Mutagenesis vol.12 no.5 pp.359-364, 1997
Characterization of hprt mutations following l,2-epoxy-3-butene
exposure of human TK6 cells
Ann-Marie Steen, Kathy G.Meyer and Leslie Redo 1
Chemical Industry Institute of Toxicology, 6 Davis Drive, Research Triangle
Park, NC 27709, USA
'To whom correspondence should be addressed
1,3-Butadiene (BD) is an indirect-acting mutagen that is
bioactivated in laboratory animals to at least two mutagenic metabolites, l,2-expoxy-3-butene (EB) and 1,2,3,4diepoxybutane (DEB). In the present study, the cytotoxicity,
mutagenicity and mutational spectrum at hprt were determined after EB-exposure of human TK6 lymphoblastoid
cells (TK6 cells). EB was cytotoxic at concentrations ranging
from 200 to 1000 [iMX24 h; at 400 U.MX24 h, the cell
survival relative to unexposed controls was ~10%. Exposure
of TK6 cells to EB (400 UMX24 h) resulted in a 5-9fold increase in the hprt mutant frequency. Molecular
characterization of EB-induced hprt mutants indicated that
78% of the mutations at hprt were single base substitutions.
A significant (P < 0.05) increase in A:T—>T:A transversions
was observed compared with spontaneous hprt mutants
isolated during these studies. All of the A:T—»T:A transversions in EB-induced mutants occurred with the A in the
non-transcribed strand. These data indicate that a primary
mode of genotoxicity induced by EB in human TK6 cells
is the induction of single base substitutions.
Introduction
1,3-Butadiene (BD) has been used in the production of synthetic
rubber since the 1940s. With an annual production of 1.7X 106
tons in 1995, BD is ranked among the top 20 synthetic organic
chemicals produced in the US (Chemical Week, 1996). BD is
carcinogenic in rats and mice. However, there are striking
species differences in cancer potency and spectrum of tumours,
with mice being more susceptible to tumour induction than
rats (National Toxicology Program, 1984; Huff et al, 1985;
Melnick et al, 1990a,b; Owen and Glaister, 1990). The
International Agency for Research on Cancer (IARC) classified
BD as a Group 2A carcinogen (probable human carcinogen)
based on sufficient evidence of animal carcinogenicity but
limited evidence for carcinogenicity in humans (IARC, 1992).
The epidemiology and toxicology of BD has been the subject
of a number of reviews and discussions (Adler et al, 1995a;
Jacobson-Kram and Rosenthal, 1995; Melnick and Kohn, 1995;
Bondef al, 1995; Sorsa etal, 1996; Himmelstein <?/a/., 1996).
Recent epidemiological studies on BD-exposed populations
among synthetic rubber workers have reported an increased
risk for leukaemias (Delzell etal, 1996; Macaluso et al, 1996).
BD is biotransformed in vitro and in vivo to numerous
metabolites (reviewed in Himmelstein et al, 1996). BD is
bioactivated to at least two genotoxic metabolites, 1,2-epoxy3-butene (EBj and 1, 2, 3, 4-diepoxybutane (DEB). EB and
DEB are direct-acting mutagens in bacteria and mammalian
cells (de Meester, 1988; Cochrane and Skopek, 1994).
i UK Environmental Mutagen Society/Oxford University Press 1997
Comparative studies on the in vitro mutagenicity and induction
of sister chromatid exchange (SCE) by EB and DEB have
been conducted in rodent cells, human T-lymphocytes and
human TK6 cells (Sasiadek et al, 1991a,b; Cochrane and
Skopek, 1994). In human TK6 cells, EB is mutagenic at the
hprt and tk loci at ~ 100-fold greater concentration than DEB
(Cochrane and Skopek, 1994a). While DEB is mutagenic in
human TK6 cells at levels that occur in the blood of mice
exposed to 625-1250 p.p.m. BD, the levels of EB that are
mutagenic in human TK6 cells are ~100 times the levels of
EB detected in mice exposed to 625-1250 p.p.m. BD by
inhalation (Himmelstein et al, 1994).
Molecular characterizations of EB and DEB-induced hprt
mutants in human TK6 cells have been reported (Cochrane
and Skopek, 1994a; Steen et al, 1997). Southern blot analysis
of hprt mutants isolated from EB-exposed human TK6 cells
indicated that a substantial fraction of the EB mutants compared
with mutants isolated from DEB-exposed TK6 cells had no
change in the hprt hybridization pattern analysed by Southern
blot (Cochrane and Skopek, 1994a). These Southern blot data
indicate that a primary mode of mutagenicity at hprt induced
by EB was either through small deletions or point mutations
that were not detectable by Southern blot analysis.
In the present study, we examined the mutational spectrum
for EB at hprt in human TK6 cells and compared it with the
mutational spectrum from a set of spontaneous hprt mutants
isolated from the same stock cultures (reported previously in
Steen et al, 1997) to identify EB-induced mutations. By
characterizing the types of DNA changes induced by EB,
these studies will enable further insights into the genotoxic
mechanisms induced by the parent compound BD.
Materials and methods
Cell culture and exposure
TK6 cells are a human B lymphoblastoid cell line, described earlier by Skopek
et al. (1978). TK6 cells do not contain active P450 2E1 (Cochrane and
Skopek, 1994). TK6 cells were grown in Roswell Park Memorial Institute
(RPMI) 1640 medium supplemented with 2 mM L-glutamine, 60 U/ml
penicillin, 60 mg/ml streptomycin and 109c heat-inactivated equine serum at
37°C and 59c CO2. The TK6 cells were maintained by daily subculture at
4X10 5 cells/ml.
Prior to exposure TK6 cells were treated for 2 days with CHAT (cytidine,
10"5 M; hypoxanthine, 2X1CT4 M; aminopterin, 2X10"7 M; thymidine,
1.75X10"5 M)-supplemented medium to reduce the background hprt mutant
frequency. Following CHAT treatment, the cells were grown in THC
(thymidine, 10"5 M; hypoxanthine, 2X10"4 M; cytidine, 10~5 M (-supplemented
medium and allowed to expand for 2 days.
Exposure of TK6 cells to EB (CAS# 930-22-3; Sigma, St Louis, MO,
USA) was done in capped flasks with complete medium as described above.
As a positive control chemical, ethyl methane sulphonate (EMS; CAS#
62-50-0; Sigma) was used. Both EB and EMS were dissolved in dimethylsulphoxide (DMSO; CAS# 67-68-5; Sigma).
Mutant frequency and mutant isolation
Cytotoxicity of EB was assessed following a 24 h exposure to 0, 200, 400,
600, 800 or 1000 uM EB in TK6 cells (4X105 cells/ml in 10 ml). Solvent
control (DMSO) and unexposed cultures of TK6 cells were included with
every experiment. An exposure of 20 U.M EMSX24 h was included with each
experiment as a positive control. Post-exposure, cells were placed in fresh
359
A.-M.Steen, K.G.Meyer and L.Recio
medium and plated to estimate relative cell survival by cloning efficiency in
96-well microtitre plates (2 cells/well for DMSO and unexposed cells; 8 cells/
well for cells exposed to EB or EMS). Cultures were counted and diluted
daily to 4X10 5 cells/ml for 10 days to allow phenotypic expression of induced
hprt mutations and also to estimate cytotoxicity by growth curve extrapolation
to the day of exposure.
An experiment to determine the hprt mutant frequency (MF) was set up in
parallel to collect hprt mutants for molecular analysis of EB-exposed (400
HMX24 h) TK6 cells. TK6 cells were exposed to EB as described above (EMS
was used as a positive control). After the hprt phenotypic expression time, cells
were seeded at 40 000 cells/well in 96-well microtitre plates in the presence of
1 (ig/ml 6-thioguanine (6-TG: CAS# 154-42-7; Sigma), and at 2 cells/well
without 6-TG to determine cloning efficiency values. The plates were counted
for growing colonies 10 days after plating. Cloning efficiency with and without
6-TG was calculated, assuming a Poisson distribution for colony formation, to
determine the hprt mutant frequency.
For hprt mutant collection, six 75 ml flasks containing human TK6 cells
(4X10 5 cells/ml in 50 ml) were exposed to 400 uM EBX24 h. After the EB
exposure, the cells were placed in fresh medium, and each 50 ml flask was
divided into 10X 10 ml cultures in 25 ml flasks at 1 x 105 cells/ml for a total of
60 independent flasks. After 8 days of expression time, cells from each flask
were plated onto two microtitre plates at 40 000 cells/well in 6-TG (1 |Jg/ml).
After 10 days, one clone/plate was chosen and expanded into 10 ml of medium
(25 ml flasks) with 1 |lg/ml of 6-TG. To ensure that each mutant to be analysed
was independent, cells from each flask (0.5-1 X10 6 TK6 cells/ml) were then
replated onto two plates at low cell density, 0.3 cells/well in 6-TG (1 |ig/ml).
One clone was chosen per plate and placed in 10 ml media containing 6-TG
(1 (ig/ml). Following expansion to 0.5-1 X10 6 TK6 cells/ml, samples from each
were frozen for molecular analysis of hprt. One mutant clone corresponding to
one of the original 60 flasks inoculated immediately post-exposure was used for
molecular analysis of EB-exposed cultures.
RNA and cell lysate preparation
Total RNA was prepared from 5-10X10 6 cells with TRlzol™ reagent and
extracted with chloroform according to the manufacturer (Gibco BRL, Gaithersburg, MD). Following isopropanol precipitation, total RNA was washed in 75%
ethanol and dissolved in 200 JJ.1 RNase-free water.
For DNA analysis, 0.5-1 X106 TK6 cells were collected by centrifugation
and washed once in phosphate-buffered saline, and frozen at -80°C. Frozen cell
pellets were lysed as previously described (Fuscoe et ah, 1992).
PCR and DNA sequencing primers
PCR and DNA primers were synthesized using an ABI DNA synthesizer. Primers
used in the study are listed in Steen et al. (1997), or are indicated in the text.
Mutant characterization
Total RNA solution (10 u.1) was used in a final 20 \\\ reverse transcriptase (RT)
reaction including 200 U Moloney Murine Leukemia Virus (Gibco BRL), 40 U
RNAsin (Promega. Madison, WI). 200 U.M of each dNTP (Pharmacia. Piscataway, NJ), RT buffer (10 mM Tris-HCI pH 8.3, 50 mM KC1, 1.5 mM MgCl2)
and 50 pmol hprt 3' primer HPRT 2 (Steen et al., 1997). The RT reaction was
incubated in a Perkin Elmer 9600 (Foster City, CA) for 5 min at 25°C, 60 min
at 37°C, 5 min at 99°C, and then 4°C. For the subsequent PCR amplification, 80
u.1 polymerase chain reaction (PCR) mixture [200 u.M of each dNTP (Pharmacia).
PCR buffer (10 mM Tris-HCI pH 8.3, 50 mM KC1, 1.5 mM MgCl2). 5 U Taq
(Perkin Elmer) and 50 pmol of each primer; HPRT 5 and HPRT 6 (Steen el al.,
1997)] were added to the RT tubes. The PCR cycle programme was 5 min 94°C
followed by a two-step cycle: 1 min denaturation at 94°C and 1 min annealing
at 60°C for 30 cycles, with the last cycle containing a 5 min extension at 68°C.
An aliquot of the RT-PCR products was then analysed on a 1 % agarose gel. In
samples producing a poor RT-PCR product, a nested PCR amplification using
primers (5'->3') NF _19TAC GCC GGA CGG ATC CGT T_, and NR 695AGG
ACT CCA GAT GTT TCC AA678 (hprt cDNA base numbers) was done using
0.5 )il from the first reaction in a final volume of 50 u.1, with cycling conditions as above.
For determination of genomic alterations by PCR, 8 nl of a cell lysate sample
was used as template in a 50 Jil PCR reaction. The genomic PCR incubation
conditions were the same as for RT-PCR except for exon 1 PCR and multiplex
PCR, which required a different buffer system containing 6.7 mM MgCl2, 16.6
mM (NH4)2SO4, 5 mM p"-mercaptoethanol, 6.8 mM EDTA, 67 mM Tris-HCI,
pH 8.8, and 10% DMSO. The multiplex PCR amplification of hprt exons 1, 2,4
and 9 was performed to classify large genomic deletions. K-ras was used as a
DNA template control in the lysed cell extracts. Primer concentrations used in
the multiplex reaction were for K-ras and hprt exon 9, 10 pmol/reaction, hprt
exons 2 and 4, 5 pmol/reaction and hprt exon 1, 25 pmol/reaction. The PCR
cycle for the multiplex reaction was 94°C for 5 min and 33 cycles of 94°C for
30 s, 61°C for 2 min and 68°C for 2 min followed by 5 min extension at 68°C
incubated in a Perkin Elmer DNA Thermal Cycler. All primers used for genomic
PCR of hprt are listed in Steen et al. (1997); Gibbs et al. (1990); Fuscoe et al.
(1992), except for hprt ins 2S 29,97 ,TAC CTG TAT TC A AGT CTC TAA TA 29993
and hprt ins 2A 32,86sATG ATA TTT TCA ACT TCA GA32i846 (sequences are
5'-»3'; hprt genomic DNA base numbers) that were used in the characterization
of mutants B3A1 and D9A1. The primer pairs used for PCR amplification were
used as DNA sequencing primers.
For DNA sequence analysis, PCR amplification products were purified on
Wizard columns (Promega), and eluted in 50 u.1 of water (Sigma). Six ^tl of each
purified PCR product was used in a DNA cycle sequencing reaction using Taq
DyeDeoxy™FS Terminator cycle sequencing kit (Perkin Elmer). For cDNA
PCR, the primers used for DNA sequencing were the same primers as those used
in Steen et al. (1997) and indicated above. Sequencing reactions were ethanol
precipitated and analysed on a 6% denaturing gel using an Applied Biosystems
373A DNA sequencer (Applied Biosystems Inc., Foster City, CA, USA). The
sequence data was analysed using Factura™ and Autoassembler™ sequence
analysis software. Mutations were confirmed by visual inspection of each
individual histogram with respect to a wild-type sequence.
Analysis of the EB-induced mutational spectrum
The frequency and percentage of all mutational types among the EB-induced
mutants were compiled for statistical analysis. The mutational spectrum determined in a collection of spontaneous mutants isolated from the same TK6
stock cultures was used for comparison with the EB mutational spectrum for
identifying specific EB-induced mutations (Steen et al, 1997). The statistical
difference between the mutational types determined among the EB-induced
mutants compared with our previously reported data from spontaneous mutants
(Steen et al., 1997) was assessed by a one-way Fisher's exact test using P < 0.05.
Results
Cell survival and mutant frequency
Cytotoxicity of EB was assessed following a 24 h exposure
to 0, 200, 400, 600, 800 or 1000 \M EB in TK6 cells
(4X105 cells/ml in 10 ml). Cytotoxicity was assessed by
cloning efficiency and growth curve extrapolation. A 7%
relative survival was observed after an exposure of 400
(iMX24 h EB (data not shown). These data are consistent
with those reported in a previously published study (Cochrane
and Skopek, 1994). The 400 |iM EBX24 h exposure that
was used to generate hprt mutants for molecular analysis
induced a 5-9-fold increase in the hprt mutant frequency
Table I. Mutagenicity of 1,2-epoxy-3-butene (EB) at the hprt locus in human TK6 cells (400 uM EBX24 h)
Exposure
Spontaneous
DMSO
EB b
EMSC
Relative percentage survival (%)
hprt mutant frequency (X 10" 6 )
TK6-l a
TK6-3a
TK6-l a
100
122
7
60
100
96
7
57
5.2
3.9
24.9
16.2
TK6-1 and TK6-3 are two subcultures treated independently.
b
Four independently exposed TK6-1 flasks and six independently exposed TK6-3 flasks.
Ethyl methane sulphonate (20 uMX24 h).
c
360
±
±
±
±
TK6-3a
1.5
2.0
8.7
1.3
3.6
3.9
35.3
16.4
±
±
±
±
1.1
3.7
9.4
4.4
Characterization of hprt mutations following l,2-epoxy-3-butene exposure
(in two independent cultures) above the spontaneous hprt
mutant frequency (Table I).
Mutant characterization
All EB mutants were initially analysed by hprt cDNA-specific
RT-PCR and agarose gel electrophoresis. Hprt specific RTPCR amplified products were obtained in 47/49 (96%) of the
mutants isolated. Two mutants, A9A1 and E7 A1, did not produce
an RT-PCR product and were then analysed for genomic deletions by multiplex PCR of hprt exons 1, 2, 4 and 9 using K-ras
as a DNA template control. Genomic DNA from both A9A1 and
E7A1 produced a PCR product for K-ras but not for the hprt
exons tested (1, 2, 4 and 9). A9A1 and E7A1 were classified as
large hprt deletions.
Of 49 EB mutants analysed 39 (78%) were point mutations
and 11 (22%) were deletions that ranged in size from single
deletions to large deletions of hprt (Tables II and III). In the
49 mutants analysed, we observed 50 mutations; one mutant
D3A1 had two point mutations (Table II). Point mutations
were evenly distributed between G:C base pairs (18/39) and
Table II. DNA sequence analysis of hprt mutant clones isolated from epoxybutene-exposed (EB; 400 p.MX24 h) human TK6 cells
Mutant
no.
Base substitution mutations
A7B1
D6A1
A2A1
E2A1
C2A1
B6A1
B5A1
C4A1
E9A1
C3B1
E6A1
F3A1
F9A1
D8A2
A8A1
C10B1
A10A1
D3A1
E1A1
F4A1
C5A1
C6A1
D10A1
B7A1
D4A1
C9B1
C1A1
C7A1
B4A1
D5A1
D7A1
F8A1
B2A1
D2A1
F6B2
E4A1
F5A1
D3A1
E10A1
Deletions
E5A1
B1A1
F10A1
B3A1
D9A1
D1A1
B9A1
B10A1
F7A2
A9A1
E7A1
Sequence
alteration
Base pair
no. a
Predicted amino
acid change
DNA sequence
5'^3'»
Exon(s)
affected
G-»A
G->A
G->C
C-»T
G->A
C->T
G->A
G-»A
G-»A
G-»A
G->A
G->A
G->A
G->A
G->T
G->C
G->A
G-»T
A->T
A->T
A-*T
A->T
A->T
T->G
A-»T
A->T
A-»T
T-»G
A->G
A-»T
A->G
A->G
A->G
A->G
A-)T
A-»T
A->T
A->T
A-»G
3
209
255
325
400
454
538
538
539
568
580
IN3:1 (16,787)
IN3:1 (16,787)
IN4:1 (27,958)
IN4:-1 (31,617)
IN4:-1 (31,617)
IN6:1 (35,021)
539
124
215
301
301
343
374
404
523
611
618
IN2:-2 (16,601)
IN2:-2 (16,601,)
IN2-2 (16,601)
IN2:-2 (16,601)
IN4:-2 (31,616)
IN4:-2 (31,616)
IN7:3 (39,865)
IN8:4 (40,114)
IN8:4 (40,114)
552
IN8:-2 (41,453)
met—»ile
gly-»glu
leu—>leu
gin—»stop
glu—»lys
gln->stop
gly-»arg
gly-»arg
gly->glu
gly-»arg
asp-»asn
in frame deletion
frameshift
in frame deletion
in frame deletion
in frame deletion
frameshift
frameshift
ile—>phe
tyr—>cys
arg—>stop
arg—>stop
lys—»stop
leu-»stop
asp—>val
lys-»stop
his—>leu
cys-»trp
in frame deletion
in frame deletion
frameshift
frameshift
in frame deletion
in frame deletion
frameshift
frameshift
frameshift
frameshift
frameshift
GTT ATG GCG
AAG GGG GGC
GCA CTG AAT
GAC CAG TCA
GTG GAA GAT
AGG CAG TAT
GTT GGA TTT
GTT GGA TTT
GTT GGA TTT
GTA GGA TAT
CTT GAC TAT
TATTGT] gtgact
TATTGT] gtgact
GGGAAG] gtatgt
ttctag [AATGTC
ttctag [AATGTC
CGCAAG] gtatgt
TTG GAT TTG
CTA ATT ATG
GGC TAT AAA
ATC AGA CTG
ATC AGA CTG
ATA AAA GTA
ACT TTA ACT
GAA GAT ATA
TAT AAG CCA
AAT CAT GTT
GTT TGT GTC
ctgtag [GACTGA
ctgtag [GACTGA
ctgtag [GACTGA
ctgtag [GACTGA
ttctag [AATGTC
ttctag [AATGTC
CAGACT] gtaagt
TTGAAT] gtaagt
TTGAAT] gtaagt
ATT CCA GAC
ttatag [CATGTT
1
3
3
4
5
6
8
8
8
8
8
2,3C
3C
4C
5C
-G
-G
-C
-2680 bp
-2680 bp
-39 bp
-exon 2
-exon 4
-exon 6
154
640
648
IN4/5:(30,146-32,825)
(30,146-32,825)
(34,920-34,958)
genomic deletion
genomic deletion
genomic deletion
large deletion
large deletion
frameshift
frameshift
frameshift
deletion
deletion/insertion
frameshift
frameshift
in frame deletion
frameshift
CGA GAT GTG
AAA GCA AAA
AAATAC AAA
3
9
9
5
5
6C
2
4
6
5°
6C
8C
2
3
3
3
4
4
6
7
9
9
2,3C
2,3C
3C
3C
5C
5C
7C
8C
8C
8C
8C
"For hprt cDNA, base number 1 is the A in the AUG start codon. For genomic DXA, designation such as 1X3:1 refers to the firbt base of intron 3,
and IN4:-1 to the last base of intron 4. The Edwards et al. (1990) numbering system for the entire gene is given in parenthesis.
''Uppercase letters are coding sequence, and lowercase letters are intron sequence.
c
Exon skipped at the cDNA level.
361
A.-M.Steen, K.G.Meyer and L.Recio
A) wild type
.5'
3'
5'tttaactag [ Exon 4 latatqtat —ttttcttctag I Exon 5 Igtaagttca
3'
5'
• tttgaaag | Exon 6 | gtatgtatg 3 '
1
splicing
Exon 4 1 Exon 5
[ Exon 6
B) exon 5 deletion ( B3A1, D9A1)
intron 4 sequence
intron 5 sequence
.5'
3V
3'
5 ' tttaactag | Exon 4 [qtatqtat —ttttttag
intron 5|qtaagttggata-tttgaaag I Exon 6 | gtatgtatg 3 '
30119 30145 32826
32935
deletion breakpoint
intron 4 seq.
Exon 5
I
lExon 4
intron 5 seq.
deleted sequence
splicing
I intron 4 I intron 5 I Exon 6
I
Fig. 1. Deletion mutation of exon 5 in mutants B3A1, D9A1 and proposed mechanism of intron 4/5 sequence insertion ('pseudo exon') in hprt cDNA.
Table III. A summary of the hprt mutational spectrum from 1,2epoxybutene-exposed (400 |iMX24 h) human TK6 cells (50 mutations were
analysed from 49 mutants)
Mutational type
Spontaneous i
Epoxybutene (%)
Base substitutions
Transitions
A:T-4G:C
G:C^A:T
Transversions
A:T->C:G
22(51)
39 (78)b
3(7)
10(23)
7(14)
14 (28)
3(7)
2(5)
1 (2)
3(7)
2 (4)
12 (24)c
2(4)
2(4)
0(<2)
21 (49)
1 (2)
8(19)
10(23)
2(5)
0«2)
11 (22)b
0(<2)
0(<2)
9(18)
2(4)
A:T-H>T:A
G:C->C:G
G:C->T:A
Other alterations
Insertions
Genomic hprt deletions
Partial 5' deletions
Partial 3' deletions
Internal
Total deletions
"Spontaneous data are from Steen el al. (1997): n = 43 mutations.
b
EB mutations that were significantly different (one-way Fisher's exact test.
P < 0.05) compared with spontaneous hprt mutants in TK6 cells (Steen
et al., 1997).
C
P < 0.01 (one-way Fisher's exact test).
A:T base pairs (21/39). The proportion of transitions and
transversions was 21/50 and 18/50 respectively. In three
mutants (E5A1, B1A1 and F10A1), a single base pair deletion
resulted in frame-shift mutations in the hprt cDNA (Table II).
hprt RT-PCR products with internal exon(s) missing were
obtained in 21/49 mutants. Genomic PCR of the corresponding
missing exon(s) in the hprt cDNA resulted in products for 18/
21 mutants. After DNA sequence analysis of the PCR amplified
products from genomic DNA, 16/21 mutants had point
362
mutations in the splice acceptor/donor sites that were probably
responsible for the aberrant splicing. Mutant D3A1, a mutant
with exon 8 skipping in the hprt cDNA, had two point
mutations within exon 8, and mutant D1A1 had a 39 bp
deletion extending 21 bp into exon 6 that resulted in the
splicing out of exon 6 from hprt mRNA. Genomic deletions
of entire exons were found in three mutants (B9A1, B10A1 and
F7A2), that produced short hprt RT-PCR products (Table II).
The hprt cDNA from two mutants, B3A1 and D9A1 (Table
II), contained sequences from introns 4 and 5 that were inserted
between exons 4 and 6 (exon 5 sequence was absent). Genomic
PCR amplification with exon 5-specific PCR primers did
not result in a corresponding PCR product. PCR primers
downstream from the intron 4 and 5 insert observed in the
hprt cDNA were synthesized and used to PCR amplify
genomic DNA from TK6 cell wild type DNA and from the
genomic DNA of mutants B3A1 and D9A1. A genomic
deletion of 2679 bp that included exon 5 was confirmed with
genomic PCR and DNA sequencing. At the breakpoint junction,
there is a CA dinucleotide that could come from either end of
the intron 4 or intron 5 insert. DNA sequence analysis revealed
the presence of cryptic splice sites surrounding the deletion
that are nearly identical to the exon 5 splice site sequences.
These splice site sequences in mutants B3A1 and D9A1 likely
resulted in a hprt pseudo exon (the intron 4 and 5 hprt cDNA
insert) that was a result of a hprt genomic deletion (Figure 1).
The EB mutational spectrum obtained in the present study
was compared with the spontaneous mutational spectrum
determined from the same TK6 stock cell cultures (data in
Steen et al.. 1997). There was a significant (P < 0.01) increase
in A:T—>T:A transversions among the EB-induced mutants
(12/50; 24%) compared with spontaneous mutants (2/43; 5%)
(Steen et al.. 1997). Examination of the DNA sequence context
Characterization of hprt mutations following l,2-epoxy-3-butene exposure
of the A:T—>T:A transversions demonstrated that all the As
were located on the non-transcribed strand (Table II).
Discussion
BD is a mutagenic animal carcinogen bioactivated to at least
two mutagenic metabolites, EB and DEB, that may mediate
many of the biological effects of BD. Following inhalation
exposures of BD, blood levels of EB and DEB are greater in
mice than in rats (Himmelstein et al, 1994). The bioactivation
of EB to DEB occurs in purified human CYP2E1 and CYP3A4
enzyme preparations and in human, mouse and rat liver
microsomes (Seaton et al, 1995). Mice are susceptible to the
genotoxic effects of BD at exposure concentrations that are
not genotoxic in rats. The increased levels of the genotoxic
metabolites EB and DEB in mice compared with rats is
postulated to account in part for the increased susceptibility
of mice to the carcinogenic and genotoxic effects of BD (Bond
et al, 1995). The present study was performed to develop a
further understanding of the mechanisms of genotoxicity
induced by EB, a genotoxic metabolite of BD.
The EB mutational spectrum (400 |iMX24 h) at hprt in
human TK6 cells was analysed to provide data to assess its
potential role in the in vivo mutagenicity of the parent
compound BD. The alterations found in the EB-mutational
spectrum in human TK6 cells at hprt were statistically compared with spontaneous hprt mutants (Steen et al, 1997)
collected from the same stock cultures. The EB mutational
spectrum in this study demonstrated a significant increase in
single base substitutions as compared to the spontaneous
hprt spectrum (Steen et al, 1997). Three EB-induced G->A
transitions were also observed as part of the spontaneous
mutational spectrum. Genomic deletions affecting hprt
occurred less frequently among the EB mutants analysed
compared with the spontaneous mutational spectrum. In two
mutants, a 2679 genomic deletion affecting hprt exon 5 resulted
in an insertion of intron sequences into the hprt cDNA. The
cDNA insert was flanked by cryptic splice site sequences, and
at the breakpoint junction a CA dinucleotide repeat was
observed. The presence of dinucleotide repeats at the
breakpoints of hprt deletion mutants has been previously
reported (Rainville et al, 1995).
The most striking difference in the type of base substitution
mutations determined between EB-exposed and unexposed
TK6 cells was the increase in A:T->T:A transversions (P <
0.01). An increased frequency of A:T—»T:A transversions at
hprt has also been observed in DEB-exposed human TK6 cells
(Steen et al, 1997). In B6C3F1 lad transgenic mice exposed
to the parent compound BD at carcinogencity bioassay levels
of 625 and 1250 p.p.m., an increased frequency of mutations
occurs at A:T base pairs at the lad transgene (Sisk et al,
1994; Recio and Meyer, 1995).
The DNA sequence context of the mutations at A:T base
pairs among the EB-induced mutants showed strand bias at
a frequency that is similar to that observed for UV light. In
19/21 (90%) of the mutations observed at A:T base pairs
among the EB-induced mutants reported in this study, the A
was located in the nontranscribed strand; all the A:T—>T:A
transversions occurred with the A in the nontranscribed strand.
This strand specificity has been described for UV-induced
lesions at hprt in Chinese hamster cells and is associated
with the formation of pyrimidine dimers in the nontranscribed
strand (Vrieling et al, 1991). These data suggest a potential
role for adenine-derived adducts in the induction of mutation
in EB-exposed human cells. These data also suggest that
relevant biomarkers for BD exposure in humans should include
BD-derived adducts at adenine.
Although a number of DNA adducts have been described
following exposure to either BD or EB, a comprehensive
evaluation of BD-derived DNA adducts either in vitro or
in vivo is not available. However, both adenine and guanine
adducts have been observed after EB exposures both in vitro
and in vivo (Citti et al, 1993; Koivisto et al, 1995, 1996;
Neagu et al, 1995; Selzer and Elfara, 1996; Tretyakova et al,
1996). Following inhalation exposure in rats exposed to BD,
A^-adenine adducts have been detected in liver (Sorsa et al,
1996). In rats exposed to EB (30 and 60 mg/kg i.p. injection),
a dose-response for stable A^-adenine adducts in the liver was
observed (Koivisto et al, 1995). Although A^-adenine adducts
can occur following BD and EB exposure, whether these
adducts are responsible for the increased frequency of
A:T->T:A transversions observed in EB-exposed human TK6
cells is at present uncertain. The formation of M-ethenoadenine
adducts has been postulated to cause specific A:T—>T:A
transversions found in the tumour suppressor gene p53 in liver
angiosarcomas from vinyl chloride exposed factory workers
(Hollstein et al, 1994; Marion et al, 1996). These data and
the observations reported here suggest that the role of BDderived A^-adenine adducts in mediating the induction of
mutations induced by BD and its metabolites should be further
investigated.
Since BD bioactivation can produce two genotoxic
metabolites, it is necessary to evaluate the mutagenicity and
mutational spectrum of each metabolite independently to assess
its role in the in vivo mutagenicity and mutational spectrum
of the parent compound BD. Previously, we determined the
hprt mutational spectrum for DEB in human cells (Steen et al,
1997), and in the present study we determined the hprt
mutational spectrum for EB in human cells. These data show
that EB and DEB differ in mutagenic potency and mechanism
of action. On a molar basis, DEB is a more effective in vitro
mutagen than EB (Cochrane and Skopek, 1994). EB is more
effective at inducing single base substitutions, while DEB can
induce both single base substitutions and deletions. Although
which BD metabolite is ultimately responsible for the in vivo
genotoxic effects of BD is uncertain, the analysis of the
mutational spectrum of each metabolite compared with that
obtained for the parent compound in vivo is an approach
towards identifying the ultimate mutagenic metabolite or
metabolites of BD. These studies may also provide mechanistic
insights into BD-induced genotoxicity and provide a basis for
developing relevant biomarkers for BD-induced genotoxic
effects.
Acknowledgements
This study was supported in part by the Health Effects Institute, agreement
number HEI 94-08; and the Swedish Council For Work Life Research, Number
93-1580. We thank Drs R.J.Preston and James A.Bond for reviewing the
manuscript. We thank Dr Barbara Kuyper for editorial assistance.
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Received on January 31. 1997: accepted on April 3. 1997