A method for rapid, sensitive quantitation of short-patch DNA repair in cultured rat hepatocytes

Mutation Research, 119 (1983) 381-386 Elsevier Biomedical Press

381

A method for rapid, sensitive quantitation of short-patch DNA repair in cultured rat hepatocytes Michael J. Olson*, Daniel A. Casciano and Joel G. P o u n d s National Center f o r Toxicological Research, Jefferson, A R 72079 and the Division o f Interdisciplinary Toxicology, University o f Arkansas f o r Medical Sciences, 4301 West Markham Street, Little Rock, A R 72201 (U,S.A.) (Accepted 7 September 1982)

Summary The sensitivity of DNA-repair detection after incubation of cultured rat hepatocytes with dimethylnitrosamine (DMN) and [Me-l',2'-3H]thymidine ([3H]TdR) was enhanced by the preparation of isolated nuclei. Nuclear isolationliquid scintillation counting (NI-LSC) provided a rapid method for the determination of unscheduled DNA synthesis (UDS). DMN-induced UDS detected by autoradiography and NI-LSC correlated well and provided similar dose-response curves, indicating the utility of the NI-LSC method for the quantitation of shortpatch DNA repair.

Isolated rat hepatocytes metabolize chemical carcinogens to reactive forms and the spectrum of DNA adducts generated and the kinetics of adduct removal are similar to that found in the liver in vivo (Howard et al., 1981; Umbenhauer and Pegg, 1981). Primary cultures of rat hepatocytes have been used for the detection of DNA repair following a variety of chemical and physical insults (Casciano et al., 1978; Yager and Miller, 1978; Williams and Laspia, 1979). Attempts at rapid LSC quantitation of UDS in response to agents inducing short-patch D N A repair (Michalopoulos et al., 1978) had limited sensitivity due to thymidine catabolism and binding of thymidine to nonnuclear elements (Yager and Miller, 1978). Modifications designed to improve the sensitivity of the LSC technique for UDS have included density gradient isolation of DNA from cellular homogenates (Michalopoulos et al., 1978; Yager and Miller, 1978) and isolation of nuclei from broken cells followed by extraction of DNA (Hsia and Kreamer, 1979; Althaus et * Present address: Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC 27514 (U.S.A.). 0165-7992/83/0000-0000/$03.00 © Elsevier Science Publishers

382 al., 1980). Isolation of nuclei by centrifugation techniques provided a high yield of DNA from small amounts of tissue homogenate (Blobel and Potter, 1966) and may be performed more quickly than gradient purification or chemical extraction methods. The detection of UDS induced by low doses of DMN has proven difficult owing to the small quantity of [3H]TdR inserted during the repair of DMN-induced DNA lesions (Michalopoulos et al., 1978). Our report details the improved sensitivity of UDS detection by the NI-LSC method using DMN as a model short-patch repair inducing agent which requires metabolic activation.

Materials and methods

Reagents. [3H]TdR (71.8 Ci/mmole) was supplied by New England Nuclear, Boston (MA). Nuclear track emulsion, NTB-2, was purchased from Eastman Kodak, Rochester (NY). Bio-Labs, Northbrook (IL), supplied Thermonax tissue culture cover slips. Fetal bovine serum (FBS) from KC Biologicals, Lenexa (KS) was used in hepatocyte culture medium. Other cell culture media were obtained from Gibco Laboratories, Grand Island (NY). Gentamicin sulfate was obtained from Schering Corporation, Kenilworth (N J). DMN [CAS 62-75-9] and all other reagents were purchased from Sigma Chemical Co., St. Louis (MO). Rat hepatocyte culture. Hepatocytes were isolated from male Fischer 344 rats (200-300 g) by the procedure of Oldham et al. (1979). Monolayer cultures were established by plating 10 × 106 viable cells/100-mm culture dish in William's medium E (containing 17070 FBS, 2 mM glutamine, 2 mU/ml insulin, 50 #g/ml gentamicin and 10 mM HU; WE). After 1 h of incubation, the medium was changed to WE with 1007o FBS and 1 /~Ci [3H]TdR/ml. DMN in saline solution was added as required to yield concentrations ranging from 0.1 to 10.0 mM. Preparation of nuclei. The hepatocytes were scraped from the entire surface of each dish at 4 or 24 h of culture and pelleted by centrifugation. The cell pellets were washed twice with 5 ml of cold 1 × Hank's balanced salt solution containing 0.1 mM thymidine (HBSS + TdR) and suspended in 3 ml of 10 mM Tris-HCl, 5 mM MgCI2, pH 8.0. After 15 min on ice, the cell suspensions were subjected to two homogenizations of 10 sec each (Polytron Kinematica GMBH, Lucerne, Switzerland; power setting 7). Three 0. l-ml aliquots of the whole cell homogenate were removed for quantitation of DNA and [3H]TdR. The remaining homogenate was centrifuged at 800 g for 10 min to produce a crude nuclear fraction. The 800 g pellet was suspended in 3.3 ml of 1.62 M sucrose (buffered with 50 mM Tris-HC1, 25 mM KC1, 5 mM MgCI2, pH 8.0; TKM) and sampled for quantitation of DNA and [3H]TdR as before. Purified nuclei were then prepared by the discontinuous sucrose gradient method of Blobel and Potter (1966). Briefly, 3 ml of the suspension prepared from the 800 g pellet was placed in a 1/2 × 2 in polyallomer tube and 1 ml of buffered 2.3 M sucrose was injected beneath the 1.62 M sucrose solution. The gradients were

383 then centrifuged at 124 000 g for 30 min. The pellet resulting from this centrifugation was suspended in 1 ml of TKM buffer and 0.1 ml aliquots were assayed for [3H]TdR incorporation into DNA. LSC determination of UDS. 0.1 ml of a 1o70 sodium dodecyl sulfate solution was added to each of the 0.1-ml samples collected for DNA and [3H]TdR determination. Following a 30-min incubation at 37 °, the samples were made 10010 perchloroacetic acid (PCA) and placed on ice for 30 min. The resulting precip!tate was collected by centrifugation at 1000 g for 15 min, washed twice with 1 ml of 5°70 PCA, once with 1 ml of 70°70 ethanol and dried under nitrogen. The dried precipitate was suspended in 1 ml of 5°70 PCA ano hydrolyzed in a 90 ° water bath for 20 min. The hydrolysate was cooled to room temperature and clarified by centrifugation at 1000 g for 15 min. 0.25-ml aliquots of the clear PCA hydrolysate were taken for LSC with 10 ml of Scintisol (Isolab, Inc., Akron, OH) and the DNA content of the hydrolysate was determined by the method of Burton (1956). Results were calculated as DNA specific activity (dpm 3H/ttg DNA). Autoradiographic determination of UDS. Monolayer cultures of hepatocytes on Thermanox coverslips were prepared for autoradiography as described by Oldham et al. (1980), with the exception that the emulsion exposure period was 10 days. After developing the silver grains and Giemsa staining, the grains over 50 randomly selected cells per slide were counted. The data were derived by averaging the cytoplasmic grains appearing in 2 areas the diameter of a nucleus and subtracting this value from the number of nuclear grains to obtain the corrected nuclear grain count.

Results and discussion

DNA yield. Analysis of the DNA yield at each step of preparation indicated that when 1 × 106-2.5 × 106 cells from the same dish are used, the recovery of DNA in the 800 g fraction was 91 _+ 907o of the amount in the whole cell homogenate (x ___ SD of 3 Expts.). The final nuclear pellet contained 71 + 6°7o of the initial DNA. The DNA recovery decreased rapidly when more than 2.5 × 106 cells were applied to the gradients (application of 5 × 106 hepatocytes resulted in only 21 + 4°7orecovery of DNA). Therefore, in experiments using the NI-LSC method for detecting UDS (Table 1, Figs. 1 and 2), the hepatocytes from one dish were divided into aliquots containing 2.5 × 106 cells before preparation of nuclei. Effect of nuclear preparation on detection of UDS. Each step in the preparation of nuclei resulted in the loss of non-DNA associated [3H]TdR (Table 1). The sensitivity of detecting DNA repair was increased in isolated nuclei compared to the whole cell homogenate, as noted by the increase in the relative change in thymidine incorporation with respect to the untreated control for each treatment. In addition, experiments performed in suspension culture (data not shown) and at early time points in monolayer culture (Fig. 1, 4-h curve), suggested that the ability to detect short-

384 TABLE 1 EFFECT OF NUCLEAR PREPARATION ON QUANTITATION OF UDS IN CULTURED RAT HEPATOCYTES The data are expressed as dpm//~g DNA and represent the x + SD of 3 determinations from one typical experiment. Values in parentheses are dpm//~g DNA expressed as % control with 10 mM HU (+ HU). Samples were taken 24 h after addition of DMN and [3H]TdR. Treatment

Whole cell homogenate

800 g pellet

Isolated nuclei

Control ( - HU)

688 + 19 (227)

453 ± 39 (214)

184 + 8 (202)

Control (+ HU)

303 ± 24 (100)

212 ± 16 (100)

91 ± 11 (100)

0.1 mM DMN ( + H U )

288± 14 (95)

202± 12 (95)

115± 4 (126)

1.0mMDMN(+HU)

336±23(111)

258± 9(122)

214± 9(235)

1015±31 (335)

924± 13 (436)

541±21 (595)

10.0 mM DMN ( + H U )

900

1400

.T p

800 0

_/

~ 300 Z

/:

~ 200

lO

1000

TY

Z 0

5 600

6

"o o~

200 100 ~ o

0"/I

I

0 0.1

I

I

1

Concentration

I-

10 (raM)

0.1

I

I

Concentration

10 (mM)

Fig. 1. The dose-dependent induction of UDS by DMN in rat hepatocyte cultures after 4 h (C)) or 24 h ( 0 ) of DMN exposure. UDS was determined in nuclei as described in Materials and Methods and is expressed as a percent of the control ( + HU) specific activity. Each point represents the mean or mean + SEM of 2-5 Expts. Fig. 2. Comparison of NI-LSC ( I ) and autoradiographic ([]) quantitation of UDS induced by DMN in hepatocyte cultures after 24 h of exposure to DMN and [3H]TdR. UDS is expressed as DNA specific activity for the NI-LSC method and corrected grains per nucleus for the autoradiographic method. The data represent 1 Expt. typical of 5 and are expressed as the mean _+ SD of 3 determinations.

385 patch DNA repair resulting from exposure to DMN was increased with longer incubation times. Williams and Laspia (1979) reported a similar effect and ascribed increased UDS to provision for longer repair time and not prolonged DMN metabolism. Inclusion of 10 mM HU in control cultures (Table 1) decreased specific activity 50-60070, consistent with the results of Michalopoulos et al. (1978). Suppression of scheduled DNA synthesis enhanced the sensitivity of repair detection by decreasing the control specific activity to which comparisons were made. DMN-induced UDS. DMN was selected as a model for the induction of shortpatch DNA repair since detection of UDS induced by DMN in previously reported studies was possible only at doses of 1 mM or greater. Thus, if the sensitivity of repair detection following DMN exposure could be enhanced, it would represent a significant improvement in the LSC method for quantitating UDS. In addition, DMN requires metabolic activation to exert its genotoxic effect which makes it appropriate in the metabolically capable primary hepatocyte culture system. The concentration dependence of UDS was evaluated by the NI-LSC method following exposure of hepatocytes to 0.1-10.0 mM DMN for 4 or 24 h (Fig. 1). This range of DMN concentration was not overtly cytotoxic to hepatocytes in primary culture, as defined by the release of the cytosolic enzyme lactate dehydrogenase (LDH) (control LDH release was 23 _+ 4°7o of the total cellular LDH at 24 h, cultures treated with 10 mM DMN had released 26 _ 2°70 of their total LDH following 24 h of DMN exposure; no significant difference) which correlated well with trypan blue dye exclusion and maintenance of cellular viability (Jauregui et al., 1981). In contrast to earlier reports, the repair of DMN-induced DNA damage was detected at the lowest dose tested, 0.1 mM (Michalopoulos et al., 1978; Williams and Laspia, 1979). The ability to quantitate repair at a DMN concentration 10-100-fold lower than previously reported was most probably the result of (1) increased sensitivity due to the use of isolated nuclei and (2) the relatively long exposure time to DMN and [3H]TdR in culture. The correlation between the induction of UDS in these cells and hepatocarcinogenic potential is not exact, as noted by Michalopoulos et al. (1978). This is due to carcinogen-specific differences in the size of the DNA region repaired and to the fact that some DMN-induced DNA alkylation is repaired by mechanisms not involving base excision (Umbenhauer and Pegg, 1981). Thus, the low levels of UDS induced by relatively high concentrations of DMN do not accurately reflect the genotoxic activity of DMN. Comparison o f LSC and autoradiography. Fig. 2 further illustrates the utility and sensitivity of the NI-LSC method for the detection of UDS. Although the autoradiographic technique has the advantage of exclusion of cells in S phase (Oldham et al., 1980), the NI-LSC method produced dose-response curves for DMN which were very similar in slope to those derived by autoradiography. The effect of HU was especially important in making these comparisons since a very small percentage of cells in S may contribute to a large increase in control specific activity and obscure repair of low dose damage by the LSC method.

386

Conclusion The NI-LSC method proved valuable in determining the UDS dose-response for DMN and quantitating this response at lower doses of DMN than previously reported. The sensitivity of short-patch repair detection was improved by NI-LSC when compared to LSC of whole cell homogenates and the results obtained with DMN closely corresponded to those obtained by the autoradiographic technique. The NI-LSC technique for determining UDS provides a rapid alternative to autoradiography with sufficient sensitivity for use with agents that induce shortpatch repair.

References

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