A recombination-based transgenic mouse system for genotoxicity testing

Fundamental end Molecular Mechanisms of Mutagenesis

ELSEVIER

Mutation Research 307 (1994) 583-595

A recombination-based transgenic mouse system for genotoxicity testing J. Ramana Murti, Kerry J. Schimenti, John C. Schimenti * The Jackson Laboratory, Bar Harbor, ME 04609, USA (Received 16 April 1993; Revision received 7 October 1993; Accepted 11 October 1993)

Abstract It is well established that mutagens induce recombination in cultured cells and experimental organisms. Presumably, this is a consequence of the DNA-damage-triggering cellular-repair mechanisms. The relationship between recombination and mutagenicity has been exploited in submammalian organisms, such as yeast, to assay the ability of chemical agents and radiation to induce a form of recombination called gene conversion - the non-reciprocal transfer of genetic information. This work has demonstrated the efficacy of predicting mutagenicity on the basis of recombination induction. Here, we describe the utilization of a transgenic mouse system for efficient detection of germ-line gene-conversion events as a mutagen-screening tool. These mice contain two mutually defective reporter (lacZ) genes under the regulatory control of a spermatogenesis-specific promoter. A particular intrachromosomal gene conversion event must occur for the generation of functional lacZ activity. Conversion events are visualized by histochemical staining or flow cytometric analysis of transgenic spermatids. The highly mutagenic compound chlorambucil induced a several fold percentage-wise increase of lacZ--positive spermatids, whereas acrylamide, a weak genotoxin, produced no marked increase in converted spermatids. The results indicate that recombination-based transgenic mouse models for genotoxin screening present a viable option for inexpensive and rapid whole-animal mutagen testing. The particular mice we describe may ultimately prove to be a useful tool for identifying agents which can cause heritable genetic mutations in humans. Key words: Transgenic mouse system; Recombination; Cellular repair mechanisms; D N A damage

I. Introduction In t h e p a s t few years, t h e r e has b e e n a d r a m a t i c i n c r e a s e in e n v i r o n m e n t a l a w a r e n e s s . T h e r e

* Corresponding author, Tel. 207 288 3371; Fax 207 288 5079.

is l e g i t i m a t e c o n c e r n o v e r t h e safety o f synthetic, d i e t a r y a n d e n v i r o n m e n t a l l y i n t r o d u c e d substances. T h e i m p l e m e n t a t i o n o f novel t h e r a p e u t i c d r u g s is c o n f o u n d e d by t h e extensive t i m e req u i r e d to d e t e r m i n e t h e i r l o n g - t e r m safety. T h e e x p a n d i n g use o f novel s y n t h e t i c s u b s t a n c e s in all facets o f i n d u s t r i a l i z e d society m a y r e p r e s e n t a

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significant risk to exposed individuals and their future children. Conventional, whole-animal bioassays are prolonged, cost-prohibitive and impractical to perform on a routine, widespread basis. The lack of efficient and rapid means to assess the potential genotoxicity of these agents is therefore a serious problem. The genotoxic status of a chemical has usually been inferred from tests designed to assess genetic damage. More than 200 assays have been developed (Nesnow et al., 1987), each designed for specific endpoints such as detection of germcell mutagens, carcinogens, specific-locus mutations, biotransformation capability, sister-chromatid exchanges (SCE), etc. Based on the principle that DNA-damaging agents are universally deleterious or genotoxic, simple mutation assays have been developed in prokaryotic organisms, lower eukaryotes or-cultured mammalian cells. The advantages of these systems are that they are performed rapidly and inexpensively. The major drawback is that none can perfectly reflect the mutational or carcinogenic potential of an agent introduced into a whole animal. Thus, a universally applicable genotoxicity test has remained elusive and governmental regulatory agencies require hazards to be inferred from a battery of tests (Butterworth et al., 1984). The paucity of cost-effective, highly accurate short-term tests has several consequences. Most importantly, agents may be released into the environment or to the population for some time before they are realized to be genotoxic or carcinogenic. The process of testing experimental therapeutic drugs for detrimental long-range effects is currently lengthy and frustrating to those who urgently require treatment. Another obvious consideration is that the conventional tests are extremely expensive, thereby contributing to the high cost of drugs and other agents.

Recombination as an indicator of genotoxicity A common characteristic of DNA-damaging agents is that they induce genetic recombination. Recombination induction appears to be a broad indicator of an agent's mutagenicity, reflecting repair of induced D N A damage (Zimmermann, 1971; Hellgren, 1992; Wiirgler, 1992). DNA re-

pair is generally template-directed; lesions are corrected using an homologous sequence as a guide. Enzymes involved in this process include those which have roles in recombination. Some agents, such as ionizing radiation, may stimulate recombination by inducing DNA-strand breakage. Double-strand breaks are thought to be an initiating step in recombination (Ray et al., 1988; Sun et al., 1989; Szostak et al., 1983). However, mitotic recombination is stimulated by a wide spectrum of mutagenic agents, including those which cause point changes, deletions and frameshifts. Hence, it appears that recombination is a cellular response to DNA insults (Zimmermann, 1971). This property is a major advantage in using recombination induction - intrachromosomal gene conversion in particular - as an indicator of mutagenicity. This is in contrast to methods such as the Ames test, for example, in which a variety of tester strains must be used to detect different types of mutation (point changes, frameshifts). Stimulation of recombination by chemical agents has been exploited to develop an efficient screening system in yeast, which is capable of detecting mutagens and carcinogens (Schiestl, 1989; Schiestl et al., 1989), some of which are negative in the Ames test. In this system, intrachromosomal recombination causes deletion of a selectable marker (HIS). An assay called S M A R T (Somatic Mutation And Recombination Test) was developed which detects mutagen-induced mosaicism, such as "twin spots," in the fruitfly Drosophila melanogaster (Graf et al., 1984). Chemicals can also induce recombination in mammalian cells (Therman and Kuhn, 1976; Wang et al., 1988). At present, it is not clear whether mutagenstimulated recombination in eukaryotic cells is a consequence of generalized cellular repair induction, or specific repair of damaged DNA sequences (Hellegren, 1992; Schiestl and Wintersberger, 1992). It is difficult to discriminate between these two possibilities, since most assays for recombination examine a particular locus in treated cells, and it is impossible to determine whether the recombination was initiated by direct mutagen damage of the target genes. It is possi-

J.R. Murti et al. / Mutation Research 307 (1994) 583-595

ble that both occur, considering that bacteria and mammalian cells undergo protein synthesis and DNA-repair activities in response to DNA damage (Elespuro, 1987). Indirect experimental paradigms may be required to answer this question.

Recombination can be mutagenic Unfavorable illegitimate recombination events include translocations, mitotic crossing-over, gene conversion, and deletions/duplications via interor intra-chromosomal recombination between homologous but non-allelic sequences. Mitotic crossing-over, and possibly gene conversion, can cause loss of heterozygosity at "tumor-suppressor" gene loci (such as retinoblastoma), and is therefore an important mechanism in tumorigenesis (Koufos et al., 1985; Okamoto et al., 1988). Recombination between repetitive elements appears to be responsible for deletions in the human/3-globin locus (Gilman, 1987),/3-hexaminase A gene (Myerowitz and Hogikyan, 1987), human LDL receptor gene (Lehrman et al., 1985), and other loci (Wiirgler, 1992). Gene conversion appears to underlie some mutations at the steroid 21-hydroxylase locus, by transferring homologous sequence from a pseudogene to the functional gene (Morel et al., 1989). Since the mammalian genome is replete with repetitive and duplicated DNA sequences, unequal or illegitimate recombination is a potentially major form of mutagenesis. Hence, recombination-based mutagen screening systems are unique in their ability to detect such agents. However, it is not clear whether agents that induce recombination but do not damage DNA exist to any significant degree. In this report, we describe experiments with a recombination-based transgenic mouse system for mutagen screening. These mice allow efficient identification of a specific gene conversion event in the male germ-line. Treatment of these mice with chlorambucil caused a striking increase in the gene-conversion frequency. These initial results indicate that this recombination-based transgenic mouse system (or variations thereof) has the potential to be a rapid and inexpensive short-term screening tool for detecting germ-line mutagens.

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2. Methods

Mice The transgenic mouse lines used in these studies, COR3 and OPP2, have been described (Murti et al., 1992). The transgenic constructs are described in the Results section. Preparation of spermatids With minor modifications, the standard method for preparation of spermatogenic cells was adopted (Romrell et al., 1976). Briefly, after the testes were stripped of their outer tunic, the tubules were dispersed by collagenase (1 mg/ml) in 20 mM sperm medium (1 x SpM = 2 mM CaCI 2 and MgC12, 0.3% glucose and fructose, in 10 mM phosphate-buffered saline) at 33°C for 20 min with frequent vigorous mixing. The fragmented tubules were pelleted at 200 g for 2 min at 25°C. The resuspended ceils (in 5 ml) were treated with trypsin (0.25%) at room temperature for 3-5 min with periodic shaking. To this mixture, DNAasel (10 ng/ml) and Soybean Trypsin Inhibitor (SBTI, 1 mg/ml) were added in 5 ml SpM and incubated at 33°C for 10 min. The ceils were pelleted and washed 3 times with SpM. The pelleted cells were resuspended 1 ml of SpM and filtered through double-layered fine nylon mesh. At this stage, a portion of the filtered ceils were either used for (a) standard microwave-accelerated glutaraldehyde fixation (Murti and Schimenti, 1991), or (b) prepared for flow-cytometric analysis. PCR of sperm DNA About 2000 epididymal sperm were used as substrate for PCR as described (Cui et al., 1989). The primers (Fig. 1) generate a 1.2-kb amplified fragment. The lacZ primer was 32p end-labelled by kinasing prior to PCR. The amplified fragments were gel purified, followed by digestion with either ClaI or NruI. The digested DNA was electrophoresed on 12% acrylamide non-denaturing gels, which were dried and exposed to X-ray film (shown), or imaged on an AMBIS blot analysis system for direct quantitation of radioactive emissions.

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Staining methods (i) Propidium iodide staining. 2 x 106 cells were suspended in 50 /zl of ice-cold PBS. 500 /zl of -20°C methanol was gently overlaid. The two layers were vigorously vortexed for 10 sec to completely fix the cells. 500/zl of PBS was added, the cells were pelleted and rinsed twice in PBS. The cells were treated with RNAase (10/xg/ml) at 37°C for 30 min. Finally, 500/zl of the staining solution (100 /xg/ml propidium iodide in PBS containing 0.1% NP-40 and 0.01% sodium azide) was added and the mixture was maintained thereafter on ice for flow-cytometric DNA analysis. Oi) Immunochemical analysis. Both polyclonal (mouse anti-C3-galactosidase, Sigma Product No. G-4644, 1:1000 dilution) and monoclonal antibodies (Sigma Product No. G-8021, 1:2000 dilution) were used as the primary antibody with equal success, though the fold-increase of the specific signal using the monoclonal antibody was marginally better (data not shown). Hence for most analyses, polyclonal antibody was used at the recommended titers. All steps were performed at 37°C for 30 min each. All washes were done in (1:1) PBS:normal goat serum (NGS) followed by pelleting the cells at 400 g for 1 min at 10°C. 2 x 106 cells were pre-incubated in 300/zl

Recipient

Prm-5'

Nrul

P

I

I

/ L.

Flow cytometry and data analysis. Fluorescence measurements were made with an Ortho Cytofluorograph IIs system (Westwood, MA) equipped with a 5 W argon laser operating at 250 mW at 488 nm. Fluorescein and propidium fluorescence were collected through standard Ortho filters (red > 640 nm) with a 540 nm short pass added to the standard 530/45 band pass supplied by Ortho. Data were acquired with a linear amplifier for the integrated and peak signal from propidium (red) fluorescence and a logarithmic amplifier for the integrated signal for immunofluorescence (green). A bivariate plot of peak versus integrated red signals was employed to estimate single-cell, cell-aggregate and debris content. Immunofluorescence data were analyzed using the Ortho 2151 system. Estimates of cell cycle phases were made with the Modfit DNA modeling software (Verity Software House, Topsham, ME) using a broadened trapezoid model for S-phase fit.

Donor

P

L a c Z

PBS: NGS. Following two rinses, the ceils were incubated with ImmunoPure (Pierce, Rockford, IL) biotinylated-Goat anti-mouse IgG antibody. After incubation, the cells were rinsed twice and incubated with Immunopure Avidin (FITC-conjugated), in the dark to prevent bleaching. The cells were washed thrice and resuspended in 0.5 ml PBS before being subjected to flow cytometric analysis.

~*

Clal

,~

I I Prrn-3'

I L a c Z

IOCRprime~

Fig. 1. Structure of transgene constructs. As indicated, the black boxes represent mouse protamine 1 (Prm-1) sequences and shaded boxes are lacZ sequences. Transcriptional orientations of the lacZ genes are indicated by the arrows. The black vertical stripe in the recipient lacZ gene is a 2-bp insertion mutation, which converted a ClaI site into a NruI site. P = PuuII. The crossed-out " P " s correspond to the PvuII sites in the recipient gene, but were destroyed during cloning. The donor lacZ gene is deleted for the first 36 and last 136 amino acids of the enzyme. The construct is 7.5 kb in length. To reconstruct a functional lacZ gene, unmutated sequences from the donor gene must be transferred to the recipient in a gene conversion event which corrects the frameshift mutation. See Murti et al., (1992) for details.

Drug-administration regimens Chlorambucil (CAS No. 305-03-3). 60-90-day-old transgenic males were administered intraperitoneally 3 doses (10 m g/ kg body weight; stock prepared in 70% ethanol and diluted 10 times in PBS) spaced 7 days apart. This regimen was based upon those previously used to induce germ-line mutations in mice (Flaherty et al., 1992; Rinchik, 1991; Russell et al., 1989). The animals were sacrificed 3 weeks following the last injection. Control males were injected with an equal volume of 7% ethanol in PBS. Acrylamide (CAS No. 79-06-1). 60-90-day-old male mice were treated with a total of 250 mg

J.R. Murti et al. / Mutation Research 307 (1994) 583-595

acrylamide/kg body weight in a 5-day regimen of 50 mg/kg daily intraperitoneal administration (Russell et al., 1991). Each batch of the chemical was prepared fresh, filter-sterilized through 0.22/zm membrane filters and used within 30 min of preparation. All animals were sacrificed 21-23 days following the last i.p. administration.

3. Results

Assay system for quantitating germ-line gene conversion We have previously described a system for efficient detection and quantitation of intrachromosomal gene conversion events in the germ-line of mice (Murti et al., 1992). It is based upon transgenic mice containing two mutually defective lacZ (bacterial fl-galactosidase) reporter genes under the regulatory control of a spermatogenesis-specific promoter, mouse protamine-1 (Prm1). lacZ enzymatically cleaves the colorless substrate X-gal into a visually prominent, water-insoluble blue product. A specific intrachromosomal gene conversion event must occur to generate a functional active lacZ gene. Conversion events are visualized by histochemical staining of transgenic spermatids with X-gal; those spermatids that underwent the planned conversion stain blue. The basic construct we have used for generating transgenic mice is shown in Fig. 1. The construct consists of two lacZ genes with different mutations. One of these genes, called the "recipient," is under the transcriptional control of the Prm-1 promoter. A central 2-bp insertion was introduced into the recipient gene, thereby abolishing /3-galactosidase activity by frameshift mutation. The second lacZ gene, called the "donor", does not possess a promoter. It has two lesions: 36 and 136 amino acid truncations at the amino and carboxy termini, respectively. These two separate deletions in the donor gene were designed such that a single intrachromosomal reciprocal crossover between the recipient and donor genes could not restore lacZ function. Male mice carrying the entire construct transcribe the recipient gene specifically in haploid germ cells, but will not produce functional lacZ

587

unless a spontaneous mutation or recombination event corrects the 2-bp frameshift mutation. To measure the frequency of gene converted spermatids, haploid cells from transgenics are purified from whole testicular cell preparations followed by fixation and X-gal staining (Murti et al., 1992). The spontaneous recombination frequency in the two transgenic lines examined here was 1-2% (see Results; Murti et al., 1992). Control transgenics containing only the recipient portion of the transgene (see Fig. 1) yield virtually no blue ceils (Murti et al., 1992). To obtain molecular corroboration that the presumed gene conversions responsible for generating lacZ-positive ceils indeed involve the planned sequence transfer, PCR amplification of the Recipient gene from COR3 hemizygotes was performed using the primers shown in Fig. 1. The amplified fragments were digested with either NruI or ClaI. As described in the Fig. 1 legend, the recipient gene mutation was created by changing a wild-type ClaI site into a NruI site.

a

b

c

d

Fig. 2. P C R assay for conversion events in transgenic sperm. Sperm from transgenic males were PCR-amplified as described in Methods. Lane a, C O R 3 digested with NruI. Lane b, C O R 3 digested (in duplicate) with ClaI. Lane c, ClaI digest of amplified D N A from a mixture of sperm from two animals carrying only the recipient lacZ gene (Fig. 1; Murti et al, 1992), or only the donor gene. Lane d, same as lane c, but a Nru I digest.

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588

Cleavage with ClaI is indicative of a gene conversion event which replaced the mutant NruI site in the recipient gene, and NruI cleaves the vastly predominant non-recombinant products. The resuits are shown in Fig. 2. Whereas amplified material from a control mixture of sperm from mice carrying only the recipient and only the donor transgenes showed no digestion with ClaI (Fig. 2), approximately 1.9% (determined by direct quantitation of radioactive emissions [Fig. 2 legend]) of the product from a COR3 hemizygote was cleaved to the expected size. To normalize this percentage for comparison to the lacZ activity staining results, it must be divided by 2 since only half the sperm contain a transgene. The resulting percentage of 0.95% is in reasonably close agreement with that determined by lacZ staining (about 1.75%; Table 1; Murti et al., 1992).

Table 1 Chlorambucil treatment of transgenic mice The m e t h o d s for drug administration and preparation of quantitation, the dispersed cells were examined (400 x ) individual cells were counted from the prints developed. samples with the highest fold-induction (No. 2 and No. 9) by PI staining) only were scored.

Induction of gene conversion by the potent mutagen chlorambucil To determine whether gene conversion frequency could be elevated by exposure to genotoxic chemicals, we treated our transgenic mice with chlorambucil, a fat-soluble compound that induces mutations at a high frequency in postspermatogonial germ cells. It has been used to create germ-line mutations in mice, which are primarily chromosomal deletions and translocations (Flaherty et al., 1992; Rinchik, 1991; Russell et al., 1989). Due to the disastrous effects at the genetic level, this drug has been classified as an extremely potent carcinogen with a high genotoxic risk. Mice were administered with a regimen previously established for the generation of mutations in the mouse (see Methods). Any increase in the percentage of blue spermatids (the transgene ex-

total seminiferous tubular cells are described in Methods. For manual by light microscopy. Selected fields were photomicrographed and the Classification of cell types was based upon morphology and size. T h e were used for parallel flow-cytometric analysis. Haploid cells (identified Flow cytometry

Manual quantitation Mouse

Total

COR3 transgenic line Non-transgenic Un-treated No. 6

199 1 540

Treated No. 7 Treated No. 8 Treated No. 9 Total, treated

% blue

N u m b e r of haploid

0 27

0.00% 1.75%

50 000

17

1 300 1 080 2100

59 61 147

4.54% 5.65% 7.00%

6 436

353

5.48%

4 480

267

5.96%

700

10

1.43%

2 3 4 5

2250 840 1 560 1 225

171 18 3 52

7.60% 2.14% 0.19% 4.24%

10 588

1 440

13.60%

Overall Without No. 4

5 875 4440

244 241

4.15% 5.42%

OPP2 transgenic line Un-treated No. 1 Treated Treated Treated Treated

No. No. No. No.

N u m b e r of blue

N u m b e r of positive

% blue 0.034%

J.R. Murti et al. / Mutation Research 307 (1994) 583-595

pression is limited to haploid cells) compared to controls is attributable to the mutagen's effects, presumably reflecting the degree to which it has induced DNA damage. Chlorambucil treatment in all mice of both transgenic lines drastically curtailed spermatogenesis in general, decreasing the spermatid count. This is concordant with the known effects of this compound (Russell et al., 1989). Despite this, the percentage of lacZ -positive spermatids was substantially elevated over baseline values (Table 1). Quantitation of gene conversion frequency in treated and untreated transgenics was determined in two ways: by standard activity staining for lacZ, and by flow cytometry. Using the latter method, an unpurified preparation of testicular cells was co-stained with propidium iodide (PI) and an anti-lacZ antibody (see Methods). The PI staining, for determination of DNA content, allowed selective quantitation of haploid cells in the crude preparation, which contains germ cells at all stages of spermatogenesis. Haploid cells (spermatids) could also be distinguished on the

589

basis of light scatter, once their complexity was correlated with PI staining. The results demonstrate a clear, striking induction of gene conversion with chlorambucil (Table 1). Whereas untreated samples showed the typical basal levels of gene conversion (1.75% for COR3 mice, 1.43% for OPP2), individual treated animals exhibited up to a nearly 10-fold induction by flow cytometric analysis (13.6% in the case of OPP2 mouse No. 2). The combined data from histochemical staining demonstrates a 3.4-fold and 2.9-fold induction in the COR3 and OPP2 mice, respectively (Table 1). The overall measured stimulus in the OPP2 line may be artificially low since one of the mice (No. 4) displayed only 0.19% positive cells, which is far below the untreated level. Since this is the only time we have ever observed such a low frequency from an individual in this line, it is possible that this animal was either not a true transgenic, or that an experimental error was made in the histochemical analysis. The OPP2 line carries the transgene insertion on the Y chromosome, since only males inherit it. Conse-

Table 2 Acrylamide treatment of transgenic mice The samples were processed for manual quantitation in a manner similar to that described in the legend to Table 1. For flow cytometric analysis, distinction of haploid cells was based on light scatter complexity: haploid spermatids were in the 3-5 g m diametric range as determined by PI staining of control cells." Manual quantitation Mouse

Number of haploid

Flow cytometry Number of Blue

% Blue

Number of haploid

Number of positive

% blue

COR3 transgenic line Un-treated A Treated Treated Treated Treated Treated

B C D E F

Total, treated

490

7

1.43%

50 000

142

0.28%

650 3 400 650 1855 1 260

26 52 15 22 21

4.00% 1.53% 2.31% 1.19% 1.67%

50 000 50 000 50000 50 000 50 000

2 181 775 1 137 855 864

4.36% 1.55% 2.27% 1.71% 1.73%

7815

136

1.74%

250000

5 812

2.32%

OPP2 transgenic line Un-treated G

3 740

62

1.66%

50 000

1031

2.06%

Treated Treated Treated Treated Treated

3 100 1 140 1 570 1216 1 280

36 12 17 17 28

1.16% 1.05% 1.08% 1.40% 2.19%

50000 50000 50 000 50 000 50000

1390 866 763 1417 1 753

2.78% 1.73% 1.53% 2.83% 3.51%

8306

110

1.32%

250000

6 189

2.47%

H I J K L

Total, treated

a This percentage is suspiciously low. Typical percentages on independent experiments are 1.5-2%.

a

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J.R. Murti et al. / Mutation Research 307 (1994) 583-595

quently, we do not perform D N A typing for this transgene, and assume all males are positive. It is possible that a rare recombination during parental meiosis resulted in this individual inheriting a transgene-negative Y. If this animal is ignored, the percent induction becomes 3.8.

Effects of acrylamide upon gene conuersion We observed no obvious effect of acrylamide upon the percentage of lacZ-positive ceils in total testicular cell preps in either transgenic line (Table 2). However, several notable observations were made during microscopic examination of the treated testicular cells. While acrylamide monomer did not seriously curtail the spermatid or

Untreated

total testicular cell count, the presence of numerous unusually large ceils, some of which stained blue, were immediately apparent in testicular preps from treated animals compared to vehicletreated controls (Fig. 3). The origin of these large cells was initially puzzling and their identity could only be speculated. While their morphology (size, in particular) relative to the other cells in the same field of observation suggested that they were late spermatogonial or spermatocyte stage cells, the protamine promoter is inactive at these stages. We speculated that these cells arose as a consequence of the gross cellular damage inflicted by acrylamide at the dosage employed, or were multinucleate symplasts arising as a byprod-

Acrylamide--treated

Fig. 3. Acrylamide was administered to transgenic hemizygous male mice at regimens described in Methods. Total cells from the seminiferous tubules were prepared, fixed and stained for fl-galactosidase activity as described (Murti and Schimenti, 1991). The left panel (A) and the right panel (B) shows the spermatogenic cells from untreated and treated mice respectively. The arrows draw attention to the abnormally large-sized cells, the plausible origin and identity of which are discussed in the text.

J.R. Murti et al. / Mutation Research 307 (1994) 583-595

uct of the trypsin dissociation procedure (Romrell et al., 1976), exacerbated by the acrylamide. An alternative and more plausible explanation is based on a recent report that acrylamide monomer perturbs cell division in vitro and in vivo (Adler et al., 1993). The abnormally large cells we observed may be due to interkinetic delay during the first meiotic division. This would artificially cause an underestimate of the extent gene conversion events, since only haploid ceils (flow cytometry) or spermatids (visual quantitation), are scored in these analyses.

4. Discussion Induction o f gene conversion in germ cells by mutagens

The striking induction of gene conversion by the potent mutagen chlorambucil indicates that murine male germ cells undergo a stimulation in recombination activity in response to DNA damage as do cultured mammalian cells and other experimental organisms. This is not an unexpected finding, since DNA repair is a fundamental cellular activity likely to be similar among eukaryotic cells. Nevertheless, our observations suggest the potential usage of recombination as a marker for DNA damage in whole animals. A major question not addressed in the present study concerns the cell types in which the recombination has been induced. Recombination induction systems implemented in fungi detect mitotic gene conversion (Zimmermann, 1971), as do those in mammalian cultured cells (Hellegren, 1992). The transgenics used here undergo both meiotic and pre-meiotic (mitotic) conversion events at the transgene locus (Murti et al., 1992). Both events result in lacZ-positive spermatids which cannot be distinguished from one another. The occurrence of premeiotic conversion events was inferred from the presence of lacZ-positive spermatid clusters in undissociated seminiferous tubules (Murti et al., 1992). Since only dissociated cells were examined in this study, we could not determine in which cell types recombination was induced.

591

One way to distinguish between the possibilities depends upon the time at which analysis is performed following drug administration. It takes approximately 27 days for spermatocytes to progress to mature spermatids in mice (Oakberg, 1956). If meiotic gene conversion is primarily induced, any elevation in lacZ-positive cells would disappear 27 days following the final drug administration (assuming a very short drug half-life in the body), as the meiotically converted cells are cleared from the seminiferous tubules. Induced conversions in spermatogonia would have 2 effects: first, up to 3 weeks would be required for a primary, converted spermatogonium to progress to the spermatid stage where lacZ could be expressed. Second, increased conversion in primitive spermatogonia would cause a long-term elevation of positive spermatids. Transgenic lines in which the reporter is expressed pre-meiotically would more directly address this issue. Affect o f acrylamide upon gene conversion

The water-soluble chemical monomeric acrylamide has found extensive applications in the industry, agriculture and common laboratory. It has been used in the production of polymers, and was reported to be biologically inert as soil stabilizers, etc (Dearfield et al., 1988). Besides its well documented neurotoxic properties, the genotoxic, carcinogenic, reproductive and developmental effects are only now beginning to be evaluated. Sperm-head abnormalities, degeneration of testicular tissue, and dominant lethal effects have been attributed to acrylamide (Sega et al., 1989; Shelby et al., 1987). This chemical has been recommended to be a "B2" (limited) carcinogen due to tentative epidemiological data, confusing independent risk assessments and far-from-conclusive genotoxic assessment studies (Dearfield et al., 1988). Most mammalian genotoxic studies on the acrylamide monomer have been performed with specific experimental end-points in mind. Results in gene mutation assays such as the Ames test (1985), the mammalian C H O / H P R T cell culture assay (Wayne, 1983b), and the Drosophila sex-linked recessive lethal assay (Wayne, 1983a) are largely inconclusive. Chromosomal assays, such as

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those monitoring spermatocyte chromosomal aberrations (Shiraisi, 1978), the mouse micronucleus assay, and the rodent dominant lethal test (Dearfield et al., 1988), have either directly demonstrated that acrylamide monomer inflicts gross chromosomal damage or have indirectly suggested its effects are heritable in the form of reciprocal D N A translocations. Our analysis of acrylamide in this report did not reveal any clear induction of gene conversion. However, as described earlier, this may be due to the complicating factor of possible meiotic arrest being induced by the high levels of administered acrylamide. Alternatively, the transgenic system in its current state is not sufficiently sensitive to detect marginal genotoxins. Comparison to other transgenic systems in toxicological analyses Several transgenic mouse systems for detecting genotoxic agents have been introduced, each utilizing lacZ as a reporter molecule (reviewed in Gossen and Vijg, 1993). The two most popular systems carry a target gene in a lambda shuttle vector. In each case, the vector is present in a tandem array of numerous copies. The Big Blue mouse marketed by Stratagene (Kohler et al., 1991) is a lacI transgenic mouse. Following mutagen exposure, DNA is extracted from any tissue, packaged in vitro into bacteriophage particles which are then plated on bacteria to form plaques. lacZ-positive plaques contain a mutated lacI repressor gene. The " M u t a M o u s e " contains normal copies of the lacZ gene. Mutations are detected in a manner similar to "Big Blue," except that lacZ-negative plaques represent mutants (Myhr, 1991). The initial data indicate that there is a dose-dependent increase in the number of mutations detected by both systems (Gossen et al., 1989; Kohler et al., 1990, 1991; Malling and Burkhart, 1992; Myhr, 1991), although very high dosages are needed to induce striking changes. The disadvantage of these systems are the analytical processes Of D N A extraction, in vitro phage packaging, and plating from each tissue of each treated animal. Furthermore, since the "rescued" D N A must be passed through a bacterial host, there are questions as to whether DNA adducts

formed in the mouse are converted into mutations by the bacteria and not the animal (Mailing and Burkhart, 1992; Provost et al., 1992). However, the nature of recovered mutations has not indicated this to be a problem (Provost et al., 1992). Interestingly, testis and germ cells show the lowest mutation frequencies in these systems (Gossen and Vijg, 1993). The advantages are that any tissue can be assayed, and recovery of the mutated reporter gene reveals the nature of the lesion through D N A sequencing. In comparison, whereas the recombinationbased system we describe here does not determine the-type of mutations induced, the analytical process is extremely rapid and specialized for analysis of chemicals which can cause heritable mutations. It avoids potential complications associated with passing damaged mouse DNA through another organism (bacteria). Results are obtained the same day of sacrifice. Furthermore, this system can detect "recombinogens", which may induce illegitimate recombination but not create point mutations or other subtle lesions. At this point in time, we have not noticed any obvious genetic instability in either transgenic line. The COR3 line contains a single copy of the conversion construct (Fig. 1), and is currently maintained in the hemizygous state while in the process of placing the trangenes on an inbred C 5 7 B L / 6 J background. All DNA-identified transgenics continue to display typical spontaneous conversion levels 3 years after being generated. As described in the Results section, the OPP2 line, in which the integration site is on the Y, is necessarily maintained in the hemizygous state and is also being bred onto the C57BL/6J background. Sensitivity o f the system Clearly, an extension of these initial experiments is required to determine the general applicability of these transgenic lines to detect germline genotoxic agents. Such studies are under way. Preliminary results indicate that the mutagens ENU and EMS induce a marked response in these mice. Nevertheless, it is likely that the transgenic lines used in this study can be improved upon. For example, it may be possible to

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increase the sensitivity by reducing the background frequency of gene conversion using modified transgene constructs. The sensitivity of this gene conversion assay is a primary factor in determining whether it will be useful as a genotoxicity screening tool. A major advantage of this approach is the sheer number of events which can be scored. Using manual quantitation and standard histochemical staining for lacZ, over 10000 cells can be scored in one day by examining photomicrographs of the stained sample. With a background frequency of about 2% positive, this corresponds to 200 blue cells. This high baseline number actually renders the system highly sensitive to rather small proportional variations. For example, if a drug treatment elevated the percentage of blue cells to 3%, this would represent 300 blue cells out of 10 000 screened. The increase of 100 cells translates into a Chi square value of 100, P < 0.00001. With the application of flow cytometry to the screening process, a much larger number of cells, from many individual mice, can be scored more rapidly. In conclusion, because of the high throughput of this system, a relatively small increase in the percentage of blue cells can translate into a highly significant deviation from basal levels. A cautionary note, however, is that the data-indicate that there may be substantial mouse-to-mouse variation in either conversion frequencies or quantitation accuracy (Table 1). This variation should be compensated for by the use of several animals in a chemical mutagenesis test.

Future prospects

Our experimental strategy provides the flexibility and rapidity of short-term tests and the predictive power associated with contemporary long-term whole animal assays. For most compounds, relevant data do not exist for germ cell mutagenicity, which is an area of considerable concern. The E P A Guidelines for Mutagenicity Risk Assessment place greater emphasis on assays that are (1) performed in germ cells rather than somatic cells, (2) on tests performed in vivo rather than in vitro, and (3) on mammalian species rather than submammalian species. Our genotox-

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icity screening system has the potential to be a rapid and inexpensive short-term rodent genotoxicity indicator, and as such, would likely be of substantial medical and economic benefit.

Acknowledgements We thank Joe Nadeau, Beth Simpson and an anonymous reviewer for critical review of the manuscript. This work was supported by an exploratory grant from the Cleveland chapter of the American Cancer Society, and an N I H grant to JCS from the Institute of General Medical Sciences. The flow cytometric analysis was performed in the Flow Cytometry facility at Case Western Reserve University, which is under the direction of J. Jacobberger.

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