ethylnitrosourea in germ cells isolated from seminiferous - PNAS

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7485-7489, August 1995 Genetics

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice (sequence spectra/risk assessment)

GEORGE R. DOUGLAS*t, JIANLI JIAO*, JOHN D. GINGERICH*, JAN A. GOSSENt, AND LYNDA M. SOPER* *Mutagenesis Section, Environmental Health Centre, Health Canada, Tunney's Pasture Ottawa, ON Canada KlA 0L2; and lDepartment of Immunology, NV Organon, P.O. Box 20, 5340 BH Oss, The Netherlands Communicated by James V Neel, University of Michigan Medical School, Ann Arbor, MI, April 14, 1995

ABSTRACT The lacZ transgenic mouse (Muta mouse) model was used to examine the timing of ethylnitrosourea (ENU)-induced mutations in germ cells. The spectrum of mutations was also determined. Animals received five daily treatments with ENU at 50 mg/kg and were sampled at times up to 55 days after treatment. In mixed germ-cell populations isolated from seminiferous tubules, there was little increase in the mutant frequency 5 days after treatment; subsequently, there was a continuous increase until the maximum (17.5-fold above background) was reached by -35 days. In the spermatozoa, an increase in mutant frequency was not seen until 20 days after treatment, with the maximum (4.3-fold above background) being achieved no sooner than -35 days. Based on the timing of sampling, these data demonstrate the detection of both spermatogonial and postspermatogonial mutations. The most prominent feature of the ENU-induced basepair mutations in testicular germ cells sampled 55 days after treatment is that 70%o are induced in AT base pairs, compared to only 16% in spontaneous mutations. These findings are consistent with comparable data from ENU studies using assays for inherited germ-cell mutations in mice. This study has demonstrated the utility and potential of the transgenic mouse lacZ model (Muta mouse) for the detection and study of germ-cell mutations and provides guidance in the selection of simplified treatment and sampling protocols.

in bacteria, A-T base pairs are the predominate source of mutations in mammalian species in vivo (13). This change is related to the reduced capacity of DNA alkyltransferase to remove ethyl adducts other than 06-ethylguanine from mammalian cells (13). ENU induces both spermatogonial stem cell and postspermatogonial mutations in mice (14). The former are characterized by base-substitution mutations in A-T base pairs (15-19); the latter have been shown to result in relatively more multilocus lesions (20) and, if adducts are induced late in development, mosaic mutations in the progeny result (21). In this study we have used a lacZ transgenic mouse (Muta mouse) model to determine the timing of ENU-induced mutation induction in a mixed population of germ cells isolated from seminiferous tubules and in spermatozoa isolated from the vas deferens. The mutation spectrum for mutations from mixed germ cells was also determined. It is anticipated that such information will be influential in determining the acceptability of transgenic mouse models for the study of germ-cell mutagenicity, in suggesting appropriate experimental protocols for future studies, and in alleviating the need to conduct costly experiments on the offspring of exposed animals when it is not necessary. It is envisaged that such a transgenic mouse germ-cell assay, once validated, may become a companion to the dominant lethal test in terms of its role in hazard identification.

The establishment of transgenic mouse models for the quantitative detection of gene mutations (1, 2) has provided unprecedented access to the study of mutagenesis in diverse tissues (3-5) and provided the basis for the establishment of a gene mutation assay in germ cells that would predict the transmission of induced mutations to subsequent generations. The process of accepting a transgenic mouse model for the detection and enumeration of induced gene mutations must involve the determination that the mutant frequencies (4, 6) and the molecular characteristics of the mutations detected are representative of endogenous genes (6). Attainment of this goal is facilitated by the detailed knowledge that exists on the biology and timing of mouse male germ-cell development (7, 8) and by the considerable information available on the mutagenicity of ethylnitrosourea (ENU) in male germ cells. The mutagenicity of ENU, the DNA damage it induces, and the repair mechanisms involved have been reviewed in detail (9, 10). Since its first discovery as a potent direct-acting germ-cell mutagen in mice (11), much has been learned about ENU. It is capable of ethylation of all the nitrogens and oxygens of DNA and produces a significant proportion of alkylphosphotriesters (12). While mutations in GC base pairs are primarily responsible for ENU mutagenesis

MATERIALS AND METHODS Transgenic Animals. The transgenic mouse line 40.6 (Muta mouse; Corning Hazleton, Vienna, VA) used in this study has been described in detail by Gossen et al. (1). Briefly, the transgene is based on a recombinant AgtlO vector containing a 3096-bp Escherichia coli lacZ gene, which was injected into the male pronucleus of a CD-2 F1 x CD-2 F1 mouse single-cell embryo. Subsequent selective breeding of progeny led to line 40.6 (1), which is disomic for the "40-copy single-locus insert (2n = =80) located in the B band of chromosome 3 (22). Animals were bred and maintained in Health Canada facilities under conditions approved by the Health Protection Branch Animal Care Committee. Treatment of Animals. Animals used in this study were 9-10 weeks old and were housed five animals per cage. ENU was dissolved in phosphate-buffered saline (PBS) containing 5% (vol/vol) dimethyl sulfoxide and administered i.p. in five consecutive daily doses of 50 mg/kg. Subsequently, animals were euthanized by cervical dislocation at various times to detect mutations induced through the entire germ-cell development process. Tissue Collection and DNA Isolation. The testes were removed, placed in cryotubes, flash frozen in liquid nitrogen,

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Abbreviation: ENU, ethylnitrosourea. tTo whom reprint requests should be addressed. 7485

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and stored at - 80°C until needed. Prior to freezing, the section of the vas deferens from the cauda epididymis to the seminal vesicle was removed. The tissue was placed on a ground glass plate, and while grasping the cauda epididymis with forceps, a 5-mm-diameter roller was used to strip the spermatozoa from the vas deferens. The expelled tissue was resuspended in PBS, transferred to a cryotube, frozen, and stored at -80°C as indicated above. In preparation for DNA isolation, testes were thawed, seminiferous tubules were separated from other structures, and germ cells were released from tubules by gently rolling the mass of tubules using the roller described above (4). The cell population so isolated contains very few spermatogonia or Sertoli cells and is enriched for spermatocytes through to the spermatid stages, as determined by prior microscopic examination (G.R.D., unpublished data). DNA was isolated from seminiferous tubule germ cells as detailed (4). For isolation of DNA from spermatozoa, cells were thawed at 37°C with 0.75 ,ul of 10% (wt/vol) SDS at 37°C for 15 min, washed in PBS, lysed in hypotonic buffer (10 mM Tris HCl, pH 8.4/50 mM KCI/2.5 mM MgCl2/4 mM dithiothreitol/0.05% SDS) (23) along with 5 ,ul of a stock solution of proteinase K at 25 mg/ml. After incubation at 37°C for 2 h, 1.5 ml of isotonic lysis buffer was added with 40 ,ul of 5 M NaCl, 50 ,ul of proteinase K at 25 mg/ml, and 50 ,ul of 10% SDS, and the temperature was raised to 42°C for 1.5 h. Recovery of A Phage and Detection of lacZ Mutants. Single copies of the AgtlOlacZ recombinant phage vector were rescued from genomic DNA by in vitro packaging into phage particles with commercial packaging extract (Gigapack II Gold) under conditions recommended by the manufacturer (Stratagene). Phage particles were adsorbed to E. coli C AlacZ- (1), mixed with top agarose containing 5-bromo-4chloro-3-indolyl 13-D-galactopyranoside (BRL; 0.9 ,tg/ml), and plated on 22.6 cm x 22.6 cm screening plates containing nutrient LB agar. The mutant frequency for each animal was calculated as the number of mutant (i.e., clear or light blue) plaques divided by the total number of plaques. All mutants were confirmed by picking and replating plaques. DNA Sequencing of Mutants. A complementation assay was used to presumptively localize mutations to one of the three

Proc. Natl. Acad. Sci. USA 92 (1995) complementation regions of the lacZ gene. Mutants sequenced subsequently as described (4, 24).

were

RESULTS In mixed germ-cell populations isolated from seminiferous tubules, there was little increase in the mutant frequency 5 days after treatment; subsequently, there was a continuous increase until the maximum was reached by -35 days (Table 1 and Fig. 1). In the spermatozoa, no increase in mutant frequency was seen until 20 days after treatment, with the maximum being achieved no sooner than -35 days. The apparent lower mutant frequency observed at 55 days is not statistically different from that at 35 days and probably results from the fact that there were only data from two animals available at 55 days. Ex vivo mutations (i.e., premutations that escape fixation in the mouse but are converted subsequently by processes in the host bacteria and are detected as mosaic plaques) were not observed in this study. One of the five mice sampled 10 days after treatment had a very high spermatozoa mutant frequency relative to the other animals in that group (40.1 x 10-5 vs. 2.4 x 10-5; see Table 1). To determine whether this "outlier" was the result of a jackpot mutation (25), DNA from five mutants was sequenced. All five mutants sequenced were identical (containing a C3066 A

transversion), indicating

that

a

jackpot

mutant

clonal

subpopulation was present among the cells sampled. Accordingly, the data on spermatozoa from this animal were excluded from subsequent analysis. This jackpot mutation is tissuespecific, since other tissues from this animals did not yield a similar high response (data not shown). The characteristics of the 31 testicular germ-cell mutants obtained 55 days after ENU treatment for which mutant DNA sequences were obtained are described in Table 2. Although this transgenic mouse model readily detects insertions and deletions up to 550 bp (4), none were detected in this study; only base-pair substitutions were observed. There was one double mutation at adjacent base pairs (bp 556 and 557). No more than one mutation was observed at any base position. Table 3 summarizes the mutations according to their type. Information on spontaneous mutations in male germ cells

Table 1. Time-related recovery of lacZ mutations in transgenic mouse germ cells isolated from seminiferous tubules and the vas deferens Mutant frequency Days after (x 105) Total Tissue treatment Treatment (i.p.) SEM Animals, no. Mutants, no. Mean plaques, no. Seminiferous tubules ENU 5 5 27 5.8 1.4 477,000 10 5 21 7.4 2.1 322,800 15 5 58 14.1 1.0 414,000 4* 35 157 40.4 2.3 387,600 45 5 176 45.6 5.8 394,800 55 5 171 45.0 8.1 380,148 5 4* Dimethyl sulfoxide 7 2.6 0.2 262,800 55 5 3 1.1 0.7 308,406 Vas deferens ENU 10 4t 15 2.4 0.9 609,560 15 5 10 3.4 0.7 313,500 20 5 34 6.9 6.3 496,000 25 3* 9 12.5 6.3 88,000 2*: 35 20 19.0 5.3 104,700 55 4t 26 14.9 3.2 187,200 5 4* 10 4.4 3.0 Dimethyl sulfoxide 288,000 55 5 17 3.1 0.5 476,300 ENU was given in five consecutive daily doses of 50 mg/kg; 10% dimethyl sulfoxide was given in PBS. *An animal died. tData from one "outlier" animal were excluded because it was a jackpot confirmed by DNA sequence analysis. The mutant frequency of the excluded animal was 40.1 X 10-5 (68/169,400). tSample lost.

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0r

CL3

Ca

20

co

10

0 Sperm

/

0

Spermatids Meiosis Spermat- Spermatogonial t / / ogonia Stem Celns 20 40 60

Time after ENU treatment, days FIG. 1. Temporal response of testicular germ cells isolated from seminiferous tubules and spermatozoa isolated from the vas deferens to ENU in lacZ transgenic mice. Mice were treated with five daily i.p. doses of ENU at 50 mg/kg and subsequently sampled at various times. Data are the mean ± SEM. Sperm, spermatozoa. *Estimated cell population exposed at time of treatment that contributed mutations to the seminiferous germ cells sampled. tEstimated cell population exposed at time of treatment that contributed mutations to the vas deferens spermatozoa sampled.

from a previous publication (4) is provided for comparison. The most prominent feature of the ENU-induced base-pair mutations is that 70% are induced in A'T base pairs, compared to only 16% in spontaneous mutations from this tissue (4). Only 3 of the 10 (30%) ENU treatment-induced G-C -> A-T mutations occurred at 5'-CpG sites, compared to 7 of the 8 (87.5%) spontaneous mutations. These former 3 mutations may be spontaneously derived.

DISCUSSION The timing of sampling is critical for the determination of mutant frequency in germ-line tissues, particularly if a determination of the most sensitive stage of germ-cell development is to be made. While the timing of the progression of germ-cell differentiation for untreated mice is well-known (7, 8), delays caused by chemical-induced germ-cell toxicity can complicate such determinations. If there is no delay in cell development due to toxicity, then mutations arising in spermatogonial stem cells at the end of the treatment period would not appear in vas deferens spermatozoa until approximately the last time sampled. The mutations in spermatozoa observed prior to this would be predominantly post-spermatogonial in origin. In fact, the maximum mutant frequency in spermatozoa (Fig. 1) corresponds to the time of the last scheduled DNA synthesis and, accordingly, the last opportunity for fixation of mutations

Proc. Natl. Acad. Sci. USA 92 (1995)

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through errors in DNA replication. DNA adducts induced after this point in germ-cell development (i.e., present at earlier times after treatment) would have less likelihood of being fixed as mutations prior to fertilization, since only DNA repair-based mechanisms of mutation fixation would be prevalent. By the same logic, because DNA repair ceases at the midspermatid stage (26, 27), the probability of observing mutations in spermatozoa T TS TGTTfGCAT C E7 61 1213 T A TV GTGGTACAC 4 LB Gl 1265 ATTG&AACC A -G TS C 37 G18 1267 TGAAACCCA A C TV C E13 37 1393 G A TS GCTGGGGAA LB 31 E5 1511 T A TV GATATTATT El 61 LB 1523 CCGATGTAC T C TS C E18 61 1576 GTCCLTCAA A->T TV LB G17 37 1655 T A TV AGTCTTGGC 31 LB G16 1673 AAATACCCA A G TS C 37 1734 G8 T A TV TGGATCAGT 61 LB E15 1826 T A TV TGTAGAAC C 5 G3 1826 T G TV TGTATGAAC C 6 -->A E22 5 2543 GCGTGGCAG TS GIO 61 LB 2729 A T TV CAAGAAAAC C E21 4 2775 T A TV GGGATCTGC C 61 2804 A G G19 TS CCGTACGTC 5 A- T LB 2822 TV G4 GAAAACGGT 5 LB G14 2891 A T TV TTCAACATC LB 61 2940 T A TV G20 GCCATCTGC C 4 2999 G12 G A TS ATTGOTGGC 4 GIl LB 3013 T G TV CTCCTGGAG 31 LB 3022 T C G6 TS CCCGTCAGT C 61 3023 G9 C -T TS CCGTCAGTA C, clear; LB, light blue; TS, transition; TV, transversion. In the target sequences, the target base highlighted by shading.

[90% (29)] and monkey [94% (30)], although any differences may simply be the result of different experimental conditions including dose (31). The proportion of mutations in AT vs. G-C base pairs in the present study is nearly identical to that reported for the lacI transgenic model [i.e., 68% (5)]. Correction for the slightly lower proportion of A-T base pairs in the lacZ gene results in a revised estimate of 73% mutations in A-T base pairs, which would not affect these comparisons. The most prominent classes of mutations induced by ENU in this study (Table 3) are (i) AT -* TA transversions, presunmably induced by 02-ethylthymine (32), (ii) G-C -- AT transitions in Table 3. Summary of ENU-induced lacZ- mutations in testicular germ cells of transgenic mouse obtained 55 days after treatment Mutations Base

Mutation type Transition

change G-C A- T

Spontaneous

ENU induced

n % % 9 (6) 10 (3) 37.5 33.3 A-T-G-C 1 4.2 5 16.1 15 Subtotal 10 41.7 49.4 Transversion 5 (5) C-G A-T 20.8 1 3.3 T-A G-C 1 2 4.2 6.5 A-T T-A 1 4.2 13 41.9 3 (2) G*C >C-G 12.5 0 0 10 Subtotal 16 41.7 51.7 3 Deletion 12.5 0 0 1 Insertion 4.2 0 0 24 Total 100 31 100 Data for the number of spontaneous mutations are from ref. 4, obtained by using the same methods as the present study. n indicates the number of mutations. Data in parentheses are the number of mutations in CpG sites. Data in boldface type are totals or subtotals. n

Protein change Gln Stop Leu Stop

Cys Tyr Arg Stop Gly Stop

Cys Stop Ser Leu

Tyr Asn Glu Gly Thr

Pro

Gly Ser Ile >Asn Met Thr Ile Phe Leu His

Tyr Cys Asp Glu Met

Lys

Met

Trp

Arg Stop

Glu

Val

Asp Glu Tyr Cys Asn Asn His

Ile Ile Gln

Gly Asp Trp Gly Ser Thr Ser Leu

non-5'-CpG sites, thought to be caused by 06-ethylguanine adducts (27), and (iii) A-T -- G-C transitions, reported to be caused by 04-ethylthymine adducts (33). Mutant frequency is a function of a number of interrelated variables including the level of adduct deposition, DNA repair, and DNA replication (13). 06-Ethylguanine adducts are removed/repaired in mammalian cells by both 06-alkylguanine transferase and nucleotide excision repair (34); whereas, 02-ethylthymine and 04ethylthymine have been reported to be not removed by either mechanism (34, 35). The greater opportunity for correction of 06-ethylthymine adducts suggests, as seen in this study in Table 3, that o2- and 04-ethylthymine would be more persistent, resulting in a greater proportion of mutations in A-T than GC base pairs through other processes, such as misincorporation during DNA replication (34). The lacZ transgene contains no exogenous mammalian promoters, and if there is no translation mediated by endogenous cryptic promoters, then DNA repair strand bias (36) should not occur. The data in Table 2 indicate that for 06-guanine, 04- and 02-thymine ethylation, for which the adducted strand is identified, or can be inferred, 15 of 24 (62.5%; estimate excludes G-C -* AT mutations in 5'-CpG sites) of the mutations occurred in the sense strand. While this data does suggest some strand bias, it only takes into account a subset of the possible damage leading to mutations. Among the mutations sequenced, there was no more than one at any base position, indicating that there were no detectable "hot spots" as was observed with spontaneous mutations in the lacZ mouse (4), and in contrast to results seen in ENU-treated germ cells from lacI transgenic mice (5). The fact that there were no duplicate mutations also suggests that there was no detectable preferential clonal expansion of mutant

spermatogonia.

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The occurrence of jackpot mutations has been observed previously in both lacZ and lacI transgenic mice, and their occurrence is not rare. Both tissue-specific, in which clonal expansion of a mutant occurs within a tissue (37, 38), and whole-animal jackpots (38), which result from a mutation in a transgene in a germ cell, have been identified. While the latter can be distinguished by a mutant frequency that is equivalent to one mutation divided by 80 (i.e., the number of transgene copies per cell), the latter must be distinguished by DNA sequencing or through comparison of mutation frequencies from discrete organ sections or organs (e.g., liver lobes or lungs and kidneys). Careful interpretation of outlier mutation frequencies leading to exclusion of jackpot data can be helpful in reducing extraneous variation and improving the precision of the transgenic mouse mutation data. This study has shown the temporal and molecular characteristics of ENU-induced gene mutations in the male germ cells of the transgenic lacZ mouse (Muta mouse) and demonstrated the utility and potential of this system for the detection and study of spermatogonial and postspermatogonial germ-cell mutations. The spermatogonial mutations detected in this lacZ transgenic mouse model are predominantly in AT base pairs, entirely consistent with the spectrum of ENU-induced germ-cell mutations induced in mammalian endogenous genes, providing further evidence of the applicability of this mouse model to the study of gene mutations induced by chemicals in rodent germ cells. We are grateful for the helpful and stimulating discussions with Drs. D. Blakey, S. Lewis, H. Malling, G. Sega, and V. Seligy in the completion of this paper.

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