and kanamycin from Gibco, Grand Island, NY (U.S.A.); Eagle's minimal medium from Flow Laboratories, Ayrshire (United Kingdom); Dulbecco's modified Eagle medium and horse serum from The National Institute of Public Health, Oslo (Norway); dexamethazone, insulin, collagenase (IV), GSH, BSA (V), &aminolevulinic acid, Nonidet P-40, hydroxyurea, deoxyribonucleic acid (I), hypoxanthine, aminopterin and thymidine from Sigma, St. Louis, MO (U,S.A.); [Me-3H]thymidine (45-49 Ci/mmole) from Amersham International, Amersham, Buckinghamshire (United Kingdom); Dimilume scintillation fluid from Packard, Groningen (The Netherlands); DEM and DMSO from Merck-Schuchardt, Darmstadt (F.R.G.); metyrapone from Ciba-Geigy A.G., Basel (Switzerland). Other chemicals were reagent grade. The Salmonella typhimurium strains, TA98 and TA100, were kindly provided by Dr. Bruce N. Ames, University of California, Berkeley, CA (U.S.A.). Isolation and culture of hepatocytes. Male Wistar rats, weighing 180 300 g, were obtained from Mollegard Breeding Centre, Ejby (Denmark). They were given a standard pellet diet (Norwegian Standard No. 3155, Mollesentralen) and water ad libitum. Hepatocytes were prepared by the method of collagenase perfusion (Berry and Friend, 1969; Seglen, 1975; Holme et al., 1982). Cell viability was always greater than 90% as determined by trypan-blue exclusion. The hepatocytes were suspended (0.5 × 10 6 cells/ml) in Dulbecco's modified Eagle medium without cysteine and supplemented with 15% horse serum, 2.5% foetal calf
207 serum, 17 ffg/ml 8-aminolevulinic acid, 0.5 m g / m l asparagine, 0.17 m g / m l leucine, 4 x 10 -5 M insulin, 2 . 6 x 10 - 7 M dexamethazone, 100 U / m l penicillin, 0.1 m g / m l streptomycin and 60 U / m l mycostatin (Hoime et al., 1983b). The cells were then incubated at 37°C as stationary monolayers (7 X 1 0 4 cells/cm 2) in 60-mm or 100-mm Falcon tissue culture dishes in the Salmonella/hepatocyte and DNA-repair test system, respectively. Salmonella / hepatocyte assay. Hepatocyte monolayers were rinsed, 2 h after plating, with Hanks-Hepes buffer, pH 7.4, supplemented with 1% BSA and then added to the same buffer containing test substance and 0.1 ml of an overnight culture of S. typhimurium (Holme et al., 1983a). After 2 h co-incubation, the bacteria were collected by centrifugation and plated to determine the number of revertants. The number of spontaneous revertants in each experiment was subtracted from the number with test substance. Hepatocyte cytotoxicity. Cytotoxicity was determined by the measurement of lactate dehydrogenase (LDH) released into the culture medium (Anuforo et al., 1978; Holme et al., 1983c) after centrifuging the medium at 350 x g for 5 min. Hepatocyte unscheduled DNA synthesis. Unscheduled DNA synthesis was measured by liquid scintillation counting of 3H-TdR incorporated into nuclear DNA (Althaus et al., 1982). Monolayers of hepatocytes were incubated with 10 mM HU for 1 h before the experiment started. Usually, cell-culture medium containing HU (10 mM), 3H-TdR (1.25 ffCi/ml) and test substance was then added to the hepatocytes. In some experiments the hepatocytes were exposed to test substance in Hanks-Hepes buffer, pH 7.4, with 1% BSA for 3 h before the cell culture medium containing HU and 3H-TdR was added. H U was added 1 h before medium change. Additions, such as AAF, Tris-BP, metyrapone ANF, were first dissolved in DMSO, not exceeding 0.7% of the medium, whereas other compounds (N-acetylcysteine, GSH) were dissolved directly in the medium and the pH was corrected, if necessary. DEM dissolved in the Hanks-Hepes buffer was added to the cells 1 h before the start of the experiments. The experiments were terminated after an incubation time of 18-19 h by placing the dishes on ice, whereafter they were rinsed with 10 ml of
T r i s - H C l - b u f f e r e d saline, pH 7.4, containing 2.0 mM non-radioactive thymidine. The hepatocytes were scraped off and transferred to centrifuge tubes. The nuclei were then isolated by a combination of the methods of Hildebrand and Okinaka (1976) and Aithaus et al. (1982) and kept at 4°C, if not otherwise stated. Cells pooled from two dishes were treated with Nonidet P-40 (1%) and vortexed vigorously for 30 sec. After 15 min, the suspension was vortexed as above, and centrifuged at 800 x g for 5 min. The pellet was resuspended in 1 ml of 0.25 M sucrose buffer (50 mM Tris-HC1, 25 mM KC1, 15 mM MgC12, pH 8.0) and 1 ml of 0.88 M sucrose buffer was layered underneath. The nuclei were then centrifuged at 1000 x g for 10 min. After addition of 1 ml 10 mM Tris-HC1, pH 8.0 and 0.5 ml 1 M KOH, the nuclei were incubated for 45 min at room temperature and neutralized with 0.5 ml 1 M HC1. DNA was precipitated in the presence of 10% TCA and 0.5% BSA. The precipitate was collected by centrifugation and DNA was hydrolysed by heating in 5% TCA at 90°C for 20 min. Remaining precipitates were removed by centrifugation at 2000 x g for 15 min. The amounts of radioactivity incorporated into the DNA were determined by liquid-scintillation counting in 7.5 ml Dimilume and DNA concentrations were determined according to Burton (1956). Cooalent binding. Monolayers of hepatocytes, 2 h after plating, were incubated with various concentrations and time intervals with 3H-Tris-BP. The total amount of covalent protein binding was determined by scraping off the cells and washing them once by centrifugation for 1 min at 55 x g. In one experiment the amounts of 3H-Tris-BP and 14C-AAF covalently bound to protein were determined in hepatocytes which were still attached after washing the plates. After washing the cells, protein was precipitated with TCA and washed with TCA, methanol and ethanol:ether, as described (Dybing et al., 1979). Protein was measured by the method of Lowry et al. (1951). Zero time values were subtracted. Results and discussion
The formation of reactive electrophiles capable of binding to cellular molecules are considered to be important events in cytotoxic, genotoxic and
208
carcinogenic effects of both Tris-BP (Prival et al., 1977; Blum and Ames, 1977; Soderlund et al., 1981) and AAF (Miller, 1978; Weisburger, 1981). We wanted to compare cytotoxic and genotoxic effects of these compounds using monolayer cultures of rat hepatocytes. The hepatocytes activated 0.05 mM 3H-Tris-BP and 0.25 mM 14C-AAF to reactive electrophiles binding to cellular protein at a similar rate, being 727 and 760 pmoles bound per mg protein per 3 h, respectively (data not shown). Both Tris-BP and AAF caused a concentration-dependent mutagenicity in the Salmonella/hepatocyte test, Tris-BP being more potent under these experimental conditions (Fig. 1). The genotoxic products were thus sufficiently stable and lipophilic to traverse the various membranes from the cytoplasm of the hepatocytes and to react with the bacterial DNA. At concentrations of TrisBP which were cytotoxic to the hepatocytes, i.e. > 0.05 mM (Fig. 2), genotoxic metabolites were still formed and released in increased quantity into the medium (Fig. 1). In an in vivo situation, such metabolites could cause DNA damage in cells outside the liver, even if such cells were unable to
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CONCENTRATION (raM) Fig. 1. Concentration-dependent mutagenicity of AAF and Tris-BP in the Salmonella/hepatocyte test. 2 h after plating, monolayers of hepatocytes were co-cultured with S. typhimurium tester strain TA98 or TA100 and various concentrations of AAF or Tris-BP, respectively. After 2 h co-incubation, the bacteria were collected and plated to determine the number of revertants. The data represent a typical experiment with hepatocytes isolated from one rat liver and each point is the mean + S.D. of 3 dishes.
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Fig. 2. Concentration-dependent LDH leakage and unscheduled DNA synthesis of AAF and Tris-BP in monolayer cultures of hepatocytes. 3 h after plating, hepatocytes were incubated in the presence of 10 mM HU and various concentrations of AAF or Tris-BP for 18 h after which LDH leakage into the medium and 3H-TdR incorporation in DNA extracted from isolated nuclei were measured. The values represent the means_+ S.D. of 3 Expts.
metabolize Tris-BP into its ultimate reactive form(s). While Tris-BP was markedly cytotoxic, as indicated by LDH activity released into the incubation medium, no cytotoxic effects of AAF were seen in monolayers of hepatocytes at concentrations up to 0.5 mM (Fig. 2). The reactive metabolites formed from Tris-BP and AAF caused a concentration-dependent increase in DNA repair measured by liquid-scintillation counting of 3H-TdR incorporated into DNA isolated from the hepatocyte nuclei (Fig. 2). At a concentration of 0.05 mM, increases of a similar magnitude in unscheduled DNA synthesis were seen with Tris-BP and AAF (Fig. 2). Higher concentrations of AAF resulted in increased DNA repair, whereas cytotoxic effects of increasing Tris-BP concentrations obscured repair. Clearly, neither cytotoxic nor genotoxic effects of carcinogenic chemicals such as Tris-BP and AAF are directly related to the amount of test substance covalently bound to cellular protein. Differences in the nature of their ultimate reactive metabolites with respect to stability and lipophilicity, a n d / o r
209
their interactions with nucleophilic groups in proteins or vital low molecular weight molecules compared to D N A must be taken into account when explaining these effects. The DNA-repair synthesis caused by Tris-BP and AAF were examined in some detail in order to elucidate factors that could influence this response. The sensitivity of quantification of DNArepair synthesis by biochemical methods depends on the extent to which the unspecific background values can be reduced. Isolation of rat nuclei before the extraction of DNA from the hepatocytes decreased the background radioactivity and thus increased the relative degree of DNA-repair synthesis caused by Tris-BP and AAF (Table 1). Similar findings with different test compounds have been reported by Althaus et al. (1982). The use of Nonidet P-40 to break up the cells before separating the nuclei from the other cellular components by centrifugation resulted in similar DNA recovery (about 75%) and relative increase in DNA-repair synthesis as thorough mechanical homogenization of the hepatocytes (data not shown), and was a more convenient method. The low replicative DNA synthesis in monolayer cultures of rat hepatocytes (Sirica et al., 1980) can be further inhibited by about 50% by the addition of HU (Table 1). A variable effect of HU on the
background incorporation of 3H-TdR radioactivity in isolated hepatocytes has been reported (Michalopoulos et al., 1978; Yager and Miller, 1978; Sirica et al., 1980), probably due to variations in the culture conditions employed. In the present study, HU was generally added to the cultures 1 h before and during carcinogen exposure. However, the same effect was seen by simply adding HU during the carcinogen exposure period (data not shown). A stimulation of 3H-TdR incorporation into DNA upon medium change has been reported (Yager and Miller, 1978), but we did not find such an effect (data not shown). Increasing hepatocyte concentrations decreased the genotoxic response of AAF, whereas the cytotoxic response of Tris-BP decreased when cell density was increased above 4 million ceils per dish (Fig. 3). In addition to increasing the amount of test substance per cell, a decreasing cell concentration also increases the relative amount of 3H-TdR per cell, but changes in the amount of 3H-TdR between 2.5 and 20/~Ci per dish did not have any marked effects on the genotoxic responses of TrisBP and AAF in the hepatocytes (data not shown). When hepatocytes were exposed to 0.05 mM Tris-BP for 18 h, about 25% of the hepatocytes were detached from the plates, as judged from the recovery of DNA (Table 1). Using all the hepato-
TABLE 1 T H E Q U A N T I F I C A T I O N OF DNA-REPAIR SYNTHESIS U S I N G DNA EXTRACTED FROM WHOLE CELL HOMOGENATES OR NUCLEI ISOLATED FROM HEPATOCYTES INCUBATED WITH OR W I T H O U T H Y D R O X Y U R E A Preparation
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% of control
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181 + 5 279_+ 7 559+ 27
100 154 309
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D M S O + HU T r i s - B P + HU A A F + HU
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4 8 9
100 187 497
Isolated nuclei
DMSO Tris-BP AAF
69+6 60_+ 2 70 _+2
248_+ 9 243 + 17 693 + 108
100 98 279
Monolayers of hepatocytes, 3 h after plating, were incubated in the presence of HU (10 mM) 3H-TdR (1.25 /LCi/ml) and Tris-BP (0.05 mM) or AAF (0.25 raM) for 18 h. DNA was then extracted from whole cell preparations or isolated nuclei and 3H-TdR incorporated into D N A determined as described under Materials and Methods. Means + S.D. of 3 determinations each with cells pooled from 2 dishes.
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Fig. 3. LDH leakage and unscheduled D N A synthesis as a function of hepatocyte concentration. 3 h after plating, monolayers of hepatocytes from the same liver were incubated with 0.25 mM AAF or 0.05 mM T r i s - B P for 18 h whereafter LDH leakage and unscheduled D N A synthesis (UDS) were measured. Values are means+ S,D. of 3 determinations, each from cells pooled from 2 dishes.
cytes plated, and not only those attached at the end of the experiments, a larger increase in DNArepair synthesis with Tris-BP was seen (data not shown). No such differences were seen with these two methods using non-cytotoxic concentrations of AAF. Consequently, there seems to have been a larger increase in DNA-repair synthesis in the hepatocytes that eventually died and detached than
in the surviving ones. Since, however, only genotoxic effects occurring in surviving cells can cause mutations and/or cancer, we chose to use only attached hepatocytes in the determination of DNA-repair synthesis. The relative DNA-repair synthesis caused by an 18 h AAF exposure decreased with time after plating, while Tris-BP first caused an increased DNA-repair synthesis up to 27 h, whereafter the response declined towards control values (Fig. 4). The decrease in relative genotoxic effects of AAF and Tris-BP seen with cultures at longer times after plating do not automatically implicate a decreased formation of reactive metabolites. In parallel with the decreased relative response in DNA-repair synthesis, the background radioactivity in isolated nuclei increased both when the hepatocytes were incubated with (Fig. 4, Table 2) and without (Table 2) HU. Thus, an increased background can also partly explain the decrease in the relative DNA-repair synthesis induced by test chemicals. These findings further indicate that both DNA-repair and replication synthesis in the hepatocytes increases with increasing time after plating. Similar increases in DNA-repair synthesis at longer times after plating have been observed by Kitagawa et al. (1975). The increased DNA-repair synthesis under control conditions can be demonstrated long before cell death is evident;
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Fig. 4. LDH leakage and unscheduled D N A synthesis in rat hepatocytes as a function of time after plating. Various times after plating, monolayers of hepatocytes were incubated with DMSO, 0.25 mM A A F or 0.05 mM T r i s - B P for 18 h after which LDH leakage and unscheduled D N A synthesis were measured. Values are means + S.D. of 3 Expts.
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3H-TdR was added to monolayers of hepatocytes at various
times after plating, and incorporation of radiolabel into the D N A extracted from isolated nuclei was determined as described in Materials and Methods. Mean_+S.D. of 3 determinations from cells pooled from 2 dishes.
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this c o u l d reflect i m p o r t a n t d e f i c i e n c i e s in the hepatocyte culture conditions eventually causing t h e g r a d u a l d e t e r i o r a t i o n u s u a l l y seen u p o n l o n g e r t e r m c u l t u r e of h e p a t o c y t e s (Bissel et al., 1973; W i l l i a m s , 1976; S c h w a r z e et al., 1982; H o l m e et al., 1983b).
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T h e t i m e p a t t e r n of the c y t o t o x i c effects seen w i t h T r i s - B P did n o t f o l l o w t h a t for the g e n o t o x i c r e s p o n s e s , in that c y t o t o x i c i t y s h o w e d a n a d i r b o t h at 27 a n d 97 h (Fig. 4). A s i m i l a r lack o f a d i r e c t c o r r e l a t i o n b e t w e e n c y t o t o x i c a n d g e n o t o x i c effects was f o u n d w h e n testing p o s s i b l e m e t a b o l i t e s o f T r i s - B P ( H o l m e et al., 1983c), s u g g e s t i n g t h a t d i f f e r e n t m e t a b o l i t e s of T r i s - B P m a y b e i n v o l v e d in these t w o c e l l u l a r responses.
INCUBATION TIME (hours)
Fig. 5. Time course of LDH leakage and unscheduled DNA synthesis in hepatocytes. 3 h after plating, monolayers of isolated hepatocytes from the same liver were incubated with 0.25 mM AAF or 0.05 mM Tris-BP for various time intervals after which LDH leakage and unscheduled DNA synthesis were measured. Values are means + S.D. of 3 determinations, each with cells pooled from 2 dishes.
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Fig. 6. Modulation of LDH leakage and unscheduled DNA synthesis in the hepatocytes with inhibitors of cytochrome P-450. 3 h after plating, monolayers of hepatocytes isolated from the same liver were exposed to 0.25 mM AAF or 0.05 mM Tris-BP in the presence of 1.0 mM metyrapone or 0.1 mM ANF for 3 h in Hanks-Hepes buffer, whereafter the cell medium was removed and replaced with complete cell-culture medium containing 3H-TdR. Both LDH leakage into the Hanks-Hepes buffer (left column) and the complete cell-culture medium (right column) were measured. Unscheduled DNA synthesis was measured 21 h after plating. Values are means + S.D. of 3 determinations, each with cells pooled from 2 dishes.
212
The early time course of the induced genotoxic and cytotoxic responses in the hepatocytes in relationship to Tris-BP and AAF exposure were exanained in some more detail (Fig. 5). Increased DNA-repair synthesis was demonstrated as early as 3 h after starting the exposure to the test substances. While the relative DNA-repair synthesis caused by Tris-BP remained constant after 6 h of exposure, the response caused by AAF still increased after this time. The maximum in cytotoxicity due to Tris-BP, as determined by the increased L D H leakage, was seen already after 3 h of exposure. The more detailed study of the
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DNA-repair synthesis shows additional differences in the genotoxic effects of Tris-BP and AAF. Furthermore, it seems to be generally recommendable to start the experiment 3 h after plating 4 × 10 6 hepatocytes per plate, by adding cell-culture medium containing 10 mM HU, 1.25 ~Ci, H3-TdR per ml and test substance, and to terminate the experiment 18-20 h thereafter. Changes in carcinogen metabolism can be major determinants for the observed alterations in tumor formation seen when animals are coadministered a carcinogen and a second chemical. To assess the role of different metabolic pathways in the geno-
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Fig. 7. M o d u l a t i o n of L D H leakage and unscheduled D N A synthesis in the hepatocytes with s u l f h y d r y l - c o n t a i n i n g substances. 2 h after plating, h e p a t o c y t e s were depleted by the a d d i t i o n of 0.6 m M D E M . 3 h after plating, hepatocytes isolated from the same liver were exposed to 0.25 m M A A F or 0.025 m M T r i s - B P in the presence of 10 m M G S H or 2 m M N-acetylcysteine for 3 h, and then further i n c u b a t e d for 15 h as described in Materials and Methods. Both L D H leakage into the H a n k s - H e p e s buffer (left column) and c o m p l e t e cell culture m e d i u m (right column) were measured. U n s c h e d u l e d D N A synthesis was measured 21 h after plating. Values are m e a n s + S.D. of 3 d e t e r m i n a t i o n s , each with cells pooled from 2 dishes.
213
toxic and cytotoxic effects of Tris-BP and AAF, the hepatocytes were exposed to test substances in the presence of various metabolic inhibitors for 3 h, whereafter the cell medium was removed and replaced by the complete cell-culture medium containing 3H-TdR and HU, but without inhibitors. No further covalent binding of radiolabelled TrisBP and AAF to cellular protein occurred under these conditions (data not shown), suggesting that changing the medium removed the test substances efficiently. When the hepatocytes were exposed to Tris-BP and AAF for 3 h, DNA damage was demonstrated by increased incorporation of 3HTdR into DNA after the carcinogen exposure period (Fig. 6). The well known inhibitors of microsomal mono-oxygenases, metyrapone and ANF, both reduced the genotoxic effects of Tris-BP, whereas only metyrapone was effective in reducing the cytotoxic effects of Tris-BP. In contrast, only ANF reduced the genotoxic effects of AAF. These results are in agreement with the postulated role of cytochrome P-450 in the activation of these compounds (Soderlund et al., 1979; Miller and Miller, 1969). The intracellular level of GSH, the nucleophilic sulfhydryl-containing tripeptide, could be depleted to under 10% of control values by pretreatment of cells with DEM. This treatment increased dramatically the cytotoxic effect of Tris-BP (Fig. 7). No genotoxic effect of Tris-BP could be demonstrated under these conditions due to the increase in cytotoxicity. Furthermore, the addition of GSH or N-acetylcysteine decreased both the genotoxic and cytotoxic effects of Tris-BP. These finding~ are in accordance with the postulated role of GSH as an important defense mechanism against toxic metabolites of Tris-BP (Soderlund et al., 1979; Soderlund et al., 1981). The role of GSH in detoxification of the reactive intermediates generated from AAF is still unclear. On the one hand, AAF administration to mice does not deplete liver GSH levels (Thorgeirsson and Nebert, 1977) and AAF or AF are not substrates for GSH-S-transferases (Ketterer, 1982). On the other hand, GSH conjugates of AAF have recently been identified (Meerman et al., 1982) and GSH seems to give some protection against the bacterial mutagenicity of AAF (Hongslo et al., 1983; Holme et al., 1983a). However, manipulations in the level of sulf-
hydryl-containing substances in the hepatocytes by the above agents had little effect on the DNA-repair synthesis caused by AAF (Fig. 7). Clearly, while sulfhydryl-containing compounds seem to be of major importance in the detoxification of TrisBP, GSH affords little protection against the intracellular genotoxic effects of AAF.
Acknowledgements The authors wish to thank Dr. E. Dybing for helpful discussions, Ms. B. Sevaldson and Ms. L.T. Haug for technical assistance and Ms. I. St~ren for typing the manuscript.
References Althaus, F.R., S.D. Lawrence, G.L. Sattler, D.G. Longfellow and H.C. Pitot (1982) Chemical quantification of unscheduled DNA synthesis in cultured hepatocytes as an assay for the rapid screening of potential chemical carcinogens, Cancer Res., 42, 3010-3015. Anuforo, D.C., D. Acosta and R.V. Smith (1978) Hepatotoxic studies with primary cultures of rat liver cells, In Vitro, 14, 981-987. Berry, M., and D. Friend (1969) High-yield preparation of isolated rat liver parenchymal cells, J. Cell Biol., 43, 506-520. Bissel, D.M., L.E. Hammaker and V.A. Meyer (1973) Parenchymal cells from adult rat liver in nonproliferating monolayer culture, I. Functional studies, J. Cell Biol., 59, 722-734. Blum, A., and B.N. Ames (1977) Flame retardant additives as possible cancer hazards. The main flame retardant in children's pyjamas is a mutagen and should not be used. Science, 195, 17-23. Burton, K. (1956) Determination of DNA concentration with diphenylamine, Biochem. J., 62, 315-323. Dybing, E., E. Soderlund, L.T. Haug and S.S. Thorgeirsson (1979) Metabolism and activation of 2-acetylaminofluorene in isolated rat hepatocytes, Cancer Res., 39, 3268-3275. Glatt, H.R., R. Billings, K.L. Platt and F. Oesch (1981) Improvement of the correlation of bacterial mutagenicity with carcinogenicity of benzo[a]pyrene and four of its major metabolites by activation with intact liver cells instead of cell homogenate, Cancer Res., 41,270-277. Hildebrand, C.E., and R. Okinaka (1976) A rapid method for preparation of nuclear membranes from mammalian cells, Anal. Biochem., 75, 290-3.00. Holme, J.A., P.J. Wirth, E. Dybing and S.S. Thorgeirsson (1982) Cytotoxic effects of N-hydroxyparacetamol in suspensions of isolated rat hepatocytes, Acta Pharmacol. Toxicol., 51, 87-95. Holme, J.A., L.T., Haug and E. Dybing (1983a) Modulation of aromatic amine mutagenicity inSalmonella typhimurium with
214 rat-liver 9000 g supernatant or monolayers of rat hepatocytes as an activation system, Mutation Res., 117, 113-125. Holme, J.A., E. Soderlund and E. Dybing (1983b) Drug metabolism activities of isolated rat hepatocytes in monolayer culture, Acta Pharmacol. Toxicol., 52, 348-356. Holme, J.A., E.J. Soderlund, J.K. Hongslo, S.D. Nelson and E. Dybing (1983c) Comparative genotoxicity studies of the flame retardant tris(2,3-dibromopropyl)phosphate and possible metabolites, Mutation Res., 124, 213-224. Hongslo, J., L.T. Haug, P.J. Wirth, M. Moller, E. Dybing and S.S. Thorgeirsson (1983) The influence of conjugation reactions on the mutagenicity of aromatic amines, Mutation Res., 107, 239 247. Hsia, M.T.S., B.L. Kreamer and P. Dolara (1983) Quantitation of chemically-induced D N A damage and repair in isolated rat hepatocytes by a filter elution method, Studies on the anti-juvenile hormone precocene, II. Toxicol. Lett., 18, suppl. 1, 116. Ketterer, B. (1982) The role of nonenzymatic reactions of glutathione in xenobiotic metabolism, Drug Met. Rev., 13, 161-187. Kitagawa, T., G. Michalopoulos and H.C. Pitot (1975) Unscheduled D N A synthesis in cells from N-2-fluorenylacetamide-induced hyperplastic nodules of rat liver maintained in a primary culture system, Cancer Res.. 35, 3682- 3692. Lowry, O., N.J., Rosebrough, A.L. Farr and R.L. Randall (1951) Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193, 265-275. Meerman, J.H.N., F.A. Beland, B. Ketterer, S.K.A. Srai, A.P. Bruins and G.J. Mulder (1982) Identification of glutathione conjugates formed from N-hydroxy-2-acetylaminofluorene in the rat, Chem.-Biol. Interact., 39, 149-168. Michalopoulos, G., G.L. Sattler, L. O'Connor and H.C. Pitot (1978) Unscheduled D N A synthesis induced by procarcinogens in suspensions and primary cultures of hepatocytes on collagen membranes, Cancer Res., 38, 1866-1871. Miller, E.C. (1978) Some current perspectives on chemical carcinogenesis in h u m a n s and experimental animals: presidential address, Cancer Res., 38, 1479 1496. Miller, J.A., and E.C. Miller (1969) The metabolic activation of carcinogenic aromatic amines and amides, Prog. Exp. Tumor Res., 11, 273 301. Prival, M.J., E.C. McCoy, B. Gutter and H.S. Rosenkranz
(1977) Tris(2,3-dibromopropyl)phosphate: Mutagenicity of a widely used flame retardant, Science, 195, 76-78. Schwarze, P.E., A.E. Solheim and P.O. Seglen (1982) Aminoacid and energy requirements for rat hepatocytes in primary culture, In Vitro, 18, 43-54. Seglen, P.O. (1975) Preparation of isolated rat liver cells, Meth. Cell Biol., 13, 29-83. Sirica, A.E., and H.C. Pitot (1980) Drug metabolism and effects of carcinogens in cultured hepatic cells, Pharmacol. Res., 31,205-228. Sirica, A.E., C.G. Hwang, G.L. Sattler and H.C. Pitot (1980) Use of primary cultures of adult rat hepatocytes on collagen gel-nylon mesh to evaluate carcinogen induced D N A synthesis, Cancer Res., 40, 3259-3267. Stich, H.F., R.H.C. San and H.J. Freeman (1981) D N A repair synthesis (UDS) as an in vitro and in vivo bioassay to detect precarcinogens, ultimate carcinogens, and organotropic carcinogens, in: H.F. Stich and R.H.C. San (Eds.), Short-Term Tests for Chemical Carcinogens, Springer, New York, pp. 65-82. Soderlund, E.J., S.D. Nelson and E. Dybing (1979) Mutagenic activation of tris(2,3-dibromopropyl)phosphate: The role of microsomal oxidative metabolism, Acta Pharmacol. Toxicol., 45, 112 121. S~derlund, E.J., S.D. Nelson and E. Dybing (1981) In vitro and in vivo covalent binding of the kidney toxicant and carcinogen tris(2,3-dibromopropyl)phosphate, Toxicology, 21,291-304. Thorgeirsson, S.S., and D.W. Nebert (1977) The Ah locus and the metabolism of chemical carcinogens and other foreign compounds, Adv. Cancer Res., 25, 149 193. Weisburger, E.K. (1981) N-Substituted aryl compounds in carcinogenesis and mutagenesis, Natl. Cancer Inst. Monogr. 58, 1-10. Weisburger, J.H., and G.M. Williams (1982) Metabolism of chemical carcinogens, in: F.F. Becker (Ed.), Cancer, Vol. 1, Plenum, New York, pp. 241 333. Williams, G.M. (1976) Primary and long-term culture of adult rat liver epithelial cells, Methods Cells Biol., 14, 357-364. Williams, G.M. (1977) The detection of chemical carcinogens by unscheduled D N A synthesis in rat liver primary cell cultures, Cancer Res., 37, 1845-1851. Yager, J.D., and E.C. Miller (1978) D N A repair in primary cultures of rat hepatocytes, Cancer Res., 38, 4385-4394.
205
MTR 03853
Unscheduled DNA synthesis of rat hepatocytes in monolayer culture Jorn A. H o l m e a n d E r i k J. S o d e r l u n d Department of Toxicology, National Institute of Public' Health, Postuttak, Oslo 1 (Norway) (Received 10 October 1983) (Revision received 13 December 1983) (Accepted 15 December 1983)
Summa~ Monolayer cultures of rat hepatocytes activated tris(2,3-dibromopropyl)phosphate (Tris-BP) more efficiently than 2-acetylaminofluorene (AAF), to genotoxic products which caused mutations in co-cultures of S. typhimuriurn. In contrast, AAF caused a greater genotoxic response in the hepatocytes than Tris-BP, as judged by the increase in DNA-repair synthesis measured by liquid scintillation counting of 3H-TdR incorporated into DNA isolated from the nuclei of the hepatocytes. Covalent binding of 0.05 mM 3H-Tris-BP to cellular proteins occurred at a similar rate as covalent binding of 0.25 mM a4C-AAF. Tris-BP was the more cytotoxic of the two compounds as determined by leakage of cellular lactate dehydrogenase into the culture medium. The observed differences in the cytotoxic and genotoxic responses between Tris-BP and AAF were probably caused by differences in the nature of their reactive metabolites with respect to stability, lipophilicity a n d / o r their interactions with various cellular nucleophilic sites. The relative DNA-repair synthesis induced by an AAF exposure for 18 h decreased with time after plating of isolated hepatocytes. Tris-BP first caused an increase in the relative DNA-repair synthesis up to 27 h after plating, whereafter the response declined reaching control values using cultures 75 h after plating. In parallel with the decreased relative response in DNA-repair synthesis with time, the background radioactivity in isolated nuclei from untreated cells increased both when the hepatocytes were incubated in the presence or absence of hydroxyurea to inhibit replicative DNA synthesis. Increased DNA-repair synthesis was demonstrated as early as 3 h after commencing exposure to the test substances. While the induced DNA-repair synthesis caused by Tris-BP remained constant after 6 h of exposure, the response caused by AAF increased with increased exposure time beyond 6 h. To assess the role of different metabolic pathways in the genotoxic and cytotoxic responses of Tris-BP and AAF, the hepatocytes were exposed to test substances in the presence of various metabolic inhibitors for 3 h, whereafter the cell medium was removed and replaced by cell-culture medium containing 3H-TdR and hydroxyurea. The cytochrome P-450 inhibitor metyrapone decreased both the genotoxic and cytotoxic effects of Tris-BP, while a-naphthoflavone reduced the genotoxic effect of AAF. The addition of glutathione (GSH) or N-acetylcysteine decreased both the cytotoxic and genotoxic effects of Tris-BP, while cellular depletion of GSH by diethylmaleate increased these effects. Manipulations in the cellular levels of sulfhydryl-containing substances in the hepatocytes by these agents had little effects on the DNA-repair synthesis caused by AAF. The results indicate that such a hepatocyte culture system may be very useful as a tool to study mechanisms involved in the formation of cytotoxic a n d / o r genotoxic metabolites from various xenobiotics. Abbreuiations: Tris-BP, tris(2,3-dibromopropyl)phosphate; AAF, 2-acetylaminofluorene; 3H-TdR, [Me-3H]thymidine; DMSO, dimethyl sulfoxide; LDH, lactate dehydrogenase; GSH, glutathione; BSA, bovine serum albumin; DEM, dieth-
ylmaleate; ANF, a-naphthoflavone; HU, hydroxyurea; cytochrome P-450, a collective term for all forms of the cytochrome P-450 polysubstrate mono-oxygenases.
206 Most chemical mutagens and carcinogens as well as many cytotoxic compounds require metabolic activation before they are converted to their ultimate reactive forms responsible for cellular damage (Miller, 1978; Weisburger and Williams, 1982). Nonreplicating cultures of primary rat hepatocytes in suspensions or as monolayers, possess a considerable capacity to metabolize both cytotoxic and genotoxic substances (Sirica and Pitot, 1980). The balance between activation and detoxification pathways of the toxic chemicals in such hepatocyte systems more closely resemble the in vivo situation than that occurring using liver subcellular fractions (Sirica and Pitot, 1980; Glatt et al., 1981; Holme et al., 1983a). Furthermore, well established methods make it easy to obtain large quantities of homogeneous liver parenchymal cells and to culture these in suspensions (Berry and Friend, 1969; Seglen, 1975; Holme et al., 1982) or as monolayers (Bissel et al., 1973; Williams, 1976; Holme et al., 1983b). These are some of the main reasons why hepatocyte systems are very valuable for both in vitro screening for cytotoxic and genotoxic chemicals and for studying the mechanisms of action of such compounds. Isolated hepatocytes in culture have been shown to exhibit DNA repair, measured as induced unscheduled DNA synthesis after exposure to genotoxic chemicals (Williams, 1977; Stich et al., 1981). Unscheduled DNA synthesis has been detected by incorporation of radiolabelled thymidine into hepatocyte DNA, measured either by autoradiography (Williams, 1977; Stich et al., 1981) or by liquid-scintillation counting of DNA isolated on cesium chloride gradients (Sirica et al., 1980; Michalopoulos et al., 1978), on polycarbonate filters (Hsia et al., 1983) or by other analytical techniques (Yager and Miller, 1978). Recently, Althaus et al. (1982) measured unscheduled DNA synthesis by liquid-scintillation counting of 3HTdR incorporated into the DNA of isolated ratliver nuclei. We used this method to characterize unscheduled DNA synthesis and to study further cytotoxic effects caused by the flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) and the aromatic amine 2-acetylaminofluorene (AAF) in monolayers of hepatocytes. To assess the role of different metabolic pathways in the formation of toxic metabolites from
these precarcinogens, we determined their cytotoxic and genotoxic responses in hepatocytes in the presence of various metabolic inhibitors. Materials and methods
Chemicals. 3H-Tris-BP was prepared as described previously (Soderlund et al., 1981). 9-14CAAF (47.6 mCi/mmole) was purchased from New England Nuclear Chemicals GmbH (Dreieichenhain, West-Germany). Other chemicals were obtained from the following sources: Tris-BP and ANF from Aldrich Europe, Beerse (Belgium); AAF, N-acetyl-L-cysteine, from Koch-Light, Coinbrook (Great Britain); mycostatin from Squibb, Twickenham (United Kingdom); foetal calf serum and kanamycin from Gibco, Grand Island, NY (U.S.A.); Eagle's minimal medium from Flow Laboratories, Ayrshire (United Kingdom); Dulbecco's modified Eagle medium and horse serum from The National Institute of Public Health, Oslo (Norway); dexamethazone, insulin, collagenase (IV), GSH, BSA (V), &aminolevulinic acid, Nonidet P-40, hydroxyurea, deoxyribonucleic acid (I), hypoxanthine, aminopterin and thymidine from Sigma, St. Louis, MO (U,S.A.); [Me-3H]thymidine (45-49 Ci/mmole) from Amersham International, Amersham, Buckinghamshire (United Kingdom); Dimilume scintillation fluid from Packard, Groningen (The Netherlands); DEM and DMSO from Merck-Schuchardt, Darmstadt (F.R.G.); metyrapone from Ciba-Geigy A.G., Basel (Switzerland). Other chemicals were reagent grade. The Salmonella typhimurium strains, TA98 and TA100, were kindly provided by Dr. Bruce N. Ames, University of California, Berkeley, CA (U.S.A.). Isolation and culture of hepatocytes. Male Wistar rats, weighing 180 300 g, were obtained from Mollegard Breeding Centre, Ejby (Denmark). They were given a standard pellet diet (Norwegian Standard No. 3155, Mollesentralen) and water ad libitum. Hepatocytes were prepared by the method of collagenase perfusion (Berry and Friend, 1969; Seglen, 1975; Holme et al., 1982). Cell viability was always greater than 90% as determined by trypan-blue exclusion. The hepatocytes were suspended (0.5 × 10 6 cells/ml) in Dulbecco's modified Eagle medium without cysteine and supplemented with 15% horse serum, 2.5% foetal calf
207 serum, 17 ffg/ml 8-aminolevulinic acid, 0.5 m g / m l asparagine, 0.17 m g / m l leucine, 4 x 10 -5 M insulin, 2 . 6 x 10 - 7 M dexamethazone, 100 U / m l penicillin, 0.1 m g / m l streptomycin and 60 U / m l mycostatin (Hoime et al., 1983b). The cells were then incubated at 37°C as stationary monolayers (7 X 1 0 4 cells/cm 2) in 60-mm or 100-mm Falcon tissue culture dishes in the Salmonella/hepatocyte and DNA-repair test system, respectively. Salmonella / hepatocyte assay. Hepatocyte monolayers were rinsed, 2 h after plating, with Hanks-Hepes buffer, pH 7.4, supplemented with 1% BSA and then added to the same buffer containing test substance and 0.1 ml of an overnight culture of S. typhimurium (Holme et al., 1983a). After 2 h co-incubation, the bacteria were collected by centrifugation and plated to determine the number of revertants. The number of spontaneous revertants in each experiment was subtracted from the number with test substance. Hepatocyte cytotoxicity. Cytotoxicity was determined by the measurement of lactate dehydrogenase (LDH) released into the culture medium (Anuforo et al., 1978; Holme et al., 1983c) after centrifuging the medium at 350 x g for 5 min. Hepatocyte unscheduled DNA synthesis. Unscheduled DNA synthesis was measured by liquid scintillation counting of 3H-TdR incorporated into nuclear DNA (Althaus et al., 1982). Monolayers of hepatocytes were incubated with 10 mM HU for 1 h before the experiment started. Usually, cell-culture medium containing HU (10 mM), 3H-TdR (1.25 ffCi/ml) and test substance was then added to the hepatocytes. In some experiments the hepatocytes were exposed to test substance in Hanks-Hepes buffer, pH 7.4, with 1% BSA for 3 h before the cell culture medium containing HU and 3H-TdR was added. H U was added 1 h before medium change. Additions, such as AAF, Tris-BP, metyrapone ANF, were first dissolved in DMSO, not exceeding 0.7% of the medium, whereas other compounds (N-acetylcysteine, GSH) were dissolved directly in the medium and the pH was corrected, if necessary. DEM dissolved in the Hanks-Hepes buffer was added to the cells 1 h before the start of the experiments. The experiments were terminated after an incubation time of 18-19 h by placing the dishes on ice, whereafter they were rinsed with 10 ml of
T r i s - H C l - b u f f e r e d saline, pH 7.4, containing 2.0 mM non-radioactive thymidine. The hepatocytes were scraped off and transferred to centrifuge tubes. The nuclei were then isolated by a combination of the methods of Hildebrand and Okinaka (1976) and Aithaus et al. (1982) and kept at 4°C, if not otherwise stated. Cells pooled from two dishes were treated with Nonidet P-40 (1%) and vortexed vigorously for 30 sec. After 15 min, the suspension was vortexed as above, and centrifuged at 800 x g for 5 min. The pellet was resuspended in 1 ml of 0.25 M sucrose buffer (50 mM Tris-HC1, 25 mM KC1, 15 mM MgC12, pH 8.0) and 1 ml of 0.88 M sucrose buffer was layered underneath. The nuclei were then centrifuged at 1000 x g for 10 min. After addition of 1 ml 10 mM Tris-HC1, pH 8.0 and 0.5 ml 1 M KOH, the nuclei were incubated for 45 min at room temperature and neutralized with 0.5 ml 1 M HC1. DNA was precipitated in the presence of 10% TCA and 0.5% BSA. The precipitate was collected by centrifugation and DNA was hydrolysed by heating in 5% TCA at 90°C for 20 min. Remaining precipitates were removed by centrifugation at 2000 x g for 15 min. The amounts of radioactivity incorporated into the DNA were determined by liquid-scintillation counting in 7.5 ml Dimilume and DNA concentrations were determined according to Burton (1956). Cooalent binding. Monolayers of hepatocytes, 2 h after plating, were incubated with various concentrations and time intervals with 3H-Tris-BP. The total amount of covalent protein binding was determined by scraping off the cells and washing them once by centrifugation for 1 min at 55 x g. In one experiment the amounts of 3H-Tris-BP and 14C-AAF covalently bound to protein were determined in hepatocytes which were still attached after washing the plates. After washing the cells, protein was precipitated with TCA and washed with TCA, methanol and ethanol:ether, as described (Dybing et al., 1979). Protein was measured by the method of Lowry et al. (1951). Zero time values were subtracted. Results and discussion
The formation of reactive electrophiles capable of binding to cellular molecules are considered to be important events in cytotoxic, genotoxic and
208
carcinogenic effects of both Tris-BP (Prival et al., 1977; Blum and Ames, 1977; Soderlund et al., 1981) and AAF (Miller, 1978; Weisburger, 1981). We wanted to compare cytotoxic and genotoxic effects of these compounds using monolayer cultures of rat hepatocytes. The hepatocytes activated 0.05 mM 3H-Tris-BP and 0.25 mM 14C-AAF to reactive electrophiles binding to cellular protein at a similar rate, being 727 and 760 pmoles bound per mg protein per 3 h, respectively (data not shown). Both Tris-BP and AAF caused a concentration-dependent mutagenicity in the Salmonella/hepatocyte test, Tris-BP being more potent under these experimental conditions (Fig. 1). The genotoxic products were thus sufficiently stable and lipophilic to traverse the various membranes from the cytoplasm of the hepatocytes and to react with the bacterial DNA. At concentrations of TrisBP which were cytotoxic to the hepatocytes, i.e. > 0.05 mM (Fig. 2), genotoxic metabolites were still formed and released in increased quantity into the medium (Fig. 1). In an in vivo situation, such metabolites could cause DNA damage in cells outside the liver, even if such cells were unable to
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0.025
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0.2
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CONCENTRATION (raM) Fig. 1. Concentration-dependent mutagenicity of AAF and Tris-BP in the Salmonella/hepatocyte test. 2 h after plating, monolayers of hepatocytes were co-cultured with S. typhimurium tester strain TA98 or TA100 and various concentrations of AAF or Tris-BP, respectively. After 2 h co-incubation, the bacteria were collected and plated to determine the number of revertants. The data represent a typical experiment with hepatocytes isolated from one rat liver and each point is the mean + S.D. of 3 dishes.
500
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CONCENTRATION
(mM)
Fig. 2. Concentration-dependent LDH leakage and unscheduled DNA synthesis of AAF and Tris-BP in monolayer cultures of hepatocytes. 3 h after plating, hepatocytes were incubated in the presence of 10 mM HU and various concentrations of AAF or Tris-BP for 18 h after which LDH leakage into the medium and 3H-TdR incorporation in DNA extracted from isolated nuclei were measured. The values represent the means_+ S.D. of 3 Expts.
metabolize Tris-BP into its ultimate reactive form(s). While Tris-BP was markedly cytotoxic, as indicated by LDH activity released into the incubation medium, no cytotoxic effects of AAF were seen in monolayers of hepatocytes at concentrations up to 0.5 mM (Fig. 2). The reactive metabolites formed from Tris-BP and AAF caused a concentration-dependent increase in DNA repair measured by liquid-scintillation counting of 3H-TdR incorporated into DNA isolated from the hepatocyte nuclei (Fig. 2). At a concentration of 0.05 mM, increases of a similar magnitude in unscheduled DNA synthesis were seen with Tris-BP and AAF (Fig. 2). Higher concentrations of AAF resulted in increased DNA repair, whereas cytotoxic effects of increasing Tris-BP concentrations obscured repair. Clearly, neither cytotoxic nor genotoxic effects of carcinogenic chemicals such as Tris-BP and AAF are directly related to the amount of test substance covalently bound to cellular protein. Differences in the nature of their ultimate reactive metabolites with respect to stability and lipophilicity, a n d / o r
209
their interactions with nucleophilic groups in proteins or vital low molecular weight molecules compared to D N A must be taken into account when explaining these effects. The DNA-repair synthesis caused by Tris-BP and AAF were examined in some detail in order to elucidate factors that could influence this response. The sensitivity of quantification of DNArepair synthesis by biochemical methods depends on the extent to which the unspecific background values can be reduced. Isolation of rat nuclei before the extraction of DNA from the hepatocytes decreased the background radioactivity and thus increased the relative degree of DNA-repair synthesis caused by Tris-BP and AAF (Table 1). Similar findings with different test compounds have been reported by Althaus et al. (1982). The use of Nonidet P-40 to break up the cells before separating the nuclei from the other cellular components by centrifugation resulted in similar DNA recovery (about 75%) and relative increase in DNA-repair synthesis as thorough mechanical homogenization of the hepatocytes (data not shown), and was a more convenient method. The low replicative DNA synthesis in monolayer cultures of rat hepatocytes (Sirica et al., 1980) can be further inhibited by about 50% by the addition of HU (Table 1). A variable effect of HU on the
background incorporation of 3H-TdR radioactivity in isolated hepatocytes has been reported (Michalopoulos et al., 1978; Yager and Miller, 1978; Sirica et al., 1980), probably due to variations in the culture conditions employed. In the present study, HU was generally added to the cultures 1 h before and during carcinogen exposure. However, the same effect was seen by simply adding HU during the carcinogen exposure period (data not shown). A stimulation of 3H-TdR incorporation into DNA upon medium change has been reported (Yager and Miller, 1978), but we did not find such an effect (data not shown). Increasing hepatocyte concentrations decreased the genotoxic response of AAF, whereas the cytotoxic response of Tris-BP decreased when cell density was increased above 4 million ceils per dish (Fig. 3). In addition to increasing the amount of test substance per cell, a decreasing cell concentration also increases the relative amount of 3H-TdR per cell, but changes in the amount of 3H-TdR between 2.5 and 20/~Ci per dish did not have any marked effects on the genotoxic responses of TrisBP and AAF in the hepatocytes (data not shown). When hepatocytes were exposed to 0.05 mM Tris-BP for 18 h, about 25% of the hepatocytes were detached from the plates, as judged from the recovery of DNA (Table 1). Using all the hepato-
TABLE 1 T H E Q U A N T I F I C A T I O N OF DNA-REPAIR SYNTHESIS U S I N G DNA EXTRACTED FROM WHOLE CELL HOMOGENATES OR NUCLEI ISOLATED FROM HEPATOCYTES INCUBATED WITH OR W I T H O U T H Y D R O X Y U R E A Preparation
Additions
Yield DNA
UDS
(t~g)
cpm/t.tg DNA
% of control
Whole cell
DMSO + HU Tris-BP + HU AAF+HU
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181 + 5 279_+ 7 559+ 27
100 154 309
Isolated nuclei
D M S O + HU T r i s - B P + HU A A F + HU
70_+4 50+_5 70_+2
94_+ 176+464_+
4 8 9
100 187 497
Isolated nuclei
DMSO Tris-BP AAF
69+6 60_+ 2 70 _+2
248_+ 9 243 + 17 693 + 108
100 98 279
Monolayers of hepatocytes, 3 h after plating, were incubated in the presence of HU (10 mM) 3H-TdR (1.25 /LCi/ml) and Tris-BP (0.05 mM) or AAF (0.25 raM) for 18 h. DNA was then extracted from whole cell preparations or isolated nuclei and 3H-TdR incorporated into D N A determined as described under Materials and Methods. Means + S.D. of 3 determinations each with cells pooled from 2 dishes.
210 i
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CELL CONCENTRATION (x 10 e hepatoeytes per dish)
Fig. 3. LDH leakage and unscheduled D N A synthesis as a function of hepatocyte concentration. 3 h after plating, monolayers of hepatocytes from the same liver were incubated with 0.25 mM AAF or 0.05 mM T r i s - B P for 18 h whereafter LDH leakage and unscheduled D N A synthesis (UDS) were measured. Values are means+ S,D. of 3 determinations, each from cells pooled from 2 dishes.
cytes plated, and not only those attached at the end of the experiments, a larger increase in DNArepair synthesis with Tris-BP was seen (data not shown). No such differences were seen with these two methods using non-cytotoxic concentrations of AAF. Consequently, there seems to have been a larger increase in DNA-repair synthesis in the hepatocytes that eventually died and detached than
in the surviving ones. Since, however, only genotoxic effects occurring in surviving cells can cause mutations and/or cancer, we chose to use only attached hepatocytes in the determination of DNA-repair synthesis. The relative DNA-repair synthesis caused by an 18 h AAF exposure decreased with time after plating, while Tris-BP first caused an increased DNA-repair synthesis up to 27 h, whereafter the response declined towards control values (Fig. 4). The decrease in relative genotoxic effects of AAF and Tris-BP seen with cultures at longer times after plating do not automatically implicate a decreased formation of reactive metabolites. In parallel with the decreased relative response in DNA-repair synthesis, the background radioactivity in isolated nuclei increased both when the hepatocytes were incubated with (Fig. 4, Table 2) and without (Table 2) HU. Thus, an increased background can also partly explain the decrease in the relative DNA-repair synthesis induced by test chemicals. These findings further indicate that both DNA-repair and replication synthesis in the hepatocytes increases with increasing time after plating. Similar increases in DNA-repair synthesis at longer times after plating have been observed by Kitagawa et al. (1975). The increased DNA-repair synthesis under control conditions can be demonstrated long before cell death is evident;
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Fig. 4. LDH leakage and unscheduled D N A synthesis in rat hepatocytes as a function of time after plating. Various times after plating, monolayers of hepatocytes were incubated with DMSO, 0.25 mM A A F or 0.05 mM T r i s - B P for 18 h after which LDH leakage and unscheduled D N A synthesis were measured. Values are means + S.D. of 3 Expts.
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3H-TdR was added to monolayers of hepatocytes at various
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i
this c o u l d reflect i m p o r t a n t d e f i c i e n c i e s in the hepatocyte culture conditions eventually causing t h e g r a d u a l d e t e r i o r a t i o n u s u a l l y seen u p o n l o n g e r t e r m c u l t u r e of h e p a t o c y t e s (Bissel et al., 1973; W i l l i a m s , 1976; S c h w a r z e et al., 1982; H o l m e et al., 1983b).
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T h e t i m e p a t t e r n of the c y t o t o x i c effects seen w i t h T r i s - B P did n o t f o l l o w t h a t for the g e n o t o x i c r e s p o n s e s , in that c y t o t o x i c i t y s h o w e d a n a d i r b o t h at 27 a n d 97 h (Fig. 4). A s i m i l a r lack o f a d i r e c t c o r r e l a t i o n b e t w e e n c y t o t o x i c a n d g e n o t o x i c effects was f o u n d w h e n testing p o s s i b l e m e t a b o l i t e s o f T r i s - B P ( H o l m e et al., 1983c), s u g g e s t i n g t h a t d i f f e r e n t m e t a b o l i t e s of T r i s - B P m a y b e i n v o l v e d in these t w o c e l l u l a r responses.
INCUBATION TIME (hours)
Fig. 5. Time course of LDH leakage and unscheduled DNA synthesis in hepatocytes. 3 h after plating, monolayers of isolated hepatocytes from the same liver were incubated with 0.25 mM AAF or 0.05 mM Tris-BP for various time intervals after which LDH leakage and unscheduled DNA synthesis were measured. Values are means + S.D. of 3 determinations, each with cells pooled from 2 dishes.
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Fig. 6. Modulation of LDH leakage and unscheduled DNA synthesis in the hepatocytes with inhibitors of cytochrome P-450. 3 h after plating, monolayers of hepatocytes isolated from the same liver were exposed to 0.25 mM AAF or 0.05 mM Tris-BP in the presence of 1.0 mM metyrapone or 0.1 mM ANF for 3 h in Hanks-Hepes buffer, whereafter the cell medium was removed and replaced with complete cell-culture medium containing 3H-TdR. Both LDH leakage into the Hanks-Hepes buffer (left column) and the complete cell-culture medium (right column) were measured. Unscheduled DNA synthesis was measured 21 h after plating. Values are means + S.D. of 3 determinations, each with cells pooled from 2 dishes.
212
The early time course of the induced genotoxic and cytotoxic responses in the hepatocytes in relationship to Tris-BP and AAF exposure were exanained in some more detail (Fig. 5). Increased DNA-repair synthesis was demonstrated as early as 3 h after starting the exposure to the test substances. While the relative DNA-repair synthesis caused by Tris-BP remained constant after 6 h of exposure, the response caused by AAF still increased after this time. The maximum in cytotoxicity due to Tris-BP, as determined by the increased L D H leakage, was seen already after 3 h of exposure. The more detailed study of the
3200
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LDH
DNA-repair synthesis shows additional differences in the genotoxic effects of Tris-BP and AAF. Furthermore, it seems to be generally recommendable to start the experiment 3 h after plating 4 × 10 6 hepatocytes per plate, by adding cell-culture medium containing 10 mM HU, 1.25 ~Ci, H3-TdR per ml and test substance, and to terminate the experiment 18-20 h thereafter. Changes in carcinogen metabolism can be major determinants for the observed alterations in tumor formation seen when animals are coadministered a carcinogen and a second chemical. To assess the role of different metabolic pathways in the geno-
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Fig. 7. M o d u l a t i o n of L D H leakage and unscheduled D N A synthesis in the hepatocytes with s u l f h y d r y l - c o n t a i n i n g substances. 2 h after plating, h e p a t o c y t e s were depleted by the a d d i t i o n of 0.6 m M D E M . 3 h after plating, hepatocytes isolated from the same liver were exposed to 0.25 m M A A F or 0.025 m M T r i s - B P in the presence of 10 m M G S H or 2 m M N-acetylcysteine for 3 h, and then further i n c u b a t e d for 15 h as described in Materials and Methods. Both L D H leakage into the H a n k s - H e p e s buffer (left column) and c o m p l e t e cell culture m e d i u m (right column) were measured. U n s c h e d u l e d D N A synthesis was measured 21 h after plating. Values are m e a n s + S.D. of 3 d e t e r m i n a t i o n s , each with cells pooled from 2 dishes.
213
toxic and cytotoxic effects of Tris-BP and AAF, the hepatocytes were exposed to test substances in the presence of various metabolic inhibitors for 3 h, whereafter the cell medium was removed and replaced by the complete cell-culture medium containing 3H-TdR and HU, but without inhibitors. No further covalent binding of radiolabelled TrisBP and AAF to cellular protein occurred under these conditions (data not shown), suggesting that changing the medium removed the test substances efficiently. When the hepatocytes were exposed to Tris-BP and AAF for 3 h, DNA damage was demonstrated by increased incorporation of 3HTdR into DNA after the carcinogen exposure period (Fig. 6). The well known inhibitors of microsomal mono-oxygenases, metyrapone and ANF, both reduced the genotoxic effects of Tris-BP, whereas only metyrapone was effective in reducing the cytotoxic effects of Tris-BP. In contrast, only ANF reduced the genotoxic effects of AAF. These results are in agreement with the postulated role of cytochrome P-450 in the activation of these compounds (Soderlund et al., 1979; Miller and Miller, 1969). The intracellular level of GSH, the nucleophilic sulfhydryl-containing tripeptide, could be depleted to under 10% of control values by pretreatment of cells with DEM. This treatment increased dramatically the cytotoxic effect of Tris-BP (Fig. 7). No genotoxic effect of Tris-BP could be demonstrated under these conditions due to the increase in cytotoxicity. Furthermore, the addition of GSH or N-acetylcysteine decreased both the genotoxic and cytotoxic effects of Tris-BP. These finding~ are in accordance with the postulated role of GSH as an important defense mechanism against toxic metabolites of Tris-BP (Soderlund et al., 1979; Soderlund et al., 1981). The role of GSH in detoxification of the reactive intermediates generated from AAF is still unclear. On the one hand, AAF administration to mice does not deplete liver GSH levels (Thorgeirsson and Nebert, 1977) and AAF or AF are not substrates for GSH-S-transferases (Ketterer, 1982). On the other hand, GSH conjugates of AAF have recently been identified (Meerman et al., 1982) and GSH seems to give some protection against the bacterial mutagenicity of AAF (Hongslo et al., 1983; Holme et al., 1983a). However, manipulations in the level of sulf-
hydryl-containing substances in the hepatocytes by the above agents had little effect on the DNA-repair synthesis caused by AAF (Fig. 7). Clearly, while sulfhydryl-containing compounds seem to be of major importance in the detoxification of TrisBP, GSH affords little protection against the intracellular genotoxic effects of AAF.
Acknowledgements The authors wish to thank Dr. E. Dybing for helpful discussions, Ms. B. Sevaldson and Ms. L.T. Haug for technical assistance and Ms. I. St~ren for typing the manuscript.
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(1977) Tris(2,3-dibromopropyl)phosphate: Mutagenicity of a widely used flame retardant, Science, 195, 76-78. Schwarze, P.E., A.E. Solheim and P.O. Seglen (1982) Aminoacid and energy requirements for rat hepatocytes in primary culture, In Vitro, 18, 43-54. Seglen, P.O. (1975) Preparation of isolated rat liver cells, Meth. Cell Biol., 13, 29-83. Sirica, A.E., and H.C. Pitot (1980) Drug metabolism and effects of carcinogens in cultured hepatic cells, Pharmacol. Res., 31,205-228. Sirica, A.E., C.G. Hwang, G.L. Sattler and H.C. Pitot (1980) Use of primary cultures of adult rat hepatocytes on collagen gel-nylon mesh to evaluate carcinogen induced D N A synthesis, Cancer Res., 40, 3259-3267. Stich, H.F., R.H.C. San and H.J. Freeman (1981) D N A repair synthesis (UDS) as an in vitro and in vivo bioassay to detect precarcinogens, ultimate carcinogens, and organotropic carcinogens, in: H.F. Stich and R.H.C. San (Eds.), Short-Term Tests for Chemical Carcinogens, Springer, New York, pp. 65-82. Soderlund, E.J., S.D. Nelson and E. Dybing (1979) Mutagenic activation of tris(2,3-dibromopropyl)phosphate: The role of microsomal oxidative metabolism, Acta Pharmacol. Toxicol., 45, 112 121. S~derlund, E.J., S.D. Nelson and E. Dybing (1981) In vitro and in vivo covalent binding of the kidney toxicant and carcinogen tris(2,3-dibromopropyl)phosphate, Toxicology, 21,291-304. Thorgeirsson, S.S., and D.W. Nebert (1977) The Ah locus and the metabolism of chemical carcinogens and other foreign compounds, Adv. Cancer Res., 25, 149 193. Weisburger, E.K. (1981) N-Substituted aryl compounds in carcinogenesis and mutagenesis, Natl. Cancer Inst. Monogr. 58, 1-10. Weisburger, J.H., and G.M. Williams (1982) Metabolism of chemical carcinogens, in: F.F. Becker (Ed.), Cancer, Vol. 1, Plenum, New York, pp. 241 333. Williams, G.M. (1976) Primary and long-term culture of adult rat liver epithelial cells, Methods Cells Biol., 14, 357-364. Williams, G.M. (1977) The detection of chemical carcinogens by unscheduled D N A synthesis in rat liver primary cell cultures, Cancer Res., 37, 1845-1851. Yager, J.D., and E.C. Miller (1978) D N A repair in primary cultures of rat hepatocytes, Cancer Res., 38, 4385-4394.