The in vivo mutagenicity and mutational spectrum at the lacI transgene recovered from the spleens of B6C3F1 lacI transgenic mice following a 4-week inhalation exposure to 1,3-butadiene

Mutation Research 401 Ž1998. 99–110

The in vivo mutagenicity and mutational spectrum at the lacI transgene recovered from the spleens of B6C3F1 lacI transgenic mice following a 4-week inhalation exposure to 1,3-butadiene Leslie Recio ) , Linda J. Pluta, Kathy G. Meyer Chemical Industry Institute of Toxicology, 6 DaÕis Dr., P.O. Box 12137, Research Triangle Park, NC 27709, USA Received 26 June 1997; revised 27 November 1997; accepted 8 December 1997

Abstract 1,3-Butadiene ŽBD. is carcinogenic and mutagenic in B6C3F1 mice. We determined the lacI mutant frequency and mutational spectrum in spleen following inhalation exposure to BD at levels that are known to induce tumors. B6C3F1 lacI transgenic mice were exposed to air or to 62.5, 625, or 1250 ppm BD for 4 weeks Ž6 hrday, 5 daysrweek. and euthanized 14 days after the last exposure. BD increased the lacI mutant frequency in spleen at all levels of BD examined. In BD-exposed mice, an increased frequency of G:C ™ A:T transitions occurred at non-5X-CpG-3X sites. Exposure to BD in B6C3F1 lacI transgenic mice also increased the frequency of base substitution mutations that occurred at A:T base pairs when compared to air controls. The increased frequency of specific mutations at G:C base pairs in spleen was not observed in our previous studies in bone marrow and indicates tissue-specific differences in the BD-induced mutational spectrum. These data demonstrate that in vivo transgenic mouse mutagenicity assays can identify tissue-specific mutagenicity and mutational spectrum responses of genotoxic carcinogens at exposure levels that are known to induce tumors. q 1998 Elsevier Science B.V. All rights reserved. Keywords: 1,3-Butadiene; lacI transgenic mouse; Mutational spectrum; Spleen

1. Introduction 1,3-Butadiene ŽBD. is used in the production of synthetic rubber and has a worldwide consumption of 6.1 million metric tons w1x. BD is among the top 50 chemicals produced in the US w2x and is regulated as a hazardous air pollutant under the 1990 Clean Air Act Amendment w3x. BD is carcinogenic in rodents at multiple sites, with mice more susceptible than rats to the carcinogenic effects w4–8x. ) Corresponding author. Tel.: q1-919-558-1329; fax: q1-919558-1300; E-mail: [email protected]

The toxicology and epidemiology of BD have been the subject of a number of reviews and discussions w9–15x. The International Agency for Research on Cancer ŽIARC. has classified BD as a Group 2A carcinogen Žprobable human carcinogen. w16x. This 1992 IARC classification for BD was based on sufficient evidence of animal carcinogenicity and limited evidence for carcinogenicity in humans. Epidemiological studies on synthetic rubber workers have reported an association between BD exposure and risk for leukemia w17,18x. BD is biotransformed in vitro and in vivo to numerous metabolites w15,19x. BD is bioactivated to

0027-5107r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 3 1 9 - 9

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L. Recio et al.r Mutation Research 401 (1998) 99–110

at least two genotoxic metabolites, 1,2-epoxybutene ŽEB. and 1,2,3,4-diepoxybutane ŽDEB.. Following inhalation exposures of BD, blood levels of EB and DEB are greater in mice than in rats w20x. The bioactivation of EB to DEB occurs in purified human CYP2E1 and CYP3A4 enzyme preparations and in human, mouse and liver microsomes w21x. EB and DEB are direct-acting mutagens in human cells w22x. The increased levels of the genotoxic metabolites EB and DEB in mice compared with rats is hypothesized to account for the increased susceptibility of mice to the carcinogenic effects of BD compared with rats w12x. BD is genotoxic in a number of in vitro and in vivo experimental systems w9,10,15,16,23x. Mice show an increased susceptibility to the genotoxic effects of BD Žmicronuclei. compared with rats that is also likely due to the differences in blood levels of EB and DEB in mice and rats w24,25x. BD is mutagenic after inhalation exposures at hprt in mouse T-lymphocytes w26,27x, and at the lacI transgene in the bone marrow of B6C3F1 lacI transgenic mice w28x. BD is a germ cell mutagen in mice that induces dominant lethal and heritable translocations following inhalation exposure w23,29,30x. In the present article, we report the lacI mutant frequency and mutational spectrum at the lacI transgene recovered from the spleens of B6C3F1 lacI transgenic mice exposed by inhalation to BD. The exposure levels of BD used for these mutagenicity studies Ž62.5, 625 and 1250 ppm. were used in BD cancer bioassays in B6C3F1 mice w5,6x. The analysis of the lacI mutant frequency and mutational spectrum from spleen reported in this article was done from the same BD exposures and group of animals used previously to assess the lacI mutant frequency and mutational spectrum in bone marrow w28,31x. These studies were done to assess tissue-specific differences in mutational response and mutational spectrum following inhalation exposure to BD.

2. Materials and methods 2.1. lacI mutant frequency in spleen The mutagenicity experiment utilized B6C3F1 lacI transgenic mice ŽBig Bluee. purchased from

Stratagene Cloning Systems ŽLa Jolla, CA. and obtained from Taconic Farms ŽGermantown, NY.. Details on the experimental design, including chemicals, animals, and exposure system used for this mutagenicity experiment, are reported elsewhere w28x. Briefly, whole body exposures of male B6C3F1 lacI transgenic mice Ž6–8 weeks of age. to 0, 62.5, 625, or 1250 ppm BD Ž"5%. were for 4 weeks, 5 daysrweek, 6 hrday. Animals were euthanized 14 days after the last exposure. Spleens were collected, initially frozen in liquid nitrogen and then stored at y808C until processed for DNA extraction and lacI mutant frequency determination. The lacI mutant frequency was determined as described in detail previously w28,32x. The lacI mutant frequency was determined using 25-cm2 nutrient agar plates containing X-gal. All lacI mutant plaques were restreaked on plates containing X-gal to confirm the lacIy phenotype. The number of confirmed lacI mutant Žblue. plaques was divided by the total number of non-mutant Žcolorless. plaques scored to estimate the lacI mutant frequency Ž=10y5 .. The lacI mutant frequency was determined in three animals each from the air control and BD-exposed groups. The mutational spectrum was determined from lacI mutant plaques that were re-isolated from the same plates used to determine the lacI mutant frequency in air control animals and animals exposed to 1250 ppm BD. 2.2. Isolation and DNA sequence analysis of lacI mutants lacI mutants cored from the plates used to determine the lacI mutant frequency were confirmed by restreaking on plates containing X-gal and were then replated at low density to select a single isolated lacI plaque for DNA sequence analysis. The l phage lacI mutants were adsorbed to log-growth cultures of SCS-8 Escherichia coli cells, and l phage DNA was extracted and purified from the supernatant using Magic Lambda Preps ŽPromega, Madison, WI.. The lacI gene was amplified by the polymerase chain reaction ŽPCR. for DNA sequence analysis. Oligonucleotide primers used for PCR amplification, PCR methods, and DNA sequencing have been reported w28,31x. The full length lacI PCR product

L. Recio et al.r Mutation Research 401 (1998) 99–110

was purified using Magic PCR Preps ŽPromega. and the lacI gene was sequenced using the Taq DyeDeoxye Terminator sequencing kit and an Applied Biosystems 373A DNA sequencer ŽApplied Biosystems, Foster City, CA.. The lacI gene was sequenced in its entirety for each mutant. All mutations were determined by at least two primer reactions. The first G in the GTG start codon is base number 29 w33x. To identify potential sites of mutation, the sequence data obtained from each primer were initially analyzed using the SeqEde 675 DNA sequence analysis software ŽApplied Biosystems.. Potential sites of mutation identified by the SeqEde 675 software were examined visually on the DNA histogram from each primer reaction to confirm the mutation with respect to the wild type sequence. 2.3. Statistical analysis of lacI mutant frequency and mutational spectra data The lacI mutant frequency was log-transformed and tested for significant differences Žair control compared to 625 ppm BD-exposed group. using Student’s t-test Ž P - 0.05. w34x. To examine the mutational spectrum data in air control and BD-exposed mice for differences, the percentage of inde-

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pendent mutations determined for each animal was used to calculate a mutation frequency for the air control and 1250 ppm BD-exposed group; lacI mutant plaques with identical mutations in spleen isolated from a single animal were considered to be mutant siblings arising from one in vivo mutational event w31x. The mutation frequencies were used to calculate the contribution of each mutational class to the lacI mutation frequency determined for each group Žpoint mutations at G:C or A:T base pairs; the contribution of deletions, insertions, and tandem changes were considered together as complex mutations.. Fisher’s exact test ŽOne-tailed; P - 0.05. was used to test for significant differences between groups using the mutation frequency calculated for each particular genotypic change within the air control and 1250 ppm BD-exposed groups Že.g., mutation frequency at G:C base pairs in air controls vs. mutation frequency at G:C base pairs in animal exposed to 1250 ppm BD. w31x. 3. Results 3.1. lacI mutant frequency The lacI mutant frequency was determined in DNA isolated from the spleens of animals exposed

Table 1 Mutant frequency at the lacI transgene recovered from the spleens of B6C3F1 lacI transgenic mice following inhalation exposure to 1,3-butadiene Animal no. 1 2 5 mean " SD 6 9 10 mean " SD 11 12 14 15 mean " SD 16 18 19 mean " SD )

lacIy plaquesrtotal plaques

lacIy mutant frequency Ž=10y5 .

0.0 0.0 0.0

16r581,514 24r573,206 21r529,161

62.5 62.5 62.5

36r295,466 28r307,458 81r298,725

2.8 4.2 4.0 3.6 " 0.8 12.2 9.1 27.1 16.1 " 9.6 ) 10.6 11.8 15.8 11.1 12.3 " 2.4 ) 11.0 15.8 19.1 15.3 " 4.1)

1,3-Butadiene exposure level Žppm.

625 625 625 625

32r302,146 40r337,892 42r265,722 38r342,244

1250 1250 1250

37r337,374 48r304,065 60r314,306

Significantly greater than air controls Žone-tailed Student’s t-test, P - 0.05..

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Table 2 Results of the DNA sequence analysis of lacI mutants recovered from the spleens of air control and 1,3-butadiene-exposed Ž1250 ppm. B6C3F1 lacI transgenic mice X

X

DNA sequence 5 ™ 3

Mutant codon

Base change

Amino acid change

Mutants

42 92 95 95 99 173 180 210 341 381 630 791 867 936 49 86 92 95 180 195 198 273 287 375 375 381 y8 56 93 186 198 269 284 308 330 530 882

GTA ACG TTA TCC CGC GTG CGC GTG GTG CGC GTG GTG GTG GTG AAC ATT CCC AAC AAC CGC GTG CAG TCG TTG GTC GAA GCC CAA CGC GTC GGC TGG CAT ATG CGC GCC AGC TCA TGT GAC CGC TTG TTA TAC GAT ACC GTT TCC TCC CGC GTG CGC GTG GTG AAC CGC GTG CAA CTG GCG CTG GCG GGC GCG GCG ATT CGC GCC GAT CTC GCG CAA CTC GCG CAA CAA CGC GTC GCA TGA TAG GTC GCA GAG TCC CGC GTG GTG GCA CAA CTG GCG GGC GTC GCG GCG TCT CGC GCC AGC GTG GTG GAA CGA AGC ACG CGA CTG CCG CCG TTA

ATG TGC ATG CTG GGG GCC CAC TAG TAA CAC TAG TGC TGA CCC TAA TTT TGC ATG CAC CCG GTG GAG ACC GGG GTG CAC TGG ACA CAC GTA GTG ACG TGC ATG CAA TGA CTG

C™T C™T G™A G™C T™G C™G G™A C™A G™T G™A G™A C™T C™G G™C C™A G™T C™T G™A G™A T™C C™T C™A G™A C™G C™T G™A A™G G™A G™A C™T C™T G™A C™T G™A G™A C™T C™T

thr ™ met arg ™ cys val ™ met val ™ leu val ™ gly pro ™ ala arg ™ his ser ™ stop glu ™ stop arg ™ his trp ™ stop arg ™ cys ser ™ stop arg ™ pro tyr ™ stop val ™ phe arg ™ cys val ™ met arg ™ his leu ™ pro ala ™ val ala ™ glu ala ™ thr ala ™ gly ala ™ val arg ™ his ala ™ thr arg ™ his ala ™ val ala ™ val ala ™ thr arg ™ cys val ™ met arg ™ gln arg ™ stop pro ™ leu

2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 2 1 1 2 1 1 1 1 1 2

1,3-Butadiene exposed 16 y34 49 83 86 90 93 95 186 273

TGG TGC AAA TTA TAC GAT CAG ACC GTT ACC GTT TCC GTT TCC CGC TCC CGC GTG CGC GTG GTG GTG GCA CAA GCG GCG ATT

AGC TAA GCC TTT TAC CAC ATG GAA GAG

T™A C™A A™G G™T C™A G™A G™A C™A C™A

tyr ™ stop thr ™ ala val ™ phe ser ™ tyr arg ™ his val ™ met ala ™ glu ala ™ glu

3 1 1 1 2 1 1 1 1

Animal no. Air control 1

2

5

Base no.

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Table 2 Žcontinued. Animal no.

18

19

X

X

Base no.

DNA sequence 5 ™ 3

Mutant codon

Base change

Amino acid change

Mutants

324 707 710 723 792 857 42 54 56 57 72 95 103 198 311 318 378 843 864 882 944 1079 1188 1200 1206 20 56 82 84 153 169 198 269 273 350 381 783 792 843 1011 1206

ATG GTA GAA TTT CAA CAA CAA CAA ACC CAA ATG CTG ATG CGC GCC ACC GAA GAC GTA ACG TTA GAT GTC GCA GTC GCA GAG GTC GCA GAG GGT GTC TCT CGC GTG GTG GTG AAC CAG CTG GCG GGC GTG GTG GTG GTG TCG ATG GCG CAA CGC GTG GGA TAC GAC AGC TCA CCG CCG TTA CTG CAA CTC GCA CGA CAG TAT GTT GTG AAT TGT GAG GAG CGG ATA AGG GTG GTG GTC GCA GAG TAT CAG ACC CAG ACC GTT GCG ATG GCG AAT TAC ATT CTG GCG GGC GTC GCG GCG GCG GCG ATT TGT AAA GCG CAA CGC GTC CTG GGC GCA ATG CGC GCC GTG GGA TAC AAA ACC ACC GAG CGG ATA

GCA TAA TAA AAG CAC TAA ATG GCC TCA GTA GCC ATG AAA GTG TTG TAG CCA GAA AAC CTG TAA TGA GCT TAT CAG CTG ACA CAT ATC ACG TAA GTG ACG GAG TAA CAC GAC CAC GAA AAC CCG

T™C C™T C™T T™A G™A G™T C™T T™C G™T C™T T™C G™A C™A C™T G™T C™A A™C G™A G™A C™T C™T C™T T™C G™A G™A G™C G™A G™T C™T T™C C™A C™T G™A C™A A™T G™A G™A G™A G™A C™A G™C

val ™ ala gln ™ stop gln ™ stop met ™ lys arg ™ his glu ™ stop thr ™ met val ™ ala ala ™ ser ala ™ val val ™ ala val ™ met asn ™ lys ala ™ val val ™ leu ser ™ stop gln ™ pro gly ™ glu ser ™ asn pro ™ leu gln ™ stop arg ™ stop

2 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 2 1

to 0, 62.5, 625 or 1250 ppm BD for 4 weeks ŽTable 1.. The fixation time post exposure to BD was 2 weeks. The total number of plaques counted, confirmed mutant plaques isolated from each animal and the lacI mutant frequency are shown in Table 1. The

val ™ leu ala ™ thr gln ™ his thr ™ ile met ™ thr tyr ™ stop ala ™ val ala ™ thr ala ™ glu lys ™ stop arg ™ his gly ™ asp arg ™ his gly ™ glu thr ™ asn

lacI mutant frequency was increased four- to fivefold at all three exposure levels of BD Ž P - 0.05. above that determined in air controls. The lacI mutant frequency did not show a ‘dose-response’ with increasing BD exposure levels. The increase in the

L. Recio et al.r Mutation Research 401 (1998) 99–110

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lacI mutant frequency at 62.5 ppm BD was equivalent to the increase in the lacI mutant frequency observed at 1250 ppm. 3.2. Spleen lacI mutational spectrum: air control and 1250 ppm BD-exposed mice The lacI mutational spectrum was determined from lacI mutant plaques that were recovered from the spleens of air control mice and mice exposed to 1250 ppm BD. Approximately 20 mutant plaques were isolated and sequenced from each of the three mice used to determine the lacI mutant frequency. The lacI mutational spectrum determined in air control animals was compared to that determined in mice exposed to 1250 ppm BD to assess differences among BD-induced mutations from those that occurred in air control mice. 3.3. lacI mutational spectrum in air control mice From the air control mice, 47 mutants were analyzed by DNA sequencing. The types and locations of base substitution mutations identified from each mouse in the air control group are shown in Table 2

Žindicated bases are the coding strand for lacI .. Other alterations Žsmall deletions and tandem changes. identified in air control mice are shown in Table 3. These data are summarized in Table 4. From 47 mutants sequenced, 41 Ž87%. were considered to be independent mutations. Six mutants among the air control mice were considered to be mutant siblings w31x. From the 41 mutations analyzed from the spleens of air control mice, four small deletions were recovered. Among the deletion mutants in air control mice ŽTable 3., a four-base deletion that occurred in a triple tandem repeat Žbase pairs 620–623., two single base deletions and a 21-bp deletion were recovered. The four-base deletion that occurred at bp 620–623 has been observed previously w28,35x. Single base substitution mutations accounted for 90% of all the mutations recovered from the spleen DNA of air control mice ŽTable 4.. The lacI mutational spectrum in air control mice was dominated by base substitution mutations at G:C base pairs 34r41 Ž83%.. G:C ™ A:T transitions accounted for 24r41 Ž58%. of the mutations in air control mice with 22r24 Ž92%. of the G:C ™ A:T transitions occurring at 5X-CpG-3X sites. Among the 41 mutations

Table 3 Deletions and tandem change lacI mutations recovered from the spleens of B6C3F1 lacI transgenic mice: air control and 1,3-butadiene-exposed Ž1250 ppm. Deletions Animal no. Air control 1 2 5 BD-exposed 16

18 19

Site

Alteration

Sequence content

No. of mutants

620–623 71 691 995–1016

–CTGG –G –T y21 bp

CGT CTG GCT GGC TGG CAT GGT GTC TCT TGG AGT GCC TCA . . . CTG

1 1 1 1

358–359 426–427 703–706 81–82 483–484 9–38

–G –C –T –AG –AC y29 bp

GCG GCG GTG GAT GCC ATT GGT TTT CAA TAT CAG ACC TCT GAC CAG GAG AGT . . . GTA

1 1 1 1 1 5

886

A™CqC

CCG TTA ACC

1

Tandem change BD-exposed 16

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Table 4 A summary of lacI mutations recovered from the spleens of B6C3F1 lacI transgenic mice: air controls and 1250 ppm 1,3-butadiene exposure groups Air control

At G:C base pairs G:C ™ A:T G:C ™ A:T @ CpG sites G:C ™ A:T @ non-CpG sites G:C ™ T:A G:C ™ C:G At A:T base pairs A:T ™ G:C A:T ™ C:G A:T ™ T:A Multibase alterations Tandem Insertions Deletions Totals

BD-exposed, 1250 ppm

Mutants

Mutations Ž%.

Mutants

Mutations Ž%.

30

26

5 5

24 Ž59. 22 Ž54. 2 Ž5. 5 Ž12. 5 Ž12.

16 2

24 Ž42. 14 Ž25. 10 Ž17. 14 Ž24. 2 Ž4.

2 1 0

2 Ž5. 1 Ž2. 0

7 1 5

6 Ž10. 1 Ž2. 3 Ž5.

0 0 4 47

0 0 4 Ž10. 41

1 0 10 68

1 Ž2. 0 6 Ž10. 57

identified from air control mice, three Ž7%. occurred at A:T base pairs; no A:T ™ T:A transversions were recovered from air control mice.

3.4. lacI mutational spectrum in 1250 ppm BD-exposed mice From the mice exposed to 1250 ppm BD, 68 mutants were analyzed by DNA sequencing. The types and locations of base substitution mutations identified from the mice in the BD-exposed group are shown in Table 2 Žindicated bases are the coding strand for lacI .. Other alterations Žsmall deletions and tandem changes. identified from the mice in the BD-exposed group are shown in Table 3. These data are summarized in Table 4. From 68 mutants in the BD-exposed group sequenced, 57 Ž84%. were considered to be independent mutations. Of the 57 mutations analyzed from the spleens of mice exposed to 1250 ppm BD, seven deletions were recovered. Among the seven deletion mutants, three were single base deletions, two were tandem deletion events, and one was a deletion of 29 bp. In one of the BD-exposed mice Žanimal number 16 in Table

3., there was a single base substitution and the insertion of a C at the same site. Of the 57 mutations analyzed from the spleens of mice exposed to 1250 ppm BD, 50 Ž88%. were single base substitutions ŽTable 4.. In the BD-exposed mice, 40r57 Ž70%. of the mutations analyzed were single base substitution mutations at G:C base pairs. G:C ™ A:T transitions accounted for 24r57 Ž42%. of the mutations in BD-exposed mice; 14r24 Ž58%. of the GC:AT transitions occurred at 5X-CpG-3X sites, while 10r24 Ž42%. were GC ™ A:T transitions that occurred at non-5X-CpG-3X sites. A total of 14 from 57 Ž25%. mutations in the BD-exposed group were G:C ™ T:A transversions; eight occurred at 5X-CpG-3X sites, while six of these G:C ™ T:A transversions occurred at non-5X-CpG-3X sites. Among the 57 mutations identified in BD-exposed mice, 10 Ž18%. occurred at A:T base pairs.

3.5. Statistical analysis of the mutational spectrum in BD-exposed mice compared to air control mice The lacI mutation frequency w31x was calculated for each animal from the lacI mutant frequency and

L. Recio et al.r Mutation Research 401 (1998) 99–110

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Table 5 lacI mutation frequency for each class of mutation recovered from the spleens of B6C3F1 lacI transgenic mice Air control lacI mutation frequency Ž=10y5 .

BD-exposed, 1250 ppm

No. of mutations

lacI mutation frequency Ž=10y5 .

No. of mutations

Single base substitutions At G:C base pairs G:C ™ A:T G:C ™ A:T @ CpG sites G:C ™ A:T @ non-CpG sites G:C ™ T:A G:C ™ C:G At A:T base pairs A:T ™ G:C A:T ™ C:G A:T ™ T:A

1.8 1.7 0.1 0.4 0.4

24 22 2 5 5

5.4 3.1 2.3 3.1 0.5

26 14 12 ) 14 2

0.2 0.1 - 0.1

2 1 0

1.3 0.2 0.6

6) 1 3)

Multibase alterations Tandem change Insertions Deletions Total

- 0.1 - 0.1 0.3 3.1

0 0 4 41

0.2 - 0.2 1.3 12.8

1 0 6 57

)

P - 0.05, one-tailed Fisher’s exact test.

the percentage of independent mutational events determined Žsee Tables 2 and 3.. The calculated lacI mutation frequency in air control mice was 3.1 = 10y5 Ž3.6 = 10y5 lacI mutant frequency= 87% independent mutationss 3.1 = 10y5 .; the calculated lacI mutation frequency in BD-exposed mice was 12.8 = 10y5 Ž15.3 = 10y5 lacI mutant frequency= 84% independent mutationss 12.8 = 10y5 .. The contribution of each mutational class ŽTable 4. to the lacI mutation frequency was calculated for air control mice and BD-exposed mice. To compare specific BD-induced mutational events with air controls, the contribution of each mutational class in the air control mice was compared to the contribution of each mutational type in the BD-exposed mice Ž1250 ppm. using Fisher’s exact test Žone-tailed; P - 0.05; Table 5.. Among BD-induced mutations, there was a significant increase in base substitution mutations at G:C base pairs that occurred at non-5X-CpG-3X sites ŽG:C ™ A:T transitions and G:C ™ T:A transversions.. In air control mice, there were 2r41 Ž5%. G:C ™ T:A transitions and no G:C ™ T:A transversions Ž- 2%. that occurred at non-5X-CpG-3X sites. This was compared to 10r57 Ž18%. G:C ™ A:T transitions and 6r57 Ž11%. G:C ™ T:A transversions that occurred at non-5X-CpG-3X sites in BD-ex-

posed mice. Among BD-exposed mice, there was also a significant increase in single base substitution mutations occurring at A:T base pairs. BD exposure increased the frequency of A:T ™ C:G transitions and A:T ™ T:A transversions but did not increase the frequency of A:T ™ C:G transversions. In air control mice, 3r41 Ž7%. mutations were single base substitutions at A:T base pairs compared to 10r57 Ž18%. in BD-exposed mice.

4. Discussion Transgenic mice containing shuttle vectors provide a unique opportunity for assessing the in vivo mutant frequency and the resulting mutational spectrum that occurs in the tissues of mice exposed to carcinogens. Previously, we have shown that BD is an in vivo mutagen that induced an increased lacI mutant and mutation frequency in the bone marrow w28,31,36x. Exposure to BD in B6C3F1 lacI transgenic mice resulted in an increased frequency of point mutations at A:T base pairs in the lacI transgene recovered from bone marrow compared to air controls. Mutation at A:T base pairs also occurs in

L. Recio et al.r Mutation Research 401 (1998) 99–110

hprt mutant T-lymphocytes isolated from B6C3F1 mice exposed to BD w26x. In the present study, we determined the lacI mutant frequency and assessed the mutational spectrum in the spleens of BD-exposed mice compared to air control mice. BD was mutagenic in spleen at all exposure levels examined, but did not show a doseresponse. The lack of dose-response in spleen may likely be due to clonal expansion of lacI mutants in spleen prior to sampling. In the bone marrow the lacI mutant frequency increased between 62.5 ppm and 625 ppm; however, between 625 ppm and 1250 ppm there was no difference in the lacI mutant frequency w36x. Here also, an increase in the lacI mutant frequency due to clonal expansion at the lowest exposure level 62.5 ppm would obscure evidence for a dose-response. At 62.5 ppm, the range in the lacI mutant frequency among the three animals examined is 9.1–27.1 = 10y5 ŽTable 1.; animal number 10 in the 62.5 ppm group had the highest lacI mutant frequency Ž27.1 = 10y5 . among all animals analyzed. Therefore, clonal expansion in this group would have obscured a dose-response in the lacI mutant frequency from BD exposure. DNA sequence analysis is required to establish the contribution of mutant clonal expansion to the lacI mutant frequency. BD exposure increased the frequency of specific point mutations when compared to air control mice. The mutational spectrum in spleen from air control mice was consistent with previous reports w37x and was dominated by G:C ™ A:T transitions occurring at 5X-CpG-3X sites Ž22r41; 54%.. In contrast to the low frequency of G:C ™ A:T transitions that occurred at non-5X-CpG-3X sites in air control mice Ž2r41; 5%., there was an increased frequency of G:C ™ A:T transitions Ž10r57; 18%. in BD-exposed mice that occurred at non-5X-CpG-3X sites. Exposure to BD in B6C3F1 lacI transgenic mice also increased the frequency of base substitution mutations that occurred at A:T base pairs compared to air controls. Therefore, BD is an in vivo point mutagen in spleen at exposure levels that induce tumors in this strain of mouse. Certain types of mutations that were increased by BD in spleen differ from those observed in the bone marrow among the same group of exposed B6C3F1 lacI transgenic mice. The increased frequency of

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mutations at G:C base pairs recovered from the spleens of BD-exposed B6C3F1 lacI transgenic mice was not observed in bone marrow w28,31x. The frequency of G:C ™ A:T transitions that also occurred at non-5X-CpG-3X sites in spleen for air control mice Ž2r41; 5%. was slightly lower than that observed in the bone marrow Ž5r45; 11%. from air control mice w28x. The low frequency of G:C ™ A:T transitions that occurred at non-5X-CpG-3X sites in spleen enabled the detection of an increased occurrence of this mutation in BD-exposed mice. These data indicate that there are tissue-specific differences in the types of mutations that occur at an increased frequency in the spleens and bone marrow of BD-exposed mice compared to air controls. In contrast to the spectrum of mutations observed in bone marrow from BD-exposed mice, BD also induces specific mutations at G:C base pairs in the spleens of B6C3F1 lacI transgenic mice. These differences in the mutational spectrum observed in spleen vs. bone marrow likely represent tissue-specific differences in the DNA repair of BD-induced lesions. In similarity to the mutational spectrum observed in the bone marrow of BD-exposed mice, exposure to BD also increased the frequency of specific mutations at A:T base pairs. Although BD exposure did increase the frequency of A:T ™ T:A transversions, exposure to BD did not increase the frequency of A:T ™ C:G transversions. This specificity of mutations at A:T base pairs in spleen is consistent with the mutational spectrum observed in the bone marrow of BD-exposed mice w28,31,36x. Numerous DNA adducts have been described following exposure in vitro and in vivo to BD or to its genotoxic metabolites, EB and DEB. Adenine and guanine adducts have been observed after exposure to BD and its metabolites using in vitro and in vivo experimental systems w38–45x. Following inhalation exposure in rats exposed to BD, N 6-adenine and N 7-guanine adducts have been detected in liver w13,40x. In one study, up to six adducts Žfour at adenine and two at guanine. were detected following reaction of EB with calf thymus DNA w44,45x. In another study, reaction of EB with guanosine resulted in eight guanosine adducts that consisted of four diastereomeric pairs w43x. Specific adenine and guanine adducts have also been described for the in vitro reaction of DEB with adenosine and guanosine

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w41,44x. Invoking a specific adduct or adducts for the observed mutational spectrum resulting from exposure to BD and its genotoxic metabolites is premature due to the numerous DNA adducts described thus far for BD and its genotoxic metabolites, EB and DEB. Mutational spectrum studies integrated with a comprehensive evaluation of DNA adducts from BD exposure in vitro or in vivo is an approach to begin assessing the relevance of specific BD-derived DNA adducts as biomarkers for BD-induced genotoxic responses.

5. Conclusion These studies have extended our previous studies and show that BD is mutagenic in spleen following inhalation exposure in B6C3F1 lacI transgenic mice. Exposure to BD also resulted in a shift in the lacI mutational spectrum as indicated by an increased frequency of specific point mutations at both G:C and A:T base pairs compared to air controls. The increased frequency of specific mutations at G:C base pairs in spleen was not observed in our previous studies in bone marrow and indicates tissue-specific differences in the BD-induced mutational spectrum. Although these studies primarily focused on gene mutation events, BD exposure can induce a variety of chromosomal alterations in mice that are likely to be involved in induction of tumors by BD w24,25,46x. These studies demonstrate that in vivo transgenic mouse mutagenicity assays can identify tissuespecific mutagenicity and mutational spectrum responses of genotoxic carcinogens at exposure levels that are known to induce tumors.

Acknowledgements We thank Drs. James A. Bond and R. Julian Preston for reviewing the manuscript and Dr. Barbara J. Kuyper for editorial assistance. We also thank the personnel in the CIIT Inhalation Exposure Facility for establishing and monitoring BD exposure levels and the personnel in the CIIT Animal Care Facility for maintenance of animals during these studies.

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