(CANCER RESEARCH 58, 2857-2862. July 1. Õ998]

Chemically Induced Mutations in Mitochondrial DNA of Human Cells: MutationalSpectrum of W-Methyl-W-nitro-Af-nitrosoguanidine1

Luisa A. Marcelino, Paulo C. André,Konstantin Khrapko, Hilary A. Coller, Jacqueline Griffith, andWilliam G. Thilly2

Division of Bioenxineering and Environmental Health. Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02/.Õ9

ABSTRACT

We have observed a reproducible mitochondria! mutational spectrumin the MT1 human lymphoblastoid line treated with .V-nirllijl-.V'-iiilro-

yv-nitrosoguanidine (MNNG). The MNNG spectrum was distinct from the

spontaneous mutational spectrum. However, our ability to observeMNNG-induced mitochondria! mutations above the high level of accumulated spontaneous mutations was dependent on the M l'I phenotype. Mil

cells are markedly resistant to the cytotoxicity but not the mutagenicity ofMNNG, presumably as a result of inactivation of both copies of theli.MSIIfi (GTBP) mismatch repair gene. Thus, we were able to use condi

tions of treatment that yielded induced mitochondria! mutant fractionsbeyond the practical limits for human cell experiments in mismatch-

proficient human cell lines. In contradistinction, when Mil cells weretreated repeatedly with maximum tolerated concentrations of (±) anti-benzo(a)pyrene diol-epoxide, no induced mitochondria! mutations above

the spontaneous background were observed.A single dose of 4 ¿IMMNNG (survival, 0.85) induced a mutant fraction

of 8 x 10 ' in the nuclear hypoxanthine-guanine phosphoribosyltrans-

ferase gene, and a clear and reproducible pattern of seven MNNG-induced

hotspot mutations was observed within the mitochondria! DNA targetsequence studied (mitochondria! bp 10,030-10,130). All of the MNNG-

induced hotspot mutations were G:C to A: I transitions present at frequencies between 6 x 10~s and 30 x 10~5.

Additional experiments supported the conclusion that MNNG-induced

hotspot mutations observed were generated in living cells as a result ofMNNG treatment and not from mismatch intermediates or DNA adductsconverted into mutations during the PCR process.

INTRODUCTION

Mitochondrial point mutations are the cause of a variety of severelydebilitating diseases (1, 2). These point mutations could result fromendogenous factors, such as DNA replication errors or damage byendogenous cellular metabolites, or induction by exogenous compounds.

Using a physical separation of mutant DNA sequences from wildtype, we have found the same set of mutational hotspots in a 100-bp

mitochondria! DNA target sequence of different human organs, theirderived tumors, and human cells grown in the laboratory (3, 4). Thedata indicated to us that these most frequent mitochondrial pointmutations are spontaneous and do not result from reactions withexogenous chemicals. This we have found to be true even for bronchial epithelial samples drawn from identical twins discordant forcigarette smoking (5).

These findings seemed paradoxical because mitochondrial DNAhad been found to react with exogenous chemicals to an extentsignificantly higher than nuclear DNA. This had been found to be true

Received 12/15/97; accepted 4/30/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate Ihis fact.

' This work was supported by Grant PO1-E503926 from the National Institute for

Environmental Health Sciences. Genetics and Toxicology Program. Grant P42-ES04675

from the Superfund Hazardous Substances Basic Research Program, and Grants NIHR01-CA 77044-01 and P30 AG 133I4.

2 To whom requests for reprints should be addressed, at Center for Environmental

Health Sciences, Room 16-743, MIT. 21 Ames Street. Cambridge. MA 02139. Phone:(617)253-6220; Fax: (617)258-5424; E-mail: thilly@mit.edu.

for alkylating agents (6, 7), polycyclic aromatic hydrocarbons (8, 9),aflatoxin Bl (10), and cisplatin (11). However, treatment of humancells with ethyl methanesulfonate. MNNG,3 and BPDE at concentra

tions that increased mutant fractions for nuclear genes showed noinduction of mitochondrial mutations (12). Ethyl methanesulfonatedid induce A:T to T:A transversions in mitochondrial DNA of yeast(13).

Two possible explanations have been formally considered. DNAadducts might simply block replication, thus automatically preventingmutation (12). Alternatively, the fact that spontaneous mitochondrialpoint mutation rates appear to be more than 100-fold the nuclear point

mutation rates would be expected to create a high background ofspontaneous point mutations, preventing observation of mitochondrialmutations induced in human cell experiments limited by the cytotoxicity of the treatment (4).

To test these hypotheses, we used the MT1 cell line, which isresistant to the cytotoxic but not the mutagenic effects of MNNGtreatment, presumably due to deficiency in the mismatch repair geneHMSH6 (14-16). When we treated MT1 cells with 4 /XMMNNG, we

observed a reproducible set of point mutations above the spontaneousbackground in the mitochondrial sequence studied (bp 10,030-

10130). In this case, it is clear that the related DNA adducts did notblock DNA replication.

However, when we treated MT1 cells with a maximally toleratedconcentration of 3.2 JU.MBPDE through eight sequential treatments,we confirmed and extended, in terms of sensitivity, the absence ofdetectable BPDE-induced mutations originally reported by Mita et al.

(12). Thus, we cannot exclude the possibility that BPDE adductsblock DNA replication. But we note that had we used MNNG at aconcentration equimutagenic to nuclear genes with sequential BPDEtreatments, we would not have reached a detectable level of MNNGinduced mitochondrial mutations.

MATERIALS AND METHODS

Human Cells. MT1 is an alkylation-resistant lymphoblastoid cell linederived from TK6 cells treated with acridine ICR-191 and subsequently

screened for mutants able to survive the cytotoxic action of MNNG ( 14). TK6cells are an immortal human B cell line that maintains ploidy except for severalrearrangements among the C group chromosomes (17). Cells were maintainedin exponential growth at 37°Cin a humidified atmosphere containing 5% CO2by daily dilution to 4 x IO5 cells/ml with RPMI 1640 supplemented with 10%

horse serum (Life Technologies. Inc.. Gaithersburg. MD).Cell Treatment and hprt Mutation Assay. MT1 cells were treated with

MNNG (Sigma Chemical Co.. St. Louis, MO) as described previously (14).Two cultures of 8 X IO7 cells each were treated in duplicate, whereas two

others were left untreated as controls. Mutant fractions induced by MNNGtreatment were measured in the hprl gene, as 6-thioguanine-resistant colonies, as described previously (18). The /iprf-induced mutant fraction after

a 4 JAM MNNG treatment, with 95% confidence intervals, was(8.6 ±3.9) x 10~3.

' The abbreviations used are: MNNG. W-methyl-W-nitro-A'-nitrosoguanidine: BPDE, (±)

anti-benzol«)pyrene-7.8-dihydrodiol-9.10-oxide-r-7.f-8-dihydroxy-(-9.10-epoxy-7.8.9.10-tct-rahydroben7.o|«]pyrene |<±)anti-benra(ii)pyrene diol-epoxide]; CDCE. constant dénaturant

capillary electrophoresis; hprt, hypoxanthine guanine phosphoribosyl transferase; bp. basepair.

2857

CHEMICALLY INDUCED MUTATIONS IN HUMAN MITOCHONDRIAL DNA

MT1 cells were treated with BPDE using the same conditions as describedpreviously for the parental TK6 cells (19). Duplicate cultures of 8 X IO7 cells

each were treated with 0.4 JJ.MBPDE every 6-8 days up to 8 times, or left

untreated as controls. The total treatment induced a mutant fraction, with 95%confidence intervals, of (9.0 ±2.0) X 10~4.

Mutational Spectra Analysis in Mitochondria! DNA. Our mutationalspectrometry used constant dénaturantgel electrophoresis (20) to initiallyisolate a fraction enriched in mutant sequences, and CDCE (21) coupled tohigh-fidelity PCR amplification (22) to recognize, enumerate, and isolate

individual mutant sequences. This methodology requires the target sequence tobe about 200-bp in length with a 100-bp isomelting domain adjacent to another

isomelting domain of significantly higher melting temperature (23). We useda 100-bp spanning part of the genes coding for tRNAgIy and the NADH

dehydrogenase subunit 3, bp 10,030-10,130, as our target sequence.

Sequences with single bp changes in the low melting domain will havelower melting temperatures as compared with the wild type when they are

converted to heteroduplex molecules after melting and annealing in the presence of excess wild-type sequences. Such heteroduplexes invariably migrate

more slowly than wild-type homoduplexes during constant dénaturantelectro

phoresis.Mutational spectra analysis was performed as described (3). Briefly, cellular

DNA was isolated from treated and untreated cultures and digested by therestriction enzymes Rsal and Ddel to excise the mitochondrial target sequence.The number of copies of the mitochondrial DNA target sequence present in thesamples was quantified by competitive PCR with a known amount of a mutantPCR product of the same sequence and determined to be —¿�1000mitochondrial

target sequences per MT1 cell. Purified mutants used as internal standardswere introduced in each DNA sample at precise fractions of the total copynumber of the target sequence. The mutants originally present in the DNAsample, together with the internal standards, were then separated from the largeexcess of wild-type sequences in two steps. The DNA samples, consisting ofa total of 2 X IO8 mitochondrial DNA copies, were first subjected to constant

dénaturantgel electrophoresis. Areas of the gel enriched in mutant sequenceswere extracted, amplified with Pfu DNA polymerase (Stratagene, La Jolla,CA) and the primers: forward, CW7 (5'-ACCGTTAACTTCCAATTAAC-3';bp 10011-10031 of the human mitochondrial DNA) and reverse, 5'-fluores-cein labeled J3 (5'-TGGAGAAAGGGACGCGGGCG-3', complementary to

mitochondrial bp 10196-10215; Synthetic Genetics, San Diego, CA). These

primers were selected by iterative experimentation to avoid inclusion of anynuclear pseudogene sequences that are invariably present in human cells athigh (—1000) copies. After amplification, the mutant-enriched samples were

then further enriched in mutants by CDCE. The fractions containing theenriched mutants were collected from the capillary column and subsequently

amplified by a final round of PCR. Mutants were identified as peaks usinglaser-induced fluorescence. The mutant fractions that each mutant peak rep

resented in the initial DNA sample were calculated by comparing the area ofeach peak to the areas of the mutant peaks used as internal standards. Individual mutant peaks were purified by further rounds of collection and PCR andsequenced. As the mutant peaks were isolated from the samples and se-quenced, they were included in a "standard set of mutants." Labeling the

standard set of mutants differently from the sample under analysis and coin-

jecting both samples in the same capillary column allowed peaks from bothsamples to be visualized on a dual-dye detector CDCE (3). This step provided

for preliminary identification of mutant peaks based on comigration. Toconfirm that a sample peak was identical to a peak in the standard set, withwhich it comigrated, the samples were subjected to hybridization "in-capillary"

as described (3).

RESULTS

Human MT1 Cells Treated with BPDE. MT1 cells were treatedwith eight sequential doses of 0.4 JUM(survival, 0.20 per dose) ofBPDE, which induced a mutant fraction of approximately 9 X 10~4

in the nuclear hprt gene. Fig. 1 shows the set of mutant peaksobserved during CDCE when mutational spectra analysis was appliedto the mitochondrial DNA of untreated MT1 controls (Fig. la) and toMT1 cells treated with eight sequential treatments of 0.4 JUMBPDE(Fig. \b). No differences in the set of mitochondrial mutant peakswere observed between BPDE-treated and untreated cells. Based on

the sensitivity in reconstruction experiments, this observation indicates that no single mutation has been induced in this sequence at alevel greater than 5 X 10~6. Had a hotspot mutation equal to orgreater than 1% of the nuclear hprt mutant fraction, or 9 X 10~6,

occurred, it would have been detected.Human MT1 Cells Treated with MNNG. A single treatment of 4

/j,MMNNG (survival, 0.85) induced a mutant fraction of 8 X 10~3 in

the hprt gene of MT1 cells. This same treatment generated a set ofmutant peaks in the mitochondrial DNA target sequence, which wasdifferent from the background set of mutant peaks observed in untreated controls (Fig. 2). Mutant peaks present only in the MNNG-

treated cultures, designated Ml to M7 in Fig. 2a, were identical inassays of replicate cultures. By comparing the areas of each MNNG-

induced peak with the area of an internal standard peak, multipleMNNG-induced hotspots were estimated to be present at frequenciesbetween 6 X 10~5 and 30 X 10~5. These values are about equal to orgreater than 1% of the nuclear hprt mutant fraction, or 8 X 10~5, andwell above the detection limit of 5 X 10~6.

We specifically tested whether the mutants observed in MT1 cells

Fig. 1. Spectra of mitochondrial mutations ofMT1 cells, a, duplicated cultures of MT1 cellsuntreated controls; b, duplicated cultures of MT1cells treated with eight doses of 0.4 ¡MBPDE.Both mutants with higher melting temperature thanthe wild type (left panel) and lower melting temperature than the wild type (right panel} are shown.Internal standards, designated as artificial mutantam (C:G to T:A transition, mitochondria bp10,040) and 13 (T:A to C:G transition, mitochondrial bp 10,072), were added to the initial samplesat fractions of 3 X I0~4 and 10~4, respectively, of

the initial number of mitochondrial copies. Theposition and type of mutation of the numberedpeaks are shown in Fig. 4.

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MT1 ceilsUNTREATED

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2858

CHEMICALLY INDUCED MUTATIONS IN HUMAN MITOCHONDRIAL DNA

Fig. 2. Spectra of mitochondrial mutations ofMT1 cells, a, duplicated cultures of MT1 cellstreated with 4 (¿MMNNG; h, duplicated cultures ofuntreated MTI cells that served as controls. Internal standards, designated as am (C:G to T:A transition, mitochondria bp 10.040) and 13 (T:A to C:Gtransition, mitochondrial hp 10.072), were added tothe initial samples at fractions of 3 X I0~4 and10~4, respectively, of the initial number of milo-

chondrial copies. Both mutants with higher meltingtemperature than wild type (left panel) and lowermelting temperature than wild type (righi panel)are shown. Numbers and tellers indicate the specific mutation represented by each peak. Peaksnumbered Ml to M7 were identified by sequencingor by comigration with tetramethylrhodamine-la-

beled standards recorded in a separate channel (notshown) and further confirmation by hybridization"in capillary." The position in the sequence and the

type of mutation are displayed in Fig. 4.

IC

were actually present as mutations in the mitochondrial DNA aftertreatment with MNNG or whether they resulted from DNA polymer-ase bypass of MNNG-adducts during PCR amplification. Previously,

it was shown that nearly all of the spontaneous mutant peaks observed, when this same method was applied to human tissue samples,did not arise from chemical reaction products in cellular DNA, mismatch intermediates, or PCR errors (3). The treated and untreatedcultures initially grown for 7 generations were allowed to grow for anadditional 14 generations. Any adduci present in the DNA afterMNNG treatment would be diminished by dilution by at least 214-

fold. As shown in Fig. 3, after 14 generations of additional growth, nochange in any of the MNNG-induced mutant peak areas was observed.

We concluded that the mitochondrial mutations observed representmutations generated by the cells after MNNG treatment.

The seven most frequent MNNG-induced mutant peaks (peaks M l

to M7) were eluted from the capillary column and sequenced. Allwere found to be G:C to A:T transitions (Ml at bp 10,094; M2 at bp10,090; M3 at bp 10,091; M4 at bp 10,098; MS at bp 10,099, M6 atbp 10,056, and M7 at bp 10,085). Fig. 4a shows the frequency of eachMNNG-induced hotspot mutation in the 100-bp target sequence spanning part of the genes coding for tRNAgly and the NADH dehydro-

genase subunit 3. The spontaneous mutational spectrum in humancells and tissues for this sequence is also shown, in Fig. 4b, forcomparison. The spontaneous point mutations consist primarily ofG:C to A:T and A:T to G:C transitions in approximately equalnumbers (3, 4).

DISCUSSION

Human MTI Cells Treated with BPDE. No significant mitochondrial mutations were seen in the BPDE-treated cultures. Eight

sequential treatments with 0.4 /AMBPDE induced a mutant fraction ofabout 9 X 10~4 in the hprt gene of MTI cells, but no induced

mutations were observed in the mitochondrial DNA of the same cells.

2859

There are several possible explanations. Mita et al. (12) first reportedthe absence of BPDE-induced mutations in the mitochondrial DNA ofhuman HeLa cells in the D-loop region. They suggested that BPDE

adducts might block mitochondrial DNA replication, yielding no

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Fig. 3. Spectra of MNNG-induced mutations in the mitochondrial DNA of MTI cellsgrown for 7 and 21 generations after exposure to 4 pM MNNG. Numbers and lettersindicate the specific mutation represented by each peak. The position in the sequence andthe type of mutation are displayed in Fig. 4. The artificial mutant used as internal standardam (C:G to T:A transition, mitochondria! bp 10.040) was added to the samples at a mutantfraction of either 3 X I0~4 or 6 X 10~4.

CHEMICALLY INDUCED MUTATIONS IN HUMAN MITOCHONDRIAL DNA

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Fig. 4. Display of the kinds, positions, and frequencies of hotspot mutations observed in the 1000-bp mitochondria! DNA target sequence (mitochondrial bp 10,030-10,130). Thejunction between the tRNAgly sequence and the beginning of the NADH dehydrogenase subunit 3 sequence are indicated, a, MNNG-induced hotspot mutations (black columns)

observed in the mitochondrial DNA of MT1 cells (the height of each bar representing MNNG); induced hotspots reflect the average mutant fraction of each hotspot in two replicateMT1 cultures treated simultaneously, b, set of hotspot mutations (while columns) observed previously in human tissues their derived tumors (4); the height of each column representsa hotspot peak observed in vivo and reflects the average mutant fraction of each hotspot in most of the lung samples analyzed previously (4). Note that mutants 1 and 2 are differentsubstitutions at the same bp. * and #, identical sites of mutation in the two mutational spectra.

surviving mutant copies, and our data do not eliminate this possibility,extending their observations to a greater degree of BPDE exposureand using a mutation assay of greater sensitivity. Another possibilityis that our 100-bp mitochondrial DNA target sequence is "vacant" for

BPDE-induced mutations. Our lab has observed previously that in—¿�15% of cases, any particular mutagen fails to induce mutations inany particular 100-bp sequence of the nuclear hprt gene (19, 24-26).

Finally, it is possible that the total dose of BPDE used to treat MT1cells might not have been high enough to induce mutations above themitochondrial spontaneous background. We did our best to create amaximum frequency of BPDE-induced mutations in nuclear DNA

with an experiment lasting 8 weeks with daily dilution of cultures. Toreach a level 10 times higher, equaling the mutant fraction inducedwith a single MNNG treatment, would have involved an 80-week

experiment. This length of growth is more than 100 cell divisions,during which time selection for faster growing or even BPDE-resist-

ant variants would occur. These would not be expected to carrymutant mitochondrial copies (27).

Human MT1 Cells Treated with MNNG. The data are primafacie evidence of MNNG induction of a series of G:C to A:T transitions in the mitochondrial sequence (bp 10,030-10,130) of human

MT1 cells. Some other peaks of lower area were observed but notcollected for sequencing. All seven hotspot mutants induced byMNNG were G:C to A:T mutations and were consistent with previousobservations of MNNG-induced mutational hotspots in prokaryoticDNA and eukaryotic nuclear DNA (28-30). However, we did not

perform analyses of the kinds of DNA adducts created at the mutatedsites and cannot conclude that these arise from 06-methylguanine, as

has been shown for nuclear DNA sequences (31-32).

What is clear is that under conditions causing minimal toxicity inMT1 cells, MNNG treatment created a reproducible set of pointmutations. Obviously, the MNNG-damaged mitochondrial DNA se

quences were replicated, and thus the premutagenic lesion for thesemutations was not a block to DNA replication. Because such extraordinary measures were required to induce a detectable mitochondrialmutational spectrum, these data are consistent with our prior conclusion that it is unlikely that in vivo human mitochondrial mutations areinduced in this sequence by exogenous mutagens (4).

This is the first demonstration of mutation induction in the mitochondrial DNA of human or other mammalian cells by a chemicalagent. However, Pascucci el al. (33) concluded recently that theyobserved UV-C mutations induced in a 5-bp sequence (a Nci\ recog

nition site) of the mitochondrial DNA of human embryonic kidney2860

CHEMICALLY INDUCED MUTATIONS IN HUMAN MITOCHONDRIAL DNA

293 cells, using a restriction site mutation assay (34). Although theirreported mitochondrial mutants were consistent with the G:C to A:TUV light-induced mutations observed in nuclear DNA sequences, few

mutants were attributable to UV exposure (33 mutants). These did notseem to be distributed as a nonrandom spectrum within the 5-bp target

sequence. These reported observations involved isolating mutant sequences one at a time and sequencing them. In contradistinction, themethod used herein to study MNNG-induced mutations in a defined100-bp sequence resulted in an observed total mutant fraction of about3 X 10~3 over 8 X IO7 treated, surviving cells, containing about 1000

mitochondrial copies/cell, in a total of 8 X 10'°mitochondrial copies.

This means that our reported spectrum consisted of about 240,000,000independently induced mutations distributed over seven hotspot positions. Such high numbers of induced mutants account for the highreproducibility among our replicate experiments.

The MNNG induced mitochondrial spectrum for this 100-bp target

(Fig. 4ij) were compared with mutational spectra observed previouslyin human tissues, such as colon, muscle, and lung, their derivedtumors, and TK6 cells in culture (Fig. 4h). Because the same mitochondrial hotspot mutations were observed in vivo and in cells inculture, the most frequent mitochondrial mutations were hypothesizedto be spontaneous in origin (4). The spontaneous hotspots, mainly G:Cto A:T or A:T to G:C transitions, were found distributed over theentire target sequence and occurred at very high rates, more than100-fold the nuclear point mutation rates (4). Five of the MNNG-

induced hotspot mutations were distinct from the spontaneous hotspotset, whereas two of the MNNG-induced hotspots were also observed

in tissues but greatly increased in frequency by MNNG treatmentwhen compared with the mutational spectrum of untreated MT1 cells.

We have observed previously the spontaneous and MNNG-inducednuclear (hprt gene) mutations in the parental TK6 human lympho-blasts as well as in the TK6-derived mismatch repair-deficient MT1

cell line (14, 24, 25). We have also determined the spontaneousmutational spectra in the mitochondrial DNA of MT1 (this work) andTK6 cells (4). The spontaneous mutation rate in nuclear DNA of MT1is about 15-fold higher than the parental mutation rate, as expected ofa mismatch repair-deficient cell line (14). The spontaneous mutation

rate in mitochondrial DNA, however, is similar in MT1 and TK6 cells.These data suggest that the mismatch repair system that operates onthe nuclear DNA does not operate on mitochondrial DNA.

MNNG induces nuclear gene mutations at about the same frequency in MT1 and in TK6 (14). Interestingly, the MNNG-induced

mutant fraction per bp in the mitochondrial DNA of MT1 cells iswithin a factor of three of that induced in nuclear DNA (an MNNG-induced mutant fraction of 2.9 X 10~3 in the 100-bp mitochondrialsequence, or 2.9 X 10~5 per bp may be compared with an MNNG-induced mutant fraction of 8.6 X 10~3 in the 1000-bp hprt locus, or8.6 X 10~6 per bp). This difference may simply be due to the

approximately 3-7-fold higher alkylation in mitochondrial DNA thanin nuclear DNA, measured as levels of A^-methylguanine and O6-

methylguanine (6, 7).To observe MNNG-induced mutations above the large spontaneous

background, we were required to treat MNNG-resistant MT1 cells

with an amount of MNNG that would have killed all normal parentcells (TK6) in an experiment of practical dimensions. That suchextreme measures were required to observe a significant increase inhuman cell mitochondrial mutation above the high spontaneous accumulated background are consistent with, and extend, our previousfinding that mitochondrial mutations accumulated during an individual's lifetime are more likely to result from endogenous processes

than as a direct result of exposure to environmental mutagens.

ACKNOWLEDGMENTS

We thank Dr. R. Wasserkort, A. Tornita, and D. McGaffigan for criticalreading and careful editing of the manuscript. We also thank B. Turnquist forcomputer expertise.

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