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Mutagenesis Advance Access first published online on July 14, 2007
This version published online on July 19, 2007
Mutagenesis, doi:10.1093/mutage/gem023
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© The Author 2007. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

The instability of (GpT)n and (ApC)n microsatellites induced by formaldehyde in Escherichia coli

Wei Wang, Jun Xu, Liu Xu1, Bisong Yue and Fangdong Zou*

Key Laboratory of Bio-resources and Eco-environment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu 610064, People's Republic of China 1College of Bioengineering, Southwest Jiaotong University, Chengdu 610031, People's Republic of China

Formaldehyde, a potential human nasal carcinogen, has been reported to induce DNA lesions. However, the effect of formaldehyde on microsatellite instability has not previously been reported. Plasmids containing different lengths of complementary (ApC)n or (GpT)n dinucleotide repeats on the leading strand were constructed to investigate whether the mutagenesis by formaldehyde can contribute to microsatellite instability. We observed that exposure of Escherichia coli to 2.5 mM formaldehyde increased the frequency of expansions and deletions of the dinucleotide repetitive sequences. After being induced by formaldehyde, the microsatellite mutation frequencies of (GpT)n and (ApC)n were 2- to 24-fold higher than those in the control. Although complementary to each other, (ApC)n and (GpT)n had different mutation frequencies when they were on the leading strand: mutation frequencies of (GpT)n were 13- to 24-fold higher than the control group, whereas frequencies of (ApC)n were only 2- to 3-fold higher the control group. Sequencing of the repetitive and flanking sequences in mutant clones showed that all mutants displayed expansions or deletions of dinucleotide repeats. These results clearly suggest that formaldehyde can increase micrsosatellite instability by affecting the fidelity of microsatellite maintenance. We presumed that a mutagenic mechanism of formaldehyde and the temporal formation of left-handed helix Z-DNA might be related to the microsatellite instability.


   Introduction
Top
 Introduction
Materials and methods
 Results
 Discussion
 Conclusion
 References
 
Microsatellites are tandem repeats of DNA units, usually 1–6 bp per repeat unit (motif). They are located in both protein-coding and noncoding regions and commonly distributed in both eukaryotes and prokaryotes (1Go). Microsatellites are highly polymorphic with mutation rates 100 times that of nonrepetitive sequences (2Go). Instability of microsatellites both in intronic and coding regions is a hallmark of genetic instability in human cancers (3Go). The predominant model of microsatellite instability is DNA replication slippage—a transient dissociation of the replicating DNA strands followed by misaligned reassociation (4Go). This model predicts that microsatellites are susceptible to slipped-strand mispairing during replication, resulting in deletions or additions. Instability is attributed to loss of mismatch repair function or other factors, such as unusual DNA secondary structures, including hairpins, left-handed Z-DNA and intramolecular triplexes or H-DNA which can induce not only expansions and deletions but also DNA strand breaks and rearrangements (5Go–9Go). In Escherichia coli, for example, (GC)n dinucleotide repeats, which bring about hairpins in vitro, are quite labile (10Go). In addition, microsatellite instability can be induced by endogenous or exogenous mutagens. It has been demonstrated that oxidative damage to bacterial cells significantly increases the frequency of microsatellite instability (11Go,12Go).

Formaldehyde is a reactive, water-soluble molecule and is normally maintained at an acceptably low concentration in vivo. Formaldehyde carcinogenicity has been studied extensively over the last 30 years and it is considered to be a potential human nasal carcinogen based on the results from recent studies (13Go). Furthermore, several studies suggested a possible association between formaldehyde exposure and leukemia (14Go,15Go). The potential impact of these findings, particularly for risk assessment, is subject to controversy in the scientific community. Results of previous in vitro and in vivo studies, which have attempted to estimate the genotoxic effects of formaldehyde using the micronucleus test and other means to assess chromosomal aberrations have been inconsistent (16Go,17Go). In general, it has been difficult to assess the genotoxicity of formaldehyde on the basis of the available data and to draw meaningful conclusions with regard to a dose–effect relationship for risk estimation. Given their high mutation rate and wide distribution in genome, dinucleotide repeats of microsatellite might offer a realistic means to study the genotoxic effects of environmental mutagens. Furthermore, the relationship between formaldehyde and microsatellite instability has not yet been reported to date. Therefore, we applied a plasmid-based assay involving examination of microsatellite sequences to detect the genotoxicity of formaldehyde. Briefly, (ApC)n and (GpT)n dinucleotide repeats, two of the most abundant microsatellite motifs in the genome (1Go) were inserted in the plasmid to study the genotoxicity of formaldehyde in E. coli. We found that the damage of formaldehyde to wild-type bacterial cells significantly increased the frequency of microsatellite instability.


   Materials and methods
Top
 Introduction
 Materials and methods
Results
 Discussion
 Conclusion
 References
 
Strains, plasmids and media
Wild JM109 strain (E14– (McrA–) recA1 endA1 gyrA96 thi-1 hsdR17 (rK– mK+) supE44 relA1 {Delta} (lac-proAB) [F' traD36 proAB lacIq ZDM15]) was routinely cultivated in LB (Luria-Bertani medium) which was supplemented with tetracycline (15 mg/ml) or carbenicillin (100 mg/ml). The plasmid pAJ19 (tetR carbR) was a generous gift from Lawrence A. Loeb (University of Washington) (12Go). Dinucleotide repeats of three different lengths—(ApC)32 or (GpT)32, (ApC)25 or (GpT)25, (ApC)16 or (GpT)16—were synthesized artificially with the SacI and BglII recognition sites at their terminus. These dinucleotide repeats were digested with SacI and BglII and ligated into carbenicillin resistance gene of pAJ19, which was digested with same restriction enzymes as shown in Figure 1. After being transformed with recombinant plasmids, JM109 cells were plated on LBA (Luria Bertani Agar) plus 15 mg/ml tetracycline. Positive clones with different dinucleotide repeats were identified by polymerase chain reaction with the following primers: 5'-ATCCAGTTCGATGTAACCCACTCG-3' and 5'-ATGTGCGCGGAACCCCTATTTGT-3'.


Figure 1
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  		Fig. 1. Description of insert mode of (GpT)n and (ApC)n on the leading strand of pAJ19. In plasmids, the complementary dinucleotide sequence was inserted between the SacI and BglII sites of the linker. Different position of SacI and BglII sites on inserted sequences (GpT/ApC)n results in two possible orientations. The (GpT)n sequence on the left corresponds to the sequence of the leading template strand in the 5' to 3' orientation. The insert of the plasmid containing the repeat sequence in this particular orientation is referred to as in the (GpT) orientation. On the other hand, insertion of the oligonucleotides in the other orientation will lead to plasmids pAJ19 (ApC)n, which will be referred to as in the (ApC) orientation.

 
Formaldehyde exposure
E. coli strains JM109 with different microsatellites were grown overnight in LB supplemented with 15 mg/ml tetracycline. Fresh overnight cultures were diluted with LB and grew for another 3 h. Then, cells were collected by centrifugation at 13 200 x g, and washed with an equal volume of 1x M9 salt and resuspended in an equal volume of 1 M9 salt. Cells (300 µl) were divided equally into two parts. One was exposed to an equal volume of solution of formaldehyde, which was freshly diluted in 1x M9 salt (The final concentration of formaldehyde in a 300 µl reaction was 2.5 mM). The other was the control group with an equal volume of 1x M9 salt added. Formaldehyde exposure was performed at 37°C with shaking at 400 rpm for 30 min. The exposure was terminated by centrifugation of the cells followed by resuspension with 300 µl of LB.

Mutation frequency analyses
After exposure to formaldehyde, cells were diluted and immediately plated on LBA plates (containing 15 mg/ml tetracycline or 15 mg/ml tetracycline plus 100 mg/ml carbenicillin) and grew overnight. The number of colonies recovered on each plate was normalized for the volume plated, and the frequency of mutation was estimated by dividing the number of carbenicillin-mutant colonies (tetR carbR) per milliliter by the number of tetracycline-resistant survivor per milliliter. We employed the same method to the control group, using 1x M9 salt in lieu of formaldehyde. The experiments described herein were repeated five times. Mutation frequencies were compared among different treatment groups using the two-tailed Student's t-test. Confidence intervals for mutation frequencies were calculated using an exact Poisson method.

Sequencing
To verify that the enhanced frequency of mutations was result of expansions or deletions of dinucleotide repeats in E. coli, we sequenced the microsatellite frame of recovered plasmids with the Thermosequenase kit (Amersham) using the primer: 5'-ACAATAACCCTGATAAATGC-3'.


   Results
Top
 Introduction
 Materials and methods
 Results
Discussion
 Conclusion
 References
 
When (GpT)n or (ApC)n repeats were inserted into the carbenicillin resistance gene of the pAJ19 plasmid, they caused a frameshift in the carbenicillin resistance gene, rendering it nonfunctional. Thus, this recombinant plasmid was employed to identify microsatellite frameshift mutations that restored the reading frame of the carbenicillin resistance gene and conferred resistance to carbenicillin. E. coli strains with pAJ19 were treated by formaldehyde or 1x M9 salt. Colonies grown on tetracycline-containing plates were indicators of plasmid maintenance, whereas colonies grown on tetracycline plus carbenicillin were indicators of expansions or deletions of dinucleotide repeats that restored the reading frame of the carbenicillin resistance gene. Mutation frequency was calculated as the number of carbenicillin-resistant mutants recovered per tetracycline-resistant survivor.

The dose of formaldehyde in the present study was critical: lower dose did not saturate the repair capacity of the cell, and higher dose resulted in extensive damage to the cell. We investigated the instability of (GpT)32 repeats and cell survival affected by formaldehyde at different doses—1.25, 2.5, 5 and 10 mM. The mutation frequency induced by the 2.5, 5 and 10 mM formaldehyde did not show significant difference (P > 0.05), while the mutation frequency induced by 1.25 mM formaldehyde was merely 1.3 x 10–4 (Figure 2A). In addition, the 1.25 and 2.5 mM formaldehyde treatment corresponded to 44 and 21% survival relative to untreated E. coli strains, as measured by tertracycline-resistant transformants, whereas the 5 and 10 mM formaldehyde treatment corresponded to 13 and 7% survival, respectively (Figure 2B). Therefore, 2.5 mM, the optimal concentration, was used in our study.


Figure 2
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  		Fig. 2. A dose-dependent instability of (GpT)32 repeat and survival affected by formaldehyde (FA). Mutation frequency was calculated as the ratio of carbenicillin-resistant mutants recovered per tetracycline-resistant survivor damaged by formaldehyde. Survival rate is calculated as relative to survival in absence of formaldehyde. Mean ± SEM of five independent tests with the different dose of formaldehyde (FA). **P < 0.01; *P < 0.05.

 
The mutation frequencies of out of frame (GpT)32, (GpT)25 and (GpT)16 induced by 2.5 mM formaldehyde were approximately 10–3, while (ApC)32, (ApC)25 and (ApC)16 were 10–4 (Figure 3). Microsatellite mutation frequencies of (GpT)n and (ApC)n were 2- to 24-fold higher than those of the control group after induction with formaldehyde (Table I). As we expected, cells exposed to 2.5 mM formaldehyde experienced a greater frequency of mutation induction than cells that were with 1x M9 salt exposure (P < 0.05). In addition, when compared with the data from the formaldehyde treated group, the mutation frequency increased with an increasing number of repeat units. Expansions or deletions of dinucleotide repeats were much more frequent in long repeat sequences than in short ones. As shown in Figure 3, the mutation frequency of (GpT)32 was about three times higher than that of (GpT)16. The complementary sequences (GpT)n and (ApC)n inserted on the leading strand of pAJ19 had significantly different mutation frequencies (P < 0.05). The mutation frequencies of formaldehyde-exposed (GpT)n were 13- to 24-fold higher than those of the control group, while those of (ApC)n were only 2- to 3-fold higher (Table I).


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  		Table I. Formaldehyde (FA)-induced microsatellite instability (Mean ± SEM)

 


Figure 3
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  		Fig. 3. Instability spectra of plasmids containing the (GpT/ApC)n insert. The (GpT) orientation of every (GpT/ApC)n group is on the left side and the (ApC) orientation is on the right. For the same insert orientation of microsatellites, the mutation frequency increased with an increasing number of repeat units. Black bars represent mutation frequency induced by 2.5 mM of formaldehyde; light gray bars by 1x M9 salt. Mean ± SEM of five independent tests with the different length of microsatellites. **P < 0.01; *P < 0.05.

 
Recovered plasmids were sequenced to determine the locations of the induced mutations and to verify that the mutations induced by formaldehyde were consistent with the expectation for microsatellite instability. The results demonstrated that the phenotype resulted from compensating frameshift mutations in the carbenicillin-resistant gene. Moreover, they showed that all frameshifts occurred within microsatellite repeat sequences. All of 112 mutated plasmids sequenced in the present study contained different mutation modes of insertion or deletion within the microsatellite that restored the reading frame. The majority of frameshift mutations in (GpT)32 and (ApC)32 were 2-bp deletions, while most mutations in (GpT)16 and (ApC)16 were 2-bp insertions (Table II). Eighty percent of the mutations in the (GpT)32 and (ApC)32 repeats consisted of deletions of repeat units, whereas 5 and 14% of the mutations in the (GT)16 and (AC)16 repeats, respectively, were deletions. Furthermore, the formaldehyde (2.5 mM) exposure produced a small percentage of larger expansions or deletions. The alterations of microsatellite length in all mutants were no more than ±4 bp (Table II).


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  		Table II. Number of the expansions or deletions within microsatellite repeats induced by formaldehyde

 

   Discussion
Top
 Introduction
 Materials and methods
 Results
 Discussion
Conclusion
 References
 
Formaldehyde, which is a genotoxic agent in vitro and naturally occurs in tissues, cells and body fluids, is widely used in industries and hospitals. The normal endogenous concentration of formaldehyde in the blood is ~0.1 mM in rats, monkeys and humans (18Go). However, formaldehyde is a potent nasal irritant, being cytotoxic at high doses (13Go). Several studies have reported a carcinogenic effect in humans after inhalation of formaldehyde, in particular an increased risk for nasopharyngeal cancer (19Go,20Go). While many such studies have been conducted, conflicting results regarding the genotoxic potential of formaldehyde have been reported (18Go). Part of the reason for the controversy associated with the mutagenesis of formaldehyde may be due to methodological limitations of previous studies, which cannot reveal subtle molecular alterations, perhaps involving the change of one or a few nucleotide bases. Therefore, we used a plasmid-based system for the detection of microsatellite instability. In this system, the carbenicillin resistance gene of pAJ19 plasmid in E. coli was disrupted by (GpT)n or (ApC)n repeats. After the treatment of E. coli with formaldehyde, expansions or deletions of dinucleotide repeats restored the carbenicillin resistance gene reading frame, enabling selection for the carbenicillin resistance phenotype. Our results demonstrate that the treatment of (GpT)n and (ApC)n microsatellites with 2.5 mM formaldehyde in E. coli induced more mutations than the untreated control groups. The possible reason for the mutagenesis of formaldehyde is that formaldehyde can easily penetrate the cell membrane and rapidly bind to thiols and macromolecules at the contact site (21Go,22Go). Subsequently, formaldehyde can initiate DNA–protein crosslinks that can temporally arrest DNA replication (23Go), which would cause the nucleotide strands to generate a transient dissociation followed by a misaligned reassociation, a DNA replication slippage process, resulting in an increase of the microsatellite instability. Furthermore, the microsatellite mutation frequency increased despite the fact that JM109 cells contained an intact mismatch repair system, which suggests that the damage induced by formaldehyde at 2.5 mM concentration to cells could not be efficiently repaired by the mismatch repair system in cell.

Interestingly, the difference in the mutation frequency observed between the (GpT)32 and the (ApC)32 orientation may involve the transient formation of a Z-DNA structure. It has been demonstrated that specific sequences with alternating purine–pyrimidine regions, i.e. GC and GT repeats, are typically readily converted into Z-DNA structures both in vitro and in vivo (24Go). In this secondary structure, damage to the DNA in a Z-DNA conformation was harder to repair by DNA repair enzymes. For example, alkylating damage, such as N7-methylguanine, which was typically removed by a DNA glycosylase, was not efficiently repaired in Z-DNA (25Go,26Go). Furthermore, Klysik et al. (27Go,28Go) discovered that deletions of plasmids containing CpG repeats, a kind of Z-DNA-forming sequence, occurred at a frequency substantially greater than that of plasmids without the CpG repeats. Later, Freund et al. measured the stability of CpG repeats in a lacZ mutation-reporter plasmid in bacteria and found that CpG repeats underwent small deletions and that the most common mutation event was a small deletion of a single repeat unit. The authors proposed that these small deletions within the repeats are most likely due to slippage events (29Go), which corroborates with our result that the plasmids containing (GpT/ApC) microsatellites, a kind of Z-DNA-forming sequence, had a much higher mutation frequency than that of the control group (Figure 3). Moreover, the guanosine nucleotides were in a syn position where the bases were located over the sugar without protection and thus were more exposed to DNA-damaging factors (30Go), such as formaldehyde. This in turn could explain the difference of mutation frequencies of the (GpT)n and (ApC)n orientations in the present study.

Two additional phenomena are also worth mentioning. First, the mutation frequencies of microsatellites were positively correlated with the length of the repeat sequence. The mutation frequency of (GpT)32 and (ApC)32 was significantly higher than that of the other two repeat lengths (P < 0.05) (Table I). This phenomenon was consistent with previous findings (31Go). In yeast, GT microsatellite tracts ≥51 bp had significantly higher mutation frequencies than tracts ≤33 bp (32Go). Second, after exposure to formaldehyde, deletions were more frequent than insertions in strains containing (GpT/ApC)32, whereas the opposite was true for strains containing (GpT/ApC)16. In other words, short microsatellites tended to increase in length, whereas long microsatellites tended to decrease in length. This length-dependent model has also been observed in different organisms (33Go).


   Conclusion
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
References
 
Microsatellite sequences are highly polymorphic regions of the genome. The results clearly demonstrate that the genotoxic effects of formaldehyde can increase microsatellite instability. Furthermore, (GpT)n and (ApC)n microsatellites, which are complementary dinucleotide repeats, have different mutation frequencies when exposed to formaldehyde. We hypothesize that the mutagenic mechanism of formaldehyde and the formation of Z-DNA may account for such microsatellite instability. Our results also suggest that the mutation frequencies of microsatellites are positively correlated with the length of the repeat sequence and that the model of microsatellite mutation depends on their lengths. Testing microsatellite instability as described in this study could help assess the mutagenic effects of other environmental toxic compounds.


   Acknowledgments
 
We are grateful to Lawrence A. Loeb in the University of Washington for a generous gift of pAJ19 plasmid. We thank Emily H. King and Ying Du at Sichuan University, Xiaojing Wang and John shaffer at the University of Pittsburgh for improving the manuscript.


   Notes
 
* To whom correspondence should be addressed. Tel: +86 28 85412488; Fax: +86 28 85414886; Email: fundzou{at}scu.edu.cn

This version has the correct addresses of the authors.


   References
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 

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Received on January 7, 2007; revised on April 30, 2007; accepted on May 17, 2007.


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