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Mutagenesis Advance Access originally published online on April 11, 2008
Mutagenesis 2008 23(4):309-315; doi:10.1093/mutage/gen016
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© The Author 2008. 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.

Acrylamide-induced molecular mutation spectra at HPRT locus in human promyelocytic leukaemia HL-60 and NB4 cell lines

Lin Ao, Sheng-Xue Liu, Meng-Su Yang1, Chi-Chun Fong1, Hui An and Jia Cao*

Department of Hygienic Toxicology, Preventive Medical College, Third Military Medical University, Chongqing 400038, People's Republic of China 1Department of Biology and Chemistry, City University of Hong Kong, Kowloon 83, Hong Kong, People's Republic of China

Acrylamide (AA) is a compound widely used in many industries around the world. The recent finding that it is formed naturally in foods by heating raises human health concerns. AA is a proven carcinogen in animals and a probable carcinogen in humans, while its mutagenicity detected using in vitro mammalian gene mutation assays is still inconsistent in different cell systems. In the present study, we investigated the mutagenicity of AA in human promyelocytic leukaemia cells, HL-60 and NB4 cells, by examining the mutations at the hypoxanthine–guanine phosphoribosyltransferase (HPRT) gene locus. In a 6-h treatment without the exogenous activation, AA exerted a weak mutagenic effect at the highest concentration used in the study (700 mg/l) in HL-60 cells (P < 0.01) as well as in NB4 cells (P < 0.05). Molecular analysis of AA-induced mutants revealed a different mutation spectrum, when compared to that of spontaneous mutants. The most frequent spontaneous mutations were point mutations, whereas AA-induced mutations were mainly single exon deletions besides point mutations, and an increase in the proportion of partial deletion was associated with the increase of AA treatment. There was no obvious difference in the mutation spectra observed between the HL-60 and NB4 cell lines. These results showed that AA has a weak mutagenic effect at HPRT gene locus in human promyelocytic leukaemia HL-60 and NB4 cell lines and those molecular mutation spectra (single exon deletions and point mutations) may be related to some specific and precise mechanism.


   Introduction
Top
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Acrylamide (AA) is a water-soluble, vinyl monomer that has multiple chemical and industrial applications around the world. More recently, it was found to form naturally during the frying, roasting or baking of a variety of foods at very high temperatures (1Go,2Go).

AA is a well-documented neurotoxicant in both humans and laboratory animals. In addition to neurotoxicity, there are considerable experimental data from rodent studies that AA produces reproductive toxicity (e.g. reduced litter size, DNA strand breaks and dominant lethal mutation) (3Go–6Go). Furthermore, studies of AA-exposed laboratory animals (primarily rodents) have revealed an increased incidence of tumours in certain tissues (e.g. mammary gland fibroadenomas in female rats, tunica vaginalis mesotheliomas in female and male rats) (7Go–10Go). AA has been classified as a probable human carcinogen by IARC (group 2A), based on the significant evidence of carcinogenicity in experimental rodent models (11Go) and DNA adduct formation in human bronchial cells (12Go). However, epidemiological studies of occupationally exposed human populations have failed to establish a relationship between AA exposure and an increased risk for cancer (13Go,14Go), mainly because of the limitations of the present epidemiological studies of human industrial exposures used in the cancer risk assessment (15Go).

Although the mechanisms underlying AA-induced carcinogenesis have not yet been fully elucidated, the genotoxicity in cells of in vivo and in vitro systems should contribute to it. A large number of studies and updated reviews on the genotoxicity of AA have been reported (12Go,16Go,17Go). In the comet assay, AA could induce DNA damage in the FRTL5 and PC Cl3 rat thyroid cell lines (18Go), as well as in human lymphoblastoid TK6 cells (19Go) and human HepG2 cells (20Go). In the micronucleus (MN) test, positive results were observed in rodents and mammalian cell lines (21Go–23Go). Meanwhile, several studies reported the induction of chromosomal aberrations and mitotic disruptions following AA exposure of cultured mammalian cells, mostly Chinese hamster cell strains and lines (16Go). However, there are also some disputed results from point mutation assays. AA has consistently exhibited negative results in bacterial gene mutation assays with and without activation (16Go,24Go). In in vitro mammalian gene mutation tests, AA showed negative activity at the hypoxanthine–guanine phosphoribosyltransferase (HPRT) locus in V79 cells even at relative high dose levels (25Go,26Go), but positive activity in cultured Chinese hamster ovary cells in the presence or the absence of exogenous activating systems (12Go). AA was also mildly mutagenic at the thymidine kinase (TK) locus (19Go) and HPRT locus in mouse lymphoma cells (24Go,27Go). Moreover, there was evidence which suggested that AA produced increased lymphocyte HPRT mutant frequencies in Big Blue mice, with the high doses producing responses 16- to 25-fold greater than that of the respective control (28Go). These disputed results indicated that the genotoxic effects of AA induced have not been adequately evaluated, especially for the effect on the HPRT gene.

In this paper, two kinds of human promyelocytic leukaemia cells, HL-60 and NB4 cells, were used for the first time to determine the mutagenicity induced by AA at the HPRT gene locus. The obtained results will be helpful in understanding the mutagenic effects of AA in human cell lines. Furthermore, a multiplex polymerase chain reaction (PCR) analysis method for HPRT gene mutants was used to analyse the molecular mutational spectrum of AA-induced mutants. The studies on the induction of HPRT mutations and the involved molecular mechanism by AA may help to evaluate whether the use of this industrial chemical carries a carcinogenic risk.


   Materials and methods
Top
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Chemicals
AA (99% purity), 6-thioguanine (6-TG, 98% purity), hypoxanthine (99% purity), aminopterin (98% purity) and thymidine (99–100% purity) were all purchased from Sigma (Sigma Chemical Co., MO, USA).

Cell culture
HL-60 and NB4 cells are two human acute promyelocytic leukaemia cell lines described earlier by Collins et al. (13Go) and Lanotte et al. (29Go). These cells were provided by Cell Bank, Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China), and were, respectively, maintained as an asynchronous, exponentially growing population in RPMI 1640 medium (Sigma Chemical Co.) supplemented with 10% foetal bovine serum (SJQ, Hangzhou, China), 100 U/ml penicillin (Sigma Chemical Co.), 100 µg/ml streptomycin (Sigma Chemical Co.) and 2 mM L-glutamine (Gibco/BRL Life Technologies Inc., MD, USA) at 37°C in an atmosphere of 5% CO2. Before treatment, the cells were incubated for 1 day in complete medium supplemented with 10–6 M aminopterin, 10–4 M hypoxanthine and 10–5 M thymidine (HAT culture medium) to remove the pre-existing HPRT mutants that cannot live in HAT culture medium. Then the medium was replaced with complete medium supplemented with 10–5 M thymidine and 10–4 M hypoxanthine. Two days later, this medium was removed and the cells were incubated in normal medium for 7–10 days before treatment.

Cytotoxicity
For measuring the cytotoxicity of AA, exponentially growing HL-60 and NB4 cells were seeded at 5.0 x 106 viable cells and treated with different concentrations of AA in culture medium for 6 h. Sterile distilled water was used as negative control, and N-ethyl-N-nitrosourea (Sigma Chemical Co.) was used as positive control. At the end of treatment, the cells were harvested and washed twice with D-Hank's medium (Hank's buffer without Ca++ and Mg++) at 37°C in normal culture medium. The cells were counted, diluted and transferred to 96 microwell plates (Gibco/BRL Life Technologies Inc.) at an average of one cell in 200 µl medium per well. After incubating for 7 days, wells containing clones were counted and the plating efficiency (PE) was calculated. Three 96-well plates were used for the PE calculation in every group:

Formula

Mutation experiments
After treatment with different concentrations of chemicals, cells were used for mutation detection. Cells were incubated for 8 days and passaged every 3–4 days for the expression of gene mutation. To determine the cloning efficiencies (CE), cells were diluted and added in the 96-well microwell plates to ensure that one cell was inoculated per well. After incubating at 37°C, 5% CO2 for 7 days, wells with colony formation were counted as positive wells for CE calculation. To determine the mutation frequency (MF), cells were added in other 96-well micro-well plates to ensure that each well received 1 x 104 cells in 200 µl medium with 1 µg/ml 6-TG. Resistance to the lethal effects of the purine analogue 6-TG was used as the mutagenic marker. After incubating at 37°C, 5% CO2 for 8 days, positive wells were counted. MF was calculated and corrected for cell survival. In this study, three plates were used for CE and MF calculation in each treatment:

Formula

Mutant screening, expansion and DNA isolation
A single positive clone was transferred from the 96-well plate to a 24-well microwell plate to culture for additional 1–2 days. Each well contained 1 ml screening medium supplemented with 2 µg/ml 6-TG according to the Liu et al. (30Go). To exclude the false-positive mutant and reverse mutant, a part of cells in a clone was then transferred to a new 24-well microwell plate which contained HAT culture medium with 1 x 103 cells per well and cultured for 1–3 days. If the cells in a clone were obviously dead, these cloned cells were identified as mutant and the remaining cloned cells in the original 24-well microwells were transferred into culture bottles for cell expansion. DNA isolation and purification from wild-type cells and HPRT mutant cells were performed as previously described (30Go–32Go).

Design, synthesis and appraisal of exon primers
Eight pairs of oligonucleotide primers were designed by computer software according to Wei et al. (33Go) with a small modification, in which the difference was mainly in the design of exon 1 as described previously (30Go). The synthesis and appraisal of the eight pairs of primers were completed by different laboratories of Beckman Company in Beijing, Cybersyn B.J. in American and the Institute of Cellular Biology of Chinese Academy of Science in Shanghai, respectively.

The designed sequences of eight pairs of oligonucleotide primers are shown in Table I. Exons 7 and 8 were amplified simultaneously using the same pair of primers, because they are only 163 bp apart. The primers were located at a distance that was close to the immediate splice junctions. In our pre-experiments with several primer pairs in a single PCR reaction, it was difficult to include exon 1 primers within the remaining set of all primers without having a spurious synthesis of non-specific signal and to control and optimize the reaction conditions. In addition, insertions and deletions within exons could occur. Therefore, we restricted the number of primer pairs in a single PCR reaction in order to confirm the distances of PCR products according to their molecular weights. This reduced the number of false-negative or false-positive results. After several pre-experiments, eight pairs of primers were divided into three groups: one multiplex PCR included exons 2, 5, 6 and 7/8; the second one included exons 3, 4 and 9 and exon 1 was amplified separately (typical electrophoresis pattern given in Figure 1).


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  		Table I. Oligonucleotide primers for the multiplex PCR of the human HPRT locus

 


Figure 1
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  		Fig. 1. Detection of multiplex PCR products in human HPRT gene from normal cells by 3% agarose gel electrophoresis. Lanes 1 and 5: DNA marker; lane 2: group 1 including exon 1; lane 3: group 2 including exons 2, 5, 6 and 7/8 and lane 4: group 3 including exons 3, 4 and 9.

 
PCR analysis
For amplification of HPRT exons, genomic DNA template (36–50 ng) was mixed with 50 pmol of each primer pair in a total reaction volume of 50 µl containing 50 mM KCl, 10 mM Tris–HCl (pH 8.8), 0.3–1.05 mM MgCl2, 0.2 mM dNTPs and 2.5 U of Amplitaq DNA polymerase (Shenggong, Shanghai, China). After initial denaturation of the template DNA at 98°C for 7 min, a total of 40 PCR cycles were performed with denaturation at 94°C for 1.5 min, annealing at 52°C for 1.5 min and extension at 72°C for 2.0 min. Exon 1 was synthesized individually with a modified condition: a total of 30 PCR cycles were performed with denaturation at 95°C for 0.5 min, annealing at 64°C for 1.0 min and extension at 72°C for 1 min. The last cycle was finished with a 7-min extension at 72°C. The PCR product (10 µl) was analysed by 3% agarose gel electrophoresis or 5% polyacrylamide gel electrophoresis.

Category of mutations
Mutants are divided into several categories based on their multiplex PCR patterns. ‘Total deletion’ mutants include mutants for which no representative exon fragment is synthesized. ‘Partial deletion’ mutants consist of either intragenic or end deletions. Intragenic deletion mutants contain two or more breakpoints within the HPRT gene, which include deletions of one exon and more (less than nine exons). End-deletion mutants have only one breakpoint within the HPRT gene. ‘Normal pattern’ represents those which contain all exon PCR products of expected sizes. Mutants with normal pattern could have transitions, transversions, frameshifts or small deletions which could not be detected by PCR analysis (31Go,32Go).


   Results
Top
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Cytotoxicity and mutagenicity of AA
The cytotoxicity and mutagenicity of AA to HL-60 and NB4 cells are shown in Table II. The PE gradually decreased with the increasing concentrations of AA. The results indicated a significant toxic effect of AA on PE at the concentrations ≥50 mg/l in HL-60 and ≥300 mg/l in NB4 cells.


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  		Table II. The PE, CE and MF in HL-60 cells and NB4 cells treated with AA at different concentrations

 
A linear increase of the MF with increasing concentrations of AA was found. This dose–response relationship could be expressed with the equation: Y = 8.17e2.07x (P < 0.01), although the significant difference occurred only at the highest concentration of AA (700 mg/l) in HL-60 cells (P < 0.01) and NB4 cells (P < 0.05), respectively, where the MF was about five times higher than that of the control cultures in both cell lines.

Exon deletions detected by multiplex PCR analysis
The number of mutants and the relative percentage of mutants between HL-60 and NB4 cells in each group are summarized in Table III. There was a total of 107 HPRT mutants analysed by multiplex PCR, including 26 mutants selected from the control group and 81 mutants from the AA treated group. According to the electrophoresis pattern of PCR products, 74 out of 107 mutants analysed were not found to exhibit abnormal bands in any of the nine exons, which indicated that these mutants had potential point mutations but not exon deletions and insertion mutations. The remaining 33 mutants showed less than eight bands for each locus, which indicated partial deletions of exons. However, in all the mutants analysed, there was no mutant that did not amplify any PCR product, which meant that all of the spontaneous and AA-induced mutants did not show total exon deletions.


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  		Table III. Summary of multiplex PCR analysis of spontaneous and AA-induced HPRT mutants in HL-60 cells and NB4 cells

 
It was also shown in the results that the proportions of deletion mutations were different between spontaneous and AA-induced mutants. Among all of the 81 mutants induced by AA, there were 31 mutants with deletion mutations, the proportions were about 14.3–54.5% in HL-60 and 0–60.0% in NB4 cells at different treatments of AA. In contrast, there were only two mutants which showed the deletion mutations among the 26 spontaneous mutants, the proportions were 10.0% in HL-60 and 0% in NB4 cells, respectively. These results suggested that AA could increase the percentage of exon deletion mutation. Moreover, there was a clear relationship between the dose of AA and percentage of mutations. With the increasing treatment of AA, the percentage of partial deletion mutation increased gradually while the percentage of point mutation decreased inversely.

Distribution analysis of exon deletions
The schematic diagram of the distribution of deletions in the nine exons of HPRT gene from the spontaneous and AA-induced mutants is also exhibited in detail in Figure 2. Most of the deletions were single exon deletion. In AA-induced mutants with deletion mutations, the proportions of single exon deletion were 75% (12/16) in HL-60 cells and 66.7% (10/15) in NB4 cells, respectively. Only nine mutants had the multiple exon deletions, among them there were two mutants with continuous deletion in exons 4 and 5. In addition, the results showed that deletions could be found in any exon except 7/8. No obvious difference of absolute numbers of mutations was found among each of the eight exons; thus, there were no apparent mutational hot-spot positions observed in HPRT mutants from either spontaneous or AA-induced mutants.


Figure 2
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  		Fig. 2. Schematic diagram of the distribution of exon deletion within the nine exons of the human HPRT gene in HL-60 cells and NB4 cells. Black bar indicates deletion of an exon.

 

   Discussion
Top
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Among the techniques developed for the detection of HPRT gene mutation, the cell cloning assay has been proven to be valuable due to the advantages of allowing recovery and further analysis of gene component or structure of HPRT mutant (location and qualitation of gene mutation) (34Go,35Go). With the HPRT cloning assay, mutant frequency could be determined by measuring cell CE in the absence and presence of 6-TG. In the present study, PE decreased significantly at concentrations of AA >50 mg/l in HL-60 cells and 300 mg/l in NB4 cells. At the highest concentrations (700 mg/l), PE were only 1.2% in HL-60 cells and 2.3% in NB4 cells, while Baum et al. (26Go) reported that AA cytotoxicity was not observed in V79 cells at concentrations <5 mM (equal to 505 mg/l). Our results suggest a severe cytotoxicity of AA in human promyelocytic leukaemia cells, especially in HL-60 cells. Additionally, the CE did not show a concentration-dependent decrease in our study. In HL-60 cells, it even increased gradually with the increasing concentration of AA. The reason for this effect can probably be attributed to the exclusion of damaged cells from the cell population during the 8 days mutation expression period.

Although a linear increase of the MF with increasing concentrations of AA was found in this study, the significant difference of MF compared with the control group occurred only at the highest treatment (700 mg/l) in HL-60 cells as well as in NB4 cells. Our result indicated a weak mutagenicity of AA in HPRT gene in human promyelocytic leukaemia cells. The genotoxicity of AA has been studied extensively, but previously studies have exhibited inconsistent mutagenic effects in the HPRT gene in different cell models. In V79 cells, no significant induction of HPRT mutations was observed with AA up to a concentration of 10 mM (equal to 1010 mg/l) (26Go), while positive results were obtained in mouse lymphoma cells (24Go,27Go) as well as in Big Blue mice (28Go). In addition, AA induced weak genotoxicity in human lymphoblastoid TK6 cells by TK assay determination, while its cytotoxicity was strong at high concentrations (>10 mM) (19Go). According to these results, we suppose that the inconsistencies in mutagenicity induced by AA should be mainly due to the use of different cell lines in these tests.

The carcinogenicity of AA has been well documented in experimental rodent models while disputed results of genotoxic effect were received from in vitro gene mutation assays, implying a possible action of metabolic transversion for this chemical. There is substantial evidence that glycidamide (GA), metabolically generated from AA by CYP450 2E1-mediated epoxidation, acts as the ultimate genotoxic metabolite in mice, rats and humans (36Go,37Go). GA can react with cellular DNA, leading to the formation of several DNA adducts. A number GA–DNA adducts have been characterized including the two major adducts with N-7 of guanine and N-3 of adenine (38Go). GA induces mutations in bacteria (39Go) and, when applied intraperitoneally to mice, it was found to induce MN in a dose-dependent manner in peripheral erythrocytes (40Go). Baum et al. (26Go) compared the mutagenic potential of AA and GA in V79 cells by using the HPRT mutation assay in the absence of metabolic activation, and found a concentration-dependent induction of mutation for GA at concentrations of 800 µM, whereas AA was inactive up to a concentration of 10 mM. Some other studies also demonstrated that GA is more potent when the mutagenicity of AA and GA are compared (19Go,28Go), which indicates that the metabolic conversion of AA to GA appears to be critical for the genotoxicity of AA. However, Dearfield et al. (16Go) pointed out that there were at least two theories on the mutagenic effect of AA. The first is the metabolic conversion of AA to GA that has been mentioned above. Another possible mechanism is a direct Michael-type reactivity of AA with nucleophiles in DNA and proteins, such as the interaction with glutathione (GSH), a molecule protecting the cell against endogenous and exogenous oxidants and electrophiles. Recently, there have been increasing data that suggest the possibility that the latter mechanism is responsible for the genotoxicity of AA in some experiments. AA has been reported to be mildly genotoxic in the MN and TK assays of human lymphoblastoid TK6 cells without metabolic activation (19Go). However, the cytotoxicity and genotoxicity of AA were not enhanced by metabolic activation (rat liver S9), and molecular analysis of the TK mutants revealed that AA predominantly induced loss of heterozygosity mutation while GA induced primarily point mutations. Those results indicate that the rat liver S9 did not activate AA and the genotoxic characteristics of AA and GA were distinctly different (19Go). Moreover, AA has been proved to be positive at the TK locus and HPRT locus in mouse lymphoma cells without metabolic activation (24Go,27Go). Detailed study showed that no GA-derived DNA adducts were found after AA treatment, consistent with a lack of metabolic conversion of AA to GA. Analysis of the mutational spectra revealed a significant difference between the types of mutations induced by AA and GA treatments (27Go). That study suggests that AA induces mutations probably through an increase of cellular oxidative stress. Thus AA was clastogenic and GA was mutagenic. In our system, AA appears to exert a weak genotoxic activity in the absence of metabolic activation. This result is similar with that obtained in mouse lymphoma cells. Because of the high expression of CYP450 2E1 found in human myelocytic leukaemia cell lines including HL-60 cells (41Go), whether the mutagenic effect of AA results from the generation of GA, or from the interaction between AA and DNA, is worth investigating in the future.

Based on the 107 HPRT mutants characterized by multiplex PCR, a few differences in the mutation spectra were found between control and AA-induced mutants. Among the 26 spontaneous mutants, only two (7.7%) mutants showed a partial deletion while the remaining 24 (92.3%) mutants had the normal electrophoresis pattern, implying point mutation. Especially, all of the six spontaneous mutants in NB4 cells showed the point mutation (Table III). This result is in agreement with literature previously reported (42Go), which indicated that spontaneous mutation at the HPRT locus in some types of cell includes mainly point mutations, which cannot be distinguished by using the multiplex PCR method only. Among the 81 mutants induced by AA at different concentrations, however, 14.3–54.5% (HL-60) and 0–60.0% (NB4) of mutants had exon deletions while the other mutants had point mutations. The percentage of exon deletion in AA-induce mutants was much higher than that in spontaneous mutants, and an increase in the proportion of partial deletion was associated with increase in AA concentration. Moreover, the results revealed AA predominantly induced single exon deletion. Only nine mutants showed two exons deletion and there was no mutant with deletions in more than two exons. Deletion of all exons was not induced by AA even at the highest concentration. Our results suggest that the spectra of spontaneous and AA-induced mutations are distinctly different, and the smaller changes (the nature of the point mutations and single exon deletions) in genetic structure (on the exon level) have something to do with AA-induced mutation mechanism.

It was interesting that several mutants (four from HL-60 cells and five from NB4 cells) had a deletion of more than one exon (Figure 2). These mutants with deletions of two exons were all isolated after treatment of human promyelocytic leukaemia cells with the two highest doses of AA (500 or 700 mg/l, data not shown). Our results suggest that the size of genetic alteration appears to be dependent on dose, although these results are not conclusive and indicate that further experiments at higher doses and analysis of statistically significant numbers of mutants will be necessary to draw more useful conclusions. Other investigators have also reported a dose dependence of the mutation spectrum in response to radiation damage (31Go,43Go). It was observed that ‘multi-locus’ deletions were induced at a frequency dependent on the square of the exposure, whereas smaller intragenic changes were in direct proportion to the dose. It was concluded that these two types of mutational events are unlikely to result from the same initial damage and that the ‘two-hit’ kinetics of multi-locus deletions probably reflects two independent damage events leading to DNA loss. Another possibility could be factors such as the packaging of DNA and its attachment to a matrix which may allow the association of an apparently distant region (35Go,44Go).

In this study, both of human promyelocytic leukaemia HL-60 and NB4 cells were used to evaluate the cytotoxicity and genotoxicity of AA. One of our purposes is to compare the results obtained in the different human promyelocytic leukaemia cell lines, to find some consistency or difference and to use this information to make some conclusions concerning the mutagenicity of AA. The results showed that PE determination indicates a slightly higher susceptibility of HL-60 cells to AA-induced cytotoxic effects than that of NB4 cells. But this result still suggests a severe cytotoxicity of AA in both of the human promyelocytic leukaemia cell lines, when compared with the result obtained in another study (26Go). Additionally, the MF induced by AA in HL-60 cells is similar to that in NB4 cells, and no obvious difference on molecular mutation spectra of HPRT gene was found between these two cell lines. Thus in our experimental conditions, the results indicate a consistency in the mutagenicity of AA between the two human promyelocytic leukaemia cell lines, although the HL-60 looks a little more sensitive in cytotoxicity than NB4.

In our laboratory, we have found that after treatment of mouse cells with AA (100, 200 and 400 mg/l) both MN containing whole chromosomes and MN containing acentric fragments increase in a dose-dependent manner, demonstrating that AA is not only a clastogen but also an aneugen (45Go). The results of the present study show for the first time the positive response of AA to HPRT gene. We intend to further characterize the AA-induced mutants with point mutations by DNA sequencing, to better understand the mutagenic mechanism of AA. It would, therefore, be important to study whether genotoxic effects can be detected in humans occupationally exposed to AA.


   Funding
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
The National Natural Science Foundation of China (No. 39970650, 30100153 and 30471476) and by the National Key Basic Research and Development Program of China (973) (No. 2002CB512901).


   Acknowledgments
 
The authors would like to thank Dr Li You from Chemical Industry Institute of Toxicology (CIIT) Centers for Health Research for his helpful comments on the manuscript.

Conflict of interest statement: None declared.


   Notes
 
* To whom correspondence should be addressed. Tel: +86 023 68752271; Fax: +86 023 68752277; Email: caojia{at}mail.tmmu.com.cn


   References
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received on January 22, 2007; revised on February 26, 2008; accepted on February 27, 2008.


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