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A comparison of DNA repair synthesis in primary hepatocytes from young and old rats
Mechanisms of Ageing and Development, 29 (1985) 283-298 Elsevier Scientific Publishers Ireland Ltd.
283
A COMPARISON OF DNA REPAIR SYNTHESIS IN PRIMARY HEPATOCYTES FROM YOUNG AND OLD RATS
HAROLD E. KENNAH, II, MONA L. COETZEE and PETER eVE Department of Anatomy and Cell Biology, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261 (U.S.A.)
(Received August 20th, 1984) (Revision received October 30th, 1984) SUMMARY DN~A repair synthesis has been compared in primary hepatocyte cultures obtained from 3-month-old and 16-20-month-old rats. Several morphological and metabolic characteristics were determined to assure cultures of comparable quality. DNA damage was induced by the addition of bleomycin or the exposure of the culture to UV irradiation. DNA repair (unscheduled DI~A synthesis) was determined by measuring [3H]thymidine incorporation. After UV irradiation, there was almost twice as much [aH]thymidine incorporation in ceils obtained from young rats as in those obtained from old rats. Equal amounts of bleomycin resulted in substantially greater damage to DNA in cells from old rats than from young rats. For equal amounts of DNA damage there was again diminished [aH] thymidine incorporation in cells obtained from old rats. Finally equal amounts of bleomycin resulted in equal damage to DNA when the bleomycin was added to isolated rat liver nuclei from young or old rats. Bleomycin treated nuclei from young rats incorporated substantially more [3H]thymidine triphosphate (TTP) than bleomycin treated nuclei from old rats. The results indicate that hepatocytes from old rats are much more susceptible to bleomycin than hepatocytes from young rats and that the capacity for DNA repair synthesis is impaired in hepatocytes from old rats.
Key words: DNA repair; Primary hepatocytes
INTRODUCTION Since the early work of Curtis [ 1] dealing with chromosomal alterations in aging and the .proposal by Szilard [2] that the accumulation of DNA damage in somatic cells might be a significant event in the aging process there have been many investigations dealing 0047-6374/85/$03.30 Printed and Published in Ireland
© 1985 ElsevierScientific Publishers Ireland Ltd.
284 with quantitative and qualitative alterations of proteins as a consequence of aging [3--9]. Changes in protein synthesis may result due to chromosomal alterations or errors accumulating in aging organisms. Such errors or alterations in chromosomes, in cells of old organisms may in turn accumulate as a consequence of impaired repair synthesis of DNA. It is well established that such a reduced DNA repair capacity occurs in cells allowed to age in vitro [ i 0 - 1 3 ] . The work of Williams et al. [14] indicates that in vitro aged Syrian hamster cells are less efficient in repair synthesis than young cells. Similar results were reported by Little et al. [12] using aged human diploid cells. Regan and Setlow [15], on the other hand, found no inherent defect in DNA repair in human diploid fibroblast cell strains developed from patients with "progeroid" syndromes. Lambert et al. [ 16] found a significant age-related decline in DNA repair of peripheral lymphocytes from human subjects exposed to UV irradiation, even though both the correlation coefficient and the rate of decline were low. More recently Plesko and Richardson [17] have expanded those studies to in vivo aging. They have shown that there is a significant decline of UV-induced unscheduled DNA synthesis in hepatocytes isolated from old rats as opposed to young rats. In this report, we confirm the finding of Plesko and Richardson and show in addition that bleomycin-induced unscheduled DNA synthesis is likewise decreased in hepatocytes isolated from old rats despite the finding that hepatocytes isolated from old rats are much more susceptible to bleomycin-induced DNA damage than hepatocytes isolated from young rats. MATERIALS AND METHODS Materials
I_eibovitz L-15 and Swim's S-77 Media, fetal calf serum (FCS) and antibiotic/antimycotic were obtained from GIBCO Grand Island, NY. Collagenase Types I and IV, Soybean trypsin inhibitor, insulin, dexamethasone, 3,3,5'-triiodothyronine (T3), bovine serum albumin (BSA), rat liver albumin (RLA), hydroxyurea (HU) and N-ethylmaleimide (NEM) were from Sigma Chemical Company, St. Louis, MO. [methyl-aH]thymidine (31 Ci/mmol), [8 -3H] deoxyadenosine 5'-triphosphate (17 Ci/mmol) and L-[4,5-aH]leucine (35 Ci/mmol) were obtained from ICN Inc., Irvine CA. Blenoxane (bleomycin sulfate) was manufactured by Bristol Laboratories, Syracuse NY. Poly(dA-dT) was obtained from Miles Laboratories, Elkhart, IN and Micro¢occus luteus DNA polymerase (2.7,7.7) from Pharmacia P-L Biochemicals, Inc. Milwaukee, WI. Animals
Young Sprague-Dawley male rats weighing between 150 and 170 g were purchased from Zivic-Miller, Allison Park, PA. Old male Sprague-Dawley rats were 16-20 months old, weighed between 800 and 1200 g and were from an NIH Aging Colony, Charles River, MA. New Zealand white rabbits were purchased from Hilltop Lab Animals, Inc. Scottdale, PA. Animals were kept in a temperature and light-controlled room and received food and water ad libitum.
285
Isolation o f hepatocytes Hepatc~.ytes were isolated by the in situ 2-step collagenase perfusion technique essentially as described by Seglen [18]. The liver was perfused with 150 ml Swim's S-77 medium (pH = 7.4), containing 10 -6 M insulin, 0.5 mM EGTA and 0.4 U/ml heparin, followed by 200 ml of Swim's S-77 (pH = 7.4) containing 10 -6 M insulin, 10 -3 M CaC12, 0.4 mg/ml collagenase, and 0.2 mg/ml soybean trypsin inhibitor. The medium was gassed with 95% O2/5% CO2. The cells were dispersed with gentle mincing in 100 ml L-15 medium, 20 mM HEPES (pH 7.4), 0.15% glucose, 1% antibiotic and centrifuged at 5 0 g for 3 min. The ceils were washed twice and suspended in the same medium containing in addition 5% FCS, 10 -6 M Ta, 10 -s M dexamethasone and 0.2% BSA. Viability was determined by the trypan blue exclusion test and cell number with a hemocytometer. Only preparations with a viability greater than 80% were used. Cells were suspended at a ceil density of 1-1.5 × 106 cells/ml and 7 ml added per lO0-mm dish. The cultures were maintained at 37°C in an air incubator. At 12-20 h the medium was changed to serum free L-I 5 medium supplemented as above and medium changes were made every 24 h.
, [3H]Thymidine incorporation and DNA determination At 48 h the medium was changed and 5/aCi [aH]thyrnidine/ml medium were added to each dish. At 1 or 2 h later the medium was removed and the cells washed three times with 10 ml cold PBS. The cells were then scraped into a known volume of PBS and 0 . 1 0.2-ml aliquots removed for cell number determination using a Coulter counter. All samples were counted in triplicate with the mean value used in all calculations. The remaining cells were pelleted and 1 ml 1 M NaOH was added and heated at 80°C for 10 min. The samples were cooled, brought to 5% trichloroacetic acid and centrifuged at 10 0 0 0 g for 10 min. The pellet was redissolved in 2.0 ml 1 M NaOH and heated at 80°C for 10 min. One milliliter was used for the determination of radioactivity as previously described [19] and 1 ml for DNA determination by the diphenylamine method as described by Burton [20].
Bleomycin-induced [ 3H] thymidine incorporation At 4 0 - 4 8 h hepatocyte cultures were given a serum-free medium change with the following additions; 10 mM hydroxyurea, 0.01 units bleomycin and 5/aCi [3H]thymidine/ml. The cells were incubated at 37°C in an air incubator for various time periods. At the completion of the incubation period, the medium was removed and the cells washed twice with 10 ml PBS containing 10 mM NEM. [aH]Thymidine incorporation and DNA determination were performed as described above.
UV irradiation-induced [ 3H] thymidine incorporation At 4 0 - 4 8 h, the medium was removed and the hepatocyte monolayers were exposed to 480/~W/cm 2 of UV irradiation at 254 nm f o r 4 0 sec [21]. The UV source was a UVS54 Mineralight (Ultraviolet Products Inc., San Gabriel, CA). Following irradiation, fresh
286 serum-free medium containing 10 mM HU and 5 taCi/ml [3H]thymidine was added. Control cultures were deprived of medium for the prescribed exposure period. Following incubation for the specified time the cells were harvested for [3H]thymidine incorporation and DNA determination as described above.
[ 3H]L eucine incogporation and pro tein content determination At 48 h, the medium was changed and 1.0 taCi [3H]leucine was added per ml and the ceils harvested 1-2 h later. Aliquots were removed for cell number determination. The remaining cells were pelleted and suspended in 2.0 ml 1 M NaOH and heated at 80°C for 10 min. One milliliter was brought to 5% trichloroacetic acid for the determination of radioactivity as previously described [20] using instead a chloroform/methanol (2: 1) wash. The remaining 1 ml was brought to 5% trichloroacetic acid for protein determination. The celite filter pad was heated at 80°C for 20 min in 1 ml 1 M NaOH and an aliquot was used in the Bio-Rad Protein assay [22]. Determination of secreted albumin Albumin secretion from cultured hepatocytes was quantitated by Rocket Immunoelectrophoresis [23]. The method utilizes 1.0% agarose gels, cast and run at 2 V/cm in Tris-barbital-sodium barbital buffer (pH = 8.8). The gels contained a monospecific antibody (0.05 tag/ml) against rat liver albumin (RLA). Anti-serum was produced in New Zealand white rabbits by bi-weekly intradermal injections of 500 tag/ml RLA. Sera which produced precipitation lines on Ouchterlony double diffusion was pooled and precipitated by 40% ammonium sulfate saturation [24]. Monospecific RLA-antibody was prepared by affinity chromatography. Sigma RLA was purified by Sephadex G-100 and DEAE cellulose chromatography to yield a single band on SDS polyacrylamide gel electrophoresis. The purified RLA was bound to cyanogen bromide activated Sepharose 4B and the antisera applied in 10 mM NaPO4 (pH 9.0)/ 150 mM NaC1. Following extensive washing of the column, the monospecific rabbit IgG anti-RLA was eluted using 200 mM glycine/500 mM NaC1 (pH = 2.8). The sample was dialyzed, lyophilized, and stored at -20°C. Measurement of dTTP pool size An enzymatic assay, utilizing DNA polymerase and an alternating co-polymer of poly(dA-dT) as a template was used to determine the intraceUular TTP pools in the presence of excess [3H]dATP [25]. The reaction mixture contained (200 tal final vol.) 0.1 tag poly(dA-dT); 44 tamol MgC12; 11 tam Tris-HC1 (pH = 8.3); 1.0 Unit DNA polymerase; 140 tamol [3H]dATP (0.5 taCi) and 0 - 2 0 pmol dTTP. Assay mixtures were incubated at 37°C for 2 5 - 3 0 min and the reaction terminated by placing the tubes in ice. A lO0-tal aliquot was spotted onto 5-cm2 Whatman 3MM filter paper and allowed to dry at room temperature. The filter papers were subsequently thoroughly washed with 5% trichloroacetic acid containing 1% inorganic pyrophosphate, 95% ethanol and ether and placed in
287 scintillation vials. Radioactivity was determined using OCS (Amersham Corp. Arlington Heights, IL). The deoxyribonucleotides were extracted from the hepatocytes by the addition of 400 mM perchloric acid immediately after medium aspiration and incubated at 0°C for 20 min [26]. Following a 10-min centrifugation at 3000 g the supernatant was neutralized by the addition of 0.5 M alamine 336 in freon 113. The aqueous phase was separated by centrifugation at 500 g for 20 min, lyophilized and stored at --20°C.
Measurement o f DNA damage by alkaline elution Following the exposure to bleomycin the hepatocytes were washed with PBS: 10 mM NEM and suspended in 100 mM citric acid at a concentration of 2 - 4 X l0 s cells/ml. The suspension was homogenized and underlaid with an equal volume of 340 mM sucrose. The nuclei were sedimented by centrifugation at 5000 g for 10 min and resuspended in 10 ml PBS. 14C-Labelled calf thymus DNA was added as a control for DNase degradation during elution. The nuclei were sedimented onto 47-mm Nucleopore filters (2-tam pore size) and washed with 20 ml PBS at a flow rate of 12 ml/h [27]. Lysis of nuclei was executed at room temperature and elution of damaged DNA was performed as described [28]. The DNA content of the fractions and amount of DNA remaining on the filters was assayed by a fluorometric procedure using Hoechst dye 33258 and a Turner fluorometer [291.
Isolation o f hepatocyte nuclei Nuclei were isolated from young and old rat livers as previously described [30]. The washed nuclei were suspended in 0.25 M sucrose and used immediately for assay. Aliquots of nuclei were removed for DNA determination.
Incorporation of[ all] TTP by isolated nuclei The reaction mixture, 0.5 ml final volume, contained 50 lamol Tris-HC1 (pH 7.5); 2 /amol MgC12; 2 #mol 2-mercaptoethanol; 80/~mol KC1; 1/amol ATP; 0.04 #mol each dATP, dCTP, and dGTP; and 0.02/amol [aH]TTP (50/aCi//amol). Nuclei (75-100/ag DNA) were added after preincubation of the tubes for 2 min at 37°C. The reaction was stopped by the addition of 1 ml 1 M NaOH and radioactivity determined as previously described [30].
DNA extraction and sucrose density centrifugation Nuclei were incubated in groups of eight identical tubes, each containing 150-200/ag DNA in 1.0 ml reaction mixture in the presence or absence of bleomycin as previously described [30]. DNA was extracted essentially according to Zamenhof [31]. Linear 5-20% neutral sucrose gradients were prepared according to Saito and Andoh [32]. Three gradients were prepared simultaneously and 2.5-3.0 absorbance units of DNA were layered on each gradient. Centrifugation and collection of fractions were as previously described [30]. Recovery of DNA was 80%.
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289 RESULTS The preparation of primary hepatocyte cultures in monolayers from young or adult rats has been well established [ 1 8 , 3 3 - 3 6 ] . Few comparative data are available, however, on hepatocytes isolated from old rats. In order to obtain meaningful results depending on a comparison of hepatocytes from young and old rats, morphological and biochemical characteristics have to be compared. Typical hepatocyte cultures from young and old rats are shown in Fig, 1. The cultures were essentially free of fibroblasts or Kupffer cells as 99% o f the cells stained for glucose-6-phosphatase [37]. Some biochemical characteristics are shown in Table I. Total protein synthesis, dTTP pool sizes, and [3H]thymidine incorporation in the presence o f 10 mM hydroxyurea were not statistically different in young and old cells. Protein content, DNA content, DNA synthesis in the absence of hydroxyurea, and albumin synthesis on the other hand were significantly different. Both the DNA and protein concentration values may be a reflection o f a higher proportion o f multinucleated hepatocytes in old rats. An increase in albumin synthesis in hepatocytes from old rats has previously been reported by several investigators [ 3 - 5 , 3 8 ] . The addition o f bleomycin to the hepatocyte cultures in the presence o f hydroxyurea resulted in a dramatic increase in [3H]thymidine incorporation as shown in Fig. 2. Increased incorporation was linear for at least 2 h in the presence of bleomycin and hepatocytes from old rats incorporated more label than cells from young rats suggesting
TABLE I BIOCHEMICAL CHARACTERISTICS OF PRIMARY HEPATOCYTES FROM YOUNG AND OLD RATS 48 h AFTER ISOLATION The methods used for the indicated determinations have been described in "Materials and Methods." All determinations were done on at least 3 different hepatocyte cultures + S.E. Young Protein content (mg/10* cells) Protein synthesis (nmol [SH]leucine/h/10* cells Albumin synthesis (#g/mg protein) DNA content 0zg/10* cells) DNA synthesis (pmol [SH]thymidine/h/10* cells) DNA synthesis + HU (pmol [ SH]thymidine/h/10' cells) TTP pool size (pmol TTP/2 X l0 s cells)
3.60 + 0.3 a
Old 2.3 _+0.4 a
22.00 + 1.0
21.00 + 1.0
82.00 + 8.0 a
112.00 + 7.0 a
33.00 + 8.0 a
45.00 _+6.0a
1.00 _+0.3 a
0.60 _+0.2 a
0.33 _+0.1
0.28 _+0.1
3.20 + 0.3
2.80 _+0.5
a Different values for young and old hepatocyte cultures axe statistically significant with P < 0.01.
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Fig. 2. Bleomycin-induced incorporation of [SH]thymidine in young and old hepatocytes. At 48 h after hepatocyte isolation the medium was replaced. One set of dishes received medium containing 0.01 units/ml bleomycin, 10 mM hydroxyurea and 5 ~Ci/ml [~H]thymidine. In the control dishes bleomycin was omitted. The plotted values were derived by substracting the incorporation without bleomyein from incorporation in the presence of bleomycin. • R, young hepatocytes; o - - - o , old hepatocytes. Individual values are the averages of at least 6 determinations +-S.E.
increased unscheduled DNA synthesis (UDS) in old hepatocytes. The dTTP pool size did not change significantly during bleomycin exposure nor between cells from young or old rats. After 60 rnin of bleomycin exposure we found 3.2 and 3.3 pmol/2 X l0 s cells for old and young hepatocytes respectively. After 2 h of exposure to bleomycin the value
291 was 3.4 pmol/2 × l0 s ceils for both hepatocyte preparations. Since unscheduled DNA synthesis is induced by bleomycin-caused damage to DNA it must be shown that equal amounts of bleomycin cause equal damage to DNA before a comparison of the ability of old or young hepatocytes to respond with unscheduled DNA synthesis to DNA damage can be made. The extent of bleomycin-induced damage was determined by the alkaline elution method [28] and the results are shown in Fig. 3. It is apparent from these results that equal amounts of bleomycin induce many more breaks in the DNA of old hepatocytes than in DNA of young hepatocytes. When the results with [3H]thymidine incorporation in the presence of bleomycin and the results of bleomycin induced breaks were compared and analyzed it was quite apparent that DNA repair synthesis was less efficient in old hepatocytes than in young hepatocytes. These results are shown in Table II. Equal amounts of bleomycin present in young hepatocyte cultures for 2 h caused 1.4 times the damage that it caused when present for 1 h in old hepatocyte cultures. Incorporation of [3H]thymidine following the same exposure conditions to bleomycin was 1.7 times higher in young ceils than in old cells indicating approximately 35% more UDS in young cells than in old cells. The elution curves in Fig. 3 are linear only for the first 30 rain. The reason might be that small DNA fragments elute rapidly and larger fragments more slowly during the last 30 rain. Although we have not tested the effect of increased doses of bleomycin, it seems probable that both the elution rate and the amount of fragments eluted would be greater. To substantiate these findings, we also compared unscheduled DNA synthesis in young and old hepatocytes following UV irradiation. The results of these experiments are shown in Fig. 4 and again indicate a diminished repair capacity for old hepatocytes.
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Fig. 3. Alkaline elution pattern of DNA isolated from young or old hepatocytes exposed to bleomycin. Conditions were as described for Fig. I except that [SH]thymld/ne was omitted. Exposure to bleomycin was for 1 h (I) or 2 h (2), -%DNA from young hepatocytes; o - - - o , DNA from old hepatocytes. The concentration of bleomycin was 0.01 units/mL
292 TABLE II A COMPARISON OF BLEOMYCIN-INDUCED BREAKS IN DNA AND [3H]THYMID1NE INCORPORATION IN YOUNG AND OLD HEPATOCYTES The procedures for the alkaline elution and [SH]thymidine incorporation determination have been described in "Materials and Methods." The values were derived from the data shown in Figs. 2 and 3 for a comparison of DNA damage with unscheduled DNA synthesis.
Age
7~me o f incubation /hi
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[3H] Thymidine incorporation :pmol/mg DNA)
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Fig. 4. Incorporation of [3Hlthymidine into young and old hepatocytes following UV irradiation. The procedure for UV irradiation has been described in "Materials and Methods". Incorporation of control cultures was subtracted from incorporation in UV irradiated cells. • =, young hepatocytes; o - - - o , old hepatocytes. Individual values are the averages of at least 6 diffezent determinations ± S.E.
293 To obtain some insight into the greater susceptibility of old hepatocytes to bleomycin we isolated sucrose nuclei from young and old rat liver and exposed these nuclei to bleomycin thus eliminating possible differences in cellular uptake or cytoplasmic degradation of bleomycin. Hepatocyte nuclei responded with increased DNA synthesis to the presence of bleomycin as can be seen in Fig. 5. Nuclei from young rats responded significantly better than nuclei from old rats, confirming diminished repair capacity in old hepatocytes. The amount of DNA damage induced by bleomycin in nuclei from young and old rats was also compared. As shown in Fig. 6, the addition of bleomycin to nuclei resulted in equal damage in nuclei derived from liver of young or old rats. Our results suggest that, due to increased membrane permeability to bleomycin or a decrease in bleomycin degradation in the cytoplasm, old hepatocytes are more susceptible to bleomycin-induced breaks. Possible differences in DNA or chromatin between
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Fig. 5. Bleomycin-induced incorporation of [SH]thymidine triphosphate into isolated nuclei from young and old rat hepatocytes. The nuclear incorporating system and bleomycin exposure has been described in "Materisls and Methods". -=, nuclei from hepatocytes of young rats; o - - - o , nuclei from hepatocytes of old rats. Individual points are the averages of at least 5 different determinations ± S.E.
294
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Fig. 6. Sucrose density gradient prof'fles of DNA extracted from young and old hepatocyte nuclei incubated with and without bleomycin. The assay, extraction, and gradient procedures have been described in "Materials and Methods". Gradients were centrifuged at 60 000 g for 20 h. A, DNA from nuclei of young animals; B, DNA from nuclei of old rats. X X, incubated for 30 min no additions; v - - - o , incubated for 30 rain in the presence of 50 pg bleomycin/0.5 ml reaction mixture. young and old hepatocytes do not appear to be responsible for the increased susceptibility of old hepatocytes to bleomycin. Our results also provide evidence, on the basis of three independent procedures, that hepatocytes from old rats are less efficient in DNA repair synthesis than hepatocytes from young rats. DISCUSSION Diminished capacity to repair DNA damage as an organism ages may contribute to altered metabolic events associated with aging as well as to an increased risk of cancer. In the majority of studies DNA repair capacity has been compared in early and late passage cells (in vitro aging) [39--43]. We have used the primary hepatocyte culture system to study DNA repair capacity as related to in vivo aging. Our results clearly indicate a decline in DNA repair capacity of hepatocytes with age. To eliminate early developmental considerations, we have chosen young adult rats, 2 - 3 months old, and compared their hepatocytes to those of old adults, 1 6 - 2 0 months
295 old. Published reports indicate that the greatest change in rat liver cell function and ploidy occurred during the first year of life and our old rats were well beyond that stage. That the hepatocytes isolated from old rats retained some if not all of the characteristics associated with liver cells from old rats was indicated by several biochemical parameters. Old hepatocytes contained less protein and there was more DNA in an equal number of cells. Similar findings for protein content have been reported by Coniglio et aL [44]. An increase in ploidy with age has also been described by Van Bezooijen [9] and Shima and Sugahara [45]. In addition, the old hepatocytes synthesize more albumin than young cells, confirming our previous in rive findings [3-5] and those of Bezooijen in vitro [38]. Total protein synthesis on the other hand was similar in cultures of young and old hepatocytes, again confirming our previous in rive findings [3-5]. In addition to protein synthesis, several other parameters were similar in ceils from young and old rats. This indicates that the two cell preparations are metabolically active and retain many if not all of their in rive characteristics. In all of our culture conditions, when we measured incorporation of [3H]thymidine, the medium was 10 mM in hydroxyurea, According to Trosko and Yager [46], the addition of hydroxyurea at a concentration between 5 and 20 mM inhibits semiconservarive DNA synthesis but does not affect DNA repair synthesis. Hydroxyurea levels as high as 100 mM had only a slight effect on UDS [21]. Under these conditions, background incorporation in the absence of bleomycin or UV irradiation was similar in young and old cultures. Furthermore, there was no statistically significant difference in the dTTP pool size of young and old hepatocytes. In the absence of hydroxyurea and without bleomycin or UV irradiation there was considerably more [aH]thymidine incorporation in young hepatocyte culture (1.0/am/h/lO 6 cells) than in old hepatocytes (0.6/am/h/lO 6 cells). The UV irradiation dose used in our experiments was 192 J/m 2. This is higher than a dose of 75 J/m 2 reported by Yager and Miller [21] but considerably lower than the dose of 1680 J/m 2 used by Plesko and Richardson [17]. Both studies were done with hepatocytes in culture. In fact the latter authors reported that age-related changes in UDS appeared greater at higher doses of UV, e.g. 840 J/m 2 and greater. Our results obtained when we measured UDS in response to bleomycin were at first surprising. As can be seen in Fig. 2, treatment of cells with identical bleomycin concentrations for the same time period resulted in more label being incorporated into old hepatocytes. It was not until we compared the amount of breakage causod by bleomyein in the DNA of young and old cells in culture that the results in Fig. 2 took on a different meaning. It was apparent that bleomycin caused much more damage to DNA of old hepatocytes than of young hepatocytes. As can be seen in Table II it took only 1 h for the same amount of bleomycin to cause approximately the same amount of damage in old cells for which a 2-h exposure was necessary for young cells. If the UDS results are correlated with DNA breaks it becomes apparent that there is less UDS in old cells than in young cells per unit of breaks. At present, we do not know the reason for the more extensive DNA damage in old hepatocytes. One explanation could be a more rapid uptake of bleomycin into old
296 hepatocytes. This seems unlikely since we did not detect a more rapid uptake of the labeled precursors used in our studies and in addition there are several reports indicating an age-related decrease in the uptake and retention of bilirubin, several drugs, steroids, and bromsulfopthalein [ 4 7 - 4 9 ] . Another possibility could be that due to changes in chromatin structure or composition, DNA in old hepatocytes becomes more susceptible to bleomycin. There are several reports on changes in chromatin with age [50,51]. Our results with isolated nuclei seem to rule out this possibility, however. Nuclei, which have their chromatin intact, incurred equal damage to DNA to equal exposure time and equal amounts of bleomycin whether isolated from liver of young or old rats. It appears that a cytoplasmic effect of bleomycin is the most likely explanation. The existence of a hydrolase able to degrade bleomycin has been reported [52]. It is possible that there are higher or more active levels of this enzyme in young hepatocytes. Our results agree with and confirm the findings of several other investigators. Plesko and Richardson [ 17] have reported a decline in UDS in old hepatocytes using short-term suspension cultures from rats of different ages and inducing UDS by UV irradiation. Niedemuller [53] has done in vivo studies inducing DNA repair by the administration of several carcinogens and reported decreased DNA repair in older animals. At present, we do not know what structural or chemical changes in old hepatocytes are responsible for the decline in DNA repair capacity. Hart and Setlow [ 10], using late passage human cells, found a correlation between the fraction of cells able to carry out replicative synthesis and the fraction of cells doing repair synthesis. As semiconservative synthesis is less in the older hepatocytes than in the young cells and repair synthesis is also less in the older cells, a similar association may exist for hepatocytes. To fmd the answers to these questions remains an important objective in aging research. ACKNOWLEDEGMENT This work was supported by National Institute of Health Grant AG 01453. REFERENCES 1 H.J. Curtis, Biological mechanisms underlying aging. Science, 141 (1963) 686-694. 2 L. Szilard, On the nature of the aging process. Proc. Natl. Acad. Sci. USA, 45 (1959) 30-45. 3 P. Ove, M. Obertrader and A. Lansing, Synthesis and degradation of liver proteins in young and old rats. Biochim. Biophys. Acta, 277 (1972) 211-221. 4 J.C. Chen, P. Ove and A.I. Lansing, In Vitro synthesis of microsomal protein and albumin in young and old rats. Biochim. Biophys. Acta, 312 (1973) 593-607. 5 M. Obenrader, J. Chen, P. Ore and A.I. Lansing, Etiology of increased albumin synthesis in old rats. Exp. GerontoL, 9 (1974) 173-180. 6 M. Obenrader, J. Chem, P. Ore and A.I. Lansing, Functional regeneration in liver of old rats after partial hepateetomy. Exp. GerontoL, 9 (1974) 181-190. 7 W.I.P. Mainwaring, The effect of age on protein synthesis in mouse liver. Biochern. J., 113 (1969) 869-878.
297 8 R.E. Beauchene, L.M. Roeder and C.H. Barrows, Jr., The interrelationships of age, tissue protein synthesis and proteinurea. J. Gerontol., 25 (1970) 359-363. 9 C.F.A. Van Bezooijen, Influence of age-related changes in rodent liver morphology and physiology on drug metabolism - A review. Mech. Ageing Dee., 25 (1984) 1 - 2 2 . 10 R.W. Hart and R.B. Seflow, DNA repair in late passage human cells. Mech. Ageing Dee., .5 (1976) 67-77. 11 P.D. Bowman, R.L. Meek and C.W. Daniel, Decreased unscheduled DNA synthesis in nondividing aged WI 38 cells. Mech. Ageing Dee., 5 (1976) 251-257. 12 J.B. Little, J. Epstein and J.R. Williams, Repair of DNA strand breaks in progeric fibroblasts and aging human diploid cells. In P.C. Hanawalt and R.B. Setlow (eds.), Molecular Mechanisms for Repair of DNA, Part B, Plenum Press, New York, 1975, pp. 7 9 3 - 8 0 0 . 13 R.W. Hart and R.B. Setlow, Correlation between deoxyribonucleic acid excision-repair and lifespan in a number of mammalian species. Proc. Natl. Acad. Sci. USA, 71 (1974) 2169-2173. 14 J.R. Williams, J.B. Little, J. Epstein and W. Brown, Altered DNA metabolism in aged and progeric fibroblasts. Adv. Exp. Med. Biol., 61 (1974) 268. 15 LD. Regan and R.B. Setlow, DNA repair in human progeroid cells. Biochem. Biophys. Res. Commun., 59 (1974) 8 5 8 - 8 6 5 . 16 B. Lambert, U. Ringborg and L. Skoog, Age-related decrease of ultraviolet light-induced DNA repair synthesis in human peripheral leukocytes. Cancer Res., 39 (1979) 2792-2795. 17 M.M. Plesko and A. Richardson, Age-related changes in unscheduled DNA synthesis by rat hepatocytes. Biochem. Biophys. Res. Commun., 118 ( 1 9 8 4 ) 7 3 0 - 7 3 5 . 18 P. Seglen, Prepartion of isolated rat liver cells. Methods Cells Biol., 13 (1976) 2 9 - 8 3 . 19 P. Ove, M.L. Coetzee and H.P. Morris, DNA synthesis and the effect of sucrose in nuclei of host liver and Morris hepatomas. Cancer Res., 31 (1971) 1389-1395. 20 K. Burton, Determination of DNA concentration with diphenylamine. Methods EnzymoL, 12B (1968) 163-166. 21 J.D. Yager, Jr. and J.A. Miller, Jr., DNA repair in primary cultures of rat hepatocytes. Cancer Res., 38 (1978) 4385-4394. 22 Bio-Rad Protein assay, Bulletin 1069, February, 1979. 23 C-B. Laurell, Quantitative estimation of protein by electrophoresis in agarose gels containing antibodies. Anal. Biochem., 15 (1966) 4 5 - 5 2 . 24 J. Porath, K. Aspberg, H. Drevin and R. Axen, Preparation of cyanogen-bromide-activated gels. J. Chromatog., 86 (1973) 5 3 - 5 6 . 25 U. Lindberg and L. Skoog, A method for the determination of dATP and dTTP in picomole amounts. Anal. Biochem., 34 (1970) 152-160. 26 D. Hunting and J.F. Henderson, Methods for the determination of deoxyribonucleoside triphosphate concentrations. Methods Cancer Res., 20 (1982) 245-284. 27 Z.M. Iqbai, K.W. Kohn, R.A.G. Ewig and A.J. Fornace, Jr., Single-strand scission and repair of DNA in mammalian cells by bleomycin. Cancer Res., 36 (1976) 3834-3838. 28 K.W. Kohn, L.C. Erickson, R.A.G. Ewig and C.A. Friedman, Fractlonation of DNA from mammalian cells by alkaline elution. Biochemistry, 15 (1976) 4629-4637. 29 D.L. Stout and F.F. Becker, Fluorometric quantitation of single-stranded DNA: A method applicable to the technique of alkaline elution. Anal Biochem., 127 (1982) 302-307. 30 P. Ove and M.L. Coetzee, A difference in bleomycin-induced DNA synthesis between liver nuclei from mature and old rats. Mech. Ageing Dev., 8 (1976) 363-375. 31 S. Zamenhof, Preparation and assay of deoxyribonucleic acid from animal tissues. Methods Enzymol., 3 (1957) 6 9 6 - 7 0 4 . 32 M. Saito and T. Andoh, Breakage of a DNA-protein complex induced by bleomycin and their repair in cultured mouse fibroblasts. Cancer Res., 33 (1973) 1696-1700. 33 G. Michalopoulos, H.D. Cianciulli, A.R. Novotny, A.D. Kligerman, S.C. Strom and R.L. Jirtle, Liver regeneration studies with rat hepatocytes in culture. Cancer Res., 42 (1982) 4673-4682. 34 J.A. McGowan and N.L.R. Bucher, Pyruvate promotion of DNA synthesis in serum-free primary cultures of adult rat hepatocytes. In vitro, 19 (1983) 159-166.
298 35 K. Hasegawa, K. Namai and M. Koga, Induction of DNA synthesis in adult rat hepatocytes cultured in a serum-free medium. Biochem. Biophys. Res., Commun., 95 (1980) 2 4 3 - 2 4 9 . 36 A.E. Sixiea, W. Richards, Y. Tsukada, C.A. Sattler and H.C. Pitot, Fetal phenotypic expression by adult rat hepatocytes on collagen gel/nylon meshes. Proc. Natl. Acad. Sci. USA, 76 (1979) 2 8 3 287. 37 M. Wachstein and E. Meisel, Histochemistry of hepatic phosphatases at physiologic pH. Am. J. Clin. Pathol., 27 (1957) 1 3 - 2 3 . 38 C.F.A. Van Bezooijen, T. Greil and D.L. Knook, Albumin synthesis by liver parenchymal cells isolated from young, adult and old rats. Biochem. Biophys. Res. Commun., 71 (1976) 5 1 3 - 5 1 9 . 39 R.T. Dell'Orco and P.L. Guthrie, Altered protein metabolism in arrested populations of aging human diploid fibroblasts. Mech. Ageing Dev., 5 (1976) 3 9 9 - 4 0 7 . 40 S. Goldstein, The role of DNA repair in aging of cultured fibroblasts from xeroderma pigrnentosum and normal (35655). Proc. Soc. Exp. Biol. Med., 137 (1971) 7 3 0 - 7 3 4 . 41 G.M. Hahn, D. King and S.J. Yang, Quantitative changes in unscheduled DNA synthesis in rat muscle ceils after differentiation. Nature, 230 (1971) 2 4 2 - 2 4 4 . 42 K.T. Wheeler and J.T. Lett, On the possibility that DNA repair is related to age in non-dividing cells. Proc. Natl. Acad. Sci, USA, 71 (1974) 1862-1865. 43 V. Paffenholz, Correlation between DNA repair of embryonic fibroblasts and different life span of 3 inbred mouse strains. Mech. Ageing Dev., 7 (1978) 131-150. 44 J.J. Coniglio, D.S. Liu and A. Richardson, A comparison of protein synthesis by liver parenchymal cells isolated from Fischer F344 rats of various ages. Mech. Ageing Dev., 11 (1979) 7 7 - 9 0 . 45 A. Shima and T. Sugahara, Age-dependent ploidy class changes in mouse hepatocyte nuclei as revealed by feulgen-DNA cytofluorometry. Exp. Gerontol., l l (1976) 193-203. 46 J.E. Trosko and J.D. Yager, A sensitive method to measure physical and chemical carcinogeninduced "unscheduled DNA synthesis" in rapidly dividing eukaryotic cells. Exp. Cell Res., 88 (1974) 4 7 - 5 5 . 47 C.F.A. Van Bezooijen, T. Grell and D.L. Knook, Bromsultophthalein uptake by isolated parenchymal ceils. Biochem. Biophys. Res. Commun., 69 (1976) 3 5 4 - 3 6 1 . 48 F.R. DeLeeuw-lsrael, C.F. Hollander and J.M. Arp-Neeqes, Hepatic storage and maximal biliary excretion of bromosulfophthalein in young and old rats. J. Gerontol., 24 (1969) 140-142. 49 A. Richardson, M.A. Sutter, A. Jayaraj and J.W. Webb, Age-related changes in the ability of hepatocytes to metabolize aflatoxin B-1. In K. Kitani (ed.), Liver and Aging-1982, Liver and Drugs, Elsevier Biomedical Press, Amsterdam, 1982, pp. 119-132. 50 Z.A. Medvedev, M.N. Medvedev and L. Robson, Age-related changes of the pattern of non-histone chromatin proteins from rat and mouse chromatin. Gerontology, 25 (1979) 2 1 9 - 2 2 7 . 51 C. Pieri, I. Zs-Nagy, G. Mazzufferi and C. Giuli, The aging of rat fiver revealed by electron microscopic morphometry. I-Basic parameters. Exp. Gerontol, 10 (1975) 2 9 1 - 3 0 4 . 52 J.S. Lazo and C.J. Humphreys, Lack of metabolism as the biochemical basis of bleomycin-induced pulmonary toxicity. Proc. Natl, Acad. Sci. USA, 80 (1983) 3064-3068. 53 H. Niedermuller, Age dependency of DNA repair in rats after DNA damage by carcinogens. Mech. Ageing Dev., 19 (1982) 2 5 9 - 2 7 1 .
283
A COMPARISON OF DNA REPAIR SYNTHESIS IN PRIMARY HEPATOCYTES FROM YOUNG AND OLD RATS
HAROLD E. KENNAH, II, MONA L. COETZEE and PETER eVE Department of Anatomy and Cell Biology, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261 (U.S.A.)
(Received August 20th, 1984) (Revision received October 30th, 1984) SUMMARY DN~A repair synthesis has been compared in primary hepatocyte cultures obtained from 3-month-old and 16-20-month-old rats. Several morphological and metabolic characteristics were determined to assure cultures of comparable quality. DNA damage was induced by the addition of bleomycin or the exposure of the culture to UV irradiation. DNA repair (unscheduled DI~A synthesis) was determined by measuring [3H]thymidine incorporation. After UV irradiation, there was almost twice as much [aH]thymidine incorporation in ceils obtained from young rats as in those obtained from old rats. Equal amounts of bleomycin resulted in substantially greater damage to DNA in cells from old rats than from young rats. For equal amounts of DNA damage there was again diminished [aH] thymidine incorporation in cells obtained from old rats. Finally equal amounts of bleomycin resulted in equal damage to DNA when the bleomycin was added to isolated rat liver nuclei from young or old rats. Bleomycin treated nuclei from young rats incorporated substantially more [3H]thymidine triphosphate (TTP) than bleomycin treated nuclei from old rats. The results indicate that hepatocytes from old rats are much more susceptible to bleomycin than hepatocytes from young rats and that the capacity for DNA repair synthesis is impaired in hepatocytes from old rats.
Key words: DNA repair; Primary hepatocytes
INTRODUCTION Since the early work of Curtis [ 1] dealing with chromosomal alterations in aging and the .proposal by Szilard [2] that the accumulation of DNA damage in somatic cells might be a significant event in the aging process there have been many investigations dealing 0047-6374/85/$03.30 Printed and Published in Ireland
© 1985 ElsevierScientific Publishers Ireland Ltd.
284 with quantitative and qualitative alterations of proteins as a consequence of aging [3--9]. Changes in protein synthesis may result due to chromosomal alterations or errors accumulating in aging organisms. Such errors or alterations in chromosomes, in cells of old organisms may in turn accumulate as a consequence of impaired repair synthesis of DNA. It is well established that such a reduced DNA repair capacity occurs in cells allowed to age in vitro [ i 0 - 1 3 ] . The work of Williams et al. [14] indicates that in vitro aged Syrian hamster cells are less efficient in repair synthesis than young cells. Similar results were reported by Little et al. [12] using aged human diploid cells. Regan and Setlow [15], on the other hand, found no inherent defect in DNA repair in human diploid fibroblast cell strains developed from patients with "progeroid" syndromes. Lambert et al. [ 16] found a significant age-related decline in DNA repair of peripheral lymphocytes from human subjects exposed to UV irradiation, even though both the correlation coefficient and the rate of decline were low. More recently Plesko and Richardson [17] have expanded those studies to in vivo aging. They have shown that there is a significant decline of UV-induced unscheduled DNA synthesis in hepatocytes isolated from old rats as opposed to young rats. In this report, we confirm the finding of Plesko and Richardson and show in addition that bleomycin-induced unscheduled DNA synthesis is likewise decreased in hepatocytes isolated from old rats despite the finding that hepatocytes isolated from old rats are much more susceptible to bleomycin-induced DNA damage than hepatocytes isolated from young rats. MATERIALS AND METHODS Materials
I_eibovitz L-15 and Swim's S-77 Media, fetal calf serum (FCS) and antibiotic/antimycotic were obtained from GIBCO Grand Island, NY. Collagenase Types I and IV, Soybean trypsin inhibitor, insulin, dexamethasone, 3,3,5'-triiodothyronine (T3), bovine serum albumin (BSA), rat liver albumin (RLA), hydroxyurea (HU) and N-ethylmaleimide (NEM) were from Sigma Chemical Company, St. Louis, MO. [methyl-aH]thymidine (31 Ci/mmol), [8 -3H] deoxyadenosine 5'-triphosphate (17 Ci/mmol) and L-[4,5-aH]leucine (35 Ci/mmol) were obtained from ICN Inc., Irvine CA. Blenoxane (bleomycin sulfate) was manufactured by Bristol Laboratories, Syracuse NY. Poly(dA-dT) was obtained from Miles Laboratories, Elkhart, IN and Micro¢occus luteus DNA polymerase (2.7,7.7) from Pharmacia P-L Biochemicals, Inc. Milwaukee, WI. Animals
Young Sprague-Dawley male rats weighing between 150 and 170 g were purchased from Zivic-Miller, Allison Park, PA. Old male Sprague-Dawley rats were 16-20 months old, weighed between 800 and 1200 g and were from an NIH Aging Colony, Charles River, MA. New Zealand white rabbits were purchased from Hilltop Lab Animals, Inc. Scottdale, PA. Animals were kept in a temperature and light-controlled room and received food and water ad libitum.
285
Isolation o f hepatocytes Hepatc~.ytes were isolated by the in situ 2-step collagenase perfusion technique essentially as described by Seglen [18]. The liver was perfused with 150 ml Swim's S-77 medium (pH = 7.4), containing 10 -6 M insulin, 0.5 mM EGTA and 0.4 U/ml heparin, followed by 200 ml of Swim's S-77 (pH = 7.4) containing 10 -6 M insulin, 10 -3 M CaC12, 0.4 mg/ml collagenase, and 0.2 mg/ml soybean trypsin inhibitor. The medium was gassed with 95% O2/5% CO2. The cells were dispersed with gentle mincing in 100 ml L-15 medium, 20 mM HEPES (pH 7.4), 0.15% glucose, 1% antibiotic and centrifuged at 5 0 g for 3 min. The ceils were washed twice and suspended in the same medium containing in addition 5% FCS, 10 -6 M Ta, 10 -s M dexamethasone and 0.2% BSA. Viability was determined by the trypan blue exclusion test and cell number with a hemocytometer. Only preparations with a viability greater than 80% were used. Cells were suspended at a ceil density of 1-1.5 × 106 cells/ml and 7 ml added per lO0-mm dish. The cultures were maintained at 37°C in an air incubator. At 12-20 h the medium was changed to serum free L-I 5 medium supplemented as above and medium changes were made every 24 h.
, [3H]Thymidine incorporation and DNA determination At 48 h the medium was changed and 5/aCi [aH]thyrnidine/ml medium were added to each dish. At 1 or 2 h later the medium was removed and the cells washed three times with 10 ml cold PBS. The cells were then scraped into a known volume of PBS and 0 . 1 0.2-ml aliquots removed for cell number determination using a Coulter counter. All samples were counted in triplicate with the mean value used in all calculations. The remaining cells were pelleted and 1 ml 1 M NaOH was added and heated at 80°C for 10 min. The samples were cooled, brought to 5% trichloroacetic acid and centrifuged at 10 0 0 0 g for 10 min. The pellet was redissolved in 2.0 ml 1 M NaOH and heated at 80°C for 10 min. One milliliter was used for the determination of radioactivity as previously described [19] and 1 ml for DNA determination by the diphenylamine method as described by Burton [20].
Bleomycin-induced [ 3H] thymidine incorporation At 4 0 - 4 8 h hepatocyte cultures were given a serum-free medium change with the following additions; 10 mM hydroxyurea, 0.01 units bleomycin and 5/aCi [3H]thymidine/ml. The cells were incubated at 37°C in an air incubator for various time periods. At the completion of the incubation period, the medium was removed and the cells washed twice with 10 ml PBS containing 10 mM NEM. [aH]Thymidine incorporation and DNA determination were performed as described above.
UV irradiation-induced [ 3H] thymidine incorporation At 4 0 - 4 8 h, the medium was removed and the hepatocyte monolayers were exposed to 480/~W/cm 2 of UV irradiation at 254 nm f o r 4 0 sec [21]. The UV source was a UVS54 Mineralight (Ultraviolet Products Inc., San Gabriel, CA). Following irradiation, fresh
286 serum-free medium containing 10 mM HU and 5 taCi/ml [3H]thymidine was added. Control cultures were deprived of medium for the prescribed exposure period. Following incubation for the specified time the cells were harvested for [3H]thymidine incorporation and DNA determination as described above.
[ 3H]L eucine incogporation and pro tein content determination At 48 h, the medium was changed and 1.0 taCi [3H]leucine was added per ml and the ceils harvested 1-2 h later. Aliquots were removed for cell number determination. The remaining cells were pelleted and suspended in 2.0 ml 1 M NaOH and heated at 80°C for 10 min. One milliliter was brought to 5% trichloroacetic acid for the determination of radioactivity as previously described [20] using instead a chloroform/methanol (2: 1) wash. The remaining 1 ml was brought to 5% trichloroacetic acid for protein determination. The celite filter pad was heated at 80°C for 20 min in 1 ml 1 M NaOH and an aliquot was used in the Bio-Rad Protein assay [22]. Determination of secreted albumin Albumin secretion from cultured hepatocytes was quantitated by Rocket Immunoelectrophoresis [23]. The method utilizes 1.0% agarose gels, cast and run at 2 V/cm in Tris-barbital-sodium barbital buffer (pH = 8.8). The gels contained a monospecific antibody (0.05 tag/ml) against rat liver albumin (RLA). Anti-serum was produced in New Zealand white rabbits by bi-weekly intradermal injections of 500 tag/ml RLA. Sera which produced precipitation lines on Ouchterlony double diffusion was pooled and precipitated by 40% ammonium sulfate saturation [24]. Monospecific RLA-antibody was prepared by affinity chromatography. Sigma RLA was purified by Sephadex G-100 and DEAE cellulose chromatography to yield a single band on SDS polyacrylamide gel electrophoresis. The purified RLA was bound to cyanogen bromide activated Sepharose 4B and the antisera applied in 10 mM NaPO4 (pH 9.0)/ 150 mM NaC1. Following extensive washing of the column, the monospecific rabbit IgG anti-RLA was eluted using 200 mM glycine/500 mM NaC1 (pH = 2.8). The sample was dialyzed, lyophilized, and stored at -20°C. Measurement of dTTP pool size An enzymatic assay, utilizing DNA polymerase and an alternating co-polymer of poly(dA-dT) as a template was used to determine the intraceUular TTP pools in the presence of excess [3H]dATP [25]. The reaction mixture contained (200 tal final vol.) 0.1 tag poly(dA-dT); 44 tamol MgC12; 11 tam Tris-HC1 (pH = 8.3); 1.0 Unit DNA polymerase; 140 tamol [3H]dATP (0.5 taCi) and 0 - 2 0 pmol dTTP. Assay mixtures were incubated at 37°C for 2 5 - 3 0 min and the reaction terminated by placing the tubes in ice. A lO0-tal aliquot was spotted onto 5-cm2 Whatman 3MM filter paper and allowed to dry at room temperature. The filter papers were subsequently thoroughly washed with 5% trichloroacetic acid containing 1% inorganic pyrophosphate, 95% ethanol and ether and placed in
287 scintillation vials. Radioactivity was determined using OCS (Amersham Corp. Arlington Heights, IL). The deoxyribonucleotides were extracted from the hepatocytes by the addition of 400 mM perchloric acid immediately after medium aspiration and incubated at 0°C for 20 min [26]. Following a 10-min centrifugation at 3000 g the supernatant was neutralized by the addition of 0.5 M alamine 336 in freon 113. The aqueous phase was separated by centrifugation at 500 g for 20 min, lyophilized and stored at --20°C.
Measurement o f DNA damage by alkaline elution Following the exposure to bleomycin the hepatocytes were washed with PBS: 10 mM NEM and suspended in 100 mM citric acid at a concentration of 2 - 4 X l0 s cells/ml. The suspension was homogenized and underlaid with an equal volume of 340 mM sucrose. The nuclei were sedimented by centrifugation at 5000 g for 10 min and resuspended in 10 ml PBS. 14C-Labelled calf thymus DNA was added as a control for DNase degradation during elution. The nuclei were sedimented onto 47-mm Nucleopore filters (2-tam pore size) and washed with 20 ml PBS at a flow rate of 12 ml/h [27]. Lysis of nuclei was executed at room temperature and elution of damaged DNA was performed as described [28]. The DNA content of the fractions and amount of DNA remaining on the filters was assayed by a fluorometric procedure using Hoechst dye 33258 and a Turner fluorometer [291.
Isolation o f hepatocyte nuclei Nuclei were isolated from young and old rat livers as previously described [30]. The washed nuclei were suspended in 0.25 M sucrose and used immediately for assay. Aliquots of nuclei were removed for DNA determination.
Incorporation of[ all] TTP by isolated nuclei The reaction mixture, 0.5 ml final volume, contained 50 lamol Tris-HC1 (pH 7.5); 2 /amol MgC12; 2 #mol 2-mercaptoethanol; 80/~mol KC1; 1/amol ATP; 0.04 #mol each dATP, dCTP, and dGTP; and 0.02/amol [aH]TTP (50/aCi//amol). Nuclei (75-100/ag DNA) were added after preincubation of the tubes for 2 min at 37°C. The reaction was stopped by the addition of 1 ml 1 M NaOH and radioactivity determined as previously described [30].
DNA extraction and sucrose density centrifugation Nuclei were incubated in groups of eight identical tubes, each containing 150-200/ag DNA in 1.0 ml reaction mixture in the presence or absence of bleomycin as previously described [30]. DNA was extracted essentially according to Zamenhof [31]. Linear 5-20% neutral sucrose gradients were prepared according to Saito and Andoh [32]. Three gradients were prepared simultaneously and 2.5-3.0 absorbance units of DNA were layered on each gradient. Centrifugation and collection of fractions were as previously described [30]. Recovery of DNA was 80%.
288
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289 RESULTS The preparation of primary hepatocyte cultures in monolayers from young or adult rats has been well established [ 1 8 , 3 3 - 3 6 ] . Few comparative data are available, however, on hepatocytes isolated from old rats. In order to obtain meaningful results depending on a comparison of hepatocytes from young and old rats, morphological and biochemical characteristics have to be compared. Typical hepatocyte cultures from young and old rats are shown in Fig, 1. The cultures were essentially free of fibroblasts or Kupffer cells as 99% o f the cells stained for glucose-6-phosphatase [37]. Some biochemical characteristics are shown in Table I. Total protein synthesis, dTTP pool sizes, and [3H]thymidine incorporation in the presence o f 10 mM hydroxyurea were not statistically different in young and old cells. Protein content, DNA content, DNA synthesis in the absence of hydroxyurea, and albumin synthesis on the other hand were significantly different. Both the DNA and protein concentration values may be a reflection o f a higher proportion o f multinucleated hepatocytes in old rats. An increase in albumin synthesis in hepatocytes from old rats has previously been reported by several investigators [ 3 - 5 , 3 8 ] . The addition o f bleomycin to the hepatocyte cultures in the presence o f hydroxyurea resulted in a dramatic increase in [3H]thymidine incorporation as shown in Fig. 2. Increased incorporation was linear for at least 2 h in the presence of bleomycin and hepatocytes from old rats incorporated more label than cells from young rats suggesting
TABLE I BIOCHEMICAL CHARACTERISTICS OF PRIMARY HEPATOCYTES FROM YOUNG AND OLD RATS 48 h AFTER ISOLATION The methods used for the indicated determinations have been described in "Materials and Methods." All determinations were done on at least 3 different hepatocyte cultures + S.E. Young Protein content (mg/10* cells) Protein synthesis (nmol [SH]leucine/h/10* cells Albumin synthesis (#g/mg protein) DNA content 0zg/10* cells) DNA synthesis (pmol [SH]thymidine/h/10* cells) DNA synthesis + HU (pmol [ SH]thymidine/h/10' cells) TTP pool size (pmol TTP/2 X l0 s cells)
3.60 + 0.3 a
Old 2.3 _+0.4 a
22.00 + 1.0
21.00 + 1.0
82.00 + 8.0 a
112.00 + 7.0 a
33.00 + 8.0 a
45.00 _+6.0a
1.00 _+0.3 a
0.60 _+0.2 a
0.33 _+0.1
0.28 _+0.1
3.20 + 0.3
2.80 _+0.5
a Different values for young and old hepatocyte cultures axe statistically significant with P < 0.01.
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Fig. 2. Bleomycin-induced incorporation of [SH]thymidine in young and old hepatocytes. At 48 h after hepatocyte isolation the medium was replaced. One set of dishes received medium containing 0.01 units/ml bleomycin, 10 mM hydroxyurea and 5 ~Ci/ml [~H]thymidine. In the control dishes bleomycin was omitted. The plotted values were derived by substracting the incorporation without bleomyein from incorporation in the presence of bleomycin. • R, young hepatocytes; o - - - o , old hepatocytes. Individual values are the averages of at least 6 determinations +-S.E.
increased unscheduled DNA synthesis (UDS) in old hepatocytes. The dTTP pool size did not change significantly during bleomycin exposure nor between cells from young or old rats. After 60 rnin of bleomycin exposure we found 3.2 and 3.3 pmol/2 X l0 s cells for old and young hepatocytes respectively. After 2 h of exposure to bleomycin the value
291 was 3.4 pmol/2 × l0 s ceils for both hepatocyte preparations. Since unscheduled DNA synthesis is induced by bleomycin-caused damage to DNA it must be shown that equal amounts of bleomycin cause equal damage to DNA before a comparison of the ability of old or young hepatocytes to respond with unscheduled DNA synthesis to DNA damage can be made. The extent of bleomycin-induced damage was determined by the alkaline elution method [28] and the results are shown in Fig. 3. It is apparent from these results that equal amounts of bleomycin induce many more breaks in the DNA of old hepatocytes than in DNA of young hepatocytes. When the results with [3H]thymidine incorporation in the presence of bleomycin and the results of bleomycin induced breaks were compared and analyzed it was quite apparent that DNA repair synthesis was less efficient in old hepatocytes than in young hepatocytes. These results are shown in Table II. Equal amounts of bleomycin present in young hepatocyte cultures for 2 h caused 1.4 times the damage that it caused when present for 1 h in old hepatocyte cultures. Incorporation of [3H]thymidine following the same exposure conditions to bleomycin was 1.7 times higher in young ceils than in old cells indicating approximately 35% more UDS in young cells than in old cells. The elution curves in Fig. 3 are linear only for the first 30 rain. The reason might be that small DNA fragments elute rapidly and larger fragments more slowly during the last 30 rain. Although we have not tested the effect of increased doses of bleomycin, it seems probable that both the elution rate and the amount of fragments eluted would be greater. To substantiate these findings, we also compared unscheduled DNA synthesis in young and old hepatocytes following UV irradiation. The results of these experiments are shown in Fig. 4 and again indicate a diminished repair capacity for old hepatocytes.
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Fig. 3. Alkaline elution pattern of DNA isolated from young or old hepatocytes exposed to bleomycin. Conditions were as described for Fig. I except that [SH]thymld/ne was omitted. Exposure to bleomycin was for 1 h (I) or 2 h (2), -%DNA from young hepatocytes; o - - - o , DNA from old hepatocytes. The concentration of bleomycin was 0.01 units/mL
292 TABLE II A COMPARISON OF BLEOMYCIN-INDUCED BREAKS IN DNA AND [3H]THYMID1NE INCORPORATION IN YOUNG AND OLD HEPATOCYTES The procedures for the alkaline elution and [SH]thymidine incorporation determination have been described in "Materials and Methods." The values were derived from the data shown in Figs. 2 and 3 for a comparison of DNA damage with unscheduled DNA synthesis.
Age
7~me o f incubation /hi
% DNA eluted in 60 min
[3H] Thymidine incorporation :pmol/mg DNA)
Young Young Old Old
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0 10.5 7.6 21.8
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Fig. 4. Incorporation of [3Hlthymidine into young and old hepatocytes following UV irradiation. The procedure for UV irradiation has been described in "Materials and Methods". Incorporation of control cultures was subtracted from incorporation in UV irradiated cells. • =, young hepatocytes; o - - - o , old hepatocytes. Individual values are the averages of at least 6 diffezent determinations ± S.E.
293 To obtain some insight into the greater susceptibility of old hepatocytes to bleomycin we isolated sucrose nuclei from young and old rat liver and exposed these nuclei to bleomycin thus eliminating possible differences in cellular uptake or cytoplasmic degradation of bleomycin. Hepatocyte nuclei responded with increased DNA synthesis to the presence of bleomycin as can be seen in Fig. 5. Nuclei from young rats responded significantly better than nuclei from old rats, confirming diminished repair capacity in old hepatocytes. The amount of DNA damage induced by bleomycin in nuclei from young and old rats was also compared. As shown in Fig. 6, the addition of bleomycin to nuclei resulted in equal damage in nuclei derived from liver of young or old rats. Our results suggest that, due to increased membrane permeability to bleomycin or a decrease in bleomycin degradation in the cytoplasm, old hepatocytes are more susceptible to bleomycin-induced breaks. Possible differences in DNA or chromatin between
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Fig. 5. Bleomycin-induced incorporation of [SH]thymidine triphosphate into isolated nuclei from young and old rat hepatocytes. The nuclear incorporating system and bleomycin exposure has been described in "Materisls and Methods". -=, nuclei from hepatocytes of young rats; o - - - o , nuclei from hepatocytes of old rats. Individual points are the averages of at least 5 different determinations ± S.E.
294
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Fig. 6. Sucrose density gradient prof'fles of DNA extracted from young and old hepatocyte nuclei incubated with and without bleomycin. The assay, extraction, and gradient procedures have been described in "Materials and Methods". Gradients were centrifuged at 60 000 g for 20 h. A, DNA from nuclei of young animals; B, DNA from nuclei of old rats. X X, incubated for 30 min no additions; v - - - o , incubated for 30 rain in the presence of 50 pg bleomycin/0.5 ml reaction mixture. young and old hepatocytes do not appear to be responsible for the increased susceptibility of old hepatocytes to bleomycin. Our results also provide evidence, on the basis of three independent procedures, that hepatocytes from old rats are less efficient in DNA repair synthesis than hepatocytes from young rats. DISCUSSION Diminished capacity to repair DNA damage as an organism ages may contribute to altered metabolic events associated with aging as well as to an increased risk of cancer. In the majority of studies DNA repair capacity has been compared in early and late passage cells (in vitro aging) [39--43]. We have used the primary hepatocyte culture system to study DNA repair capacity as related to in vivo aging. Our results clearly indicate a decline in DNA repair capacity of hepatocytes with age. To eliminate early developmental considerations, we have chosen young adult rats, 2 - 3 months old, and compared their hepatocytes to those of old adults, 1 6 - 2 0 months
295 old. Published reports indicate that the greatest change in rat liver cell function and ploidy occurred during the first year of life and our old rats were well beyond that stage. That the hepatocytes isolated from old rats retained some if not all of the characteristics associated with liver cells from old rats was indicated by several biochemical parameters. Old hepatocytes contained less protein and there was more DNA in an equal number of cells. Similar findings for protein content have been reported by Coniglio et aL [44]. An increase in ploidy with age has also been described by Van Bezooijen [9] and Shima and Sugahara [45]. In addition, the old hepatocytes synthesize more albumin than young cells, confirming our previous in rive findings [3-5] and those of Bezooijen in vitro [38]. Total protein synthesis on the other hand was similar in cultures of young and old hepatocytes, again confirming our previous in rive findings [3-5]. In addition to protein synthesis, several other parameters were similar in ceils from young and old rats. This indicates that the two cell preparations are metabolically active and retain many if not all of their in rive characteristics. In all of our culture conditions, when we measured incorporation of [3H]thymidine, the medium was 10 mM in hydroxyurea, According to Trosko and Yager [46], the addition of hydroxyurea at a concentration between 5 and 20 mM inhibits semiconservarive DNA synthesis but does not affect DNA repair synthesis. Hydroxyurea levels as high as 100 mM had only a slight effect on UDS [21]. Under these conditions, background incorporation in the absence of bleomycin or UV irradiation was similar in young and old cultures. Furthermore, there was no statistically significant difference in the dTTP pool size of young and old hepatocytes. In the absence of hydroxyurea and without bleomycin or UV irradiation there was considerably more [aH]thymidine incorporation in young hepatocyte culture (1.0/am/h/lO 6 cells) than in old hepatocytes (0.6/am/h/lO 6 cells). The UV irradiation dose used in our experiments was 192 J/m 2. This is higher than a dose of 75 J/m 2 reported by Yager and Miller [21] but considerably lower than the dose of 1680 J/m 2 used by Plesko and Richardson [17]. Both studies were done with hepatocytes in culture. In fact the latter authors reported that age-related changes in UDS appeared greater at higher doses of UV, e.g. 840 J/m 2 and greater. Our results obtained when we measured UDS in response to bleomycin were at first surprising. As can be seen in Fig. 2, treatment of cells with identical bleomycin concentrations for the same time period resulted in more label being incorporated into old hepatocytes. It was not until we compared the amount of breakage causod by bleomyein in the DNA of young and old cells in culture that the results in Fig. 2 took on a different meaning. It was apparent that bleomycin caused much more damage to DNA of old hepatocytes than of young hepatocytes. As can be seen in Table II it took only 1 h for the same amount of bleomycin to cause approximately the same amount of damage in old cells for which a 2-h exposure was necessary for young cells. If the UDS results are correlated with DNA breaks it becomes apparent that there is less UDS in old cells than in young cells per unit of breaks. At present, we do not know the reason for the more extensive DNA damage in old hepatocytes. One explanation could be a more rapid uptake of bleomycin into old
296 hepatocytes. This seems unlikely since we did not detect a more rapid uptake of the labeled precursors used in our studies and in addition there are several reports indicating an age-related decrease in the uptake and retention of bilirubin, several drugs, steroids, and bromsulfopthalein [ 4 7 - 4 9 ] . Another possibility could be that due to changes in chromatin structure or composition, DNA in old hepatocytes becomes more susceptible to bleomycin. There are several reports on changes in chromatin with age [50,51]. Our results with isolated nuclei seem to rule out this possibility, however. Nuclei, which have their chromatin intact, incurred equal damage to DNA to equal exposure time and equal amounts of bleomycin whether isolated from liver of young or old rats. It appears that a cytoplasmic effect of bleomycin is the most likely explanation. The existence of a hydrolase able to degrade bleomycin has been reported [52]. It is possible that there are higher or more active levels of this enzyme in young hepatocytes. Our results agree with and confirm the findings of several other investigators. Plesko and Richardson [ 17] have reported a decline in UDS in old hepatocytes using short-term suspension cultures from rats of different ages and inducing UDS by UV irradiation. Niedemuller [53] has done in vivo studies inducing DNA repair by the administration of several carcinogens and reported decreased DNA repair in older animals. At present, we do not know what structural or chemical changes in old hepatocytes are responsible for the decline in DNA repair capacity. Hart and Setlow [ 10], using late passage human cells, found a correlation between the fraction of cells able to carry out replicative synthesis and the fraction of cells doing repair synthesis. As semiconservative synthesis is less in the older hepatocytes than in the young cells and repair synthesis is also less in the older cells, a similar association may exist for hepatocytes. To fmd the answers to these questions remains an important objective in aging research. ACKNOWLEDEGMENT This work was supported by National Institute of Health Grant AG 01453. REFERENCES 1 H.J. Curtis, Biological mechanisms underlying aging. Science, 141 (1963) 686-694. 2 L. Szilard, On the nature of the aging process. Proc. Natl. Acad. Sci. USA, 45 (1959) 30-45. 3 P. Ove, M. Obertrader and A. Lansing, Synthesis and degradation of liver proteins in young and old rats. Biochim. Biophys. Acta, 277 (1972) 211-221. 4 J.C. Chen, P. Ove and A.I. Lansing, In Vitro synthesis of microsomal protein and albumin in young and old rats. Biochim. Biophys. Acta, 312 (1973) 593-607. 5 M. Obenrader, J. Chen, P. Ore and A.I. Lansing, Etiology of increased albumin synthesis in old rats. Exp. GerontoL, 9 (1974) 173-180. 6 M. Obenrader, J. Chem, P. Ore and A.I. Lansing, Functional regeneration in liver of old rats after partial hepateetomy. Exp. GerontoL, 9 (1974) 181-190. 7 W.I.P. Mainwaring, The effect of age on protein synthesis in mouse liver. Biochern. J., 113 (1969) 869-878.
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