6 lacI transgenic mice

Mutation Research 388 Ž1997. 175–178

Analysis of the mutagenic potential of ENU and MMS in germ cells of male C57BLr6 lacI transgenic mice Richard A. Winegar b

a,)

, Greg Carr b, Jon C. Mirsalis

a

a SRI International, 333 RaÕenswood AÕenue, Menlo Park, CA 94025, USA Proctor and Gamble, Miami Valley Laboratories, P.O. Box 398707, Cincinnati, OH 45239, USA

Abstract Mutant frequencies in male germ cells were determined in mice 3 days after exposure to saline, methylmethane sulfonate ŽMMS., or ethylnitrosourea ŽENU.. DNA was isolated from seminiferous tubules by a modified version of the drop dialysis method. A 5-fold increase in mutant frequency was observed in mice treated with ENU. No statistically significant increase was observed in mice treated with MMS. Keywords: Germ cell mutagenesis; Transgenic mutation assay; Ethylnitrosourea; Methylmethane sulfonate

1. Introduction This brief communication presents the results of a small-scale study conducted as part of the collaborative Transgenic Germ Cell Mutagenesis Study.

2. Materials and methods 2.1. Animals Mice were housed and cared for in a facility accredited by the American Association for Accreditation of Laboratory Animal Care. The 1970 Animal Welfare Act and its amendments ŽP.L. 89-544 and P.L. 91-579. and the principles promulgated by the National Institutes of Health in its Guide for the )

Corresponding author. SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025, USA. Tel.: 415-859-6457; Fax: 415-859-2889; E-mail: [email protected]

Care and Use of Laboratory Animals ŽNIH Publication No. 85–23. were followed at all times. Male C57BLr6 lacI transgenic mice were obtained from Stratagene Cloning Systems ŽLa Jolla, CA. and were approximately 8 weeks of age at the time of dosing. Animals were quarantined for 7 days before use. Mice were housed five per cage in polycarbonate, solid-bottom, suspended drawer-type cages containing Sani-Chips hardwood bedding ŽP. J. Murphy Forest Products, Montville, NJ.; they were allowed free access to Purina Certified Rodent Chow and deionized, UV-exposed water. The light cycle was 12-h lightr12-h dark. 2.2. Chemical exposures Dose groups of five mice were exposed to the test agents by intraperitoneal injection Ž10 mlrkg body weight.. The dose groups were given phosphatebuffered saline ŽPBS., MMS Ž40 mgrkg. or ENU Ž150 mgrkg. ŽAldrich Chemical Co., Milwaukee,

1383-5718r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 2 1 8 Ž 9 6 . 0 0 1 1 4 - 0

176

R.A. Winegar et al.r Mutation Research 388 (1997) 175–178

WI.. Three days after exposure, mice were euthanized by administration of 60 mgrkg sodium pentobarbital. 2.3. Isolation of seminiferous tubules After euthanasia, the testes were removed and one testis from each animal was frozen for later use. One testis was placed in a petri dish and the epithelial capsule removed. The aggregate of seminiferous tubules was gently broken up using forceps. The tubules were transfered to a 50-ml centrifuge tube containing 10 ml collagenase solution Žcollagenase type II, 1 mgrml, in Dulbecco PBS supplemented with 2 mM CaCl 2 .. The centrifuge tubes containing this mixture were incubated for 10 min at 378C in a rolling drum rotating at 200–250 rpm for DNA isolation. The seminiferous tubules were allowed to settle to the bottom of the tube, and the supernatant was decanted. The tubules were washed with 10 ml of PBS and allowed to settle again, and the supernatant was decanted. Two separate isolations were conducted, the first using a fresh testis and the second using the remaining testis, which had been quick frozen in liquid nitrogen and stored at y808C until isolation. 2.4. Isolation and packaging of DNA DNA was isolated using a modification of the drop dialysis procedure ŽMonforte et al., 1994, 1996.. Briefly, the tubules were suspended in 0.5 ml of PBS with EDTA Ž12.3 mM Na 2 HPO4 , 1.4 mM KH 2 PO4 , 13.7 mM NaCl, 2.7 mM KCl, 10 mM EDTA, pH 8.0.. Then 0.5 ml of 0.5 mgrkg proteinase K sodium dodecyl sulfate ŽSDS. solution Ž2 mgrml proteinase K, 2% SDS, 100 mM EDTA. and 20 ml b-mercaptoethanol were added to the tubules. The tubules were gently mixed, then allowed to incubate at 508C. The incubation time was 15 min in the first isolation and 3 hr in the second isolation. The solution was then transferred to a 0.45-mm pore size filter Ž47-mm type HA cellulose, Millipore, Bedford, MA. floating on 35 ml of TE buffer. Samples were allowed to dialyze for 24 hr at room temperature, then transferred to 1.5-ml microcentrifuge tubes and stored at 48C until DNA packaging was performed. Lambda phage packaging was performed using Transpack

ŽStratagene, La Jolla, CA. according to the manufacturer’s protocol, and reactions were plated on 25-cm2 NZY agar plates. Plaque-forming units ŽPFU. were quantitated by counting the number of plaques in an area representing 1% of the assay plate. 2.5. Statistical analysis Statistical analysis was perfomed using the generalized score test described by Carr and Gorelick Ž1994, Carr and Gorelick, 1995.. The one-sided pvalue for an increase in mutant frequency ŽMF. over that in control animals was chosen, since a decrease in MF was of little interest, and the one-sided test is more sensitive to increases in MF. To account for the fact that different animals contribute different numbers of plaques to the analysis, the standard error of the MF is reported rather than the standard deviation.

3. Results and discussion The small experiment reported here was conducted as part of a collaborative study among several laboratories to evaluate the utility of transgenic mutation assays for detection of germ cell mutagens. The test chemicals, doses, and dosing regimens were preassigned; however, each laboratory was given considerable flexibility in their DNA isolation and packaging methodologies. A significant difference between our study and others in the collaborative study is that we attempted to adapt the drop dialysis method ŽMonforte et al., 1994, 1996. for isolation of DNA from seminiferous tubules. We have previously shown that this easy-touse method yields 150 000 or more plaques per packaging reaction when DNA is isolated from kidney, spleen, and liver by using a 15-min digestion phase. In this study, two modifications to the drop dialysis procedure were made. First, isolated seminiferous tubules rather than isolated nuclei were prepared for digestion with proteinase K. Second, because seminiferous tubules are more resistant to digestion than most somatic tissues, the length of time for digestion was increased from the usual 15 min to 3 h and b-mercaptoethanol was added to the digestion buffer. The first isolation, using a 15-min digestion period with freshly isolated tubules, re-

8 8 8 8 8

8 8 8 8 8

6

7

8

9

10

11

12

13

14

15

ENU Ž150 mgrkg. ENU Ž150 mgrkg. ENU Ž150 mgrkg. ENU Ž150 mgrkg. ENU Ž150 mgrkg.

MMS Ž40 mgrkg. MMS Ž40 mgrkg. MMS Ž40 mgrkg. MMS Ž40 mgrkg. MMS Ž40 mgrkg.

Saline Saline Saline Saline Saline

Treatment

3

3

3

3

3

3

3

3

3

3

3 3 3 3 3

Ždays.

Expression time

Sem.tubules

Sem.tubules

Sem.tubules

Sem.tubules

Sem.tubules

Sem.tubules

Sem.tubules

Sem.tubules

Sem.tubules

Sem.tubules

Sem.tubules Sem.tubules Sem.tubules Sem.tubules Sem.tubules

Cell type

Statistically significant increase over control group

8 8 8 8 8

1 2 3 4 5

)

Animal age at start Žweeks.

Animal code

Table 1 Summary of mutational frequency data

Not calc.

Not calc.

Not calc.

Not calc.

Not calc.

Not calc.

Not calc.

Not calc.

Not calc.

Not calc.

Not calc. Not calc. Not calc. Not calc. Not calc.

-Amt. DNA recovered per mouse

2

2

2

4

2

2

2

3

2

2

2 4 2 2 2

Packages per animal

2

1

2

3

1

0 3 2 0 2

Total mutant PFU per animal

Dose group summary

61 302

38,535

39 699

14 446

14

9

7

2

Dose group summary 103 200 12

44,222

47 198

16 381

74 018

13 767 94 075 30 335 7 835 61 725 Dose group summary 11 275

Total PFU per animal

22.84

23.36

17.63

13.84

11.63

4.52

2.12

12.21

4.05

8.87

0.00 3.19 6.59 0.00 3.24

MF Ž=10y5 . per animal

17.12

4.66

3.37

MF Ž=10y5 . for dose group

2.76

0.95

0.54

SE of MF

0.007

0.12

NA

)

p-value

R.A. Winegar et al.r Mutation Research 388 (1997) 175–178 177

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R.A. Winegar et al.r Mutation Research 388 (1997) 175–178

sulted in very poor DNA yield and unacceptably low packaging efficiencies. The second isolation, using a 3-h digestion with tubules isolated from a frozen testis, gave improved DNA yields; however, the samples were resuspended in an excessive volume of TE buffer Ža volume more consistent with isolations from liver or kidney routinely done in our laboratory., so these samples were ethanol precipitated and redissolved in TE buffer before packaging. These additional manipulations resulted in packaging efficiencies that were about one-fourth the efficiencies generally observed with the drop dialysis method. Clearly, the use of the drop dialysis method for male germ cells will require further optimization. Despite the low yield of plaques, a highly significant Ž5-fold. increase in the MF was observed in mice treated with ENU ŽTable 1.. This result demonstrates the relative robustness of the assay for detection of strong mutagens. In contrast, no increase in MF was observed in MMS-treated mice. The design for this study is clearly less than optimum. Provost and Short Ž1994. demonstrated very significant Ž20-fold. increases in MF in seminiferous tubules 90 days after treatment with ENU, whereas increases after 3 days were small Žapproximately 3-fold.. The 5-fold elevation observed in our study is less impressive when one realizes that 150 mgrkg of one of the most potent germ cell mutagens ever identified was required to elicit this

response. We therefore recommend that a repeateddose regimen with an exposure period of 1–3 weeks, followed by at least a 60-day expression period, be used in the evaluation of germ cell mutagens in transgenic animal systems.

Acknowledgements The authors gratefully acknowledge Dr. Joseph A. Monforte, Kathleen O’Loughlin, and Judy Loken for providing expert technical assistance.

References Carr, G.J. and N.J. Gorelick Ž1994. Statistical tests of significance in transgenic mutation assays: Considerations on the experimental unit. Environ. Mol. Mutagen., 24, 276–282 Carr, G.J. and N.J. Gorelick Ž1995. Statistical design and analysis of mutation studies in transgenic mice. Environ. Mol. Mutagen., 25, 246–255. Monforte, J.A., R.A. Winegar and C.J. Rudd Ž1994. Megabase genomic DNA isolation procedure for use in transgenic mutagenesis assays. Environ. Mol. Mutagen., 23 ŽSuppl. 23., 46. Monforte, J.A., R.A. Winegar and C.J. Rudd Ž1996. Megabase genomic DNA isolation for use in transgenic mutagenesis assays, in preparation. Provost, G.S. and J.M. Short Ž1994. Characterization of mutations induced by ethylnitrosourea in seminiferous tubule germ cells of transgenic B6C3F1 mice. Proc. Natl. Acad. Sci. USA, 91, 6564–6568.