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Carcinogenesis Advance Access originally published online on April 29, 2007
Carcinogenesis 2007 28(8):1703-1709; doi:10.1093/carcin/bgm102
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Dietary flavonoids induce MLL translocations in primary human CD34+ cells

Sahar Barjesteh van Waalwijk van Doorn-Khosrovani*, Jannie Janssen1, Lou M. Maas, Roger W.L. Godschalk, Jan G. Nijhuis2 and Frederik J. van Schooten

1 Department of Health Risk Analysis and Toxicology, Nutrition and Toxicology Research Institute Maastricht, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands, Department of Clinical Genetics
2 Department of Obstetrics and Gynecology, Academic Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands

* To whom correspondence should be addressed. Tel: +31 43 3881103; Fax: +31 43 3884146;Email: s.khosrovani{at}grat.unimaas.nl


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Genetic abnormalities leading to infant leukemias already occur during fetal development and often involve rearrangements of the mixed-lineage leukemia (MLL) gene. These rearrangements resemble the aberrations observed in therapy-related leukemias following treatment with topoisomerase II (topoII)-inhibiting agents such as etoposide. Since flavonoids are potent topoII inhibitors, we examined the role of three widely consumed dietary flavonoids (quercetin, genistein and kaempferol) on the development of MLL rearrangements in primary human CD34+ cells. Using the neutral Comet assay, we demonstrated a dose-dependent double-strand break (DSB) formation after exposure to flavonoids. An incorrect repair of these DSBs resulted in chromosomal translocations that co-localized with those identified in infant leukemias. Most of these translocations were formed by microhomology-mediated end joining. Moreover, in all but one translocation, SINE/Alu or LINE/L1 repetitive elements were present in at least one side of the breakpoint junction. Beside MLL translocations, fluorescence in situ hybridization analysis demonstrated monosomy or trisomy of MLL in 8–10% of the quercetin-exposed CD34+ cells. Our study demonstrates that biologically relevant concentrations of flavonoids can induce MLL abnormalities in primary hematopoietic progenitor cells. This is particularly alarming knowing that the differences in metabolism and excretion rate between mother and fetus can lead to a higher flavonoid concentration on the fetal side. Therefore, it is important to raise public awareness and set guidelines for marketing flavonoid supplements to reduce the risk of infant leukemias.

Abbreviations: DMSO, dimethylsulfoxide; DSB, double-strand breaks; FISH, fluorescence in situ hybridization; MLL, mixed-lineage leukemia; PCR, polymerase chain reaction; topoII, topoisomerase II; wt, wild type


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Chromosomal abnormalities associated with infant leukemias originate during fetal development (1,2) and often involve rearrangements of the mixed-lineage leukemia (MLL) gene, an essential transcription factor for hematopoiesis (3). MLL is affected in 80% of infant acute lymphoblastic leukemia and 65% of infant acute myelogenous leukemia (4–6). The finding that similar abnormalities develop in children and adults treated with inhibitors of topoisomerase II (topoII) (e.g. etoposide) (7,8) has led to the hypothesis that maternal exposure to topoII inhibitors during pregnancy might induce infant leukemias (9,10).

TopoII, an important enzyme for replication and recombination, resolves knots and tangles of DNA by inducing transient double-strand breaks (DSBs) (11,12). Under normal conditions, the cleaved DNA ends are immediately religated by topoII. However, in the presence of certain topoII-inhibiting agents, like etoposide, the topoII–DNA cleavage complex is stabilized and increase the risk of chromosomal translocations (13). Indeed some recent studies have demonstrated that etoposide-induced DSBs can lead to MLL translocations in primary stem cells (14–16).

Several epidemiological studies investigated the contribution of potent topoII inhibitors in the development of MLL+ leukemias. In a case (n = 136) control (n = 266) study, Alexander et al. (17) demonstrated that ingestion of herbal medicines, dipyrone or exposure to pesticides elevates the risk of MLL+ infant leukemias. As flavonoids present in soy, vegetables, fruits, cereals, nuts and beverages (red wine, beer, tea and cocoa) are potent topoII inhibitors and induce topoII-mediated DSBs (18–21), Spector et al. (22) investigated the effects of maternal diet in a case (n = 240) control (n = 255) study. Although maternal fruit and vegetable intake is associated with decreased overall risk of infant leukemia, consumption of food containing topoII inhibitors, like onions, apples and canned beans seemed to increase the risk of MLL+ acute myelogenous leukemia (22). An earlier study by the same group in a rather limited population (84 cases and 97 matched controls) had demonstrated that maternal consumption of topoII inhibitor-containing food is associated with a 10-fold higher risk of infant acute myelogenous leukemia (23).

Although epidemiological studies provide some evidence for the involvement of topoII inhibitors in leukemogenesis, the complex nature of human diet and recall bias makes it difficult to elucidate the contribution of flavonoids. In order to provide direct evidence for the contribution of flavonoids in the development of leukemia-associated MLL translocations, we examined the clastogenic effect of three widely consumed dietary flavonoids in primary human CD34+ cells isolated from umbilical cord blood. Given the fact that most MLL+ leukemias originate in CD34+ progenitors (24,25), these cells are the most relevant model for studying the clastogenic effect of flavonoids (15). In this study, the effect of flavonoid exposure is compared with that of etoposide and a metabolite of benzene, hydroquinone. Exposure to both etoposide and hydroquinone can lead to leukemia-associated chromosomal abnormalities.

For the first time, our results demonstrate that exposure of human CD34+ cells to high but biologically relevant concentrations of flavonoids induce chromosomal translocations similar to those associated with infant leukemias.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
CD34+ cell culture and exposure to topoII inhibitors
Mononuclear cells were isolated from umbilical cord blood by density gradient centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway). Subsequently the CD34+ fraction was selected by magnetic-activated cell sorting CD34+ isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. CD34+ cells were expanded for 6 days in Iscove's modified Dulbecco's media supplemented with 10% fetal calf serum, Stem Cell Factor 50 ng/ml, Flt3-L 75 U/ml, Transforming growth factor-beta 1 100 U/ml (PeproTech, Tebu-bio, Heerhugowaard, The Netherlands) and IL-3 300 U/ml (Imgen, ITK diagnostics, Uithoorn, The Netherlands). After expansion, the cells were treated overnight with concentrations of 1–100 µM of etoposide (Sigma–Aldrich, St Louis, MO), hydroquinone (Fluka Chemika, Buchs, Switzerland), quercetin (Sigma–Aldrich, Steinheim, Germany), genistein (Alexis, Carlsbad, CA), kaempferol (ICN Biomedicals, Eschwege, Germany) or the vehicle dimethylsulfoxide (DMSO). After overnight exposure, the cells were washed twice and either immediately fixed for the Comet assay in low melting point agarose or were allowed to recover and expand for 48 h in the presence of cytokines. The latter were used for inverse polymerase chain reaction (PCR) assay and fluorescence in situ hybridization (FISH) analysis. All treatments were performed in at least two independent experiments. Cell counts and viability were determined by a hemocytometer and trypan blue dye.

Neutral Comet assay
CD34+ cells were gently suspended in 0.5% of low melting point agarose in phosphate-buffered saline at 37°C and dispersed on microscope slides coated with 1.5% normal melting point agarose (Sigma–Aldrich). The slides were protected with cover slips and allowed to solidify at 4°C for 30 min. After careful removal of the cover slips, the slides were incubated overnight at 4°C in lysis buffer (2.5 M NaCl, 100 mM ethylenediaminetetraacetic acid, 10 mM Tris, 250 mM NaOH, 1% sodium lauryl sarcosinate, 10% DMSO and 1% Triton X-100, pH = 10). Subsequently, the slides were subjected to electrophoresis (25 V, 300 mA,) at 4°C for 20 min (buffer: Tris 90 mM, boric acid 90 mM and ethylenediaminetetraacetic acid 2 mM, pH = 7.5), fixed with 100% ethanol and stained with 20 µg/ml ethidium bromide. The cells were then scored with fluorescence microscopy using Comet assay III software program (Perceptive Instruments, Haverhill, UK). The assay was performed in triplicate and 50 cells were scored on each slide. The mean Comet tail moment (the product of DNA in the tail and the mean distance of migration in the tail) was used as measure of DSB induction.

Inverse PCR assay and sequencing
Genomic DNA was isolated using DNAZOL reagent (Invitrogen, Carlsbad, CA). One microgram DNA was incubated with 1 U shrimp alkaline phosphatase (Promega, Madison, WI) for 1 h at 37°C and subsequently inactivated at 75°C for 30 min. Next, the DNA was digested with 24 U Xba I (Promega) for 4 h at 37°C. After 1 h heat inactivation at 75°C, the DNA was circularized overnight at 4°C using 3 U T4 DNA ligase (Promega) in a final volume of 50 µl. A fraction of the ligation product was heat inactivated and digested with Pvu II (Promega) to abolish amplification of the wild-type (wt) MLL. Approximately, 40 ng of the circularized DNA (either Pvu II treated or untreated) was used for the first PCR. The PCR was carried out using conditions and primers are described earlier by Libura et al. (15). The amplified fragments were diluted and applied for the nested PCR. The nested PCR products were separated by electrophoresis on a 1% agarose gel, excised and purified by QIAquick Gel Extraction Kit (Qiagen, Venlo, The Netherlands). The purified PCR products were subsequently sequenced by ABI Prism 3100 Genetic Analyzer (PE Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands) using 5 pmol of MLL-specific primers (5'-tgataaactctcctccatgcga-3', 5'-aggtttgaccaattgtccca-3' and 5'-gaaacaaataaaggtcaccca-3'). All primers were synthesized by Eurogentec (Liege, Belgium).

Sequence acquisition and analysis
MLL genomic sequence was obtained from National Center for Biotechnology Information database (gi:34305634, provided by Environmental Genome Project, NIEHS ES15478, Department of Genome Sciences, Seattle, WA). Detection of highly repetitive DNA sequences was performed using the RepeatMasker software (Smit,AFA, Hubley,R and Green,P. RepeatMaskerOpen-3.0.1996-2004, http://www.repeatmasker.org). The palindromic sequences were identified by BioPHP (http://www.biophp.org).

FISH
FISH analysis was performed using LSI MLL Dual Color, Break Apart Rearrangement Probe (Vysis, Abbott Molecular Inc, IL, USA) according to the manufacturer's protocol. The frequency of MLL chromosomal rearrangements was determined in interphase nuclei of CD34+ cells. Images were acquired on a Zeiss Axioplan II microscope using a Cohu camera and software from Applied Imaging (Newcastle, UK). Subsequently, the percentage of MLL abnormalities in quercetin-exposed cells (50 and 100 µM) was compared with DMSO-exposed cells in two independent experiments. Combined data from these experiments were analyzed for statistical significance (P < 0.05) using Fisher's exact test.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
DSB formation in primary CD34+ cells after exposure to flavonoids
It has previously been demonstrated that etoposide induces DNA-strand breaks in CD34+ cells (16). To examine whether flavonoids have similar potentials, we compared the extent of DNA DSB formation by quercetin and etoposide. Primary CD34+ cells were incubated overnight with concentrations of 1, 10 and 50 µM of quercetin or etoposide and were subsequently prepared for the neutral Comet assay. In contrast to the customary alkaline Comet, the neutral Comet exclusively detects DSBs (26). A comparison of the mean tail moments (Figure 1A) demonstrated that the amount of DSBs is significantly higher in cells exposed to quercetin 50 µM than those exposed to equal concentration of etoposide (P = 0.02). Subsequently, we compared the DSB formation potential of quercetin with genistein and kaempferol at concentrations of 1, 10, 25, 50 and 100 µM. The neutral Comet assay could detect an overall trend of increase in the mean tail moment after exposure to high concentrations of flavonoids. Especially, quercetin and genistein at concentration of 25 µM or higher were strong inducers of DSBs in hematopoietic CD34+ cells (Figure 1B).


Figure 1
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  		Fig. 1. The neutral Comet assay. Double-strand break formation following overnight exposure of CD34+ cells to various concentrations of etoposide (A), quercetin (A and B), kaempferol and genistein (B) or the vehicle 0.05% DMSO (A and B) is indicated as mean tail moment.

 
Exposure to flavonoids quercetin, kaempferol and genistein induces MLL translocations in primary human CD34+ cells
As flavonoids induce DSBs, we assumed that exposure to flavonoids may also induce MLL translocations. To examine this, CD34+ cells were incubated overnight with either different concentrations (0, 1, 10 and 50 µM) of flavonoids (quercetin, genistein and kaempferol) or to the well-known clastogenic compounds etoposide (27–31) and hydroquinone (14–16). The percentages of viable cells were assessed by trypan blue exclusion (Figure 2). Both etoposide and hydroquinone resulted in a considerable amount of cell death at the concentration of 50 µM. On the contrary, ~80% of the cells were still viable after overnight incubation with 50 µM of flavonoids. After incubations, cells were allowed to repair the DSBs and proliferate for 48 h in a drug-free medium, supplemented with cytokines. The occurrence of MLL abnormalities due to illegitimate repair was subsequently investigated using an inverse PCR approach (Figure 3A), which detects various MLL translocations irrespective of the fusion partners (15). Using this method, we were able to detect rearrangements in intron 11 and exon 12 of the MLL gene, which is a translocation breakpoint hotspot in infant- (32) and treatment-related leukemias (33).


Figure 2
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  		Fig. 2. Viability of CD34+ cells. Viability of cells was determined after overnight exposure to various concentrations (1, 10 and 50 µM) of flavonoids, etoposide, hydroquinone and the vehicle DMSO (0.05%) by trypan blue exclusion.

 


Figure 3
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  		Fig. 3. Inverse PCR on genomic DNA of CD34+ cells. Genomic DNA was digested by XbaI restriction enzyme and circularized by self-ligation. The circularized ligation products were used for first PCR and nested PCR using primer pairs that point away from each other. Amplification of the wt MLL by inverse PCR generates a 1.8 kb product. MLL translocations, however, generate alternative sized PCR products. The size of each PCR product is determined by both the location of the breakpoint junction in MLL and the position of the XbaI site in the fusion partner (A). The inverse PCR products were visualized by gel electrophoresis. The cells exposed to the vehicle DMSO 0.05% (B) showed no abnormal PCR products. In contrast, exposure to concentrations of 1, 10 and 50 µM of flavonoids (C) or 50 and 100 µM of etoposide (D) generated alternative sized bands. Lanes 1–5 in (B), lanes 1–5, 6–10 and 11–15 in (C) and lanes 1–4 and 5–8 in (D) demonstrate representative products of parallel inverse PCR on equal aliquots of DNA (~40 ng) isolated from single exposures. *The amplification of the wt MLL is visible in (B), lane 1 and (C) lane 1, 6 and 11. In all the other lanes [(B) lanes 2–5, (C) lanes 2–5, 7–10 and 12–15 and (D) lanes 1–8], the wt MLL is eliminated prior to PCR to favor amplification of MLL translocations.

 
Inverse PCR on the DNA isolated from untreated samples or DMSO-treated samples resulted in amplification of the wt MLL only (1.8 kb) (Figure 3B), which was confirmed by sequence analysis. Similarly, the hydroquinone-exposed cells generated one single band of 1.8 kb, as visualized by agarose gel electrophoresis (data not shown). In contrast, exposure of the CD34+ cells to quercetin, kaempferol, genistein and etoposide generated multiple bands of different sizes, along with the wt MLL (Figure 3C). The variety in the size of PCR products was determined by both the location of the breakpoint junction in MLL and the position of the XbaI site in the fusion partner. The amplification of the alternative sized bands became more prominent when the wt transcript was eliminated prior to PCR by enzymatic digestion. The nucleotide sequence analysis of the abnormal-sized products demonstrated fusion of the MLL gene with different chromosomes. Table I demonstrates the nucleotide sequence of the breakpoint junction of 23 distinct MLL translocations induced by flavonoids. The induction of genomic MLL fusions as a result of exposure to flavonoid was demonstrated in at least three independent experiments in cells isolated from different umbilical cord blood samples. Remarkably, quercetin and genistein appeared to result in a higher number of MLL translocations than kaempferol and etoposide (Figure 3C and D). Each aliquot of DNA that was used for PCR (40 ng DNA corresponding to 6000 cells) contained an average of three MLL translocations, when the cells were treated with a concentration of 50 µM quercetin or genistein (~500 translocations per 106 cells). The frequency of MLL translocations in kaempferol- or etoposide-treated cells was considerably lower (<200 translocation per 106 cells). As the inverse PCR method favors amplification of smaller fusion products (15), we mainly recovered fusions partners that possessed an XbaI recognition site, in the vicinity of the breakpoint junction. Therefore, we assume that the frequency of MLL translocations in flavonoid-exposed cells might be even higher.


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  		Table I. Analysis of the breakpoint junctions of distinct flavonoid-induced MLL translocations

 
Similar to the MLL translocations observed in childhood leukemias, most flavonoid-induced breakpoints occurred in intron 11 within the SINE/Alu or LINE/L1 elements (Figure 4). In all but one translocation, SINE or LINE repetitive elements were involved in at least one side of the breakpoint junction. Four translocations displayed LINE/L1-LINE/L1 and two Alu-Alu recombinations. Both Alu-Alu recombinations were associated with extensive sequence homologies between the fusion partners. Furthermore, short segments of overlapping nucleotides (1–12 nucleotides) were present in 19/23 translocations, indicating microhomology-mediated end joining. In addition, 15/23 translocations exhibited short palindromic sequences (4–10 nucleotides) at the breakpoint junction (Table I).


Figure 4
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  		Fig. 4. The flavonoid-induced MLL translocation junction co-localize with those identified in childhood leukemias. The genomic localization of MLL breakpoints identified in this study and those detected in childhood leukemias are designated with arrowheads, respectively, above and below the figure. The breakpoints occurred in MLL intron 11, mainly within the repetitive elements LINE/L1 and SINE/Alu. The location of MLL breakpoints in childhood leukemias is obtained from the National Center for Biotechnology Information database (gi:57471673, gi:31505705, gi:57471692, gi:2605918, gi:37954733, gi:57471684, gi:5921386, gi:57471691, gi:71794113, gi:71794126, gi:57471665, gi:71794112, gi:71794111, gi:101914504, gi:71794114, gi:57471678 and gi:71794123).

 
FISH
In order to show MLL aberrations at chromosomal levels, we applied dual-color FISH analysis on interphase nuclei. Cells were incubated with either DMSO or quercetin and allowed to recover and proliferate for 48 h. When exposed to concentrations of 50 and 100 µM of quercetin, cleavage of the MLL gene (split signal) was observed in, respectively, 0.7% (4/599) and 0.5% (2/404) of the cells (Table II), whereas none of the DMSO-exposed cells (n = 988) demonstrated a split signal. Monosomy or trisomy of MLL, displayed as simultaneous gain or loss of both green and red signals, was observed in 8.0% (37/599 gain and 11/599 loss of both signals) and 10.1% (19/404 gain and 22/404 loss of both signals) of the cells treated with, respectively, 50 and 100 µM quercetin. The percentage of cells with monosomy (20/988) or trisomy (16/988) of MLL in the control group was significantly lower (3.6%, Fisher's exact test, P < 0.001). Gain or loss of either a red or a green signal was observed in 1.7% (7/599 gain of the red signal, 1/599 loss of the red signal and 2/599 loss of the green signal) and 4.5% (13/404 gain of the red signal, 4/404 loss of the red signal and 1/404 gain of the green signal) of the cells treated with 50 and 100 µM quercetin, respectively, comparing to 0.9% (6/988 gain and 3/988 loss of the red signal) in the DMSO-exposed cells (Fisher's exact test, P = 0.15 and P < 0.001, respectively).


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  		Table II. The percentages of MLL abnormalities detected by FISH analysis

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Several studies have reported that exposure to the topoII-inhibiting drug etoposide induces MLL translocations in CD34+ cells (14–16), and may ultimately give rise to leukemic clones (7,8). In this study, we report for the first time that flavonoids induce similar MLL translocations in stem cell-enriched population. Surprisingly, the number of MLL translocations that were detected by inverse PCR was higher in quercetin- and genistein-exposed cells than in cells treated with etoposide.

Strick et al. (21) had previously demonstrated that certain flavonoids induce a reversible break in the MLL gene. To examine the extent of such flavonoid-induced DNA strand breaks in CD34+ cells, we carried out the neutral Comet assay. Using this assay, we demonstrated dose-dependent DSB formation due to flavonoid exposure. Significant levels of DSBs were particularly detected at concentrations higher than 10–25 µM of flavonoids, presumably due to the detection limit of the Comet assay. Likewise, Comet assay could not detect a significant level of DSBs in cells exposed to concentrations of 1–10 µM of etoposide. At higher concentration, the level of DSBs induced by etoposide was significantly lower than that induced by quercetin (P = 0.02). This may explain the lower number of etoposide-induced MLL translocations detected by the inverse PCR assay. A correct repair of these DSBs by either homology-dependent or non-homologous end joining is essential for the maintenance of genomic integrity, as an aberrant repair can result in chromosomal abnormalities (34). To examine whether flavonoid-induced DSBs can lead to MLL abnormalities, CD34+ cells were allowed to recover for 48 h after flavonoid exposure. During this recovery period most dead and apoptotic cells were cleared and the cells that managed to repair the DSBs continued to proliferate. Subsequently, both inverse PCR and FISH analysis were applied to determine the frequency of MLL abnormalities. The inverse PCR approach demonstrated that flavonoid exposure results in fusion of the MLL gene to a variety of different partners in the genome. Some fusion partners were genes that encode functionally important proteins, for instance histone deacetylase 3 that regulates the access of transcription factors to DNA by deacetylation of histone proteins (35). Another fusion partner was a tumor suppressor gene that encodes protein tyrosine phosphatase receptor type T and is mutated in some cases of colorectal cancer (36). Interestingly, one of the fusion partners of MLL, the gene that codes for Fragile X mental retardation syndrome protein (FMR1), is one of the five folate-sensitive fragile sites in the genome. Another folate-sensitive fragile site, FMR2 is a member of the AF4/LAF4/AF5q31 family of genes (37,38), which is frequently involved in MLL translocations (39–41). We hypothesize that the anti-folate activity of some flavonoids (42) may contribute to DSB formation in common fragile sites of the genome. Our hypothesis is supported by the finding that folate supplementation during pregnancy (43) as well as a polymorphism in the methylentetrahydrofolate reductase gene protect against the development of MLL+ childhood leukemias (44).

Further analysis of the breakpoint sequences demonstrated that most MLL translocations were generated by microhomology-mediated end joining, suggesting that a short nucleotide sequence overlap may facilitate the religation of the cleaved DNA ends. Two translocations exhibited extensive sequence homologies at the breakpoint junctions indicating the involvement of the homology-mediated single-strand annealing repair pathway (45). It is interesting that both translocations were generated due to Alu-Alu recombination. Alu elements are known to be involved in a wide range of chromosomal translocations (46), including MLL abnormalities (47). In addition to the Alu repeats, other repetitive elements were also observed at the breakpoint junctions. LINE–LINE fusion, for instance, was observed in four distinct translocations. Furthermore, we detected short palindromic sequences in the vicinity of most breakpoint junctions. The possible contribution of these palindromes to the development of chromosomal translocations needs further investigation.

The inverse PCR analysis demonstrated that genistein and quercetin are strong inducers of MLL translocations. We estimated the frequency of MLL translocation detected by inverse PCR as 500 per 106 cells exposed to 50 µM quercetin or genistein. Strikingly, MLL abnormalities were less frequent in kaempferol- and etoposide-treated cells (<200 per 106 cells). Nonetheless, the estimated frequency of etoposide-induced MLL rearrangements in our study was in accordance with that reported by Libura et al. (150 per 106 cells) (15). Although these estimations do not reflect the exact frequency of MLL aberrations, they can be used to broadly compare the clastogenic effect of different compounds. We did not detect any MLL abnormalities following exposure of CD34+ cells to DMSO or hydroquinone. Hydroquinone seems to be involved in the development of other leukemia-associated abnormalities, including deletions and monosomies of chromosomes 5 and 7 (27,29).

As the inverse PCR approach only detects translocations involving the breakpoint hotspot in intron 11 and exon 12 (15), we carried out FISH analysis to detect a broader range of MLL aberrations. A split signal was detected in 0.5–0.7% of the CD34+ cells after quercetin exposure. Surprisingly, 8–10% of the quercetin-exposed CD34+ cells demonstrated monosomy or trisomy of MLL. It is possible that quercetin also induces DSBs in the centromeric part of 11q23, which may result in asymmetric distribution of the telomeric segment in daughter cells. This phenomenon has been described earlier, in Ataxia Telangiectasia Mutated-deficient fibroblasts following etoposide exposure (48).

Previous reports demonstrated development of MLL rearrangements in a lymphoblastoid cell line after treatment with the apoptotic stimulus anti-CD95 (49,50). Nevertheless, we believe that the MLL translocations detected in primary CD34+ stem cells in our study were not induced by an apoptotic trigger. One important argument is that the frequency of MLL translocations did not correlate with apoptotic cell death. For instance, hydroquinone resulted in more apoptotic cell death than flavonoids, whereas no MLL translocations could be detected following exposure to this agent. The fact that topoII-targeting agents give rise to MLL+ leukemias (7,8) also confirms the specific role of flavonoids in inducing such translocations.

In our study, a flavonoid concentration as low as 1 µM already resulted in MLL translocations in CD34+ cells. Consumption of 220 g onions results in plasma quercetin concentration of 1.5 µM (51). As our food contains multiple flavonoids, the total topoII-inhibiting effect should be in proportion to the sum of flavonoid-rich food consumption. Recently, Todaka et al. (52) have demonstrated that the levels of flavonoids are higher in human umbilical cord blood than in maternal serum, possibly due to differences in metabolism and excretion rates between mother and fetus. It seems that some flavonoids incline to stay longer on the fetal side than on the maternal side. This is particularly alarming knowing that fetal cells have high topoII activity (53) due to their rapid proliferation and are therefore likely to be more sensitive to the topoII-inhibiting effect of flavonoids. Currently, the marketing of flavonoids as dietary supplement raises concern about their adverse consequences. Worldwide, flavonoid supplements are available on over-the-counter basis in all pharmacies, drugstores and even supermarkets. The manufacturers recommend a daily dose of up to 1200 mg/day of these supplements. Strikingly, plasma quercetin concentration after ingestion of a dose of 300 mg quercetin already reaches a clastogenic concentration of 10 µM (54). Currently, several clinical trials are investigating the therapeutic potentials of flavonoids in the treatment of different types of cancer including leukemias (55). The clastogenic effect of certain flavonoids may, however, confound their potential clinical usefulness (56).

As prenatal exposure to high concentration of flavonoids might contribute to the development of infant leukemia, it is important to raise public awareness about their potential health hazards and set guidelines for marketing herbal medicines and flavonoid supplements.


   Acknowledgments
 
We sincerely thank Jill Hagelstein for the Comet data, Bertien Hollanders-Crombach and Karien Mebis-Verhees for their assistance in cytogenetic analysis and Dr Geja Hageman and Liesbeth Geraets for providing kaempferol and genistein. We are also grateful to the personnel of the Department of Obstetrics and Gynecology, especially Dr Florien ten Cate for kindly providing umbilical cord blood for this study. This work was supported by grant number 06A031 from the American Institute for Cancer Research.

Conflict of Interest statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 7, 2006; revised April 17, 2007; accepted April 19, 2007.


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