Mutation Research, 212 (1989) 231-239 231 Elsevier

MTR 04756

The relationship between DNA damage and mutation frequency in mammalian cell lines treated with N-nitroso-N-2-fluorenylacetamide

M i n g - L i a n g K u o a n d J e n - K u n L i n

Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan (Republic of China)

(Received 15 August 1988) (Revision received 10 December 1988)

(Accepted 18 January 1989)

Keywords: N-Nitroso-N-2-fluorenylacetamide; Mutation; DNA damage; C3H10T1/2 cells; CHO ceils

Summary

The induction of DNA single-strand breaks in C3H10T1/2 mouse fibroblasts and Chinese hamster ovary (CHO) cells by N-nitroso-N-2-fluorenylacetamide (N-NO-2-FAA) was demonstrated by the alkaline elution technique. Without metabolic activating system (i.e., rat liver $9 fraction), N-NO-2-FAA exhibits more direct and strong damaging effects on DNA than its parent compound, 2-FAA, at equal concentra- tion in both ceil lines. To compare the DNA-damaging potency of N-NO-2-FAA with other well-known carcinogens, such as benzo[a]pyrene, 2-nitrofluorene, and N-methyl-N'-nitro-N-nitrosoguarfidine (MNNG), the order of potency is as follows: M N N G (5 t~M) > N-NO-2-FAA (150/zM) > benzo[a]pyrene (20 t~M) at equitoxic concentrations, LD3v, in the same cell system. Another parallel experiment indicated that N-NO-2-FAA could disrupt the superhelicity of circular plasmid DNA (pBR 322) at a dose range of 0.1-50 raM; however, a complete conversion to form III linear DNA was found at the highest concentration (50 mM). After treatment with various concentrations of N-NO-2-FAA, ouabain resistance (oua r) was induced in C3H10T1/2 cells, while both oua r and 6-thioguanine resistance (6-TG r) were induced in CHO cells. The mutation frequency in the Na+/K+-ATPase locus in CHO cells (1.5 × 10 - 6

mutants//zM) is higher than that in C3H10T1/2 cells (1.0 × 10 - 6 mutants//~M). The maximal mutation frequency at the Na+/K+-ATPase gene locus was attained with 30 rain of

exposure in C3H10T1/2 cells, whereas the mutation frequency in CHO cells continued to increase up to 80 min of treatment. Similarly, the maximal mutation frequency at the H P RT locus also continued to increase up to 80 min of treatment. Finally, a linear plot of alkali-labile lesions versus 6-TG r mutations was obtained; but the same relationship was not observed in the case of oua r mutation.

A large body of experimental evidence has sug- gested that the occurrence of mutation in cells could result from the accumulation of DNA

Correspondence: Dr. Jen-Kun Lin, Institute of Biochemistry, College of Medicine, National Taiwan University, NO. 1 Sec- tion 1, Jen-Ai Road, Taipei, Taiwan (R.O.C,).

damages, including the gain, loss, or substitution of one or more nucleotide bases (Lawley, 1973; Lawley and Jarman, 1975; Cox and Irving, 1976; Erickson et al., 1977; Sarma et al., 1975). Some reports also have shown a good correlation be- tween the carcinogenic potential and DNA- damaging activities of numerous chemicals in mammalian cells (Irving, 1973; Swenberg et al.,

0027-5107/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

232

1976; Cesarone et al., 1982). But in fact, the process of carcinogenesis in animals has been proven to involve multiple steps; the first step, initiation, is produced by DNA-damaging agents, and may or may not result in cancer (Lasne et al., 1977; Barrett and Ts'O, 1978). Hence, mutation events should be more closely associated with DNA damages than carcinogenesis.

The alkaline elution assay has been utilized for monitoring DNA damage (i.e., alkali-labile le- sions) in cultured mammalian cells and in various organs of laboratory animals treated with single doses of carcinogens (Kohn at al., 1976, 1981; Cesarone et al., 1983; Pala et al., 1982; Parodi et al., 1981). Extensive evidence has been presented that the elution rate is determined by the DNA single-strand breakage (SSB) frequency (Petzold and Swenberg, 1978; Tsapakos et al., 1981); this includes the observation that the molecular weight of DNA single strands eluting early during al- kaline elution is lower than the molecular weight of DNA eluting at later elution times, as measured by alkaline sucrose gradient sedimentation. The alkaline elution assay therefore seemed to be a useful pre-screening test for identifying certain suspected genotoxic carcinogens (Parodi et al., 1978).

Both C H O / H P R T and C 3 H 1 0 T 1 / 2 / N a + / K+-ATPase mutation assays are commonly used systems in evaluating the mutagenic potential of various types of carcinogens (Landolph and Heidelberger, 1979; O'Neill et al., 1977). Funda- mentally, these 2 types of resistance mutants (oua ~ and 6-TG r) are markedly different in their induc- tion mechanisms; ouabain resistance is likely to occur as a result of relatively small, localized genetic changes affecting the DNA segment en- coding the binding site for ouabain. Certain muta- tions in this domain presumably prevent binding of the cardiac toxin, ouabain, but maintain the functionality for the Na + / K +-ATPase. The HPRT locus, in contrast, is sensitive to a broader spec- trum of mutational changes, in that 6-TG r can result from base-pair substitution, large deletions or insertions.

N-Nitroso-N-2-fluorenylacetamide (N-NO-2- FAA), a new direct-acting mutagen and teratogen (Lin and Kuo, 1988), has been shown to react readily with different biological molecules, includ-

ing nucleotides, amino acids, etc., in physiological conditions. In the present study, we tried to in- vestigate the relationship between DNA damages and mutations at 2 genetic loci in their respective cells, treated either with various concentrations of N-NO-2-FAA for a fixed time period or with a fixed concentration of N-NO-2-FAA and varied exposure time.

Material and method

Cell cultures Chinese hamster ovary (CHO) cells were cul-

tured in minimal essential medium (Gibco) sup- plemented with 10% fetal calf serum, and C3H10T1/2 cells were grown in basal Eagle's medium supplemented with 10% fetal calf serum. Both cell lines were cultured in a controlled en- vironment of 5% CO2 and 95% relative humidity.

Chemicals 2-FAA, 2-nitrofluorene, MNNG, and benzo[a]-

pyrene (B(a)P) were obtained from Aldrich Chem- ical Co., Milwaukee, WI. N-NO-2-FAA was synthesized by the nitrosation of 2-FAA according to a process described earlier (Lin and Kuo, 1988). Ouabain (Sigma) was dissolved in medium, in- cubated at 56°C for 1 h and sterilized by filtra- tion through a 0.2-/zm filter. The final concentra- tion in the medium was 3 raM. 6-Thioguanine (Sigma) was dissolved in 0.1 N NaOH and added to the medium at a final concentration of 50/~M.

Mutation assay Aliquots of both CHO and 10T1/2 cells (5 x

l0 s) were plated in 100-mm dishes with 10 ml of complete medium, allowed to attach for 18 h, and then the cultures were treated with N-NO-2-FAA either for a fixed time with increasing doses or with a fixed dose for increasing time periods. Cells were then washed twice with cold phosphate- buffered saline (PBS) and incubated in fresh medium for an expression time of 48 h for ouabain resistance in 10T1/2 cells or CHO cells and of 7 days for 6-thioguanine resistance in CHO cells.

During this period, cells were subcultured when necessary to maintain exponential growth. After the appropriate expression time, cells were trypsinized and replated at a density of 1 x 105

cells per dish, for oua r and 6-TG r, respectively, in 100-mm dishes with 10 ml of selective medium, which was changed after 1 week. After a total of 10 days the cells were fixed and stained. The same cell suspension was plated in parallel at clonal density (500 cel ls /60-mm dish, 3 dishes per point) to determine the cloning efficiency (CE). The mu- tation frequency was calculated as the ratio of the observed number of mutant colonies to the num- ber of cells able to form colonies.

AIkaline elution assay The number of alkali-labile lesions was de-

termined by alkaline elution as previously de- scribed (Kohn et al., 1976). Briefly, 105 cells/60- m m dish were labeled for a 24-h incubation time with 3H-thymidine (0.5 /xCi/ml, ICN). Cultures were treated with a fixed dose of N-NO-2-FAA for increasing exposure times, or for a fixed ex- posure time with increasing doses of N-NO-2- FAA. Control and treated cells were deposited onto a millipore mixed-esters cellulose filter (25 m m diameter; 5 t~m pore size), washed with cold PBS and lysed with 0.2% sodium lauroyl sarcosinate (Sarkosyl), 2 M NaC1, 0.02 M N a z E D T A (pH = 10). The lysing solution was rinsed off with 0.02 M N a 2 E D T A (pH = 10), and single-strand D N A was eluted from the filter in the dark with 15 ml of 0.06 M tetraethylam- monium hydroxide, 0.02 M N a 2 E D T A ( p H = 12.3), at a controlled flow rate of 0.13 ml /min . The D N A content of the 10 eluted fractions and that remaining on the filter were estimated radio- metrically by liquid scintillation counting. The values of 3H radioactivity for each eluted fraction were used to calculate the amount of damaged DNA, and the values of the fractions of treated and control D N A remaining on the filters were also determined.

Treatment of plasmid DNA with N-NO-2-FAA and gel electrophoresis

Plasmid D N A pBR322 was isolated from Escherichia coli strain RRI by standard proce- dures (Maniatis et al., 1982). The D N A was in- cubated with various concentrations of N-NO-2- FAA in 20 mM Tris-HC1 buffer (pH = 7.4) for 2 h at 37°C. After this incubation, the reaction buffer was extracted several times with CHCI 3 to

233

remove the unreacted N-NO-2-FAA and then the nucleic acids were precipitated by 2 vols. of 95% cold ethanol. Finally, the D N A samples were analyzed with non-denaturing agarose gel electro- phoresis as described (Maniatis et al., 1982).

Results

Fig. 1A,B shows typical alkaline elution pat- terns of D N A from C H O and C3H10T1/2 cells, respectively. Both cell lines were treated with 0.05-0.2 mM of N-NO-2-FAA and a 2-h exposure to this N-nitroso compound caused a dose-depen- dent increase in the rate of 3H-DNA elution from the filters. Almost 100% of 3H-DNA eluted from the filters when both cell lines were exposed to 0.2 mM N-NO-2-FAA, as compared to 10% elution for the control group. Under these dose levels, it was apparent that no marked difference in the production of alkali-labile lesions of DNA for the

loo; ~'~O-Lo . . . . . . . .

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" 2o x,.... A " ~ x"'~r"~ x ~ x O. 15 m M

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\ \ \ . ,...,._ e \ ~ x.~ x.......x ,._..x N-NO-2" FAA

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B x-.-.~ x x ~ x ~ x O. 15 mM 0 , . , , ~ x - - , . - . x Q.2Om, M ,

0 4 8 12 16 20

Volume of elution, m!

Fig. 1. Alkaline elution pattern of DNA from (A) CHO and (B) C3H10T1/2 cells, treated with various concentrations of N-NO-2-FAA for 2 h. Every determination was performed at

least twice, and the symbol represents the mean value.

234

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Fig. 2. Alkaline elution patterns of DNA from (A) CHO and (B) C3H10T1/2 cells exposed to various chemical carcinogens, including MNNG, B(a)P, 2-FAA, 2-nitrofluorene and N-NO- 2-FAA. The treatment protocols are as follows. (A) Pro- carcinogens: B(a)P (0.02 mM), 2-FAA (0.15 mM), and 2- nitrofluorene (0.15 raM) were incubated with cultures for 24 h. (B) Direct-acting agents: MNNG (0.005 mM) and N*NO-2- FAA (0.15 mM) were only incubated for 2 h in the same

cultures.

2 cell lines was observed. In agreement with this, Table 1 shows that the cytotoxic potency of N- NO-2-FAA in CHO and 10T1/2 cells was quite similar, indicating that the degree of DNA damage might be associated with the cytotoxicity in both cell fines induced by this new N-nitroso com- pound. To evaluate the potency of DNA damage (single-strand breakage) produced by N-NO-2- FAA and other well-known carcinogens, such as M N N G and B(a)P, we chose the LD37 (the con- centration at which 37% of the cells survived) as the comparison point. At this equitoxic dose level, the order of DNA-damaging potency was: M N N G (5 #M) > N-NO-2-FAA (150 /~M) > B(a)P (20 /~M) in C3H10T1/2 mouse fibroblasts (Fig. 2B); but, since the CHO cells did not have any recta-

bolic enzymes to activate the B(a)P, they exhibited only 20% DNA elution, slightly greater than the control group (as shown in Fig. 2A). In addition, the 2 fluorenyl compounds, 2-nitrofluorene and 2-fluorenylacetamide (the parent compound of N-NO-2-FAA) used as positive controls in this assay did not show any marked DNA-damaging activities, as compared to the N-NO-2-FAA treat- ment groups at equimolar concentrations.

In order to study the kinetics of induction of alkali-labile lesions of DNA samples from both cell lines treated with N-NO-2-FAA for varying time periods, the percentage of DNA eluted from the filters for every time point was calculated (Fig. 3). The results showed that a maximal level of DNA elution from the filters appeared when CHO cells were exposed to N-NO-2-FAA (150/~M) for 20 min and 10T1/2 cells for 30 min. However, beyond this maximal DNA elution point, a sharp decline in DNA elution was detected in 10T1/2 cells, whereas a steady decrease of elution rate was found in CHO cells. Furthermore, in agreement with those results attained in the alkaline elution assay, we also found the superhelicity of plasmid pBR322 could be disrupted by treatment with a high concentration of N-NO-2-FAA. A repre- sentative photograph of the gel electrophoresis of DNA samples treated with increasing concentra-

I 0 0

J

80

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Fig. 3. Elution kinetics of CHO and 10Tl/2 cell DNA, as determined by alkaline elution, after increasing exposure time to 0.15 mM N-NO-2-FAA. The values obtained in CHO and 10T1/2 cells are indicated by • and m, respectively. Each

symbol represents the mean of 2 independent observations.

I 2 3 4 5 6 7 8

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Fig. 4. Detection of strand breaks in N-NO-2-FAA-treated plasmid D N A under non-denaturing conditions. About 2 /~g of pBR322 D N A was treated with varying concentrations of N-NO-2-FAA at pH 7.4 for 2 h at 37 o C: the concentration of N-NO-2-FAA was 0 in lane 1, 0.1 m M in lane 2, 0.2 m M in lane 3, 0.5 m M in lane 4, 1 m M in lane 5, 5 m M in lane 6, 10 m M in lane 7, and 50 m M in lane 8. Each sample was electrophoresed on a 0.8% agarose gel at 3 V / c m for 16 h. In the left margin, the position of linear D N A size markers is shown, and on the fight, the position of forms I, II and III

plasmid DNA.

tions of N-NO-2-FAA is shown in Fig. 4. One single-strand breakage results in conversion of the plasmid DNA from form I (supercoiled) DNA to form II (relaxed circular) DNA; this can be easily detected under non-denaturing conditions. As seen in this Fig., complete conversion of form I to form II occurs at the highest concentration of 50 mM N-NO-2-FAA after a 2-h incubation at 37 ° C. At this highest concentration, form III (linear plas- mid) DNA could also be observed, indicating that a large amount of N-NO-2-FAA could completely

235

disrupt the topological structure of plasmid DNA. Because the sample of DNA was isolated from E. coli RRI by the mini-lysate method (as described in Material and method), a small amount of form II (relaxed circular) D N A and dimer of plasmid DNA could be detected in the control lane (Fig. 4).

When 10T1/2 and CHO cells were exposed to 0.05-0.2 mM N-NO-2-FAA the mutation fre- quencies were measured at 2 genetic loci, the Na+/K÷-ATPase gene and the HPRT gene, re- spectively. As shown in Table 1, after treatment with N-NO-2-FAA the 6-TG r mutation frequen- cies (mutated at the H P RT locus in CHO) were increased in a linear dose-responsive manner; in contrast, the oua r mutation frequencies showed a ' plateau' condition when the dose of N-NO-2-FAA was higher than 0.1 mM. Based upon these find- ings, we propose that these 2 mutagenic responses for the respective cell lines are mediated through different biological processes, which include dif- ferent types of D N A damage, xenobiotic metabo- lism or different cellular repair systems induced by N-NO-2-FAA in these 2 cell lines.

To investigate the induction kinetics at both genetic loci required to attain maximal mutation frequencies, both cell lines were exposed to 0.15 mM N-NO-2-FAA for increasing periods of time, and oua r and 6-TG r mutation frequencies were measured in aliquots of the respective cells at the appropriate expression times. Fig. 5 shows that the maximal induction of oua r mutation in 10T1/2 cells was reached within 30 min of exposure;

TABLE 1

COMPARISON OF M U T A T I O N FREQUENCIES IN C3H10T1 /2 A N D CHO CELLS

N-NO-2-FAA a Survival C3H10T1 /2 ( x 105) Survival ceils b cells b

mutat ion (%) freq. of oua r (%)

CHO ( x l0 s)

mutat ion freq. of 6-TG r

mutat ion freq. of oua r

Control (0.1% DMSO) 100 0 100 0.05 m M 78 5.5 80 0.1 m M 57 14.8 55 0.15 m M 40 15.2 37 0.2 m M 21 15.4 18

0 13.7 21.2 24.0 27.5

23.3

a Cells were treated with various doses of N-NO-2-FA.A for 2 h. b Survival cells was expressed as follows:

. plating efficiency of treated groups x 100% cell survival (%) ~ groups

236

beyond this time point no further increase in mutation frequency was observed. By contrast, the 6-TG r mutation in CHO cells reached almost 90% (2.1 × 10 -4 mutants /survivor) of the maximum level within 20 rain of exposure, and continued to increase up to the maximum (2.45 × 10 -4 m u -

t a n t s / s u r v i v o r s ) until 80 rnin after exposure. Ad- ditional experimental results indicated that the induction of oua r in CHO cells reached 80% of the maximum (1.75 × 10 -4 mutants /survivor) within 20 min of exposure and continued to in- crease up to the m a x i m u m (2.37 × 10 -4 mutants/survivor) until 180 rain after exposure (Fig. 5, broken line). However, the rate of induc- tion of mutations at these 2 loci was very fast for the first 20-30 rain of exposure, 0.5 × 10 -s mutants / ra in for oua r and 1.05 × 10-s m u t an t s / rain for 6-TG r.

Finally, a plot of alkali-labile lesions versus mutations at the H P R T locus gave a straight line with the intercept at 0 (Fig. 6), suggesting an association between mutagenesis in the HPRT gene and alkali-labile D N A damage in CHO cells. But in the same plot we could not obtain a straight line between oua r mutation frequency and alkali- labile lesions in 10T1/2 cells, indicating that the outcome of oua r mutation was not correlated with

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0

m c 0

~ eo

CHO cells (HPRT gene loci)

. CliO cells(No -K ATPose gene loci)

t

lOT I /2 cells(No+-K + ATPose gene loci I m-_..,_ m e l / I i/,c r - • =

O i i f i i

0 80 160 240

T;me , rain

Fig. 5. Kinetic inductions of oua r in 10T1/2 and CHO cells and 6-TG r in CHO cells by treatment with 0.15 mM N-NO-2- FAA for increasing time periods. Each symbol represents the

mean of 2 or more independent observations.

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o= 12 (o+r 5) (o.z)

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( 0 . 0 5 ]

0 2 0 4 0 60 80 I 0 0

DNA eluled from the f i l l e r , %

Fig. 6, The relationship between alkali-labile lesions and muta- tions to oua r in 10T1/2 cells (m) and 6-TG r in CHO cells (A) treated for 2 h with varying concentrations of N-NO-2-FAA. Frequency of mutations for each dose level of N-NO-2-FAA is plotted against % of DNA elution for the same dose level. Every point represents 2 or more independent observations. Bars indicate the standard deviation. The values in parentheses

represent the doses of N-NO-2-FAA.

alkali-labile lesions of D N A in this cell system, when treated with N-NO-2-FAA.

Discussion

The rate of elution in the alkaline assays de- pends on the length of single-stranded DNA; the shorter the strand, the faster it elutes (Swenberg et al., 1976). The sensitivity of the assay can be increased over that reported in previous studies (Fornace et al., 1986) by increasing the time of elution and decreasing the vo lume/uni t time. However, in this study, we used the unmodified standard procedure as described by Kohn et al. (1981) and repeatedly obtained typical and marked elution data from both cell lines after exposure to N-NO-2-FAA. In this experiment, even when the dose level of N-NO-2-FAA was very low (0.01 raM), we could also obtain detectable 3H-DNA elution from the filters (data not shown). But using the same low dose in the mutagenicity assay, no mutants were detectable. It has been proposed that the alkali-labile lesions might result from depurination or depyrimidination of D N A (Verly, 1974) or from the formation of phosphotriesters

(Lawley, 1973). Since an analytical technique such as alkaline elution only measures the end product, i.e., a DNA break, we cannot actually define which type of DNA damage is induced by N-NO- 2-FAA. Reports have shown that the end products of alkaline elution might include single- and dou- ble-strand breakage, alkali-labile damage, etc. (Lloyd et al., 1978). Therefore we used supercoiled DNA (pBR322) as a model to inspect whether N-NO-2-FAA could 'break' the model DNA without any alkali treatment in vitro. As expected, the superhelicity of plasmid DNA was relaxed by N-NO-2-FAA in neutral conditions. Hence we suggested that, except for the alkali-sensitive le- sions, N-NO-2-FAA seemed to exhibit diversified types of DNA-damaging activities. This proposal may be supported by a previous study (Lin and Kuo, 1988), which reported that N-NO-2-FAA could cause mutations in Salmonella strains in- cluding TA98, TA97, TA100, and TA1538.

In investigating the extent of single-strand breaks induced by N-NO-2-FAA at varying ex- posure times, maximal 3H-DNA elution was ap- parent during the first 20-30 min of exposure to N-NO-2-FAA in CHO and 10T1/2 cells. Beyond this maximal point, the elution rate of 3H-DNA declined with different slopes for the 2 cell lines. It appears then, that in the first 20-30 min N-NO- 2-FAA could readily cross the cellular and nucleic membrane and create a large amount of DNA damage. However, after that (limited by its half-life of 30 min), no more DNA damage could be created and the damage might be suppressed or even repaired by cellular repair systems, which could be induced by N-NO-2-FAA treatment. Therefore we suggested that the maximal point of alkaline elution should be the equilibrium point between DNA damage and repair.

In dose-response assays of mutation at 2 genetic loci (the HPRT locus in CHO and the Na+/K+-ATPase locus in 10T1/2 ceils) we found that dose dependence exists at the HPRT locus after treatment with N-NO-2-FAA; in contrast, the mutation at the Na+/K+-ATPase locus reached a plateau when the dose went up to 0.1 mM. A possible interpretation for this finding is that the phenotype of mutants at the N a + / K +- ATPase gene is based on a 'critical' point muta- tion, which results in an altered Na+/K+-ATPase

237

that no longer binds ouabain or binds it less tightly than the wild-type enzyme does. Most im- portantly, this mutated enzyme must maintain its vital function as well as the wild-type enzyme. Hence we propose that there may be specific DNA sequences in the Na+/K+-ATPase locus to fit this 'critical' point mutation. Then, if the dose level of N-NO-2-FAA was too high (over 0.1 raM), leading to devitalization of the function of the enzyme and to cell death, the phenotype of the mutant would no longer be expressed.

Different induction kinetics were demonstrated for mutations at those 2 genetic loci upon ex- posure to N-NO-2-FAA for varying time periods. Two time-dependent processes, i.e., chemical deg- radation of the mutagen and DNA repair on a damaged template, were considered in relation to the mutation frequency in exposure-duration ex- periments. The present data show that the frequency of both the 6-TG r mutation and the oua r mutation increases rapidly almost to the maximum over the first 30 rain (Fig. 5), but be- yond this time point the expression frequency of 6-TG r and oua r in CHO cells still increases, though with a slower induction rate. In contrast to this, with 10T1/2 cells, after the oua r mutation frequency reached the maximum, a plateau fol- lowed. The time courses for mutation induction in the Na+/K+-ATPase locus (resistance to oua) in CHO cells and C 3 H 1 0 T I / 2 cells are somewhat different (Fig. 5). These differences may be due to the genetic differences between the two cell lines used (hamster and mouse). In view of the results obtained from kinetic induction of alkaline elution and mutagenesis at 2 genetic loci in CHO and 10T1/2 cells, we found that there is an unknown but close relationship between these 2 molecular processes. A possible explanation for this relation- ship may be that N-NO-2-FAA-induced alkali- labile lesions could result in heritable genetic al- terations, which at a constant proportion may produce mutations; this could be proved by the fact that the maximal response for mutation and alkaline elution occurred at the same time period of exposure. Furthermore, based upon the differ- ent slope for the second stage of kinetic induction (after the 30-min time point) of alkaline elution, we proposed that the repair potency of 10T1/2 mouse cells is more efficient than that of CHO

238

cells induced by this N-nitroso compound. This could also explain why the mutagenic responses induced by N-NO-2-FAA at HPRT and N a + / K+-ATPase in CHO were more potent than those at Na+/K+-ATPase in 10T1/2. Another hypothe- sis may also explain this result, one that is related to the HPRT and Na+/K+-ATPase gene struc- tures. Our knowledge of DNA lesions in mam- malian cells derives from measurements that pro- vide an average estimate of the events occurring in the entire genome, whereas mutation frequency determinations are related to damage of individual specific segments of the genome. Although HPRT and N a + / K ÷ - A T P a s e can be cons idered 'housekeeping' genes, being expressed in most cell types (Melton et al., 1986; Shull et al., 1985), too little is known about the precise structure of the 2 genes during the cell cycle which could affect the amount of damage and contribute to the dif- ference in mutational kinetics.

Further characterization of the N-NO-2- F A A - D N A adducts from cells is now in progress. Studies of this kind will allow definition and reali- zation concerning the molecular mechanisms of repair processes and mutation responses at the gene level.

Acknowledgement

This study was supported by the National Sci- ence Council, NSC 78-0412-B002 (02), Taipei, Taiwan (R.O.C.).

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