Molecular analysis of Tripterygium hypoglaucum (level) Hutch-induced mutations at the HPRT locus in human promyelocytic leukemia cells by multiplex polymerase chain reaction

Mutagenesis, Jan 2003

The genotoxicity and cytotoxicity of a Chinese medicinal herb, Tripterygium hypoglaucum (level) Hutch (THH), was investigated in human promyelocytic leukemia (HL-60) cells using the hypoxanthine-guanine phosphoribosyltransferase mutation assay. THH showed clear cytotoxicity and mutagenicity in HL-60 cells at concentrations between 6.7 and 20.0 mg/ml. When the mutants were characterized by techniques based on multiplex PCR, 46.6% of induced mutants were found to have deletions, whereas only 7.7% of spontaneous mutants showed deletions. The rest were not characterized, but were assumed to be mainly point mutations. Mapping of all intragenic deletion breakpoints showed a random distribution of breakpoints in nine exons. Deletion of exon 1 appeared as the only whole gene deletion, while deletions of exon 7/8 and 9 often occurred concomitantly (71.4%). It is concluded that THH is mutagenic in HL-60 cells, predominantly inducing deletions. Since this herb is widely used as a traditional medicine, its genotoxicity should be assessed in vivo in treated humans.

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Molecular analysis of Tripterygium hypoglaucum (level) Hutch-induced mutations at the HPRT locus in human promyelocytic leukemia cells by multiplex polymerase chain reaction

Sheng Xue Liu 0 Jia Cao 0 Hui An 0 0 Department of Hygiene Toxicology, College of Preventive Medicine, Third Military Medical University , Chongqing 400038 , People's Republic of China The genotoxicity and cytotoxicity of a Chinese medicinal herb, Tripterygium hypoglaucum (level) Hutch (THH), was investigated in human promyelocytic leukemia (HL-60) cells using the hypoxanthine-guanine phosphoribosyltransferase mutation assay. THH showed clear cytotoxicity and mutagenicity in HL-60 cells at concentrations between 6.7 and 20.0 mg/ml. When the mutants were characterized by techniques based on multiplex PCR, 46.6% of induced mutants were found to have deletions, whereas only 7.7% of spontaneous mutants showed deletions. The rest were not characterized, but were assumed to be mainly point mutations. Mapping of all intragenic deletion breakpoints showed a random distribution of breakpoints in nine exons. Deletion of exon 1 appeared as the only whole gene deletion, while deletions of exon 7/8 and 9 often occurred concomitantly (71.4%). It is concluded that THH is mutagenic in HL-60 cells, predominantly inducing deletions. Since this herb is widely used as a traditional medicine, its genotoxicity should be assessed in vivo in treated humans. Introduction PCR (Saiki et al., 1985; Park et al., 1995) has been used for the analysis of exon deletions in different human cells (Gibbs et al., 1989, 1990), Chinese hamster cells (Yang,J.-L. et al., 1989; Rossiter et al., 1991) and other cell lines (Yu et al., 1992; Elisabetta et al., 1995; Pluth et al., 1998). These studies have indicated a wide hypoxanthine-guanine phosphoribosyltransferase gene (hprt) mutation spectrum in various mammalian cell lines induced by physical and chemical mutagens. This technique is, therefore, a good method to understand in more detail the molecular mutation spectrum. Tripterygium hypoglaucum (level) Hutch (THH) is a traditional Chinese herb belonging to the genus Celastraceae. Its main chemical components are alkaloids, terpenes and pigments. THH has been used widely in traditional Chinese medicine for the treatment of various human autoimmune diseases, such as rheumatic arthritis, lupus erythematosus, hyperthyroidism, psoriasis and so on. It has also been reported that THH shows antitumor activity (Luo et al., 1988; Wang,S.M. et al., 1989). In their basic studies on THH, Wang,S.M. et al. (1989) confirmed that THH has a strong ability to induce chromosomal non-disjunction, chromosomal aberrations and aneuploidy in mice. Wang et al. (1993) found, in addition, that THH can induce C-mitotis, malsegregation and sister chromatid exchange (SCE) in mice. In our laboratory we used fluorescent in situ hybridization (FISH) with mouse minor centromeric and telomeric DNA probes and CREST antibodies to study the chromosomal composition of micronuclei (MN). We found that 6070% of MN induced by THH contained whole chromosomes and, in addition, that THH showed a very strong ability to induce apoptosis in Chinese hamster embryo, mouse NIH3T3 and human lymphoma Jurkat cell lines (Cao et al., 1997, 1998; Cao and Nusse, 1999). It is interesting that Jurkat tumor cells were found to be more sensitive (~10- to 20-fold) in terms of apoptosis as compared with non-tumor cells. All these results indicated that THH has an ability to induce chromosomal damage and aneuploidy and that it is also an inducer of apoptosis. There is, however, no previous information on the ability of THH to induce gene mutations in mammalian cells. In this paper, the multiplex PCR molecular analysis method for HPRT gene mutations in human promyelocytic leukemia cells (HL-60) was used to analyze the mutation spectra induced by THH and the mechanism of genotoxicity. Materials and methods Water extracts from THH The dry root of THH was provided by Kunming Medicine Co. (Yunnan, Peoples Republic of China). Samples of 20 g of the herb were kept in 400 ml of distilled water overnight and then boiled three times, the extract was concentracted to 30 ml and the sediment was removed by centrifugation and filtration (1 ml of water extract is equal to 0.67 g THH). At present, the chemical components of this water extract are thought to include only alkaloids, terpenes and pigments. Cell culture HL-60 is a human acute promyelocytic leukemia cell line described earlier by Collins et al. (1978). HL-60 cells were maintained as an asynchronous, exponentially growing population in RPMI 1640 medium (Sigma, St Louis, MO) supplemented with 10% fetal bovine serum (SJQ, Hangzhou, Peoples Republic of China), 100 U/ml penicillin (Sigma), 100 g/ml streptomycin (Sigma) and 2 mM L-glutamine (Gibco, Carlsbad, USA) at 37C in an atmosphere of 5% CO2. Before treatment the cells were incubated for 1 day in complete medium supplemented with 106 M aminopterin (Gibco), 104 M hypoxanthine (Sigma) and 105 M thymidine (Sigma) (HAT culture medium) to remove pre-existing HPRT mutants that cannot live in HAT culture medium. Then the medium was replaced with complete medium supplemented with 105 M thymidine and 104 M hypoxanthine. Two days later, this medium was removed and the cells were incubated in normal medium for 710 days before treatment. Cytotoxicity To measure the cytotoxicity of THH, exponentially growing HL-60 cells were treated with different concentrations of THH in culture medium for 4 h. Initial cell numbers per treatment were fixed at 5.010 6 cells. Sterile distilled water was used as a negative control and N-ethyl-N-nitrosourea (Shanren, Tokyo, Japan) was used as a positive control. At the sampling time the cells were harvested and washed twice with D-Hanks medium (Hanks buffer without Ca2 and Mg2 ) at 37C and afterwards diluted in normal culture medium. The cells were counted, diluted and transferred to 96-well microwell plates (Gibco) at an average of 1 cell/200 l medium/well. After incubation for 7 days, wells containing colonies were counted and the plating efficiency (PE) was calculated: PE [ln(no. of negative wells/no. of all wells)]/[no. of cells per well]. Mutation experiments After expression of gene mutations (8 days), approximate cell numbers per treatment dose were 4.8510 7, 3.6510 7, 3.1210 7, 2.3110 7, 1.0110 7 Primer sequence (53) Fragment size (bp) and 0.6910 7 in the 0, 3.33, 6.67, 10.00, 13.33 and 20.00 mg/ml groups, respectively. For cloning efficiency (CE) 1 cell/well was transferred to the 96-well plates and for assay of mutant frequency (MF) 110 4 cells were added to each well in 200 l medium with 1 g/ml 6-thioguanine (6-TG) (Sigma). Plates were analyzed for colony presence 7 days after seeding for CE and 8 days after for MF. [ln(no. of negative wells/no. of all wells)]/[no. of cells per wellCE]. Screening, extension and DNA isolation A single positive clone was transferred from the 96-well plate to a 24-well microwell plate (Gibco) to continue culture for an additional 12 days. Each well contained 1 ml screening medium including 2 g/ml 6-TG. Some of the cloned cells were then transferred to a new 24-well plate which contained HAT culture medium at 103 cells/well and cultured for 13 days. If the cloned cells in a well were obviously dead they were identified as mutated clones and the remaining cloned cells in the original 24 wells were transferred to culture bottles for extension expression. DNA isolation and purification from wild-type cells and HPRT mutant cells were performed according to conventional methods. Design, synthesis and appraisal of primers Eight pairs of oligonucleotide primers were designed by computer software with a small modification according to Wei et al. (1996). The synthesis and appraisal of the eight pairs of primers were completed by different laboratories (Beckman Co., Beijing; Cybersyn B.J., USA; Institute of Cellular Biology of Chinese Academy of Science, Shanghai). Table I shows the sequences of the eight pairs of oligonucleotide primers. Exons 7 and 8 were amplified simultaneously using the same primers, because they are only 163 bp apart. All primers except the exon 1-specific ones enabled amplification of the corresponding exons by multiplex PCR. It was, however, difficult to include exon 1 primers within the remaining set of all primers without spurious synthesis of a non-specific signal. In our preliminary experiments with several primer pairs in one PCR reaction it was difficult 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 and false positive results. Therefore, after several preliminary experiments, the eight pairs of primers were divided into three groups: one multiplex PCR included exons 2, 5, 6 and 7/8, the second included exons 3, 4 and 9 and exon 1 was amplified separately. Seventy-one mutants were analyzed by this multiplex PCR method. PCR analysis For amplification of HPRT exons, genomic DNA template (3650 ng) was mixed with 50 pmol of each primer pair in a total reaction volume of 50 l containing 50 mM KCl, 10 mM TrisHCl (pH 8.8), 0.31.05 mM MgCl2, 0.2 mM dNTPs and 2.5 U AmpliTaq DNA polymerase (Shenggong, Shanghai, Peoples Republic of China). After initial denaturation of the template DNA at 98C for 7 min, a total of 40 PCR cycles were performed with denaturation at 94C for 1.5 min, annealing at 52C for 1.5 min and extension at 72C for 2.0 min. Exon 1 was synthesized individually under modified conditions: a total of 30 PCR cycles were performed with denaturation at 95C for 0.5 min, Fig. 1. The relationships between plating efficiency (PE), mutant frequency (MF), cloning efficiency (CE), deletion percentage (DP) and THH dose. annealing at 64C for 1.0 min and extension at 72C for 1 min. The last cycle was finished with a 7 min extension at 72C. The PCR product (10 l) was used for analysis by 3% agarose gel electrophoresis or by PAGE. Results Cytotoxicity and mutagenicity of THH Figure 1A shows the cytotoxicity of THH to HL-60 cells. The PE gradually decreased with increasing concentration of THH. The doseresponse relationship could be expressed by the equation y 96.3e0.03x (P 0.01). There was a significant effect on PE at THH concentrations of 5 mg/ml. Figure 1B shows the mutagenicity of THH in HL-60 cells. A linear increase in MF with increasing concentration of THH was found. This doseresponse relationship could be expressed by the equation y 3.34 0.76x (P 0.01). At 6.67 20 mg/ml THH, MF was 10.6- to 43.8-fold that in the control cultures. Tripterygium hypoglaucum Hutch-induced mutations Treatment No. analyzed No. showing PCR changes Deletion (%) No. showing no change Total deletion Partial deletion 1/13 (7.7%) 1/4 (25.0%) 3/9 (33.3%) 4/10 (40.0%) 5/14 (35.7%) 7/21 (33.3%) 12/13 (92.3%) 3/4 (75.0%) 5/9 (55.6%)* 5/10 (50.0%)* 7/14 (50.0%)* 11/21 (52.4%)* Spontaneous THH (mg/ml) 3.33 6.67 0.05 versus control group. Multiplex PCR analysis Thirteen spontaneous and 58 THH-induced HPRT mutants were characterized by multiplex PCR. According to the electrophoresis pattern of PCR products, 43 (60.6%) of 71 mutants analyzed were found to exhibit no abnormal band in any of the nine exons. This indicated that these mutants had point mutations and not exon deletion or insertion. In 21 of 71 mutants there were less than eight bands for each locus, which showed partial deletion of exons. The remaining seven mutants had no PCR products, which meant that all exons studied were deleted. Of all mutants analyzed, 39.4% (28 of 71) had partial or whole deletions. Molecular spectrum of HPRT gene Table II shows the changes of spontaneously derived and THH-induced mutants at the HPRT locus. The electrophoresis patterns of mutants mainly consisted of three types: the normal pattern including point mutations, total deletions and partial deletions. THH-induced mutant cells (6.6720 mg/ml) showed mutation spectra that were significantly different from the spectra of spontaneous mutations. No spontaneous mutants showed total exon deletions, while THH-induced mutants did. The proportions of deletion mutations were very different between spontaneous and THH-induced mutants. About 25 50% of mutations found in THH-induced mutants were deletions while the proportion in spontaneous mutants was only 7.7%. The proportion of the normal pattern was very high (92.3%) in spontaneous mutants, compared with only 5075% in THH-induced mutants. A clearer doseresponse relationship was seen in induction of partial and whole deletion mutation than in induction of total mutations. Analysis of deletion breakpoints Figure 2 shows the distribution of the deletions in the nine exons of the HPRT gene found in the 71 mutants analyzed during this experiment. Neither an obvious difference among absolute numbers of mutations in the nine exons nor a clear hot-spot were found. Deletion mutations were found in all nine exons of the HPRT gene, while single deletions per mutant only in exon 1, 7/8 or 9 were not found. Deletion in exon 1 was observed only when total gene deletion occurred, and most of the deletions in exons 7/8 and 9 were concomitant (linked deletions, 71.4%). Discussion It has been shown that THH not only affects tumor growth, but also induces non-disjunction, aneuploidy and chromosomal aberrations. Recently, Cao and Nusse (1999) reported that a water extract of THH could also induce apoptosis in cultured cells in vitro. However, no evidence has so far been reported Fig. 2. Schematic diagram of the distribution of deletions (black bars) within the nine exons of the human HPRT gene. (A) Distribution of deleted exons in spontaneous mutants; (B) distribution of deleted exons in THHinduced mutants. *, Intragenic deletion; **, intragenic insertion. on the mutagenicity of THH. Our study has demonstrated that water extracts of THH are cytotoxic and mutagenic to cultured HL-60 cells in vitro. The present results on the induction of HPRT mutations by THH may help in understanding the pharmacological and toxicological effects of THH and further suggest that the use of this herb may have a genotoxic risk. Based on 13 spontaneous and 58 THH-induced HPRT mutants characterized by multiplex PCR, distinct differences in the mutation spectra were found between control and induced mutants. Among the 13 spontaneous mutants no total deletion mutations were found and only one mutant showed a partial deletion. It is known that spontaneous mutation at the HPRT locus in many kinds of cells mainly involves point mutations (Tomita et al., 2000), which could not be distinguished using the multiplex PCR method alone. However, 2550% of THH-induced mutants had exon deletions. It is of interest that the highest fractions of partial and whole deletions were not found at the highest concentration (20 mg/ml) but at lower concentrations (1013.3 mg/ml). Yamada et al. (1996) also reported a similar effect when studying X-ray-induced HPRT gene mutations in primary human skin fibroblasts. They found that the highest fraction of whole deletions did not appear at the highest dose (4 Gy) but at a lower dose (2 Gy). The reason for this effect is probably that higher doses (or in our case higher concentrations of THH) induce serious exon deletions so that these cells are not able to survive. We have reported that THH induces a high frequency of MN harboring whole chromosomes at all concentrations tested (3.33, 6.67 and 13.33 mg/ml) and produces a dose-dependent increase in fragment-containing MN, indicating that THH has both aneugenic and clastogenic potential (Yang and Cao, 2001). We intend to further characterize the THH-induced mutants by DNA sequencing, to better understand the mutagenic mechanism of THH. Various studies have shown that THH has multiple genotoxic potential, inducing gene mutations, chromosome breakage and aneugenic events. It is, therefore, important to study whether genotoxic effects can be detected in patients treated with THH. Acknowledgements The authors would like to thank Dr. Makoto Hayashi of NIHS, Japan and Dr. J. Fitzgerald of Department of Human Services of South Australia for their helpful comments on this manuscript. This research was supported by NSFC contract 39970650, 30100153 and 30100241.


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Sheng Xue Liu, Jia Cao, Hui An. Molecular analysis of Tripterygium hypoglaucum (level) Hutch-induced mutations at the HPRT locus in human promyelocytic leukemia cells by multiplex polymerase chain reaction, Mutagenesis, 2003, 77-80, DOI: 10.1093/mutage/18.1.77