Mutageneds vol.11 no.3 pp.229-233, 1996
DNA sequence analysis of methylene chloride-induced HPRT
mutations in Chinese hamster ovary cells: comparison with the
mutation spectrum obtained for 1,2-dibromoethane and
formaldehyde
RJ.Graves1-3, P.Trueman2, SJones 2 and T.Green14
'Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield,
Cheshire, SK10 4TJ, UK and 2Zeneca Pharmaceuticals, Alderley Park,
Macclesfield, Cheshire, UK
^Present address: Amersham International pic, Cardiff Laboratories,
Forest Farm, Whitchurch, Cardiff CF4 7YT, UK
•"To whom correspondence should be addressed
Glutathione-S-transferase-mediated metabolism of methylene chloride (MC) generates 5-chloromethylglutathione,
which has the potential to react with DNA, and formaldehyde, which is a known mutagen. MC-induced mutations
in the HPRT gene of Chinese hamster ovary cells have
been sequenced and compared with the mutations induced
by 1,2-dibromoethane (1,2-DBE), which is known to act
through a glutathione conjugate, and formaldehyde. All
three compounds induced primarily point mutations, with
a small number of insertion and deletion events. The most
common point mutations induced by MC were GC—>AT
transitions (4/8), with two GC-»CG transversions and two
AT—»TA transversions. This pattern of mutations showed
greater similarity with 1,2-DBE, where the dominant point
mutations were GC—»AT transitions (7/9), than formaldehyde, where all mutations were single base transversions
and 5/6 occurred from AT base pairs. The mutation
sequence results for MC suggest that S-chloromethylglutathione plays a major role in MC mutagenesis, with only a
limited contribution from formaldehyde. The involvement
of a glutathione (GSH) conjugate in MC mutagenicity
would be analogous to the well-characterized pathway of
activation of 1,2-DBE.
Introduction
Methylene chloride (MC) is a widely used industrial chemical
which is also used in aerosol preparations and in paint stripper.
In chronic bioassay exposure, MC induces liver and lung
tumours in mice, but not in rats or hamsters (Burek et ai, 1984;
NTP, 1986). A major effort from this and other laboratories has
focused on a mechanistic understanding of the observed species
specificity, leading to an assessment of the potential for human
risk from occupational or consumer exposure (ECETOC,
1988). Previous studies have identified significant interspecies
differences in the metabolism of MC. As the glutathione-5transferase (GST) pathway is much more active in the mouse
than in other species (Green, 1989; Reitz et ai, 1989), these
results have implicated an involvement of GST metabolites of
MC in the initiation of carcinogenicity in the mouse.
Further evidence for this hypothesis comes from recent
studies in this laboratory which have shown that GST metabolites of MC cause DNA single-strand breaks in the target
organs of the mouse but not in other species (Graves et ai,
1994, 1995). These results support studies showing that the
cause of bacterial mutagenicity of MC is endogenous GSTmediated metabolism (Dillon et ai, 1992; Graves et ai, 1993).
© UK Environmental Mutagen Society/Oxford University Press 1996
Metabolism of MC by mouse liver cystosol enriched for GSTs
(S100 fraction) also causes an increase in mutations at the
HPRT locus in Chinese hamster ovary (CHO) cells (Graves
and Green, 1996). In this study we have analysed the induced
CHO HPRT mutations by sequence analysis of the cDNA.
The potentially reactive products of GST-mediated metabolism of MC are 5-chloromethylglutathione and formaldehyde
(Ahmed and Anders, 1976, 1978). Formaldehyde is known to
cause DNA damage and mutations in bacteria and mammalian
cells (reviewed by Ma and Harris, 1988), but 5-chloromethylglutathione is rapidly hydrolysed in the cell, and to date no
interaction with DNA has been reported (Hashmi et ai, 1994).
However, 5-(l-acetoxymethyl)glutathione, prepared as an
analogue of the MC metabolite, reacted with 2-deoxyguanosine
in vitro to yield 5-[l-(A72-deoxyguanosinyl)methyl]glutathione
(Thier et ai, 1993). Furthermore, structurally analogous glutathione conjugates are implicated as causative agents in the
mutagenicity of 1,2-dihaloethanes (Ozawa and Guengerich,
1983).
The objectives of the present study were to characterize the
mutations seen at the HPRT locus after exposure of CHO cells
to MC in the presence of mouse liver cytosol. The mutation
spectra were compared with those induced by formaldehyde
and by a reactive glutathione conjugate [from dibromoethane
(DBE)] in order to determine which of the MC metabolites
was responsible for the observed mutagenicity.
Materials and methods
CHO Kl cells (from Zeneca Pharmaceuticals) were grown at 37°C in a
humidified 5% CO2 incubator in Ham's F12 medium (Gibco/BRL, Paisley,
Scotland) containing 10% fetal calf serum, supplemented with 50 U/ml
penicillin, 0.1 mg/ml streptomycin sulphate and 2 mM glutamine. Mutagenicity
experiments are described in full in a separate publication (Graves and Green,
1996) and followed established protocols (Nestmann et ai, 1991).
Briefly 2X10* cells were exposed as attached cultures to 5 mM 1,2-DBE
(99% v/v; Aldrich, Gillingham, Dorset, UK) for 4 h, or 5X106 cells in
suspension were exposed for 1 h to either 1 mM HCHO (40% aqueous
solution, Analar grade; Fisons, Loughborough, UK) or 0.3% (v/v) MC (99.8%
v/v, HPLC grade, BDH, Lutterworth, Leicester, UK) with 20% liver S100
fraction (from male B6C3F1 mice, Charles River Supplies, Maidstone, Kent,
UK) and 5 mM glutathione. Survival was estimated by colony forming ability,
and for mutation expression the exposed cells were grown in T175 flasks for
8 days, with subcultunng every 2-3 days. Each mutation experiment was
repeated at least three times. For mutant selection, each culture of exposed
cells was plated on 6X9 cm dishes (2X1O5 cells/dish) in hypoxanthine-free
Harris's F12 medium (Imperial Laboratories, Andover, Hants, UK) containing
10% dialysed fetal calf serum (Gibco) and 20 |lM 6-thioguanine (Sigma,
Poole, Dorset, UK). Plating efficiency was determined by colony forming
ability in selective medium without 6-thioguanine.
After 10 days of growth, the mutant colonies were counted and samples
picked for growth to -10* cells. All the DBE-induced mutant colonies were
derived from independently treated cultures. The cells were harvested by
trypsinization into 0.5 ml Hank's balanced salt solution (Gibco/BRL) and
pelleted in a microfuge, and mRNA prepared using a Pharmacia micro mRNA
purification kit. Following ethanol precipitation and resuspension in 16 |il
water, the mRNA was copied into cDNA in duplicate reactions using a
Pharmacia first-strand cDNA synthesis kit and dN6 primer as supplied. HPRT
sequences were PCR amplified using the nested set of outer primers described
by Yang et at. (1992). The first strand cDNA synthesis mixture (15 \i\) was
diluted to 50 (ll in vent R DNA polymerase reaction buffer containing 40 pmol
229
RJ.Graves et aL
of outer primers and 2 U of vent R DNA polymerase (New England
Biolabs, Hitchen, Herts, UK), and amplified for 30 cycles as described (Yang
et al., 1989).
The 0.8 kb product was purified from agarose gels using Promega Wizard
Prep™ DNA purification columns, and 1 nl aliquots of the eluate reamplified
in multiple reactions using the inner set of outer primers. Following purification
from solution using Gibco Glassmax™ spin columns, the DNA products were
quantified by A 260onl absorbance using a Pharmacia GeneQuant™. For DNA
sequencing, six internal primers were synthesized:
H
2)ACCGATTCCGTCATGGCGACC (9 )
( l70)TGAGGACATAATTGACACTGG ( i90)
( 399 ) TGGGAGGCCATCACATTGTGG ( 4i9 )
(672 )ACTTGAACTC'rCATCTTAGGC (6 52)
(464) AGGTTGTACCGCTTGACCAGG ( 444 )
( 246)AATGTAATCCAGCAGGTCAGC ( 2 2 6 )
DNA sequencing was carried out with an Applied Biosystems (Abi Division,
Warrington, UK) 373A 'stretch' DNA Sequencer controlled with an Apple
Macintosh Quadra 650. The sequencing reactions (containing 0.5 |ig template
and 1.2 pmol of pnmer) were performed via AmpliTaq R thermal cycle
sequencing in a Perkin Elmer GeneAmp PCR system 9600, as per Abi's
standard protocol for PRISM™ Ready Reaction Dideoxy terminator sequencing kit. The polyacrylamide sequencing gel was 4.75% acrylamide in 1 XTBE
buffer and 8.3 M urea, and was electrophoresed for 12 h. The HPRT cDNA
sequences were compared with the published sequence for untreated Chinese
hamster V79 RJK159 cells (Konecki et al., 1982). The identification of each
putative mutation was confirmed by bidirectional sequencing and, if necessary,
by sequencing of the duplicate first strand cDNA synthesis template.
Results
HPRT (-) mutations were induced in CHO cells by single
exposures to 1,2-DBE, formaldehyde and MC. The mean
Table I. HPRT mutations induced in CHO Kl cells following exposure to
1,2-dibromoethane, formaldehyde and methylene chloride
Treatment
Mutation frequency
(X 1CT6)1
Plating
efficiency
Fold
increase
Control
5 mM 1,2-DBE
1 mM HCHO
0.25% MC b
3.5
86.5
16.3
27.9
82
46
80
67
1.0
24.7
4.7
8.0
± 1.2
±21.3
± 0.7
± 3.9
±
±
±
±
3
26
10
15
"Values given are means (± SEM) from 2-3 experiments,
incubations also included 20% SI00 fraction from mouse liver
homogenate.
increases above background mutation frequency from 2-3
experiments were 24.7-fold for 1,2-DBE, 4.7-fold for formaldehyde and 8-fold for MC (Table I). Approximately 60% of the
gel-purified HPRT products reamplified to give sufficient
amounts of template for DNA sequence analysis. In total, 28
HPRT mutants were sequenced, of which 21 involved single
base substitutions. Only one of these 21 sequences contained
more than one point mutation, suggesting that PCR copying
errors did not contribute to the observed mutations. Also,
the distinct mutation spectrum for each compound implies
chemically induced sequence alterations rather than template
copying errors. A change from the published control cDNA
sequence was observed at position 464, with a C residue
replacing a T.
Results of cDNA sequence analysis of 13 HPRT mutations
induced by 1,2-DBE are shown in Table n. Nine of these
mutations were single base pair changes, seven being GC—>AT
transitions with two GC—»TA transversions. All the point
mutations occurred at unique sites except for two G-»A
transitions at position 119 and two G—>A transitions of position
539. These represent possible hotspots for DBE-induced mutagenesis since these mutant colonies were derived from independently treated cultures. All the G residues in the GC—^AT
transitions were located on the non-transcribed strand and a
sequence bias was apparent, with 5/7 mutations occurring at
the middle G is the sequence GGA. Of the remaining 1,2DBE-induced mutations, two involved deletions of exon 6 and
one a deletion of exon 2. The remaining mutation was a 17 bp
deletion in exon 9, with the deletion site flanked by a 4 bp
tandem repeat.
DNA sequence analysis of six formaldehyde-induced HPRT
(-) mutations is shown in Table HI. All were single base pair
mutations, and all were transversions. The three AT—>TA
mutations occurred at position 548 of exon 8, with a single
GC—»TA transversion and two AT—>CG transversions at
unique sites.
Of the eleven MC-induced HPRT (-) mutations sequenced,
eight were single base mutations (Table IV), four being
GC-»AT transitions, with two each of AT-»TA and GC->CG
transversions. All these mutations occurred at unique sites,
Table II. Types and locations of mutations induced by 1,2-DBE in the HPRT gene of CHO Kl cells
Mutant
(single base substitutions)
Position
Exon
Mutation
Target sequence (5'-3')"
Amino acid change
D17, D13
D15
D8, D16
D14
D2
D5
D4
119
400
539
569
599
132
2
5
8
8
8
2
G->A
G->A
G->A
G-»A
G->A
C->A
CAT GG.A GTG
GTT GAG GAC
GTTGGATTT
GTT GGA TAT
TTC AGG GAT
ATG GAC AGG
Gly-»Glu
Glu-»Lys
Gly->G!u
Gly->Glu
Arg-»Lys
Asp—»Glu
135
3
G->T
GAC AGG ACT
Arg-»Ser
Splice site mutations
D1.D3
Dll
403^185
28-134
6
2
splice defect
splice defect
exon 6 missing
exon 2 missing
Deletions
DI2
610-626
9
17 bp deletion with
4 bp tandem repeat
T ITG AA IG... ITGAA IACT
CAT ATT TGT GTC ATT AG
frameshift
"The sequence of the non-transcribed strand is shown. Underlined bases represent position of point mutations.
230
DNA sequence analysis of HPRT mutations
the 1,2-DBE-GSH conjugate, and replication of the damaged
DNA in Salmonella TA100 resulted primarily in base substitutions (Cmarik et al, 1992). Like the CHO results, GC->AT
transitions accounted for most of the bacteriophage point
mutations, although in contrast to the CHO results, every class
of point mutation except GC—>CG transversions was recovered.
These differences probably result from the smaller number of
samples sequenced in this study, and the use of repair-deficient
bacteria versus repair-proficient CHO cells. The 1,2-DBEinduced HPRT mutations also included two deletions of exon
6, one deletion of exon 2 and a 17 bp deletion with a 4 bp
tandem repeat flanking the deleted sequence.
Of six formaldehyde-induced HPRT mutants sequenced, all
were single base transversions, with three AT-»TA, two
AT-»CG and one GC-»TA. The predominance of mutations
at AT base pairs is consistent with the results of Liber et al.
(1989), in which formaldehyde-induced HPRT mutations in
human lymphoblasts were at AT base pairs, 5/6 being AT—»CG
transversions with one AT—»GC transition. Differences in the
exposure regimen [a single exposure at 1 mM in this study
compared with eight repetitive daily treatments at 0.15 mM
(Liber et al, 1989)] may be responsible for the subtle differences in the mutation spectrum. The single GC—>TA transversion mutation in this study could represent the influence of
the background mutation spectrum, since the formaldehydeinduced mutation frequency was only 4.7-fold above background.
Three out of six formaldehyde-induced mutants in this study
and 4/6 in the previous study occurred at a unique site,
although different ones. In neither study were all the mutants
although three were recovered from a single mutant clone,
implicating multiple hits at this sequence. The remaining three
mutations were two deletions of exon 6 and an 18 bp duplication
insertion. A comparison by class of the types of mutation
induced by all three compounds is shown in Table V.
Discussion
The precise nature of the DNA damage leading to MC
mutagenicity had not been identified prior to the current work,
although there was considerable evidence for involvement of
the reactive metabolites generated by GSH-mediated metabolism of MC, namely 5-chloromethyl GSH and formaldehyde
(Dillon et al, 1992; Graves et al, 1993, 1994, 1995; Graves
and Green, 1996). In an attempt to further characterize this
mutagenic DNA damage and determined which of the MC
metabolites was primarily responsible, we have compared the
spectrum of MC-induced mutations at the HPRT locus in CHO
cells with the mutations induced by formaldehyde and 1,2DBE. The results provide the first DNA sequence analysis of
MC and 1,2-DBE-induced mutations in mammalian cells,
and extend the previous sequence analysis of formaldehyde
mutagenesis in human lymphoblasts (Liber et al, 1989).
The CHO mutations induced by 1,2-DBE were mainly point
mutations and were dominated by GC-»AT transitions (7/9),
with two GC-»TA transversions. It is likely that 1,2-DBE
mutations result from formation of 5-[2-(A^-guanyl)ethyl]glutathione, which is the major adduct formed in DNA (>95% of
total) (Guengerich et al, 1987). Treatment of bacteriophage
M13mpl8 DNA with 5-(2-chloroethyl)GSH, an analogue of
Table III. Types and locations of mutations induced by formaldehyde in the HPRT gene of CHO Kl cells
Mutant
(single base substitution)
Position
Exon
Mutation
Target sequence
(5'-3') a
Amino acid change
F9
F7
Fl, F5, F10
F8
205
223
548
550
3
3
8
8
A->C
T->G
T->A
C->A
CTG AAGGGG
TTCTTTGCT
GAA ATT CCA
ATT CCA GAC
Lys->Glu
Phe-»Val
Ile-»Asn
Pro-»Thr
T h e sequence of the non-transcribed strand is shown. Underlined bases represent position of point mutations.
Table IV. Types and locations of mutations induced by methylene chloride in the HPRT gene of CHO Kl cells
Mutant
Position
Exon
Mutation
Target sequence
(5'-3')«
Amino acid change
Single base substitutions
MC13
MC7
MC14
MC6
MCI2
74
551
115
600
466
2
8
2
8
6
C->T
C->T
C->G
G-*C
A->T
ATT CCT AAT
ATT CCA GAC
CCT CAT GGA
TTC AGG GAT
CCC AAA ATG
Pro->Leu
Pro -»Leu
His-»Asp
Arg->Ser
Lys->STOP
131
490
525
2
7
7
A->T
C->T
G->A
ATG GAC AGG
TTG CTG GTG
TAT AGG CCA
Asp-»Val
none
none
403-485
6
splice defect
exon 6 missing
401^118
5-6
insertion of 18 bp duplicate
sequence (underlined) al
position marked by arrow
GGA AAG AAT GTC TTG ATT GTT GAG GAC
Multiple base substitutions
MC9
Splice site mutations
MC3, MC5
Insertion
MCI
t
231
RJ.Graves et aL
Table V. Types of mutations induced in the HPRT gene of CHO Kl cells
treated with 1,2-DBE, methylene chloride and formaldehyde
Mutation
No. of mutations
1,2-DBE
Methylene
chloride
Formaldehyde
References
Transversions
AT->TA
AT->CG
GC->CG
GC->TA
0
0
0
2
2
0
2
0
3
2
0
1
Transitions
AT->GC
GC->AT
Splice mutations
Deletions
Insertions
Total
0
7
3
1
0
13
0
4
2
0
1
11
0
0
0
0
0
6
isolated from independently treated cultures, so the possibility
that some of these mutants were sibs cannot be discounted.
However, as previously discussed (Liber et aL, 1989), it is
likely that these sites represent hotspots for formaldehyde
mutagenesis. Multiple mutations at unique sites were observed
with the DBE mutants, which were derived from independently
treated cultures.
The most common point mutation induced by MC was the
GC-»AT transition (4/8), as seen with 1,2-DBE. Of the
remaining point mutations, AT—»TA transversions (2/8) were
also seen with formaldehyde, whereas the two GC—»CG
transversions were unique to MC. The remaining MC mutations
were two deletions of exon 6, as seen with 1,2-DBE, and a
unique duplication insertion event. The results are comparable
to the DNA sequence analysis of MC mutations in Escherichia
coli by Zielenska et al. (1993). In repair-proficient bacteria,
every class of point mutation was recovered, with GC—»AT
transitions dominating, followed by GC—>CG transversions. Of
the bacterial DNA rearrangement mutations, only duplication
insertion mutations were found. The fact that MC-induced
mutations were similar, but not identical to, those of DBE
may reflect the different target site on the guanine bases for
the respective glutathione conjugates (Ozawa and Guengerich,
1983; Thier et aL, 1993).
In conclusion, although the number of mutants analysed in
this study was small, making statistical analysis difficult, the
mutation spectra obtained with DBE (9/9 at GC base pairs)
and with formaldehyde (5/6 at AT base pairs) are entirely
consistent with published work in bacteria and mammalian
cells (Liber et al., 1989). In our study, 678 MC-induced
mutations were at GC base pairs, including two which were
unique to MC, leading to the conclusion that these mutations
were not induced by formaldehyde. It is also relevant to note
that formaldehyde mutations are accompanied by extensive
DNA-protein crosslinking, which was lacking in CHO cells
exposed to MC (Green and Graves, 19%). On the other hand,
the similarity of the mutation spectrum of MC to that of DBE,
the structural similarity of the reactive glutathione conjugates
of MC and DBE, and the common base for the DNA adduct
(Ozawa and Guengerich, 1983; Thier et aL, 1993) lead to the
conclusion that the MC induced mutagenesis in CHO cells is
caused primarily by its metabolite, S-chloromethylglutathione.
232
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Received on July 28, 1995; accepted on January 16, 1996
233