Mutagenesis vol.15 no.1 pp.77–83, 2000
Assessment of the adaptive response induced by quercetin using the MNCB peripheral blood human lymphocytes assay
N.G.Oliveira1,2, M.Neves1, A.S.Rodrigues1,3, O.Monteiro Gil1,4, T.Chaveca1,2 and J.Rueff1,5 1Department
of Genetics, Faculty of Medical Sciences, New University of Lisbon, R. da Junqueira 96, P-1349-008 Lisbon, 2Faculty of Pharmacy, University of Lisbon, Lisbon, 3University Luso´fona, Lisbon and 4Nuclear and Technological Institute, Department of Radiological Protection and Nuclear Safety, Sacave´m, Lisbon, Portugal
Over more than two decades the existence of an adaptive response (AR) has been reported in several cell types and extensively studied with low doses of radiation. Besides radiation, some chemicals [alkylating compounds, mitomycin C (MMC), bleomycin, hydrogen peroxide and metals] may also induce an adaptive response. We have recently reported that the food mutagen quercetin can also induce an adaptive response in V79 Chinese hamster cells. In this work we have studied the effect of low doses of quercetin on the genotoxicity of MMC and bleomycin assessed by the formation of micronuclei in cytokinesisblocked (MNCB) human peripheral blood lymphocytes. Our results suggest the existence of an AR induced by quercetin in human lymphocytes. Seven of the nine donors studied showed in at least one independent experiment a significant decrease in the frequency of MNCB induced by MMC. The range of these decreases varied between 31 and 58%. In addition, we observed an AR induced by quercetin towards challenging doses of bleomycin. In accordance with other studies with ionizing radiation in which heterogeneity of the AR in the population has been extensively observed, the response here reported also showed some degree of variability between the different donors studied. In view of the results obtained one cannot rule out a possible protective effect of low doses of quercetin leading to adaptation to further exposure to mutagens or carcinogens. Introduction Over more than two decades the existence of an adaptive response (AR) has been reported by several authors and there is growing evidence of its importance in the protection of cells and organisms against genotoxic injury (Stecca and Gerber, 1998; for a review see Wolff, 1998). An AR has been reported both in prokaryotes (Samson and Cairns, 1977; Lindahl et al., 1988) and in eukaryotes (Samson and Schwartz, 1980; Olivieri et al., 1984; Ikushima, 1987; Cai and Liu, 1990; Corte´s et al., 1990; Wojcik and Tuschl, 1990). This phenomenon has been studied mainly with low doses of radiation (Olivieri et al., 1984; Ikushima, 1987; Boothman et al., 1996; Ueno et al., 1996; Wojcik et al., 1996; Wolff, 1998). However, alkylating compounds (Samson and Schwartz, 1980; Laval and Laval, 1984; Frosina and Abbondandolo, 1985; Morimoto et al., 1986; Mahmood and Vasudev, 1993; Nikolova and Huttner, 1996), mitomycin C (MMC) (Moquet et al., 1989; Madrigal-Bujaidar et al., 1994), bleomycin (Wolff 5To
et al., 1988; Vijayalaxmi and Burkart, 1989a,b; Mozdarani and Saberi, 1994; Ne´me´thova´ et al., 1995), hydrogen peroxide (Laval, 1988; Wolff et al., 1988; Corte´s et al., 1990; Domı´nguez et al., 1993; Wiese et al., 1995; Flores et al., 1996; Anuszewska et al., 1997), metals (Cai and Cherian, 1996) and monoepoxybutene (Sasiadek and Paprocka-Borowicz, 1997) may also induce an AR. Quercetin is the major dietary flavonoid and is ingested in edible fruits and vegetables (Hertog et al., 1995) at levels up to 15 mg/day in Western diets (Rimm et al., 1996). The genotoxicity of this compound may occur by different pathways but the generation of reactive oxygen species (ROS) seems to be the most important mechanism in mammalian cells (Rueff et al., 1992; Gaspar et al., 1994; Caria et al., 1995). Hydroxyl radicals could be the ultimate responsible species for the similarity of patterns of genotoxicity between quercetin and ionizing radiation. Recently we reported that the food mutagen quercetin could also induce an AR in mammalian cells (Oliveira et al., 1997). Low doses of quercetin were able to induce an AR towards challenging doses of the same compound and other mutagens, namely hydrogen peroxide and MMC, using the induction of chromosomal aberrations in V79 cells as the end-point. In the present work we report on the induction of an AR by low doses of quercetin in human lymphocytes. We have studied the effect of low doses of quercetin on the genotoxicity of MMC (3.0 µM, treatment for 3 h) and bleomycin (bleomycin sulphate, BS) (50 and 100 µg/ml, treatment for 3 h) as assessed by the formation of micronuclei in cytokinesis-blocked (MNCB) human peripheral blood lymphocytes. Materials and methods Chemicals and culture media Quercetin, MMC, foetal calf serum, Ham’s F-10 medium, cytochalasin B, Lglutamine, penicillin and streptomycin were purchased from Sigma (St Louis, MO). Acetic acid, dimethylsulphoxide (DMSO), methanol, potassium chloride and Giemsa dye were obtained from Merck (Darmstadt, Germany). Phytohaemagglutinin (PHA) (HA 15) was purchased from Murex (Dartford, UK) and reconstituted in 5 ml of sterile water. Heparin was obtained from Braun (Melsungen, Germany). BS was obtained from Laborato´rios Delta (Portugal). Lymphocyte cultures Heparinized peripheral blood samples were obtained from nine healthy nonsmoker donors not exposed to radiation or drugs. The replicate experiments for each donor were carried out with blood samples collected at different time intervals over a 2 year period. Aliquots of 500 µl of whole blood were cultured in 4.5 ml Ham’s F-10 medium supplemented with 24% foetal calf serum, penicillin (100 IU/ml), streptomycin (100 µg/ml), 1% L-glutamine and 1% heparin (50 IU/ml). Lymphocytes were stimulated using 25 µl of PHA and incubated at 37°C. Adaptation protocol Independent experiments were performed to assess the AR induced by quercetin towards MMC and bleomycin genotoxicity. Nineteen hour cultures growing in tissue culture tubes (Greiner; Frickenhausen, Germany) were exposed to an adaptive dose (AD) of quercetin (3.3 µM) dissolved in DMSO. After 5 h incubation a challenge dose (CD) of MMC (3.0 µM) or BS (50 and
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© UK Environmental Mutagen Society/Oxford University Press 2000
77
N.G.Oliveira et al.
Table I. Adaptive response induced by low doses of quercetin in human lymphocytes towards MMC genotoxicity assessed by the MNCB assay Donor/exp.
Age/sex
Treatment
CB (%)
PoliN (%)
Met (%)
PI (%)
PI’ (%)
MNCB (‰) Observed
A/1
22/F
A/2
A/3
A/4
B/1
22/F
B/2
C/1
22/M
C/2
D/1
22/F
D/2
E/1
35/F
E/2
E/3
E/4
F/1
23/F
F/2
G/1
G/2
78
23/M
DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO
54.3 77.6 30.5 41.2 65.2 54.6 39.4 44.0 50.2 45.0 40.7 42.9 65.2 64.2 33.4 30.7 61.8 75.6 54.5 48.2 52.7 60.3 44.3 58.1 54.7 74.5 24.7 34.8 53.7 56.0 43.4 45.9 68.9 71.3 57.9 57.7 49.6 45.0 36.3 36.9 59.8 59.3 22.7 10.5 48.1 57.9 32.1 28.8 57.4 50.1 9.6 17.5 52.3 46.9 26.4 35.5 43.4 47.9 32.4 30.4 58.3 64.5 43.7 44.7 51.9 65.7 40.5 49.1 59.0
21.0 3.0 2.1 5.4 5.9 9.7 8.2 2.6 19.4 20.5 5.8 5.4 5.9 8.8 2.0 2.7 16.5 5.0 5.8 6.5 17.6 11.8 3.9 4.3 16.4 4.3 1.3 2.6 25.2 20.2 6.1 20.3 10.2 13.4 4.7 8.9 18.0 15.4 15.9 9.9 1.5 5.8 4.1 3.1 18.8 3.5 11.9 11.1 8.2 4.3 0.6 1.7 16.3 19.8 3.9 6.3 13.9 13.2 6.2 6.8 21.2 22.1 8.4 10.8 1.1 10.5 3.9 4.0 6.1
1.7 1.9 2.4 0.7 1.8 1.2 1.9 1.5 1.6 0.9 3.9 1.8 0.7 1.0 0.3 0.3 1.2 1.4 1.4 1.2 1.9 1.8 1.4 1.7 1.1 2.1 0.4 0.8 3.0 0.8 1.1 3.7 1.1 1.4 1.2 1.9 4.8 5.1 1.6 3.4 0.8 0.6 2.6 0.8 3.7 2.0 2.2 2.5 1.7 5.1 1.1 2.0 1.7 1.5 0 0.6 0.7 1.6 1.6 2.1 2.0 0.5 1.2 1.6 0.1 1.2 1.0 1.0 2.0
100.0 85.7 61.9 71.4 100.0 100.0 83.3 83.3 100.0 100.0 70.0 75.0 100.0 100.0 72.2 72.2 100.0 95.0 80.0 80.0 100.0 94.7 73.7 89.5 100.0 90.0 65.0 70.0 100.0 95.4 72.7 86.4 100.0 105.3 89.5 94.7 100.0 89.5 89.5 78.9 100.0 106.3 81.2 75.0 100.0 89.5 84.2 78.9 100.0 83.3 61.1 61.1 100.0 105.3 68.4 78.9 100.0 100.0 77.8 77.8 100.0 104.8 76.2 81.0 100.0 126.7 93.3 100.0 100.0
100.0 94.7 68.4 78.9 100.0 100.0 88.2 82.4 100.0 90.0 70.0 75.0 100.0 100.0 72.2 72.2 100.0 94.7 84.2 84.2 100.0 100.0 77.8 88.9 100.0 94.7 68.4 73.7 100.0 100.0 75.0 90.0 100.0 105.3 89.5 89.5 100.0 94.4 94.4 83.3 100.0 106.3 81.2 75.0 100.0 88.9 83.3 83.3 100.0 88.2 64.7 64.7 100.0 100.0 66.7 77.8 100.0 100.0 82.4 82.4 100.0 110.5 84.2 84.2 100.0 126.7 93.3 100.0 100.0
0.0 1.0 205.1 117.4 2.0 3.0 131.4 65.0 0 4.0 124.5 126.9 1.2 2.9 89.9 74.8 3.1 2.8 69.8 53.5 1.1 3.5 53.1 27.0 0.0 0.0 87.5 59.2 1.0 2.0 71.2 52.6 4.0 6.9 114.5 127.4 2.0 4.0 80.9 76.9 1.0 2.0 186.7 78.5 3.0 4.0 125.9 79.0 1.0 5.0 158.3 135.8 1.0 4.9 163.2 104.8 3.9 5.0 96.9 64.1 1.9 5.0 76.8 50.9 0.0 3.0 88.0 48.9 3.0
Expected
Decrease (%) P value
206.1
43.0
⬍0.001
132.4
50.9
⬍0.001
128.5
1.3
NS
91.6
18.3
NS
69.5
23.0
NS
55.5
51.4
⬍0.01
87.5
32.3
⬍0.01
72.2
27.1
⫽0.06
117.4
–8.6
NS
82.9
7.2
NS
187.7
58.0
⬍0.001
126.9
37.7
⬍0.001
162.3
16.3
⫽0.09
167.1
37.3
⬍0.001
98.0
34.6
⬍0.01
79.9
36.3
⬍0.01
91.0
46.3
⬍0.001
Adaptive response induced by quercetin
Table I. Continued Donor/exp.
Age/sex
Treatment
CB (%)
PoliN (%)
Met (%)
PI (%)
PI’ (%)
MNCB (‰) Observed
G/3
H/1
23/F
H/2
I/1
I/2
23/F
AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD DMSO AD CD AD⫹CD
39.6 36.1 44.1 67.6 69.8 31.4 33.6 58.7 49.1 37.9 36.0 51.8 58.3 43.2 38.2 59.8 56.8 26.9 45.6 57.0 52.5 50.5 53.2
3.4 0.6 2.8 5.9 9.3 0.5 0.4 11.9 25.3 8.5 12.5 16.2 11.8 5.6 4.1 13.1 4.6 3.1 3.8 8.3 10.9 6.3 7.1
2.1 1.6 1.6 1.7 0.8 0.4 0.7 0.7 1.9 1.0 1.5 2.0 1.0 1.3 1.7 0.5 1.7 1.4 0.8 1.7 3.1 2.0 2.0
88.2 76.5 88.2 100.0 105.6 72.2 72.2 100.0 122.2 88.9 88.9 100.0 94.7 78.9 73.7 100.0 85.0 70.0 80.0 100.0 100.0 94.1 100.0
82.4 76.5 88.2 100.0 105.6 72.2 72.2 100.0 111.1 83.3 88.9 100.0 100.0 83.3 77.8 100.0 84.2 73.7 84.2 100.0 100.0 94.1 100.0
2.0 41.3 30.5 1.9 2.0 132.1 124.8 3.9 4.9 69.2 105.8 1.0 1.0 95.9 110.7 3.0 4.0 160.7 111.1 1.4 1.9 76.6 51.2
Expected
Decrease (%) P value
40.3
24.3
NS
132.2
5.6
NS
70.2
–50.7
⬍0.01
95.9
–15.4
NS
161.7
31.3
⬍0.01
77.1
33.6
⬍0.05
DMSO, solvent control; AD, quercetin 3.3 µM, pretreatment for 5 h; CD, MMC 3.0 µM, incubation for 3 h; AD ⫹CD, AD followed by CD; NS, not significant.
100 µg/ml) was added to the culture medium. The cultures were grown for a further 3 h and then washed and fresh culture medium added. Micronucleus (MN) assay in vitro Cytochalasin B (stock solution 8.34 mM prepared in DMSO) was added to 44 h cultures at a final concentration of 12.5 µM. At 72 h the cultures were harvested by centrifugation at 120 g for 10 min. The supernatants were removed and the pellet was gently treated with 3 ml of 0.11 M KCl supplemented with 2% foetal calf serum and immediately centrifuged at 120 g for 5 min. Cells were fixed with freshly prepared methanol:acetic acid (3:1) for 10 min and then centrifuged at 120 g for 5 min. This protocol was repeated twice. Cell suspensions were carefully dropped onto two to four wet slides. After air drying for 1–2 days the slides were stained with 4% Giemsa in phosphate buffer, pH 7.3, for 10 min. At least 1000 cytokinesis-blocked (CB) (binucleated) human lymphocytes with preserved cytoplasm were scored whenever possible for each dose treatment. MN were identified in accordance with the criteria followed by Caria et al. (1995). Control cells included a DMSO control (solvent only), an AD control (quercetin, 3.3 µM) and a CD control (MMC, 3.0 µM or BS, 50 or 100 µg/ml). DMSO concentration for both adapted and control cells did not exceed 15 mM. Cytotoxicity was evaluated by classifying at least 1000 lymphocytes according to the number of nuclei and two indices besides per cent CB cells were used. The proliferating index PI is the ratio of the nuclear division index (NDI) calculated according to Eastmond and Tucker (1989) between treated samples and DMSO control samples. The proliferating index PI’ is the ratio of CB proliferating index (CBPI) proposed by Surralle´ s et al. (1995) between treated samples and DMSO control samples. NDI and CBPI were calculated according to the following formulae: NDI ⫽ (MI ⫹ 2MII ⫹ 3MIII ⫹ 4MIV)/ total and CBPI ⫽ [MI ⫹ 2MII ⫹ 3(MIII ⫹ MIV)]/total, where MI–MIV represent the number of human lymphocytes with one to four nuclei. Expected values are considered to be the sum of MNCB human lymphocytes induced by the AD and those induced by the CD, subtracting the DMSO control values. Expected and observed values (MNCB) were compared using the χ2 test (Tables I and II).
Results Table I presents the frequencies of MNCB induced by MMC (3.0 µM, 3 h) in human lymphocytes when the cells were exposed to a single dose of quercetin (3.3 µM) for 5 h prior
to MMC treatment. These represent the results from two to four independent experiments carried out for each donor. Figure 1 presents the percentage decrease in the frequencies of MNCB human lymphocytes after quercetin pretreatment. This figure shows the average results from two to four independent experiments carried out for each donor. Seven donors presented in at least one independent experiment a significant decrease between expected and observed frequencies of MNCB human lymphocytes (donors A, B, C, E, F, G and I). The range of these decreases varies between 31 and 58%. In four of these donors this response was consistently reproduced (C, E, F and I), although in the third experiment with donor E and in the second experiment with donor C there was only a statistical trend towards a decrease in the response. Concerning donors A, B and G, despite some important decreases in the frequencies of MNCB, these were not consistent. We have observed different patterns of cytogenetic response to quercetin pretreatment. Heterogeneity of the response from different donors is observed in Figure 1. Donor D presented no decrease between expected and observed frequencies of MNCB in both experiments and donor H showed an important synergistic cytogenetic response to quercetin. Table II presents data from experiments performed with lymphocytes from three donors (A, C and E) in order to study the existence of AR induced by quercetin towards bleomycin clastogenicity. According to Table II one can observe a decrease in the frequencies of MNCB induced by both CDs of BS (50 and 100 µg/ml, 3 h) in human lymphocytes when the cells were exposed to an AD of quercetin (3.3 µM). The AD of quercetin, 3.3 µM (Tables I and II), did not lead to a difference in the three cytotoxicity indices [CB (%), PI and PI’] when compared with control cells. Moreover, in all 79
N.G.Oliveira et al.
Table II. Adaptive response induced by low doses of quercetin in human lymphocytes towards BS genotoxicity assessed by the MNCB assay Donor
Treatment
CB (%)
PoliN (%)
Met (%)
PI (%)
PI⬘ (%)
MNCB (‰) Observed
E
A
C
DMSO AD CD1 AD⫹CD1 CD2 AD⫹CD2 DMSO AD CD1 AD⫹CD1 CD2 AD⫹CD2 DMSO AD CD2 AD⫹CD2
53.2 45.3 40.9 46.0 43.3 48.4 48.3 52.1 48.6 47.3 37.6 37.2 49.8 52.6 36.8 43.9
15.8 10.8 7.5 9.5 6.0 6.7 11.0 11.3 10.2 15.3 9.1 3.2 6.1 7.9 5.6 5.6
0.5 2.2 1.5 2.1 1.2 1.2 0.4 0.6 1.1 1.3 0.2 0.2 0.8 0.6 0.6 0.5
100.0 88.5 82.2 86.7 81.3 85.3 100.0 103.1 98.2 104.0 90.5 81.8 100.0 104.2 90.0 94.7
100.0 89.3 83.7 88.4 83.5 87.1 100.0 102.4 98.9 103.9 91.6 84.4 100.0 104.1 91.4 96.0
13 13 100 75 104 74 2 5 90 61 107 58 5 4 129 92
Expected
Decrease (%)
P value
100
25.0
⬍0.05
104
28.8
⬍0.05
93
34.4
⬍0.01
110
47.3
⬍0.001
128
28.1
⬍0.05
DMSO, solvent control; AD, adaptive dose of quercetin (3.3 µM), pretreatment for 5 h; CD, challenge dose of BS, treatment for 3 h; CD1, 50 µg/ml; CD2, 100 µg/ml; AD⫹CD, AD followed by CD.
Fig. 1. Mean percentage and standard deviation of the decrease in the frequencies of MMC-induced MNCB human lymphocytes (MMC, 3.0 µM, 3 h) from nine donors in two to four independent experiments after quercetin pretreatment of the lymphocytes (3.3 µM) for 5 h.
the experiments, no marked trend was observed in these indices in challenged cells when compared with conditioned and challenged cells. The results presented clearly suggest the existence of an AR induced by low doses of quercetin in human lymphocytes. Discussion We have recently reported that quercetin could induce an AR in V79 Chinese hamster cells (Oliveira et al., 1997). Low doses of this compound were able to reduce the cytogenetic damage induced by challenging doses of this compound and other mutagens, namely hydrogen peroxide and MMC. In the present study a similar protocol was carried out in order to investigate this response in human lymphocytes from nine donors. In these experiments we have chosen an AD of quercetin (3.3 µM) which induced an AR in the previous study with 80
V79 Chinese hamster cells (Oliveira et al., 1997). This dose was chosen because it is a very low dose that does not induce MN in human lymphocytes, as shown in Tables I and II, and also because it does not lead to differences in the three cytotoxicity indices [CB (%), PI and PI’] when compared with control cells. In fact, higher doses of quercetin could cause cell cycle arrest (Yoshida et al., 1992) and be rather cytotoxic. Although quercetin significantly induces MN in human lymphocytes at doses ⬎10 µM (Caria et al., 1995), we have chosen two well-studied human lymphocyte MN inducers, MMC and bleomycin, as the challenging agents since their genotoxicities are higher than that of quercetin in human lymphocytes, thus rendering the detection of an AR easier. These challenging agents were also selected because their mechanisms of genotoxicity, which are apparently rather different, include, however, a similar pathway, namely the generation of ROS. MMC is a crosslinker, but its genotoxicity
Adaptive response induced by quercetin
can also be explained by other mechanisms, including a oneelectron reduction to a semiquinone free radical (Halliwell and Gutteridge, 1995) in which some reductases are involved (Vromans et al., 1990; Goeptar, 1993). This reduction pathway is dependent on the presence of oxygen, with production of superoxide anion, hydrogen peroxide and hydroxyl radicals (Goeptar, 1993). Quercetin and MMC may have a partially similar DNA damage mechanism, which may be important but not indispensable for the outcome of a cross-adaptation. There are some reports of cross-adaptation between MMC and different agents that generate ROS. Wolff et al. (1988) have reported an AR with low doses of β-rays (tritiated thymidine) against MMC-induced chromatid aberrations. An AR was also present in other studies that reported similar in vitro experiments with both X-rays (Moquet et al., 1989) and γ-rays (Ikushima, 1989; Osmak and Horvat, 1992) and even with α-rays (Tuschl et al., 1983) as adaptive agents towards MMC genotoxicity. We have studied a group of nine donors in order to assess the existence of an AR induced by a low dose of quercetin (3.3 µM) towards a CD of MMC (3.0 µM). The results presented show the existence of an AR induced by quercetin in human lymphocytes against MMC genotoxicity using the MNCB assay as the end-point. This AR was not always present in the experiments performed, suggesting an important degree of variability between donors. Heterogeneity of the AR in human lymphocytes to ionizing radiation as well as to other agents has been reported and discussed by several authors (Morimoto et al., 1986; Bosi and Olivieri, 1989; Sankaranarayanan et al., 1989; Wojcik and Tuschl, 1990; Olivieri et al., 1994; Vijayalaxmi et al., 1995; Zhang, 1995; Nikolova and Hutnner, 1996; Salone et al., 1996). We have observed the three types of response described by others in previous experiments, namely the presence of AR, absence of AR and synergism (Bosi and Olivieri, 1989; Sankaranarayanan et al., 1989; Salone et al., 1996). Four of the nine donors displayed a clear decrease in the frequencies of MMC-induced MN in repeated experiments (C, E, F and I), while donor D presented absence of an AR to quercetin and donor H showed an important synergism with this flavonoid. The existence of a characteristic type of response for both the presence of AR (Bosi and Olivieri, 1989; Sankaranarayanan et al., 1989; Vijayalaxmi et al., 1995) and for its absence in some individuals (Bosi and Olivieri, 1989; Bauchinger et al., 1989; Hain et al., 1992) suggests that the AR might not be entirely due to transient factors but also to constitutive parameters, although both types of response in the same donor have been reported (Olivieri et al., 1994). The results from donors B and G and, especially, donor A seem to belong to the former. Donor A in the first two experiments showed a strong decrease in MMC-induced DNA damage and in the third experiment a clear absence of adaptation. These results seem to suggest that other unknown factors could be involved in this phenomenon. Temporal variations in some important intermediate factor(s) for the appearance of the AR or even in some external factors such as environmental agents or diet could explain these discrepant results. Some authors point out that a real understanding of the causes of human heterogeneity in the AR will only become clear when the mechanisms behind the phenomenon are revealed (Wojewdozka et al., 1994; Salone et al., 1996). The variability in the type of pattern exhibited by each donor has been explained on different grounds, including the
genetic constitution of the donor (Bosi and Olivieri, 1989; Sankaranarayanan et al., 1989; Wojcik and Tuschl, 1990; Vijayalaxmi et al., 1995; Nikolova and Hutnner, 1996; Salone et al., 1996), the experimental protocol used (Olivieri et al., 1994; Salone et al., 1996), the end-point selected (Salone et al., 1996), the experimental conditions of culture (Bosi et al., 1991; Salone et al., 1996), random alterations in cell cycle kinetics (Wojcik et al, 1996), age (Gadhia, 1998) and previous exposure to environmental agents (Nikolova and Hutnner, 1996). The antioxidant status of the donor (enzymatic and non-enzymatic) could also be an important factor for the AR. The experiments carried out with bleomycin as the challenging agent were performed in order to evaluate whether the protection provided by low doses of quercetin was also present towards two very high doses of a peculiar genotoxicant, bleomycin, in different donors using the MNCB assay. In fact, concerning bleomycin’s mechanism of genotoxicity, this natural antibiotic is considered to be a S-independent genotoxicant and is regarded as a true radiomimetic compound, resembling almost completely the genetic effects of ionizing radiation (Dresp et al., 1978; Hoffmann et al., 1993; Halliwell and Gutteridge, 1995). Therefore, the patterns of genotoxicity of quercetin and bleomycin should be quite similar. In addition, bleomycin has been used as the challenging agent in former studies of adaptation with low doses of ionizing radiation (Mozdarani and Saberi, 1994; Barquinero et al., 1996; Tedeschi et al., 1996). The results show that low doses of quercetin were also able to protect the cells against bleomycin-induced DNA damage. Finally, it is important to mention some recent perspectives on low dose exposure to toxic agents and risk assessment. Pollycove (1998), from the US Nuclear Regulatory Commission, reviewed data related to some beneficial health effects of low level radiation exposure. The report focused on the interest of these data in order to assess in a more realistic manner the environmental risks of radiation. The US Food and Drug Administration, as discussed in a comprehensive study by Gaylor et al. (1998), in the future intend to take into account data concerning both physiological ARs and other beneficial effects of low level exposure. In that report carcinogenic contaminants as well as radiation from medical devices were carefully excluded. For the latter these authors considered potential cancer risk at low levels of exposure. In the light of the results presented in this study one cannot rule out a possible protective effect of low doses of quercetin leading to adaptation to further exposure to mutagens or carcinogens. However, these results concern only in vitro experiments with human cells, which means that additional animal studies should be performed in order to assess the AR in vivo and thus clarify this point. Acknowledgements We gratefully acknowledge Dr P. Aguiar for the help in the statistical analysis. We also acknowledge Dr Ana Amaral from Laborato´ rios Delta. N.G.O. was supported by a fellowship from PRAXIS XXI programme. This work was supported in the framework of INTAS-KZ 95-32.
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