DEVELQPMENT AND APPLICATIQNS OF MUTATIQNAL SPECTRA TECHNQLQCY
7
xcept for cigarette smoking and lung cancer, the major . causes of cancer death, as derived from National Cancer Institute statisascribed otics, specific haveexposures not been to environmental agents. Nevertheless, the assumption that environmental agents are responsible for a large percentage of cancer deaths remains widespread. The idea that cancer arises as a result of genetic changes ( 1 ) having broad effects on cell growth and biochemistry (2, 3) resulted in the demonstration that tumors are monoclonal and the present elucidation of many oncogenes and tumor suppressor genes. In parallel, the finding that radiation and chemicals could bcarcinogenic in humans an experimental animals was folowed by the demonstration that these agents are capable of causing the kinds of genetic damage, point mutations, and chromosomal rearrangements now found as human and animal oncomutations. (See definitions, p. 486A.3 Together these parallel lines of inquiry established what we have called the central premise of genetic toxicology as applied to human cancer: Human tumors carry oncomutations, the environment is replete with many mutagenic agents, therefore environmental mutagens are the primary causes of human cancer. This hypothesis, however, is far from proven. For example, no one has shown that the carcinogenic effect of ultraviolet light on the skin or of cigarette smoke on the lung epithelium proceeds via induction of oncomutations. Plausible alternatives exist. Increasing the absolute number of basal cells at risk would
MUTATIONAL SPECTROSCOPY PROVIDE
raise the slope of the age-specific death rate curve as surely as would raising the oncomutation rate. Without being responsible for inducing oncomutations, sunlight or cigarette smoke constituents could also induce tumors by altering the mutational yield from replication error, endogenous mutagens, or other exogenous mutagens.
A complex of uncertainties Given the lack of certainty about the most common pathways to cancer, it is not surprising that linking any specific mutation or cancer
478 A Environ. Sci. Technoi., Vol. 28. No. l l , 1994
H I L A R Y A. COLLER WILLIAM G. THILLY Massachusetts Institute of Technology Cambridge, MA 02239
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with particular environmental agents has not generally been possible. This problem is even more comnlex when we realize that within our daily experience, there are many potential causes of human mutations. A complete list must include even those that exist in the absence of exposure to any environmental agents. For instance, alterations i n the base sequence of DNA can be produced by errors made by the cell's own enzymatic machinery for replicating DNA ( 4 ) . Such induced mistakes could theoretically be responsible for some of the mutations along the pathway from a normal to a tumorigenic cell. In addition, when cells metabolize food for energy and perform other vital functions, a wide variety of chemically reactive substances are created. Any of these can mutate the cell that created the metabolite or the DNA of surrounding cells. In considering environmental mutagens, candidates include all those to which an individual has been exposed over several decades of life. Food, drugs, tobacco, sunlight, air, and water are all vectors of exposure. The dose level received for every candidate agent would be hopelessly difficult to ascertain. Not only the total dose but also the distribution of the dose rates over time might be relevant for determining the associated mutation rate. The problem of estimating the genetic effects of environmental mixtures in humans is intensified by the action of enzymatic metabolism of xenobiotics, primarily oxidation or reduction and subsequent conjugation reactions. These reactions, applied in series and in parallel, produce many different chemicals from a single environmentally de-
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0013-936W94/0927-478A$04.50/0 0 1994 American Chemical Society
rived parent molecule. Even if the identity and mutagenic activity of all environmental agents and their metabolites that could affect a particular cell were determined, it would still be necessary to consider the dynamics of metabolism in the 40 different cell types in the human body from which most lethal tumors are derived. Each of these cell types contains a unique set of drugmetabolizing enzymes and thus handles environmental chemicals in a qualitatively and quantitatively different way. After a chemical reaction has occurred with DNA, a cascade of enzymes comprising the DNA repair pathways will repair most of the lesions formed. Those adducts not repaired or misrepaired are then poised to induce mutations. The pattern of expressed DNA repair enzymes must be considered unique for each cell type as well. An important final dimension must be considered because humans are an outbred population of animals. Reasonably large differences among humans with regard to both the biochemistry of xenobiotic metabolism and DNA repair can therefore be expected. With so many layers of complexity, it is no surprise that few plaintiffs have been able to convince a judge or jury that it was “more probable than not” that their exposure to a specific occupational, environmental, or pharmaceutical agent caused their particular tumor ( 5 , 6). Development of the technology This morass may be greatly simplified by the emerging technology of mutational spectrometry, an approach built on the observation that for an homogeneous cell population exposed to a given mutagen at a particular condition of exposure, there exists a specific, reproducible pattern of genetic change: the mutational spectrum. Although any specific mutation may still have several sources, mutational spectra may reveal an overall pattern that defines the most prominent mutagen for a given cell population. Mutational spectra were discovered by Seymour Benzer and Ernest Freese ( 7 ) , who showed that the position and frequency of mutations in the genome of the T4 bacterial virus were different for untreated and chemically treated virus populations. Each mutagen was observed to induce a set of characteristic mutagenic “hotspots”: specific mutations, base substitutions, or small 480 A
deletions or insertions, which occur within a treated population significantly more frequently than would be expected by chance. In the following 35 years, Benzer’s observations were confirmed and extended to bacteria, yeast, rodents, and human cells. Researchers, notably Jeffrey Miller, simplified the laborious techniques originally used to assess the mutational spectra of particular compounds (8).Currently, most investigators treat cells with a mutagen, select individual mutant colonies thereafter, and determine the DNA sequence of each mutant. However, obtaining a spectrum with sufficient numbers of separate mutations in genes of about 1000 base pairs (bp) requires a daunting amount of sequencing to generate statistically reproducible results. To overcome this tactical barrier, our laboratory has used the technique of denaturing gradient gel electrophoresis (DGGE), which allows us to analyze the equivalent of 10,000 to 100,000mutants at a time. We are therefore able to detect the major hotspots in replicate experiments. Developed in the early 1980s by Stuart Fischer and Leonard Lerman, molecular biologists then at State University of New York at Albany, this technique allows separation of DNA based on differences in the melting temperature of the sequences (9). Even alterations of a single base pair in a 150-bp strand of DNA can have a significant effect on the melting temperature of the strand. Thus, instead of sequencing each mutant one at a time, it is possible to separate a heterogeneous mixture containing several mutant sequences from each other and from an excess of wild type. Visualization of 0.1% to 20% hotspots is possible on an autoradiogram (see Figure 1).The hotspot bands are then isolated and sequenced to reveal the point mutational spectrum of the test agent. Mutational spectra in selectable genes
The procedure for determining mutational spectra in the hypoxanthine-guanine phosphoribosyl transferase (HPRT)gene of human B cells grown in culture has been described in detail (4, 10-22). This strategy can easily be extended to any selectable locus in growing mammalian cells, bacteria, yeast, or virus populations but, it should be noted, it is limited to detecting only point mutations including base sub-
Environ. Sci. Technol., Vol. 28,No. 11, 1994
stitutions and small insertions and deletions; large-scale chromosomal rearrangements including deletions are not detected. First, one makes sure there are enough mutants. In the case of the HPRT gene, which encodes an enzyme necessary for salvaging purines from the cell’s microenvironment and incorporating them into DNA, null mutants may be selected by their ability to grow in the presence of guanine analogues, such as 6-thioguanine (6TG) or 8-azaguanine (8AG), that are toxic when incorporated into the cell’s nucleic acids (23). There are about 1000 bps at which mutations might yield an HPRT- cell. Ten thousand (10,000) initial mutants allows one to obtain a sufficiently reproducible spectrum. These numbers permit observation of hotspots constituting as low as 1% of the total with 95% confidence limits that represent only *2O% of the expected value, because 1% of 10,000 is 100 mutants, and the 95% confidence limits on 100 independent events are 80 and 120. If we expose the cells to a mutagen that induces a mutant fraction of 2 x while killing one half the cells, the number of cells that must be treated in order to obtain 10,000 surviving mutants: (104)/[(2 x x (0.5)l = lo9 cells. In the case of human B cells, which grow exponentially at l o 6 cells/milliliter, a culture size of one liter is required. For bacterial experiments, 10’ cells would be easily encompassed in one milliliter. These 5 x lo8 surviving cells containing 10,000 HPRT mutants are grown and diluted daily for the number of generations necessary to degrade and dilute previously existing normal HPRT molecules and thus become 6-thioguanine resistant (6TG’) ( 2 4 ) . This compound is then added to the culture. In 48 h, the 6TG kills all nonmutant cells, and these cells disintegrate with time. In 1 0 days, the HPRT mutants have increased in number from 10 cells/mL to about 3 x lo5 cells/ mL. Most of the cells are frozen for further study, but replicate samples of l o 6 cells are required for DNA isolation and further analysis. Isolation and purification The DNA is isolated from the cells on an anion-exchange resin that exploits DNA’s negative charge, The next step is to create many copies of only the portion of the genome of interest. For us, one
Once these copies of the sequence are purified, amplification occurs ectrometry using clone-by-clone again. This time, internal primers g gradient gel electrophoresis are used for amplification to ensure that only the desired sequence has Polymerase chain reactionbeen amplified and purified. In this denaturing gradient gel second PCR procedure, one of the electrophoresis primers is labeled with 3zP on its end. This permits visualization of the DNA with X-ray film. Choice of the primers for the second round of amplification requires the investigator to consider the ensuing step, DGGE. The ability of DNA to form cooperative melting domains (19) allows calculation of melting temperature as a function of position within any DNA sequence. In pursuing mutational spectra studies, melting maps as shown in Figure 2 are created for all target sequences. The ideal sequence for denaturing gradient gel analysis contains a region of DNA that melts at a relatively high temperature adjacent to a region of 100with P .) 200 bps that melts at a constant, lower temperature. Figure 2 shows the melting map of a 400 base pair section of the HPRT gene containanalysis ing exon 3, a sequence with these properties. If an isothermal low melting domain is not present adjacent to a high melting domain, then ‘“Barcode” Sequence each clone a GC clamp, that is, a sequence with a very high melting temperature, Mutational spectrum bands can be added to one of the primers synthesized for the second round of PCR. By adding a high melting region, it is possible to convert sequences not immediately suitable for DGGE separation into appropriate sequences. Sequence The radioactive DNA is purified and prepared for analysis on a denaSequence turing gradient gel. A denaturing gradient gel is a polyacrylamide gel that contains an increasing gradient of urea-formamide, a chemical comof the exons of the HPRT gene is the kind and rate of errors varying position that, like heat, denatures selected target. The polymerase among specific DNA polymerases DNA. Strands of DNA that differ in chain reaction (PCR), a technique and reaction conditions used. A sequence will migrate to different that allows an investigator to make 106-fold amplification (20 duplica- positions in the urea-formamide many copies of one small section of tions) of a 100 base pair sequence gradient because of the associated the genome, is employed (15, 16). using a DNA polymerase with an er- differences in their melting temperThis technique permits rapid syn- ror rate of 2 x IO-‘ mutations per atures. Therefore, the specific mutathesis of desired sequences from base per duplication would result tions that were hotspots for mugenomic DNA by chain extension in product DNA in which 20% of tagenesis by the original chemical catalyzed by a DNA polymerase oc- the single stranded copies con- will be present as sharp “bands” on curring simultaneously from two tained a mutation. For our work in an autoradiogram of the denaturing opposing primers. From lo5 copies determining mutations present as gradient gel. The series of bands obof genomic DNA, lo1’ copies of the mutant fractions less than (and served will be characteristic of the sequence of interest are created. as low as lo-’), these PCR-generated specific mutagen just like the bar Although the development of errors create serious background code on products in supermarkets PCR has greatly facilitated the abil- problems. To minimize PCR-gener- (see Figure 1). ity to analyze point mutations, it ated “noise,” we have been actively The procedure outlined (treatalso has a significant disadvantage. seeking more faithful enzymes and ment of cells, selection with 6TG, Any DNA polymerase will make er- improved reaction conditions (17, PCR, and DGGE) has been used to rors during DNA synthesis with the 18). determine the spectrum of muta-
1
Environ. Sci. Technol., VoI. 28. No. 11, 1994 481 A
Hypoxanthene-guaninephosphoribosyl transferase exon 3 meltin map showing the natural melting map and the melting map w en a GC clamp is added to analyze the high melting domain
9,
92
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260
300
340
380
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bp-position source: Reference 12
tions induced by a number of differ- quency of 0.6% and 0.470, respecent agents on the HPRT gene in a tively; and a five bp deletion (bp human B cell line. In one such ex- 274-278) was present at an average periment, the spectra of oxygen- frequency of 0.3%. The AT to TA and hydrogen peroxide-induced transversion was detected in all mutations at the HPRT locus has three replicate cultures; the other been compared to the spontaneous three mutations were detected in spectrum ( 4 ) . Two spontaneous two out of three replicate cultures. hotspots in exon 3 were detected in Therefore, the point mutational triplicate cultures: a 1 bp deletion spectra of both oxygen and hydro(-A) and a 2 bp deletion (-a). Each gen peroxide were significantly difhotspot comprised about 1%of the ferent from the spontaneous spec6TG' mutants. trum and were different from each Analysis of the oxygen-induced other. This implies that neither oxy6TG' mutants (48 h, 910 mM 0,) re- gen nor hydrogen peroxide was the vealed none of the spontaneous major cause of spontaneous mutapoint mutation hotspots. Two point tion in this particular human cell mutations in common with the culture. However, it is important to H,O,-treated cultures were found in remember that the mutation rate inone of the three oxygen-treated cul- duced in 0,- and H,O,-treated cultures, while in the other two there tures was 10 to 100 times greater were no observed hotspots. Hydro- than in untreated cultures. gen peroxide-induced mutations Specific chemicals' effect (1 h, 20 mM) also differed from the spontaneous spectrum. Four H,O,The mutational spectrum of beninduced hotspots were observed in zo[olpyrene diol epoxide (BPDE), exon 3 of HPRT An AT to TA trans- an activated mutagen produced by version was present at an average combustion products, has also been frequency of 3% of all 6TG' mu- investigated using this approach tants; two GC to CG transversions (11).This work is an excellent exwere detected at an average fre- ample of the high reproducibility of 482 A Environ. Sci. Technol., VoI. 28, No. 11. 1994
mutational spectra induced in independent cell cultures [see Figure 3). Each of four independent cultures contained the same 16 different BPDE-induced mutations in the third exon of the HPRT gene. The mutations were found to be predominantly G to T transversions. Six of these mutations occurred within a run of 6 guanines, and 5 occurred in the sequence 5'-GAAGAG-3'. Figure 3 represents a DGGE analysis of the mutations in the high melting temperature domain of exon 3. The four treated cultures, lanes 3 , 4 , 6 , and 7 represent the independently derived spectra of mutations induced by BPDE. Figure 4 is a synthesis of the results of several experiments on the third exon of the hypoxanthineguanine phosphoribosyl transferase gene. In addition to the spectrum of BPDE, it also provides the results from treatment of cells with ICR191, MNNG ( l o ) ,and ultraviolet light (12). As is obvious from inspection, these mutational spectra are reproducible and distinct for different chemical treatments. ICR191,a substituted acridine hypothesized to intercalate between the stacked bases of DNA, produced both +1 frameshifts in the GGGGGG sequences of exon 3 and +1 frameshifts in a CCC sequence. MNNG, an alkylating agent, produced a spectrum characterized by G to A transitions at the same run of six guanines (20). Finally, ultraviolet light has also been tested and found to induce a set of mutants that is distinct from those induced by any of the other compounds (11).In five independent ultraviolet light-treated cultures, eight predominant mutations of exon 3 were detected. Three transitions, including two GC to AT and one AT to GC, and two AT to CG transversions appeared in the treated hut not the untreated cultures. Four hotspots induced by chromium (VI) have also been detected by this method (21). These hotspots include two large CG to AT transversions representing 4.5% and 4.0% of 6TG' mutants, an AT to TA mutation representing 2.0% of the mutants and a 2.5% GC to AT transition. Two of the mutations shared the common local sequence 5'TACA-3' on the nontranscribed strafid and 5'-TGTA-3' on the transcribed strand, Suggesting that this quadruplet may be especially susceptible to chromium mutagens. This same approach has also been performed to analyze a complex
We believe that in the real world this dispersion is so great that one mutagen would be expected to produce the plurality, majority, or nearly all of the mutations induced by the mixture. This can be represented as:
FIGURE 3
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iggrr ’‘ ith be
ONA electrophoresed in lanes 3, 4, 6, and 7 were treated Source: Keohavong andThilly. 1991.
mixture. While complex mixtures are of obvious environmental significance (humans are exposed not to one specific mutagen but to an extremely complex mixture of mutagens), that a clear mutational spectrum would be produced by exposure to a mixture is not necessarily apparent. To explain why we consider such an outcome probable, we must explain why we believe that in any mixture of mutagens there will be one or two most prominent. First, we know that at very low concentrations, elements in mixtures are less likely to interact with each other directly or via induction of cellular responses. In photochemistry this concept is expressed as the Beer-Lambert law, which states that at infinite dilution the absorbance of a transparent solution is equal to the sum of the absorbances of the individual solutes. In mutational chemistry we note that there is a broad range of chemical concentrations “seen” by a typical cell. Similarly, experience has
taught us that the mutagenic potential of chemicals is distributed over many orders of magnitude. The distribution of the product of concentration and mutagenic potential for each chemical in the human body, which is proportional to its mutational effect, will be broader than either the distribution of concentration or mutagenic potential. This leads us to expect that at the extreme upper end of this distribution will be a mutagen or two with mutagenic effects far above that of the sum of the rest. In algebraic terms we are arguing that as a first approximation, the mutagenicity of a dilute mixture is the sum of the products of the specific activity, A , and the concentration, Ci, of its constituent chemicals. Mixture mutagenicity = A,C, + A & + . . A.C. = X A& We note that the product A,C, will be distributed with a much greater dispersion than Ai or C,.
.
AiC, = A& where x is the mutagen most responsible for the mutagenic properties of the mixture. Experimentation has supported this theory to some extent. The mutagenicity to bacteria or human B cells of combustion exhausts has been determined (22,23). In this work, soot extracts were fractionated and the mutagenicity of many different compounds was compared with the mutagenicity of the soot itself. Greater than 90% of the mutagenic activity at the lowest concentration studied was produced by a single compound, cyclopenta[c,dlpyrene. More recently, Lata Shirname-More has determined that the human cell mutational spectrum induced by aqueous extracts of smokeless tobacco reveals a clear and simple specinnn. This further supports the hypothesis that even complex mixtures can have characteristic mutational spectra produced by a single most prominent mutagen. By analogy to the larger environment, this lays the theoretical framework for the prediction that a human’s tissue will yield a simple, clear pattern of mutations that can be assigned to one particular mutagen. Unselected mutational spectramdry These arguments-the existence of unique mutational spectra characteristic of a given mutagen and the broad distribution of environmental chemical concentrations and mutagenic activities-serve as the theoretical basis for the application of mutational spectrometry to human populations. If the most important mutagen for our target gene is also the most important mutagen for genes involved in tumor initiation, then the results of these studies may be directly related to determining the causes of cancer. Other researchers have attempted to define the spectra induced in human blood by exposure to specific exogenous agents, including smoking (24)and ethylene oxide (251,using the clone-by-clone methodology described above to identify HPRT mutations present in T-lymphocytes. These studies, like those performed in vitro, result in a small
Environ. Sci. Technol., VoI. 28, No. 11, 1994 483 A
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Patterns of 6TGr mutations induced by four mutagens ii exon 3 of the hprt gene
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Base-pair position ‘Benzo[a iene diol epoxide. Source: &havong and Thilly, 1991.
number of mutants distributed over the entire 1000 bp coding region of HF’RT, making statistically significant analysis of hotspots difficult. We are pursuing three analytical tactics to determine the mutational spectrum present in human tissues. In the first, constant denaturant capillary electrophoresis (CDCE) coupled with high fidelity PCR, we separate mutants from nonmutants based on melting temperature. In the second, mismatch amplification mutation assay (MAMA),we use allele-specific PCR. In the third, highefficiency restriction assay (HEM) we first destroy wild type sequences by restriction digestion. The size of the target region differs dramatically for these techniques (see Table 1). CDCE allows detection of essentially all point muta-
tions occurring in a 100-150 bp low melting domain adjacent to a naturally occurring high melting domain. The target for HERA includes all point mutations in a 6 bp restriction site. Finally, MAMA can detect the presence or absence of a single mutation of interest. CDCE-Hi Fi PCR CDCE extends the in vitro approach described above to genes for which it is not possible to enrich for mutant cells. This approach is necessary because it is not possible to obtain sufficient numbers of cells from a living person to perform the experiments outlined above. If there are to be on average 10 mutations per base pair, for a 1000 base pair gene, io4 independently created mutant molecules must be analyzed.
484 A Environ. Sci. Technol., Val. 28, No. 11, 1994
Given that the HPRT mutant fraction in actual human blood is approximately 5 x lod (261,2 x 10’ cells are required. This translates to 2 L of blood or 10 g of tissue. One way we are trying to avoid this problem is to choose a target gene present as many copies per cell. Genes encoded in the cell’s mitochondria, the source of energy for the cell, are particularly appropriate targets because there are, on average, io3 to lo4 copies of the mitochondrial genome in each cell. To obtain 2 x IO9 independently created mutants of a mitochondrial gene, one milliliter of blood is more than sufficient. When mitochondrial genes are targeted, it is not possible to select for those with mutations in a target gene. A cell that has a mutation in one of its mitochondrial genes would have io4 normal copies to compensate for the associated deficiency. Therefore, it is necessary to find an alternative approach to enrich the population for mutants. If enrichment does not occur, and mutants are present in the entire population as a 5 x i O P fraction when the DNA is subjected to the PCR, then the mutations induced by the DNA polymerase in vitro during PCR will overwhelm those few mutants originally present. Another strategy is therefore necessary. CDCE is used to enrich for mutant copies based on differences in elution time from a capillary gel (27).In this method, fluorescently labeled DNA fragments are electrophoresed through a capillary of 75 pm in diameter filled with viscous polyacrylamide solution. A portion of the capillary is heated to permit separation of mutant versus wild type sequences based on differences in the melting temperature. The DNA is in an equilibrium between an “open” form in which the low melting portion of the molecule is denatured and a “closed” form in which the entire molecule is double stranded. At a specific temperature range, sequences with a lower melting temperature will exist in the open form more of the time than sequences with a higher melting temperature. These differences cause the velocity and elution time of the DNA to be highly sequence specific. Therefore, by plotting the amount of fluorescein-labeled DNA detected by the laser as a function of elution time, the mutational hotspots can be observed as “peaks.” DNA present in the peaks can be eluted and subjected to further analysis.
The use of microcapillaries enables the investigator to increase the speed of separation about 30 time as compared with DGGE. The first step in the unselecte( protocol is to separate the wild typi from mutant copies by electrophoresing the DNA through the capillary and collecting the DNA that migrates in the slower moving region. After this initial separation the protocol proceeds with high fidelity DNA amplification, anothe cycle of CDCE separation and highfidelity DNA amplification, and visualization of the peaks on a final CDCE. Individual peaks can then be isolated, purified, and sequenced. Reconstruction experiments with purified mutant sequences spiked into wild type sequences have demonstrated that sequences originally present as lo-' or even 10" of the original DNA can be detected (Figure 5). We are confident that this technology will be able to identify point mutations present in human tissue samples. Mismatch amplification mutation assay A number of laboratories have attempted to use PCR to amplify only mutant sequences when they are mixed with a large excess of wild type copies. The MAMA approach (28) has defined conditions that allow direct measurement of specific point mutations in animal tissues. Using MAMA, Cha a n d colleagues have detected mutations as infrequent as one in io5 copies in cell systems and in animal tissues (29).One specific mutation investigated, the G to A transition at the second nucleotide of the 1 2 t h codon of the ras gene, is frequently found mutated in rat mammary tumors. MAMA was used to test whether exposure to the mutagen nitrosomethylurea (NMU) actually induced mutations in the ras gene previously observed in tumors of rats treated with NMU. Cha and colleagues measured the number of ras alleles with a mutation in the 12th codon before and after NMU exposure and determined that treatment with a carcinogenic dose of NMU did not increase the number of mutants or the number of organ sectors containing mutants. This contradicted previous interpretations that treatment with NMU was directly inducing ras mutations that were then responsible for tumor induction. The observed clustering of mutants within sectors of the mammary gland is consistent with their
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origin as mutations arising during early organ development as opposed to independent mutational events. MAMA has also been used to test whether mutational hotspots char-
acteristic of a given mutagen are present in a cell culture sample. MNNG-induced hotspots i n the HPRT gene of human cells have been confirmed using this technique (21).
Environ. Sci. Technol., Vol. 28, No. 11, 1994 485 A
Mutational Spectra Technol Definitions from A to 2 :X-ray film that h a s been e x p e d to ioactivity (often 32Por ‘*S) has been efore provides a visual depiction of
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any of numerous proteins produced by living or biological catalysts in living organisms. e portion of a gene that actually codes for pr ns, which are the portions that are excised before th : a mutation involving a small insertion or de ‘reading frame”of a gene. Because every th e for an amino acid, an insertion or deletion mu
e nuc eotides that follow to code for incorrect amin
in will therefore be nonfunctional. ome: a complete haploid set of chromosomes. loid: Having the number of chromosomes of a g ber in a normal somatic cell thelium: Membranous tissue, usually in a s ranged cells separated by little intercellular ing of most internal surfaces, such as the Iu drion: a microscopic body occurring in the csil o sms and containing enzymes responsible for th
en: An agent, as a radioactive element or ultra
I
n: the act or process of being changed. In bi
of genes or chromosome of an organism. ogene: a gene believed to be utated, is believed to contribute t cogenesis: formation or development of comutations: mutations in a gene believ gulation and thus contributing to the cancer ph iatric tumors: tumors in young children.
High-efficiency restriction assay Another approach to detecting mutations that are present in a low frequency is by using restriction enzymes. Restriction enzymes cleave the sugar-phosphate backbone of DNA at the site of specific, usually palindromic, sequences. If there is but a single mutation in the recognition site, most enzymes will fail to cleave. Therefore, restriction enzyme digestion can greatly enrich a mixed population for mutant sequences. By subjecting the restricted DNA to PCR with primers spanning the restriction enzyme site, it should be possible to determine the original number of mutants in the sample. In preliminary studies with this approach, two samples of bronchial epithelial cells isolated from anatomically distinct regions of a lung have been analyzed. The known pseudogenes, copies of DNA highly homologous to the target mitochondrial se486A
quence but present in the nucleus, were eliminated. Next, the DNA was digested with high-efficiency restriction enzymes. The digested DNA was amplified with PCR primers flanking the restriction site and separated on a denaturing gradient gel. DNA from both portions of the lung contained the same single base pair mutation with the wild type sequence. Further experiments are being conducted t o determine whether the sequence represents a previously undetected pseudogene. If the sequence represents a mitochondrial mutation, its presence in both anatomically distinct sections of the lung makes it unlikely to have resulted from clonal expansion of a single stem cell population. Mutational spectrometry: The first human trials The first job for the human mutational spectrometrist is to observe what has actually happened in humans. A variety of DNA sequences
Environ. Sci. Technol.. Vol. 28, No. 11. 1994
should be chosen to maximize the kinds of information to be gained. Certainly, transcribed and untranscribed nuclear sequences and a mitochondrial sequence should be chosen. Genes relevant for cancer research, specific oncogenes and tumor suppressors, are important targets for establishing a link between environmental exposures and cancer. To determine the spectra of single copy nuclear genes, autopsy or surgical s ecimens are required to , of the target seobtain 10g copies quence ( 3 4 grams of tissue). For multicopy genes such as the ribosomal RNA genes or mitochondrial genomes, the problem of a large tissue sample is eliminated as they occur at about 500 or 5000 copies per cell, respectively. The first several humans studied should reveal whether clear mutational spectra exist. If useful spectra are found to exist, lifetime studies in multiple organs would be performed with a focus on the primary organs of lethal cancers: lung, colon, breast, and prostate. If T cells are used, the possibility of clonal expansion of a particular subpopulation must be considered as an explanation for specific hotspots. Spermatogonia and oogonia would be another priority. The small number of oogonia will necessarily require the study of multicopy sequences. The goal of such studies would be to determine which environmental agents, if any, are primary mutagens for various human organs. We have dedicated our efforts to these technical goals because we believe that mutational spectrometry may provide a means to establish cause-and-effect relationships be-
Hilary A. Colleris ogroduotestudentin the Division of Toxicology and the Center for Environmentol Health Sciences, Massachusetts Institute of Technology.
William G. Thilly is the director of the Center for Environmental Health Sciences, Whitoker College, Massochusetts Institute of Technology, ond on MZTprofessor of applied biology and civil engineering.
tween chemicals a n d genetic change. If such relationships exist, they may be exploited in the process of regulating chemicals and in attributing causation in a court of law. If such relationships do not exist, the technology for measuring mutations in the human body will be necessary for defining the endogenous pathways of human mutations and, perhaps, approaches to reduce life-long mutation rates. References (1) Boveri, T. In Zurfrage derhtstehung maligner Tumoren; Gustav Fischer: Jena, Germany, 1914; pp. S. 1-64. (2) Warburg, 0.;Posener, K.; Negelein, E.
Uber den Stoffwechsel der Carcinomzell Biochem Z . 1924,152, 309-344. (3) Warburg, 0. Science 1956,123, 30914. (4) Oller, A.; Thilly, W. J. Mol. Biol. 1992, 228, 813-26. (5) Besheda v. Johns-Manville Products Corp., 90 N.J. 191, 1982. (6) Sindell v. Abbott Labs, 163 Cal. Rptr. 132,1980. (7) Benzer, S.: Freese, E . Proc. N a t l . Acad. Sei. USA 1958,44,112-19. (8) Coulondre, C.; Miller, J. J. Mol. B i d . 1977,117,577-606. (9) Fischer, S.; Lerman, L. Proc. Natl. Acad. Sei., USA 1983,80, 1579-83. (10) Cariello, N. et al. Mutat. Res. 1990, 231, 165-76. (11) Keohavong, P.; Thilly, W. Proc. Natl. Acad. Sei. USA 1992,89,4623-27. (12) Keohavong, P.; Liu, V.; Thilly, W. Mutat. Res. 1991,249, 147-59. (13) Szybalski, W.; Szybalski, E. H. Univ. Mich. Med. Bull. 1962,28, 277. (14) Thilly, W. et al. Chem.-Bio. Interact. 1976,25, 33-50, (15) Kleppe, K. et al. J. Mol. Biol. 1971,56, 341-61. (16) Mullis, K. B.; Faloona, F. A. Methods Enzymol. 1987,155, 335-50. (17) Keohavong, P.; Thilly, W. Proc. Natl. Acad. Sei. USA 1989,86,9253-57. (18) Ling, L. et al. PCR Meth. App. 1991,1, 63-69. (19) P o l a n d , D.; Scheraga, H. J . Chem Phys. 1966,45,1464-69. ( 2 0 ) Kat, A. G. Ph.D. Thesis, Massachusetts Institute of Technology Cambridge, MA, 1991. (21) Chen, J.; Thilly, W. Mutat. Res. 1994, 323, 21-27. (22) Skopek, T.et al. J. Natl. Cancer. Inst. 1979,63, 309-12. (23) Kaden, D.; Hites, R.; Thilly, W. Cancer Res. 1979,39, 4152-59. (24) Vrieling, H. et al. Carcinogenesis 1992,13, 3625-31. (25) Bastlova, T. et al. Mutat. Res. 1993, 287, 283-92. (26) Albertini, R. et al. Mutat. Res. 1988, 204,481-92. (27) Khrapko, K. et al. Prog. Nucleic Acid Res. Mol. Biol., in press. (28) Cha, R. et al. PCRMeth. App. 1992,2, 14-20. (29) Cha, R.; Thilly, W.; Zarbl, H. Proc. Natl. Acad. Sei. USA 1994,91, 374953.
ES&Twill be introducing a new monthly department in 1995 that highlights important and interesting research papers in the current literature. This feature will contain brief (1 00-word) summaries of cited material from a wide range of journals in environmentally related disciplines. We are looking for contributors who have current knowledge of relevant scientific and technical fields, the ability to pinpoint significant research resuIts, and the writing skill to concisely interpret that significance to an audience of nonspecialists. Contributors will submit 4-8 items each month. Send a resume and a list of those scientific and technical publications you read regularly to ES&T Managing Editor, 1155 16th St., N.W., Washington, DC 20036.
Environ. Sci. Technol., Vol. 28, No. 11, 1994 487 A
